The Impact of Low-Level Iron Supplements on the Faecal Microbiota of Irritable Bowel Syndrome and Healthy Donors Using in Vitro Batch Cultures

nutrients

Article The Impact of Low-Level Iron Supplements on the Faecal Microbiota of Irritable Bowel Syndrome and Healthy Donors Using In Vitro Batch Cultures

Carlos Poveda 1, Dora I. A. Pereira 2, Marie C. Lewis 1 and Gemma E. Walton 1,* 1 Department of Food and Nutritional Sciences, University of Reading, P.O. Box 226, Whiteknights, Reading RG6 6AP, UK; [email protected] (C.P.); [email protected] (M.C.L.) 2 Department of Pathology, Tennis Court Road, University of Cambridge, Cambridge CB2 1QP, UK; [email protected] * Correspondence: [email protected]; Tel.: +44-118-378-6652

Received: 4 November 2020; Accepted: 8 December 2020; Published: 14 December 2020

Abstract: Ferrous iron supplementation has been reported to adversely alter the gut microbiota in infants. To date, the impact of iron on the adult microbiota is limited, particularly at low supplementary concentrations. The aim of this research was to explore the impact of low-level iron supplementation on the gut microbiota of healthy and Irritable Bowel Syndrome (IBS) volunteers. Anaerobic, pH-controlled in vitro batch cultures were inoculated with faeces from healthy or IBS 1 donors along with iron (ferrous sulphate, nanoparticulate iron and pea ferritin (50 µmol− iron)). The microbiota were explored by fluorescence in situ hybridisation coupled with flow cytometry. Furthermore, metabolite production was assessed by gas chromatography. IBS volunteers had different starting microbial profiles to healthy controls. The sources of iron did not negatively impact the microbial population, with results of pea ferritin supplementation being similar to nanoparticulate iron, whilst ferrous sulphate led to enhanced Bacteroides spp. The metabolite data suggested no shift to potentially negative proteolysis. The results indicate that low doses of iron from the three sources were not detrimental to the gut microbiota. This is the first time that pea ferritin fermentation has been tested and indicates that low dose supplementation of iron is unlikely to be detrimental to the gut microbiota.

Keywords: gut microbiota; iron; irritable bowel syndrome; nanoparticulate iron; pea ferritin

1. Introduction In 2015, it was reported that about a third of the World’s population suffered from anaemia, and in half of these cases, iron deficiency was the cause; there is no indication that this prevalence is decreasing [1]. Iron deficiency anaemia (IDA) is more prevalent in low-income countries, due to poor nutrient availability, reliance on plant-based foods and the impact of infection on iron absorption [2]. However, IDA is the main nutrient deficiency disorder affecting high-income countries [3]. The body absorbs iron from dietary sources; however, when dietary iron is insufficient, IDA can result, with the common symptoms being tiredness, fatigue, lethargy and breathlessness [4]. To help prevent development of IDA, low dose iron supplementation is commonplace. Iron supplements frequently take the form of ferrous (Fe2+) salts (e.g., ferrous sulphate, fumarate and gluconate) and ferric compounds (e.g., ferric ammonium citrate) [5]. However, solubility and bioavailability of these compounds differ; the ferrous form is more soluble and more readily absorbed, as well as cheaper to manufacture, and therefore, is the most commonly used [6,7]. Unfortunately, oral iron supplementation, particularly at high doses, is associated with negative side-effects, such as nausea, vomiting, constipation, diarrhoea, black stools and bloating, which often leads to non-adherence

Nutrients 2020, 12, 3819; doi:10.3390/nu12123819 www.mdpi.com/journal/nutrients Nutrients 2020, 12, 3819 2 of 21 with a treatment plan [8,9]. In addition to the observable side effects, iron supplementation has been shown to affect the community of microbes living in the gastrointestinal tract of humans [2,10]. After consumption of macro and micronutrients, those that are not absorbed in the small intestine by the human host are available to gut microbes in the colon [11]. When high dose ferrous salt supplements are taken, the body is commonly unable to absorb all the iron and there is an increased amount that passes through to the colon [12]. Iron is essential for the growth of many microbes and when in limited supply some microorganisms do not grow optimally [13]. Lactobacillus, a genus associated with positive health effects, does not require iron to grow, and therefore, has a selective advantage when iron availability is low [10,14]. Conversely, when iron availability is high (e.g., following excess supplementation), it has been shown that bacterial growth is increased, including some potentially negative genera, such as Escherichia/Shigella and Clostridium [10,15]. In addition, high dose iron supplementation studies have resulted in increased gut inflammation, as demonstrated by an increase in calprotectin (protein marker of inflammation) [10,16]. The impact of iron on the gut microbiota has been reviewed in detail by Yilmaz and Li, 2018 [17]. This review indicates the potential of ferrous sulphate to promote the growth of potentially pathogenic micro-organisms. However, caution should be observed when considering findings, as many of the studies performed to date have been undertaken in infants [10,16,18–20], and only limited studies have investigated the effects of iron on the microbiota in adults. Adults are major supplement consumers; therefore, it is important to determine what iron does to their microbiota and whether there are less disruptive alternative supplementary approaches. There is a symbiotic relationship between gut microbes and the human host and furthermore a bi-directional relationship between the gut microbiota and luminal iron absorption [21]. Some carbohydrates and proteins that cannot be digested and absorbed by the host in the upper gastrointestinal tract can be fermented by microbes populating the colon to produce metabolites such as short chain fatty acids (SCFA) [22]. SFCAs can be absorbed by the host, and often have positive systemic effects around the body. Protein fermentation additionally results in the production of branched-chain fatty acids (BCFA), such as iso-valerate, along with some unfavourable amino acid metabolites, such as p-cresol and indole [23]. Therefore, changes in the microbial fermentation profile can alter the bacterial metabolites produced and could impact the host [22,24]. Research of Dostal et al. (2013) showed that reducing the ferrous sulphate concentration leads to decreased levels of acetate and BCFA in vitro, which were restored in the presence of ferrous sulphate [25]. As such, this could be indicative of increased protein fermentation upon utilisation of this iron source. Whilst the impact of iron on the microbiota has focused on ferrous sulphate at high doses, lower iron doses are often used as supplementation. Within the gut, 10–20 µM Fe represents the typical luminal concentration of iron from the diet [26,27], whilst supplementation might increase this to 50–200 µM. As such, it is important to consider disruption caused by lower, more commonly consumed supplementary doses [28]. Ferritin (an iron storage molecule) present in the diet, has been shown to be well-absorbed by the enterocytes, and therefore, may be less available to pathogenic bacteria in the distal colon [2,29]. Peas are a rich source of ferritin; however, the high phytic acid content within peas can lead to a low level of iron bioavailability [30]. Purified pea-ferritin iron could indeed provide a suitable alternative, and highly bioavailable, iron source [30,31]. The molecular structure of ferritin has provided inspiration for innovative nanoparticulate forms of iron [32,33]. These iron nanoparticles (Nano Fe(III)) mimic a ferrihydrite-like core, similar to that of ferritin but without the protein shell. The Nano Fe(III) molecule is modified by incorporating tartatic and adipic ligands into the construct (named IHAT). IHAT has been observed to be highly bioavailable in humans (~80% of ferrous sulphate), with low toxicity (in cellular and murine models), and has promoted positive bacterial changes in anaemic murine mice [32–34]. When investigating changes in the microbiota because of iron, it is also worth considering the health status of the test subjects. Healthy individuals have been shown over time to maintain a relatively consistent community of gut bacteria and when subjected to environmental changes (e.g., changes in Nutrients 2020, 12, 3819 3 of 21 nutrients or condition), they will tend towards that steady state. However, after continued exposure, the bacterial equilibrium will move away from that previously consistent community, which raises the possibility that the microbiota from those with other conditions may not respond in the same way [35]. Irritable bowel syndrome (IBS) is a relatively common, debilitating condition defined by abdominal pain, bloating and excess wind, with diarrhoea or constipation, or a combination of the two. Perturbations in the composition and/or activity of the gut microbiota are associated with IBS [36], whilst the microbial communities appear to be distinct in the two IBS sub-types: diarrhoea-predominant IBS (IBS-D) and constipation-predominant IBS (IBS-C). Therefore, there is the possibility that modulation of the microbiota by iron would be more apparent whilst using the microbiota from those with IBS. The majority of consumed iron is not absorbed; as such, it remains available and can have an impact on the gut microbial community [21,33,34]. In vitro batch cultures held under anaerobic, pH-controlled conditions provide a useful approach to studying gut microbial communities and to determine how they are influenced by the presence of different substrates and supplements. Macfarlane et al. showed how a complex media could support the growth of gut micro-organisms to provide a microbial community similar to that within the gut of sudden death victims [37]. As such, these models provide a valuable source of experimentation, and have previously been used to identify how a microbial community is likely to respond before carrying out studies in more complex gut models or even human trials [38,39]. As such, the approach outlined in this manuscript allows exploration of how different iron sources impact on the gut microbial community. The objective of this study was to observe the effects of three different forms of iron supplementation at low doses, namely, ferrous sulphate, pea ferritin and IHAT, on the microbiota and subsequent metabolites of healthy and irritable bowel syndrome donors using in vitro, pH controlled, anaerobic, faecal batch culture models.

2. Materials and Methods

2.1. Participants Fifteen participants in total, aged between 25 and 52 years old, were recruited from the local Reading area (UK) to donate fresh faecal samples to use within fermentation models. There were five healthy donors (two male, three female, age range 23–38 years), five constipation-predominant IBS (IBS-C) donors (one male, four female, age range 22–55 years), and five diarrhoea-predominant IBS (IBS-D) donors (one male, four female, age range 23–50 years). Donors were excluded if they had taken antibiotics within 3 months of sample donation. Additional exclusion criteria were regular consumption of supplements containing iron, prebiotics or probiotics; consumers of medication active on the gastrointestinal tract (e.g., proton pump inhibitors) and those with gastrointestinal illness (other than IBS). IBS volunteers were diagnosed to be suffering from IBS by their GP.

2.2. Ethics This study was conducted according to the guidelines laid down in the Declaration of Helsinki following Good Clinical Practice. It was approved for conduct by the University of Reading’s Research Ethical Committee (ethics reference UREC 1520). All volunteers provided informed written consent before providing stool samples.

2.3. Materials Unless otherwise stated, all chemicals and reagents were obtained from Sigma-Aldrich Co Ltd. (Poole, UK). Ferrous sulphate and the Ammonia assay kit (53659 FluoroSELECT™ Ammonia Kit) were also obtained from Sigma-Aldrich Co Ltd. Pea ferritin was purified from marrowfat peas as previously described [30]. All nucleotide probes used for fluorescence in situ hybridisation (FISH) were commercially synthesised and labelled with the fluorescent dye Alexa Fluoro 488 or Alexa Fluoro Nutrients 2020, 12, 3819 4 of 21

647 at the 50 end (Euroﬁns, Wolverhampton, UK). Sterilisation of media and instruments was carried out by autoclaving at 121 ◦C for 15 min.

2.4. Faecal Sample Preparation Faecal samples were collected and used in the experiment within 2 h of production. Anaerobic conditions were maintained in the collection containers through the use of anaerobic sachets (Thermo Scientiﬁc™, Basingstoke, UK, Oxoid AnaeroGen 2.5 L). Prior to use whole faecal samples were diluted 1:10 (w/v) with anaerobic phosphate buﬀered saline (0.1 M PBS, pH 7.4) and homogenised in a stomacher for 2 min (460 paddle beats/min). Prior to inoculation, a sample was taken to assess the baseline faecal microbial characteristics of the volunteers.

2.5. Faecal Batch Culture Fermentation Fifteen separate fermentation experiments were carried out, with a sample from a different donor used for each experiment. Batch culture fermentation vessels were autoclaved and aseptically filled with 135 mL of low iron gut model medium (peptone water (10 g/L), casein (3 g/L), pectin (2 g/L), xylan (2 g/L), arabinogalactan (2 g/L), starch (5 g/L), guar gum (1 g/L), inulin (1 g/L), KH2PO4 (0.5 g/L), K HPO 3H O (0.5 g/L), NaHCO (1.5 g/L), KCl (4.5 g/L), NaCl (4.5 g/L), MgSO 7H O 2 4· 2 3 4· 2 (1.25 g/L), CaCl 2H O (0.15 g/L), Cysteine-HCl (0.8 g/L), ox-bile (0.4 g/L), vitamin K (10 µL/L), tween 2· 2 80 (1 mL/L) and resazurin (4 mg/L of a 0.024% (w/v) resazurin solution). The mineral solution consisted of (Na Citrate 2H O (pH 6.5) (21 g/L), MnSO 2H O (5 g/L), CoSO 6H O (1 g/L), ZnSO 7H O (1 g/L), 3 · 2 4· 2 4· 2 4· 2 CuSO 5H O (1 g/L) AlK(SO ) (0.1 g/L), H BO (0.1 g/L), Na2MoO 2H2O (1 g/L), NiCl 6H O 4· 2 4 2 3 4 4· 2· 2 (0.25 g/L), Na SeO (2 g/L), V(III)Cl (0.1 g/) and Na WO 2H O (0.1 g/L)). The vitamin solution 2 3 2 4· 2 consisted of (biotin (2 mg/L), folic acid (2 mg/L), pyridoxine HCl (10 mg/L), thiamine HCl (5 mg/L), riboflavin (5 mg/L), nicotinic acid (5 mg/L), DL-calcium pantothenate (10 mg/L), vitamin B12 (0.5 mg/L), p-aminobenzoic acid (5 mg/L), lipoic acid (5 mg/L) and menadione (1 mg/L)), which was prepared and filtered (1% v/v), and each solution was added to the media in the autoclaved vessel. The media was a reduced iron version of the gut model media validated by Macfarlane et al. 1998 [37]. Anaerobic conditions were created by gassing vessels overnight with N2 (15 mL/min). The vessels were maintained at 37 ◦C via a circulating water bath and the pH within the vessels was maintained between 6.3 and 6.5 using a pH controller connected to 0.5 N solutions of HCl and NaOH (Electrolab, Tewkesbury, UK). Immediately prior to faecal sample inoculation, test substrates were added to the vessels; these consisted of the equivalent of 50 µM iron i.e., 2.085 mg iron sulphate (ferrous II sulphate, Sigma, UK), 1.5 mL of 2.2 mg/mL pea ferritin (containing 5 mM Fe) and 1.745 mg IHAT (Nemysis, UK) or prebiotic β-galactooligosaccharide (B-GOS) (1.5 g) (Clasado, Reading, UK) and a control vessel with no additional substrate (negative). The vessels were inoculated with 15 mL of faecal slurry (1:10, w/w), and anaerobic conditions at 37 C and pH 6.3 6.5 were maintained for 24 h ◦ − to try to recreate some of the conditions between the transverse and distal regions of the large intestine. Samples were collected at three time points (0, 8 and 24 h).

2.6. Preparation of the Samples for Bacterial Metabolite Analysis, Ammonia Analysis and Flow Cytometry-Fluorescent In Situ Hybridisation (FISH) Samples were taken at 0, 8 and 24 h post inoculation. A total of 1 mL was centrifuged (in a micro centrifuge tube) at 11,337 g for 5 min the supernatant was stored at 20 C for metabolite analysis. × − ◦ For FISH analysis 0.75 mL was placed in a micro centrifuge tube and centrifuged for 5 min at 11,337 g. The supernatant was removed, 375 µL PBS was added and mixed and 1125 µL 4% × paraformaldehyde solution was added. The sample was stored at 4 ◦C for 4 h, then washed twice with PBS. The pellet was resuspended in 150 µL PBS, to which 150 µL ethanol was added, which was then mixed and stored at 20 C, for future ﬂow-FISH analysis. A further 1 mL sample was collected and − ◦ stored at 20 C, for ammonia analysis. − ◦ Nutrients 2020, 12, 3819 5 of 21

2.7. Flow-FISH Analysis The samples were prepared for flow-FISH analysis as indicated by Rigottier-Gois et al. (2003) and Rochet et al. (2004) [40,41], modified for analysis with the Accuri C6 flow cytometer. Briefly, samples were removed from storage at 20 C, and once defrosted, they were vortexed for 10 s. A total of 75 µL − ◦ of the sample was added to 500 µL PBS in an Eppendorf tube (1.5 mL), vortexed and centrifuged at 11,337 g for 3 min. The supernatant was removed, and 100 µL Tris-EDTA buffer containing lysozyme × (1 mg/mL) was added to the tube, mixed using a pipette and incubated in the dark at room temperature for 10 min. The samples were vortexed, centrifuged at 11,337 g for 3 min and the supernatant removed. × The pellet was resuspended in 500 µL of PBS, then vortexed and centrifuged at 11,337 g for 3 min and × lastly, the was supernatant removed. The pellet was resuspended in 150 µL hybridisation buffer (0.9 M NaCl, 0.2 M Tris-HCl (pH 8.0), 0.01% sodium dodecyl sulphate, 30% formamide), vortexed and centrifuged at 11,337 g for × 3 min. The supernatant was removed and pellet resuspended in 1 mL hybridisation buffer. Four µL of the oligonucleotide probe solutions (50 ng/µL) (refer to section below) was added to 50 µL of the sample in Eppendorf tubes (1.5 mL), vortexed and incubated at 36 ◦C overnight, conditions previously determined by ourselves to be optimum with the probes selected [42]. Following hybridisation, 125 µL of hybridisation buffer was added to each tube, vortexed and centrifuged at 11,337 g for 3 min. × The supernatant was removed and the pellet was resuspended in 175 µL washing buffer solution (0.064 M NaCl, 0.02 M Tris/HCl (pH 8.0), 0.5 M EDTA (pH 8.0), 0.01% sodium dodecyl sulphate), vortexed and incubated for 30 min at 35 ◦C in the dark, to remove any non-specific binding of the probe. The samples were then centrifuged at 11,337 g for 3 min, the supernatant was removed and 300 µL × PBS was added, then vortexed. The samples were stored at 4 ◦C in the dark prior to flow cytometry. Fluorescence measurements were performed by a BD Accuri™ C6 flow cytometer, BD, Erembodegem, Brussels, measuring at 488 nm and 640 nm and analysed used the Accuri CFlow Sampler software.

2.8. Bacterial Quantification Using Flow-Fluorescent In Situ Hybridisation (FISH) Differences in bacterial populations were assessed using flow-FISH analysis with oligonucleotide probes designed to target specific regions of 16S rRNA. The probes used were Bif164 for Bifidobacterium spp. [43], Bac303 for Bacteroides–Prevotella group [44], Lab158 for Lactobacillus/Enterococcus [45], Erec482 for Eubacterium rectale–Clostridium coccoides group, Chis 150 for the Clostridium histolyticum group [46], Rrec584 for Roseburia–E. rectale group, Prop853 for clostridial cluster IX [47], Ato291 for Atopobium cluster [48], Fprau 645 for Faecalibacterium prausnitzii spp. [49] and Dsv687 for most Desulfovibrionales (excluding Lawsonia) and many Desulfuromonales [50]. Three probes (Eub338, Eub338II and Eub338III) formed the Eub 338 probe mix to detect the total bacterial count by targeting the bacteria domain [51]. The probe Non-Eub was used as a negative control to control for non-specific Eub338 binding [52]. The individual probe sequences can be found in Table1.

2.9. Gas Chromatography (GC) for Short Chain and Branched Chain Fatty Acid Analysis

2.9.1. Standard Solution Preparation Individual solution standards at 5 mM were prepared for acetate, iso-butyrate, butyrate, propionate, valerate, iso-valerate and lactate. The external standard solution contained acetate (30 mM), iso-butyrate (5 mM), n-butyrate (20 mM), propionate (20 mM), n-valerate (5 mM), iso-valerate (5 mM) and lactate (10 mM). The internal standard consisted 2-ethylbutyric acid (100 mM) as described by Richardson et al. (1989) [53]. Nutrients 2020, 12, 3819 6 of 21

Table 1. Oligonucleotide probe sequences.

Probe Name Sequence Non-Eub ACTCCTAGGGAGGCAGA Eub338 ‡ GCTGCCTCCCGTAGGAGT Eub338 ‡ GCAGCCACCCGTAGGTGT Eub338 ‡ GCTGCCACCCGTAGGTGT Bif164 CATCCGGCATTACCACCC Lab158 GGTATTAGCAYCTGTTTGGA Bac303 CCAATGTGGGGGACCTT Erec482 GCTTCTTAGTCARGTACCG Rrec584 TCAGACTTGCCGYACCGC Ato291 GGTCGGTCTCTCAACCC Prop853 ATTGCGTTAACTCCGGCAC Fprau655 CGCCTACCTCTGCACTAC DSV687 TACGGATTTCACTCCT Chis150 TTATGCGGTATTAATCTYCCTTT

‡ These probes are used together in equimolar concentrations (all at 50 ng/µL).

Briefly, 1 mL of sample was mixed with 50 µL internal standard, 0.5 mL concentrated HCl and 2 mL diethyl ether. The sample was vortexed for 1 min at 1500 rpm and then centrifuged at 865 g for × 20 min. The top organic (diethyl ether) layer was extracted and retained; where less than 1 mL was extracted, the steps were repeated. In total, 400 µL of the ether extract was added to 50 µL MTBSTFA (N-Methyl-N-tert-butyldimethylsilyltrifluoroacetamide) and stored at room temperature for at least 48 h prior to GC analysis. The samples were analysed using an HP GC 5690 fitted with a flame ionisation detector and a HP-5ms column (30 0.25 mm, 0.25 µm film thickness (Agilent, Cheschire, UK)). Injector and detector × temperatures were 275 ◦C with the column temperature programmed to increase from 63 ◦C to 190 ◦C by 5 ◦C/min and held at 190 ◦C for 30 min. Helium was the carrier gas (flow rate 1.9 mL/min and head pressure 139.8 kPa). One µL samples were injected. Peak areas were recorded using HPChem software.

2.9.2. Ammonia Assay The procedure was followed as detailed in the FluoroSELECT™ ammonia kit instructions. Brieﬂy, 1 mM ammonia standard was prepared by mixing 5 µL of the ammonia kit solution with 20 mM NH4Cl and 95 µLH2O in an Eppendorf tube. The working reagent was prepared by combining 100 µL assay buﬀer, 4 µL Reagent A (kit) and 4 µL Reagent B (kit) for each sample. To a multiwell plate, a range of calibration standards were added at concentrations of 0.1 µM to 50 µM, with the sample added to the remaining wells. To this, 100 µL working reagent was added. The plate was mixed and incubated for 15 min at room temperature in the dark. Readings were taken at 460 nm on a tecan plate reader, (Tecan, Theale, UK). The ammonia concentration of each sample was calculated from the calibration curve.

2.9.3. Statistical Methods Comparisons between substrates and within each substrate for the different time-points were carried out using repeated-measures two-way ANOVA with the Dunnett’s multiple comparisons test. Reported p-values are multiplicity adjusted. Statistical comparisons were performed using GraphPad for Prism 7 for Mac. Results are presented as means with standard deviations (SD), unless otherwise stated. The significance level was set at p < 0.05. Beta diversity or between-community diversity indicates similarity (or difference) in organismal composition between samples and acts as a similarity (or dissimilarity) score between groups. Here, we show beta diversity using dimension reduction ordination analysis [54]. Principal component analysis was carried out using IBM SPSS Statistics 25. When comparing the full data set, an unpaired students t-test was used to compare results at a given time point with those of the control. Nutrients 2020, 12, x FOR PEER REVIEW 7 of 21

IBS-D had significantly more bacteria within the Clostridium histolyticum (CHIS) group within their Nutrients 2020, 12, 3819 7 of 21 samples (Figure 1).

3. Results 10 Healthy IBS_D IBS_C 3.1. Bacterial Population Enumeration by Flow-FISH Differences in bacterial populations were observed between the three different donor groups. Healthy donorsa a hada significantly more bifidobacteria (BIF) than IBS-D, who had more than IBS-C donors; the same pattern was also observed for the Clostridium group IX (PROP). Additionally, those Nutrients 20208, 12, x FOR PEER REVIEW a a a 7 of 21 with IBS-C had significantly fewer lactobacilli (LAB) than IBS-D and healthy donors. Donors with a a b IBS-D had significantlysignificantly more bacteria within b the Clostridium histolyticum (CHIS)a group within their a a ac a a samples (Figure 1 1).). b c c b a a c b 610 a a Healthy IBS_Db IBS_C a b a b c

bacterial cells per ml culture c

10 a a a 8 a a a Log 4 b a b a a a a a a al IF b C acC c O IS ot B AB E E b SV T Lc BAC ATO PR D CH ER RR a c PRAU a F b 6 a a b a Figure 1. Baseline bacteria profile asb detected by flow-FISH from faeces of healthya b donors (n = 5) and c

IBS constipation-predominant bacterial cells per ml culture IBS (IBS-C) (n = 5) and diarrhoea-predominant IBSc (IBS-D) (n = 5)

donors. Different10 letters indicate statistically significant differences within each bacteria group (p < 0.05). Log 4 al IF C C O IS Increases in totalot bacterialB ABnumbers wereE obseE rved following all treatmentsSV and volunteer T L BAC ATO PR D groups (p < 0.05) (Figures 2–4). Furthermore,ER forRR all volunteer groupsPRAU and treatments,CH significant F increases in Bifidobacterium and Atopobium–Coriobacterium spp. (ATO) were observed following 8 and 24 h of fermentation.Figure 1. BaselineBaseline bacteria profile profile as detected by flow-FISH flow-FISH from faeces of healthy donors ((nn == 5) and IBS constipation-predominantconstipation-predominant IBS IBS (IBS-C) (IBS-C) (n =(n5) = and5) and diarrhoea-predominant diarrhoea-predominan IBS (IBS-D)t IBS (IBS-D) (n = 5) (n donors. = 5) Significant increases in Clostridium coccoides–Eubacterium rectale group (EREC) were observed donors.Different Different letters indicate letters statisticallyindicate statistically significant significant differences differences within each within bacteria each group bacteria (p < group0.05). (p < within the healthy and IBS-D batch cultures following 24 h fermentation for all substrates, apart from 0.05). IHAT, whereIncreases there in was total just bacterial a significant numbers increase were observed in the following case of the all treatmentshealthy donors and volunteer (Figures groups 2 and 3). In the(p

p=0.01 * * IHAT, where* * there* * was* *just *a* significant increase in the case of the healthy donors (Figures 2 and 3). 8 8 8 * In the presence of ferrous sulphate, the IBS-C donor samples also significantly* * * increased* * * * in Clostridium coccoides–Eubacterium rectale (Figure 4). cells per ml culture cells per ml culture cells per ml culture 6 6 6 10 10 10 Log Log Log

4 BIF 4 LAB 4 BAC L 4 L S n T in O O4 A t O4 AT O OS 0h 8h 24h O riti 0h 8h 24h OS ri 0h 8h 24h G SO HAT R G S IH G r S IH TR p=0.04 e I 10 - e 10 e e N 10 B- Ferritin F B Fer F NTROL B- F F O p=0.04 O C CONT C p=0.01 * * * * * * * * * * 8 8 8 * Figure 2. Cont. * * * * * * * cells per ml culture cells per ml culture cells per ml culture 6 6 6 10 10 10 Log Log Log

4 4 4 L 4 L S n T in O O4 A t O4 AT O OS O riti OS ri G SO HAT R G S IH G r S IH TR e I - e e e N B- Ferritin F B Fer F NTROL B- F F O O C CONT C

Nutrients 2020, 12, x FOR PEER REVIEW 8 of 21

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4 4 4 * * * * n 4 L n T * S n 4 * * cells per ml culture 6 O cells per ml culture 6 O4 A cells per ml culture 6 O IHAT IH S IHAT 10 -GOS erriti eS 10 -GOS 10 TROL NTROL B F F B Ferriti FeS B-GO Ferriti Fe ONTRO CO C CON Log Log Log 4 4 4 n 4 L n T S n 4 O O4 A O IHAT IH S IHAT -GOS erriti eS -GOS TROL NTROL B F CHIS F B Ferriti FeS B-GO Ferriti Fe ONTRO CO C CON 0h 8h 24h 10

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cells per culturecells per ml 6 10 8 Log

4 L S n T ti A cells per culturecells per ml 6 GO IH

10 TRO - eSO4 B Ferri F ON C Log 4 L S n T ti A TRO -GO eSO4 IH B Ferri F FigureON 2. Effect of different substrates on bacterial groups detected by flow-FISH in pH-controlled C batch cultures inoculated with faeces from healthy donors. Samples were collected at 0 h (baseline), 8 Figure 2. Effect of different substrates on bacterial groups detected by flow-FISH in pH-controlled hFigure and 24 2. h. Effect Target of bacteria: different Bifidobacterium substrates on spp.bacterial (BIF), gr Lactobacillusoups detected spp. by (LAB), flow -FISHmost Bacteroidaceae in pH-controlled and batch cultures inoculated with faeces from healthy donors. Samples were collected at 0 h (baseline), 8 h Prevotellaceae (BAC), Clostridium coccoides–Eubacterium rectale group (EREC), Roseburia subcluster batchand cultures 24 h. Target inoculated bacteria: withBifidobacterium faeces fromspp. healthy (BIF), donors.Lactobacillus Samplesspp. were (LAB), collected most Bacteroidaceae at 0 h (baseline),and 8 (RREC), Faecalibacterium prausnitzii (FPRAU), Clostridium cluster IX (PRO), Atopobium–Coriobacterium h andPrevotellaceae 24 h. Target(BAC), bacteria:Clostridium Bifidobacterium coccoides–Eubacterium spp. (BIF), rectale Lactobacillusgroup (EREC), spp. (LAB),Roseburia mostsubcluster Bacteroidaceae (RREC), and spp.Prevotellaceae Faecalibacterium(ATO), Desulfovibrio (BAC), prausnitzii Clostridium (DSV)(FPRAU), and coccoides Clostridium–Eubacteriumcluster histolyticum IX (PRO),rectale (CHIS).Atopobium group Values (EREC),–Coriobacterium are Roseburiathe meanspp. (ATO),subclusterfrom five independent(RREC),Desulfovibrio Faecalibacterium experiments(DSV) and prausnitzii Clostridium± SD. Differences (FPRAU), histolyticum Clostridiumbetween(CHIS). Valuessubstrates cluster are IX the (PRO),are mean indicated fromAtopobium five with independent–Coriobacterium p-values. *, significantexperiments differencesSD. Di fromfferences the baseline between substratescondition are (i.e., indicated time-point with p0-values. h) for each *, significant substrate, diff erencesp < 0.05. spp. (ATO), Desulfovibrio± (DSV) and Clostridium histolyticum (CHIS). Values are the mean from five independentfrom the baselineexperiments condition ± (i.e.,SD. time-pointDifferences 0 h) between for each substrate,substratesp < are0.05. indicated with p-values. *,

significant differencesBIF (IBS-D) from the baseline conditioLABn (i.e.,(IBS-D) time-point 0 h) for each substrate,BAC (IBS-D) p < 0.05.

0h 8h 24h 0h 8h 24h 0h 8h 24h 10 10 10

* * * * BIF (IBS-D)* * * * * * LAB (IBS-D) BAC (IBS-D)

8 0h 8h 24h 8 0h 8h 24h 8 0h 8h 24h 10 10 10

* * * * * * * * *

cells per ml culture * cells per ml culture 6 cells per ml culture 6 6 10 10 8 10 8 8 Log Log Log

4 4 4 L S L 4 L S

cells per ml culture cells per ml culture AT 6 O4 cells per ml culture 6 O 6 S IHAT IHAT IH 10 10 TRO 10 eS B-GO Ferritin Fe B-GOS Ferritin F NTRO B-GO Ferritin FeSO4 CON CONTRO CO Log Log Log 4 4 4 L S L 4 L S O4 O AT S Figure 3. Cont. HAT H TRO IHAT eS I I B-GO Ferritin Fe B-GOS Ferritin F NTRO B-GO Ferritin FeSO4 CON CONTRO CO

Nutrients 2020, 12, x FOR PEER REVIEW 9 of 21

NutrientsNutrients 2020,2020 12, ,x12 FOR, 3819 PEER REVIEW 9 of 219 of 21 EREC (IBS-D) RREC (IBS-D) ATO (IBS-D)

0h 8h 24h 0h 8h 24h 0h 8h 24h 10 10 10

* * EREC (IBS-D)* * RREC (IBS-D) ATO (IBS-D) 8 8 8 0h 8h 24h 0h 8h 24h * * * * 0h* * 8h * * 24h 10 10 10 * * * * * cells per ml culture per ml cells 6 * * cells per ml culture 6 cells per ml culture 6 10 10 8 8 10 8 * * * * * * * * * * Log Log Log * 4 4 4 4 L 4 cells per ml culture per ml cells 6 S

cells per ml culture in OL OS 6 O t cells per ml culture 6 OL O O4

10 R G rritin IHAT S 10 - GOS IHAT 10 IHAT T e FeSO TR - TR N B F N B Ferri FeSO B-G Ferritin Fe CO O Log C

Log CON Log 4 4 4 4 L in 4 S OL OS O t OL O O4 R -G rritin IHAT GOS IHAT S IHAT T e FeSO TR - TR N B F N B Ferri FeSO B-G Ferritin Fe PRO (IBS-D) O FPRAU (IBS-D) DSV (IBS-D) CO C CON 0h 8h 24h 0h 8h 24h 0h 8h 24h 10 10 10

PRO (IBS-D) FPRAU (IBS-D) DSV (IBS-D) * * 8 0h 8h 24h 8 0h 8h 24h 8 0h 8h 24h 10 10 10 * * * *

cells per cells culture ml 6 *

cells per ml culture ml per cells 6 * cells per ml culture ml per cells 6 10 10 8 8 10 8 Log Log Log 4 * 4 4 * * *

cells per cells culture ml 6 L n T OS culture ml per cells 6 n HAT O ti O4 A culture ml per cells 6 S i 10 rritin I O t O4 AT -G 10 S e FeSO4 rri e IH 10 ri H NTROL B F TR e F -G I O B-GOS F B FeS C NTROL Fer Log

Log CON Log CO 4 4 4 OS L n T HAT O ti O4 A S in -G rritin I S O t O4 AT e FeSO4 rri e IH ri H NTROL B F TR e F -G I O B-GOS F B FeS C NTROL Fer CHIS (IBS-D) CON CO 0h 8h 24h 10

CHIS (IBS-D)

8 0h 8h 24h 10

cells per ml culture ml per cells 6

10 8 Log

4 n 4 cells per ml culture ml per cells 6 S i T OL it O A

10 r S -GO IH NTR B Fer Fe Log CO 4 S n 4 T OL ti O A ri S -GO IH FigureNTR 3. EffectB Ferof differentFe substrates on bacterial groups detected by flow-FISH in pH-controlled CO batch cultures inoculated with faeces from diarrhoea-predominant IBS (IBS-D) donors. Samples were Figure 3. Effect of different substrates on bacterial groups detected by flow-FISH in pH-controlled collectedFigure 3. atEffect 0 h (baseline),of different 8 hsubstrates and 24 h. on Target bacterial bacteria: groups Bifidobacterium detected by spp.flow -FISH(BIF), Lactobacillusin pH-controlled spp. batch cultures inoculated with faeces from diarrhoea-predominant IBS (IBS-D) donors. Samples were (LAB),batch cultures most Bacteroidaceae inoculated with and faeces Prevotellaceae from dia (BAC),rrhoea-predominant Clostridium coccoides IBS (IBS–Eubacterium-D) donors. Samplesrectale group were collected at 0 h (baseline), 8 h and 24 h. Target bacteria: Bifidobacterium spp. (BIF), Lactobacillus spp. (EREC), Roseburia subcluster (RREC), Faecalibacterium prausnitzii (FPRAU), Clostridium cluster IX collected(LAB), at most 0 h (baseline),Bacteroidaceae 8 hand andPrevotellaceae 24 h. Target(BAC), bacteria:Clostridium Bifidobacterium coccoides– spp.Eubacterium (BIF), Lactobacillus rectale group spp. (PRO),(LAB),(EREC), AtopobiummostRoseburia Bacteroidaceae–Coriobacteriumsubcluster and (RREC), Prevotellaceae spp. (ATO),Faecalibacterium Desulfovibrio(BAC), prausnitzii Clostridium (DSV)(FPRAU), andcoccoides ClostridiumClostridium–Eubacterium histolyticumcluster IXrectale (PRO), (CHIS). group Values(EREC),Atopobium are Roseburia the– meanCoriobacterium subcluster from fivespp. independent(RREC), (ATO), Desulfovibrio Faecalibacterium experi(DSV)ments and ±prausnitzii SD.Clostridium Differences (FPRAU), histolyticum between Clostridium(CHIS). substrates Values cluster areare not IX statistically(PRO),the Atopobium mean significant. from– fiveCoriobacterium independent *, significant spp. experiments differences (ATO), DesulfovibrioSD. from Diff theerences baseline (DSV) between and condition Clostridium substrates (i.e., are histolyticumtime-point not statistically 0(CHIS). h) for ± eachValues significant.substrate, are the mean *,p significant< 0.05. from five differences independent from the experi baselinements condition ± SD. (i.e.,Differences time-point between 0 h) for eachsubstrates substrate, are not statisticallyp < 0.05. significant. *, significant differences from the baseline condition (i.e., time-point 0 h) for each substrate, p < 0.05. BIF (IBS-C) LAB (IBS-C) BAC (IBS-C)

0h 8h 24h 0h 8h 24h 0h 8h 24h 10 10 10 * * * * * * * * * * BIF (IBS-C) LAB (IBS-C) BAC (IBS-C)

8 0h 8h 24h 8 0h 8h 24h 8 0h 8h 24h 10 10 10 * * * * * * * * * * *

cells per ml culture 6 cells per ml culture ml per cells 6 culture ml per cells 6 10 10 8 10 8 8 Log Log Log

4 4 * 4 S S n 4 T S 4 tin cells per ml culture 6 O4 cells per ml culture ml per cells 6 O O culture ml per cells 6 O SO GO rri IHAT G IHA G rritin IHAT 10 eS 10 TROL - rriti 10 TROL - TROL e B e FeS B e Fe N B- F F F N F O C

CON CO Log Log Log 4 4 4 S S n 4 T S 4 tin O O O O4 G FigureG 4. ContSO GO rri eS IHAT TROL - rriti IHA TROL - rritin . IHAT TROL e B e FeS B e Fe N B- F F F N F O CON CO C

NutrientsNutrients 2020,2020 12, ,x12 FOR, 3819 PEER REVIEW 10 of 2110 of 21

EREC (IBS-C) RREC (IBS-C) ATO (IBS-C)

0h 8h 24h 0h 8h 24h 0h 8h 24h 10 10 10

* * * * * 8 8 8 * * * * cells per ml culture cells per ml culture cells per mlculture 6 6 6 10 10 10 Log Log Log

4 4 4 n 4 n n T ti i O4 GOS IHAT IHAT GOS rrit IHA TROL rri TROL -GOS - e eS N B- Fe FeSO N B Ferriti FeSO4 B F F O O C C CONTROL

PRO (IBS-C) FPRAU (IBS-C) DSV (IBS-C)

0h 8h 24h 0h 8h 24h 0h 8h 24h 10 10 10

* * * * * * * 8 8 * * * 8 * * * * * * * * * cells per ml culture cells per ml culture 6 culture ml per cells 6 6 10 10 10 Log Log Log

4 4 4 L S 4 S L in T O OL O4 OS t A S HAT S HAT G IH GO rritin I -GO rritin I erri B- Fe Fe B Fe Fe NTRO B- F FeSO4 ONTRO ONTR C C CO

CHIS (IBS-C)

0h 8h 24h 10

8 * *

cells per mlculture 6 10 Log

4 L S 4 O O G IHAT B- Ferritin FeS CONTRO Figure 4. Effect of different substrates on bacterial groups detected by flow-FISH in pH-controlled Figurebatch 4. culturesEffect of inoculated different withsubstrates faeces from on bacterial diarrhoea-predominant groups detected IBS (IBS-C)by flow donors.-FISH Samplesin pH-controlled were batchcollected cultures at inoculated 0 h (baseline), with 8 hfaeces and 24 from h. Target diarrhoea-predominant bacteria: Bifidobacterium IBS spp.(IBS-C) (BIF), donors.Lactobacillus Samplesspp. were collected(LAB), at most 0 h (baseline),Bacteroidaceae 8 hand andPrevotellaceae 24 h. Target(BAC), bacteria:Clostridium Bifidobacterium coccoides– spp.Eubacterium (BIF), Lactobacillus rectale group spp. (LAB),(EREC), mostRoseburia Bacteroidaceaesubcluster and (RREC), PrevotellaceaeFaecalibacterium (BAC), prausnitzii Clostridium(FPRAU), coccoidesClostridium–Eubacteriumcluster IXrectale (PRO), group (EREC),Atopobium Roseburia–Coriobacterium subclusterspp. (RREC), (ATO), Desulfovibrio Faecalibacterium(DSV) andprausnitziiClostridium (FPRAU), histolyticum Clostridium(CHIS). Values cluster are IX the mean from five independent experiments SD. Differences between substrates are not statistically (PRO), Atopobium–Coriobacterium spp. (ATO), ±Desulfovibrio (DSV) and Clostridium histolyticum (CHIS). Valuessignificant. are the mean *, significant from five differences independent from the experi baselinements condition ± SD. (i.e.,Differences time-point between 0 h) for eachsubstrates substrate, are not p < 0.05. statistically significant. *, significant differences from the baseline condition (i.e., time-point 0 h) for eachSignificant substrate, p increases < 0.05. in Clostridium coccoides–Eubacterium rectale group (EREC) were observed within the healthy and IBS-D batch cultures following 24 h fermentation for all substrates, apart from IHAT,The levels where of thereClostridium was just cluster a significant IX increased increase within in the the case vessels of the of healthy the healthy donors and (Figures IBS-C2 batch culturesand3 following). In the presence 8 and 24 of ferroush of fermentation sulphate, the fo IBS-Cr all donorsubstrates, samples apart also from significantly IHAT, where increased there in was just aClostridium significant coccoides increase–Eubacterium in the case rectale of the(Figure healthy4). donors (Figures 2 and 4). There was a significant increase inThe the levels numbers of Clostridium of lactobacillicluster following IX increased the within fermentation the vessels of of B-GOS the healthy in the and vessels IBS-C inoculated batch withcultures the IBS-C following samples, 8 and 24whilst h of fermentation Faecalibacterium for all substrates,prausnitzii apart (FPRAU) from IHAT, levels where increased there was justafter a the fermentationsignificant of increase all substrates. in the case of the healthy donors (Figures2 and4). There was a significant increase in the numbers of lactobacilli following the fermentation of B-GOS in the vessels inoculated with the The levels of Desulfovibrio (DSV) were close to our limit of detection, but tended to increase IBS-C samples, whilst Faecalibacterium prausnitzii (FPRAU) levels increased after the fermentation of during the fermentations. all substrates. WhenThe considering levels of Desulfovibrio the healthy(DSV) donors, were close there to our was limit also of detection,significantly but tended more toBacteroides-Prevotella increase during spp. the(BAC) fermentations. following fermentation of all substrates after 8 and 24 h fermentation. This was not the case for the control vessel, or for the IBS volunteer samples. Furthermore, a decrease in the Roseburia subcluster (RREC) was observed at 24 h across all substrates, but this only reached statistical significance in the control, B-GOS and ferritin-supplemented cultures (Figure 2). In relation to the differences between the test substrates and the control, for healthy donors there was significantly more Bifidobacterium following the fermentation of B-GOS after 8 and 24 h of fermentation. There was also significantly more Bacteroides-Prevotella spp. (BAC) at 8 h in the batch

Nutrients 2020, 12, 3819 11 of 21

When considering the healthy donors, there was also significantly more Bacteroides-Prevotella spp. (BAC) following fermentation of all substrates after 8 and 24 h fermentation. This was not the case for the control vessel, or for the IBS volunteer samples. Furthermore, a decrease in the Roseburia subcluster Nutrients 2020(RREC), 12, x FOR was observedPEER REVIEW at 24 h across all substrates, but this only reached statistical significance in the 11 of 21 control, B-GOS and ferritin-supplemented cultures (Figure2). cultures with B-GOS,In relation compared to the differences to the between control the vess test substratesel. No other and thesignificant control, for differences healthy donors were detected between thethere test was substrates significantly (Figure more Bifidobacterium 2). following the fermentation of B-GOS after 8 and 24 h of fermentation. There was also significantly more Bacteroides-Prevotella spp. (BAC) at 8 h in the batch In thecultures IBS batch with B-GOS,cultures, compared there to were the control no vessel.signific Noant other differences significant di betweenfferences were the detected test substrates for any time pointbetween of thefermentation test substrates (Figures (Figure2). 3 and 4). When allIn batch the IBS culture batch cultures, data therewere were combined no significant (hea difflthyerences and between IBS), the the test ferr substratesous sulphate for any treatment time point of fermentation (Figures3 and4). resulted in a significantlyWhen all batch culturemore dataBacteroides were combined spp. (healthyas compared and IBS), to the the ferrous negative sulphate control treatment (p = 0.038). A principal componentresulted in a significantlyscore plot moreof between-communityBacteroides spp. as compared bacterial to the diversity negative control (beta-diversity) (p = 0.038). at the start of fermentationA principal and component after 24 scoreh showed plot of between-community that the clustering bacterial of samples diversity (beta-diversity) was driven atby the faecal donor, rather thanstart substrate. of fermentation As such, and after there 24 h showedwas no that eviden the clusteringt clustering of samples by was substrate driven by faecalor by donor, the iron sources rather than substrate. As such, there was no evident clustering by substrate or by the iron sources (V3-V5 in Figure(V3–V5 in 5) Figure and5 non-iron) and non-iron sources sources (V1-V2 (V1–V2 in in Figure Figure5). 5).

(A) T 0h Healthy T 24h Healthy 2 2 V4 V5 V1 V5 V2 V4 V3 V4 V1 V1 1 V5 V3V2 1 V1 V2 V4 V5 V3 V2 V3 V3 V5 0 0 V4 V4 V4 V5 V1 V3 V2 V1 V2 V5 V4 V3 V3 V5 V1 V2 V3 V2 PC2 (23%) PC2 PC2 (29%) PC2 -1 -1 V4 V5 V1 V1 V4 V2 V5 V2 V3 V1 -2 -2

-3 -3 -4 -2 0 2 -4 -2 0 2 PC1 (57%) PC1 (37%) (B) T 0h IBS-D T 24h IBS-D 2 3

V2 V4 2 V5 V5 V4 V2 V1 1 V3 V5 V2V1 V1 V3 V3 V4 V4 V5 1 V3 V2 V5 0 V1 V1 V3 PC2 (30%) V2 V2 PC2 (25%) PC2 0 V1 V2 V4 V4 V1 V4 V5 V3 V3 V4 -1 V3 V4 V5 V2 V5 V4 V1 V2 -1 V1 V3 V1 V2 V5 V3 V5

-2 -2 -1 0 1 2 -2 -3 -2 -1 0 1 2 PC1 (51.6%) PC1 (33.9%) (C) Figure 5. Cont. T 0h IBS-C T 24h IBS-C 2 2

Figure 5. Score plot of the principal component analysis representing the variation in bacterial composition at 0 and 24 h post-inoculation with fresh faecal material collected from (A), healthy donors (n = 5); (B), diarrhoea-predominant IBS (IBS-D, n = 5) and (C), constipation-predominant IBS (IBS-C, n = 5) donors. The five independent donors are colour coded (red, blue, green, pink and black clusters) and the different experimental conditions are labelled as V1 (Control, only gut model

medium), V2 (B-GOS), V3 (ferritin), V4 (FeSO4) and V5 (IHAT). The percentage variance values accounted for by the two first components (PC1 and PC2) are shown in parenthesis.

Nutrients 2020, 12, x FOR PEER REVIEW 11 of 21 cultures with B-GOS, compared to the control vessel. No other significant differences were detected between the test substrates (Figure 2). In the IBS batch cultures, there were no significant differences between the test substrates for any time point of fermentation (Figures 3 and 4). When all batch culture data were combined (healthy and IBS), the ferrous sulphate treatment resulted in a significantly more Bacteroides spp. as compared to the negative control (p = 0.038). A principal component score plot of between-community bacterial diversity (beta-diversity) at the start of fermentation and after 24 h showed that the clustering of samples was driven by faecal donor, rather than substrate. As such, there was no evident clustering by substrate or by the iron sources (V3-V5 in Figure 5) and non-iron sources (V1-V2 in Figure 5).

-3 -3 -4 -2 0 2 -4 -2 0 2 PC1 (57%) PC1 (37%) (B) T 0h IBS-D T 24h IBS-D 2 3

V2 V4 2 V5 V5 V4 V2 V1 1 V3 V5 V2V1 V1 V3 V3 V4 V4 V5 1 V3 V2 V5 0 V1 V1 V3 PC2 (30%) V2 V2 PC2 (25%) PC2 0 V1 V2 V4 V4 V1 V4 V5 V3 V3 V4 -1 V3 V4 V5 V2 V5 V4 V1 V2 -1 V1 V3 V1 V2 V5 V3 V5

-2 -2 -1 0 1 2 -2 Nutrients 2020, 12, 3819 -3 -2 -1 0 112 2 of 21 PC1 (51.6%) PC1 (33.9%) (C) T 0h IBS-C T 24h IBS-C 2 2

V3 V5 V1V4 1 V1V2 V3 1 V2 V3 V4 V4 V2 V3 V2 V5 V4 V1 V1 V1 V4 V4 V3 V3 V1 V1 V5 V5 0 V5 V2 V2 V5 V3 0 V2 V5 V4 V5 PC2(23%) PC2 (18%) PC2 V4 -1 V4 -1 V5 V5 V3 V3 V2 V4 V1 V1 V2 -2 V1 V2 V3 -2 -3 -2 -1 0 1 2 -2 -1 0 1 2 PC1 (41%) PC1 (56%) Figure 5. Score plot of the principal component analysis representing the variation in bacterial Figure 5. compositionScore plot at 0of and the 24 hprincipal post-inoculation component with fresh faecal analys materialis representing collected from (A the), healthy variation donors in bacterial composition(n = at5); (0B ),and diarrhoea-predominant 24 h post-inoculation IBS (IBS-D, win th= 5) fresh and (C faecal), constipation-predominant material collected IBS (IBS-C,from (A), healthy n = 5) donors. The ﬁve independent donors are colour coded (red, blue, green, pink and black clusters) Nutrientsdonors 2020 (n ,= 12 5);, x FOR(B), PEERdiarrhoea-predominant REVIEW IBS (IBS-D, n = 5) and (C), constipation-predominant12 of 21 IBS and the diﬀerent experimental conditions are labelled as V1 (Control, only gut model medium), V2

(IBS-C, n =(B-GOS), 5) donors. V3 (ferritin), The five V4 (FeSO independent4) and V5 (IHAT). donors The are percentage colour variance coded values (red, accounted blue, green, for by the pink and black 3.2. Bacterial Metabolites clusters) andtwo ﬁrstthe components different (PC1 experimental and PC2) are shown conditions in parenthesis. are labelled as V1 (Control, only gut model medium),Concentrations3.2. Bacterial V2 (B-GOS), Metabolites of acetate, V3 (ferritin), propionate V4 and(FeSO butyra4) andte wereV5 (IHAT). significantly The percentage higher following variance 24 hvalues of fermentation when compared with 8 h for all conditions tested and for healthy and IBS donors accountedConcentrations for by the two of first acetate, components propionate (PC1 and butyrateand PC2) were are signiﬁcantly shown in parenthesis. higher following 24 h (Figuresof 6–8). fermentation Acetate when was compared the dominant with 8 hSCFA for all prod conditionsuced testedin all andthe forbatch healthy cultures and IBS (Figures donors 6–8). Branched(Figures chain6– 8fatty). Acetate acids was(BCFA), the dominant such as SCFAiso-buty producedrate and in all iso-valerate, the batch cultures indicative (Figures of 6proteolytic–8). fermentation,Branched were chain present fatty acids at very (BCFA), low suchlevels as for iso-butyrate all test conditions. and iso-valerate, indicative of proteolytic fermentation, were present at very low levels for all test conditions.

0 h 8 h 24 h Control Ferritin 2.0 60 Control Ferritin 100 Control Ferritin B-GOS FeSO4 * 90 B-GOS FeSO IHAT * 4 * B-GOS FeSO4 80 ** 1.5 * IHAT * IHAT * 70 40 60

1.0 * 50 20

20 15 0.5 10

5 Bacteria metabolites concentration (mM) concentration metabolites Bacteria Bacteria metabolites concentration (mM) concentration metabolites Bacteria 0.0 10 (mM) concentration metabolites Bacteria 0 te te te te te ate a ate a te te te te te te te r r t ta a rate a a a le eta n yr cta aceta alera a lac ce ionate o ty buty v v a p alerate alerate lactate ac ut aler la o butyrate butyrate v v bu b v valer propion r o o iso butyra iso p propi iso is iso is

Figure 6. SCFA production at baseline (0 h), 8 and 24 h of fermentation in pH-controlled batch Figure 6. SCFA production at baseline (0 h), 8 and 24 h of fermentation in pH-controlled batch cultures cultures inoculated with faeces from healthy adult donors. Values are mean SD of five independent ± inoculatedexperiments with faeces (five donors). from healthy *, significant adult diff erencesdonors. from Values the control are conditionmean ± (i.e.,SD gutof five model independent media experimentsonly, with (five no donors). additional *, testsign substrate),ificant differencesp < 0.05. from the control condition (i.e., gut model media only, with no additional test substrate), p < 0.05.

0 h 8 h 24 h

60 100 5 Control Ferritin Control Ferritin Control Ferritin 90 B-GOS FeSO B-GOS FeSO4 4 B-GOS FeSO4 4 80 * * IHAT IHAT IHAT * 70 40 3 60 * 50 20 2 20 15 10 1 5 Bacteria metabolites concentration (mM) 0 Bacteria concentration metabolites(mM) 0 (mM) concentration metabolites Bacteria 0 te te te e e e e e a t t te t t t n ta a a a a ra rate rate o tyrate tyrate lera era e e nate y erate i a lactate c o tyrate t le l acetate p u u val a lactate acetate a a lactate b v pion butyr val bu bu v v o ro o pro so b is p o butyr propi i is iso valer iso is

Figure 7. SCFA production at baseline (0 h), 8 and 24 h of fermentation in pH-controlled batch cultures inoculated with faeces from diarrhoea-predominant IBS (IBS-D) donors. Values are mean ± SD of five independent experiments (five IBS-D donors). *, significant differences from the control condition (i.e., gut model media only, with no additional test substrate), p < 0.05.

0 h 8 h 24 h

Control Ferritin 60 100 * 5 Control Ferritin Control Ferritin B-GOS FeSO 4 90 B-GOS FeSO ** IHAT 4 B-GOS FeSO4 * 80 4 IHAT * IHAT 70 40 3 60 50 20 2 20 15 1 10 5 Bacteria metabolites concentration (mM) 0 Bacteria metabolites concentration (mM) 0 0 e e e te te t te e e e e e tate a ra a rat ta t te t n e ate a ate at ate ate tat rate tat Bacteriametabolites concentration(mM) o ty l er i aler lac tyr tyr act ac -1ace u v va acet u aler l ace utyra l b butyrat bu b val v butyrate b valerate o ropiona vale prop p o propionateo is iso is iso is iso

Figure 8. SCFA production at baseline (0 h), 8 and 24 h of fermentation in pH-controlled batch cultures inoculated with faeces from constipation-predominant IBS (IBS-C) donors. Values are mean ± SD of five independent experiments (five IBS-C donors). *, significant differences from the control condition (i.e., gut model media only, with no additional test substrate), p < 0.05.

Nutrients 2020, 12, x FOR PEER REVIEW 12 of 21 Nutrients 2020, 12, x FOR PEER REVIEW 12 of 21 3.2. Bacterial Metabolites 3.2. Bacterial Metabolites Concentrations of acetate, propionate and butyrate were significantly higher following 24 h of Concentrations of acetate, propionate and butyrate were significantly higher following 24 h of fermentation when compared with 8 h for all conditions tested and for healthy and IBS donors fermentation(Figures 6–8). when Acetate compared was the with dominant 8 h for SCFA all conditionsproduced intested all the and batch for healthycultures and(Figures IBS donors6–8). (FiguresBranched 6–8). chain Acetate fatty acidswas the(BCFA), dominant such asSCFA iso-buty prodrateuced and in iso-valerate, all the batch indicative cultures of (Figures proteolytic 6–8). Branchedfermentation, chain were fatty present acids (BCFA),at very low such levels as iso-butyfor all testrate conditions. and iso-valerate, indicative of proteolytic fermentation, were present at very low levels for all test conditions.

0 h 8 h 24 h Control Ferritin 2.0 0 h 60 8 h Control Ferritin 100 24 hControl Ferritin B-GOS FeSO4 Control Ferritin * 90 IHAT B-GOS FeSO B-GOS FeSO 2.0 60 * 4 100* 4 B-GOS FeSO Control Ferritin 80 * Control Ferritin 4 * IHAT ** IHAT 1.5 * 90 * B-GOS FeSO 70 B-GOS FeSO IHAT 40 * 4 * 4 60 80 ** 1.5 * IHAT * IHAT * 70 1.0 * 50 40 20 60 15 1.0 20 * 50 20 0.5 10 15 20 5

0.5 (mM) concentration metabolites Bacteria Bacteria metabolites concentration (mM) concentration metabolites Bacteria Bacteria metabolites concentration (mM) concentration metabolites Bacteria 10 0.0 10 0 e te te t te te te te te te te te te ate ra rate ta a a a a a e t 5 n rate l onate eta yr cta aceta alera a lac ce i lactate o ty la Bacteria metabolites concentration (mM) concentration metabolites Bacteria buty v v a p alerate alerate ac ut aler Bacteria metabolites concentration (mM) concentration metabolites Bacteria o butyrate butyrate v v (mM) concentration metabolites Bacteria bu b v valer 0.0 propion 1 r o o iso butyra iso 0 p 0 propi iso is iso is te te te te te e e e ate a ate a t t te te te te t r r t ta a rate a a a le eta n yr cta aceta alera a lac ce ionate o ty buty v v a p alerate alerate lactate ac ut aler la o butyrate butyrate v v bu b v valer propion r o o iso butyra iso p propi iso is iso is Figure 6. SCFA production at baseline (0 h), 8 and 24 h of fermentation in pH-controlled batch cultures Figureinoculated 6. SCFA with production faeces from at baseline healthy (0 adult h), 8 anddono 24rs. h ofValues fermentation are mean in pH-controlled± SD of five independent batch cultures experiments (five donors). *, significant differences from the control condition (i.e., gut model media inoculated with faeces from healthy adult donors. Values are mean ± SD of five independent only, with no additional test substrate), p < 0.05. experiments (five donors). *, significant differences from the control condition (i.e., gut model media

only,Nutrients with2020 no, 12additional, 3819 test substrate), p < 0.05. 13 of 21 0 h 8 h 24 h

60 100 5 Control Ferritin Control Ferritin Control Ferritin 8 h 90 0 h B-GOS FeSO 24 h B-GOS FeSO4 4 B-GOS FeSO4 4 80 * * IHAT IHAT IHAT 60 100 5 Control Ferritin * Control Ferritin 70 Control Ferritin 40 90 3 B-GOS FeSO 60 B-GOS FeSO B-GOS FeSO4 * 4 4 4 5080 * * IHAT IHAT 20 IHAT * 70 2 40 15 3 20 60 * 1050 1 20 2 5 15 Bacteria metabolites concentration (mM) Bacteria concentration metabolites(mM) 20 (mM) concentration metabolites Bacteria 0 0 0 te te te e e e e e a t t te t t t 10 1 n ta a a a a ra rate rate o tyrate tyrate lera era e e nate y erate i a lactate c o tyrate t le l acetate p u u val a lactate acetate a a lactate b v pion butyr val bu bu v v o ro 5 o pro so b is p o butyr propi i is iso valer iso is Bacteria metabolites concentration (mM) Bacteria concentration metabolites(mM) (mM) concentration metabolites Bacteria 0 0 0 e e te t t te te te te te te a a a a a a a n era t r nate rate rate io tyrate tyrate lera e e o tyrate ty le lerate acetate p u u a lactate c lactate a a lactate b v val a pion val acetate v v o butyr bu bu pro so b ro o butyr o Figure 7. SCFAi productionis at baseline (0p h), 8 and 24iso valer h of fermentation in pH-controlledpropi is batch cultures is iso inoculated with faeces from diarrhoea-predominant IBS (IBS-D) donors. Values are mean ± SD of five Figure 7. SCFA production at baseline (0 h), 8 and 24 h of fermentation in pH-controlled batch cultures independent experiments (five IBS-D donors). *, significant differences from the control condition Figureinoculated 7. SCFA production with faeces from at baseline diarrhoea-predominant (0 h), 8 and 24 IBSh of (IBS-D) fermentation donors. Values in pH-controlled are mean SD batch of five cultures ± inoculated(i.e., independentgut model with faeces media experiments fromonly, diawith (fiverrhoea-predominant IBS-Dno additional donors). *,test significant substrate), IBS (IBS-D) differences p < donors 0.05. from . theValues control are condition mean ± (i.e.,SD of five independentgut model experiments media only, with(five no IBS- additionalD donors). test substrate), *, significantp < 0.05. differences from the control condition (i.e., gut model media only, with no additional test substrate), p < 0.05. 0 h 8 h 24 h

Control Ferritin 60 100 * 5 Control Ferritin Control Ferritin B-GOS FeSO 4 90 B-GOS FeSO ** IHAT 4 B-GOS FeSO4 * 80 4 0 h 8 h IHAT * 24 h IHAT 70 40 Control Ferritin 60 60100 * 5 3 Control Ferritin Control Ferritin B-GOS FeSO 4 50 90 B-GOS FeSO 20 ** IHAT 4 B-GOS FeSO4 2 * 80 4 IHAT 15 * IHAT 20 70 40 1 10 3 60 5 50 20 Bacteria metabolites concentration (mM) 0 Bacteria metabolites concentration (mM) 0 0 2 e e e te te t te e e e e e tate a ra a rat ta t te 15 t n e 20 ate a ate at ate ate tat rate tat Bacteriametabolites concentration(mM) o ty l er i aler lac tyr tyr act ac -1ace u v va acet u aler l ace utyra l b butyrat bu b val v butyrate b valerate o ropiona 10 vale 1 prop p o propionateo is iso is iso is iso 5 Bacteria metabolites concentration (mM) 0 Bacteria metabolites concentration (mM) 0 0 Figure 8. SCFAe productione e at baseline (0 h), 8 and 24 h of fermentation in pH-controlled batch cultures te te t te e e e e e tate a ra a rat ta t te t n e ate a ate at ate ate tat rate tat FigureBacteriametabolites concentration(mM) 8.o SCFAty productionl at baseline (0 h), 8 and 24 h ofer fermentation in pH-controlled batch cultures i u aler lac tyr tyr act ac -1ace b butyrat v va acet u aler l ace utyra l inoculated with faeces from constipation-predominantbu b val IBSv (IBS-C) donors. Values are butyrate meanb vale SDvalerate of prop o ropiona o o is iso p is iso propionates iso ± inoculatedfive independent with faeces experiments from cons (fivetipation-predominant IBS-C donors). *, significant IBS (IBS-C) differences donors. from Values thei control are mean condition ± SD of five independent(i.e., gut model experiments media only, with(five no IBS-C additional donors). test *, substrate), significantp < diffe0.05.rences from the control condition Figure(i.e., gut 8. SCFA model production media only, at with baseline no additional (0 h), 8 and test 24 substrate), h of fermentation p < 0.05. in pH-controlled batch cultures inoculatedLooking with at faeces the data from for cons volunteertipation-predominant groups separately, IBS the (IBS-C) different donors. added Values test substrates are mean did ± SD not of

fiveappear independent to significantly experiments impact (five SCFA IBS-C or BCFAdonors). production *, significant by the diffe microbiotarences from present the control in the condition culture (i.e.,vessels gut model inoculated media with only, faecal with material no additional from healthy test substrate), or IBS donors. p < 0.05. When viewed as a whole dataset, however, all substrates led to enhanced acetate levels (Table2) and fermentation in the ferrous sulphate vessel led to enhanced levels of propionate (p = 0.027). B-GOS fermentation led to enhanced levels of lactate at 8 h (p = 0.007) and reduced levels of ammonia after 24 h (p = 0.012). Within the iron vessels, lactate levels were significantly lower than within the negative control. Ammonia concentration increased over time during fermentation but this was only statistically significant at the 24 h time-point (Figure9). The only statistically significant di fference between test substrates was lower ammonia production in the B-GOS condition for the IBS-C donor culture vessels after 24 h of fermentation. There were no other differences observed between the test compounds in terms of ammonia production for any of the time-points investigated. Nutrients 2020, 12, 3819 14 of 21

Table 2. Metabolite production at baseline (0 h), 8 and 24 h of fermentation in pH-controlled batch cultures inoculated with faeces from 15 donors (5 with IBS-D, 5 with IBS-C and 5 healthy controls, data combined).

Time Ammonia Acetate Propionate Butyrate Iso Butyrate Iso Valerate Valerate Lactate BCFA (hours) mM B-GOS 0 2.20 1.05 0.21 0.09 0.2 0.11 0.01 0.01 0.01 0.01 0.01 0.01 0.72 0.13 0.02 0.03 18.9 3 4.38 ± ± ± ± ± ± ± ± ± FeSO 0 2.26 1.08 0.23 0.11 0.2 0.12 0.01 0.01 0.01 0.01 0.01 0.02 0.8 0.21 0.02 0.03 20.4 4.27 4 ± ± ± ± ± ± ± ± ± IHAT 0 2.24 1.06 0.22 0.09 0.18 0.11 0.01 0.01 0 0.01 0.01 0.01 0.76 0.13 0.02 0.03 20.14 5.29 ± ± ± ± ± ± ± ± ± negative 0 2.26 1.01 0.24 0.11 0.2 3 0.10 0.01 0.01 0.01 0.01 0.01 0.02 0.8 0.20 0.03 0.03 19.01 4.59 ± ± ± ± ± ± ± ± ± pea ferritin 0 2.18 1.11 0.21 0.09 0.2 0.12 0 0.01 0.01 0.01 0.01 0.01 0.73 0.15 0.02 0.03 19.62 5.01 ± ± ± ± ± ± ± ± ± B-GOS 8 39.37 15.44 3.18 2.57 1.95 1.60 0.01 0.01 0.01 0.02 0.0 6 0.06 20.46 10.59 * 0.08 0.08 23.99 6.71 ± ± ± ± ± ± ± ± ± FeSO 8 36.92 8.05 * 3.96 2.04 2.28 1.58 0.02 0.02 0.02 0.03 0.1 0.08 11.14 5.51 0.13 0.11 29.29 7.36 4 ± ± ± ± ± ± ± ± ± IHAT 8 38.85 9.31 * 4.55 2.46 2.47 1.75 0.02 0.04 0.03 0.06 0.09 0.10 10.59 5.28 0.14 0.16 30.44 8.69 ± ± ± ± ± ± ± ± ± negative 8 30.28 8.71 3.39 2.81 2.19 1.58 0.01 0.02 0.01 0.02 0.09 0.08 11.52 5.51 0.11 0.11 29.38 9.47 ± ± ± ± ± ± ± ± ± pea ferritin 8 34.79 11.42 4.24 2.58 2.48 1.73 0.01 0.02 0.02 0.02 0.09 0.08 10.86 5.29 0.12 0.11 28.57 8.67 ± ± ± ± ± ± ± ± ± B-GOS 24 71.96 9.58 * 14.71 6.73 12.03 7.18 0.09 0.10 0.13 0.15 0.83 1.02 10.2 11.55 1.04 1.06 44.68 14.93 * ± ± ± ± ± ± ± ± ± FeSO 24 70.56 11.81 * 16.34 4.43 * 11.53 3.64 0.29 0.36 0.36 0.41 1.95 2.23 1.55 2.66 * 2.59 2.57 67.72 24.62 4 ± ± ± ± ± ± ± ± ± IHAT 24 70.8 19.00 * 14.93 3.66 11.61 4.15 0.34 0.43 0.4 0.53 1.57 1.86 0.91 2.38 * 2.31 1.96 64.24 21.95 ± ± ± ± ± ± ± ± ± negative 24 56.95 5.56 12.39 4.84 9.39 4.23 0.12 0.16 0.18 0.22 0.89 1.39 5.27 6.24 1.19 1.49 60.52 17.46 ± ± ± ± ± ± ± ± ± pea ferritin 24 65.89 12.21 * 15.3 5.07 11.45 4.24 0.27 0.39 0.32 0.43 1.63 2.09 1.35 2.49 * 2.21 2.41 65.9 9 23.85 ± ± ± ± ± ± ± ± ± Values are mean SD of 15 independent experiments. *, signiﬁcant diﬀerences from the control condition (i.e., gut model media only, with no additional test substrate), p < 0.05. ± Nutrients 2020,, 12,, x 3819 FOR PEER REVIEW 1415 of 21

0h 8h 24h 0h 8h 24h 0h 8h 24h 120 b 100 100 b b b b b b 100 b 80 b 80 b 80 b b 60 60 b b 60 a c a a a a a aa 40 40 a a a a 40 a a a a a a a a a a a a a a a a a a 20 20 20 Ammonia comcentration(mM) Ammonia comcentration (mM) Ammonia comcentration (mM) comcentration Ammonia 0 0 0 S in T S n O t AT i H OL A OL -G eSO4 I R rritin IH GO SO4 IHAT F -GOS e eSO4 - e B Ferr B F F B Ferriti F ONT CONTROL C CONTR (A) Healthy (B) IBS-D (C) IBS-C

Figure 9. AmmoniaAmmonia production production in in pH-controlled pH-controlled batch batch cultures cultures inoculated inoculated with with faeces faeces from from ( (AA),), Healthy donors; ( B), IBS-D donors and ( C), IBS-C donors. Values Values are mean ± SDSD of five five independent ± experiments. Different Different label label letters letters (a, b) indicate significant significant diffe differencesrences between time-points. There were no significant significant differences differences between the test co compoundsmpounds and the control condition (i.e., gut model media only, with no additional test substrate), pp << 0.05.

4. Discussion 4. Discussion Low dose oral iron supplements are often taken to help avoid iron deficiency anaemia; however, Low dose oral iron supplements are often taken to help avoid iron deficiency anaemia; however, previous studies have indicated that supplementation with ferrous salts can result in undesirable previous studies have indicated that supplementation with ferrous salts can result in undesirable side-effects [8–10], often associated with changes to the microbiome. The current study has explored side-effects [8–10], often associated with changes to the microbiome. The current study has explored the impact of a low supplementary dose of ferrous sulphate, pea ferritin and IHAT (a nanoparticulate the impact of a low supplementary dose of ferrous sulphate, pea ferritin and IHAT (a nanoparticulate iron) on the faecal microbiota and subsequent metabolites using in vitro batch cultures inoculated with iron) on the faecal microbiota and subsequent metabolites using in vitro batch cultures inoculated faecal bacteria from adults with IBS and healthy controls. with faecal bacteria from adults with IBS and healthy controls. Within all experiments, it could be seen that the media used had an impact on the results. Within all experiments, it could be seen that the media used had an impact on the results. For For example, for all substrates and all donor groups, increases in bifidobacteria were observed. Such a example, for all substrates and all donor groups, increases in bifidobacteria were observed. Such a change can be explained by the complexity of the media used. The gut model media, although low in change can be explained by the complexity of the media used. The gut model media, although low iron, contained a variety of carbohydrate and protein sources required by bacteria for growth. These in iron, contained a variety of carbohydrate and protein sources required by bacteria for growth. Theseconditions conditions selected selected were designed were designed to mimic to mimic conditions conditions found withinfound within the proximal the proximal colon, andcolon, would, and would,therefore, therefore, be likely be to encouragelikely to encourage the growth the of gut grow bacteriath of [37gut]. Thisbacteria allows [37]. for This better allows modelling for better of the modellinggut community, of the allowing gut community, microbial allowing changes microbial to be assessed changes in the to presencebe assessed of a in background the presence healthy of a backgrounddiet. However, healthy as the diet. conditions However, were as plentiful the conditions large changes were plentiful attributable large to changes the iron attributable supplementations to the ironcould supplementations not be seen. The modelcould isnot still be a seen. useful The tool, model and indicatesis still a theuseful likely tool, changes and indicates that might the be likely seen changeswhilst consuming that might a regularbe seen diet. whilst An inconsuming vitro study a ofregular Kortman diet. and An coworkers in vitro study (2016), of using Kortman a low ironand coworkersmedia with (2016), a different using carbohydrate a low iron media composition, with a different showed carbohydrate that ferrous sulphatecomposition, was disruptiveshowed that to ferrousthe microbiota sulphate [28 was], leading disruptive to decreases to the microbiota in bifidobacteria [28], leading and lactobacilli; to decreases the einff ectsbifidobacteria observedwere and greater with 250 µmol 1 ferrous sulphate supplementation as compared to 50 µmol 1. In the current lactobacilli; the effects− observed were greater with 250 μmol−1 ferrous sulphate supplementation− as study, the lower dose of iron used, along with the altered media, is likely to have mitigated some of compared to 50 μmol−1. In the current study, the lower dose of iron used, along with the altered media,negative is elikelyffects Kortmanto have mitigated observed, some supporting of nega thetive idea effects that theKortman lower doseobserved, of iron supporting is less disruptive the idea to thatthe microbiota.the lower dose of iron is less disruptive to the microbiota. One consistent observation observation from from inin vivo studies is a reduction in commensals with low iron Lactobacillus Bifidobacterium requirement (such as Lactobacillus or Bifidobacterium species [[55]).55]). Animal Animal models models and and inin vitro vitro experiments indicate indicate that that increasing increasing iron iron availability availability in inthe the gastrointestinal gastrointestinal tract tract may may alter alter the gut the microbiota,gut microbiota, enabling enabling potentially potentially pathogenic pathogenic microorganisms microorganisms to tohave have a acompetitive competitive advantage advantage [55]. [55]. In thethe currentcurrent experiment,experiment, when when the the pooled pooled data data was was looked looked at, increasesat, increases in Bacteroides in Bacteroides/Prevotella/Prevotellawere wereapparent apparent following following fermentation fermentation of ferrous of ferro sulphate,us sulphate, but notbut thenot otherthe other iron iron sources. sources.Bacteroides Bacteroidesare areknown known to beto goodbe good at scavengingat scavenging for for iron iron [56 [56].]. Dostal Dostal et et al. al. (2013) (2013) observedobserved reductionsreductions in levels of Bacteroides spp. inin vitro vitro following the introduction of an iron chelatorchelator toto thethe mediamedia [[25].25]. Previous research of Mevisenn-Verhage et al. (1985) (1985) has has shown shown milk milk fortified fortified with with iron iron to to lead to increased levelslevels of Bacteroides spp. in in the the faeces of infants [57]. [57]. In In the the current current experiment, the the media used was designed to to be be low low in in iron; iron; as as such, such, the the environmen environmentt would would have have been been limiting limiting for iron for ironutilisers, utilisers, and therefore,and therefore, the addition the addition of iron of enabled iron enabled their growth their growth.. Such increases Such increases were not were observed not observed in the presence in the of the IHAT or pea ferritin iron sources. IHAT is an engineered analogue of natural food iron

Nutrients 2020, 12, 3819 16 of 21 presence of the IHAT or pea ferritin iron sources. IHAT is an engineered analogue of natural food iron (ferritin), which has recently been tested in a large Phase II clinical trial as an alternative iron supplement [58]. It is distinct from currently available oral iron supplements in that it is not soluble and does not require solubilisation in the stomach to be absorbed [33,34]. Conversely, IHAT is taken up as whole nanoparticles. This means that the unabsorbed IHAT fraction will remain nanoparticulate, suggesting no ‘free’ or labile ionic iron is released into the intestinal lumen and onto the intestinal mucosa, beyond its endocytotic absorption site in the duodenum [5,33,59]. The lack of increase in the presence of Bacteroides/Prevotella with IHAT and pea ferritin may suggest that lower levels of iron are available to the microbiota from these iron sources. Indeed, research by Pereira et al. (2015, 2014) in murine studies has indicated that IHAT is not available to gut bacteria when compared to ferrous sulphate, indicating that this source of iron may not be as disruptive to the microbiota as ferrous sulphate [32,34], the current gold standard for iron supplementation. The authors are not aware of previous work of the impact of pea-ferritin on the microbiota. Plant ferritin is a natural source of dietary iron, one of the main sources from a diet rich in pulses, and the results from the current study support that low-dose pea ferritin iron supplementation does not impact the gut microbiota. The three iron supplements all led to lower levels of lactate compared to the control. This could be due to the fact that lactobacilli, a lactate producer, do not require iron for growth. As such, in the low iron condition of the control vessel (no supplementary iron) lactobacilli had a competitive advantage, whereas in the iron vessels, other members of the microbiota were able to compete, resulting in less lactate [10,14]. This does indicate a degree of available iron to the microbiota with all sources. However, the iron treatments did not have a large impact on the microbiota overall. This could in part be explained by the low dose used, which corresponds with the fact that the side-effects and gastrointestinal symptoms are observed when high dose iron supplements are administered. Typical anaemia treatment consists of 180 mg ferrous sulphate in tablet form, which is about 10 times 1 the amount consumed during a standard meal, equating to 350 µmol− lumenal concentration [60]. The dose used in the current study, however, is relevant as a low dose supplement; if it is assumed that the iron intake per meal is 6 mg (equivalent of 18 mg/day), the jejunal lumenal iron concentration 1 would be 35 µmol− following a meal [26], which could then be boosted by a low dose supplement 1 leading to the 50 µmol− dose tested here. It is important to test iron at low doses to observe the effects of iron on the microbiota at the concentrations more typically consumed. In the current study, the presence of iron (all sources) and the presence of B-GOS lead to increased levels of acetate as compared to the control. Whilst this would be expected for B-GOS fermentation due to the additional carbohydrate available, the reason for this occurrence in the iron fermentations may be different. Many organisms within the gut require iron for growth; as such, iron can enable enhanced microbial fermentation, resulting in increases in SCFA, as also observed by Dostal et al. (2013) [25]. Both proteolytic and saccharolytic fermentation can lead to increased levels of SCFA; however, in the absence of increased levels of branched chain fatty acids (BCFA), there is a likelihood that no significant rise in proteolytic fermentation occurs. This means that at the levels used, relating to a low dose supplement, the iron sources microbiota from the IBS-C, IBS-D and healthy adults did not have a negative impact on the fermentation profile. Lee et al. (2017) looked at the impact of ferrous sulphate supplementation on those with inflammatory bowel disease (IBD) and on non-IBD anaemic controls. It was observed that the control volunteers had a more stable microbiota, whereas those with IBD experienced more changes to the microbiota when consuming iron 600 mg ferrous sulphate [61]. This is a much higher dose than what was used in the current study, but indicates that the microbiota of healthy volunteers may be more stable to iron supplementation. As such, there is merit in using volunteers in different clinical groups, who may have a different microbial profile to healthy adults, such as those with IBS. A strength of this research is the exploration of healthy adults alongside IBS-D and IBS-C donor groups. When donor pre-fermentation characteristics were looked at, different microbial patterns between the different donors were revealed; these included more bifidobacteria and lactobacilli in the healthy donors than Nutrients 2020, 12, 3819 17 of 21 those with IBS. This matches well with previous research, where lower levels of bifidobacteria have been observed in IBS-D and IBS-C volunteers [62–65]. Moreover, in the current study, higher levels of bifidobacteria and lactobacilli were observed in those with IBS-D, as compared to IBS-C. Interestingly, the IBS-D group had more bacteria from the Clostridium histolyticum group, which can include toxin producers. Clostridium IX were more abundant in healthy donors, which may inversely correlate to lower levels of propionate that have been observed in those with IBS [66]. However, as there were only five volunteers per group, caution must be exercised when interpreting these data. The prebiotic B-GOS provided an additional carbohydrate source, resulting in reduced ammonia production within this fermentation. The data of Kortman et al. (2016) indicates that iron supplementation leads to increased proteolytic fermentation; furthermore, ammonia production is associated with protein fermentation, and often negative effects to the host [28]. Enhanced protein fermentation, using ammonia as a marker of this process, was not evident in the current study with the three iron sources as compared to the negative control. It is of note that fermentation of B-GOS resulted in reduced ammonia, compared to the other sources, highlighting the impact of having more fibre available. Determination of changes in functional groups of the microbiota by Flow FISH is a useful tool for assessing the impact of treatments highlighting groups of bacteria with functional relevance. This means that the full community and changes on a species basis cannot be determined. However, within the experiment, the results provide important information about which groups of bacteria the iron sources impact on within the model. This information combined with SCFA and ammonia data can help determine whether the changes induced by the supplementation are likely to lead to positive or negative changes to the microbial community and their end products. Frequently the probe-set (of 10 bacterial groups) used by the researchers covers more than 80% of the microbiome domain bacteria as detected by EUB-338I, II and III oligonucleotide probes. In the current study, however, despite all volunteer groups (healthy volunteers, IBS-D and IBS-C) having similar total bacterial numbers, for the IBS-C group, less of the microbiome was accounted for, indicating the presence of other bacteria not covered within this assessment. When sequencing has been applied by previous authors for healthy and IBS donors, a lower diversity has been observed in the latter group [64]. However, with the quantification of the 10 bacterial groups studied, the intra group variability observed was greater in IBS donors as compared with healthy donors. Nevertheless, following fermentation of all three groups, a similar bacterial community was observed after 24 h of fermentation. This demonstrates that the selected media composition and in vitro conditions were favourable for the growth of bacteria and boosted the analysed bacterial groups in a similar manner. Despite the richness of the media composition, which includes inulin, vessels where B-GOS was added led to a faster and significant increment in lactate, which demonstrates that the media alone was not the only factor impacting on the microbiota. From studies on iron supplementation in IBD patients, it is clear that there is a need to find alternative iron sources or regimens that are less disruptive to the microbiota [62]. Furthermore, in murine IBD studies, it has been observed that genotype and inflammatory status influence shifts in the gut microbiota during dietary iron intake [67,68]; as such, the information taken here, whilst showing little change to the microbial community, should be further explored in vivo to determine how the health and inflammatory status of the host could further impact this. To conclude, the microbiota of IBS donors differed in baseline populations; however, with the conditions of the experiment, these changes decreased during the fermentation. The addition of iron supplementations led to alterations to the fermentation profile, in terms of lower levels of lactate; however, the changes observed were not in combination with changes in microbial numbers. This experiment has shown that within the current model for low dietary iron supplementation and a background media, there were no adverse effects of ferrous sulphate, IHAT or pea ferritin to the faecal microbiota of healthy and IBS volunteers. Nutrients 2020, 12, 3819 18 of 21

Author Contributions: Conceptualisation, D.I.A.P., M.C.L., G.E.W. and C.P.; methodology, C.P. and G.E.W.; software, C.P.; formal analysis, D.I.A.P.; investigation, C.P.; data curation, D.I.A.P., G.E.W. and C.P.; writing—original draft preparation, C.P., D.I.A.P. and G.E.W.; writing—review and editing, D.I.A.P. and M.C.L. All authors have read and agreed to the published version of the manuscript. Funding: This project was made possible by the financial support of Nemysis Limited, who provided IHAT for use within this project. The funders had no role in the design, conduct or decision to publish this study. Acknowledgments: The authors would like to thank Simon Andrews for his advice on iron dose and media used within this work. Katharina Kessler is thanked for information provided on IHAT. The pea protein iron was provided by Jorge Rodríguez-Celma and Janneke Balk of the John Innes Centre, Norwich, UK. Conflicts of Interest: D.I.A.P. is one of the inventors of the IHAT iron supplementation technology, for which she could receive future awards to inventors through the MRC Awards to Inventor scheme. D.I.A.P. has served as a consultant for Vifor Pharma UK, Shield Therapeutics, Entia Ltd., Danone Nutritia, UN Food and Agriculture Organisation (FAO) and Nemysis Ltd. D.I.A.P. has since moved to full employment with Vifor Pharma UK. Notwithstanding, the authors declare no potential conflicts of interest with respect to the research, authorship and/or publication of this article.

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