Prebiotics

The in silico reconstruction of the genome-scale metabolic networks of two human-derived L. reuteri strains was performed by implementing the AUTOGRAPH method [73] (link). This semi-automatic method combines orthology predictions with available curated metabolic networks to infer gene-reaction associations. Using this same methodology, a metabolic model was recently constructed for the type strain L. reuteri JCM1112 [56] , based on the networks of L. plantarum[74] (link), Lactococcus lactis[75] (link), Bacillus subtilis[76] (link), and E. coli[77] (link). Due to the obvious close proximity between all human-derived L. reuteri strains relative to members of different taxa, the manually curated metabolic network of JCM1112 was used as a template for the development of the genome-scale models for L. reuteri ATCC PTA 6475 and ATCC 55730. Pair-wise orthologous relationships between the query species and JCM1112 were established by comparing their genome sequences (retrieved in May 2009 from GenBank), resorting to the stand-alone version of Inparanoid (version 3.0) using BLOSUM80 as the substitution matrix [78] (link). The original gene-reaction association of the genes considered to be orthologous between the two strains was then transferred to the corresponding genes of the query species.
The fully automated version of the model was further curated by manual inspection of the list of gene-reaction associations, incorporating experimental evidence regarding carbohydrate utilization. With this purpose, the growth of L. reuteri 55730 and 6475 on different carbohydrates was measured for 24 h in LDMIII at 600 nm (OD_{600 nm}) using commercially available sugars and well established prebiotics as previously described [79] (link). Simple carbohydrates tested consist of glucose, sucrose, lactose, raffinose, fructose, arabinose, maltose, mannose, arabinogalactan, starch and 1,2 propanediol (Sigma, St Louis, MO). Growth on following prebiotics as the sole carbon source were also tested: fructooligosaccharides (FOS, Beneo™ P95, Orafti, Belgium, 5% glucose, fructose and sucrose, degree of polymerization [DP] = 2–10), short-chain fructooligosaccharides (ScFOS, Actilight 950P, Beighin-Meiji, France, 5% glucose, fructose and sucrose, DP = 2–5), high-molecular weight inulin (Beneo™ HP, Orafti, 100% inulin, average DP = 23), galactooligosaccharides (Vivinal GOS, Friesland Food, partially dried by evaporation to form a syrup containing approximately 45% galactooligosaccharides, DP = 3–8, 15% lactose, 14% glucose, and 1% galactose).
The comparison of the newly obtained genome-scale metabolic models for L. reuteri ATCC PTA 6475 and ATCC 55730, along with the visualization of experimental data was carried out within the SimPheny™ software platform (Genomatica, Inc., San Diego, CA).

Individual bioreactors were processed in parallel in a bioreactor management device (Cryptobiotix, Ghent, Belgium). Each bioreactor contained 5 ml of nutritional medium-fecal inoculum blend supplemented with 5 g prebiotic/L, then sealed individually, before being rendered anaerobic. Blend M0003 was used for the case studies and technical assessment 1 and blend M0012 for technical assessment 2 (Cryptobiotix, Ghent, Belgium). During technical assessment 2, a mucin gel was prepared by boiling a solution of 5% mucin type II (M2378; Merck, Overijse, Belgium) and 1% agar for 2 min. Upon cooling, the pH was adjusted to 6.8 ± 0.1. Mucin droplets were introduced in polyethylene carriers with volume of 0.07 cm³. After preparation, bioreactors were incubated under continuous agitation (140 rpm) at 37°C for 24 h, except for case study 2, where a more integrative time point of 48 h was implemented (MaxQ 6,000, Thermo Scientific, Thermo Fisher Scientific, Merelbeke, Belgium). Upon gas pressure measurement in the headspace, liquid samples were collected for subsequent analysis.

This was a pilot randomized double-blind crossover trial in Y-T2DM (Supplementary Figure S1). Youth aged 10–25 years, diagnosed with Y-T2DM by the American Diabetes Association criteria (27 (link)), Tanner stage IV or V, with Hemoglobin A1c (HbA1c) ≤8% were recruited to participate in this MIGHTY-Fiber Study. Exclusion criteria included: positive diabetes related autoantibodies (GAD-65 and IA-2 autoantibodies); consumption of ≥ 2 or more servings of ≥6 oz of yogurt per day; chronic GI disease; gastric bypass surgery; cancer diagnosis or auto-immune disease; chronic insulin therapy within 3 months of the study; or use of antibiotics, immunosuppressants, hormonal contraceptives, lipid-lowering agents, proton-pump inhibitors, supraphysiologic systemic steroids, cholesterol medications, prebiotics, or probiotics in the previous month at time of screening.

Participants were randomized to the prebiotic or a placebo shake to be administered with metformin (850mg tablets) prior to visit 2 (Supplemental Figure S2). Three study agents were used: metformin standard release 850mg oral tablet, BiomeBliss^® powder, and placebo powder. Metformin 850 mg tablets were used within the approved dosing regimens as follows: at the start of each period (visit 2/4), participants took metformin 850mg once daily x 3 days, and the dose increased to 850mg twice daily for the remainder of the study period. The placebo or prebiotic supplement was dispensed as packets, for which participants received 1 packet once daily x 3 days and 1 packet twice daily for the remainder of the study period. All study medications were taken together. The macronutrient composition of the prebiotic supplement and placebo composition are illustrated in Supplemental Table S1. Study randomization was performed by an independent statistician with 1:1 allocation ratio. Blinding of the investigators and participants was maintained throughout the study. Medication adherence was determined by co-author (LM) who adjudicated pill and sachet counts at each visit.