CRICOS No.0014A These studies were supported by a grant from the Bill and Melinda Gates Foundation Figure 5: Characterisation of the faecal microbiome of a subset of 6 individuals from the study. Time points of each individual are indicated by acronym: Baseline (B), HAMS (1), Washout (W), and HAMSA (2). A. Heatmap shows normalised read counts of the predominant microbial taxa detected using MetaPhlAn2. The normalised abundance is indicated by colour gradient (white, not detected; blue, most abundant). B. Heatmap shows normalised read counts mapped to short-chained fatty acid biosynthesis genes from KEGG pathways. The normalised abundance is indicated by colour gradient (white, not detected; green, most abundant). Effect of native and acetylated high amylose maize starch on fecal pH and short chain fay acid concentrations in a cohort of children in southern India RAMADASS BALAMURUGAN 1,2 ; PUGAZHENDHI SRINIVASAN 1 ; GOWRI BALACHANDAR 1 ; TAMILSELVAN DHARMALINGAM 1 ; ELISSA MORTIMER 3 ; GEETHA GOPALSAMY 3 ; RICHARD WOODMAN 3 ; ROSIE MENG 3 ; MARK MANARY 4 ; HENRY BINDER 5 ; IAN BROWN 3 ; LEX LEONG 3,6 ; GERAINT ROGERS 3,6 ; GRAEME YOUNG 3 ; BALAKRISHNAN RAMAKRISHNA 1,7 1 Gastroenterology, Christian Medical College, Vellore, India; 2 School of Basic Sciences, Indian Institutes of Technology (IIT), Bhubaneswar, Odisha, India; 3 Flinders University, Adelaide, SA, Australia; 4 Washington University School of Medicine, St. Louis, MO, USA; 5 Yale University School of Medicine, New Haven, CT, USA; 6 Infection and Immunity, SAHMRI, Adelaide, SA, Australia; 7 SRM Institutes for Medical Science, Chennai, India. Objectives We undertook a non-randomized sequential crossover feeding study to determine whether short term feeding with HAMS or HAMSA would alter fecal pH and SCFA concentrations in a cohort of children in southern India. Figure 1 Background Carbohydrates that resist digestion demonstrate a prebiotic effect by altering the colonic microbiota and the metabolome. The production of short chain fatty acids (SCFA) by the microbiota in particular, influences many aspects of gastrointestinal health. Resistant starch is one category of prebiotic carbohydrates of which high amylose maize starch (HAMS) is an example. HAMS contains up to 70% amylase resistant starch that is resistant to pancreatic amylase and therefore enters the colon unabsorbed. There, it is rapidly fermented by colonic bacteria, yielding substantial amounts of SCFA including butyrate. Another type of resistant starch is high amylose maize starch acetate (HAMSA) which is HAMS esterified with acetate; the latter is likely to be rapidly released by microbial hydrolases in the colon. Methods • Twenty children, aged 2 to 5 years, (ten stunted with height for age [HAZ] lower than 2 standard deviations below the mean, and ten showing normal growth). • Informed written consent obtained from the parents. • Fed cookies containing HAMS (10 g/day) every day for two weeks (days 1-14). • Two week washout period on their regular diet (days 15-28). • Fed cookies containing HAMSA (10 g/day) for two weeks (days 29-44). • Thirteen stool samples collected - on days 0, 3, 7, 10, 15, 18, 22, 25, 29, 32, 36, 39 and 44. • Fecal pH was measured. • SCFA concentration quantitated by GC-MS. • Microbiome characterisation was determined using shotgun metagenomic sequencing of faecal samples from 6 individuals. Sequencing downstream processings were performed as per Nielsen et al. 2014 1 Nielsen HB, et al. Identification and assembly of genomes and genetic elements in complex metagenomic samples without using reference genomes.Nat Biotechnol. 2014;32(8):822-8. Participants Conclusions HAMS and HAMSA both lowered fecal pH with significantly lower pH being achieved in healthy children (Figure 1). HAMS and HAMSA significantly increased acetate in stunted participants compared to baseline and day 29 respectively, but HAMSA produced a significantly greater effect in healthy children (Figure 2). HAMS and HAMSA increased propionate in both healthy and stunted children compared to baseline but the rise was significantly greater in healthy children (Figure 3). HAMS increased butyrate in healthy and stunted children (not significantly different) while HAMSA had no effect in stunted children while it increased butyrate significantly in healthy relative to stunted children (Figure 4). There were significant differences between healthy and stunted children in the parameters of fermentation. Overall, colonic carbohydrate fermentation was significantly impaired in stunted children and HAMSA was less effective in inducing changes than was HAMS. However, neither resistant starch resulted in any significant changes in the microbiome of these children. The microbiota composition and functional genes encoding for short chain fatty acids biosynthesis appeared relatively stable throughout the feeding study for both healthy and stunted children (Figure 5). p=.03 p=.001 p=.02 p=.02 + + + + * * * * * Day0-14 HAMS Day14-28 Washout period, regular diet Day29-44 acetylated-HAMS 5 6 7 8 pH 0 3 7 11 15 18 22 25 29 32 36 40 44 day Healthy Stunted p values indicates significance for healthy versus stunted participants + indicates p<0.05 versus day 0 * indicates p<0.05 versus day 29 (baseline for HAMSA) Effect of HAMS and HAMSA on fecal pH p=.02 p=.02 p=.01 + + + + + * Day0-14 HAMS Day14-28 Washout period, regular diet Day29-44 acetylated-HAMS 40 60 80 100 120 0 3 7 11 15 18 22 25 29 32 36 40 44 day healthy stunted p values indicates significance for healthy versus stunted participants + indicates p<0.05 versus day 0 * indicates p<0.05 versus day 29 (baseline for HAMSA) Effect of HAMS and HAMSA on fecal acetate p=.04 + * + + + + Day0-14 HAMS Day14-28 Washout period, regular diet Day29-44 acetylated-HAMS 0 20 40 60 0 3 7 11 15 18 22 25 29 32 36 40 44 day healthy stunted p values indicates significance for healthy versus stunted participants + indicates p<0.05 versus day 0 * indicates p<0.05 versus day 29 (baseline for HAMSA) Effect of HAMS and HAMSA on fecal propionate p=.003 p=.03 p=.02 + * + + + + + Day0-14 HAMS Day14-28 Washout period, regular diet Day29-44 acetylated-HAMS 0 10 20 30 40 0 3 7 11 15 18 22 25 29 32 36 40 44 day healthy stunted p values indicates significance for healthy versus stunted participants + indicates p<0.05 versus day 0 * indicates p<0.05 versus day 29 (baseline for HAMSA) Effect of HAMS and HAMSA on fecal butyrate Results Figure 3 Figure 2 Figure 4 Figure 5A Figure 5B CRICOS No.0014A