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 fatty acid concentrations in a cohort of children in southern IndiaRAMADASS BALAMURUGAN1,2; PUGAZHENDHI SRINIVASAN1; GOWRI BALACHANDAR1; TAMILSELVAN DHARMALINGAM1; ELISSA MORTIMER3; GEETHA GOPALSAMY3; RICHARD WOODMAN3; ROSIE MENG3; MARK MANARY4; HENRY BINDER5; IAN BROWN3; LEX LEONG3,6; GERAINT ROGERS3,6; GRAEME YOUNG3; BALAKRISHNAN RAMAKRISHNA1,7

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.

ObjectivesWe 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

BackgroundCarbohydrates 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. 20141

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.

Effect of native and acetylated high amylose maize starch on fecal pH and short chain fatty acid concentrations in a cohort of children in southern India

Ramadass Balamurugan1; Gowri Balachandar1; Tamilselvan Dharmalingam1; Elissa Mortimer2; Geetha Gopalsamy2; Richard Woodman2; David Alpers3; Mark Manary3; Henry Binder4; Ian Brown2; Graeme Young2; Balakrishnan Ramakrishna1,5

1Gastroenterology, Christian Medical College, Vellore, India; 2Flinders University of South Australia, Adelaide, SA, Australia.; 3Washington University School of Medicine, St. Louis, MO, USA.; 4Yale University School of Medicine, New Haven, CT, USA; 5SRM Institutes for Medical Science, Chennai, India

Background

Carbohydrates that resist digestion alter the gut microbiome and the metabolome. The production of short chain fatty acids (SCFA) by the microbiome, in particular, influences many aspects of gastrointestinal health. High amylose maize starch (HAMS) contains up to 70% amylase resistant starch that is resistant to pancreatic amylase and therefore enters the colon unabsorbed. It is rapidly fermented by colonic bacteria, yielding high concentrations of SCFA including butyrate. High amylose maize starch acetate (HAMSA) is HAMS esterified with acetate; the latter is rapidly released by microbial hydrolases in the colon. 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. •  Twenty children, aged 2 to 5 years, (ten stunted with height for age [HAZ] less 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. •  Two week washout period on their regular diet. •  Fed cookies containing HAMSA (10 g/day) for two weeks. •  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.

Objectives

Methods

Results

Participants

Conclusions •   HAMS and HAMSA both lowered fecal pH. •   Both starches increased fecal SCFA concentration;

HAMSA increased acetate and propionate but not butyrate.

•   The effect on both parameters was significantly less pronounced in stunted children.

•   Colonic carbohydrate fermentation was significantly impaired in stunted children.

These studies were supported by a grant from the Bill and Melinda Gates Foundation

Participants

ConclusionsHAMS 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).

Results

Results

Figure 1

p=.03

p=.001

p=.02

p=.02

+ +++ *

*

** *

Day0-14 HAMS Day14-28 Washout period,regular diet

Day29-44 acetylated-HAMS

56

78

pH

0 3 7 11 15 18 22 25 29 32 36 40 44day

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

Figure 2

Figure 3

p=.02

p=.02

p=.01

+ ++

+ + *

Day0-14 HAMS Day14-28 Washout period,regular diet

Day29-44 acetylated-HAMS

4060

8010

012

0

acet

ate

(mm

ol/k

g)

0 3 7 11 15 18 22 25 29 32 36 40 44day

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

020

4060

prop

iona

te (m

mol

/kg)

0 3 7 11 15 18 22 25 29 32 36 40 44day

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

Figure 2

Figure 3

p=.02

p=.02

p=.01

+ ++

+ + *

Day0-14 HAMS Day14-28 Washout period,regular diet

Day29-44 acetylated-HAMS

4060

8010

012

0

acet

ate

(mm

ol/k

g)

0 3 7 11 15 18 22 25 29 32 36 40 44day

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

020

4060

prop

iona

te (m

mol

/kg)

0 3 7 11 15 18 22 25 29 32 36 40 44day

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

Figure 4

p=.003p=.03

p=.02+ *+

+

++

+

Day0-14 HAMS Day14-28 Washout period,regular diet

Day29-44 acetylated-HAMS

010

2030

40

buty

rate

(mm

ol/k

g)

0 3 7 11 15 18 22 25 29 32 36 40 44day

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

Figure 5A

Results

Figure 3

Figure 2 Figure 4

Figure 5A

Figure 5B

CRICOS No.0014A