Kiwifruit-driven Microbiota, Metabolites And Implications for Human Health by Dr Paul Blatchford

1. Kiwifruit-driven Microbiota, Metabolites and Implications For Human Health Dr Paul Blatchford Plant & Food Research, New Zealand

2. KIWIFRUIT-DRIVEN MICROBIOTA, METABOLITES AND IMPLICATIONS FOR HUMAN HEALTH Paul Blatchford

3. Health benefits Glycaemic impact Vitamin C Prebiotic effects? Laxation Immune benefits

4. Health benefits Glycaemic impact Vitamin C Prebiotic effects?Laxation Immune benefits Prebiotic definition ‘a selectively fermented ingredient that results in specific changes in the composition and/or activity of the gastrointestinal microbiota, thus conferring benefit(s) upon host health’ (Gibson et al., 2010)

5. KIWIFRUIT • Kiwifruit contain 2-3% non-starch polysaccharides (NSP) such as pectic polysaccharides, hemicellulose and cellulose • The NSP escape digestion by human enzymes and reach the colon relatively unmodified. Here they become substrates for bacterial fermentation The following work used green-fleshed Actinidia chinensis var. deliciosa `Hayward’ (Zespri® Green Kiwifruit) and gold-fleshed Actinidia chinensis var. chinensis `Zesy002’ (Zespri® SunGold Kiwifruit)

6. GUT MICROBIOTA • The human gut microbiota has strong associations with many aspects of human health • The colon is an active site of fermentation: - short chain fatty acids (SCFA) - branched chain fatty acids (BCFA) - gases (hydrogen, carbon dioxide and methane) • Acetate, propionate, butyrate as well as lactate, formate, succinate etc..

7. HOW DOES KIWIFRUIT AFFECT BACTERIAL COMPOSITION?

8. • Mimics the large bowel microbial community • Several variables can be controlled: - pH - temperature - O2 • 10% w/v faecal slurry • Typically run in triplicate with different faecal donors • Run for 24 – 48 hours IN VITRO PH-CONTROLLED BATCH CULTURE GUT MODEL

9. IN VITRO FERMENTATION STUDY 1 • Batch culture gut models (in vitro) conducted with green and gold kiwifruit - 16S rRNA gene sequencing, Gas Chromatography, NMR Spectroscopy Kiwifruit fermentation drives positive gut microbial and metabolic changes irrespective of initial microbiota composition. P Blatchford, H Stoklosinski, G Walton, J Swann, G Gibson, R Gearry, J Ansell, Bioactive Carbohydrates and Dietary Fibre, 2015.

10. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Green 0h Green 5h Green 10h Green 24h Green 48h Gold 0h Gold 5h Gold 10h Gold 24h Gold 48h Neg 0h Neg 5h Neg 10h Neg 24h Neg 48h RelativeAbundance(%)>1% Haemophilus Citrobacter Enterobacteriaceae Bilophila Sutterella Erysipelotrichaceae Veillonella Phascolarctobacterium Dialister Ruminococcus Oscillospira Faecalibacterium Ruminococcaceae Lachnospira Coprococcus Blautia Lachnospiraceae Clostridiaceae Clostridiales Streptococcus Prevotella Parabacteroides Bacteroides Slackia Collinsella Coriobacteriaceae Bifidobacterium Unassigned/Other Actinobacteria Bacteroidetes Firmicutes Proteobacteria

11. IN VITRO FERMENTATION EXPERIMENTS

12. ORGANIC ACIDS BY GAS CHROMATOGRAPHY µmol/mL µmol/mL µmol/mL 0 5 10 24 48 Time (h) 0 5 10 24 48 Time (h) 0 5 10 24 48 Time (h)

13. IN VITRO FERMENTATION STUDY 2 Batch culture gut models (in vitro) with gold kiwifruit - 16S rRNA gene sequencing, Gas Chromatography, LC-MS In vitro characterisation of the fermentation profile and prebiotic capacity of gold-fleshed kiwifruit P Blatchford, K Bentley-Hewitt, H Stoklosinski, T McGhie, R Gearry, G Gibson, J Ansell, Beneficial Microbes, 2015.

14. HEATPLOT OF THE TOP 22 BACTERIAL GENERA

15. BETA DIVERSITY UNWEIGHTED BIPLOT Donor 1 Donor 3 Donor 2

16. -2.00 8.00 18.00 28.00 38.00 48.00 58.00 Gold - Acetate Gold - Propionate Gold - Butyrate Negative - Acetate Negative - Propionate Negative - Butyrate µmol/mL Time (h) 0 5 10 24 48 After 48 hours, the ratio of the three primary short chain fatty acids was 67:26:7 for acetate, propionate and butyrate, respectively ORGANIC ACID CONCENTRATIONS

17. RELATIVE AMOUNTS OF METABOLITES IN THE ZESY002 VESSELS 0 0.1 0.2 0.3 0.4 0.5 0.6 0 5 10 15 20 25 30 35 40 45 50 NormalisedIntensity Time (h) Quinic acid - 191.0562 m/z Citric acid - 191.0211 m/z Malic acid - 133.0143 m/z Tryptophan - 203.0818 m/z E-caffeoyl-3-glucoside - 341.0877 m/z

18. • Despite notable donor inoculum differences, kiwifruit was able to drive the microbial profile towards more commensal genera • Kiwifruit supplementation in vitro promoted the commensal Bacteroides spp., Parabacteroides spp. and Bifidobacterium spp. • The kiwifruit-driven increase of commensal bacteria in the in vitro models resulted in a propionate- enriched environment • Organic acids and polyphenols can survive SGD and may act as substrates for bacterial fermentation CONCLUSIONS

19. • What fractions of kiwifruit are responsible for causing the microbial and metabolic effects presented in this work? • What is the effect of other kiwifruit-derived metabolites that escape simulated gastrointestinal digestion and reach the colon? We know polysaccharides are principal drivers of microbiota composition • Organic acids - Quinic acid, malic acid, citric acid • Polyphenols – E-caffeoyl-3-glucoside, E-caffeoyl-4-glucoside etc... • Fatty acids – Linoleic acid, linolenic acid, oleic acid UNANSWERED QUESTIONS…

20. Laxative effects Oxalate/raphides? Organic acids? Bacteria? Fibre? Actinidin? PARTING MESSAGE….

21. Unanswered questions.... ARE THE LAXATIVE EFFECTS OF KIWIFRUIT PARTLY MEDIATED BY BACTERIA?

22. Thanks! • To Riddet and Zespri for the opportunity to present at this forum • To everyone in the Food, Nutrition and Health group at PFR, Palmerston North • Supervisors – Juliet Ansell, Richard Gearry, Glenn Gibson QUESTIONS? THANK YOU Paul Blatchford Paul.Blatchford@plantandfood.co.nz 064 6 3556164