Dr Paul Blatchford, Scientist at Plant & Food Research, New Zealand, presentation at the 1st International Symposium on Kiwifruit and Health: http://www.kiwifruitsymposium.org/presentations/kiwifruit-driven-microbiota-metabolites-and-implications-for-human-health/ The research presented attempts to understand how kiwifruit impacts upon microbial composition and metabolism in the human large bowel using in vitro fermentation systems.

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