Impact of theMicrobiome on
Cocoa Polyphenolic
Compounds
Maria Moore, Mfamara Goita, Dr. John W. Finley
Department of Nutrition and Food Science
Louisiana State University

Introduction!

Chocolate!

Where does it come from?
!

!

What are the different types?
!

!

Africa, Brazil, Caribbean islands, Central America, SouthAmerica

Forastero* , Criollo, and Trintario

Cacao tree

Grivetti, L.E., & Shapiro, H. (eds). (2009). Chocolate: History,Culture, and Heritage. Hobroken, NJ: A John Wiley & Sons, Inc.,
Publication.

http://thestoryofchocolate.com/where/content.cfm?
ItemNumber=3299&navItemNumber=3255

History!

600-400 BCE first use of cacao!

250-900 CE first referred as Chocolate!

Grown and consumed in South America first!

Indigenous language- Chocolate and Cacao!

Used as a currency!

1851 first time chocolate was introduced to USA

Grivetti, L.E., & Shapiro, H. (eds). (2009). Chocolate: History, Culture, and Heritage. Hobroken, NJ: A John
Wiley & Sons, Inc., Publication.

History- Medicinal theories!

Chocolate was drunk to cure stomach and intestinal
complaints!

Cacao bark- “reduce bloody stool”!

Cacao fat- “disinfect cuts”!

Cacao flowers- treat cuts!

Cacao fruit pulp- “help facilitate delivery!

Cacao leaves- “antiseptics to wounds”!

Cacao oil/butter- “applied to wounds, burns, crackedlips”!

Cacao “healed” kidney disease, liver illnesses, andfaintness

Grivetti, L.E., & Shapiro, H. (eds). (2009). Chocolate: History, Culture, and Heritage. Hobroken, NJ: A John Wiley & Sons,Inc., Publication.

Proximate analysis!

Protein 20%!

Fat 14%!

Carbohydrates 58%!

!

Fiber 34%

Procyanidins

Composition of Cocoa powderProcyanidins
Miligrams of Catechin per Gram ofLyophilized PCE 	  Catechin 	  8.974	  Epicatechin 	  2.344	  Dimer 	  10.670	  Trimer	  1.027	  Tetramer	  0.120	  Pentamer	  0.009	  Epigallocatechin	  0.096	  Ortega, Nàdia, et al. "Obtention and characterization of phenolic extracts from different cocoa
sources." Journal of agricultural and food chemistry 56.20 (2008): 9621-9627.

Composition of Cocoa powder
Phenolic Acids
Micrograms of Standard perGram of Lypholized PCE	  Hydroxybenzoic acid	  42.77	  Vanillic acid	  41.74	  Caffeic Acid	  99.76	  Syringic Acid	  26.10	  Ferulic Acid	  Vanillin 	  3.45	  9.03	  Coumaric Acid	  30.47	  Cinnamic Acid	  91.28	  Chrlorgenic Acid	  21.91	  Protecatchuic Acid	  226.00	  Ortega, Nàdia, et al. "Obtention and characterization of phenolic extracts from different cocoa
sources." Journal of agricultural and food chemistry 56.20 (2008): 9621-9627.

Composition of Cocoa powder
Favones
Quercetin	  Quercetin-glucoside	  Micrograms of Standard perGram of Lyophilized PCE 	  93.17	  1284.00	  Quercetin-rhamnoside	  Quercetin-rutinoside	  18.15	  21.52	  Quercetin-arabinoside	  Dihydroquercetin	  Dihydroxykaempferol	  Kaempferol-rutinoside	  Kaempferol-glucoside	  Naringenin	  Naringenin-glucoside	  Myrincetin-glucoside	  Luteolin	  Luteolin-glucoside	  Apigenin	  Apigenin-glucoside 	  1622.00	  76.09	  378.70	  1144.00	  17.31	  8.78	  42.43	  71.91	  75.01	  362.00	  1.75	  1.31	  Ortega, Nàdia, et al. "Obtention andcharacterization of phenolic extracts
from different cocoa sources." Journalof agricultural and food chemistry
56.20 (2008): 9621-9627.

Major phenolic compounds in cocoa

Andújar, I., et al. "Cocoa polyphenols and their potential benefits for human health." Oxidative
medicine and cellular longevity 2012 (2012).

Major phenolic compounds in cocoa

Andújar, I., et al. "Cocoa polyphenols and their potential benefits for human health." Oxidative
medicine and cellular longevity 2012 (2012).

Major phenolic compounds in cocoa

Andújar, I., et al. "Cocoa polyphenols and their potential benefits for human health." Oxidative
medicine and cellular longevity 2012 (2012).

Major phenolic compounds in cocoa

Andújar, I., et al. "Cocoa polyphenols and their potential benefits for human health." Oxidative
medicine and cellular longevity 2012 (2012).

What is the fate of non-digestible orpoorly absorbed components ?
How are they modified by microbiome?
!

Procyanidins!

!

Fiber!

!

Anti-inflammatory (Antioxidant and color)

Acid Detergent Fiber!

Cell wall Digestibility!

High ADF% = low digestibility

Bacteria broke down the non-digestible particles

Approach! Model human digestion process andremove soluble products! Conduct a fermentation by model

microbiome system! Effects on fiber! pH change as an index of fermentation! Determine short chain fatty acid production! Effects on polyphenols! Determine changes in phenolic compounds

during fermentation! Determine breakdown of polyphenolic

compounds into smaller compounds by MADLI
and LC mass spectroscopyCocoaPolyphenols

Nitric Oxide Synthesis
Prostacyclin
Oxidation markers
Inflammation
Platelet adhesion

Fermentation
Bifidobacterium spp
Lactobacillus spp
Staphlococcus
Bacteriodes
C. Histolyticum subgroup

Improved Flow (FMD)
Vessel Flexibility

meMonod Dirs anmers

Monomers and Dimers

Phase II
Enzymes
Conjugates
Glucuronides
Sulfates

Urine
Feces
Unmodified
Metabolites

A comparison of the in vitro biotransformation of (–)-epicatechin
and procyanidin B2 by human faecal microbiota

Stoupi, Stavroula, et al. "A comparison of the invitro biotransformation of (–)-epicatechin andprocyanidin B2 by human faecal
microbiota." Molecular nutrition & foodresearch 54.6 (2010): 747-759.

Model System
Cocoa Powder(substrate)

Gastric Digestion
Pepsin pH 2.0

Rinse w.
Ethanol &Water

Microbiota
Fermentation

Freeze dry80% N2, 10% H2,10%CO2

Phenolics
HPLC-MS

Intestinal Digestion
Pancreatin pH 7.5

pH

Short Chain
Fatty AcidGCDifferent Cocoa Powders withvariation in processing

Unfermented

Regular A,B and C
pH 5.3-6.0

http://www.savoryspiceshop.com/spices/cacaopdr.html

Dutched D and E
Alkalined treated
pH 7.5-8.5

Fiber Analysis
Crude Protein and Fiber Percentages Found in Cocoa Powder
35302520151050
Unfermented

Regular B

Regular C

Crude Protein

Dutched A
ADF %Dutched B

pH over Time: Trial 1
pH of Different Incubation Times of Cocoa Powders8.40
8.20pH

8.00
7.80
7.60
7.40
7.20
7.00
01224

Restistant Starch012Regular A

24012
Regular C

Incubation Time2401224

Dutched A

pH over Time: Trial 2
pH of Differet Incubation Times of Cocoa Powders
8.50
8.00

pH

7.50
7.00
6.50
6.00
5.50
0612 18 24

Restistant Starch0612 18 24

Unfermented

0612 18 24

Regular B

Incubation Times

0612 18 24

Dutched B

Results- Sort Chain Fatty Acids
Acetateumol/g of substrate

7060
50403020100061218

Unfermented

24061218

Regular B
Incubation Time24061218

Dutched B

24

Propionate
20umol/g of substrate

151050061218

Unfermented-524061218

Regular B
Incubation Time

24061218

Dutched B

24

Butyrate
6umol/g of Substrate

5432
100-1

61218

Unfermented

24061218

Regular B
Incubation Time

24061218

Dutched B

24

Procyanidin results
1200µg/mL of sample

1000

800

600

400

200

0061218

Unfermented

2406121824

Dutched B
Time of incubation (hours)
Catechin

Epicatechin

0612Regular B

1824

CATECHIN Changes During
Anaerobic Fermentation
1200µg/mL of sample

1000
800
600
400
2000061218

Unfermented

2406121824

Dutched B
Time of incubation (hours)
Catechin

0612Regular B

1824

EPICATECHIN Changes During
Fermentation
60µg/mL of sample

50

4030

20

100061218

Unfermented

2406121824

Dutched B
Time of incubation (hours)
Epicatechin

0612Regular B

1824

Maldi Analysis	  	  Phenolic
Acid	  

MolarWeight	  Dutched B	  12hours	  Regular B	  Unfermented	  24	   12 hours 	  24 hours	  24 hours	  12 hours hours	  Vanillic
Acid	  

168. 15	  + 	  -	  + 	  =	  +	  =	  Caffeic
Acid	  

180.16	  + 	  =	  +	  =	  +	  +	  Syringic
Acid	  

198.17	  - 	  = 	  - 	  - 	  +	  +	  LCS-Ms Results
Dutched B

Regular B

Unfermented

Compound

Molar
Mass

0-12h

12-2
4h

0-12h

12-24h0-12h12-24hProtacatechuric

154.1
2/154

=+++-+

Vanillic

168.1
5/166

-++-++

Chlorogenic

354.3
1/357

=+++xxConclusions!

Polymers of catechin and epicatechin are not digested
by gastric enzymes!

Epicatechin and catechin are released through action ofmicrobiota!

Fiber of cocoa is fermented to healthy short chain fatty
acids!

Epicatechin and catachin are fermented to smaller
phenolic acids by the microbiome

Thank you!
Questions?