Impact of the Microbiome 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?

!  Africa, Brazil, Caribbean islands, Central America, South America

!  What are the different types?

!  Forastero* , Criollo, and Trintario

!  Cacao tree

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

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

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, cracked lips”

!  Cacao “healed” kidney disease, liver illnesses, and faintness

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 powder Procyanidins

Miligrams of Catechin per Gram of Lyophilized 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 per Gram of Lypholized PCE  

Hydroxybenzoic acid   42.77  

Vanillic acid   41.74  

Caffeic Acid   99.76  

Syringic Acid   26.10  

Ferulic Acid   3.45  Vanillin   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

Micrograms of Standard per Gram of Lyophilized PCE  

Quercetin   93.17  Quercetin-glucoside   1284.00  

Quercetin-rhamnoside   18.15  Quercetin-rutinoside   21.52  

Quercetin-arabinoside   1622.00  Dihydroquercetin   76.09  Dihydroxykaempferol   378.70  Kaempferol-rutinoside   1144.00  Kaempferol-glucoside   17.31  Naringenin   8.78  Naringenin-glucoside   42.43  Myrincetin-glucoside   71.91  Luteolin   75.01  Luteolin-glucoside   362.00  Apigenin   1.75  Apigenin-glucoside   1.31  

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.

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 or poorly absorbed components ? How are they modified by microbiome?

!  Procyanidins

!  Anti-inflammatory (Antioxidant and color)

!  Fiber

!  Acid Detergent Fiber

!  Cell wall Digestibility

!  High ADF% = low digestibility

!  Bacteria broke down the non-digestible particles

Approach

!  Model human digestion process and remove 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

Cocoa Polyphenols

Monomers and Dimers Phase II Enzymes

Conjugates Glucuronides Sulfates

Urine

Feces Unmodified Metabolites

Fermentation Bifidobacterium spp Lactobacillus spp Staphlococcus Bacteriodes C. Histolyticum sub group

Monomers and Dimers

Nitric Oxide Synthesis Prostacyclin Oxidation markers Inflammation Platelet adhesion

Improved Flow (FMD) Vessel Flexibility

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

Stoupi, Stavroula, et al. "A comparison of the in vitro biotransformation of (–)-epicatechin and procyanidin B2 by human faecal microbiota." Molecular nutrition & food research 54.6 (2010): 747-759.

Cocoa Powder (substrate)

Rinse w. Ethanol &

Water

Freeze dry

Microbiota Fermentation

80% N2, 10% H2, 10%CO2

Gastric Digestion Pepsin pH 2.0

Intestinal Digestion Pancreatin pH 7.5

Phenolics HPLC-MS pH

Short Chain Fatty Acid

GC

Model System

Different Cocoa Powders with variation in processing

Dutched D and E Alkalined treated pH 7.5-8.5

Regular A,B and C pH 5.3-6.0

Unfermented

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

Fiber Analysis

0

5

10

15

20

25

30

35

Unfermented Regular B Regular C Dutched A Dutched B

Crude Protein and Fiber Percentages Found in Cocoa Powder

Crude Protein ADF %

pH over Time: Trial 1

7.00

7.20

7.40

7.60

7.80

8.00

8.20

8.40

0 12 24 0 12 24 0 12 24 0 12 24

Restistant Starch Regular A Regular C Dutched A

pH

Incubation Time

pH of Different Incubation Times of Cocoa Powders

pH over Time: Trial 2

5.50

6.00

6.50

7.00

7.50

8.00

8.50

0 6 12 18 24 0 6 12 18 24 0 6 12 18 24 0 6 12 18 24

Restistant Starch Unfermented Regular B Dutched B

pH

Incubation Times

pH of Differet Incubation Times of Cocoa Powders

Results- Sort Chain Fatty Acids

0

10

20

30

40

50

60

70

0 6 12 18 24 0 6 12 18 24 0 6 12 18 24

Unfermented Regular B Dutched B

umol

/g o

f su

bstr

ate

Incubation Time

Acetate

-5

0

5

10

15

20

0 6 12 18 24 0 6 12 18 24 0 6 12 18 24

Unfermented Regular B Dutched B

umol

/g o

f su

bstr

ate

Incubation Time

Propionate

-1

0

1

2

3

4

5

6

0 6 12 18 24 0 6 12 18 24 0 6 12 18 24

Unfermented Regular B Dutched B

umol

/g o

f Su

bstr

ate

Incubation Time

Butyrate

Procyanidin results

0

200

400

600

800

1000

1200

0 6 12 18 24 0 6 12 18 24 0 6 12 18 24

Unfermented Dutched B Regular B

µg/m

L of

sam

ple

Time of incubation (hours)

Catechin Epicatechin

CATECHIN Changes During Anaerobic Fermentation

0

200

400

600

800

1000

1200

0 6 12 18 24 0 6 12 18 24 0 6 12 18 24

Unfermented Dutched B Regular B

µg/m

L of

sam

ple

Time of incubation (hours)

Catechin

EPICATECHIN Changes During Fermentation

0

10

20

30

40

50

60

0 6 12 18 24 0 6 12 18 24 0 6 12 18 24

Unfermented Dutched B Regular B

µg/m

L of

sam

ple

Time of incubation (hours)

Epicatechin

Maldi Analysis

        Dutched B   Regular B   Unfermented  

Phenolic Acid  

Molar Weight  

12 hours   24 hours  12 hours  

24 hours   12 hours  24 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

12-24h

0-12h 12-24h 0-12h 12-24h

Protacatechuric 154.12/154 = + + + - +

Vanillic 168.15/166

- + + - + +

Chlorogenic 354.31/357

= + + + x x

Conclusions

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

!  Epicatechin and catechin are released through action of microbiota

!  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?