Impact of the Microbiome on Cocoa Polyphenolic Compoundsgraphics8.nytimes.com/packages/pdf/health/ACS.pdfCacao bark- “reduce bloody stool” ! Cacao fat- “disinfect cuts” ! Cacao

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


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


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


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


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


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


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


µg/m

L of

sam

ple


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


µg/m

L of

sam

ple


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?

Documents

Impact of the Microbiome on Cocoa Polyphenolic Compoundsgraphics8.nytimes.com/packages/pdf/health/ACS.pdfCacao bark- “reduce bloody stool” ! Cacao fat- “disinfect cuts” ! Cacao