Antioxidants in Cocoa Powder

Antioxidants in Cocoa Powder
Originally Posted by Hersheys Center for Health & Nutrition HERE


Antioxidants in foods have gained much attention in recent years and cocoa powders tend to have relatively high amounts.  As cocoa makes its way from fresh beans to finished products like cocoa powder and chocolate, the concentration of antioxidant compounds can be affected by a variety of biological and processing conditions.  Genetics can cause as much as a 4-fold difference in antioxidant content of fresh cocoa beans (3, 4).   Fermentation of fresh cocoa beans, while critical for full cocoa flavor, also tends to decrease antioxidant content.  Roasting of cocoa beans and treatment of cocoa powder with alkali can also decrease the final content of antioxidants. A study of the antioxidant content of various alkalized cocoa powders reveals that the antioxidant content decreases proportionally with the amount of alkalization(2). Due to the effect of all of these factors, it has become important to identify the specific types of antioxidants in cocoa products and develop analytical measurements for their contents. 

 

Types

There are several types of antioxidant compounds found in cocoa powder. There has recently been a great deal of interest in polyphenolic compounds, particularly flavonoids, as antioxidants.  Flavonoids are synthesized by all vascular plants.  As a result, fruits, vegetables, nuts, seeds, herbs, spices, and whole grains are sources of flavonoids in our diet(5).  Cocoa beans are a concentrated source of antioxidants(6) and flavonoids with the flavan-3-ols and their derivatives being present in high concentrations(7).

 

The discovery of flavan-3-ols and their procyanidin polymeric forms in cocoa can be traced back as early as 1909 (8).  These flavan-3-ol compounds were later identified as catechins(9, 10).  In 1939, leucoanthocyanin phenolic compounds were identified(11) and, in 1955, fractionation and characterization of these compounds was reported(12).  The procyanidins in cocoa have more recently been fractionated into monomers through decamers with even higher forms existing (13).  The most abundant polyphenols present in cocoa are the flavan-3-ol monomers, epicatechin and catechin(14), which also serve as building blocks for the polymeric procyanidin forms. The makeup of the polymeric forms is determined by the structure of the flavan-3-ol starter unit and its companion compound.  Two primary forms of procyanidins occur: A-type and B-type which differ by the linkage between the individual compounds. The A-type procyanidins form 2-7 cross links and can be found in cranberries.  The B-type procyanidins form 4-8 cross links.  The B-1 through B-4 types differ only in the arrangement of their catechin and epicatechin units with procyandin B-1 found in grape, sorghum and cranberry, type B-2 in apple, cocoa and cherry, B-3 in strawberry and hops, and B-4 found in raspberry and blackberry(15).

 

Antioxidant Measurements

There are several types of measurement of food and beverages that can be made that help nutritionists and consumers.  These measurements can be divided into two general groups:  activity-based and chemical compound-based measurements.

Activity-based tests are those that measure the antioxidant activity of the food or beverage.  Activity-based tests do not measure specific phytonutrients; rather theses tests measure the total of compounds within a food/beverage that contribute to antioxidant activity.  Some of the activity can be a result of compounds that are absorbed by the body and have meaningful impact on human physiology.  Other compounds measured by activity-based tests have nothing to do with the physiological impact on human physiology. 

Activity-based tests are particularly useful when comparisons of foods/beverages are made.  This is because antioxidant activity represents the “common currency” measure.  Activity-based testing cuts through the difference in phytochemical content of differing foods/beverages permitting scientists and marketers to make rough comparisons of foods/beverages based on the results of these tests.

Activity-based tests have various four to five letter names such as ORAC, FRAP, TEAC, etc,  By far the most common measure in the US is an assay developed and perfected by U.S.D.A. scientists called ORAC(16).  ORAC stands for Oxygen Radical Absorbance Capacity.  The most recent form of the test measures water-soluble as well as oil-soluble antioxidants.

Chemistry-based tests measure the actual level of chemical compounds in foods/beverages.  General chemical tests measure broad classes of chemicals such as phenolic compounds, which are a collection of thousands of compounds or flavanoid compounds for which there are an estimated 8,000 variants.  Specific chemical tests measure individual or small groups of very specific chemicals within the food/beverages.  Specific measures focus on compounds that are either known to be important to human nutrition such as vitamins, lycopene, fiber or compounds implicated in health benefits.  Examples of chemistry-based tests that are used to measure specific classes of antioxidant compounds in cocoa include:  total polyphenols, dimethyl amino cinnamaldehyde (DMAC), flavanol monomers, and total flavanols (PAC’s).  

ORAC (Oxygen Radical Absorbance Capacity) is a measure of general antioxidant activity in the food/beverage and is commonly used in the US for comparison of different foods/beverages to one another.  USDA has published databases of foods/beverages with ORAC measurements expressed on an equal weight basis and on a per serving basis.  Cocoa powder and dark chocolate are in the top 3% and milk chocolate is in the top 10% of foods/beverages with antioxidant activity.  ORAC is typically express as “ORAC based on micrograms of TE activity” per gram or serving.

Total Polyphenols is a general chemical measure of compounds that have a phenolic group associated with them(17).  There are literally thousands of compounds identified in raw materials that will be measured by the total polyphenol test.  So this test does not measure specific compounds but rather measures a general class of compounds that contain phenol groups.  Some of the compounds measured will have bioactivity while others will not be bioactive, due to their chemical structure or lack of bioavailability.  Cocoa powder is a food that is high in polyphenols.  Other foods that have high polyphenols include spices, tea, and coffee.  Total polyphenols are typically expressed as mg of gallic acid or mg of epicatechin.

DMAC is a more specific measure of the total flavanol content and is based on the reaction of a chemistry specific reagent, dimethyl amino cinnamaldehyde, with the food or beverage sample.(18)  This is a relatively new test and data is just starting to be reported.  A variety of standards can be used with the DMAC assay but Hershey and other collaborating labs have found that the flavanol B-2 dimer is currently the best choice.  Hershey uses this test to more accurately express the total flavanol content and reports results as mg of flavanols on a flavanol B-2 dimer basis. 

Monomers
Flavanols are thought to be the chemicals that are, at least in part, responsible for some of the healthful benefits of cocoa and chocolate.  Direct measurement of the flavanol in cocoa and other foods can be done by several means, each method focusing on various size classes of the flavanols.  The basic sub-units of the flavanols are epicatechin and catechin, which can be linked into polymers.  Direct measurement of the subunits of the flavanols can be done in which epicatechin and/or catechin are isolated from the food and separated(19).  In this case, results are expressed as mg of the specific monomeric unit using pure epicatechin and catechin as standards. 

            flavanols

PAC’s
Flavanol polymers, sometimes called proanthocyanidins or PAC’s, can be measured in cocoa powder and chocolate as well as in other foods.  The flavanols can be isolated and separated into their component parts including the monomeric subunits, small polymers and long chain polymers.  The various polymeric flavanols are separated based on their size into separate peaks(20).  These peaks are then quantified using chemical standards isolated from cocoa or other flavanol-rich plants.  Data can be reported for individual polymeric units or as a sum of all monomers and polymers as mg of total flavanols or PAC’s.  The availability of specific standards for each polymer has been lacking in the past and as a result much of the PAC data in the literature are based on differently prepared standards.  This makes it difficult to compare data from different labs.  Recently, pure polymeric standards have become available from n=2-10.  Hershey has recently chosen to report all new PAC data as “mg of flavanols 1 through 10 (PAC-10).  

Content
The antioxidant contents of various cocoa powders have been measured using the measurements described above.  The Hershey’s cocoa powders listed in the table below are:  Hershey’s Regular Unsweetened Cocoa Powder, which is a natural cocoa, and Hershey’s Special Dark Cocoa, which is an alkalized cocoa.  Also listed are the antioxidant contents of commercial cocoa powders that have been alkalized to different degrees ranging from:  natural/non-alkalized (pH 5.56), to lightly alkalized (pH 6.97), to moderately alkalized (pH 7.35), and to heavily alkalized (pH 7.88).

Antioxidant Content of Cocoa Powders*

Cocoa Powder Type

ORAC

TP

DMAC

Monomers

PAC-10

PAC

 

(umolTE/g)

(mg/g)

(mgB-2/g)

(mg/g)

(mg/g)

(mg/g)

Hershey’s Regular Cocoa

628

56.36

 

2.66

 

36.21

Hershey’s Special Dark Cocoa

233

11.52

 

0.06

 

4.51

Commercial Natural Cocoa

629

52.64

 

3.11

 

34.60

Lightly Alkalized Cocoa

375

29.42

 

1.39

 

13.80

Moderately Alkalized Cocoa

279

20.77

 

0.63

 

7.82

Heavily Alkalized Cocoa

253

15.63

 

0.13

 

3.92

* all data taken from Miller et. al. in 2008(2)

Reference List

1.   Chocolate, Cocoa, and Confectionery:  Science and Technology; 3rd ed.; Chapman & Hall: New York, 1989.
2.   Miller, K. B.; Hurst, W. J.; Payne, M. J.; Stuart, D. A.; Apgar, J.; Sweigart, D. S.; Ou, B. Impact of alkalization on the antioxidant and flavanol content of commercial cocoa powders. 2008, 8527-8533.
3.   Clapperton, J.; Lockwood, R.; Romanczyk, L.; Hammerstone, J. F.  Contribution of genotype to cocoa (Theobroma cacao L.). 71 ed.; 1994; pp 303-308.
4.   Forsyth, W. G. C.; Rombouts, J. E.  Our approach to the study of cocao fermentation. 1951; pp 73-81.
5.   USDA Database for the Flavanoid Content of Selected Foods. 2003.
6.   Gu, L. W.; Kelm, M. A.; Hammerstone, J. F.; Beecher, G.; Holden, J.; Haytowitz, D.; Gebhardt, S.; Prior, R. L. Concentrations of proanthocyanidins in common foods and estimations of normal consumption. Journal of Nutrition 2004, 134 (3), 613-617.
7.   Ultee, A. J.; Van Dorsen, J.  Bijdrage tot de kennis der op java gecultiveerde cacaosooten. No. 33 ed.; 1909.
8.   Adam, W. B.; Hardy, P.; Nierenstein, M.  The catechin of the cocoa bean. 33 ed.; 1931; pp 727-728.
9.   Freudenberg, K.; Cox, R. F. B.; Braun, E.   The catechin of the cocoa bean. 54 ed.; 1932; pp 1913-1917.
10.   Knapp, A. W.; Hearne, J. F.  Teh presence of Leuco-anthocyanidins in Criollo Cacao. 64 ed.; 1939; pp 475-480.
11.   Forsyth, W. G. C.  Cacao polyphenolic substances 3: Separation and estimation on paper chromatograms. 60 ed.; 1955; pp 108-111.
12.   Hammerstone, J. F.; Lazarus, S. A.; Mitchell, A. E.; Rucker, R.; Schmitz, H. H. Identification of Procyanidins in Cocoa (Theobroma cacao) and Chocolate Using High-Performance Liquid Chromatography/Mass Spectrometry. JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 1999, 47, 490-496.
13.   KIM, H.; Keeney, P. G. (-)-Epicatechin content in fermented and unfermented cocoa beans. Journal of Food Science 1984, 49 (4), 1090-1092.
14.   Whiting, D.  Natural phenolic compounds 1900-2000: a bird's eye view of a centuries chemistry. 18 ed.; 2001; pp 583-606.
15.   Clapperton, J.; Hammerstone, J. F.; Romanczyk, R.; Yow, S.; Chau, J.; Lin, D.; Lookwood, R.   1992; pp 112-115.
16.   Ou, B.; Hampsch-Woodill, M.; Prior, R. L. Development and validation of an improved oxygen radical absorbance capacity assay using fluorescein as the fluorescent probe. JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 2001, 49 (10), 4619-4626.
17.   Singleton, V. L.; Rossi, J. A. Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Am. J. Enol. Vitic. 1965, 16 (3), 144-158.
18.   McMurrough, I.; McDowell, J. Chromatographic separation and automated analysis of flavanols. Analytical Biochemistry 1978, 91 (1), 92-100.
19.   Nelson, B. C.; Sharpless, K. E. Quantification of the predominant monomeric catechins in baking chocolate standard reference material by LC/APCI-MS. Journal of Agriculture and Food Chemistry 2003, 51 (3), 531-7.
20.   Gu, L.; Kelm, M.; Hammerstone, J. F.; Beecher, G.; Cunningham, D.; Vannozzi, D.; Prior, R.  Fractionation of polymeric procyanidins from low-bush blueberry and quantitation of procyanidins in selected foods with an optimized normal phase HPLC-MS flourescence detection method. 50 ed.; 2002; pp 4852-4860.

Category: Nutritional Characteristics
Posted: Sunday, August 1, 2010 05:12:00 PM
Views: 42843
Synopsis: Antioxidants in foods have gained much attention in recent years and cocoa powders tend to have relatively high amounts. As cocoa makes its way from fresh beans to finished products like cocoa powder and chocolate, the concentration of antioxidant compounds can be affected by a variety of biological and processing conditions.