Influence of alkalization treatment on the color quality and the total phenolic and anthocyanin contents in cocoa powder

Abstract

The effects of alkalization treatments on color, colorimetric fractions, total polyphenol content, and anthocyanin content of cocoa powder were investigated. A darker color and a lower total polyphenol content were obtained for cocoa powder alkalized using a K2CO3 solution than with an NaOH solution. A high temperature and basic pH conditions favored formation of dark components during alkalization due to sugar degradation, Maillard reactions, and anthocyanin polymerizing. The anthocyanin content decreased with an increasing alkali concentration, suggesting that more anthocyanins were transformed into brown polymers in darker cocoa powder. Cocoa powder with a heavy degree of alkalization had the lowest ratio of monomer anthocyanins to yellow/brown polymer content. OD460/OD525 values for alkalized samples were higher than for non-alkalized samples. Cocoa powder presented a better color quality after alkalization treatment.

Full text

Food Sci. Biotechnol. 23(1): 59-63 (2014)
DOI 10.1007/s10068-014-0008-5
Influence of Alkalization Treatment on the Color Quality and the
Total Phenolic and Anthocyanin Contents in Cocoa Powder
Yue Li, Song Zhu, Yun Feng, Feifei Xu, Jianguo Ma, and Fang Zhong
Received: 22 May 2013 / Revised: 31 July 2013 / Accepted: 1 August 2013 / Published Online: 28 February 2014
© KoSFoST and Springer 2014
Abstract The effects of alkalization treatments on color,
colorimetric fractions, total polyphenol content, and
anthocyanin content of cocoa powder were investigated. A
darker color and a lower total polyphenol content were
obtained for cocoa powder alkalized using a K2CO3
solution than with an NaOH solution. A high temperature
and basic pH conditions favored formation of dark
components during alkalization due to sugar degradation,
Maillard reactions, and anthocyanin polymerizing. The
anthocyanin content decreased with an increasing alkali
concentration, suggesting that more anthocyanins were
transformed into brown polymers in darker cocoa powder.
Cocoa powder with a heavy degree of alkalization had the
lowest ratio of monomer anthocyanins to yellow/brown
polymer content. OD460/OD525 values for alkalized samples
were higher than for non-alkalized samples. Cocoa powder
presented a better color quality after alkalization treatment.
Keywords: cocoa, alkalization, color, polyphenol
Introduction
Cocoa powder is the primary raw material of chocolate,
and also an important flavor in food products, such as
baked goods, icings, and beverages (1). During processing,
cocoa powder is usually subject to alkalization treatments
for purposes of browning, wetability, dispersability, flavor,
and to enhance the nutritional and biological qualities of
cocoa (2-5).
In practice, cocoa to be alkalized is first heated in a
closed mixing vessel, followed by addition of a warm
alkali solution for a certain reaction time, and excess
moisture is then driven off by heating or drying. The end
products of the alkalizing process are cocoa solids with a
higher pH and a darker color than in the natural state (6).
Alkalization is one of several routes that may be used by a
manufacturer to modify the color of cocoa with different
susceptibilities to development of o-quinones and Maillard
reactions, especially non-enzymatic brown compounds (5).
The degree of alkalization is controlled by the strength of
the alkali solution, the alkali type, the length of the reaction
stage, and the process temperature (6).
Cocoa beans are rich in polyphenols, contributing 12-
18% of the dry weight of the whole bean. Cocoa polyphenols
have been associated with flavor, color, and the nutritional
value of cocoa products (7-10). Although polyphenols in
cocoa powder develop the specific color of cocoa products
through roasting and alkalization, few studies have focused
on the relationships among the alkalizing conditions, the
color of cocoa products, and the polyphenol content.
Since alkalization treatments are commonly used in the
processing of cocoa powder, the properties of cocoa powder
(colors and polyphenol content) can be controlled using
various alkalization parameters in order to meet the demands
of customers. This study was performed to determine the
alkalization effects. Cocoa powder was treated under different
degrees of alkalization and the effect of alkali types on
color and the polyphenol content of cocoa powder was
evaluated. Influences of alkalizing conditions on color,
colorimetric fractions, and the anthocyanin content of
cocoa powder were also investigated.
Yue Li, Yun Feng, Feifei Xu, Jianguo Ma, Fang Zhong ()
Key Laboratory of Food Colloids and Biotechnology, Ministry of
Education, School of Food Science and Technology, Jiangnan University,
Wuxi 214122, China
Tel: +86-13812536912; Fax: +86-510-85197876
E-mail: fzhong@jiangnan.edu.cn
Song Zhu
State Key Laboratory of Food Science and Technology, Jiangnan
University, Wuxi 214122, China
RESEARCH ARTICLE

60 Li et al.
Materials and Methods
Material Commercial cocoa powder without alkalization
was provided by Xing Guang Cocoa Product Limited
Company (Shaoxing, China). The fat content of the cocoa
powder was approximately 10%. Sodium hydroxide,
sodium carbonate, acetone, glacial acetic acid, gallic acid,
hydrochloric acid, methanol, formic acid, and other
analytical reagents were purchased from Sinopharm Group
Chemical Reagent Co., Ltd. (Shanghai, China) and were of
analytical grade.
Alkalization of cocoa powder Initially, cocoa powder
was alkalized using either sodium hydroxide (NaOH, 2%,
w/w) or potassium carbonate (K2CO3 2%, w/w) solutions
at a pressure of 0.06 MPa for 20 min. In order to identify
the influence of alkalizing conditions on coloration of
cocoa powder, alkalization was then carried out using an
NaOH solution under pressures of 0.02, 0.04, 0.06, 0.08,
and 0.1 MPa, NaOH solution concentrations of 1, 1.5, 2,
2.5, and 3%(w/w), and alkalization times of 10, 15, 20, 25,
and 30 min.
Color measurement The color of alkalized cocoa powder
was determined using a Hunter Tristimulus Colorimeter
(UltraScan XE; HunterLab, Reston, VA, USA) based on
L*, a*, and b* values. An amount of 10 g of cocoa powder
was used. The L* value represents the lightness component
on the surface and ranges from 0 to 100 (100 =light to
0= black). The a* and b* values are chromatic components
of redness to greenness and blueness to yellowness,
respectively (11,12). Data are reported as the mean value
from measurements of 8 different samples.
Total polyphenol analysis The total polyphenol content
was determined using the Folin-Ciocalteu procedure (13)
with some modification according to Elwers et al. (14).
Defatted cocoa powder (0.5 g) was extracted 3 times using
70% acetone (v/v) under constant agitation. After each
extraction, the sample was centrifuged (5424; Eppendorf,
Hamburg, Germany) at 8,000×g for 10 min. The three
supernatants were combined in a flask, and acetone was
removed using rotary evaporation (N-1001S-WA; Tokyo
Rikakikai Co., Ltd., Tokyo, Japan) under a partial vacuum
at 40oC. The residue was transferred to a volumetric flask
containing 2 mL of glacial acetic acid and diluted to 10 mL
with Milli-Q Plus water. The crude extract was diluted 1/
10 (v/v) with 2.5% aqueous acetic acid.
The extract or standard (0.5 mL), 0.5 mL of Folin-
Ciocalteu reagent, and 1.5 mL of Na2CO3 solution (20%,
w/v) were mixed and diluted to 10 mL with Milli-Q Plus
water in a volumetric flask and allowed to stand for 60 min
at room temperature. Absorption was measured at 760 nm
using a 722 spectrophotometer (Shanghai Precision &
Scientific Instrument Co., Ltd., Shanghai, China). Gallic
acid was used as a standard and the total polyphenol
content was expressed as gallic acid equivalents in mg/g.
All samples were analyzed in triplicate.
Color and colorimetric fractions determination Color
components were extracted (45 min shaking) using 50 mL
of 12 M HCl/CH3OH (1 mL/L) from 2 g of cocoa powder.
Extracts were filtered through Whatman 541 paper (GE
Healthcare, Pittsburgh, PA, USA). An aliquot of filtrate (4
mL) was adjusted to 25 mL using 12 M HCl/CH3OH. The
absorbance was read at 460 and 525 nm (15,16) in a 722
spectrophotometer (Shanghai Precision, Shanghai, China).
The rest of the filtrate was then applied to a column
(300 mm×15 mm i.d.; Shanghai Huxi, Shanghai, China)
containing 1.5 g of a previously conditioned absorbent
mixture of Silica G, Silicagel 60, and Polyclar AT (2/14/4).
Fractions of monomer anthocyanins (F1), red polymers
(F2), and yellow/brown polymers (F3) were eluted using
12 M HCl/CH3OH (1 mL/L), 50% formic acid, and pure
formic acid, respectively (25 mL of each solvent). The
absorbance was measured at 525 nm (16) using a 722
spectrophotometer (722; Shanghai Precision & Scientific
Instrument Co., Ltd.). Measurements were made in triplicate.
Statistical analysis Mean values were calculated and
Duncan’s t-test was performed using SPSS version 13.0 for
Windows software (SPSS Institute Inc., Cary, NC, USA).
Results and Discussion
Influence of alkali type on the color and polyphenol
content of cocoa powder Several alkali solutions can be
used for alkalizing process. NaOH and K2CO3 are the most
commonly used alkalis for cocoa powder alkalization
(6,17,18). The influence of NaOH and K2CO3 solutions on
the color (∆E) of cocoa powder is shown in Fig. 1. The
L* a* b* color system can provide uniformity in color
measurement and human perception (19,20), and is useful
for expression of visual color changes during browning.
An equation for calculating the color difference (∆E)
before and after alkalization was developed by Scofield
(21) as: ∆E=((∆L)2+(∆a)2+(∆b)2)1/2. A higher ∆E value
reflects the darker color of cocoa after alkalization. The
human eye is capable of noticing small differences
between colors on the order of 0.2 ∆E (21). Cocoa powder
exhibited darker colors when a K2CO3 solution was used
for alkalization (Fig. 1). Since the two types of alkali used
have different basic strengths, Maillard reactions were
subject to different pH environments. Furthermore, Na+
and K+ ions may impact changes in anthocyanin structures
and, thus, produce different colors. It was reported that a

Alkalization Effects on Color of Cocoa Powder 61
K2CO3 solution generates a red-brown color and a
chocolate flavor in cocoa, and a NaOH solution has the
strong neutralization value of permitted alkalis, and it can
also produce red tones in press cake alkalization when
combined with an oxidation stage (17).
Polyphenols in cocoa powder are associated with human
health benefits and are involved in producing the typical
brown cocoa color and the astringent taste. The polyphenol
content was determined in cocoa powder after alkalization.
At the same concentration, alkalization with a NaOH
solution produced a higher polyphenol content than with a
K2CO3 solution (Fig. 1). Cocoa with a darker color had a
lower polyphenol content. To establish the relationship
between the color of cocoa powder and the total polyphenol
content, cocoa powder was treated with NaOH solutions
under various concentrations, reaction times, and pressures.
The relationship between the color of cocoa and the total
polyphenol content is shown in Fig. 2. The values of ∆E,
∆L, ∆a, and ∆b were negatively correlated with the total
polyphenol content, indicating that a darker color is
associated with a lower total polyphenol content. During
the alkalizing procedure, polyphenols such as anthocyanins,
procyanidins, and catechins, are changed into quinones,
which suffer polymerization or form high molecular weight
insoluble brown compounds via protein linking (22). Thus,
the less astringent taste, the darker the cocoa powder with
a decreased polyphenol content.
Influence of alkalizing conditions on coloration of cocoa
powder Cocoa powder presented different colors under
varying degrees of alkalization using a NaOH solution
(Fig. 3). With an increase in pressure, alkali concentration,
or reaction time, the color of cocoa powder became darker.
The color compounds are polymers formed in the production
process as a result of sugar degradation and reactions
between amino compounds and carbohydrates (23). These
reactions were affected by changes in both pH and
temperature. A high temperature and basic pH conditions
favored formation of dark components in cocoa powder. It
was reported that Maillard reactions under acidic or basic
conditions produce a set of reaction products, including
non-volatile colored compounds of intermediate molecular
weight, and brown substances of high molecular weight
(23).
Influence of the alkali concentration on anthocyanins
Pigment is one of the most important factors for the color
of cocoa powder. In addition to Maillard reactions, dark
colors can also be produced by a combination of anthocyanin
and sugar, and the stability of cocoa powder increases via
glycosylation. Anthocyanin absorbance at 525 nm was
detected (Fig. 4). The absorbance value at 525 nm decreased
and the ∆E value increased with an increasing alkali
concentration, suggesting that darker cocoa powder contains
less anthocyanin. The anthocyanin group of pigments
contains phenolic hydroxyl, and the D-heterocyclic oxygen
atom is quadrivalent. Thus, the pH values greatly impact
Fig. 3. Color of cocoa powder (∆L, ∆a, ∆b, and ∆E) afte
r
v
arying conditions of alkalization. A light degree of alkalizatio
n
was carried out at a pressure of 0.02 MPa with a 1% NaOH
solution and an alkalization time of 20 min. A heavy degree o
f
alkalization was carried out at a pressure of 0.1 MPa with a 3%
N
aOH solution and an alkalization time of 30 min.
Fig. 1. Influence of the alkali type (NaOH and K2CO3) on the
color difference (∆E) and total polyphenol content of cocoa
powder.
Fig. 2. Relationship between the color of cocoa (∆L, ∆a, ∆b,
and ∆E) and the total polyphenol content.

62 Li et al.
the anthocyanin structure. At different pH values, anthocyanin
has different configurations, resulting in different colors of
cocoa powder under different alkali concentrations during
alkalization. Additionally, hydroxy-furan carboxaaldehyde
may combine with anthocyanin and generate brown
pigment, resulting in a higher ∆E value.
Influence of alkalizing conditions on colorimetric
fractions Brown pigments (products of anthocyanin
conversion) were analyzed at 460 nm and 525 nm using
spectrophotometer (722; Shanghai Precision & Scientific
Instrument Co., Ltd.). Cocoa powders at different degrees
of alkalization contained different anthocyanin contents
(Table 1). The OD460/OD525 values for alkalized samples
were higher than for non-alkalized samples (Table 1).
Previous study showed that the value of OD460/OD525 for a
good quality cocoa after fermentation and roasting should
not be less than 1.1 (16,24). The OD525 and OD460 absorbance
values for all the alkalized samples were lower than for
non-alkalized counterparts, probably due to anthocyanin
polymerization.
Other indicators of cocoa quality based on color were
the monomer anthocyanin content (F1), the red polymer
content (F2), and the yellow and brown polymer content
(F3). These fractions were separated using column
chromatography (Shanghai Huxi). The ratio of F1 to F3
(F1/F3) indicates changes in pigment concentrations.
According to Serra and Ventura (3), the F1/F3 value should
not exceed 0.33 for good quality cocoa. The value of F1/
F3 for cocoa powder with a heavy degree of alkalization
was lowest, indicating that more anthocyanins were
polymerized into red polymers (Table 1). All alkalized
samples had lower F1/F3 values than cocoa powder
without alkalization, suggesting that more anthocyanins
were transformed into brown polymers, compared to non-
alkalized samples. Anthocyanins are probably hydrolyzed
and the resultant aglycones are oxidized to quinonic
compounds, contributing to the typical brown color of
cocoa powder (3,25).
Alkalization is commonly used in the processing of
cocoa powder, and the color qualities of cocoa powder can
be improved by optimizing alkalization parameters. Cocoa
powder alkalized under different conditions and degrees of
alkalization was studied for color, polyphenol content,
color fractions, and qualities. Cocoa powder alkalized with
a 2%(w/w) K2CO3 solution exhibited a darker color and
had a lower polyphenol content than powder alkalized with
a NaOH solution. Cocoa powder was observed to be darker
when the total polyphenol content was reduced. The
reaction was faster at a higher temperature and a basic pH,
resulting in a darker cocoa powder color (8,23). More
anthocyanins were transformed into brown polymers when
the alkali concentration was increased during alkalization.
Alkalized cocoa powder had a better color quality and was
less astringent, compared to non-alkalized powder.
Acknowledgments This work was financially supported
by National 125 Program 2011BAD23B02, 2013AA1022207;
SFC 31171686; NSF-Jiangsu-BK2012559; PAPD, China.
References
1. Miller KB, Hurst WJ, Payne MJ, Stuart DA, Apga J, Sweigart DS,
Ou B. Impact of Alkalization on the antioxidant and flavanol
content of commercial cocoa powders. J. Agr. Food Chem. 56:
8527-8533 (2008)
2. Wang JF, Schramm DD, Holt RR, Ensunsa JL, Fraga CG, Schmitz
HH, Keen CL. A dose-response effect from chocolate consumption
Fig. 4. ∆E and absorbance values at 525 nm for cocoa powde
r
treated under different alkali concentrations (pressure was
maintained at 0.02 MPa).
Table 1. Effect of varying degrees of alkalization on color and absorbance values of cocoa powders
Sample1) Color and absorbance value
OD525 OD460 OD460/OD525OD525 of F1 OD525 of F2 OD525 of F3 F1/F3
Non-alkalized cocoa powder 0.51±0.03a2) 0.59±0.03a1.16±0.03c0.09±0.03a0.06±0.02a0.33±0.04a0.31±0.05a
Cocoa powder with light degree of alkalization 0.12±0.02b0.17±0.03b1.45±0.02a0.04±0.01b0.02±0.01b0.14±0.03b0.25±0.06ab
Cocoa powder with heavy degree of alkalization 0.03±0.0c0.04±0.01c1.24±0.01b0.01±0.01c0.01±0.01b0.08±0.02c0.15±0.04b
1)A light degree of alkalization was carried out at a pressure of 0.02 MPa with a 1% NaOH solution and an alkalization time of 20 min. A
heavy degree of alkalization was carried out at a pressure of 0.1 MPa with a 3% NaOH solution and an alkalization time of 30 min.
2)Mean values with different superscript letters are significantly different (p<0.05).

Alkalization Effects on Color of Cocoa Powder 63
on plasma epicatechin and oxidative damage. J. Nutr. 130: 2115-
2119 (2000)
3. Serra BJ, Ventura CF. Factors affecting the formation of
alkylpyrazines during roasting treatment in natural and alkalinized
cocoa powder. J. Agr. Food Chem. 50: 3743-3750 (2002)
4. Wollgast J, Anklam E. Polyphenols in chocolate: Is there a
contribution to human health? Food Res. Int. 33: 449-459 (2002)
5. Serra Bonvehí J. Investigation of aromatic compounds in roasted
cocoa powder. Eur. Food Res. Technol. 221:19-29 (2005)
6. Kostic MJ. Cocoa alkalization. Manuf. Confect. 6: 128-130 (1997)
7. Forsyth WGC. Cacao polyphenolic substances II. Changes during
fermentation. Biochem. J. 51: 516-520 (1952)
8. Bravo L. Polyphenols: Chemistry, dietary sources, metabolism, and
nutritional significance. Nutr. Rev. 56: 317-333 (1998)
9. Subagio A, Sari P, Morita N. Simultaneous determination of (+)-
catechin and (-)-epicatechin in cacao and its products by high-
performance liquid chromatography with electrochemical detection.
Phytochem. Analysis 12: 271-276 (2001)
10. Niemenak N, Rohsius C, Elwers S, Ndoumou DO, Lieberei R.
Comparative study of different cocoa (Theobroma cacao L.) clones
in terms of their phenolics and anthocyanins contents. J. Food
Compos. Anal. 19: 612-619 (2006)
11. Papadakis SE, Abdul-Malek S, Kamdem RE, Yam KL. A versatile
and inexpensive technique for measuring colour of foods. Food
Tech. 54: 48-51 (2000)
12. Topuz A. A novel approach for color degradation kinetics of paprika
as a function of water activity. LWT-Food Sci. Technol. 41: 1672-
1677 (2008)
13. Slinkard K, Singleton VL. Total phenol analysis: automation and
comparison with manual methods. Am. J. Enol. Viticult. 28: 49-55
(1977)
14. Elwers S, Zambrano A, Rohsius C, Lieberei R. Differences between
the content of phenolic compounds in Criollo, Forastero and
Trinitario cocoa seed (Theobroma cacao L.). Eur. Food Res.
Technol. 229: 937-948 (2009)
15. Serra Bonvehí J, Ventura Coll F. Evaluation of bitterness and
astringency of polyphenolic compounds in cocoa powder. Food
Chem. 60: 365-370 (1997)
16. Krysiak W. Influence of roasting conditions on coloration of roasted
cocoa beans. J. Food Eng. 77: 449-453 (2006)
17. Wissgott U. Process of alkalization of cocoa in aqueous phase. U.S.
Patent 4,784,866 (1988)
18. Wiant MJ, Lynch WR, Lefreniere RC. Method for producing deep
red and black cocoa. U.S. Patent 5,009,917 (1991)
19. Leon K, Mery D, Pedreschi F, Leon J. Colour measurement in
L*a*b* units from RGB digital images. Food Res. Int. 39: 1084-
1091 (2006)
20. Pedreschi F, Leon J, Mery D, Moyano P. Development of a
computer vision system to measure the colour of potato chips. Food
Res. Int. 39: 1092-1098 (2006)
21. Clydesdale FM. Color measurement. pp. 95-149. In: Food Analysis:
Principles and Techniques. Gruenwedel DW, Whitaker JR (eds).
Marcel Dekker, New York, NY, USA (1984)
22. Ferrari CKB, Torres E. Biochemical pharmacology of functional
foods and prevention of chronic diseases of aging. Biomed.
Pharmacother. 57: 251-260 (2003)
23. Coca M, García MT, González G, Peña M. Study of coloured
components formed in sugar beet processing. Food Chem. 86: 421-
433 (2004)
24. Shamsuddin SB, Dimmick PS. Qualitative and quantitative
measurement of cacao bean fermentation. pp 55-78. In: Proceeding
of the Symposium Cacao Biotechnology. Dimmick PS (ed). The
Pennsylvania State University, University Park, PA, USA (1986)
25. Haslam E. The Flavonoids: Advances in Research. Chapman and
Hall, London, UK. pp. 417-447 (1982)