Influence of Alkalization Treatment on the Color Quality and the Total Phenolic and Anthocyanin Contents in Cocoa Powder

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1 Food Sci. Biotechnol. 23(1): (2014) DOI /s RESEARCH ARTICLE 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 K 2 CO 3 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. OD 460 /OD 525 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 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 , China Tel: ; Fax: fzhong@jiangnan.edu.cn Song Zhu State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi , China 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.

2 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 (K 2 CO 3 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 40 o C. 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 Na 2 CO 3 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/CH 3 OH (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/CH 3 OH. 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/CH 3 OH (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 K 2 CO 3 are the most commonly used alkalis for cocoa powder alkalization (6,17,18). The influence of NaOH and K 2 CO 3 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 K 2 CO 3 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

3 Alkalization Effects on Color of Cocoa Powder 61 Fig. 1. Influence of the alkali type (NaOH and K 2 CO 3 ) on the color difference ( E) and total polyphenol content of cocoa powder. Fig. 3. Color of cocoa powder ( L, a, b, and E) after varying conditions of alkalization. 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. 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. Fig. 2. Relationship between the color of cocoa ( L, a, b, and E) and the total polyphenol content. K 2 CO 3 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 K 2 CO 3 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, 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

4 62 Li et al. Fig. 4. E and absorbance values at 525 nm for cocoa powder treated under different alkali concentrations (pressure was maintained at 0.02 MPa). 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 OD 460 /OD 525 values for alkalized samples were higher than for non-alkalized samples (Table 1). Previous study showed that the value of OD 460 /OD 525 for a good quality cocoa after fermentation and roasting should not be less than 1.1 (16,24). The OD 525 and OD 460 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 nonalkalized 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) K 2 CO 3 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, 2013AA ; SFC ; NSF-Jiangsu-BK ; 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: (2008) 2. Wang JF, Schramm DD, Holt RR, Ensunsa JL, Fraga CG, Schmitz HH, Keen CL. A dose-response effect from chocolate consumption Table 1. Effect of varying degrees of alkalization on color and absorbance values of cocoa powders Sample 1) Color and absorbance value OD 525 OD 460 OD 460 /OD 525 OD 525 of F1OD 525 of F2OD 525 of F3 F1/F3 Non-alkalized cocoa powder 0.51±0.03 a2) 0.59±0.03 a 1.16±0.03 c 0.09±0.03 a 0.06±0.02 a 0.33±0.04 a 0.31±0.05 a Cocoa powder with light degree of alkalization 0.12±0.02 b 0.17±0.03 b 1.45±0.02 a 0.04±0.01 b 0.02±0.01 b 0.14±0.03 b 0.25±0.06 ab Cocoa powder with heavy degree of alkalization 0.03±0.0 c 0.04±0.01 c 1.24±0.01 b 0.01±0.01 c 0.01±0.01 b 0.08±0.02 c 0.15±0.04 b 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).

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