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Journal of Food Quality ISSN 1745-4557

EFFECT OF FERMENTATION AND ROASTING ON THE PHENOLIC CONCENTRATION AND ANTIOXIDANT ACTIVITY OF COCOA FROM NICARAGUA

YADER SUAZO, GABRIEL DAVIDOV-PARDO 1 and IÑIGO AROZARENA

Food Technology Department, Public University of Navarre, Campus Arrosadia s/n, Pamplona, Navarre 31006, Spain

1 Corresponding author. TEL: (34) 948169310; FAX: (34) 948169893; EMAIL: gabriel.davidov@unavarra.es

Received for Publication February 14, 2013 Accepted for Publication November 10, 2013

10.1111/jfq.12070

ABSTRACT

The aim of this work was to quantify the changes in the total phenolic content (TPC), antioxidant activity (AA) and color hue (CH) of cocoa ( Theobroma cacao) beans of group Trinitario from Nicaragua after fermentation and roasting. Cocoa beans with three different grades of fermentation (nonfermented, poorly fer- mented and fermented) were put under three different roasting temperatures (110, 130 and 150C). The kinetics of the roasting at 130C for 60 min was also studied. After each treatment, TPC, AA and CH were determined. For all the quantified parameters, significant differences (P < 0.05) were found between fer- mentations and roasting conditions. TPC and AA showed decrease with fermenta- tions and CH showed increases. Roasting decreased TPC except for the 150C treatment, while AA and CH increased. When studying the kinetics at 130C, TPC showed greater reductions with longer treatments while AA and CH showed increases.

PRACTICAL APPLICATIONS

Cocoa ( T. cacao) is a rich source of polyphenols with benefits for human health and is the main raw material for elaboration of chocolate. However, the processes to create the desirable organoleptic characteristics of the cocoa beans to produce chocolate affect the concentration of polyphenols and the AA of the beans. Cocoa beans are produced in developing countries in America, Asia and Africa. Nicara- gua has become an important producer of cocoa turning it into a popular cash crop and there are not any articles that study the profile and changes in the cocoa from Nicaragua. The results of this paper could be used among the producing cooperatives of cacao in Nicaragua, or even nongovernmental organizations involved in cocoa production, to improve the processing (fermentation and roast- ing) of cocoa to obtain cocoa beans richer in antioxidants, and therefore increase their nutritional value to create a more competitive product.

INTRODUCTION

Polyphenols from different sources such as plants, fruits, seeds and beverages like wine or tea have attracted the attention of many researchers due to the beneficial effects of these compounds to human health. Cocoa ( Theobroma cacao) is a rich source of polyphenols because they consti- tute approximately 10% of the dry weight of the bean (Rusconi and Conti 2010). The beneficial effects of cocoa polyphenols to human health are: the scavenge of free radi-

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cals and prevention of damage to DNA, chelation of metals, vasoprotective effects, improvements in the endothelial function, anti-inflammatory effects, amelioration of insulin resistance, anticarcinogenesis effects, among others (Katz et al . 2011). Nowadays, cocoa beans are known because they are the main raw material for elaboration of chocolate, which in fact after fruits and vegetables is one of the main sources of antioxidants to the diet of the United States (Rusconi and Conti 2010). Although cocoa products are consumed all

Journal of Food Quality 37 (2014) 50–56 © 2014 Wiley Periodicals, Inc.

Y. SUAZO, G. DAVIDOV-PARDO and I. AROZARENA

over the world, the beans are produced in developing coun- tries in America, Asia and Africa. Since 1991, after the civil war, Nicaragua has become an important producer of cocoa turning it into a popular cash crop. In Nicaragua, the main production of cocoa is in the municipality of Waslala in the north of the country (Trognitz et al . 2011). To develop desirable organoleptic characteristics to produce chocolate and other cocoa products, the beans have to be submitted to fermentation and roasting. These two processes affect the concentration of polyphenols and the antioxidant activity (AA) of the beans (Aculey et al . 2010). Basically, the fermentation is a spontaneous microbio- logical process that causes deep biochemical changes. The beans are fermented by a successive presence of microor-

ganisms (fungi, yeasts, lactic acid and acetic bacteria). These microorganisms are activated by the temperature changes during the fermentation process that can last a minimum of

5 days to a maximum of 7 days (Wollgast and Anklam 2000;

Schwan and Wheals 2004). The impact of the fermentation on the phenolic content of cocoa beans was studied by Efraim et al . (2010). After the first 3 days of fermentation

there was a 35% decrease in the phenolic content, and after

7 days of fermentation only 45% of the original phenolic

content remained. After fermentation, the beans are sun- dried or artificially dried, depending on the weather of the growing areas. The beans are dried to reach a moisture content of approximately 5–7% to prevent the growth of molds during transportation and storage (Wollgast and Anklam 2000). During the drying process, the loss of poly- phenols continues mainly when the beans are artificially dried (Efraim et al . 2010). After fermentation and drying, the beans are usually roasted. This process determines the final color, aroma and flavor of the cocoa products. The changes in the organolep- tic characteristics of the beans are the result of oxidation and polymerization of polyphenols, degradation of pro- teins and Maillard reactions (Ramli et al . 2006). Convective roasting is the most commonly roasting process in the cocoa industry; the raw beans are exposed to temperatures between 130 and 150C for 15–45 min (Krysiak 2006). In the light of this background, the aim of this work was to quantify the changes in the phenolic composition and AA of nonfermented cocoa (NFC), poorly fermented cocoa (PFC) and fermented cocoa (FC) beans (T. cacao) of group Trinitario from Nicaragua after roasting.

MATERIALS AND METHODS

Reagents

Methanol, n-hexane, sodium carbonate and gallic acid (GA) were purchased from Panreac (Barcelona, Spain). 2,2- Diphenyl-1-picrylhydrazyl (DPPH) and Folin–Ciocalteau

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EFFECT OF PROCESS ON COCOA FROM NICARAGUA

TABLE 1. CUT TEST FOR NONFERMENTED COCOA (NFC), POORLY FERMENTED COCOA (PFC) AND FERMENTED COCA (FC) SAMPLES

 

Nonfermented

Poorly

Fermented

Parameter

(%)

fermented (%)

(%)

Slaty

36.7

0

0.7

Lightly violet

4.7

20.2

2.6

Completely violet

31.5

0

1.5

Internal mold

0.2

0.3

0.8

White spots

0

0

0.2

reagent 6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) were purchased from Sigma-Aldrich Quimica SL. (Madrid, Spain).

Samples

NFC, PFC and FC cacao beans of the same group (Trinitario) were obtained from a collection center of Ritter Sport in Matagalpa, Waslala, Nicaragua. During fermenta- tion (in Nicaragua), the beans were turned once a day during 7 days to reach a temperature between 45 and 50C. Table 1 shows the results of the cut test for all samples. Because of the cut test results, the PFC beans were consid- ered poorly fermented. The reasons to explain the cut results for PFC were that the turn regime was not done on time and the mass of the beans did not reach the aimed temperature, as stated by the processing facility in Nicara- gua. Because of the lower quality of PFC, these kinds of batches are usually commercialized in the local market. All samples were sun-dried after fermentation until they reached a humidity of 6%, then the samples were sent to the Public University of Navarre for analyses and roasting. It was also stated by the processing facility in Nicaragua that PFC beans reached the 6% humidity faster than usual. All samples had phytosanitary certificates from the Agropecuary Ministry of Nicaragua.

Experimental Design

Table 2 shows the experimental design to analyze the changes in the polyphenolic concentration, AA and color hue of the beans after fermentation and roasting. The roast- ing temperatures were chosen based on the work by Krysiak (2006). The combination of the selected roasting tempera- tures and times is within the common temperatures used in the cocoa industry and was determined to achieve a water content of 2%, which is the optimal humidity in terms of beans grinding and fat extraction. The kinetics of the changes in the polyphenolic concentration, AA and color hue when roasting the NFC and FC beans at 130C for 20, 40 and 60 min was also studied. A sample of 100 g of the whole beans with the shells was roasted in an oven Digitronic

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EFFECT OF PROCESS ON COCOA FROM NICARAGUA

TABLE 2. EXPERIMENTAL DESIGN TO COMPARE THE POLYPHENOLIC CONCENTRATION AND ANTIOXIDANT ACTIVITY OF COCOA SAMPLES AFTER THE TYPE OF FERMENTATION AND ROASTING CONDITIONS

Cocoa samples

Roasting temperature (C)

Time (min)

Nonfermented cocoa (NFC)

Nonroasted

0

110

90

130

60

150

45

Poorly fermented cocoa (PFC)

Nonroasted

0

110

90

130

60

150

45

Fermented cocoa (FC)

Nonroasted

0

110

90

130

60

150

45

(Selecta, Barcelona, Spain). After roasting, the beans were left to cool for 1 h at ambient temperature before the phe- nolic extraction.

Extraction of Phenolic Compounds

The extraction of phenolic compounds from the cocoa beans was based on the defatted methodology employed by Summa et al . (2006) and the methanolic extraction based on the methodology employed by Jonfia-Essien et al . (2008). Briefly, the cocoa beans were winnowed by hand and grounded for 30 s in a coffee blender. To remove the fat, 5 g of the grounded beans were mixed with 25 mL of n-hexane and thoroughly mixed for 3 min. The mixture was centrifuged at 3,000 rpm during 15 min at 4C using a centrifuge Medifriger BL-S (Selecta, Barcelona, Spain). The process was repeated four times and the defatted cocoa powder was left overnight at ambient temperature to remove the solvent. To extract the phenolic compounds, 2 g of the defatted sample was mixed with 50 mL of an 80% methanol solution and shaken for 2 h at 50C using an orbital mini shaker (VWR, Barcelona, Spain). The extract was then filtered through a Whatman No. 1 filter paper and filled up to 50 mL in a volumetric flask to compensate the losses of solvent during the extraction and filtration.

Total Phenolic Content (TPC)

The Folin–Ciocalteu method was employed (EEC 1990) to obtain the TPC. In a 100-mL volumetric flask, 1 mL of the diluted extract, 50 mL of deionized water, 5 mL of the Folin– Ciocalteu reagent and 20 mL of a sodium carbonate solution 20% (w/v) were added in that order. The volumetric flask was filled to its volume with deionized water. After 30 min, the absorbance of the samples was measured at 750 nm using polystyrene cuvettes in a Cintra 20 (GBC Scientific, Braeside,

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Y. SUAZO, G. DAVIDOV-PARDO and I. AROZARENA

Australia) double beam spectrophotometer. The phenolic content was expressed in GA equivalents after the preparation of a standard curve of GA from 0 to 600 mg/L.

AA

The antiradical activity was evaluated based on the tech- nique by Rivero-Pérez et al . (2007). A 60-μ M methanolic solution of DPPH (2,940 μ L) was mixed with 60 μ L of the extract in a polystyrene cuvette. The absorbance at 515 nm was measured before adding the extract, and 60 min after adding it, using a Cintra 20 (GBC Scientific, Braeside, Aus- tralia) double beam spectrophotometer. The AA is reported as mmoles of Trolox equivalents per gram of dry cocoa powder after the elaboration of a standard curve of Trolox based on the reduction of the absorbance after 60 min.

Color Hue

The color hue of the samples was measured through the brown pigments ratio (Krysiak 2006), the ratio of the absor- bance at 460 nm and the absorbance at 520 (A460/A520). All the measures were made on the cocoa extracts using polystyrene cuvettes in a Cintra 20 (GBC, Braeside, Austra- lia) double beam spectrophotometer.

Statistical Analysis

Statistical analyses were conducted using Statgraphics Centurion XVI (StatPoint Technologies Inc., Warrenton VA, USA). Differences among the treatments were determined using an analysis of variance and a Tukey test with a confi- dence level of 95%. For the nonroasted samples, the extrac- tion was made four times and analyzed in duplicate giving an n = 8 per sample. The roasting of the cocoa beans was made in duplicate and analyzed in duplicate giving an n = 4 per treatment and sample. A bilateral Pearson correlation analysis was performed between the TPC and the AA after the roasting and fermentation treatments.

RESULTS AND DISCUSSION

Effect of Fermentation

Table 3 shows the effect of fermentation on the cocoa beans. The results obtained in this work are in agreement with the results obtained by Efraim et al . (2010) who studied the fermentation and drying of Forastero cocoa samples from Brazil. It is important to remark that there are no studies on the phenolic concentration of cocoa beans from Nicaragua to compare our results. It can be seen that for all the mea- sured parameters, there are significant differences among all treatments except for the A460/A520, in which case the differences between the two fermented samples are not

Journal of Food Quality 37 (2014) 50–56 © 2014 Wiley Periodicals, Inc.

Y. SUAZO, G. DAVIDOV-PARDO and I. AROZARENA

EFFECT OF PROCESS ON COCOA FROM NICARAGUA

TABLE 3. EFFECT OF FERMENTATION ON THE EVALUATED PARAMETERS OF COCOA BEANS

 

Phenolic content

Antioxidant activity

Color (Abs 460/520 nm)

Cocoa samples

(mg/g dw*)

(μM Trolox)

Nonfermented cocoa (NFC) Poorly fermented cocoa (PFC) Fermented cocoa (FC)

115 ± 2 c 56 ± 2 b 43 ± 1 a

709 ± 17 c 154 ± 8 b 124 ± 4 a

1.06 ± 0.03 a 1.27 ± 0.09 b 1.28 ± 0.04 b

Note: ac Different letters within a column are significantly different ( P < 0.05). * Grams of dry weight. The results are represented as means ± standard deviation.

significant. As expected, the polyphenolic concentration is lower in the fermented than in the nonfermented samples. It is known that the fermentation and drying processes cause a decrease in the polyphenolic content due to oxida- tion and condensation reactions of single polyphenols to

insoluble complex tannins that interact with proteins. These reactions can be nonenzymatic or catalyzed by oxidase enzymes. The loss of polyphenols diffused in the water per- meated from the beans during fermentation also contrib- utes to the decrease in the phenolic content (Wollgast and Anklam 2000; Kyi et al . 2005). The reductions in the AA are

a direct consequence of the decrease in the polyphenolic

content. After fermentation and drying, an increase in the brown pigments ratio A460/A520 was observed; nevertheless, the

results of the PFC and FC were not significantly different.

It is known that for a cocoa to be properly fermented in

terms of color, A460/A520 should be higher than 1 (Krysiak 2006). In this case, both fermented samples had A460/A520 values higher than 1. When cocoa beans are fermented and dried, their colors change from “slaty” and “purple” to

“brown” (Aculey et al. 2010). The changes in the color of

the beans could be explained by nonenzymatic and enzy- matic processes. Among the nonenzymatic reactions are hydrolysis of anthocyanins into anthocyanidins and the later polymerization with simple catechins to form com- plex tannins. Another nonenzymatic process that happens during fermentation and browning is Maillard reactions, which involve reducing sugar and the amino groups of pro- teins to produce brown polymeric compounds. Finally, the enzymatic browning during fermentation and drying of cocoa beans involves the formation of quinones from phenolic compounds and the later formation of brown to black polymers under the action of the polyphenol oxidase (Wollgast and Anklam 2000; Kyi et al . 2005).

Effect of Roasting

Figure 1 shows the evolution of phenolic concentration, AA and color hue of cocoa samples after the roasting conditions. It can be seen that the TPC of the samples decreased after the roasting treatments (Fig. 1A), except for the roasting at 150C during 45 min in which NFC pre- sented an increase in TPC. In general, more intense roasting

FIG. 1. IMPACT OF THE DIFFERENT ROASTING CONDITIONS ON THE THREE COCOA SAMPLES. (A) TOTAL PHENOLIC CONTENT. (B) ANTIOXIDANT ACTIVITY. (C) COLOR HUE

A460/A520

Different letters (a–d) are significantly different for each extract separately ( P < 0.05).

d b c B a A b c a a a d b c a
d
b
c
B
a
A
b
c
a
a
a
d
b
c
a
a
a
b
c c
b b
b
a a
a
b
c c
C
b
b
a
b
a
b
a
a
a

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EFFECT OF PROCESS ON COCOA FROM NICARAGUA

conditions result in a greater loss of polyphenols due to high redox activity of polyphenols at those conditions (Wollgast and Anklam 2000). The higher reductions in TPC for NFC when compared to PFC and FC are in agreement with the findings made by Kealey et al. (2001). The fact that the poly- phenols contained in the NFC sample did not diffuse out of their storage cells during fermentation could be the reason for the increase in the phenolic content with the 150C during 45-min treatment. Kim et al. (2006) proved that using similar heating conditions the phenolic content of grape seeds increased, because with the heat polyphenols were liberated out of their storage cells. The longer time of the other two treatments could cause liberation of the polyphenols at the beginning, but degradation afterward. Another possible explanation for the increase in TPC on NFC when roasting at 150C during 45 min could be the degradation of anthocyan- ins into other polyphenol monomers (Misnawi et al. 2005). Anthocyanins are only present in NFC beans because it is known that during fermentation anthocyanins are lost (Wollgast and Anklam 2000). With regard to the AA, the changes were less notorious than the changes of the TPC. The significant increment of AA with the treatment 150C during 45 min is consistent with the increment in TPC. The bilateral Pearson correla- tion between the TPC and the AA after the roasting and fermentation treatments was not significant (r = 0.467,

Y. SUAZO, G. DAVIDOV-PARDO and I. AROZARENA

P > 0.05). The fact that the changes in the AA did not have the same magnitude and tendency compared to the changes in the TPC could be explained by the findings of Summa et al. (2006). In their work, Summa et al. (2006) found that the concentration of reducing substances, such as polyphe- nols, measured by the Folin–Ciocalteau technique, does not always have a close relation with the AA measured with the DPPH technique. The color hue increased with the thermal treatments (Fig. 1C). This increment is consistent with the findings of Krysiak (2006) in which higher temperatures represented greater increases in the A460/A520 value. The increase of the brown pigments ratio induced by the heat could be explained by the continuation and acceleration of some of the reactions started during fermentation and drying mainly Maillard reactions (Beckett 2000). Another factor that contributes to the increase in color hue could be the aromatic acid reductones, which are formed as a conse- quence of reactions with phenols such as catechol and hyd- roquinone (Lee et al . 2001). To analyze the influence of roasting time, the NFC and FC samples were roasted at 130C for 20, 40 and 60 min and the kinetics of the changes for all the measured parameters was studied. As expected, the NFC had higher TPC and AA values and lower color values during the whole assay (Fig. 2). It can be seen that while TPC was reducing with

a c B A b a a a b a d c b a c
a
c
B
A
b
a
a
a
b
a
d
c
b
a
c
c
b
a
C
a
a
a
a
a,b
b b
a

FIG. 2. IMPACT OF THE DIFFERENT ROASTING TIMES AT 130C ON THE NFC AND THE FERMENTED COCOA SAMPLES. (A) TOTAL PHENOLIC CONTENT. (B) ANTIOXIDANT ACTIVITY. (C) COLOR HUE A460/A520 Different letters (a–d) are significantly different for each extract separately ( P < 0.05).

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Journal of Food Quality 37 (2014) 50–56 © 2014 Wiley Periodicals, Inc.

Y. SUAZO, G. DAVIDOV-PARDO and I. AROZARENA

longer treatment times, the AA was growing (Fig. 2A,B). The oxidation and condensation of single polyphenols to complex tannins could contribute to reduce the TPC, but also contribute to the increase in the AA, because it has been proved that tannins have a greater scavenging power than simple phenolic compounds (Hagerman et al . 1998). Another reason for the increment of the AA could be the formation of melanoidins, as a result of Maillard reactions during roasting, compensated the degradation of polyphenol monomers balancing the AA (Vignoli et al .

2011).

With regard to the evolution of color, there was a clear increment in the A460/A520 ratio at 20 min, followed by a slight but not significant decrease at longer times (Fig. 2C). The fact that the highest color hue values were reached after 20 min is in concordance with the finding of Lee et al . (2001) who optimized the roasting of cocoa beans and found that the darkest beans were obtained when roasted at 130C for 30 min. On the contrary, Krysiak (2006) found that the color of the beans increased continuously when the beans were roasted at 135C for 60 min.

CONCLUSIONS

As conclusions, it can be said that fermentation causes a reduction in the phenolic content and AA and an increment in the brown pigments ratio of the cocoa beans. The poor fermentation of the cocoa beans has a greater impact in the phenolic composition rather than the color. The similar color between the poorly fermented and well-fermented cocoa makes it possible to sell the former one in the Nicara- gua local market. In general, when the cocoa beans are sub- mitted to roasting treatments, there is a reduction in the TPC and an increment in the AA and color hue with the increase of temperature, except for the beans that are not fermented, in which case the fact that the polyphenols are still within the bean tissues plays an important part to reduce their losses or even revert the reduction tendency. Finally, it can be seen that the differences in AA, TPC and color hue after summiting the beans to 130C during 20, 40 and 60 min are smaller than the differences obtained when summiting the beans to different roasting temperatures, leading to the conclusion that the roasting temperature has a greater impact in the changes of beans than the roasting time.

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