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Cite This: J. Agric. Food Chem. XXXX, XXX, XXX-XXX

Environmental Growing Conditions in Five Production Systems Induce Stress Response and Affect Chemical Composition of Cocoa (Theobroma cacao L.) Beans Wiebke Niether,*,† Inga Smit,‡ Laura Armengot,§ Monika Schneider,§ Gerhard Gerold,† and Elke Pawelzik‡ †

University of Goettingen, Institute of Geography, Goldschmidtstrasse 5, 37077 Göttingen, Germany Institute of Crop Sciences, Carl-Sprengel-Weg 1, 37075 Göttingen, Germany § Forschungsinstitut für Biologischen Landbau (FiBL), Deparment of International Cooperation, Ackerstrasse 113, Postfach 219, 5070 Frick, Switzerland ‡

S Supporting Information *

ABSTRACT: Cocoa beans are produced all across the humid tropics under different environmental conditions provided by the region but also by the season and the type of production system. Agroforestry systems compared to monocultures buffer climate extremes and therefore provide a less stressful environment for the understory cocoa, especially under seasonally varying conditions. We measured the element concentration as well as abiotic stress indicators (polyamines and total phenolic content) in beans derived from five different production systems comparing monocultures and agroforestry systems and from two harvesting seasons. Concentrations of N, Mg, S, Fe, Mn, Na, and Zn were higher in beans produced in agroforestry systems with high stem density and leaf area index. In the dry season, the N, Fe, and Cu concentration of the beans increased. The total phenolic content increased with proceeding of the dry season while other abiotic stress indicators like spermine decreased, implying an effect of the water availability on the chemical composition of the beans. Agroforestry systems did not buffer the variability of stress indicators over the seasons compared to monocultures. The effect of environmental growing conditions on bean chemical composition was not strong but can contribute to variations in cocoa bean quality. KEYWORDS: T. cacao, total phenolic content, polyamines, abiotic stress

1. INTRODUCTION

maturation phase can therefore influence the total phenolic content10,12 and, by this, affect the bean flavor. Differences in the flavor profile of cocoa beans are attributed to genetics, but also to growing conditions (sunshine and rainfall), soil characteristics, ripening, and time of harvesting.13 Environmental conditions during pod ripening may further influence quality parameters of the beans like the nutrient composition,14 the share of bean compartments,15 and the fat content.2 Growing conditions depend on the season and region but also on the production system. Cocoa monocultures reach highest yields16 but demand large amounts of mineral fertilizers,17 in contrast to traditional and diverse cocoa agroforestry systems with timber, fruit, leguminous or forest trees, and low or no external input,18 that provide various ecosystem services including enhanced litter decomposition and internal nutrient cycling.19 Shade trees in agroforestry systems act like a shelter for the cocoa tree by intercepting high radiation, buffering temperature fluctuations, and reducing the evapotranspirative demand in the understory (Niether et al., under revision) thus reducing environmental stressful con-

In the humid tropics, cocoa trees (Theobroma cacao L.) are often exposed to environmental stressful conditions. Especially drought and high temperatures reduce cocoa yield.1,2 During the ripening period of the cocoa pods, water deficiency may affect physiological processes related to drought stress in the cocoa beans like in other seed crops.3 Physiological responses to abiotic stresses in plant organs are mediated by secondary metabolites, e.g., polyamines4 and phenolic compounds.5 Cocoa beans produce amines6,7 that have antioxidant properties and play a role in plant development and stress response.4 Bae et al.8 found increased polyamine-levels in leaves and flowers in response to drought and enhanced stress tolerance, and spermine and spermidine were also identified in beans,6 but an evaluation of stress response in beans in relation to the growing conditions is to our knowledge not yet done. Cocoa has a naturally high content of phenolic substances.9 They play a dominant role in flavor and bean quality, as they are responsible for the bitter taste and astringency,10 but they are also reported to have a function in stress response in plants due to their antioxidant properties.5 Phenolic compounds are accumulated in the cocoa bean during the phase of fruit development (maturation phase) together with other substances and proteins that are involved in biotic and abiotic stress response.11 Weather conditions during the seed © XXXX American Chemical Society

Received: September 27, 2017 Revised: November 3, 2017 Accepted: November 6, 2017

A

DOI: 10.1021/acs.jafc.7b04490 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

September 2014. Since cocoa pod production takes five to six months until maturity, flowering for the pods from harvest “wet” was in November 2013 and pods developed during the rainy season (1707 mm of rainfall in six months), while the fruits for the harvest “dry” were pollinated in April 2014 and matured during the dry season (539 mm of rainfall in six months).23 After removing the beans from the pods, they were weighted (fresh bean yield, FW) and kept in mash bags for processing. The mash bags were handled together with beans from sites that were not used for analysis. Beans were fermented in wooden boxes, one for organic beans and one for conventional beans, for 8 days and turned every 48 h. The beans were taken off the boxes and laid on wooden tables for seven (harvest “wet”) and six (harvest “dry”) days of sun-drying until moisture content decreased to approximately 8%, the acceptable moisture for long-term storage of cocoa beans. Harvest, fermentation, and drying were performed according to local costumes. Dry beans were weighted (dry weight, DW) and residual moisture (RM) of the samples was measured with a moisture meter (Aqua boy with cup electrode, KPM Moisture meters, U.K.). The dry weight with 8% residual moisture (DW8%) was calculated: DW8% = DW × RM−1 × 8. A dry bean factor for 8% residual moisture (DBF8%) was calculated as the ratio between dry and fresh bean weight: DBF8% = DW8% × FW−1. The dry bean yield per production system was calculated by multiplying the corresponding dry bean factor with the total fresh bean yield. 2.3. Chemicals. 1.7-Diaminoheptan, spermine, spermidine, and 1dimethylamino-naphthalene 5-sulfonic acid chloride were purchased from Sigma-Aldrich, Germany. Putrescine-dihydrochloride and gallic acid monohydrate were obtained from Carl Roth, Germany. FolinCiocalteu's phenol reagent was purchased from Merck, Germany. Standard reagents were of analytical grade. 2.4. Sample Preparation and Analyses. A subsample of 30 raw cocoa beans from each sample were manually deshelled and separated for cotyledons (nibs), seed coats (shells), and germs. Nibs, shells, and germs were weighted separately. The cotyledons were lyophilized and milled using a rotor mill (ZM100, Retsch, Germany) with a 2 mm sieve, shells and germs were disposed. Milled cotyledons were boiled in distilled water and the pH was measured with a pH-meter (Inolab, WTW, Germany). Ten grams of milled cotyledons were defatted (defatted cocoa powder) by acid exploration according to the Weibull-Stoldt method (200 mL 12.5% hydrogen chloride for 1 h) followed by extraction with petroleum ether for 5 h with a Soxhlet-apparatus.27 The petroleum ether was evaporated, and the remaining fat was weighted. The water content of milled cotyledons and cocoa powder was estimated by oven drying at 103 °C. The defatted cocoa powder was used for determination of polyamines. Total carbon (C) and total nitrogen (N) in milled cotyledons were determined after dry combustion by infrared spectroscopy (DIN ISO 10694, 1996) and wavelength detection (DIN ISO 13878, 1998), respectively (Truspec CHN, Leco, U.S.A.). Milled cotyledons were extracted with nitric acid to measure the mineral concentrations of phosphorus (P), potassium (K), magnesium (Mg), sulfur (S), calcium (Ca), sodium (Na), iron (Fe), zinc (Zn), manganese (Mn), and copper (Cu) by inductively coupled plasma optical emission spectrometry (ICP-OES, iCAP 7000 series, Thermo Scientific, MA, U.S.A.).28 Concentrations are expressed in mg g−1 and μg g−1 milled cotyledons. Total phenolic content of milled cotyledons was measured photometrically according to the Folin-Ciocalteu’s assay29 at 735.8 nm. A calibration curve was established with gallic acid, and the results are expressed as gallic acid equivalents (mg GAE g−1 milled cotyledons, dry matter). The extraction and derivatization of polyamines using 0.1 g of defatted cocoa powder followed the method described by Smit et al.30 with the following modification: 10 μM diaminoheptan was added to the extraction medium as an internal standard; before the precolumn derivatization of the amines with 1-dimethylamino-naphthalene 5sulfonic acid chloride and after purification of the samples, the solution was filtered through 0.45 μm membranes.

ditions and water needs of the cocoa.20 Soil moisture loss by evaporation can be reduced by leguminous soil cover crops that are used in organic cocoa production systems.21 Trognitz et al.22 pointed out that aroma and taste depend on specific environmental conditions, but information on how far the growing conditions affect the concentration of minerals and secondary metabolites is missing.13 This study aimed to evaluate the effect of the production systems and the harvesting time on the chemical composition of cocoa beans, focusing on abiotic stress indicators, e.g., total phenolic content and polyamines, as well as quality parameters such as the concentration of mineral elements, the share of bean compartments and the fat content that might be affected by growing conditions. We hypothesized (i) that a physiological response to abiotic environmental conditions during the ripening phase of the pods can be shown in the cocoa beans by an increase in stress indicators as well as a change in the fat content and mineral element concentration in the dry season, (ii) that this response to growing conditions is less pronounced in agroforestry systems compared to monocultures due to their buffer function and (iii) that the production system itself has an influence on the chemical composition of the bean. Therefore, we harvested beans from five different cocoa production systems comprising monocultures and agroforestry systems, both under conventional and organic farming conditions and a successional agroforestry system. Bean samples were collected once in the beginning and once at the end of the harvesting season, that coincided with the beginning and the end of the dry season, and finally analyzed concerning morphological and chemical parameters.

2. MATERIALS AND METHODS 2.1. Study Area, Experimental Design, and System Management. The study site Sara Ana is located in Alto Beni at the foothill of the Bolivian Andes with 1439 mm mean annual rainfall, 25.2 °C mean annual temperature, and 83.0% mean annual relative humidity. The rainy season lasts from October to April including 78% of total annual precipitation.23 The long-term trial was established at the end of 2008 with the aim to study the ecological, economic, and social performance of different cocoa production systems.24,25 The five production systems were fullsun monocultures (MONO) and agroforestry systems (AF) both under organic (ORG) and conventional (CONV) farming and a highly diverse successional agroforestry system (SAFS) under organic farming. The trial comprised four repetitions in a complete block design; the plot size was 48 by 48 m with a net-plot of 24 by 24 m for data collection, cocoa spacing was 4 by 4 m. Twelve different cocoa cultivars from the Trinitario group were spread in a pattern in each plot. Mineral fertilizer (Blaukorn, BASF, Germany, 12−8−16−3 N− P2O5−K2O−MgO) was applied in MONO CONV (112 kg ha−1 year −1) and AF CONV (56 kg ha−1 year −1 ). Further plot characteristics and management practices are shown in detail in Schneider et al.24 The number of stems per hectare (stem density) increased from 625 cocoa stems in MONO to 1536 stems of cocoa, plantains, and shade trees in AF and to 2431 stems in SAFS coinciding with an increase in leaf area index from 0.8 ± 0.1 in MONO to 1.9 ± 0.1 in AF to 2.1 ± 0.1 in SAFS.21 The soil organic matter content (0− 25 cm) was slightly higher in agroforestry systems than in the monocultures.26 2.2. Cocoa Yield, Bean Sampling, and Postharvest Processing. Cocoa was harvested from April to November 2014 every 15 days. The number of ripe pods per tree was counted and beans were taken off the pod. For the bean analysis, the sampling from three cultivars (TSH-565, IIa-22, ICS-1xIMC-67) in five production systems was repeated two times during the harvesting period, the first harvest (harvest “wet”) took place in April 2014, the second (harvest “dry”) in B


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Figure 1. Time series of climatic conditions from November 2013 to December 2014 and cocoa performance: (a) precipitation (Prec), (b) temperature (T) (dotted line indicate mean of time series), (c) relative humidity (RH) (dotted line indicate mean of time series), and (d) soil moisture (REW: spatial mean of relative extractable water) over the time of cocoa pod development (6 months) of harvests “wet” and “dry” (dashed lines in (e)). (e) Dry bean yield per harvest in 2014 separated for the production systems monoculture conventional (MONO CONV, circle), monoculture organic (MONO ORG, square), agroforestry conventional (AF CONV, diamond), agroforestry organic (AF ORG, triangle upside), and successional agroforestry system (SAFS, triangle downside). bean parameters to the fixed factors harvest and system and the interaction of both. Cultivar nested to block entered the model as a random factor. The standard deviation was used to show the variability of polyamine concentrations and total phenolic content from five production systems across beans from two harvests. A multivariate Principal Component Analysis (PCA) was performed on 99 bean samples using morphologic characteristics (DBF, weight, nibs, shell and germs), pH, fat, element concentration (C, N, P, K, Mg, S, Ca, Cu, Fe, Mn, Na, and Zn), total phenolic content, polyamine concentrations (putrescine, spermine, and spermidine) as well as the harvesting time and characteristics of the production systems, i.e., farming (ORG vs CONV), stem density, soil organic matter, and leaf area index to give a descriptive overview over the global data set and to identify correlations. We used Spearman’s rank correlation (ρ) for non-normally distributed data to evaluate bivariate relationships. When necessary, outliers were removed and data were log- or square root-transformed to meet the normality and homoscedasticity requirements. Data are shown as mean with the standard error of means. Data frames were managed with the plyr R package.34 Graphs were designed with the ggplot2 R package.35

Polyamines were analyzed via High Performance Liquid Chromatography (HPLC, LC-2000 Series, Jasco, Germany) according to Smit et al.30 with 15 μL injection volume, fluorescence was detected at 254 nm and emission wavelength was set at 510 nm. A binary gradient containing acetonitrile and Tris-Buffer (0.1 M, pH 8.5) was used for the elution of the polyamines with the following HPLC-adjustments: the flow rate was 1 mL min−1 after starting and reduced to 0.3 mL min−1 at the second minute; the concentration of acetonitrile increased over time (0 min 24%; 2 min 50%; 12 min 54.4%; 13 min 55%; 25 min 60%; 35 min 75%; 37 min 80%; and 47 min 100%) and decreased at minute 56 to 25%. Polyamines were eluted in the order of their retention times (Figure S1 of the Supporting Information, SI): putrescine, internal standard diaminoheptan, spermidine, and spermine, confirmed with the retention time of the reference compounds (standard reagents). Determination and quantification limits were defined according to Kromidas31 as 0.07 μg g−1 and 0.23 μg g−1 for putrescine, 0.03 μg g−1 and 0.09 μg g−1 for spermidine and 0.03 μg g−1 and 0.09 μg g−1 for spermine, respectively. The results are expressed in μg g−1 defatted cocoa powder (dry matter). The reproducibility of HPLC-measurements was evaluated with repeated measurements of one standard that showed a deviation of K (8524 ± 88 μg g−1) > P (4456 ± 44 μg g−1) > Mg (2506 ± 25 μg g−1) > S (1429 ± 9 E


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nibs, shells, germs) and the most abundant bean elements (N, K, S, Ca). Also the second principle component was loaded by characteristics of the production systems (stem density and leaf area index) and the bean elements Mg, P, Zn, and Cu, while the third principle component was loaded by the harvesting season, indicators for abiotic stress (total phenolic content and spermine), and the C, N, and K concentrations. The fourth principle component was dominated by the three polyamines (putrescine, spermine, and spermidine) as well as the bean compartments (nibs and shells) and the Mg, P, Fe, and Zn concentration. The fifth principle component, like the first two axes, was loaded with characteristics of the production systems (farming and stem density), the polyamines putrescine and spermidine, bean parameters (weight, nib germs, fat, and C content), and the N and P concentration. The PCA presented the influence of the production systems, shown by stem density (MONO < AF < SAFS), farming (ORG vs CONV), and an increase in leaf area index and soil organic matter, on the bean compartments, the fat and the C content and mineral elements in the beans, e.g., the concentration of N in the beans increased with the soil organic matter (Figure 4a). Also polyamines, mainly putrescine and spermine, were influenced by the production system. The total polyamine content decreased with increasing leaf area index (Figure 4b) and the lowest concentration of total polyamines was found in beans harvested in SAFS (Table 3), the systems with the highest stem density and a high leaf area index. Instead, the season with the harvesting times “wet” vs “dry” had a negative relationship with spermine and a positive relation to the total phenolic content. Consequently, the total phenolic content decreased with increasing spermine concentration (Figure 4c).

measured in beans harvested in AF CONV and AF ORG, respectively. 3.4. Stress Indicators in Cocoa Beans. The total phenolic content of the beans was not affected by the production systems, but strongly increased from harvest “wet” (5.3 μg g−1) to harvest “dry” (7.5 μg g−1) (Table 3). Among the polyamines, spermidine (4.5 ± 0.2 μg g−1) was most abundant in all cocoa bean samples followed by spermine (2.3 ± 0.1 μg g−1) and putrescine (1.2 ± 0.1 μg g−1). Total polyamine concentration in the beans was affected by the production systems (Table 3). Spermidine, the triamine in the polyamine metabolism pathway between the diamine putrescine and the tetramine spermine, was positively correlated with putrescine (Figure 2a) and spermine (Figure 2b), the correlation between putrescine and spermine was significant but the correlation coefficient was lower than that between the polyamines directly connected in the pathway (Figure 2c). The variability of the stress indicators between beans from different production systems was not significantly different (Table 4). Nonetheless, the variation of putrescine and spermidine across beans from both harvests was lower in beans that were produced in SAFS than in beans from MONO and AF. 3.5. Overview of the Correlations of Bean Parameters with the Growing Conditions. Five axes of the PCA were necessary to explain >50% of total variation in the sample (Figure 3, showing the first two principle components; Table S3). The first principle component was loaded by characteristics of the production systems (stem density and soil organic matter), variables concerning the bean compartments (weight,

4. DISCUSSION 4.1. Seasonal Climatic Variations Induce a Physiological Response in the Beans. We hypothesized that the growing conditions have an effect not only on pod production and total yield,24 but also on the morphological and chemical composition of the beans. In Alto Beni, the cocoa harvesting period starts with the transition from the rainy to the dry season and proceeds over the dry winter. Pods harvested in April had matured during the rainy season with sufficient water supply as well as constantly warm temperatures and high relative humidity. Pods that were harvested at the end of September had been exposed to dropping soil moisture, decreasing relative humidity, and low night temperatures. Especially during the last phase of pod maturity when still many physiological processes occur that are related to environmental conditions,11 the climate influenced the beans' chemical composition. We found indicators for abiotic stress, i.e., total phenol content and spermine, in the beans with changing concentration from harvest “wet” to harvest “dry” implying a relation to the climatic conditions and to the water supply of the tree during pod ripening. The total phenolic content showed the strongest increase from rainy to dry season as supposed by Camu et al.,10 Wang et al.,11 and Albertini et al.12 Phenolic compounds are antioxidants that reduce the increased level of reactive oxygen species during drought, as shown for drought tolerant shrubs.36 Even though cocoa is a drought susceptible species,37 an increased level of the total phenolic compounds in the dry season may protect the bean, the plants’ reproduction unit, during ripening from cell damage by reactive oxygen species.

Figure 3. Principal Component Analysis conducted on 99 cocoa bean samples across five production systems and two harvests, using morphological bean characteristics (DBF: dry bean factor; weight: total bean weight; nibs: weight of nibs; germs: weight of germs; shells: weight of the shells), pH, fat and C content, TPC: total phenolic content, bean element concentration (N; K; P; Mg; S; Ca; Na; Fe; Zn; Cu; Mn), polyamine concentrations (PUT: putrescine; SPN: spermine; SPD: spermidine), harvest: harvest “wet” vs harvest “dry”, and characteristics of the production systems (farming: organic vs conventional; stem density; LAI: leaf area index; SOM: soil organic matter). The first two principal components (PC) are shown that explain 27.1% of total variability. F


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Figure 4. Linear relationship between (a) soil organic matter (SOM) and bean N, (b) leaf area index (LAI) and the sum of polyamines (putrescine, spermidine and spermine), and (c) spermine (SPN) and total phenolic content (TPC) across all systems and harvests; the correlation coefficient ρ, degrees of freedom, and p-value are shown.

content with higher temperature may be explained by a decrease in photosynthetic rate with increasing temperature.45 4.2. Microenvironmental Growing Conditions and Farming Practices Affect Cocoa Beans Chemical Composition. Cocoa production systems differ in stem density, diversity and farming practice,24 and the resultant ecosystem services.19 Cocoa growth and production in agroforestry systems is influenced by shading and reduction of temperature extremes (Niether et al., under revision) and, because of the high root density in agroforestry systems, by a high soil organic matter content26 that maintains or enhances soil fertility and nutrient availability for the plants and retains soil humidity.46 N and K concentration of the beans were positive correlated with soil organic matter and in beans from agroforestry systems under organic farming we found highest concentrations of N, Mg, S, Na, and Fe, while Zn and Cu were even more concentrated in beans from the successional agroforestry systems, with even a higher stem density than the traditional agroforestry systems. When soil organic matter is oxidized by micro-organisms, Cu and other previously bound elements become more available for the cocoa tree.47 However, nutrients might be bound to complexes within the soil organic matter that make them less plant available, especially in Fe−Cacomplexes in the iron rich soil26 that can explain the decrease in Ca in the beans with increasing stem density and soil organic matter from MONO to AF and to SAFS. The dense canopy of agroforestry systems with a high leaf area index in comparison to monocultures reduces light transmission and buffer climate extremes and thereby reduces heat and water stress (Niether et al., under revision). Polyamines in the beans decreased with increasing leaf area index that might be explained by a lower exposure to stress in agroforestry systems than in monocultures. We expected a lower variability of stress indicators in beans from agroforestry systems than in beans from monocultures, since internal microclimate and soil moisture change less between dry and rainy season.21 Indeed, variability of putrescine and spermidine levels were lowest in successional agroforestry systems, but not in the traditional agroforestry system. Lowest total polyamine levels and variability in beans from successional agroforestry systems imply favorable growing conditions for the cocoa tree, even though no higher total yield was obtained. The effect of the growing conditions on the cocoa beans was not strong in comparison to postharvest processes.6,7,10,12,15,48 But it can explain the observed variations. Also the harvesting season showed an influence on the cocoa beans’ chemical

Polyamines regulate plant development and physiological processes and function as second messengers in response to abiotic stress.5,38 In drought intolerant Arabidopsis leaves, putrescine increases rapidly after the drought started, while spermidine decreases and the spermine level does not change39 or even decreases.40 Putrescine plays a role in stress response and development, e.g., it enhances germination in alfalfa seeds under drought stress.41 Germination was not observed in this study, even though cocoa beans germinate more often already within the pods in the dry season (personal communication and observation). Polyamine levels and corresponding gene expression in leaves and flowers of cocoa are elevated in response to drought after a few days.8 In cocoa beans, we observed a reduced level of spermine, while putrescine and spermidine concentration did not change. Besides the canalization from putrescine to spermine, a back-conversion from spermine to putrescine that reduces the accumulation of polyamines during stress acclimation is possible.42 During a dry season with decreasing, but still plant available soil moisture,21 (for the same sites and season) acclimation to drought might follow the back-conversion to a polyamine homeostasis. The mineral macronutrient content of cocoa leaves decreases when subjected to drought.14 We found a decrease of K, Ca, and Na content in the beans, while N concentration increased. This can be explained by the maintenance of enzymatic functioning in the beans as the plant embryo. Also Fe, Zn, Cu, and Mn content in leaves decrease with drought because the micronutrient uptake of the plants is reduced.14 Cu acts as cofactor for polyphenol oxidase that is identified also in cocoa beans.43 The increase in the total phenolic content in the beans to enhance the drought tolerance and functioning of the embryo during the dry season might be followed by an increase of polyphenol oxidase and Cu to oxidize surplus phenols. Mn is cofactor of superoxide dismutase,14 and the Mn increase in the beans might also play a role in oxidative stress reduction and drought tolerance.44 We observed an increase in the C and fat content in beans harvested in the dry season. Daymond and Hadley2 report a temperature effect during maturation phase (mean temperature over 60 days before harvest) on the bean fat content with a maximum between 23 °C and 24 °C and a subsequent decline in fat content. Mean temperature before harvest “dry” in winter (23.4 °C) coincided with a higher fat content than at harvest “wet” in fall (25.2 °C) supporting the idea of the effect of surrounding temperature on the fat content. The lower C G


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Fertilisation; Lichtfouse, E., Ed.; Springer: Netherlands/Dordrecht, 2011; pp 193−213. (4) Tuteja, N.; Sopory, S. K. Chemical signaling under abiotic stress environment in plants. Plant Signaling Behav. 2008, 3, 525−536. (5) Akula, R.; Ravishankar, G. A. Influence of abiotic stress signals on secondary metabolites in plants. Plant Signaling Behav. 2011, 6, 1720− 1731. (6) do Carmo Brito, B. de N.; Campos Chiste, R.; da Silva Pena, R.; Abreu Gloria, M. B.; Santos Lopes, A. Bioactive amines and phenolic compounds in cocoa beans are affected by fermentation. Food Chem. 2017, 228, 484−490. (7) Oracz, J.; Nebesny, E. Influence of roasting conditions on the biogenic amine content in cocoa beans of different Theobroma cacao cultivars. Food Res. Int. 2014, 55, 1−10. (8) Bae, H.; Kim, S.-H.; Kim, M. S.; Sicher, R. C.; Lary, D.; Strem, M. D.; Natarajan, S.; Bailey, B. A. The drought response of Theobroma cacao (cacao) and the regulation of genes involved in polyamine biosynthesis by drought and other stresses. Plant Physiol. Biochem. 2008, 46, 174−188. (9) Wollgast, J.; Anklam, E. Polyphenols in chocolate: is there a contribution to human health? Food Res. Int. 2000, 33, 449−459. (10) Camu, N.; De Winter, T.; Addo, S. K.; Takrama, J. S.; Bernaert, H.; De Vuyst, L. Fermentation of cocoa beans. Influence of microbial activities and polyphenol concentrations on the flavour of chocolate. J. Sci. Food Agric. 2008, 88, 2288−2297. (11) Wang, L.; Nagele, T.; Doerfler, H.; Fragner, L.; Chaturvedi, P.; Nukarinen, E.; Bellaire, A.; Huber, W.; Weiszmann, J.; Engelmeier, D.; Ramsak, Z.; Gruden, K.; Weckwerth, W. System level analysis of cacao seed ripening reveals a sequential interplay of primary and secondary metabolism leading to polyphenol accumulation and preparation of stress resistance. Plant J. 2016, 87, 318−332. (12) Albertini, B.; Schoubben, A.; Guarnaccia, D.; Pinelli, F.; Della Vecchia, M.; Ricci, M.; Di Renzo, G. C.; Blasi, P. Effect of fermentation and drying on cocoa polyphenols. J. Agric. Food Chem. 2015, 63, 9948−9953. (13) Kongor, J. E.; Hinneh, M.; van de Walle, D.; Afoakwa, E. O.; Boeckx, P.; Dewettinck, K. Factors influencing quality variation in cocoa (Theobroma cacao) bean flavour profile  A review. Food Res. Int. 2016, 82, 44−52. (14) Santos, I. C. d.; Almeida, A.-A. F. d.; Anhert, D.; Conceiçaõ , A. S. d.; Pirovani, C. P.; Pires, J. L.; Valle, R. R.; Baligar, V. C. Molecular, physiological and biochemical responses of Theobroma cacao L. genotypes to soil water deficit. PLoS One 2014, 9, e115746. (15) Afoakwa, E. O.; Quao, J.; Takrama, J.; Budu, A. S.; Saalia, F. K. Chemical composition and physical quality characteristics of Ghanaian cocoa beans as affected by pulp pre-conditioning and fermentation. J. Food Sci. Technol. 2013, 50, 1097−1105. (16) Rice, R. A.; Greenberg, R. Cacao Cultivation and the Conservation of Biological Diversity. Ambio 2000, 29, 167−173. (17) Ahenkorah, Y.; Akrofi, G. S.; Adri, A. K. The end of the first cacao shade and manurial experiment at the Cocoa Research Institute of Ghana. J. Hortic. Sci. 1974, 49, 43−51. (18) Ruf, F.; Schroth, G. Chocolate Forests and Monocultures: A Historical Review of Cocoa Growingand Its Conflicting Role in Tropical Deforestation and Forest Conservation. In Agroforestry and Biodiversity Conservation in Tropical Landscapes; Schroth, G., Ed.; Island Press: Washington, DC, 2010; pp 107−134. (19) Tscharntke, T.; Clough, Y.; Bhagwat, S. A.; Buchori, D.; Faust, H.; Hertel, D.; Hölscher, D.; Juhrbandt, J.; Kessler, M.; Perfecto, I.; Scherber, C.; Schroth, G.; Veldkamp, E.; Wanger, T. C. Multifunctional shade-tree management in tropical agroforestry landscapes - a review. J. Appl. Ecol. 2011, 48, 619−629. (20) Beer, J.; Muschler, R.; Kass, D.; Somarriba, E. Shade management in coffee and cacao plantations. For. Sci. 1998, 53, 139−164. (21) Niether, W.; Schneidewind, U.; Armengot, L.; Adamtey, N.; Schneider, M.; Gerold, G. Spatial-temporal soil moisture dynamics under different cocoa production systems. Catena 2017, 158, 340− 349.

composition. We related it to the decreasing water availability for the cocoa over the dry season that affected the building-up of structural complexes in the beans as a physiological response. Bean chemical analysis should therefore focus more on the climate during bean ripening, depending on the harvesting season or on the origin of the beans, local weather conditions and production systems. When precipitation patterns will change in tropical regions due to climate change,37 not only yield, but also bean quality may be affected.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b04490. Table S1. Reproducibility of bean sample preparation for HPLC-measurement. Eight bean samples were extracted in duplicate (extraction a and b) and measured by HPLC, results are shown as the area of the peak. Table S2. Reproducibility of HPLC-measurements. Standard reagents were repeatedly measured (run 1 to run 5) by HPLC, results are shown as the area of the peak. Table S3. Results for the first five principle components (PC) from the Principle Component Analysis conducted on 99 cocoa bean samples across five production systems and two harvests. Figure S1. Chromatogram showing the peaks of putrescine (PUT), the internal standard diaminoheptan (DAH), spermidine (SPD) and spermine (SPN) from fluorescence detection after separation by HPLC (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Phone: +49-551-3912136; e-mail: [email protected] (W.N.). ORCID

Wiebke Niether: 0000-0002-7776-1268 Funding

This study was funded by a grant from Johannes-HübnerStiftung, Giessen, Germany, with special support by Mrs. O. Riedl-Hübner. Study plots and field assistants were provided by FiBL, Switzerland, with funding from Biovision Foundation for Ecological Development, Coop Sustainability Fund, Liechtenstein Development Service (LED), and the Swiss Agency for Development and Cooperation (SDC). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Many thanks are regarded to the Ecotop-team in Sara Ana and to the laboratory teams in Goettingen. REFERENCES

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