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Bioactive Constituents, Metabolites, and Functions

Diversity in Composition of Bioactive Compounds among 26 Cocoa Genotypes Noor Febrianto, and Fan Zhu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b03448 • Publication Date (Web): 23 Jul 2019 Downloaded from pubs.acs.org on July 23, 2019

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Journal of Agricultural and Food Chemistry

Diversity in Composition of Bioactive Compounds among 26 Cocoa Genotypes Noor Ariefandie Febrianto1, 2 and Fan Zhu1,* 1) School of Chemical Sciences, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand 2) Indonesian Coffee and Cocoa Research Institute (ICCRI), Jl. PB Sudirman No. 90 Jember, East Java, Indonesia *Correspondence: [email protected]

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ABSTRACT

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Composition of bioactive compounds in cocoa beans is critical to the sensory and nutritional

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quality of cocoa based products. Twenty six cocoa bean genotypes were freshly collected from

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the same plantation location in Indonesia. The bioactive compounds in these raw cocoa

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genotypes were identified and quantified. The results showed a great diversity in the

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composition of bioactive compounds among the 26 cocoa samples. The concentrations of

7

methylxanthines, epicatechin, proanthocyanidin (PA) B-type oligomers, clovamide and

8

anthocyanins were important variables that differentiated these genotypes. MCC 01, SUL 3,

9

ICCRI 03 and ICS 60 genotypes had the highest contents of flavan-3-ols including PA and

10

have the potential to be developed for “healthy” product formulations. Some genotypes such

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as DR 1, DR 2, DR 38, ICS 13, KPC 1, KW 617, RCC 71 and TSH 858 could be favored by

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industries due to the potential to be made into end-products with brighter appearance.

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Keywords: proanthocyanidin; cocoa; clone; polyphenol; Theobroma cacao

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INTRODUCTION

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Cocoa (Theobroma cacao) beans are a key ingredient in food industries. The world’s

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demand of cocoa beans has kept increasing and has reached around 4.5 million tonnes in 2018.

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As an export commodity, cocoa provides significant economic support for its main producers

18

such as Ivory Coast, Ghana, Nigeria, Cameroon, Indonesia, Ecuador and Brazil.1

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Cocoa beans are rich in bioactive compounds. Aside from methylxanthines such as

20

theobromine and caffeine which provide cocoa-based product with myocardial stimulant

21

effect, cocoa beans are also rich in polyphenols. Previous reports showed that cocoa beans

22

contained catechin, epicatechin, anthocyanins, phenolic acids, flavonol glycosides and

23

proanthocyanidins (PAs) (B1−B5, C1 and larger oligomers and polymers).2-4 Patras et al.5 and

24

D’Souza et al.6 showed that cocoa beans also contained PA A-type dimer, PA glycosides,

25

sulfated flavonols and flavanol-flavanone adducts. The rich content of polyphenols in cocoa

26

beans has attracted great attention from the consumers with health awareness. Claimed health

27

effects related to the cocoa polyphenols have been widely published. Cocoa polyphenols have

28

been reported to have antioxidant, anti-inflammatory, anti-allergenic, anti-microbial, immune-

29

modulative and anti-carcinogenic activities.2 Understanding the polyphenol composition of

30

cocoa beans is critical for the nutritional properties of cocoa products such as chocolates.

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The composition of polyphenols in cocoa beans was also known to affect its sensory

32

and eating quality. Polyphenols largely contribute to bitter and astringent taste as well as the

33

development of the color of cocoa-derived final products.8 Previous reports concluded that the

34

composition and quantity of polyphenols in cocoa beans were affected by both intrinsic and

35

extrinsic factors and their interactions.8,9 Extrinsic factors included environmental condition

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during cocoa growing, harvesting season, post-harvest processing such as fermentation and its

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subsequent steps. On the other hand, polyphenols in cocoa were also reported to be dependent



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to the genetics of the cocoa (intrinsic factor).10 Genetics of cocoa was reported to affect the

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types and quantity of proteins in cocoa beans.8 However, there is a lack of information on the

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polyphenol composition in cocoa beans as affected by the crop genetics.

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The selection of suitable cocoa genotypes for cultivation is critical in cocoa plantation

42

industries. Cocoa is considered as a long-term crop investment which could last for about 25

43

years.8 There are numerous clones and hybrids that have been developed in cocoa-producing

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countries such as Indonesia. As a result of collective breeding, different cocoa varieties

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obtained have different characteristics.11 Genotype of cocoa has been known to affect the

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bioactive composition in cocoa beans. The study of Elwers et al.9 reported that there were

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significant differences in the composition of bioactive compounds among the cocoa beans of

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different cultivars, namely Criollo, Trinitario, Nacional, Lower and Upper Amazon Forastero.

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The study also showed that there were variations in the concentrations of bioactive compounds

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among different clones/hybrids of the same cocoa cultivar. Overall, further understanding on

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the variation of bioactive compounds in cocoa genotypes from different sources should be

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done. The development of baseline data employing uniformly processed samples from one

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location is essential to better understand of the genetic variations of cocoa quality.

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This study evaluated the composition of bioactive compounds from 26 different

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Indonesian cocoa genotypes grown in same plantation area. The results will provide a baseline

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on the variety and quantity of bioactive compounds in currently commercialized, cultivated,

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and developed cocoa genotypes. The result will be important for the industries and cocoa

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producers to ease the selection of cocoa genotypes for desired quality traits and to develop

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nutritionally enhanced cocoa products.

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MATERIALS AND METHODS 4 ACS Paragon Plus Environment

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Materials. Cocoa pods of 26 genotypes were collected from the germplasm garden of

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Indonesian Coffee and Cocoa Research Institute (ICCRI) (Jember, East Java, Indonesia).

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General information regarding the cocoa genotypes used in this study were presented in

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Supplementary Table 1. The pods were collected during the period of July to October, 2018.

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At least 5 cocoa pods were collected for each genotypes based on its availability. Wet cocoa

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beans were extracted by breaking the pod and sorted for any broken beans. Only the healthy

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cocoa beans with no signs of damages from pests and diseases were used. The beans was then

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dried at 60 °C until the moisture content reached less than 7.5%. The dried cocoa beans were

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then placed in sealed zip locked bag and stored in dry place until analyzed. HPLC standards

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such as (-)-epicatechin, theobromine, caffeine, and quercetin were purchased from Sigma-

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Aldrich Chemical Co. (St. Louis, MO, USA). Cyanidin-3-glucoside was purchased from AK

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Scientific (Union City, CA, USA). All the analysis was carried out using chemicals of

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analytical and HPLC grade.

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Cocoa Beans Sample Preparation. Approximately 50−100 g dried cocoa beans were hand-

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peeled to separate the shell and cotyledon (nibs). The nibs were then grinded (Waring

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laboratory blender 38BL40, Waring Commercial, New Hartford, NY, USA), milled, and sieved

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(35 mesh). The cocoa powder was then stored in sealed container at 4 °C until analysis.

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Fermentation Index Analysis. Fermentation index (FI) analysis was done using the method

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of Romero-Cortes et al.12 Cocoa powder (50 mg) was mixed with 5 mL of a mixture of 97%

81

methanol and 3% hydrochloric acid (v/v). The solution was vortexed and stored in refrigerator

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for 18 h. The solution was then centrifuged at 3,500 × rpm for 10 min (Heraeus Labofuge 400,

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Thermo Fisher Scientific, Waltham, MA, USA). The supernatant was analyzed using a Thermo

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Fisher Spectronic 200 (Waltham, MA, USA) at 460 and 530 nm. FI was calculated as the ratio

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of the absorbance of anthocyanidins monomer (460 nm) to that of oxidized and polymerized

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anthocyanidins (530 nm). 5 ACS Paragon Plus Environment


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Preparation of Cocoa Methanolic Extract. Cocoa extracts were prepared by means of

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methanolic extraction. Cocoa powder was first defatted using hexane with a ratio of sample to

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hexane at 1 to 15. The oil-rich hexane solution was then separated by centrifugation (2000 × g,

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10 min). A solution of 80% methanol was added to the defatted cocoa powder, which was

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followed by overnight extraction in a shaker (400 × rpm, 16 h). The solution was then

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centrifuged to obtain the supernatant referred as cocoa methanolic extract (CME) and used for

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HPLC analysis. The residue was dried under gentle nitrogen flow and stored for subsequent

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thiolysis treatment as described in the next section. For the anthocyanin analysis, the acidic

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methanol extracts used for the fermentation index determination was employed. The extract

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was referred as cocoa acidic-methanol extract (CAME).

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Thiolysis of Cocoa Residue. Thiolysis was carried out following the method of Hellström and

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Mattila.13 The residues obtained from the methanolic extraction process were mixed with an

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acidic-methanol solution (1.1% HCl) containing 5% of benzyl mercaptan in a sealable

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container. The mixture was then incubated in a waterbath at 40 °C for 30 min. The supernatant

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was obtained by centrifugation (2000 × g, 10 min) before immediately subjecting to HPLC

102

analysis for non-extractable PA (NEPA) quantification.

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Identification of Bioactive Compounds. Identification of bioactive compounds in CME and

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CAME was carried out by means of high-performance liquid chromatography on a Thermo

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Fisher Dionex Ultimate 3000 HPLC system (Waltham, MA, USA). The HPLC instrument was

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coupled with a Bruker Daltonics MicrOTOF system (Billerica, MA, USA) and an electrospray

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ion (ESI) source connected to a time-of-flight mass spectrometry (hybrid quadrupole TOF-

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MSn). A reverse phase (RP) Discovery C18 column (25 cm × 4.6 mm, 5 µm) equipped with a

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guard column with the same phase (Supelco, Bellefonte, PA, USA) was used. The column

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temperature was set at 25 °C. Mobile phases were acetonitrile (A) and 0.1% formic acid (B).

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The flow rate was 0.4 mL/min. The elution gradient of mobile phases was set as: 5 to 30% A 6 ACS Paragon Plus Environment

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(0−40 min), 30 to 100% A (40−45 min), isocratic (45−50 min), 100 to 50% A (50−55 min),

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and 50 to 5% A for the final 5 min. HPLC-ESI-MicrOTOF-MSn data was analyzed using

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Bruker Data Analysis (Bruker Daltonics, Billerica, MA, USA). Identification of cocoa

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bioactive compounds was done by comparing m/z obtained from the experiment with the

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theoretical m/z employing accurate mass measurements (Δ ppm < 5) in formula generator

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software based on ChemCalc.13 Data from previous studies regarding cocoa bioactive

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compound identification were also used as references.5,6 Identification of methylxanthines was

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carried out using the internal standards. Further verification was done using the MS2 analysis

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on pre-designated/targeted analytes (ramped collision energy from 6.5 to 20 eV) resulting in

121

fragmentation of targeted compounds. Dissecting algorithm was used to separate minor and/or

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overlapped peaks in the mass spectra.

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Quantification of Bioactive Compounds. Quantification of bioactive compounds in cocoa

124

beans was carried out using the data obtained from the analysis of CME employing RP-HPLC-

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diode array detector (DAD) (Agilent Technologies, Loveland, CO, USA). The same column,

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mobile phases and elution gradient used in the HPLC-ESI-MicrOTOF-MSn analysis above

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were used for the HPLC-DAD analysis of flavan-3-ols, methylxanthines, phenolic acids,

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flavonols, and NEPA. The detection was done at 280 for the methylxanthines and flavan-3-ols,

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320 for the phenolic acids, and 370 nm for the flavonols, respectively. Epicatechin, chlorogenic

130

acid, and quercetin were used as external standards for the quantification of flavan-3-ols,

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phenolic acid and flavonols, respectively.15-17 The results were expressed as external standard

132

equivalents (eq.) per kg of cocoa powder (dry matter basis, db).

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Quantification of Anthocyanins. The analysis was carried out on the CAME samples using

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the RP-HPLC-DAD system (Agilent Technologies, Loveland, CO, USA). Mobile phases of

135

methanol (A) and 1% acetic acid (B) were used. The flow rate was set at 0.6 mL/min. The

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elution gradient was set as follows: 20 to 70% A (0−12 min), 70 to 100% A (12−20 min), 100 7 ACS Paragon Plus Environment


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to 50% A (20−23 min), 50 to 20% A (23−25 min) and isocratic for the final 5 min. The

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detection was carried out at 520 nm. Cyanidin-3-glucoside was used as external standard.16 The

139

results were expressed as cyanidin-3-glucoside equivalents (cyanidin-3-glucoside eq.) per kg

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of cocoa powder (dry matter basis, db).

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Quantification of Non-extractable Proanthocyanidins (NEPA). The quantification of

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NEPA was carried out based on the results of RP-HPLC analysis using the method of Gao et

143

al.18 Standard of epicatechin was used to quantify the total area of flavan-3-ols monomer and

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their benzylthioethers.19 The result was expressed as epicatechin equivalents (epicatechin eq.)

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per kg of cocoa powder (dry matter basis, db). The degree of polymerization (DP) of NEPA

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was calculated by dividing the total area of benzylthioether with the area of flavan-3-ols

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monomer added by 1.20

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Statistical Analysis. All the analysis were carried out in triplicate. SPSS software (Version 24,

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IBM Corp., Armonk, NY, USA) was used to perform Box-plot analysis, Pearson correlation,

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and principal component analysis (PCA).

151 152

RESULTS AND DISCUSSION

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Fermentation Index (FI). The appearance of the cocoa bean samples from different genotypes

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was diverse (Figure 1). FI of the 26 cocoa bean samples were in range of 0.38 to 1.42. FI of the

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purple bean samples ranged from 0.38 to 0.52, whereas that of the white bean samples (DR 1,

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DR 2, and DR 38) were in range of 1.14 to 1.42. Unfermented and partially fermented cocoa

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beans were reported to have FI of < 1.21 All the purple bean samples evaluated in this study

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were included in this category. In this study, the freshly collected cocoa beans were directly

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dried without any fermentation. High FI value observed in white bean samples did not represent

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its fermented state but due to low concentrations of anthocyanins.9 Since FI values were not 8 ACS Paragon Plus Environment

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applicable to evaluate the degrees of fermentation of the white cocoa bean samples, the

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determination of its unfermented status was carried out by observing the beans for any possible

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fermentation traces such as brown color and cocoa/acidic odor.22 Overall, the 26 cocoa bean

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samples were unfermented. The use of FI to indicate the actual degrees of fermentation in

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different cocoa beans should be critically assessed.

166 167

Identification of Cocoa Bioactive Compounds. Typical chromatograms obtained from CME

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were shown (Figure 2). Twenty two resolved peaks were identified based on the highest

169

observed m/z intensity of the respective peaks. More than 85 peaks were resolved from a single

170

MS spectrum by employing the dissect algorithm. Twenty-seven compounds were successfully

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identified by means of comparing the m/z data and the references (Table 1). The majority of

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the identified compounds belonged to the groups of flavan-3-ols, phenolic acids, flavonols, and

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their derivatives (Table 1). There was a variation in the bioactive composition among different

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cocoa genotypes evaluated. Among the 27 identified compounds, catechin and epicatechin (m/z

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289 [M-H]-), caffeoyl aspartic acid (m/z 294 [M-H]-), PA B-type dimers (m/z 577 [M-H]-), PA

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B-type trimers (m/z 865 [M-H]-), PA B-type tetramers (m/z 1153 [M-H]-) and PA B-type

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pentamers (m/z 720 [M-2H]-) were obtained in all of the samples. The occurrence of these

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compounds in the cocoa beans was in agreement with the results of Wollgast and Anklam.2

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PAs with higher degrees of polymerization (DP) (hexamers and heptamer) were observed in

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several genotypes. They could only be resolved after using dissect algorithm. PAs of high DP

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were not resolved as obvious peaks in the current chromatographic conditions. This was in

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agreement with Gunaratne et al.23

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Flavan-3-ols were resolved as several peaks in the dissected MS spectra showing the

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existence of different isomers. Catechin and epicatechin were resolved with the retention times



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of 26 and 30 min, respectively. They represented the major flavan-3-ols in the cocoa bean

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samples. Dimers, trimers and tetramers of B-type PAs were resolved in several peaks in the

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dissected MS spectra. These peaks may represent the occurrence of different isomers of the

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PAs.6 Similar results were also reported previously by Patras et al.5 and D’Souza et al.6 There

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are several types of dimers of B-type PAs (e.g., B1 to B6) based on the variety of chemical

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bonds (48, and 46 in α and β positions). The occurrence of PA dimers B1 to dimers B5 in

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cocoa beans has been previously reported.2 On the other hand, limited types PA trimers and

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tetramers have been evaluated. PA C1 and C2 were previously reported as the combination of

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three epicatechin and three catechin through 4β8 and 4α8 bonding, respectively. The

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tetramer (cinnamtannin A2) was an epicatechin tetramer through 4β8 bonding.24 The

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multiple PA trimer peaks detected in this study suggested different PA trimer types formed

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from the variation in the types of monomers and bonding. PA glycosides were found in the

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cocoa bean samples. Pentoside of PA A-type dimer (m/z 707 [M-H]-), hexoside of PA A-type

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dimer (m/z 737 [M-H]-), pentoside of PA A-type trimer (m/z 995 [M-H]-) and hexoside of PA

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A-type trimer (m/z 1025 [M-H]-) were obtained in some of the samples. D’Souza et al.6 showed

200

that the concentrations of PA A-type glycosides were high in unfermented cocoa beans than

201

that of fermented samples. These PA glycosides degraded upon fermentation process.

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Flavonols such as apigenin hexoside and kaempferol 3-O-rutinoside (m/z 431 and 593

203

[M-H]-, respectively), quercetin-O-hexoside (m/z 463 [M-H]-) and quercetin-O-pentoside (m/z

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433 [M-H]-) were obtained in most of the cocoa samples. The occurrence of phenolic acids in

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cocoa beans was previously reported.6,9 Caffeoyl aspartic acid was reported as the major

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phenolic acid in cocoa beans.9 Aside from caffeoyl aspartic acid, other phenolic acids such as

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p-coumaryl aspartic acid (m/z 278 [M-H]-), feruoyl aspartic acid (m/z 308 [M-H]-), p-coumaryl

208

tyrosine (m/z 326 [M-H]-), caffeoyl tyrosine (m/z 342 [M-H]-) and clovamide (N-caffeoyl-L-

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DOPA, m/z 358 [M-H]-) were also identified in the samples. 10 ACS Paragon Plus Environment

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Methylxanthines Composition. The variation in methylxanthines composition of cocoa beans

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has been previously reported to depend on the genotype.25 Genetic variations in the

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methylxanthines composition were obtained among the 26 cocoa bean samples in this study

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(Figure 3). Cocoa beans of DR 2, ICS 60, DR 38 and ICCRI 04 had relatively high

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concentrations of caffeine compared to the others samples. The results indicated that these

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genotypes had potential to be developed into fine-flavor cocoa beans. A low theobromine to

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caffeine ratio is commonly found in fine-flavor cocoa beans.26 MCC 02 genotype had low

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concentrations of both theobromine and caffeine, whereas ICS 60 genotype had high

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concentrations of both of the methylxanthines. Methylxanthines in cocoa are related to the

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sensory characteristics of cocoa based products.10 Methylxanthines have relatively high

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stability during cocoa bean processing such as fermentation.25 At high concentrations,

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methylxanthine such as caffeine possess potential risks to sensitive consumers due to the nature

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as psychopharmacologically active compounds.27

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The theobromine concentrations of the 26 cocoa samples evaluated in this study ranged from

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19.4 to 31.7 g/kg db. The caffeine concentrations varied from 0.53 to 5.54 g/kg db. The results

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were in agreement with previous reports.8,25 Criollo cocoa beans have been reported to contain

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more caffeine compared to Forastero beans. Theobromine to caffeine ratio is used to

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differentiate between fine cocoa to bulk cocoa.26 Davrieux et al.28 showed that most Trinitario

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cocoa samples had theobromine/caffeine ratios between 2 to 6, whereas the samples with the

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ratios of > 6 were mostly of the Forastero cocoa. Theobromine to caffeine ratios of the 26

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cocoa bean samples used in this study was between 4.01 and 37.25. White cocoa beans (DR 1,

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DR 2, DR 38) and several purple cocoa beans (ICS 60, ICCRI 04, MCC 01 and MCC 02) had

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theobromine to caffeine ratios of < 10. The other samples had the ratios varied from 11.00 to

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37.25. The majority of these other cocoa samples had the ratios of 11.00−23.59 (Figure 3). 11 ACS Paragon Plus Environment


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Theobromine and caffeine concentrations of cocoa beans are considered as important factors

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that determine cocoa quality.25

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Flavan-3-ols. Flavan-3-ols are the major phenolic compounds in cocoa beans. Great variation

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in the flavan-3-ols composition among the 26 cocoa samples was obtained (Figure 4). TSH 858

240

had the lowest epicatechin content (9.12 g/kg db), whereas the highest epicatechin content was

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found in SUL 3 (22.78 g/kg db). The concentration of epicatechin was higher (9.12−22.78 g/kg

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db) than that of catechin (0.76−2.30 g epicatechin eq./kg db) in all the cocoa genotypes (Figure

243

4). These results were in agreement with previous reports.2,9

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PAs are the major flavan-3-ols in cocoa beans. The concentrations of PA dimers in the 26 cocoa

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bean samples were in the range of 6.01 (UFF 667) to 11.46 (SUL 3) g epicatechin eq./kg db of

246

cocoa powder. The concentrations of PA trimers ranged from 2.51 (TSH 858) to 7.56 g (SUL

247

3) epicatechin eq./kg db. The concentrations of PA tetramers (0.48 to 3.11 g epicatechin eq./kg

248

db) and pentamers (0.05 to 1.48 g epicatechin eq./kg db) were rather low. MCC 01 genotype

249

had the highest concentrations of PA tetramers and pentamers. Similar results were reported

250

previously on other cocoa genotypes.21 Pearson correlation analysis showed that the

251

concentration of epicatechin in the 26 cocoa bean samples correlated to that of PA B-type

252

dimers (r = 0.76, p < 0.05), trimers (r = 0.84, p < 0.05) and tetramers (r = 0.85, p < 0.05).

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Indeed, epicatechin is the main building block of PA B-type oligomers in cocoa beans.

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The oligomeric PAs significantly contribute to nutritional and flavor quality of cocoa beans.

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PAs with low DP tend to have high solubility and contribute to the astringency of cocoa

256

products.29 These PAs also have a relatively high absorption in human body with improved

257

antioxidative effect. PAs with higher DP (DP ≥ 3) are less soluble and hardly contribute to the


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astringent taste of cocoa beans.29 The oligomeric PAs in human colon is a source of dietary

259

antioxidants in the prevention towards oxidizing agents.30

260 261

Non-extractable Proanthocyanidins (NEPA). The concentrations of NEPA in the 26 cocoa

262

bean samples ranged from 11.1 to 27.4 g epicatechin eq./kg db. The highest values were

263

obtained for ICS 60, ICCRI 03, and ICCRI 06H (27.4, 23.3, and 22.7 g epicatechin eq./kg db,

264

respectively) (Figure 5D). These results were somewhat lower than that of Pérez-Jiménez et

265

al.31 who evaluated one cocoa powder sample (28.4 g/kg db). Apart from the cocoa genetics,

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the differences might be partially due to the processing of unfermented cocoa beans.

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Fermentation and drying of cocoa beans after harvesting may facilitate the interactions and the

268

complexation of PAs and proteins.32 PAs may also bind with insoluble cell wall

269

polysaccharides, increasing the amount of NEPA.13 The occurrence of PAs in plants is related

270

to the defense mechanism. In some plants, the synthesis of such compounds would likely

271

increase the resistance of the plants toward pests.33

272

Among the 26 cocoa samples, the DPs of the NEPA ranged from 3.51 to 6.15 with the

273

average of 4.94 (Figure 5E). This average value was higher than that of a previous study on

274

cocoa powder (DP = 3.90).34 The genetics play an important part in the characteristics of the

275

NEPA in cocoa beans. PAs with low DP (< 10) had less interference with protein adsorption

276

and utilization as well as fat digestion.35 Release of these PAs from the NEPA during human

277

digestion may provide antioxidants without significantly interfering with the nutrient

278

absorption.

279 280

Flavonols and Anthocyanins. Quercetin-O-hexoside and quercetin-O-pentoside were found

281

in small amounts (trace to 47.4 mg quercetin eq./kg db and trace to 49.8 mg quercetin eq./kg 13 ACS Paragon Plus Environment


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db, repectively) in the 26 cocoa beans (Figure 5B). SUL 3 and ICS 60 genotypes contained

283

more flavonols compared to the other cocoa samples. Previous studies also showed that cocoa

284

beans and cocoa powder contained 2 major hexose and pentose-based flavonol glycosides,

285

namely

286

arabinoside.2,17

quercetin-3-O-β-D-glucopyranoside

(isoquercetin)

and

quercetin-3-O-α-D-

287

The concentration of cyanidin-3-arabinoside was higher than that of cyanidin-3-

288

galactoside. The total anthocyanin concentrations in these samples ranged from trace to 918

289

mg cyanidin-3-glucoside eq./kg db. White cocoa beans (DR 1, DR 2 and DR 38) contained

290

little anthocyanin (Figure 5C). These results were in range with those reported by Elwers et al.9

291

The anthocyanin content is an important factor in the classification of different cocoa groups.9

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Distinct differences between Criollo and other groups (e.g., Lower Amazon Forastero,

293

Nacional, Upper Amazon Forastero and Trinitario) were found in anthocyanin composition

294

but not in flavan-3-ols. The absence of anthocyanin led to the formation of white to ivory color

295

of cocoa beans.8 The anthocyanin also play significant role in chocolate processing. Cocoa

296

beans with a low amount of anthocyanin tend to correlate with brighter appearance of chocolate

297

products.36

298 299

Phenolic Acids. Great diversity in the phenolic acid composition was obtained among the 26

300

cocoa bean samples (Figure 5A). The total phenolic acid contents ranged from 2.03 to 6.19 g

301

chlorogenic acid eq./kg db . The types of phenolic acids found in this study largely agreed with

302

the results of previous studies.6,9 The concentrations of caffeoyl aspartic acid (1.40−4.60 g

303

chlorogenic acid eq./kg db), p-coumaryl aspartic acid (0.24−1.28 g chlorogenic acid eq./kg db),

304

clovamide (N-caffeoyl-L-DOPA) (0.03−0.46 g chlorogenic acid eq./kg db) and p-coumaryl

305

tyrosine (trace to 0.16 g chlorogenic acid eq./kg db) varied greatly among the samples. Caffeoyl

306

aspartic acid was the major phenolic acid in these cocoa beans, whereas clovamide and p14 ACS Paragon Plus Environment

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307

coumaryl tyrosine were minor. ICCRI 7 had the highest concentration of caffeoyl aspartic acid.

308

ICCRI 7, DR 38 and TSH 858 genotypes contained more p-coumaryl aspartic acid compared

309

to the other genotypes. Phenolic acids play a role in the processing quality of cocoa beans.

310

Phenolic acids affect the chemical and physical characteristics of cocoa beans similar to their

311

effects on coffee beans during roasting.37 Phenolic acids are also a source of antioxidants in

312

cocoa-derived end-products such as chocolates.

313 314

Principal Component Analysis (PCA). PCA analysis was done to explain the variation of

315

phenolic compounds among the 26 cocoa genotypes studied here (Figure 6). Eighteen variables

316

including different types of flavan-3-ols, phenolic acids, flavonols, and anthocyanins were used

317

in the analysis. A total of 60.8% of the variances could be explained by the first two principal

318

components (PC1 = 44.1% and PC2 = 16.7%). Epicatechin (r = 0.88), PA B-type dimer 2 (r =

319

0.82), PA B-type trimer 2 (r = 0.87), PA B-type tetramer (r = 0.91), and PA B-type pentamer

320

(r = 0.85) were the main factors explaining the variance in PC1. PA B-type trimer 4, clovamide,

321

cyanidin-3-galactoside and cyanidin-3-arabinoside were the main factors explaining the

322

variance in PC2 (r = 0.80, r = 0.80, r = 0.76, and r = 0.71, respectively).

323

The PCA analysis confirmed that the phenolic composition of the 26 cocoa samples

324

was diverse. There is a great variation in the phenolic composition of these cocoa beans.

325

Majority of the 26 genotypes fell in the intersection of the quadrants, representing the average

326

characteristics of the bioactive compounds in the samples. Some cocoa genotypes exhibited

327

rather different characteristics of phenolics compared to the rest of the samples. In PCA score

328

plot, MCC 01, SUL 3, ICCRI 03 and ICS 60 resided in the quadrant I and were characterized

329

by high amounts of epicatechin and B-type PA oligomers. TSH 858 was found in quadrant III

330

and was characterized by relatively low amounts of epicatechin, B-type PA oligomers and



Page 16 of 30

331

anthocyanins. White cocoa beans including DR 1, DR 2 and DR 38 were placed in the lower

332

region of quadrant IV due to the absence of anthocyanins. In terms of major phenolic

333

compounds such as epicatechin and B-type PA oligomers, those white beans genotypes were

334

comparable to the majority of the samples. Overall, the PCA data showed the great diversity in

335

the phenolic composition of these 26 cocoa genotypes.

336 337

General Discussion on Some Specific Cocoa Bean Genotypes. In industries, DR 1, DR 2

338

and DR 38 genotypes are known to produce high quality cocoa products (Java light breaking

339

cocoa) due to its brighter color and unique flavor. However, those genotypes are not commonly

340

cultivated due to its low resistance to pod rot and vascular streak dieback disease38. They have

341

higher maintenance cost. Susilo et al.38 showed that the DR genotypes came from Criollo

342

hybrids (not a pure Criollo) with some characteristics of Criollo cocoa. Genotypes from Criollo

343

and other varieties showed significant differences in the composition of anthocyanins and

344

caffeoyl aspartate.9 Those genotypes studied here also had much less flavonols and PA

345

glycosides compared to the other genotypes. Flavonols contribute to the defense of plants

346

especially against insects and fungi.33,39 The low content of flavonoid glycosides in those

347

genotypes may partially contribute into its low resistance towards pest and diseases.

348

MCC 01 genotype was particularly interesting due to its low theobromine to caffeine

349

ratio, which implies good sensory quality. On the other hand, this genotype also contained high

350

concentrations of flavan-3-ols, implying high nutritional values. Development of end-product

351

from this genotype would be appealing for food industries in pursue of products with high

352

nutritional and sensory values. MCC 01 was reported to be a high-yielding genotype (~3.6

353

tonnes/ha/year) and had high resistance to diseases such as pod rot and vascular streak


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354

dieback.40 This genotype is also highly regarded by cocoa farmers due to its big-sized beans

355

(~1.6 g/bean) and good market value.

356

In conclusion, there were wide variations in bioactive composition in cocoa beans of

357

26 genotypes collected from Indonesia. Several genotypes such as MCC 01, SUL 3, ICCRI 03

358

and ICS 60 have potential to be developed into commercial cocoa with high concentrations of

359

flavan-3-ols including B-type PA oligomers. DR 1, DR 2, DR 38, ICS 13, KPC 1, KW 617,

360

RCC 71 and TSH 858 could be developed by the industries that pursue brighter appearance of

361

end-products. Genotypes such as MCC 01 and ICS 60 may have potential to be developed into

362

fine flavor cocoa beans due to its high phenolic content and methylxanthines composition. The

363

results will provide a basis to better understand the interactions between processing and cocoa

364

genetics on the quality of final products. The results of this study will also be of importance

365

for the breeding industries to develop new cocoa genotypes with targeted nutritional traits.

366

Possible variations on bioactive compounds of cocoa beans from the same genotypes reported

367

in this study should be considered due to the limited number of samples evaluated. Differences

368

in environmental conditions and harvesting seasons may affect the composition of bioactive

369

compounds in cocoa beans of those genotypes. Further studies should also focus on the flavor

370

characteristics of these cocoa genotypes to complement our understanding of these 26 cocoa

371

samples. The impacts of different environmental conditions on the quality of these samples

372

should be studied.

373 374

ACKNOWLEDGMENTS

375

The authors would like to thank Mrs. Fitratin from ICCRI and Tony Chen from School of

376

Chemical Sciences, University of Auckland for technical assistance.

377 17 ACS Paragon Plus Environment


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378

FUNDING

379

Noor Ariefandie Febrianto is financially supported by NZAid programme under the term of

380

NZ ASEAN Scholarship.

381 382

DECLARATION

383

The authors declare no conflict of interest.

384 385

REFERENCES

386

(1)

387 388

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502



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No 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

Experimental Theoritical Error m/z m/z (Δppm) 278.0671 289.0720 294.0617 305.0702 308.0779 326.1031 342.0983 358.0933 369.0274 393.1756 431.0970 433.0770 447.0920 451.1240 463.0887 575.1188 577.1351 593.1511 707.1594 720.1587 737.1716 864.1895 865.1975 995.2244 1008.223 1025.234 1153.261

278.0670 289.0718 294.0619 305.0701 308.0776 326.1034 342.0983 358.0932 369.0286 393.1766 431.0984 433.0776 447.0933 451.1246 463.0882 575.1195 577.1352 593.1512 707.1618 720.1590 737.1723 864.1907 865.1985 995.2252 1008.2224 1025.2357 1153.2619

-0.321 -0.824 0.766 0.328 -1.054 0.908 0.032 -0.208 -3.252 2.596 3.179 1.466 2.874 1.297 -1.080 1.217 0.086 0.158 3.335 0.443 0.981 1.404 1.200 0.750 -0.588 1.669 0.803

Ion

Compound

Formula

[M-H]- p-Coumaryl aspartic acid [M-H]- Catechin/epicatechin [M-H]- Caffeoyl aspartic acid [M-H]- Hydroxyjasmonic acid sulfate [M-H]- Feruoyl aspartic acid [M-H]- p-Coumaryl tyrosine [M-H]- Caffeoyl tyrosine [M-H]- Clovamide [M-H]- Catechin sulphonic acid [M-H]- Cis-3-hexenyl B-primeveroside [M-H]- Apigenin hexoside [M-H]- Quercetin-O-pentoside [M-H]- Kaempferol-3-O-hexoside [M-H]- (epi)-catechin hexoside [M-H]- Quercetin-O-hexoside [M-H]- PA A-type dimer [M-H]- PA B-type dimer [M-H]- Kaempferol-3-O-rutinoside [M-H]- PA A-type dimer pentoside [M-2H]- PA B-type pentamer [M-H]- PA A-type dimer hexoside [M-2H]- PA B-type hexamer [M-H]- PA B-type trimer [M-H]- PA A-type trimer pentoside [M-2H]- PA B-type heptamer [M-H]- PA A-type trimer hexoside [M-H]- PA B-type tetramer

C13H13NO6 C15H14O6 C13H13NO7 C12H18O7S C14H15NO7 C18H17NO5 C18H17NO6 C18H17NO7 C15H14O9S C17H30O10 C21H20O10 C20H18O11 C21H20O11 C21H24O11 C21H20O12 C30H24O12 C30H26O12 C27H30O15 C35H32O16 C75H62O30 C36H34O17 C90H74O36 C45H38O18 C50H44O22 C105H86O42 C51H46O23 C60H50O24

BL 4023 DR 1 DR 2 DR 38 ICCRI 3 ICCRI 4 ICCRI 06H ICCRI 7 ICS 13 ICS 60 KKM 22 KPC 1 KW 516 KW 562 KW 617 MCC 01 MCC 02 NA 32 RCC 70 RCC 71 SCA 6 SUL 1 SUL 2 SUL 3 TSH 858 UF 667

Table 1. Phenolic compounds identified in methanolic extracts of different cocoa genotypes and the number of resolved peaks in dissected MS spectra.

2 1 1 1 1

1 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1

1 1 3 3 3 3 1 1 1 1 1 1 1

1

1

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

1

1 2 4 2 1 4

1 1 2 2 2 2 3 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1

1 1 1 1 1 1 1 1 3 1 1 1 1 2 2 5 6 5 5

3 4 3 1 1 1 2 2 2 2 5 7 8 8 7 1 1 2 2 2 3 3 3 4

1 1 1 1 1 1 1 1 3 1 2 3 1 3 3 2 2 2 1 2 1 2 2 2 2 5 5 4 4 5 2 1 2 3 2 3 3 3 2 3 2 2 2 4 1 2 1 3 1 3 3 3 8 7 8 5 8 1 1 1 1 1 1 1 2 3 2 2 2 3 3 4 3 6

1 1 1 1 1 1 2 3 1 2 3 5 1 4 2 1 2 1 1 2 7 6 2 1 1 1 3 4

1 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 1 2 1 1 1 1 1 1 1 1 1 1 1 1 3 1 2 1 2 1 2 3 3 3 2 2 2 1 2 1 1 1 2 1 4 4 3 4 3 4 4 1 1 3 3 2 3 3 2 1 1 2 1 2 1 3 3 1 2 2 2 2 1 2 1 2 2 3 6 8 5 6 6 7 6 1 1 1 2 2 2 1 1 1 2 2 3 3 1 3 4 3 4 3 5 5

1 1 1 2 2 2 2 2 3 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 3 1 1 5 1 2 2 1 1 5 1

1 2 4 1 3 4

1 1 1 1 2 1 1 3 4 6 1 2 2 3 2 1 2 1 2 1 2 6 5 7 1 2 1

1 1 2 1 2 1 4 4 3 4 2 2 3

* Abbreviation: PA, proanthocyanidin; number of peaks observed were evaluated by employing dissect algorithm.


1 1 1 1 1 1 1 1 3 3 3 1 1 1 1 2 5 4 5 2 2 2 3 2 2 2 1 4 3 1 3 6 6 8 2 1

Page 25 of 30


Figure captions

Figure 1. Appearance of the different cocoa bean genotypes. Figure 2. Typical chromatograms of SUL 3 and DR 2 samples obtained using a RP-HPLCESI-MicrOTOF-MS system. Figure 3. (A) Box plots of methylxanthines concentrations in cocoa bean samples, (B) theobromine to caffeine ratios and caffeine concentration of cocoa bean samples. Figure 4. (A) Box plots of epicatechin and catechin concentration (g epicatechin eq./kg db) in samples, (B) Box plots of total epicatechin and catechin, proanthocyanidin dimer, trimer, tetramer and pentamer concentrations (g epicatechin eq./kg db). Figure 5. Box plots of phenolic acids concentrations (g chlorogenic acid eq./kg db) (A), flavonols concentration (mg quercetin eq./kg db) (B), anthocyanins concentration (mg cyanidin-3-glucoside eq./kg db) (C), non-extractable proanthocyanidin (NEPA) concentration (g epicatechin acid eq./kg db) (D) and its degree of polymerization (DP) (E) in the cocoa bean samples. Figure 6. Principal component analysis (PCA) of cocoa bean samples. (A) Loading plot. (B) Score plot showing distinct differences between cocoa bean samples. PAB, proanthocyanidin B-type; PAA, proanthocyanidin A-type; CoumarylAspAcid, p-coumaryl aspartic acid; CaffeoylAspAcid, caffeoyl aspartic acid.



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