Intra-Variety Diversity of Bioactive Compounds in Trinitario Cocoa

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Bioactive Constituents, Metabolites, and Functions

Intra-Variety Diversity of Bioactive Compounds in Trinitario Cocoa Beans with Different Degrees of Fermentation Noor Febrianto, and Fan Zhu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06418 • Publication Date (Web): 22 Feb 2019 Downloaded from http://pubs.acs.org on February 25, 2019

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Journal of Agricultural and Food Chemistry

Intra-Variety Diversity of Bioactive Compounds in Trinitario Cocoa Beans with Different Degrees of Fermentation

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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There has been increasing interest in bioactive components of cocoa beans as they are

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related to nutritional and sensory quality of cocoa products. Sulawesi 1 (Sul 1) cocoa beans

4

(Trinitario variety) with different degrees of fermentation were collected from Indonesia.

5

Quantification of bioactive compounds in these beans was done to better understand its intra-

6

variety diversity in composition of bioactive components. Epicatechin, proanthocyanidin

7

(PA) dimer, PA trimer, PA tetramer, and cyanidin glycosides were the major phenolics in Sul

8

1 cocoa beans. There was wide variation in concentrations of bioactive compounds among

9

the beans. These cocoa beans can be categorized into 4 distinct groups based on the profiles

10

of flavan-3-ol derivatives, phenolic acids, flavonols and anthocyanins. The fermentation

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index of cocoa beans could not be directly related to the polyphenol profile. This study

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provides insights into farm management using Sul 1 as planting material for quality

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improvement of cocoa-based products with targeted bioactive composition.

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Keywords: Trinitario, flavan-3-ol, epicatechin, proanthocyanidin, anthocyanin, intra-

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variety, Theobroma cacao


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INTRODUCTION

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Trinitario cocoa has received great attention among three major groups of cocoa due

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to its broad quality characteristics covering those of its parents (Criollo and Forastero).1 This

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hybrid has been proven of sturdiness to pests and diseases, which is inherited from

21

Forastero.2 In some cocoa-producing countries such as Indonesia, PBC 123 (commonly

22

known as Sulawesi 1/Sul 1) is one of the most popular Trinitario-based cocoa clones being

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cultivated. The high productivity and resistance toward pests and diseases (especially

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vascular streak dieback) of this cocoa clone has been highly regarded among cocoa

25

producers.3

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The increasing production of Sul 1 cocoa has raised the concern of the variability in

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quality of Trinitario cocoa beans. Previous reports suggested that Trinitario cocoa beans can

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be classified as less aromatic “bulk” cocoa or “fine and flavour” cocoa, depending on taste

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and chemical composition.4 Several studies reported that clones from groups of Trinitario

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cocoa, such as ICS (Imperial College Selection), CCL hybrids and Trinitario cultivated in

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Malaysia had different sensory quality.5, 6, 7 Kongor et al.1 mentioned that genotypes of cocoa

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beans were significantly related to the chemical composition. The changes during post-

33

harvest processing significantly affected the quality of final products. Further, the authors

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also mentioned that the types and the quantities of polyphenols were different among

35

different cocoa genotypes. Collective effort in breeding has led to different cocoa varieties

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with very diverse characteristics.8 However, less study has been carried out to evaluate the

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intra-variety/clones diversity of cocoa beans. This seriously hinders the effort in quality

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improvement of Trinitario as the planting material.

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Information regarding the variation of bioactive compounds in cocoa beans from the

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same varieties/clones and the impact of processing are critical for the quality of cocoa based



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products. The concentration of polyphenols and flavour quality of cocoa beans is known to

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have an inverse relationship.9 Catechins (~37%), proanthocyanidins (~58%) along with

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theobromine and caffeine contribute to the astringency and bitter taste of cocoa products.10 It

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is also well-established that the polyphenols in cocoa have strong anti-oxidative potential.

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Previous studies claimed that cocoa polyphenols possess anti-carcinogenic, anti-

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inflammatory, anti-allergenic, anti-microbial, immune-modulative, and anti-microbial

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activities.11 As consumers become health-conscious, there has been increasing demand of

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polyphenol-rich cocoa products. Fermentation process has been known to decrease

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polyphenol concentrations in cocoa beans. Polyphenol oxidase (PPO) converts polyphenols

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to quinones which are able to form complexes with proteins and peptides.12 The effect of

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fermentation on the polyphenol composition in cocoa beans has been studied extensively.1,10

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However, the effect of polyphenol diversity among cocoa beans from the same clone/variety

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during fermentation has often not been taken into consideration. Factors affecting the

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fluctuation of polyphenols in cocoa beans during fermentation process remain to be better

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understood. As far as the authors know, there has been no report on evaluating the diversity

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in polyphenol composition in a single clone of cocoa beans with different fermentation

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degrees.

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This report studied the composition of bioactive compounds (methylxanthines, flavan-

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3-ols, phenolic acids, anthocyanins, flavonols, and proanthocyanidins) in Sul 1 clone derived

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cocoa beans with different fermentation degrees. The results will be of importance to support

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the cocoa industry in developing cocoa products with targeted composition of bioactive

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compounds.

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MATERIALS AND METHODS


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Materials. Sul 1 (commonly known as PBC 123) cocoa bean samples were obtained

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from Indonesian Coffee and Cocoa Research Institute (ICCRI) in Java, Indonesia. The beans

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were harvested in early March 2018 from the seed garden of Sul 1 cocoa. The seed garden is

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located in an open farm area which allows the possibility of cross-pollination between several

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cocoa clones. A total amount of 200 kg wet beans were collected for fermentation.

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Fermentation was done immediately after the wet beans (equivalent to ~2000−2200 cocoa

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pods) were collected. The fermentation was done in a wooden fermentation box for 96 hours

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(one turning at 48 h). Then, the fermented cocoa beans were dried at 60°C until a moisture

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content of 7.5% was reached. A total of 2 kg of cocoa beans were randomly collected by

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means of coning and quartering. The beans were sealed in zip locked bags and stored in dry

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place until analysis. Chemicals such as (-)-epicatechin, (+)-catechin, theobromine, caffeine,

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quercetin, and benzyl mercaptan were purchased from Sigma-Aldrich Chemical Co. (St.

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Louis, MO). Cyanidin 3-glucoside was purchased from AK scientific (Union City, CA). All

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the chemicals were of analytical and HPLC grades.

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Cocoa Bean Sample Preparation. Approximately 200 dried cocoa beans were

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randomly selected from 2 kg of the sample. The samples were cut manually and observed for

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the color of the nibs. The beans were categorized based on the color of the nibs.

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Categorization of the color was done based on the cocoa cut test chart issued by the Cocoa

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Research Centre at the University of the West Indies (St. Augustine, Trinidad & Tobago). A

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total of 35 beans were selected for chemical analysis. Each bean was deshelled and the

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kernels were grinded, followed by sieving. The sample was then packed in a sealed container

86

and kept at 4 oC until analysis.

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Lipid Content Analysis. Determination of lipid content of cocoa beans was done using

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a mixture of the randomly selected samples to provide a conversion unit for comparison with

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the results of published references. The lipid content was analyzed using the method of 5 ACS Paragon Plus Environment


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AOAC 963.15 without acid hydrolysis before Soxhlet extraction. The lipid content of Sul 1

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cocoa beans was 42.02% resulting in conversion factor (CF) of 1,725.

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Fermentation Index Analysis. Fermentation index (FI) was determined using the

93

method of Romero-Cortes et al.13 based on the ratio of oxidized and polymerized

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anthocyanidins (λmax: 460 nm) and its monomer (λmax: 530 nm). Cocoa powder (50 mg) was

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mixed with 5 mL of a solution of methanol and hydrochloric acid (97:3, v/v). The mixture

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was vortexed and put in refrigerator for 18 h. The sample was then centrifuged (3,000 × rpm,

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10 min). The reddish/brownish supernatant was analyzed using a Thermo Fisher Spectronic

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200 spectrophotometer (Waltham, MA) at 460 nm and 530 nm. The FI was calculated by

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dividing the absorbance at 460 nm to that at 530 nm.

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Preparation of Methanolic Extract. Cocoa methanolic extract was prepared using

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the method of Patras, et al.14 with modifications. Briefly, ground cocoa bean sample (50 mg)

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was defatted using hexane with a sample to hexane ratio of 1:15. The defatted powder was

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extracted using 80% methanol with overnight shaking (400 × rpm, 16 h) before

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centrifugation. The supernatant was used for determination of methylxanthines, flavan-3-ols,

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phenolic acids, flavonols and proanthocyanidins. The residue of the extraction was dried

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under nitrogen and was further used for thiolysis treatment for non-extractable

107

proanthocyanidin (NEPA) determination. Acidic methanolic extract for the determination of

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anthocyanins was prepared based on the method of fermentation index analysis.

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Preparation of Non-extractable Proanthocyanidin (NEPA) Extract. NEPA was

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prepared using the method of Guyot et al.15 and Hellström and Mattila16. The dried residue

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was mixed with 5% of benzyl mercaptan in acidified methanol (1.1% HCl). The mixture was

112

sealed and incubated at 40°C for 30 min before centrifugation. The supernatant was used for

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HPLC analysis. The degree of polymerization (DP) of NEPA was calculated based on the


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method of Gu et al.17 The DP is the ratio between the total area of benzylthioether and the

115

monomer observed added by 1. NEPA content was calculated based on method proposed by

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Gao et al.18 with slight modification, as the total area of monomer flavan-3-ols and their

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benzylthioether equivalents to the external standard of epicatechin.

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HPLC Analysis. Analysis of bioactive compounds from methanolic extract and

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NEPA was done using a reversed-phase high performance liquid chromatography with diode

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array detection (RP-HPLC-DAD) (Agilent Technologies, Loveland, CO), utilizing a

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Discovery C18 column (25 cm × 4.6 mm, 5 µm) and its guard column (Supelco, Bellefonte,

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PA). The flow rate was 0.6 mL/min. The mobile phases were consisted of A: 1% acetic acid

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and B: Acetonitrile. The gradient was: 5 to 30% of B from 0−30 min, 30 to 100% B from 30

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to 40 min, 100% B from 40 to 50 min, 100 to 50% B from 50 to 55 min, and 50 to 5% B from

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55 to 60 min. Column temperature was 25 °C and the wavelength was set at 280 nm for

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methylxanthines and flavan-3-ols; 320 nm for phenolic acids and at 370 nm for flavonols.

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For the analysis of anthocyanins, the mobile phases were consisted of A: 1% acetic

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acid and B: methanol. The flow rate was 0.6 mL/min. The gradient was 20 to 70% of B from

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0 to 12 min, 70 to 100% B from 12 to 20 min, 100 to 50% B from 20 to 23 min, 50 to 20% B

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from 23 to 25 min, and 20% B from 25 to 30 min. The detection was at 520 nm.

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Analysis of proanthocyanidins was done using a normal-phase high performance

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liquid chromatography (NP-HPLC) with diode array detection and a Luna silica column (250

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× 4.6 mm, 5 µm, 100 Å) (Phenomenex, Torrance, CA) based on the method of Rigaud et al.19

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with the modification suggested by Peterson et al.20. Dichloromethane was replaced by an

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ethyl acetate-ethanol and heptane mixture. The mobile phase A was consisted of ethyl-

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acetate/ethanol (3:1):methanol:acetic acid:water (12:84:2:2) and mobile phase B was

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consisted of heptane:ethyl acetate:ethanol:acetic acid:water (48:36:12:2:2). The column

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temperature was 30°C. The flow rate was 1 mL/min. The elution gradient was 100 to 80% B 7 ACS Paragon Plus Environment


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from 0 to 20 min, 80 to 50% B from 20 to 35 min, 50 to 0% B from 35 to 40 min, 0% B from

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40 to 42 min, and 0 to 100% B from 42 to 47 min. The detection was done at 280 nm.

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Epicatechin and catechin were used as external standards for the quantification of

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flavan-3-ols and proanthocyanidin. Chlorogenic acid, quercetin, and cyanidin 3-glucoside

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were used for the quantification of phenolic acids, flavonols, and anthocyanins, respectively.

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HPLC-ESI-MicrOToF-MSn Analysis. Separation of bioactive compound was carried

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out utilizing Dionex Ultimate 3000 HPLC system (Thermo Fisher, Waltham, MA). The

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mobile phase composition and columns used were adopted from the aforementioned RP-

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HPLC-DAD method. Identification of phenolic compounds was done using a Bruker

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MicrOToF instrument (Bruker Daltonics, Billerica, MA) equipped with electrospray ion

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(ESI) source connected to a hybrid quadrupole time-of-flight (ToF) mass spectrometry. The

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fragmentation (MSn analysis) was done in pre-designated analyte by means of sequence mass

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analysis employing the same instrument, and carried out using ramped collision energy

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ranged from 6.5 to 20 eV. The confirmation of compound identification was evaluated based

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on the data of ToF and its fragmentation pattern obtained from MS2 analysis.

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Statistical Analysis. The analysis were done in triplicate. One way ANOVA

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(Duncan multiple range test, p < 0.05), Pearson correlation analysis, principal component

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analysis (PCA), and hierarchical cluster analysis were done using SPSS software Ver. 24

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(IBM Corp., Armonk, NY).

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RESULTS AND DISCUSSION

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Identification of Bioactive Compounds in Cocoa Beans. RP-HPLC analysis

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showed that several compounds of methylxanthines, flavan-3-ols, phenolic acids,

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proanthocyanidins (PA), flavonols and anthocyanins could be separated (Figure 1). RP-

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HPLC-DAD-TOF-MSn analysis identified some compounds from the different groups. Two 8 ACS Paragon Plus Environment

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methylxanthines (theobromine (m/z 181 [M+H]+) and caffeine (m/z 195 [M+H]+)), 2 flavan-

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3-ol monomers (epicatechin and catechin (m/z 289 [M-H]-)), 8 PAs such as 2 B-type dimers

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(m/z 577 [M-H]-), 2 A-type dimers (m/z 575 [M-H]-), 3 B-type trimers (m/z 865 [M-H]-), and

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1 B-type tetramer (m/z 1153 [M-H]-)), 3 hydroxycinnamic acids namely caffeoyl aspartic

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acid (m/z 294 [M-H]-), p-coumaryl aspartic acid (m/z 278 [M-H]-) and caffeoyl 3-

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hydroxytyrosine (m/z 358 [M-H]-), 3 flavonols namely quercetin-O-hexoside (1 & 2) (m/z

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463 [M-H]-), and quercetin-O-pentoside (m/z 433 [M-H]-), and 2 anthocyanins (cyanidin 3-

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arabinoside (m/z 419 [M+H]+) and cyanidin 3-galactoside (m/z 449 [M+H]+)) were identified

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using cross-reference of the data of TOF unique m/z number, UV-Vis spectrum with

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references to external standards, and literatures

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polymerization were hardly separated, as also reported previously by Gu et al.17 and

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Gunaratne et al.23. RP-HPLC-TOF-MS system separated and identified individual PAs up to

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B-type tetramers (m/z=1153 [M-H]-). PAs with higher degrees of polymerization such as B-

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type pentamer (m/z=1441 [M-H]-) and B-type hexamer (m/z=1729 [M-H]-) were identified,

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but could not be separated as individual peaks. They formed a hump in the chromatogram

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(21−35 min). The aforementioned oligomers were then evaluated by means of NP-HPLC.

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Overall, the identified compounds in cocoa beans from this research were in agreement to the

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results of previous reports.1,11,14

14,22.

However, PAs with higher degree of

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Diversity in Polyphenol Composition. Polyphenol types among the cocoa bean

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samples were largely similar. Great variation in concentrations of the polyphenols among the

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samples was obtained (Table 1 and Table 2). The concentrations of flavan-3-ols and PAs

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varied from < 0.1 g to 25.3 g of epicatechin eq./kg (db) of sample. This result agreed with a

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previous report.24 The similarity in the types of the phenolics could be due to the same

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genetic origin of the cocoa bean samples. Cocoa polyphenolic compounds have been known

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to be consisted mostly of flavan-3-ols monomers (catechin and epicatechin) and its 9 ACS Paragon Plus Environment


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derivatives (PAs).10 PA dimer was found to be a major phenolic compound in cocoa bean.11

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Concentrations of flavan-3-ols and its derivatives (PAs) varied among samples (Table 1). The

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concentrations of epicatechin and PA B-type dimers were among the highest in the beans,

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followed by that of PA trimer and PA tetramer. This suggested that cocoa PAs tend to have a

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good bioavailability as monomeric and dimeric PAs have higher bioavailability compared to

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PAs with a higher degree of polymerization.25

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Flavonols and phenolic acids have not been much reported in cocoa beans from

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previous studies probably due to their lower concentrations compared to that of other

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phenolics (Table 2). The concentrations of quercetin glycosides varied from trace to 138 mg

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quercetin eq./kg db of sample. Phenolic acids had concentrations ranging from trace to 2.53 g

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chlorogenic acid eq./kg db of sample. Caffeoyl and coumaryl conjugates of amino acids such

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as clovamide (caffeoyl 3-hydroxytyrosine) has been reported to possess various health

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benefits such as inhibiting platelet aggregation due to its similar structure to dobutamine and

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denopamine.27

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Anthocyanins in cocoa are responsible for the purple color in cocoa nibs. The

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concentration of cyanidin glycosides varied significantly from trace to 865.3 mg cyanidin 3-

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glucoside eq./kg db of sample. The concentration of cyanidin 3-arabinoside was higher than

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that of cyanidin 3-galactoside in all the bean samples. These results were in agreement with

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previous studies.10,28 The anthocyanin content of cocoa beans is significantly dependent on

208

the genetics and fermentation.28 Several bean samples had very low (trace) concentration of

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anthocyanins, which could be due to the effect of fermentation29, or the nature of the cocoa

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beans itself.24

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Diversity in Methylxanthines Composition. Theobromine and caffeine were the

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methylxanthines evaluated in the cocoa samples. There was great variation in the


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concentration of methylxanthines obtained. Theobromine in general, was found in higher

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concentration compared to caffeine (Table 2). Theobromine content of the bean samples

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varied from 10.4 to 26.1 g/kg db, while caffeine content ranging from 0.56 to 5.45 g/kg db.

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These results were consistent with a previous report.1 Methylxanthines content in cocoa is

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highly dependent on the cocoa genotype.4 Unfermented Forastero cocoa beans are usually

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characterized by a high content of theobromine and a low concentration of caffeine, whereas

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Criollo beans are rich in caffeine and have less theobromine.4

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Comparison of PA Quantification using RP-HPLC and NP-HPLC. The contents

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of PAs in cocoa beans from the NP-HPLC analysis tended to be somewhat higher than those

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of RP-HPLC for the same sample (Supplementary Table 1). The result of NP-HPLC in the

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quantification of PA monomer up to tetramer was in good agreement with the result of RP-

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HPLC. The PAs with a higher degree of polymerization were not well revolved using RP-

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HPLC under the current experimental conditions. NP-HPLC in this research could quantify

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the PAs up to hexamer. The limitation occurred in RP-HPLC may result in under-estimation

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of the concentrations of some larger PAs. Generally, it was observed that the concentrations

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of different PAs followed the order of monomers > dimer > trimer > tetramer > pentamer >

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hexamer > ‘DP>6 oligomer’ (Supplementary Table 2). In some of the cocoa samples, the

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concentration of PA trimer was higher than that of PA dimer. This tendency was in

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agreement with the report of Hellström and Mattila16.

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In addition to catechin and epicatechin, gallocatechin and epigallocatechin has been

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found in cocoa11. However, our RP-HPLC based method could not separate gallocatechin

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from epigallocatechin. These two compounds were identified in the MicrOToF system with

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m/z 305 ([M-H]-) representing either gallocatechin or epigallocatechin as a part of the

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unresolved peak. Several B-type PAs were found in cocoa beans previously.11 In this study,

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two major monomeric PAs (flavan-3-ol) and 4 PA dimers were identified, and the 11 ACS Paragon Plus Environment


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concentrations of these PAs from RP-HPLC and NP-HPLC methods were close. Overall,

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there are limited reports on the quantification of oligomeric PAs from cocoa using RP-HPLC.

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Despite the limitations, the use of both RP-HPLC and NP HPLC were complementary to

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each other in the quantification of phenolics in cocoa beans.

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Fermentation Index. Fermentation index (FI) is feasibly used in industry for the

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determination of the degree of fermentation in cocoa beans.9 FI is largely related to changes

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in color of cocoa nibs due to browning reactions during fermentation. Cocoa beans with FI

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lower than 1 are considered as under-fermented, whereas those with FI higher than 1 are

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regarded as sufficiently fermented.9,10 The FI of the cocoa bean samples significantly varied

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from 0.47 to 2.17 (Table 1). Nineteen samples had FI lower than 1 and 16 samples had FI

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higher than 1. The wide range of FI of the 35 cocoa samples covered most of the FI values of

249

cocoa beans previously studied.21

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Polyphenol Composition of Cocoa Beans Affected by FI. Anthocyanin content has

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been reported to decrease during cocoa fermentation due to its conversion to anthocyanidins

252

and complexes tannins.11 Our results confirmed that the anthocyanin content of fermented

253

cocoa beans was generally lower than that of less fermented beans (Table 2). It has been

254

reported that the amounts of flavan-3-ols, such as catechin and epicatechin, in cocoa beans

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could be reduced by fermentation12,24. In this study, Pearson correlation analysis showed that

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FI and the concentrations of major flavan-3-ols derivatives were significantly correlated (p
1) (Table 1). Box-plot graph showed that 12 ACS Paragon Plus Environment

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the concentrations of epicatechin and PAs fluctuated (Figure 2). A similar pattern has been

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reported previously by Albertini et al.30 who found that the concentration of epicatechin in

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Arriba Nacional variety did not decrease in a linear manner, but fluctuated during cocoa

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fermentation. The authors pointed out that that this phenomenon was probably due to the

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effect of non-uniformity of the samples or fermentation process.30 In our study, variation in

268

polyphenol concentration was recorded for the samples with similar FI. It is likely that this

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fluctuation was contributed by the intra-diversity among the samples.

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Concentrations of non-extractable PA (NEPA) varied from 5.05 to 22.8 g epicatechin eq./kg

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db, whereas the NEPA had the degree of polymerization (DP) ranging from 3.71 to 6.96

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(Supplementary Table 2 & Figure 2). Pérez-Jiménez, et al.31 found that the content of NEPA

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in defatted cocoa powder reached 28.35 g/kg db. The non-extractability of NEPA in cocoa

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bean could be due to the bonding of phenolics with insoluble plant material or protein. Cocoa

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fermentation generates lactic and acetic acids, resulting in a lower pH condition to assist the

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phenolics-protein interactions and complexation32. PAs tend to form bonding with insoluble

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parts of plant material such as cell wall polysaccharides16. The amounts of NEPA in the

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cocoa beans analyzed fluctuated for the samples with similar FI (Figure 3). This variation

279

could be due to the genetic diversity in the individual cocoa bean samples. Furthermore,

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enzymes activity in cocoa beans during fermentation may facilitate the release of PAs from

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insoluble complexes, decreasing the content of NEPA.12, 31

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Degree of polymerization (DP) of NEPA in general increased with increasing FI. DP of

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NEPA in fermented beans with FI >1 was generally higher than that of under-fermented

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beans with FI 0.9). The second principal

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component (PC2) explained 10.78% of the variances. It was consisted of 2 major variables,

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namely caffeoyl aspartic acid and p-coumaryl aspartic acid.

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PCA analysis showed that the intra-variety diversity among single cocoa clone

308

contributed to their polyphenol characteristics. It was also shown that the degree of

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fermentation of cocoa beans did not directly relate to their polyphenol characteristic.

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Combined analysis of PCA and hierarchical analysis showed that there were 4 possible

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groups of cocoa beans based on the similarity in polyphenol composition.


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Compared to the rest of the samples, sample no. 1 (FI = 0.467) had significantly

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different characteristics. It was characterized by a very high concentration of different

314

polyphenols (Figure 4, inset). Second group of samples fell across quadrant I, II and IV,

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which was characterized by relatively high contents of flavan-3-ols, PAs, anthocyanins, and

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phenolic acids. This group was consisted of samples 2, 4, 5, 9, 11, and 25. Sample no. 1

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(group 1) had high contents of phenolics, which was similar to the characteristics of pure

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Forastero and Amazon hybrid as reported previously by Elwers et al.24 In that report,

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epicatechin content of Forastero cocoa samples varied between 31 to 43 g/kg (fat free dry

320

basis), whereas that of other varieties ranged between 27 to 37 g/kg (fat free dry basis). In

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this study, the epicatechin content of sample no. 1 was 43.6 g/kg (fat free dry basis). This

322

indicated that this cocoa sample was more likely to fall into under-fermented Forastero type

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rather than other varieties. On the other hand, group 2 consisted of several under-fermented

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and fermented cocoa samples. Analysis of unfermented Sul 1 beans showed that the under-

325

fermented samples in group 2 had typical characteristics of unfermented Trinitario beans.

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Sample 25 was fermented, but had a relatively high epicatechin content (19.1 g epicatechin

327

eq./kg fat free dry basis). Indeed, previous research of Misnawi.34 showed that Trinitario and

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Forastero hybrid cocoa beans had a relatively high content of epicatechin even after

329

fermentation.

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Samples of group 3 were scattered along all the quadrants. They tended to have a

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medium amount of flavan-3-ols and PAs, while having low to medium concentrations of

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anthocyanins and phenolic acids. Group 3 had the dominant distribution of the samples.

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These samples could indicate typical characteristics of partially-fermented to highly-

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fermented Trinitario cocoa beans. The remaining samples were grouped at quadrant II and

335

III, which were characterized by considerably low concentrations of flavan-3-ols, PAs,

336

anthocyanins and phenolic acids. Surprisingly, all the groups except for group 1 were 15 ACS Paragon Plus Environment


Page 16 of 32

337

consisted of not only under-fermented or fermented cocoa samples only, but contained a mix

338

of samples with different FI. These results showed that under-fermented cocoa beans could

339

had similar characteristics and concentrations of polyphenols to that of fermented beans. This

340

result was seen to be contradictive with the common findings that the concentrations of

341

polyphenols in cocoa beans are “linearly” reduced as the degree of fermentation increase.9, 11,

342

24

343

composition among different cocoa beans.

The difference may be explained by the genetic intra-variety diversity in polyphenol

344

Group 4 was dominantly consisted of fermented samples, with the exception of

345

sample 13 (FI = 0.934). Samples from this group generally had low amount of flavan-3-ols,

346

PAs, phenolic acids and anthocyanin. Elwers et al.24 reported that the epicatechin content of

347

Criollo cocoa beans was in the range of 28 to 37 g/kg (fat free dry basis) (unfermented) and

348

reduced into around 2.6 g/kg (fat free dry basis) by fermentation. On the other hand, the

349

concentration of anthocyanins in unfermented Criollo beans was reported to be in range of

350

0−400 mg/kg (fat free dry basis).24 The results obtained from our study were in range of the

351

results from the previous report. 24 The Trinitario cocoa beans used in the present study had

352

characteristics close to Criollo cocoa beans. Smulders et al.35 previously described that cocoa

353

offspring can inherit both of the alleles of the maternal parent and pollen donor. Thus, it is

354

possible that maternal parent (Sul 1) cross-pollinated with pollen donor of Criollo or fine

355

Trinitario varieties. The offspring beans collected in this study had mixed characteristics of

356

Criollo and Trinitario. However, genetic analysis is needed to confirm this hypothesis.

357

In conclusions, great variation in bioactive composition of cocoa beans from a single

358

cocoa clone was obtained. The variation was recorded for the cocoa beans with a similar

359

fermentation index. This variation should be taken into consideration during quality

360

assessment of cocoa beans. Bean samples should be collected representatively from

361

respective batch, employing reliable sampling method. Furthermore, quality analysis using a 16 ACS Paragon Plus Environment

Page 17 of 32


362

small amount of beans should be avoided especially from Trinitario variety due to the

363

variations. This research provides useful insights into farm management using Sul 1 as

364

planting material. Increasing demand of chocolate products with enhanced health benefits

365

may provide opportunity for cocoa industries to improve the nutritional quality of the cocoa

366

bean. The results of this study may provide a basis to develop nutritionally enhanced cocoa

367

products by providing compatible pollen donor for Sul 1. Further research should be focused

368

on the genetic analysis for the quality and also flavor characteristics of cocoa beans derived

369

from Trinitario. The bioactive profiles of non-fermented cocoa beans affected by genetic

370

variations should also be studied.

371 372

ACKNOWLEDGMENTS

373

The authors would like to thank Mrs. Fitratin, technician of the post-harvest laboratory of

374

ICCRI, Indonesia. Noor Ariefandie Febrianto is financially supported by a New Zealand

375

ASEAN Scholarship as part of NZAid programme.

376 377

SUPPORTING INFORMATION

378

The comparison in proanthocyanidin quantification using RP-HPLC and NP-HPLC is

379

presented in Supplementary Table 1. Contents of extractable and non-extractable

380

proanthocyanidins of cocoa bean samples with different fermentation index (FI) are presented

381

in Supplementary Table 2.

382 383

DECLARATION

384

The authors declare no conflict of interest.

385



386

REFERENCES

387

(1)

Page 18 of 32

Kongor, J. E.; Hinneh, M.; de Walle, D. Van; Afoakwa, E. O.; Boeckx, P.;

388

Dewettinck, K. Factors influencing quality variation in cocoa (Theobroma cacao) bean

389

flavour profile - A review. Food Res. Int. 2016, 82, 44–52.

390

(2)

391 392

Gibson, M.. Chocolate/Cacao. In Food Science and the Culinary Arts; Gibson, M., Ed.; Academic Press: London, 2018; pp. 341–352.

(3)

Mcmahon, P.; Purwantara, A. Vascular Streak Dieback (Ceratobasidium theobromae):

393

History and Biology. In Cacao Diseases: A History of Old Enemies and New

394

Encounters; Bailey, B. A., Meinhardt, L. W., Eds.; Springer International Publishing

395

Switzerland: Cham, 2016; Vol. 1, pp. 307–335.

396

(4)

397 398

Aprotosoaie, A.C.; Luca, S.V.; Miron, A. Flavor chemistry of cocoa and cocoa products-An overview. Compr. Rev. Food Sci. Food Saf . 2016, 15, 73-91.

(5)

Sukha, D. A.; Bharath, S. M.; Ali, N. A.; Umaharan, P. An assessment of the quality

399

attributes of the Imperial College Selections (ICS) cacao (Theobroma cacao L.)

400

clones. In Acta Horticulturae: Proceeding of 3rd International Conference on

401

Postharvest and Quality Management of Horticultural Products of Interest for Tropical

402

Regions, Port of Spain, July 2013; Mohammed, M., Francis, J.A., Eds; ISHS: Leuven,

403

2014; Vol. 1047, pp 237–243.

404

(6)

Sukha, D. A.; Butler, D. R.; Umaharan, P.; Boult, E. The use of an optimised

405

organoleptic assessment protocol to describe and quantify different flavour attributes

406

of cocoa liquors made from Ghana and Trinitario beans. Eur. Food Res. Technol.

407

2008, 226, 405–413.

408

(7)

Othman, A. S.; Jumal, S.; Wan Aidah, W. I. The effects of cocoa clones on chocolate


Page 19 of 32


409 410

flavour. Malaysia Cocoa J. 2008, 4, 70–72. (8)

411 412

N’Zi, J. C.; Kahia, J.; Diby, L.; Kouamé, C. Compatibility of ten elite cocoa (Theobroma cacao L.) clones. Horticulturae. 2017, 3, 45.

(9)

Misnawi; Jinap, S.; Jamilah, B.; Nazamid, S. Effects of incubation and polyphenol

413

oxidase enrichment on color, fermentation index, procyanidins and astringency of

414

unfermented and partly fermented cocoa beans. Int. J. Food Sci. Technol. 2003, 285–

415

295.

416

(10) Misnawi; Selamat, J.; Bakar, J.; Saari, N. Oxidation of polyphenols in unfermented

417

and partly fermented cocoa beans by cocoa polyphenol oxidase and tyrosinase. J. Sci.

418

Food Agric. 2002, 82, 559–566.

419

(11)

Wollgast, J.; Anklam, E. Review on polyphenols in Theobroma cacao: Changes in

420

composition during the manufacture of chocolate and methodology for identification

421

and quantification. Food Res. Int. 2000, 33, 423–447.

422

(12)

Camu, N.; Winter, T. D.; Addo, S. K.; Takrama, J. S.; Bernaert, H.; Vuyst, L. D.

423

Fermentation of cocoa beans: Influence of microbial activities and polyphenol

424

concentrations on the flavour of chocolate. J. Sci. Food Agric. 2008, 88, 2288–2297.

425

(13) Romero-Cortes, T.; Salgado-Cervantes, M. A.; García-Alamilla, P.; García-Alvarado,

426

M. A.; del C Rodríguez-Jimenes, G.; Hidalgo-Morales, M.; Robles-Olvera, V.

427

Relationship between fermentation index and other biochemical changes evaluated

428

during the fermentation of Mexican cocoa (Theobroma cacao) beans. J. Sci. Food

429

Agric. 2013, 93, 2596–2604.

430 431

(14)

Patras, M. A.; Milev, B. P.; Vrancken, G.; Kuhnert, N. Identification of novel cocoa flavonoids from raw fermented cocoa beans by HPLC-MSn. Food Res. Int. 2014, 63,



432 433

Page 20 of 32

353–359. (15)

Guyot, S.; Doco, T.; Souquet, J. M.; Moutounet, M.; Drilleau, J. F. Characterization of

434

highly polymerized procyanidins in cider apple (Malus sylvestris var. Kermerrien) skin

435

and pulp. Phytochemistry. 1997, 44, 351–357.

436

(16)

437 438

Hellström, J. K.; Mattila, P. H. HPLC determination of extractable and unextractable proanthocyanidins in plant materials. J. Agric. Food Chem. 2008, 56, 7617–7624.

(17)

Gu, L.; Kelm, M.; Hammerstone, J. F.; Beecher, G.; Cunningham, D.; Vannozzi, S.;

439

Prior, R. L. Fractionation of polymeric procyanidins from lowbush blueberry and

440

quantification of procyanidins in selected foods with an optimized normal-phase

441

HPLC-MS fluorescent detection method. J. Agric. Food Chem. 2002, 50, 4852–4860.

442

(18)

Gao, C.;Cunningham, D. G.; Liu, H.; Khoo, C.; Gu, L. Development of a thiolysis

443

HPLC method for the analysis of procyanidins in cranberry products. J. Agric. Food

444

Chem. 2018, 66, 2159-2167.

445

(19)

Rigaud, J.; Escribano-Bailon, M. T.; Prieur, C.; Souquet, J. M.; Cheynier, V. Normal-

446

phase high-performance liquid chromatographic separation of procyanidins from cacao

447

beans and grape seeds. J. Chromatogr. A 1993, 654, 255–260.

448

(20)

Peterson, E. A.; Dillon, B.; Raheem, I.; Richardson, P.; Richter, D.; Schmidt, R.;

449

Sneddon, H. F. Sustainable chromatography (an oxymoron?). Green Chem. 2014, 16,

450

4060–4075.

451

(21)

Caporaso, N.; Whitworth, M. B.; Fowler, M. S.; Fisk, I. D. Hyperspectral imaging for

452

non-destructive prediction of fermentation index, polyphenol content and antioxidant

453

activity in single cocoa beans. Food Chem. 2018, 258, 343–351.

454

(22)

D’Souza, R. N.; Grimbs, S.; Behrends, B.; Bernaert, H.; Ullrich, M. S.; Kuhnert, N. 20 ACS Paragon Plus Environment

Page 21 of 32


455

Origin-based polyphenolic fingerprinting of theobroma cacao in unfermented and

456

fermented beans. Food Res. Int. 2017, 99, 550–559.

457

(23)

Gunaratne, A.; Wu, K.; Li, D.; Bentota, A.; Corke, H.; Cai, Y. Z. Antioxidant activity

458

and nutritional quality of traditional red-grained rice varieties containing

459

proanthocyanidins. Food Chem. 2013, 138, 1153–1161.

460

(24) Elwers, S.; Zambrano, A.; Rohsius, C.; Lieberei, R. Differences between the content of

461

phenolic compounds in Criollo, Forastero and Trinitario cocoa seed (Theobroma cacao

462

L.). Eur. Food Res. Technol. 2009, 229, 937–948.

463

(25)

Kammerer, D.; Kramer, M.; Carle, R. Phenolic compounds. In Food Analysis by

464

HPLC 3rd Edition; Nollet, L. M. L, Toldra, F., Eds.; CRC Press: Boca Raton, 2012, pp.

465

717-756

466

(26)

Thangasamy, T.; Sittadjody, S.; Burd, R. Quercetin: A potential complementary and

467

alternative cancer theraphy. In Complementary and alternative therapies and the aging

468

population; Watson, R. R., Ed.; Academic Press: London, 2009, pp. 563–584.

469

(27)

Park, J. B. Quantitation of clovamide-type phenylpropenoic acid amides in cells and

470

plasma using high-performance liquid chromatography with a coulometric

471

electrochemical detector. J. Agric. Food Chem. 2005, 53, 8135–8140.

472

(28)

Niemenak, N.; Rohsius, C.; Elwers, S.; Ndoumou, D. O.; Lieberei, R. Comparative

473

study of different cocoa (Theobroma Cacao L.) clones in terms of their phenolics and

474

anthocyanins contents. J. Food Compos. Anal. 2006, 19, 612–619.

475

(29)

Afoakwa, E. O.; Quao, J.; Takrama, F. S.; Budu, A. S.; Saalia, F. K. Changes in total

476

polyphenols, o-diphenols and anthocyanin concentrations during fermentation of pulp

477

pre-conditioned cocoa (Theobroma Cacao) beans. Int. Food Res. J. 2012, 19, 1071–



478 479

Page 22 of 32

1077. (30)

Albertini, B.; Schoubben, A.; Guarnaccia, D.; Pinelli, F.; Della Vecchia, M.; Ricci, M.;

480

Di Renzo, G. C.; Blasi, P. Effect of fermentation and drying on cocoa polyphenols. J.

481

Agric. Food Chem. 2015, 63, 9948–9953.

482

(31)

Pérez-Jiménez, J.; Arranz, S.; Saura-Calixto, F. Proanthocyanidin content in foods is

483

largely underestimated in the literature data: An approach to quantification of the

484

missing proanthocyanidins. Food Res. Int. 2009, 42, 1381–1388.

485

(32)

Kroll, J.; Rawel, H. M.; Rohn, S. Reactions of plant phenolics with food proteins and

486

enzymes under special consideration of covalent bonds. Food Sci. Technol. Res. 2003,

487

9, 205–218.

488

(33)

Di Mattia, C.; Martuscelli, M.; Sacchetti, G.; Scheirlinck, I.; Beheydt, B.; Mastrocola,

489

D.; Pittia, P. Effect of fermentation and drying on procyanidins, antiradical activity and

490

reducing properties of cocoa beans. Food Bioprocess Technol. 2013, 6, 3420–3432.

491

(34)

Misnawi. Influences of Cocoa Polyphenols and Enzyme Reactivation on the Flavor

492

Development of Unfermented and Under-Fermented Cocoa Beans. PhD. Dissertation,

493

Universiti Putra Malaysia, Selangor, 2010.

494

(35)

Smulders, M. J. M.; Esselink, D.; Amores, F.; Ramos, G.; Sukha, D. A; Butler, D. R.;

495

Vosman, B.; Van Loo, E. N. Identification of cocoa (Theobroma cacao L.) varieties

496

with different quality attributes and parentage analysis of their beans. INGENIC Newsl.

497

2009, 12, 1–13.

498 499


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Table 1. Composition of flavan-3-ols of cocoa samples with different fermentation index 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 28 29 30

Fermentation index 0.46 a 0.68 b 0.71 bc 0.77 cd 0.81 de 0.81 de 0.85 ef 0.86 ef 0.90 fg 0.91 fg 0.91 fg 0.93 g 0.93 g 0.94 g 0.94 g 0.94 g 0.95 gh 0.97 gh 0.97 gh 1.02 hi 1.02 hi 1.06 i 1.06 i 1.17 j 1.17 j 1.25 k 1.30 k 1.31 k 1.41 l 1.50 m

Catechin 1.73 m 0.49 k 0.30 fghij 0.28 efghij 0.21 cdefg 0.24 defgh 0.48 k 0.12 bc 0.57 kl 0.36 ij 0.66 l 0.32 ghij 0.18 cde 0.33 ghij 0.28 efghij 0.27 efghij 0.48 k 0.33 ghij 0.36 j (trace) 0.61 l 0.12 bcd 0.27 efghij (trace) 0.30 hij 0.65 l 0.21 cdefg (trace) 0.04 a 0.19 cdef

PA B-type PA B-type Epicatechin dimer (1) trimer (1) 6.75 r 25.3 s 4.32 w 4.59 q 14.9 r 3.04 v 2.91 ijk 6.8 ij 1.98 mn 3.51 lm 12.0 p 2.35 p 3.95 no 11.8 p 2.84 u 2.33 gh 6.2 gh 1.44 hi 2.35 gh 7.0 jk 1.49 i 2.63 hi 6.1 g 2.06 n 4.18 op 13.1 q 2.59 qr 4.14 nop 6.0 g 2.70 t 6.98 r 11.9 p 2.51 q 1.75 def 5.2 f 1.10 f 1.40 cd 3.1 d 0.78 d 2.64 hi 7.2 jk 1.89 lm 2.56 hi 7.1 jk 1.82 kl 2.56 hi 6.3 gh 1.73 jk 3.78 mn 8.1 l 2.04 n 3.30 kl 5.2 f 1.64 j 3.09 jk 9.1 m 1.91 lm 0.02 a 0.3 ab (trace) 3.92 no 8.1 l 1.90 lm 1.33 bc 3.4 d 0.75 cd 1.82 ef 7.1 jk 0.90 e 0.38 a 0.7 b 0.27 b 4.47 pq 11.1 o 2.66 rs 2.78 ij 10.5 n 2.24 o 1.68 cdef 5.9 g 0.93 e 0.20 a 0.5 ab 0.06 a 1.01 b 1.9 c 0.66 c 1.51 cde 3.9 e 0.83 de

PA B-type tetramer 2.39 u 1.87 rs 1.32 mno 1.35 no 1.93 t 0.91 hi 0.95 hi 1.04 ij 1.54 p 1.74 q 1.55 p 0.54 ef 0.46 e 0.96 hi 1.21 klmn 1.16 klm 1.41 o 1.14 jk 1.31 lmno 0.02 ab 1.29 lmno 0.32 d 0.14 bc 0.10 abc 1.79 qr 1.30 lmno 0.67 fg (trace) 0.49 e 0.21 cd

PA B-type trimer (2) 1.78 q 0.78 n 0.61 kl 0.51 ij 1.08 p 0.36 f 0.43 fgh 0.11 bc 0.69 lm 0.91 o 0.73 mn 0.21 de 0.08 ab (trace) 0.60 kl 0.54 j 0.65 lm 0.45 gh 0.63 l (trace) 0.64 l 0.11 b 0.36 f (trace) 1.01 p 0.64 l 0.25 e (trace) 0.17 bcde 0.19 cde

PA B-type trimer (3) 0.41 i 0.07 abcde 0.11 bcdef 0.11 bcdef 0.24 h (trace) 0.01 a 0.07 abcde 0.04 abc 0.11 cdef 0.13 defg (trace) (trace) 0.03 a 0.06 abcde 0.17 fg 0.05 abcd 0.02 a 0.06 abcde (trace) 0.05 abc (trace) (trace) (trace) 0.19 gh 0.13 efg (trace) (trace) (trace) (trace)

PA B-type dimer (2) 2.57 o 1.82 n 1.08 k 1.39 lm 1.70 n 0.88 hi 0.85 fgh 1.03 jk 1.48 m 1.48 m 1.41 lm 0.75 efg 0.38 bcd 0.86 gh 0.87 gh 0.92 hij 1.01 ijk 0.83 fgh 1.07 k (trace) 1.01 ijk 0.35 bc 0.51 d 0.13 a 1.35 l 1.30 l 0.65 e 0.03 a 0.44 cd 0.38 bcd

PA A-type dimer (1) 0.71 i 0.35 h 0.28 gh 0.26 fg 0.10 bcd 0.11 cde 0.06 abcd (trace) 0.07 abcd 0.03 abc 0.15 de 0.19 ef 0.08 abcd 0.30 gh 0.07 abcd 0.04 abc (trace) 0.02 ab 0.01 ab (trace) 0.10 bcd (trace) 0.14 de 0.14 de 0.02 ab (trace) 0.04 abc 0.05 abc (trace) (trace)

PA A-type dimer (2) 0.11 f 0.03 c (trace) 0.06 d 0.08 e (trace) (trace) 0.03 bc 0.01 a 0.09 e (trace) (trace) (trace) 0.01 a (trace) (trace) (trace) (trace) (trace) (trace) 0.01 a (trace) (trace) (trace) 0.05 d 0.08 e (trace) (trace) (trace) (trace)



501 502

31 32 33 34 35

1.52 m 1.56 m 1.68 n 1.74 n 2.17 o

0.23 cdefgh (trace) 0.24 cdefgh 0.19 cdef 0.20 cdef

3.75 mn 0.29 a 3.22 kl 2.32 gh 2.03 fg

2.2 c 0.1 a 7.3 k 6.6 hi 5.1 f

0.66 c (trace) 1.74 jk 1.26 g 1.36 gh

0.53 e (trace) 1.18 kl 0.75 g 0.90 h

0.16 bcd (trace) 0.62 l 0.36 f 0.38 fg

Page 24 of 32

(trace) (trace) 0.12 cdef 0.04 ab (trace)

0.27 b (trace) 0.89 hi 0.72 ef 0.50 d

(trace) (trace) (trace) (trace) (trace)

(trace) (trace) 0.01 a 0.01 a (trace)

Abbreviations: PA, proanthocyanidin; data of flavan-3-ols were obtained by means of RP-HPLC; the unit was g epicatechin eq./kg db, except for catechin (g catechin eq./kg db); values in the same column with different letters were significantly different (p < 0.05)


Page 25 of 32

503


Table 2. Contents of methylxanthines, phenolic acids, flavonols and anthocyanins of cocoa samples with different fermentation index Methylxanthines Fermentation No. index Theobromine Caffeine 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 28 29

0.46 a 0.68 b 0.71 bc 0.77 cd 0.81 de 0.81 de 0.85 ef 0.86 ef 0.90 fg 0.91 fg 0.91 fg 0.93 g 0.93 g 0.94 g 0.94 g 0.94 g 0.95 gh 0.97 gh 0.97 gh 1.02 hi 1.02 hi 1.06 i 1.06 i 1.17 j 1.17 j 1.25 k 1.30 k 1.31 k 1.41 l

24.9 rs 22.6 op 24.3 rs 19.8 ij 24.0 qr 21.1 lmn 22.1 nop 20.5 jkl 17.6 fg 14.5 c 20.9 klmn 21.9 lmno 17.0 ef 21.7 mno 23.9 qr 18.4 gh 19.7 ij 25.3 st 26.1 st 16.4 de 23.9 qr 14.1 c 24.2 qr 15.6 d 20.7 jklm 21.3 lmn 19.8 ijk 21.7 mno 16.4 de

4.02 q 1.06 e 2.94 n 4.04 q 1.19 f 0.56 a 0.96 d 1.62 h 1.91 j 1.76 i 5.45 t 2.11 k 0.62 a 3.96 q 0.84 bc 2.51 l 2.16 k 4.49 s 0.78 b 3.07 o 4.14 r 0.58 a 0.91 cd 0.89 cd 2.71 m 1.37 g 1.70 hi 2.68 m 0.63 a

Phenolic acids Caffeoyl p-coumaryl Caffeoyl 3- Quercetin-Oaspartic acid aspartic acid hydroxytyrosine hexoside (1) 1.33 u 0.27 o 0.13 l 45.5 i 0.29 g 0.07 e 0.14 l 32.5 h 0.46 k 0.08 f 0.27 o 27.9 f 0.61 n 0.10 h 0.16 m 29.1 g 0.29 g 0.05 c 0.12 k 20.7 b 0.34 i 0.09 g 0.10 i 23.2 d 0.48 lm 0.06 d 0.03 b (trace) 0.47 kl 0.14 j 0.08 g (trace) 0.43 j 0.11 i 0.11 j (trace) 0.66 o 0.16 k 0.07 f (trace) 2.53 x 0.67 r 0.07 f 21.4 c 0.60 n 0.11 i 0.09 h 20.1 a 0.35 i 0.08 f 0.03 b (trace) 0.90 r 0.14 j 0.06 e 24.0 e 0.26 f 0.04 b 0.12 k (trace) 0.18 d 0.04 b 0.08 g (trace) 1.66 w 0.20 m 0.07 f (trace) 0.98 s 0.22 n 0.10 i (trace) 0.49 m 0.08 f 0.09 h (trace) 0.11 a 0.06 d (trace) (trace) 0.84 p 0.16 k 0.11 j 21.3 c 0.29 g 0.03 a 0.08 g (trace) 0.34 i 0.05 c 0.12 k (trace) 0.32 h 0.09 g 0.04 c (trace) 0.88 q 0.17 l 0.05 d (trace) 1.24 t 0.34 p 0.11 j (trace) 0.34 i 0.06 d 0.08 g (trace) 0.13 b 0.06 d (trace) (trace) 0.17 d 0.04 b 0.07 f (trace)

Flavonols Quercetin-O- Quercetin-Ohexoside (2) pentoside 79.6 s 138 v 48.3 nopq 84.8 s 47.6 mnop 91.4 t 27.4 abc 85.4 s 45.7 mno 74.7 r 52.4 q 59.8 jkl 40.8 kl 59.9 jkl 43.8 lmn 52.5 hi 35.8 hij 69.7 pq 24.5 a 53.1 hi 59.4 r 128 u 31.0 cdefgh 62.8 lmno 27.7 abcd 35.3 de 49.5 opq 74.0 qr 38.0 ijk 65.5 nop 51.8 pq 62.4 lmn 35.6 ghij 72.1 qr 40.0 jkl 64.0 mno 30.8 cdefg 66.8 nop (trace) 31.1 cd 43.5 lm 67.2 op 25.7 ab 30.3 c 33.7 efghi 58.8 jk 34.0 efghi 47.8 g 51.8 pq 64.3 no 39.5 jkl 49.4 gh 32.4 defgh 69.7 pq 31.0 cdefgh 56.4 ij 29.9 bcdef 33.0 cd

Anthocyanins Cyanidin 3Cyanidin 3galactoside arabinoside 449.6 m 865.3 w 55.3 l 243.4 v 26.4 hi 142.2 t 32.7 k 172.4 u 29.8 jk 78.6 lm 25.9 ghi 91.1 opq 25.1 fghi 94.6 pqr (trace) 98.5 qr 15.3 ab 102.6 r (trace) 97.5 qr 24.5 efghi 118.5 s 22.5 defg 62.1 ij 21.7 def 77.3 klm 21.3 de 64.6 j 25.1 fghi 84.5 lmno 28.1 ij 86.1 mnop (trace) 82.5 lmno 20.7 cd 68.5 jk 21.1 de 81.6 lmn (trace) 13.1 a 24.6 efghi 77.7 lm 17.6 bc 53.8 hi 25.1 fghi 90.7 nopq (trace) 26.7 bc 16.3 b 75.4 kl 12.0 a 33.2 cde 23.0 defgh 47.4 gh 12.4 a 22.8 b (trace) 32.4 cd



504 505 506 507

30 31 32 33 34 35

1.50 m 1.52 m 1.56 m 1.68 n 1.74 n 2.17 o

19.0 hi 21.4 lmn 10.4 a 23.1 pq 21.0 lmn 11.6 b

0.90 cd 1.11 ef 0.95 d 2.44 l 3.17 p 1.33 g

0.21 e 1.35 v 0.16 c 0.48 lm 0.21 e 0.15 c

0.03 a 0.55 q 0.07 e 0.11 i 0.07 e 0.03 a

0.18 n 0.04 c 0.01 a 0.05 d (trace) 0.04 c

(trace) (trace) (trace) (trace) (trace) (trace)

Page 26 of 32

31.9 cdefgh 34.3 fghi (trace) 28.4 abcd 29.4 bcde (trace)

25.4 b 31.9 cd 40.6 f 34.6 cde 37.7 ef (trace)

(trace) (trace) (trace) (trace) (trace) (trace)

39.6 30.0 21.0 52.2 41.9 42.0

efg bc ab h fg fg

Unit: methylxanthines, g/kg db; phenolic acids, g chlorogenic acid eq./kg db; flavonols, mg quercetin eq./kg db; anthocyanins, mg cyanidin 3-glucoside eq./kg db; values in the same column with different letters were significantly different (p < 0.05)

508 509


Page 27 of 32


510

Figure captions

511

Figure 1. RP-HPLC-DAD chromatograms obtained at (A) 280, (B) 320, and (C) 370 nm.

512

Peaks: 1, theobromine; 2, caffeoyl aspartic acid; 3, catechin; 4, caffeine; 5, p-coumaryl

513

aspartic acid; 6, proanthocyanidin B-type dimer (1); 7, epicatechin; 8, proanthocyanidin B-

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type trimer (1); 9, proanthocyanidin B-type tetramer; 10, proanthocyanidin B-type trimer (2);

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11, proanthocyanidin B-type trimer (3); 12, proanthocyanidin B-type dimer (2); 13,

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proanthocyanidin A-type dimer (1); 14, proanthocyanidin A-type dimer (2); 15, caffeoyl 3-

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hydroxytyrosine; 16, quercetin-O-hexoside (1); 17, quercetin-O-hexoside (2); 18, quercetin-

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O-pentoside.

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Figure 2. (A) RP-HPLC-DAD chromatograms at 520 nm; (B) Chromatograms of thiolysed

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media of cocoa residue at 280 nm. Peaks: 1, cyanidin 3-galactoside; 2, cyanidin 3-

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arabinoside; A, theobromine; B, catechin; C, caffeine; D, epicatechin; E, epicatechin

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benzylthioether; F, benzyl mercaptan.

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Figure 3. Box plot of concentrations of bioactive compounds, (A) epicatechin, (B, C, D)

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major proanthocyanidins, (E) total non-extractable proanthocyanidin (NEPA) and (F) its

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degree of polymerization (DP), (G) theobromine and (H) caffeine among samples with

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different fermentation index (FI) (samples were grouped by similar FI).

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Figure 4. (A) Loading plot of first two principal components for flavan-3-ol and its

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derivatives, phenolic acids, flavanones, and anthocyanins from cocoa beans of different FI;

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sample no. 1 is not included in main loading plot due to its extreme value with inset showing



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the position of sample no. 1 compared to the other samples). (B) Hierarchical cluster analysis

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showing the most possible grouping of the samples as represented in PCA loading plot using

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colored markers. (Purple square, group 1; red diamond, group 2; green circle, group 3; blue

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triangle, group 4).


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Figure 1

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Figure 2


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Figure 3 31 ACS Paragon Plus Environment


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Figure 4

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Intra-Variety Diversity of Bioactive Compounds in Trinitario Cocoa

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