Characterization and Degradation of Pectic Polysaccharides in Cocoa

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Characterization and Degradation of Pectic Polysaccharides in Cocoa Pulp Esther Meersman, Nore Struyf, Clare Kyomugasho, Zahra Jamsazzadeh Kermani, Jihan Santanina Santiago, Eline Baert, Sami Hemdane, Gino Vrancken, Kevin J. Verstrepen, Christophe M. Courtin, Marc Hendrickx, and Jan Steensels J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03854 • Publication Date (Web): 16 Oct 2017 Downloaded from http://pubs.acs.org on October 17, 2017

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Journal of Agricultural and Food Chemistry

Characterization and Degradation of Pectic Polysaccharides in Cocoa Pulp Esther Meersmana,b**, Nore Struyfc**, Clare Kyomugashod, Zahra Jamsazzadeh Kermanid, Jihan Santanina Santiagod, Eline Baerta,b, Sami Hemdanec, Gino Vranckene, Kevin J. Verstrepena,b, Christophe M. Courtinc*, Marc Hendrickxd*, Jan Steenselsa,b*

a

Laboratory for Genetics and Genomics, Centre of Microbial and Plant Genetics (CMPG),

KU Leuven, Kasteelpark Arenberg 22, 3001 Leuven, Belgium. b

Lab for Systems Biology, VIB Center for Microbiology, Bio-Incubator, Gaston Geenslaan 1,

3001 Leuven, Belgium. c

Laboratory of Food Chemistry and Biochemistry, Leuven Food Science and Nutrition

Research Centre (LFoRCe), KU Leuven, Kasteelpark Arenberg 22, 3001 Leuven, Belgium. d

Laboratory of Food Technology, Leuven Food Science and Nutrition Research Centre

(LFoRCe), KU Leuven, Kasteelpark Arenberg 22, 3001 Leuven, Belgium. e

Barry Callebaut AG, Westpark, Pfingstweidstrasse 60, 8005 Zurich, Switzerland.

* Co-last authors ** EM and NS contributed equally to this paper.

*

Corresponding author:

Jan Steensels; [email protected]; Ph: +32 16 75 13 90 1

ACS Paragon Plus Environment


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1

Abstract

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Microbial fermentation of the viscous pulp surrounding cocoa beans is a crucial step

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in chocolate production. During this process, the pulp is degraded, after which the

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beans are dried and shipped to factories for further processing. Despite its central

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role in chocolate production, pulp degradation, which is assumed to be a result of

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pectin breakdown, has not been thoroughly investigated. Therefore, this study

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provides a comprehensive physicochemical analysis of cocoa pulp, focusing on

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pectic polysaccharides, and the factors influencing its degradation. Detailed analysis

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reveals that pectin in cocoa pulp largely consist of weakly bound substances, and

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that both temperature and enzyme activity play a role in its degradation. Furthermore,

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this study shows that pulp degradation by an indigenous yeast fully relies on the

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presence of a single gene (PGU1), encoding for an endo-polygalacturonase. Apart

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from their basic scientific value, these new insights could propel the selection of

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microbial starter cultures for more efficient pulp degradation.

15 16 17 18

Keywords

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Chocolate; pectin; Saccharomyces cerevisiae; Theobroma cacao; fermentation;

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PGU1

2


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Introduction

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The mucilaginous cocoa pulp that surrounds cocoa beans is degraded during a

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fermentation process that occurs spontaneously after farmers collect the beans in

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large heaps, trays or boxes.1 This spontaneous fermentation process is a complex

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interplay between various microbial species, and is crucial because it prohibits the

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growth of the plant embryo inside the beans. Moreover, fermentation also directly

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contributes to the flavor of the resulting chocolate, making it a key determinant of

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chocolate quality.2 During the spontaneous fermentation process, the cocoa pulp

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gets degraded, and the resulting cocoa juice, also known as ‘sweatings’, drains off.

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Pulp degradation serves several purposes. First, it allows air to penetrate the

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fermenting mass, which is pivotal for the typical microbial succession during the

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fermentation process. Second, the absence of pulp sticking to the cocoa bean shells

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facilitates downstream processing of the beans, making the production process of

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chocolate more efficient.

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Cocoa pulp consists of 83–86% water, 11-13% sugars (glucose, fructose and

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sucrose; concentration is a function of pod age and cultivar), 0.5%–1.2% pectin, 0.2–

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3% hemicelluloses, 0.7-0.9% cellulose, 0.1-0.3% lignin and 0.3-1.3% citric acid,

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resulting in a low pH of 3.3-3.9, but this can vary between cocoa cultivars and country

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of origin.1,3,4 Proteins, other organic acids, amino acids and minerals are also

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present, albeit in lower quantities.3 The consistency of cocoa pulp largely depends on

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the concentration and characteristics of the cell wall polysaccharides present.

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A wide variety of microbes originating from the environment ferment the cocoa pulp,

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with lactic acid bacteria (LAB), acetic acid bacteria (AAB) and yeasts as the key

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microbial groups.1,5 However, it is not known which microorganisms and enzymatic 3



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activities account for the pulp degradation, but most literature alludes to the activity of

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yeasts. More specifically, yeasts producing pectinolytic enzymes are often suggested

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to play a central role,4 but it is unclear whether this hypothesis is supported by

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sufficient

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filamentous fungi and Bacillus spp. isolated from spontaneous cocoa pulp

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fermentations, were shown to exhibit pectinolytic activities, thereby also potentially

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contributing to pulp degradation.6,7

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Despite that pectin degradation is regarded as the most crucial factor in pulp

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degradation,4,8 little is known about the basic characteristics and composition of the

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pectin fraction of cocoa pulp, and the mechanisms underlying its degradation during

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fermentation. Pectin is a complex family of polysaccharides rich in α-1,4-D-

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galacturonic acid (GalA) residues. The three main structural classes of this polymer

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are

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rhamnogalacturonan-II (RG-II), of which HG and RG-II have a galacturonan

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backbone, while the RG-I backbone consists of the repeating disaccharide [→4)-α-D-

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GalA-(1→2)-α-L-Rha-(1→] units.9 The rhamnose residues in RG-I can be linked to

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galactosyl and/or arabinosyl residues, substituted as single glycosyl units or linear or

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branched polymers. The backbone of RG-II is substituted with clusters of four

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different hetero-oligomeric side chains. GalA residues in HG or RG-II may be methyl-

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esterified at C6, whereas GalA in RG-I is presumably not methyl-esterified. Pectin

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polysaccharides are known to be covalently linked, but their exact relative position is

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not yet revealed.9

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To provide insight into pulp degradation, this study provides a detailed

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characterization of the composition and the factors influencing breakdown of the cell

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wall polysaccharides of cocoa pulp, with emphasis on pectin. Apart from providing

analytical

evidence.

homogalacturonan

Additionally,

(HG),

other

microorganisms,

rhamnogalacturonan-I

such

(RG-I),

as

and

4


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fundamental insights regarding the cocoa pulp fermentation process, the results can

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also aid in selecting or developing new microbial starter cultures for efficient pulp

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degradation or cocoa pulp juice production.

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Materials and methods Chemicals

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All chemicals, solvents, and reagents were purchased from Sigma-Aldrich (Bornem,

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Belgium) and were analytical grade, unless specified otherwise.

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Cocoa pulp

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Cocoa pulp used in this study originated from Ivory Coast, where it was obtained

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through depulping cocoa beans immediately after opening of the fresh cocoa pods

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during mid-crop (early July). Care was taken to only include fresh, undamaged pods.

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All the pulp used in this study was collected as a single batch, distributed in vessels

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of ~1 L and frozen at -20 °C. The time in between opening of the pods and freezing

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was minimized ( 50% is generally regarded as

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high DM pectin.23 In addition, the molar mass profile (slanting line) and concentration

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profile (curve) hint towards two pectin populations, a low concentration of high

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molecular weight polymers (elution time ~ 36 min) and a high concentration of lower

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molecular weight polymers with peak maxima at ~ 50 min (see Supplemental

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Information). The protein content of WEP was shown to be 12.2%.

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Lastly, also the neutral sugar content of the HF was determined. This revealed that

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Xyl and Glu are the most abundant sugars (but in low concentrations), suggesting the

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presence of xylans and/or xyloglucan. Furthermore, Ara, Gal and Man indicate the

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presence of arabinoxylan, (gluco)mannans and/or galacto(gluco)mannans in low

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concentrations. The high amount of Glu in the AIR compared to the HF fraction likely

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points to the presence of cellulose in the residue.

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Effect of pasteurization on viscosity, water content and composition of pectic cell wall polysaccharides

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In order to test the effect of various parameters on pulp degradation, the cocoa pulp

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needs to be pasteurized to inactivate contaminating microorganisms and prevent

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endogenous enzymatic activity. However, it is still unknown what effect this

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procedure has on the pulp. Therefore, the effect of the pasteurization process on

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viscosity, water content and composition of pectic cell wall polysaccharides was

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evaluated.

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Pasteurization was shown to result in a (non-significant) decrease in viscosity of 14%

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(p=0.0648, Student’s t-test). The water content also reduced slightly, from 80.9% to

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80.0% (p=0.0117, Student’s t-test). Next, the effect of pasteurization on pectic

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polysaccharides was evaluated. First, these experiments show that pasteurisation

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had no large effect on the weighted fractionation yield, as the AIR yield for

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pasteurized pulp was 0.1513 g per g pulp. Additionally, the percentage yield of the

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different fractions was similar (Table 2). On the other hand, examining the GalA

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revealed a lower amount of GalA relative to AIR of pasteurized pulp compared to AIR 16


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of unpasteurized pulp (Table 2) and a significant (p0.05, Student’s t-test) (Figure 2A). Next, the effect of temperature on the viscosity

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was analyzed by gradually increasing the temperature from 25 °C to 55 °C at a

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constant rate of 1 °C min-1 (Figure 2B). This 30 °C temperature increase corresponds

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to a typical temperature profile during spontaneous fermentation processes, and

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results in a viscosity decrease of 23%, while the control experiment (30 minutes at 25

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°C using the same stirring conditions), did not yield any difference in viscosity.

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Endo-polygalacturonase significantly decreases viscosity of pasteurized cocoa pulp

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To further investigate the role of pectin degradation in pulp viscosity, we tested the

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effect of incubation of the pasteurized pulp in presence of endopolygalacturonase

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(EPG) (Figure 3). This enzyme was selected based on the high GalA content present

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in the pulp, coupled with its ability to modify the linear pectin backbone. Moreover,

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modification of pectin by EPG is reported to be more likely to contribute significantly

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to pectin depolymerisation, potentially resulting in viscosity loss.25,26 In addition, we

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included several hemicellulose-degrading enzymes in the assay, to assess whether

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these could potentially play a role in pulp degradation as well. Only addition of EPG

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shows a significant effect on the viscosity of cocoa pulp, decreasing it by 18.8%

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(1000 U EPG: p=0.0137, Student’s t-test). No significant differences in viscosity are

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observed when AFA, BX or XYL are added (100 U AFA: p=0.1649, 1000 U BX:

404

p=0.1751 and 1000 U XYL: p=0.3590, Student’s t-test).

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To validate the significant decrease in viscosity after addition of 1000 U EPG, the

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molar mass distribution of the WEP fraction polymers was analyzed. EPG further

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clearly degrades high and lower-molecular mass pectin polymers in the WEP fraction

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(see supporting information). This is evidenced by lower concentrations of high

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molecular weight polymers (curve with maxima at 36 min) as well as the shift of the

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concentration curve of lower molecular weight polymers to longer elution times

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(indicating a decrease in molecular weight), in pulp with 1000 U of EPG. Although no

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significant reduction in pulp viscosity was observed in pulp treated with AFA, BX and

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XYL, the effect of these enzymes on the pulp composition was assessed by

414

comparing reducing end sugar contents in samples where the highest enzyme

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concentration was added to the control samples. The addition of 1000 U XYL and

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1000 U BX did not change reducing xylose concentrations (p=0.9091 and p=0.4684,

417

respectively, Student’s t-test), but a 6.6 fold increase in reducing arabinose is

418

observed when adding 100 U AFA (p4)-beta-endo-xylanase treatment on wheat bran. J. Cereal Sci. 2002, 36 (2), 253-260.

620 621 622

32. Debyser, W.; Peumans, W.; Van Damme, E.; Delcour, J. Triticum aestivum xylanase inhibitor (TAXI), a new class of enzyme inhibitor affecting breadmaking performance. J. Cereal Sci. 1999, 30 (1), 39-43.

623 624 625

33. Fierens, E. TLXI, a thaumatin-like xylanase inhibitor: isolation, characterisation and comparison with other wheat (Triticum aestivum L.) xylanase inhibiting proteins. PhD thesis. KU Leuven, Leuven, 2007.

626 627

34. Madhani, H. D.; Fink, G. R. The control of filamentous differentiation and virulence in fungi. Trends Cell Biol. 1998, 8 (9), 348-353.

628 629 630

35. Sanchez, J.; Guiraud, J.; Galzy, P. A study of the polygalacturonase activity of several yeast strains isolated from cocoa. Appl. Microbiol. Biotechnol. 1984, 20 (4), 262-267.

631 632 633

36. Schwan, R. F.; Cooper, R. M.; Wheals, A. E. Endopolygalacturonase secretion by Kluyveromyces marxianus and other cocoa pulp-degrading yeasts. Enzyme Microb. Technol. 1997, 21 (4), 234-244.

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634 635 636

37. Buamah, R.; Dzogbefia, V.; Oldham, J. Pure yeast culture fermentation of cocoa (Theobroma cacao L): effect on yield of sweatings and cocoa bean quality. World J. Microbiol. Biotechnol. 1997, 13 (4), 457-462.

637 638 639

38. Sanchez, J.; Daguenet, G.; Guiraud, J.; Vincent, J.; Galzy, P. A study of the yeast flora and the effect of pure culture seeding during the fermentation process of cocoa beans. Lebensmittel-Wissenschaft+ Technologie 1985, 18 (2), 69-75.

640

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Figure legends

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Figure 1. Average weight-based fractionation yields of WEP, CEP, NEP and HF of

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AIR from pasteurized and unpasteurized cocoa pulp.

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Figure 2. Influence of pH and temperature on viscosity of cocoa pulp. (a) Viscosity of

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pasteurized cocoa pulp at different pH-values covering the pH range observed in a

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spontaneous cocoa pulp fermentation process. Values are represented as the

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average of two replicates ± SD. (b) Viscosity of pasteurized cocoa pulp at increasing

648

temperatures. Values were corrected for stirring at high speed, and are represented

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as the average of three biological replicates ± SD.

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Figure 3. Influence of enzymatic activity on the viscosity of cocoa pulp. Data points

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are normalized to a non-enzyme treated sample at the same temperature (= value of

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“1”). Values are represented as the average of two biological replicates ± SD. *: p ≤

653

0.05. AFA = L-arabinofuranosidase, EPG = endo-polygalacturonase, XYL = endo-

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1,4-β-xylanase, BX = β-D-xylosidase.

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Figure 4. Influence of endo-polygalacturonase production by yeast on viscosity of

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cocoa pulp. (a) Viscosity of pasteurized cocoa pulp samples fermented with strains

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with different EPG activities. Values are represented as average of two biological

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replicates ± SD. (b) The effect of yeast EPG on the size exclusion elution profile of

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water-extractable pectin in cocoa pulp.

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Tables Table 1. Overview of Saccharomyces cerevisiae strains used in this study. Code L2323 Y115 Y771 Y927 Y927-1 Y927-1∆pgu1

Genotype

Origin

Wild-type Wild-type Wild-type Wild-type Haploid strain, HO replaced by G418 resistance marker Haploid strain, HO replaced by G418 resistance marker, PGU1 replaced by Hygromycin B resistance marker

22

Bioethanol industry (China) 24 Spontaneous cocoa pulp fermentation (Malaysia) Spontaneous cocoa pulp fermentation (Malaysia)18 This study This study

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Table 2. Sugar composition of the AIR in cocoa pulp, and derived pectin and hemicellulose fractions.

1

Concentration (mg g- AIR) Fuc

Rha

Ara

Gal

Glu

Xyl

Man

Unpasteurized

AIR WEP CEP NEP HF

1.99 ± 0.08 0.44 ± 0.01 0.03 ± 0.00 0.02 ± 0.00 0.08 ± 0.01

12.83 ± 0.61 8.95 ± 0.71 0.50 ± 0.01 0.15 ± 0.04 0.03 ± 0.00

20.13 ± 0.95 10.16 ± 0.42 0.59 ± 0.01 0.21 ± 0.02 0.37 ± 0.02

23.29 ± 1.36 20.17 ± 1.56 0.74 ± 0.00 0.25 ± 0.01 0.29 ± 0.02

72.48 ± 8.70 3.51 ± 0.18 0.57 ± 0.01 1.01 ± 0.48 1.21 ± 0.11

12.04 ± 0.53 3.03 ± 0.19 0.33 ± 0.00 0.47 ± 0.05 0.93 ± 0.12

6.36 ± 0.40 2.13 ± 0.06 0.17 ± 0.00 0.18 ± 0.01 0.33 ± 0.05

Pasteurized

AIR WEP CEP NEP HF

1.72 ± 0.1 0.29 ± 0.01 0.01 ± 0.00 0.01 ± 0.00 0.22 ± 0.00

9.9 ± 1.34 6.48 ± 0.26 0.33 ± 0.01 0.26 ± 0.00 0.04 ± 0.00

18.1 ± 0.31 6.45 ± 0.10 0.45 ± 0.00 0.27 ± 0.00 0.94 ± 0.02

20.09 ± 0.15 12.63 ± 0.38 0.45 ± 0.01 0.32 ± 0.01 0.60 ± 0.01

104.89 ± 1.50 2.11 ± 0.10 0.42 ± 0.01 0.36 ± 0.01 1.90 ± 0.01

9.73 ± 2.1 1.85 ± 0.45 0.19 ± 0.01 0.41 ± 0.01 2.14 ± 0.01

4.33 ± 2.02 1.00 ± 0.47 0.11 ± 0.01 0.05 ± 0.00 0.43 ± 0.00

Total pectin-related neutral sugars 70.28 ±1.85 42.74 ±1.77 2.19 ± 0.01 3.41 ± 1.58

59.54 ± 2.52 27.7 ± 0.65 1.43 ± 0.02 1.28 ± 0.02

GalA 265.77 ± 9.34 197.36 ± 11.63 22.64 ± 1.05 6.53 ± 1.17 ND 222.33 ± 1.69 182.32 ± 16.09 8.68 ± 0.87 6.65 ± 1.67 ND

Values are represented as the average of duplcate extractions ± SD. AIR: alcohol-insoluble residue, WEP: water-extractable pectin, CEP: chelator-extractable pectin, NEP: sodium carbonate-extractable pectin and HF: hemicellulose fraction. Fuc: fucose, Rha: rhamnose, Ara: arabinose, Gal: galactose, Glu: glucose, Xyl: xylose, Man: mannose and GalA: galacturonic acid. ND = not determined

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Table 3. Sugar ratios of WEP and CEP fractions in pasteurized and unpasteurized cocoa pulp.

Pectin fraction WEP

CEP

Sugar ratio 1 2 3 1 2 3

Formula GalA/(Fuc+Rha+Ara+Gal+Xyl) Rha/GalA (Ara+Gal)/Rha GalA/(Fuc+Rha+Ara+Gal+Xyl) Rha/GalA (Ara+Gal)/Rha

Unpasteurized 3.97 ± 0.21 0.05 ± 0.01 3.29 ± 0.04 8.64 ± 0.45 0.03 ± 0.00

Pasteurized 5.62 ± 0.32 0.04 ± 0.01 2.85 ± 0.08 5.05 ± 0.57 0.05 ± 0.00

2.64 ± 0.04

2.74 ± 0.08

Values represent averages of duplicate extractions ± SD. Sugar ratio 1 = pectin linearity, ratio 2: contribution of RG to the pectin population and ratio 3: degree of RG-I branching. WEP: water-extractable pectin and CEP: chelator-extractable pectin.

32


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Figures Figure 1 100 Fractionation yield (%)

Residue

80 HF NEP

60 CEP

40 20

WEP

0 Pasteurized

Unpasteurized

33



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Figure 2

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Figure 3

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Figure 4

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TOC graphic

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Characterization and Degradation of Pectic Polysaccharides in Cocoa

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