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Cocoa (Theobroma cacao L.) pod husk: Renewable source of bioactive compounds
Accepted Manuscript Cocoa (Theobroma cacao L.) pod husk: renewable source of bioactive compounds Rocio Campos-Vega, Karen H. Nieto-Figueroa, B. Dave Oomah
PII:
S0924-2244(17)30751-3
DOI:
10.1016/j.tifs.2018.09.022
Reference:
TIFS 2329
To appear in:
Trends in Food Science & Technology
Received Date: 27 November 2017 Revised Date:
20 September 2018
Accepted Date: 21 September 2018
Please cite this article as: Campos-Vega, R., Nieto-Figueroa, K.H., Oomah, B.D., Cocoa (Theobroma cacao L.) pod husk: renewable source of bioactive compounds, Trends in Food Science & Technology (2018), doi: https://doi.org/10.1016/j.tifs.2018.09.022. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Abstract Background
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Cocoa Pod Husk (CPH) is the main by-product from the coca industry constituting 67-76 % of the cocoa fruit weight. This waste represents an important, and challenging, economic, environmental renewable opportunity, since ten tons of wet CPH are generated for each ton of dry cocoa beans.
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Scope and Approach
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This review highlights the value that can be added to this industrial co-product to generate new pharmaceutical, medical, nutraceuticals or functional food products. Key Findings and Conclusions
The quality and functionality of cocoa pod husk (CPH) has being improving through processing (fermentation, enzymatic hydrolysis, and combustion, among others), guiding to
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their use as source of volatile fragrance compounds, lipase extraction, skin whitening, skin hydration and sun screening, ruminants’ food, vegetable gum, organic potash, antibacterial and nanoparticles synthesis with antioxidant and larvicidal activities. However, their
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exploration to produce high-value-added products, specially for the food industry, is limited
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as well as their potential health benefits. Cocoa pod husk, the main by-product from cacao industry (up to 76%), is an abundant, inexpensive, and renewable source of bioactive compounds like dietary fiber, pectin, antioxidant compounds, minerals and theobromine, justifying their valorization. This review highlights the value addition that can be achieved with this valuable industrial co-product to generate new pharmaceutical, medical, nutraceuticals or functional food products. Keywords: Cocoa by-product; Antioxidants; Dietary fiber; Pectin.
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Cocoa (Theobroma cacao L.) pod husk: renewable source of bioactive compounds
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Rocio Campos-Vegaa*, Karen H. Nieto-Figueroaa and B. Dave Oomahb
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Programa de Posgrado en Alimentos del Centro de la República (PROPAC), Research and Graduate Studies in Food Science, School of Chemistry, Universidad Autónoma de Querétaro, 76010 Santiago de Querétaro, Qro, Mexico. b
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Rocio Campos-Vega: [email protected] B. Dave Oomah: [email protected] Karen H. Nieto-Figueroa: [email protected] * Corresponding author: Tel.: (55) 1921200 Ext 5590.
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Retired, formerly with the National Bioproducts and Bioprocesses Program, Pacific Agri-Food Research Centre, Agriculture and Agri-Food Canada, Summerland, BC V0H 1Z0, Canada.
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Abbreviations
2
50W
Pectin obtained by aqueous extraction in a water bath at 50 °C for 90 min
3
5-ASA
5-aminosalicylic acid
4
ABTS
2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid)
5
ADF
Acid detergent fiber
6
BHT
Butylated hydroxytoluene
7
CHPFR
Cocoa hull product from fermented and roasted beans
8
CKPFR
Cocoa kernel product from fermented and roasted beans
9
CLEA
Cross-linked enzyme aggregate
10
CPE
Cocoa pod extract
11
CPH
Cocoa pod husk
12
CPHE-AgNPs
Cocoa pod husk extract synthesized with silver nanoparticles
13
DA
Degree of acetylation
14
DE
Degree of esterification
15
DPPH
2,2-diphenyl-1-picrylhydrazyl
16
EE
Epicatechin equivalent
17
FRAP
18
GAE
19
HM
20
IC
21
IL
22
LDL
Low-density lipoprotein
23
LM
Low methoxy pectin
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Ferric reducing antioxidant power
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Gallic acid equivalent
High methoxy pectin Inhibitory concentration
Interleukin
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MCC
Microcrystalline cellulose
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MIC
Minimum inhibitory activity
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NA-HYP
Nitric acid extracted (pH 3.5, 100 °C, 30 min) high performance pectin
27
NDF
Neutral detergent fiber
28
NO
Nitric oxide
29
NSP
Non-starch polysaccharides
30
OP
Optimized pectin extracted with nitric acid (pH 1.5, 100 °C, 30 min)
31
ORAC
Oxygen radical absorbance capacity
32
Pas
Proanthocyanidins
33
PDMS
Polydimetylsiloxane
34
RE
Rutin equivalent
35
ROS
Reactive oxygen species
36
SPME
Solid-phase microextraction
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TcANR
Anthocyanidin reductase
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TcANS
Anthocyanidin synthase
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TcLAR
Leucoanthocyanidin reductase
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TDF
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TE
42
TEAC
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TNF-α
Tumor necrosis factor-α
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TSH 565
Trinidad selection hybrid 565
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WAP
Weeks after pollination
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Total dietary fiber
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Trolox equivalent
Trolox equivalent antioxidant capacity
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1. Introduction
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Theobroma cacao L. is claimed to be the only commercially cultivated and most prominent in
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the market among the 22 species of the Theobroma genus (World Agriculture, 2011). The
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Theobroma cacao tree probably originated from divergent areas in Central and South America;
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the Upper Amazon region (10,000 – 15,000 years ago), the upper Orinoco region of north east
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Colombia and North West Venezuela, the Andean foothills of North West Colombia, Central
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America from southern Mexico (Chiapas-Usumacinta) to Guatemala (Young, 1994). However,
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Central and South American countries account for only about 14% of the current (2016, latest
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available data) world cocoa production compared to those from African countries (⅔ of world
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production) (FAO, 2018). Three countries, Cote D’Ivoire, Ghana and Indonesia together
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cultivate and produce 61 and 67%, respectively of globally traded cocoa, whereas the top ten
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countries account for ~93% of total world cocoa production (Table 1). Global cocoa production
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is estimated at 4.59 million tonnes for 2017/2018 (ICCO, 2018). In 2016, the annual production
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of cocoa, in decreasing order, by the eight largest cocoa producing countries were Côte D’Ivoire,
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Ghana, Indonesia, Nigeria, Ecuador, Cameroon, Brazil and Malaysia. These countries together
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produced about 4.23 million tonnes, representing ~ 95% of the world production (ICCO, 2015).
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The cocoa bean constitutes one third (33%) of the fruit weight, leaving behind 67% of the fruit as
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CPH as a waste by-product (Oddoye, Agyente-Badu & Gyedu-Akoto, 2013) (Fig.1). In other
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words, ten tons of wet CPH are generated for each ton of dry cocoa beans, thereby representing a
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serious disposal problem and an underexploited resource (Vriesmann, Amboni & Petkowicz,
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2011). Pods are fully developed (100-350 mm long, 0.2-1 kg wet weight) from pollinated
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flowers after 5-6 months. Three of the major cocoa diseases ((black pod, pod rot and cocoa pod
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borer), also known as the disease trilogy (Evans, 2007) affect the pod specifically resulting in
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significant crop loss (Fowler & Coutel, 2017). Several initiatives have therefore been undertaken
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to counter the severe crop loss, for example, development of varieties with thicker cuticle that
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are more resistant to the common black pod rot and/or other pathogens (Fowler & Coutel, 2017).
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Pod size, type and index (> 60 pods/tree, low disease incidence) are discriminating
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morphological characteristics among cocoa genotypes and therefore variation on pod traits
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(mainly pod size; length, width, thickness) may be associated with different morpho-geographic
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groups (Ballesteros, Logos & Ferney, 2015).
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The pod has been described as a natural laminated material consisting of three distinctly different
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layers: epicarp, mesocarp and endocarp (outer, middle and inner pericarp, respectively) (Fig.1).
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The endocarp is a soft whitish tissue protecting the delicate cocoa beans in a well-lubricated
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inner chamber; the mesocarp displays a hard-composite structure able to hold the cocoa beans in
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place even under high impact; and the outermost relatively soft layer is the yellow cover (when
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ripe) that is directly exposed to sunshine, after which it turns black indicating rot due to
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degradation (Babatope, 2005). These three distinct layers have been analyzed for their chemical
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composition and compared to the whole CPH when incorporated as feed component in broiler
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chick diets (Sobamiwa & Longe, 1994). High proportion of ash (47% CPH), hemicellulose
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(50%) and minerals (K, Ca and P) (41-66%) predominated in the epicarp; fiber (crude, NDF and
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ADF-44-48%) and cellulose (53 %) in the mesocarp; protein (50%), crude fat (50%) and pectin
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(59%) in the endocarp (Table 2). The epicarp was the most limiting portion of CPH in the
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feeding trial, presumably due to the antagonistic inhibitory effect of lignin and pectin on CPH
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utilization in broiler diets (Sobamiwa & Longe, 1994).
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Cocoa pod vary in color (from green [Forastero] to red [Criollo] or variable [Trinitario, the
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Forastero x Criollo hybrid]) and thickness when ripe depending on their clone. Pod color is a
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reflection of the exocarp (the outer 1-3 mm layer fruit tissue of pods harvested 18 weeks after
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pollination [18WAP]). This exocarp accumulates high levels of soluble and insoluble
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proanthocyanidins [PAs] (170 and 8 mg/g dw, respectively) compared with flowers, leaves and
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seeds due to highly expressed PAs synthesis genes [anthocyanidin synthase (TcANS),
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anthocyanidin reductase (TcANR) and leucoanthocyanidin reductase (TcLAR)]. Furthermore,
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epicatechin and catechin contents in the exocarp (18WAP) were: ~30 and 0.5 mg/g dry weight
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(dw), respectively (Liu et al., 2013); such information is unavailable for the pericarp.
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100 2. Composition
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The CPH constitutes 67-76% of the whole fruit by weight. It has been extensively investigated as
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poultry and/or livestock feed because of its protein (5.9-9.1%), fiber (22.6-35.7%), crude fat
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(1.2-10%) and mineral contents, among others (Oddoye, Agyente-Badu & Gyedu-Akpto, 2013).
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Fresh manually chopped CPH (~ 1 cm thick) had the following percent composition: organic
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matter 87, which includes crude protein, fiber, fat and nitrogen free extract (8.4, 55.7, 2.5 and
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20.6%, respectively). The fiber fraction consisted of neutral and acid detergent fibers (80.7 and
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74.6%), hemicellulose (6.0), cellulose (35.3) and lignin (38.8) with up to 40% in vitro
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digestibility (dry and organic matter) (Laconi & Jayanegara, 2015). However, total carbohydrate,
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total dietary fiber and lignin contents of CPH vary widely (Table 3). Moreover, chemical
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composition depends upon the pretreatment used to process CPH, described in the next sections.
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2.1. Drying methods
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The moisture content of fresh CPH is about 90% (Vriesmann, Amboni & Petkowicz, 2011),
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therefore quick drying is essential to prevent deterioration; this is achieved by slicing the fresh
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pods, drying to ~65% moisture, grinding into pellets and drying the pellets to 10% moisture prior
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to storage (Oddoye, Agyente-Badu & Gyedu-Akpto, 2013). However, the type and conditions of
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drying can affect CPH composition. For example, dry milled (< 1 mm) CPH has low lipids
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(1.5% db), high ash (6.7%), protein (8.6%), total dietary fiber (36.6%) composed primarily of
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insoluble fiber (74%) and soluble phenolics (4.6%) with carbohydrates and Klason lignin
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accounting for 32.3% and 21% of CPH, respectively (Vriesmann, Amboni & Petkowicz, 2011).
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CPH dried in a circulated air oven (80 °C, 1 day) and ground to a fine powder (22 µm) contained
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cellulose (26.4%), lignin (24.2%), hemicellulose (8.7%), carbohydrate (17.5%), ash (9%), crude
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protein (2.1%), fat (1.5%) and moisture (10.5%) (Chun, Husseinsyah & Yeng, 2016). Oven dried
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(40-45 °C) milled (< 1.68 mm) CPH contained ~ 60% total dietary fiber, predominantly non-
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starch polysaccharides (NSP) (42%), soluble sugars (13%), proteins (N x 6.25 = 9%), ash (8%),
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fat (2%), phenolics (7% dw) and minerals (0.5-6%, mainly K, P, Ca and Mg); the reducing
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sugars consisted mainly of fructose with some glucose (~ 64% of total soluble sugar). The ratio
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of insoluble to soluble dietary fiber (4.2) in addition to high water retention and swelling
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capacities (6.5 g/g and 7.3 mL/g) suggests that CPH may be suitable for improving bowel transit
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time (Yapo et al., 2013). In contrast, air dried, ground (0.40-0.45 mm) CPH had high cellulose
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(35.4% db), hemicellulose (37%), ash (12.3%), moisture (14%) and low lignin (14.7%) contents.
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The high (74%) hollocellulose [cellulose + hemicellulose] of air dried CPH indicates its
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potentially excellent quality performance for the pulp and paper industry (Daud et al., 2013).
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Hemicellullose and lignin contents increased (27 and 14%, respectively), whereas cellulose
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content decreased (10.7%) when boiled CPH was compared with directly dehydrated (48 °C, 48
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h) and milled CPH (< 1 mm). For boiling, pods were washed, chopped into ~2.5 cm pieces,
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completely covered with water and heated (98 °C, 10 min), drained and dehydrated (48 °C, 72 h)
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(Pérez et al., 2015).
140 2.2. Animal feed quality
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Several studies focused on improving CPH feed quality by reducing its high fiber and increasing
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protein contents. In this context, fresh anaerobically treated CPH (7 days, 3% molasses with
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Phanerochaete chrysoporium inoculum that produces lignin degrading enzymes) significantly
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increased protein content (19%, p < 0.05) concomitantly reducing crude fiber and lignin contents
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(18%, p < 0.01). Ammoniated cocoa pod with 1.5% urea increased hemicellulose content the
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most (2.2x) and reduced crude fiber content (8%), presumably by reducing its physical strength,
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disrupting the silicified cuticular barrier and cleavages of some lignin-carbohydrate bonds. Cow
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rumen exposure (3% w/w, anaerobic conditions for 7 days) reduced crude fiber, crude fat and
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lignin contents (27.5, 92 and 12.6%, respectively) of fresh CPH (Laconi & Jayanegara, 2015).
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Ground (< 250 µm) CPH treated with 0.1 M NaOH (solid to liquid ratio 50 g/L, room
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temperature, 1 h) incurred 26.6% weight loss, primarily due to lignin removal and changed its
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structure from compact and smooth to more porous and rough surface increasing its adsorption
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capacity (Pua et al., 2013). Such alkalization generally encourages dairy feed intake, increases
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buffering capacity and reduces the initial acid load of the diet thereby enabling more feed in the
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ration without triggering acidosis. CPH crude protein and total soluble carbohydrates increased
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(36%) with concomitant reduction in crude fiber, hemicellulose, cellulose, lignin and total
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tannins (17, 21, 26, 17 and 88%, respectively) on solid-state fermentation (Pleurotus ostreatus
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with 0.075% MnCl2 [w/w], 5 weeks) (Alemawor et al., 2009a). Solid state fermentation of CPH
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with Rhizopus stolonifera LAU 07 increased protein (95%) and reduced fiber (7.2%) contents
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(Lateef et al., 2008). Ten days’ fermentation with Phanerochaete chrysosporium significantly (p
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< 0.05) increased crude protein of CPH compared to unfermented CPH enabling the highest
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lignin degradation (39%) and efficient CPH bioconversion (Suparjo & Nelson, 2017). Other
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fermentation such as Rhizopus oryzae (28 °C, 6 days) of CPH and rice bran (1:9 ratio mixed
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substrate, sterilized at 121 °C, 15 min) also increased CPH protein content (14-15%), and free
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amino acids (glutamic acid 34x, aspartic acid 3.7x, alanine 4.6x and valine 1.2x) (Sriherwanto et
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al., 2016). Furthermore, enzyme cocktails (Pentopan MonoBG + Viscozyme L; Viscozyme L +
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Pectinex 5XL and Pentopan MonoBG + Viscozyme L + Pectinex 5XL) effectively maximized
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sugar release (42-53%) from CPH with a corresponding reduction (7-14%) in crude fiber and
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NSP enhancing its use in poultry feed (Alemawor et al., 2009b). In vitro natural fermentation
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(washed pods, sun-dried for 14 days and milled into powder; anaerobic fermentation for 3 days
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at room temperature) increased protein (22-116%), ash (11-31%), fat (68-75%), moisture (49-
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142%) and flavonoid (29-101%) contents of pod husk depending on cocoa clones (Criollo,
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Forastero, Trinitario). The fermentation improved CPH by reducing crude fiber (59-73%),
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phytate (20-81%) and tannin (41-87%) with negligible changes in carbohydrate contents of CPH
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(Shodehinde & Abike, 2017).
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CPH was investigated to develop and transfer appropriate technologies for animal feed
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production as part of a pilot scale production and commercialization of cocoa by-products in
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Ghana in a ten-year project (ICCO/CFC/CRIC, September 1993-July 2003). Full annual
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production capacity was about 5,500 tonnes of pelletized animal feed from CPH. The cost
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benefit and financial viability analysis indicated that the production of animal feed from CPH
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was unlikely to be economically feasible or profitable (Adomako, 2006). However, a recent
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study showed that sun dried CPH can be used at high inclusion rate (20%) to achieve similar
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feed intake and live weight gains with rabbits fed maize-based diets. In addition, CPH was
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economically beneficial compared to maize in feeding growing rabbits (Esong et al., 2015).
186 2.3. Biomass
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CPH ash has been extensively investigated particularly for its potential application as partial
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replacement of and/or combination with fertilizers, biogas and biofuels (Dias, 2014). However,
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the high ash content can inhibit the combustion process since oxygen cannot easily penetrate the
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ash to reach the burning biomass (Martínez-Ángel, Villamizar-Gallardo& Ortíz-Rodríguez,
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2015). Potassium (2.5-4% db) is the predominant CPH mineral, often up to 7% (db) in CPH from
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cocoa grown in Ghana accounting for nearly three-quarters (~70%) of the total ash weight
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(Donkoh et al., 1991). Although the high potassium concentration has been exploited for soap
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production, it can lead to potential fouling problems and consequently to formation of deposits
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and agglomerations when fired in boilers for combustion or in the gasification process
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(Martínez-Ángel, Villamizar-Gallardo& Ortíz-Rodríguez, 2015). Other important CPH minerals
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(Ca, 0.3-0.8%; Mg, 0.02-0.06%; P, 0.04-0.12%; S, 0.02-0.05% and Si, 0.5% db) together
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accounts for 19.9, 21.9 and 27.4% of ash weight (Dias, 2014; Martínez-Ángel, Villamizar-
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Gallardo& Ortíz-Rodríguez, 2015). High proportion of ash and minerals (47 and 41-66% of the
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total pod, respectively) predominate in the epicarp, the most lignified cocoa pod pericarp
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(Sobamiwa & Longe, 1994).
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The ten-year project (ICCO/CFC/CRIC) also investigated potash (ash) production from CPH for
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soft soap manufacturing. The potash from CPH is high in potassium carbonate content and as
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such makes better soap than others in the market. Potash production from CPH was deemed a
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profitable venture based on economic analysis (Adomako, 2006).
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207 2.4. Proteins
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The protein characteristics of CPH have not been adequately investigated. Reports on the protein
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characteristics of CPH are scarce and limited probably due to its reduced growth, food utilization
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efficiency and/or protein efficiency ratio observed earlier in animal diets (Donkoh, Atuahene,
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Wilson & Adomako, 1991; Fagbenro, 1995). One hundred and forty-four proteins were
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identified from 700 protein spots detected in CPH proteome by MALDI-TOF/TOF MS in
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combination with 2-DE analysis (Awang, Karim & Mitsui, 2010). Almost half of the identified
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proteins (48%) are involved in primary and energy metabolism functioning as housekeeping
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proteins. These proteins are involved in general cellular processes related to glycolysis (enolase,
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triose-phosphate isomerase, phosphoglycerate kinase), Calvin cycle, and Tricarboxylic acid cycle
218
(malate dehydrogenase). Six proteins including leucoanthocyanidin dioxygenase and
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anthocyanidin reductase, cinnamyl alcohol dehydrogenase and polyphenol oxidase pertained to
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the phenylpropanoid pathway responsible for the production of secondary metabolites (lignins,
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flavonoids, and phytoalexins). Caffeic acid 3-O-methyltransferase and polyphenol oxidase
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involved in lignin synthesis were differentially expressed in the two clones, LAF17 with smooth
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and waxier pod surface and the cocoa pod borer-resistant ICS39 clone with harder and thicker
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sclerotic layer (Awang, Karim & Mitsui, 2010).
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2.4.1 Amino acids
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The average protein content in CPH (generally ~ 8.5%) is lower than those previously reported
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in cocoa bean husk (15%) (Bonvehí & Coll, 1999). Data on amino acids is limited to a single
229
report (Donkoh et al., 1991) of CPH compared to an earlier (1984) study. CPH protein has
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higher (10-15%) levels of total and essential amino acids and above half (~ 56%) of the total
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amino acid content of bean husk (Table 4 and 5) due to asparagine, alanine, arginine, glycine,
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glutamine, lysine, serine, threonine and tyrosine concentrations. CPH had twice the acidic
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(aspartic + glutamic acids) and basic (arginine + histidine + lysine) amino acids, proline and
234
valine content of bean husk, whereas the levels of histidine, leucine and methionine were similar
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in both husks. CPH protein has higher Fischer ratio and lower content of aromatic amino acids
236
than those of cocoa bean husk. The lysine/arginine ratio, a determinant of the cholesterolaemic
237
and atherogenic effects of protein, is low for CPH protein, but higher than those of cocoa bean
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husk. A soluble cocoa fiber product (0.79 PER, contributing 28 g protein/kg diet) obtained by
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enzymatic treatment of cocoa bean husk reduced the negative effects of dietary-induced
240
hypercholesterolemia in an animal model (Ramos et al., 2008).
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The biological value (37.6%) of bean husk (Bonvehí & Coll, 1999) corresponds to the apparent
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digestibility of fresh and sun-dried cocoa pod husk (37.8 and 32.1%, respectively) reflected in
243
their nitrogen retention (17 and 12.2%, respectively) in sheep rations containing 53% (dm) CPH
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(Wong & Hassan, 1988). In addition to protein, CPH also contains theobromine (~ 0.2%), a
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commercially significant appetite stimulant for ruminants (Trout, Zoumas & Tarka, 1978).
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2.5. Pectin
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2.5.1. Extraction methods and properties
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Hot water extracted pectic material from CPH contains galacturonic acid (27%), galactose
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(21%), rhamnose and arabinose, whereas ammonium oxalate (0.5%) extraction resulted in
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depolymerized pectin (Blakemore, Dewar & Hodge, 1966). Pectin has also been extracted with
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mild acetic acid (0.2 N, pH 2.8, 1:3 w/v) from ripe CPH with higher yield (8-11%, db) than those
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reported earlier (Dittmar, 1958 cited in Adomako, 1972) from sun dried CPH (5.3-7.1%). The
254
acetic acid extracted pectin had the following composition: ash (8.9-9.8% comprising Ca and K
255
as major components), protein (1.1%), galacturonic acid (62%), galactose (4.65%), rhamnose
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(2.9%), arabinose (1.7%) and xylose (1.2%) with carbohydrate composition similar to apple
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pectin (Adomako, 1972).
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Investigation of CPH pectins intensified after a hiatus of almost four decades, particularly in a
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series of studies by the Brazilian group (Vriesmann et al., 2011-2017). Nitric acid (pH 1.5, 100
260
°C, 30 min) extraction from CPH produced highly esterified (56.5% degree of esterification
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[DE]) and acetylated (17.1% degree of acetylation [DA]) pectin (9.8%, db yield) with uronic
262
acid (66%), homogalacturonan and rhamnogalacturonans; this pectin is considered high methoxy
263
(HM) pectin (Vriesmann, Teófilo & Petkowicz, 2011). Hot aqueous extraction (50 °C [50W] and
264
100 °C [boiling water], 90 min, 1:25, w/v) was used in a follow up study to obtain 7.5 and 12.6%
265
pectin from milled and dried CPH (Vriesmann, Amboni & Petkowicz, 2011). The water soluble
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pectins were highly acetylated (19.2 and 29% DA), composed of low methoxy
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homogalacturonans (37 and 42.3% DE) with rhamnogalacturonan insertions exhibiting non-
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Newtonian shear-thinning behavior (Vriesmann, Amboni & Petkowicz, 2011). CPH treatment
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(aqueous citric acid pH 3, 95 min, 95 °C) yielded 10.1% (db) pectin with low moisture (2.7%),
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high carbohydrate (64%), protein (13.8%), low phenolics (9.4%), low-methoxy, highly
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acetylated homogalaturonan (DE, 40.3%; DA, 15.9%) containing 65% uronic acid and exerting
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non-Newtonian shear –thinning behavior (Vriesmann, Teófilo & Petkowicz, 2011). The pectin
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forms gels under low pH/high sucrose content, suggesting applications as additive in acidic
274
conditions. Similar highly acetylated (DA, 17%) CPH pectin was obtained by optimized nitric-
275
acid-mediated extraction of CPH and the best gel properties were observed at 1.32% galacturonic
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acid equivalent concentration, 60% sucrose and pH 2.7, demonstrating its use as a gelling and
277
thickening agent (Vriesmann & Petkowicz, 2013). High yield (10.7%, db) of CPH pectin (DE,
278
41%; DA, 17.6%) was obtained with nitric acid extraction (pH 3.5, 100 °C, 30 min) under reflux
279
with high uronic acid (59.2%), significant proportions of galactose (20.3%) and rhamnose
280
(11.6%). The large pectic molecule was purported to be primarily responsible for its gelling
281
properties; forming gels at low pH (< 2.5) and water activity (1% w/v; 60% v/v ethylene glycol)
282
(Vriesmann & Petkowicz, 2017). Pectin extracted with hot acid (nitric acid [pH 3, 100 °C, 30
283
min]) or citric acid [pH 3, 95 °C, 95 min]) had similar chemical composition (65% uronic acid,
284
8.2% rhamnose, 16.7% galactose, 40.3% DM and 15.9% DA) (Vriesmann & Petkowicz, 2017).
285
Extraction conditions greatly affect yield and chemical properties of CPH pectins (Table 6).
286
Thus, pectin yield from cocoa husks varied (3.4-7.6%) with uric acid content ranging from 31-
287
65% and degree of methylation (7-58%) depending on solvent and extraction conditions (Chan
288
& Choo, 2013). For example, the highest pectin yield (7.6%) is obtained with hot citric acid
289
(1.25, w/v; pH 2.5, 95 °C, 3h), whereas hot water under similar conditions (1.25 w/v; 95 °C, 3h)
290
extracts pectin with the highest uronic acid (65%) content. Furthermore, pectin yield increases
291
significantly (p < 0.05) with reduction in substrate –extractant ratio (from 1:25 to 1:10 w/v)
292
using citric acid at pH 2.5. Water and hydrochloric acid extracted low methylated (LM) pectin
293
(7.2-39.3% DM), whereas citric acid (1:25 w/v, pH 2.5, 1.5 h) produced wide degree of
294
methylation range and high methylated (HM) pectin (58%) (Chan & Choo, 2013). Similarly,
295
acidified (1N HNO3) water treatment (1:25 w/v, 75 °C, 90 min) differing in pH (1, 2 or 3)
296
produced CPH pectins varying in yield (3.7-8.6%), galacturonic acid content (51-75%),
297
galactose (5.2-13.9%), total neutral sugars (7.9-24.3%), calcium (48.6-155.7 µmol/g), degree of
298
methylation (37-52%), DA (3.2-9.8%), intrinsic viscosity (162-304 mL/g) and viscosity-average
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molecular weight (43-82 kDa) (Yapo & Koffi, 2013). Hydrochloric acid (1.58 M) extraction (62
300
°C, 4.8 h) produced CPH pectin (yield =11.7%) with high DE (58.5%) and galaturonate content
301
(49.9%) (Hutomo, Rahim & Kadir, 2016). The highest CPH pectin yield (23.3% db) was
302
obtained with hot water (1.05 g/mL) extraction in a water bath (50 °C); the pectin had low
303
moisture (0.2%), ash (1%), high DE (26.8%) and swelling (357, 275 and 360 swelling index in
304
0.1M HCl, phosphate buffer [pH 6.8], and distilled water, respectively) (Adi-Dako et al., 2016).
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305 2.5.2. Applications
307
The hot water soluble pectin had Hausner ratio of 1.17, compressibility index of 14.6% and angle
308
of repose ~38° indicating good flow properties. It was predominantly rich in potassium (2.27%),
309
magnesium (0.22%), phosphorous (0.096%) and sulphur (0.094%), copper (10.9% of minor
310
elements), zinc (8.3%) and nickel (3.7%). The pectin exhibited dose-dependent moderate
311
antimicrobial activity (minimum inhibitory activity-MIC) against Staphylococcus aureus and
312
Escherichia coli (MIC: 0.5-1 mg/mL); Pseudomonas aeruginosa, Bacillus subtilis, and
313
Salmonella typhi (MIC: 1-2 mg/mL); and Shigella spp (Adi-Dako et al., 2016). Freeze-dried hot
314
water extracted CPH pectin (26.8% DE) and 4% hot aqueous citric acid soluble pectin showed
315
no toxicity on the major haematological indices, bilirubin levels and the spleen when
316
administered up to 71.4 mg/kg in Sprague Dawley rats for 90 days (Adi-Dako et al., 2018). CPH
317
pectin can therefore be safely incorporated in pharmaceutical formulations as natural polymer
318
excipients.
319
The high methoxy pectin obtained by nitric acid was used to prepare microparticles containing 5-
320
aminosalicylic acid (5-ASA) for drug delivery to the colon. The microparticles (1:1, 2:1, 3:1 and
321
4:1) obtained by spray drying showed better drug retention with increased pectin content
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(Vriesman, 2012). Saponification of this high methoxy pectin promoted distinct increase in
323
biological activities; it triggered the secretion of pro-(NO, TNF-α and IL-12) and anti- (IL-10)
324
inflammatory mediators in peritoneal microphages demonstrating its application as an immune
325
modulator (Amorim et al., 2016).
326
CPH gums have been described as essentially acidic protein-polysaccharide containing 3-5%
327
protein with galactose, glucose, rhamnose, arabinose, galacturonic acid, and glucuronic acid in
328
1:0.21:1.9:0.2: 4.2:1.3 molar ratios with high (35-40%) uronic acid content. Crude gum yield
329
(9.1% of dry CPH) similar to reported polysaccharide yields (8-11%, dw) had high potassium ion
330
(9.6% dw) concentration similar to previously observed CPH ash content (9%) (Figueira, Janick
331
& BeMiller, 1994). Alcohol extracted gum (5.25%) (sun dried samples were boiled twice with
332
70% EtOH, then extracted twice with boiling 70% MeOH) from CPH exhibited pseudoplastic
333
behavior obeying the power law flow model with high viscosity (1053 mPas at 5%
334
concentration) similar to karaya gum (1160 mPas at 3% concentration) (Samuel, 2006). CPH
335
gum can potentially substitute costly karaya gum, at least partly in many formulations.
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2.6. Phenolics and antioxidants
338
2.6.1. Effect of the extraction methods
339
Total phenolic content of CPH ranged between 46 to 57 mg gallic acid equivalent (GAE)/g dry
340
matter (Karim et al., 2014b). However, lower total phenolic (2.1-3.7 mg GAE/g) has been
341
reported depending on the location of cocoa growth and solvent system used in extraction. For
342
example, methanol: acetone extracted higher total phenolic of CPH from two locations (Cone
343
and Taura) in Ecuador (3.53 and 3.65 mg GAE/g) than ethanol (2.07 and 2.27 mg GAE/g) that
344
was reflected in their antioxidant activities (38 and 44 µM TE/g ABTS, 34 µM TE/g DPPH and
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4.6 µM TE/g FRAP) compared to ethanol (24 µM TE/g ABTS, 18 and 21 µM TE/g DPPH and 2
346
µM TE/g FRAP) (Martínez et al., 2012). Acetone-water (7:3 v/v) and ethanol (70%) extracted
347
CPH phenolics (94.92 and 49.92 mg GAE/g extract, respectively) with the acetone extract
348
exhibiting antifungal activity against Fusarium oxysporum (Mu’nisa, Pagarra & Maulana, 2018).
349
Pods chopped into 1 cm2, sun dried and ground (1 mm) contained phenolic acid (19.6-49.5 mg
350
GAE/g) and flavonoid (4-22.4 mg rutin equivalent [RE]/g) depending on the clone (Karim et al.,
351
2014a). Several polyphenolic compounds were identified by LC-MS/MS in CPH extract (80%
352
aqueous ethanol, 40 °C, 30 min); phenolic acids (protocatechuic acid and its derivatives and p-
353
hydroxybenzoic acid), flavonoids (apigenin, rhamnetin, kaempferol derivatives, flavone
354
derivatives), luteolin, apigenin and linarin (Karim et al., 2014b). Some of these phenolics
355
quantified in fresh CPH consisted of catechin (36%), quercetin (21%), epicatechin (21%) and
356
gallic (11.3%), coumaric (6.5%) and protocatechuic (4.5% of total phenolic compounds) acids
357
(Valadez-Carmona et al., 2017). The total phenolics, flavonoid and flavonol contents of the fresh
358
CPH was 3.24 mg GAE/g, 0.97 mg and 0.34 mg epicatechin equivalent (EE)/g, dw respectively
359
(Valadez-Carmona et al., 2017).
360
Aqueous ethanol (80%) extract of the pod exhibited high antioxidant activity (0.26 and 0.57 EC50
361
for DPPH or 20.35 and 45.26 mg/ml) about four times lower than that of BHT. The extract (1 g)
362
also displays strong ferric reduction potential reducing 80 mol of Fe2+ from Fe3+ (Karim et al.,
363
2014a). Ethanol extract (50% aqueous ethanol) from a Colombian cocoa clone (TSH 565)
364
contained epicatechin (0.25-0.35 mg/g) and varied in antioxidant activity (FRAP 137-169 µM
365
TE/g; ABTS 116-230 µM TE/g; ORAC 252-343 µM TE/g) depending on the extraction process
366
(Sotelo, Alvis & Arrázola, 2015). Low- molecular weight soluble phenolics obtained by three
367
step sequential extraction exhibited strong antioxidant activities (85% DPPH inhibition, EC50 =
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25 g/g; 52 µM TE/g ABTS and 130 µM TE/g FRAP) significantly higher (p < 0.05) than those of
369
fermented and roasted cocoa hull and kernel products (Yapo et al., 2013). The antioxidant
370
capacity of fresh CPH was 30.6 and 15.1 µM TEAC/g (ABTs and DPPH assays, respectively)
371
similar to those reported earlier (38 and 33 µM TEAC/g for ABTS and DPPH, respectively,
372
(Martínez et al., 2012).
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373 2.6.2. Effect of processing method
375
Processing affects CPH polyphenolic content. For example, microwave drying (595 W, 11.5
376
min) increased total phenolics (3.4x), flavonoid (5.8x), flavonol (5.8x), phenolic compounds
377
(catechin 2.2x, quecetin 73%, epicatechin 3.3x, gallic acid 3.8x, coumaric acid 4.9x and
378
protocatechuic acid 5.9x) and antioxidant capacity (ABTS 2.9x and DPPH 3.9x) of CPH,
379
compared to hot air and freeze-drying (Valadez-Carmona et al., 2017). Pre-treatment to prevent
380
enzymatic (polyphenol oxidase) oxidation such as soaking (1% citric acid, 15 min) before sun
381
drying significantly (p < 0.05) increased total phenolic (24%) and flavonoid (13%) contents
382
compared to unsoaked sun dried CPH (Sartini, Asri & Ismail, 2017). Furthermore, ultrasound
383
(60 Hz, 2 h) extracted significantly (p < 0.05) higher phenolic than those obtained by agitation
384
(200 rpm, 6 h) (21-23 vs 16-19 mg GAE/g) (Sotelo, Alvis & Arrázola, 2015). Total phenolic
385
content (extracted by 50% (v/v) ethanol solution, 35 °C, 150 rpm, 2h) of dried (60 °C, 24 h) CPH
386
decreased 42% on sterilization (autoclave 120 °C, 20 min) and increased 37% after solid-state
387
fermentation with Rhizopus stolonifera NRRL 28169 ((30 °C, pH 5.5, 72 h), presumably due to
388
the generation of new bioactive molecules (Tiburcio, 2017). HPLC of aqueous ethanol extract of
389
CPH revealed the presence of 24 phenolic peaks with 3 major unidentified components
390
accounting for 72% of the total peak area compared to 14 peaks with 3 major peaks accounting
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for 81% of the total peak area (33% higher than CPH) in R. stolonifera fermented CPH.
392
Furthermore, CPH displayed higher concentrations of phenolic compounds compared to
393
fermented CPH although the latter exhibited superior antioxidant activity (DPPH and ORAC)
394
than CPH, presumably due to polyphenolic hydrolysis and/or transformation (Tiburcio, 2017).
395
Industrial fermentation such as those used for cocoa bean (6 days in wooden boxes) from
396
Mexican cocoa genotypes can be simulated (applied) to increase phenolic content of CPH similar
397
to those reported for cocoa bean husk (Hernández-Hernández et al., 2018). Total polyphenol and
398
tannin levels decreased by ≥ 11 and ≥ 89%, respectively after fungal treatment (Aspergillus niger
399
or Talaromyces verruculosus 0.15% w/w, moisture 1:3 w/v, 7 days) to eliminate theobromine
400
and reduce ochratoxin A content of CPH (Oduro-Mensah et al., 2018). Similar reduction in
401
phenolic concentration occurred on cocoa shell after solid state fermentation with Penicillium
402
roqueforti without affecting flavonol and anthocyanin contents, indicating significant
403
consumption of these metabolites during fermentation (Lessa et al., 2018). However, CPH
404
antioxidant capacity generally increased after solid state fermentation by Rhizopus stolonifera
405
LAU 07. The IC50 of the CPH substrates (fermented/unfermented – 5.5/14.7 mg/mL) increased
406
2.7 folds in DPPH-scavenging capacity (Lateef et al., 2008).
407
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2.7. Volatiles
409
A recent study (Tiburcio, 2017) identified 50 volatile compounds in CPH by SPME (5/95%
410
Carboxen/Polydimethylsiloxane, 75 µm) followed by desorption and separation on 95% PDMS,
411
5% phenyl GC column (30 m x 0.25 mm, 0.25 µm film thickness). These compounds consisted
412
of 16 alcohols, 11 hydrocarbons, 8 aldehydes, 7 ketones, 5 esters, 2 amines and isovaleric acid.
413
Solid-state fermentation of CPH with Rhizopus stolonifera NRRL 28169 (incubated in a rotatory
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shaker 100 rpm, 30 °C, pH 5.5, 7 days) reduced the number (from 50 to 33) of identified volatile
415
compounds. Thirteen volatile compounds [(2S,4S)-(+)-pentanediol, 1-methoxy-2-butanol, 2,3-
416
butanediol, epoxylinalol, phenylethyl alcohol, D-limonene, α-copaene, ethyl iso-allocholate,
417
methyl N-hydroxybenzenecarboximidoate, methyl salicylate, nonanal, octanal, and α-toluic
418
aldehyde] were present in both fermented and unfermented CPH (Table 7).
419
Ten volatiles in CPH are present in cocoa, five (2,3-butanediol, phenylethyl alcohol, nonanal, α-
420
toluic aldehyde and methyl salicylate) of which have been identified in both unfermented and
421
fermented CPH. 2,3-Butanediol imparts sweet chocolate notes reminiscent of the natural odor of
422
cocoa butter, whereas α-toluic acid (2-phenylacetaldehyde) and methyl salicylate confer floral
423
and nutty sensory perceptions, respectively (Aprotosoaie, Luca & Miron, 2016). Phenylethyl
424
alcohol (2-phenylethanol) is associated with the typical “floral” volatiles of dark chocolate
425
extracts resulting from microbial actions occurring during fermentation. It is also the most odor-
426
active compound in dried and fermented cocoa (Álvarez et al., 2016). Linalool, trans-linalool
427
oxide and isovaleric acid were identified only in nonfermented CPH.
428
phenylethanol are major alcohols in roasted nibs; linalool concentrations in cocoa vary with its
429
origin: West Africa (0-0.5 mg/kg), Malaysia (0-0.2 mg/kg) and Ghana (0.2-0.8 mg/kg). The
430
linalool/benzaldehyde ratio has been proposed as a flavor index for cocoa. Isovaleric acid has
431
been identified in fermented cocoa beans as a result of sugar metabolism. Benzyl alcohol, also
432
known as α-methylbenzyl alcohol and 1-hexanol were identified in the fermented CPH; the latter
433
confers fruity and green notes in cocoa (Aprotosoaie, Luca & Miron, 2016).
Linalool and 2-
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434 435
2.8. Other compounds
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The methyl ester of linoleic acid (9,12-octadecadenoic acid) is one of the three major
437
components of CPH ethanol (70%) extract (Mu’nisa, Pagarra & Maulana, 2018). Linoleic acid in
438
turn is the predominant (53% of the total peak area) component of bio-oil obtained from sun-
439
dried (1-2 weeks) and ground (< 1 mm) cocoa pod husks by pyrolysis (550-600 °C). The bio-oil
440
also contained palmitic, carboxymethoxy succinic, linolenic, hexanoic, caproic, pyruvic, azelaic
441
and caprylic acids (13.2, 5.6, 5.3, 3.8, 3.3, 2.2, and 2.0% of total peak area, respectively) (Adjin-
442
Tetteh et al., 2018). Linoleic acid was also present in petroleum extract (Soxhlet) of sundried (10
443
days) ground CPH powder along with other fatty acids (arachidic, palmitic, pentadecanoic,
444
stearic acids) and other compounds (Adewole et al., 2013).
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445 3. Uses and applications
447
3.1. Fragrance compounds
448
CPH impregnated with nitrogen sources has been used for the synthesis of volatile fragrance
449
compounds by a Rhizopus oligosporous strain. Solid-phase microextraction (SPME) coupled
450
with GC-MS revealed maximum abundance of volatile fragrance components when CPH was
451
fermented for 96 h (1 x 105 inoculum spores/g, 24 °C, 4-5 mm substrate thickness and pH 6.5).
452
The highest numbers and abundance of volatile compounds was obtained with L-phenylalanine
453
as the nitrogen source. This demonstrates CPH potential as an inert support for secondary
454
metabolite bioconversion by fungus strain, i.e. volatile fragrance components in a solid state
455
fermentation system (Norliza, 2006). Another study by the same group (Norliza & Rozita, 2006)
456
showed the release of three compounds, benzaldehyde, phenetyl alcohol and phenyl
457
acetaldehyde when CPH (1 mm) was mixed with L-phenylalanine (2.5, 3.0 and 3.5%, w/w) and
458
fermented with Rhizopus oligosporous for 4 days.
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Furthermore, many of the compound identified by Tiburcio (2017) in both fermented and
460
unfermented CPH [1-octen-3-ol, nonanal, octanal, α-terpineol, ionones, methyl salicylate, β-
461
hydroxybutyric acid methyl ester, α-methylbenzyl alcohol and rosefuran] are well-known
462
flavor/aroma compounds (Table 5).
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463 3.2. Skin & hair treatment
465
The skin whitening effect of CPH aqueous ethanol (80%) extract was demonstrated based on in
466
vitro mushroom tyrosinase assay and sun screening effect (UV-absorbance at 200-400 nm
467
wavelength) (Karim et al., 2014b). Resveratrol and fatty acids, such as linoleic acid, in CPH has
468
been reported to have in vitro skin whitening properties without adverse effects (Ohguchi et al.,
469
2003; Parvez et al., 2006). When tyrosinase enzyme activity is inhibited, melanin production is
470
reduced, resulting in a fairer skin (Karim et al., 2014b). The extract induced human fibroblast
471
cell proliferation at low concentration and displayed UVB sunscreen potential superior than
472
commercial UV-protecting agents (avenobenzone and octylmethoxycinnamate). It was also
473
highly effective against collagenase, better than kojic acid (a skin-lightening cosmetic product),
474
although lower against elastase and tyrosinase than pine bark extract (positive control, effective
475
in maintaining cellular function which affects the skin condition) (Karim et al., 2014b).
476
Application of gel from the CPE extract reduced in vivo skin wrinkles within 3 weeks and after 5
477
weeks (6.4, and 12.4%, respectively); skin hydration also increased (3%) after 3 weeks’
478
application (Karim et al., 2016).
479
Cocoa pod ash is a commercial ingredient used as a component of African Black Soap that in
480
turn is used in environmentally-free conditioning cleansing cream (Koiteh, 2013).
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3.3. Animal appetite stimulant
483
CPH stimulates appetite in ruminants due to the presence of theobromine (0.2-0.25% weight),
484
particularly at low theobromine (0.05-0.1%) inclusion in the feed. This commercially significant
485
appetite stimulation enables weight gain in a short time to bring animals to market sooner due to
486
increased feed consumption (Trout, Zoumas & Tarka, 1978). CPH contains approximately 0.15-
487
0.4% (w/w) theobromine limiting its inclusion to 13.7% (w/w) in animal feed due to the 300
488
mg/kg theobromine content limit imposed by the European Union (Oduro-Mensah et al., 2018).
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491
The pod may be used as a texturizing agent after drying (30-60% moisture) and grinding to
492
extremely small size (0.05-0.12 inch). Juice from the pods can be separated from the
493
parenchymatous tissues (cocoa fruit flesh representing 82% of the fruit) to form a pure
494
hydrocolloid derivative that can be used as conventional vegetable gum (Drevici & Drevici,
495
1980). The juice imparts high viscosity, preserves aroma, odor and color of products and
496
improves rheological properties due to its carbohydrate composition (galacturonic acid,
497
galactose, rhamnose, arabinose and xylose with galacturonic acid) (Drevici & Drevici, 1980).
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3.5. Antibacterial
500
Some CPH extracts are effective against two major bacteria responsible for most hospital
501
infections: Gram negative Salmonella choleraesuis (1 mg/mL MIC) and Gram positive
502
Staphylococcus epidermidis (2.5 mg/mL MIC). The extract also exhibited strong inhibitory
503
activity against Pseudomonas aeruginosa (Santos et al., 2014). Fractionation (solvent partition)
504
of CPH extracts enabled identification of bioactive molecules such as phenols, steroids, or
23
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terpenes. The extract was prepared by air drying (3 months) chopped pieces of husks (1.5-2.5 cm
506
wide), pouring distilled water over the husks and collecting the aqueous extract that was dried
507
(room temperature, 72 h) and ground (< 1.5 mm). The dry crude water extract represents 0.16%
508
of the fresh CPH (Santos et al., 2014).
509
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3.6. Nanoparticles larvicidal & antioxidant
511
CPH extract synthesized with silver nanoparticles (CPHE-AgNPs) exhibited antioxidant activity
512
with potent larvicidal activity (70-100% at 10-100 µg/ml; 43.5 µg/ml LC50) against Anopheles
513
mosquito larvae at 10-100 µg/ml concentration. The CPHE-AgNPs exhibited high stability,
514
antioxidant activity (33-85% and 14-84% DPPH-free radical scavenging and ferric ion reducing
515
activities at 20-100 µg/ml; 49.7 IC50) and synergistically improved (43-100%) antibacterial
516
activities of cefuroxime and ampicillin. The nanoparticles effectively in vitro inhibited multidrug
517
resistant Klebsiella pneumonia and Escherichia coli isolates at 40 µg/ml concentration and also
518
inhibited Streptococcus pyogenes, Staphylococcus aureus, Pseudomonas aeruginosa, Aspergillus
519
flavus, Aspergillus fumigatus and Aspergillus niger growth in emulsion paint (Lateef et al.,
520
2016).
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3.7. Encapsulating agent
523
CPH has also been investigated in green chemistry for the synthesis of nanoparticles against
524
multidrug resistant clinical bacteria because of its antioxidant, antimicrobial and larvicidal
525
activities (Lateef et al., 2016). Alpha-cellulose was obtained from CPH (26% yield) with 2.74%
526
ash, high crystallinity (27%), molecular weight (63,342) and 390 degree of polymerization
527
[glucose units] (Hutomo et al., 2012). CPH pectin may also serve as a suitable encapsulating
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528
agent alone or in combination with nanolignocellulose as demonstrated for citrus pectin to
529
protect probiotics in low pH fruit juice and gastrointestinal tract (Khorasani & Shojaosadati,
530
2017).
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533
Candida boidinii XM02G, isolated from cocoa cultivation areas, has been used to produce
534
xylitol from CPH. Xylitol was obtained in concentrations of 11.34 g.L-1, corresponding to a
535
yield (Yp/s) of 0.52 g.g-1 with a fermentation efficiency (ε) of 56.6% (Santana et al., 2018).
536
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3.9. Other valuable compounds
538
Ash (average yield 7.2%) from controlled combustion of CPH contained about 75% potash as
539
potassium carbonate with 1% impurities (iron, calcium and magnesium) that renders the organic
540
potash a premium product suitable for the pharmaceutical and food industries (Woode, 2015).
541
The high potassium content of CPH enables its use as a catalyst for biodiesel production due to
542
microstructural development when calcined at 700 °C for 4 h (Betiku et al., 2017). Carbonization
543
of dried and refined CPH with activated carbon reduces free fatty acid absorption (87%) of waste
544
cooking oil. This CPH can potentially be used as a potassium carbonate catalyst in the
545
transesterification process for biodiesel production from waste cooking oil (Rachmat, Mawarani
546
& Risanti, 2018). Microcrystalline cellulose (MCC - 5-10 µm rod-like shape, 11.635 nm
547
diameter and 74% crystallinity) was obtained by subjecting CPH to alkali treatment, bleaching,
548
and hydrochloric acid hydrolysis. This MCC can be used as a reinforcing agent with starch (2:8
549
mass ratio) and glycerol (20%) to produce high tensile strength (0.637 MPa) bioplastics (Lubis et
550
al., 2018).
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CPH can be hydrolyzed (1M HCl, 4 h, 75 °C) to yield maximum glucose (30.7%, w/v) that
552
produces ethanol (17.3 %, v/v) on fermentation (26 h) with Saccharomyces cerevisiae (Samah et
553
al., 2011).
554
Lipase extraction from CPH was achieved with the highest enzyme activity using response
555
surface methodology. Cross-linked enzyme aggregate (CLEA) was produced in the presence of
556
ammonium sulphate (30 mM) and stabilized/immobilized with glutaraldehyde (70 mM) and
557
bovine serum albumin (0.23 mM) (Yusof et al., 2016). The immobilized lipase had superior
558
stability in response to temperature (25-60 °C) and pH (5-10), and hydrophilic organic solvents
559
such as methanol (60%) compared to the free enzyme. The highest yield of lipase (11.43 U/ml)
560
was obtained with 7% CPH and 50 mM sodium phosphate buffer (pH 8) (Khanahmadi et al.,
561
2015).
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562
CPH can be easily converted into valuable products similar to those proposed and commercially
564
viable for cocoa shells. In this context, the pyrolysis plant (GmbH, Germany) can industrially
565
produce 3 products: charcoal (65-75%) as the main product of low temperature (250-300 °C)
566
torrefaction/carbonization; pyrolysis fuel (30-50%) and charcoal (25-35%) at medium
567
temperature (300-550 °C) vaporization; syngas (60-80%) and charcoal (15-20%) at high
568
temperature (600-1000 °C) gasification (Voigt, 2017). The three products, charcoal, pyrolysis
569
fuel and non-condensable gas account for 38, 32 and 30% of the total output volume,
570
respectively from the pyrolysis process. Thus, charcoal (280 kg/h) and biofuel (230 L/h) are
571
produced from an infeed of CPH (1 t/h) amounting to 60,000 Euros equivalent to an annual
572
saving of 504 million Euros. Furthermore, the Bühler shell burning technology can save
573
producers up to 50% in total cocoa production costs by CPH combustion system thereby
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reducing their carbon footprint by up to 2800 t/a. Fig. 2 summarizes the potential applications of
575
CPH.
576 4. Potential health benefits
578
Notwithstanding the foregoing, the utilization of CPH to enhance the quality and functionality of
579
pharmaceutical and food products is limited. Moreover, no research is available on the health
580
benefits of bioactive compounds from CPH. Therefore, in this section the health benefits of these
581
compounds from other plants are presented.
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4.1. Pectins
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The readily available CPH can be used to recover value-added compounds such as pectins.
585
Pectins extracted from several plant by-products are widely used as gelling, thickening and
586
stabilizing agents and have several positive effects on human health, including lowering
587
cholesterol and serum glucose levels, reducing cancer and stimulating the immune response
588
(Vriesmann, Amboni & Petkowicz, 2011).
589
Pectin favorably influences cholesterol levels in blood. It helps reduce blood cholesterol in a
590
wide variety of subjects and experimental conditions as comprehensively reviewed
591
(Sriamornsak, 2001). Consumption of at least 6 g/day of pectin is necessary to have a significant
592
effect in cholesterol reduction, lower amounts are ineffective (Ginter et al., 1979). Pectin acts as
593
a natural prophylactic substance against poisoning with toxic cations. It is effective in removing
594
lead and mercury from the gastrointestinal tract and respiratory organs (Kohn, 1982). Pectin’s
595
high water binding capacity provides a feeling of satiety, thus reducing food consumption.
596
Experiments showed a prolongation of the gastric emptying half-time from 23 to 50 minutes of a
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meal fortified with pectin (Holt et al., 1979). These attributes of pectin are used in the treatment
598
of overeating-related disorders (Di Lorenzo et al., 1988).
599 4.2. Minerals
601
Four minerals (K, P, Ca and Mg) detected in CPH are quantitatively important (0.5- 6.0%). The
602
presence of these CPH minerals which are required for maintaining vital functions in living
603
human cells makes it a potential source of those elements (Yapo et al., 2013).
604
Minerals are essential constituents of skeletal structures such as bones and teeth; play a key role
605
in the maintenance of osmotic pressure, and thus regulate the exchange of water and solutes
606
within the animal body; serve as structural constituents of soft tissues; they are essential for the
607
transmission of nerve impulses and muscle contraction; minerals play a vital role in the acid-base
608
equilibrium of the body, and thus regulate the pH of the blood and other body fluids; also serving
609
as essential components of many enzymes, vitamins, hormones, and respiratory pigments, or as
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cofactors in metabolism, catalysts and enzyme activators (FAO, 2017).
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611 4.3. Dietary fiber
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The total dietary fiber in CPH (59%) includes 11 and 48% of soluble and insoluble dietary fiber,
614
respectively (Yapo et al., 2013). CPH contains higher TDF compared to other dietary fiber
615
sources, such as citrus (35-37%), apple (51%) and banana (33-52%) by-products, but lower than
616
those of coconut fiber (63%), pea hull (75%) and yellow passion fruit rind (74-82%) by-products
617
(Yapo et al., 2013).
618
High dietary fiber intake increases stool weight and transit time which may deter large bowel
619
disorders such as constipation, diverticulitis and large bowel cancers (Elleuch et al., 2011). Most
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non-absorbed carbohydrates have laxative effects, both by increasing bacterial mass or osmotic
621
effects, and by water binding of unfermented fiber. The etiology of cancer involves both
622
inherited and environmental (dietary) factors. The overall evidence for an effect of total fiber
623
intake on the risk of colorectal cancer is not considered sufficient to serve as a basis for
624
guidelines on dietary fiber intake. However, individuals with lesser fiber intakes may have an
625
increased risk (Mudgil & Barak, 2013).
626
Recent observational studies consistently show an inverse association between dietary fiber
627
intake and the risk of coronary heart disease (Mudgil & Barak, 2013). Postulated mechanisms for
628
lower levels of total and low-density lipoprotein (LDL) cholesterol include alterations in
629
cholesterol absorption and bile acid re-absorption, and alterations in hepatic metabolism and
630
plasma clearance of lipoproteins (Theuwissen & Mensink, 2008). In some countries the evidence
631
for the cholesterol-lowering properties of certain viscous fibers, especially β-glucans from oats,
632
is considered sufficient for claims on the reduction of the risk of coronary heart disease (Mudgil
633
& Barak, 2013).
634
Some cohort studies inversely associate dietary fiber intake and the risk of developing type 2
635
diabetes (Chandalia, 2000). Furthermore, viscous fibers such as pectin and guar gum delay
636
gastric emptying, whereas slowly digested starch and resistant starch increase satiety in vivo
637
(Mudgil & Barak, 2013). Presumably other, as yet unidentified substances in such foods can
638
explain this; perhaps it is the overall combination of the dietary fiber, nutrients and bioactive
639
substances acting synergistically, that is critical to health. However, dietary fiber such as
640
resistant starch, non-digestible oligosaccharides and polydextrose may help to prevent and
641
alleviate bowel disorders, and decrease risk factors for coronary heart disease and type 2 diabetes
642
(Kaczmarczyk, Miller & Freund 2012).
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643 4.4. Antioxidants compounds
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Polyphenols are among the other bioactive compounds found in CPH. Polyphenolic compounds
646
usually accumulate in the outer parts of plants, such as shells, skins and husks (Vriesmann,
647
Amboni & Petkowicz, 2011). The total phenolic content of CPH is estimated at ∼7% and
648
significantly higher (p < 0.05) than in cocoa hull product from fermented and roasted beans
649
(CHPFR) (∼2-3%) and cocoa kernel product (CKPFR) (Yapo et al., 2013). Therefore, CPH can
650
be a potential source of antioxidant-dietary fiber-rich food, which may be used to compensate for
651
their shortage or complete lack in refined modern diets currently associated to various free
652
radical-induced disorders (Yapo et al., 2013).
653
Free radicals, or Reactive oxygen species (ROS), play a biochemical role in the development of
654
cancer, multiple sclerosis, Parkinson's disease, rheumatoid and inflammatory diseases. Metabolic
655
disease and environmental contaminants may increase ROS in tissues, leading to increased
656
oxidative stress in cells. However; antioxidants are able to ‘scavenge’ free radicals from the
657
body, reducing oxidative stress and the potential development or progression of many disease
658
states (Zumbé, 1998). Studies with procyanidins BI and B3 and (+)-catechin show that these
659
polyphenols are capable of trapping hydrophilic peroxyl radicals in vitro, and their radical
660
scavenging activity increases with polyphenol concentration (Ariga & Hamano, 1990).
661
Furthermore, antioxidant activity of polyphenols has been demonstrated in vitro to prevent lipid
662
peroxidation, a type of ROS induced cell injury and low density lipoprotein (LDL) oxidation
663
(Zumbé, 1998).
664
The anti-genotoxic effect of catechin, epicatechin, gallocatechin and epigallocatechin have all
665
been demonstrated in vitro, protecting organisms such as S. typheriurn and E.coli against
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mutation following exposure to various known carcinogens. This protective, anti-genotoxic
667
effect of the polyphenols has also been demonstrated in in vitro studies of mammalian cells
668
exposed to such cancer inducers as nitrosamine, benzo[a]pyrene, aflatoxin BI, tobacco smoke
669
and smoked meat extracts. Furthermore, polyphenols prevent DNA damage in vitro and in vivo
670
following exposure to these types of compound (Agarwal & Mukhtar, 1996; Yang et al., 1996).
671
Bioactive compounds extracted from CPH can exert potential health benefits and may therefore
672
be an alternative source of these important components (Fig.3).
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673 Conclusions
675
There is an urgent need for practical and innovative ideas to use this low cost CPH and exploit its
676
full potential increasing the overall sustainability of the cocoa agro-industry. Since changes
677
towards better efficiency and sustainability can also involve actions to improve the valorization
678
of by-products and of food related waste, the large amounts of organic compounds (i.e. pectin,
679
antioxidants, dietary fiber and minerals) contents in the CPH justify its valorization. CPH is a
680
good source of nutraceuticals, however their use in food industry is minimal partly due to limited
681
research. CPH potential food applications include extraction of aroma compounds, vegetable
682
gums, or texturizing agents, among others.
683
This by-product can be tailored through processing for diverse functionality and bioactivity.
684
Furthermore, CPH should be explored as a functional ingredient generating healthy and
685
innovative functional food, cosmetic and medical products.
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Conflict of interest: The authors declare no competing interests.
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Acknowledgements
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Author Karen Haydeé Nieto Figueroa, was supported by a scholarship from the Consejo
690
Nacional de Ciencia y Tecnología (CONACyT-Mexico) [grant number 854976]. The funding
691
provided by UAQ (Universidad Autónoma de Querétaro) and CONACyT-Fondos Mixtos
692
(FOMIX-QRO-279751) are appreciated.
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Table 1 Cultivation and production of cocoa from top ten countries.
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%Total 27.96 16.51 16.69 7.10 8.22 7.06 4.45 1.23 1.70 1.63
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Adapted from FAO, 2018.
%Total 32.96 19.23 14.71 6.53 5.30 4.79 3.98 2.42 1.82 1.26
Cultivation (Hectares) 2,851,084 1,683,765 1,701,351 723,853 838,046 720,053 454,257 125,580 172,940 165,844
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Country Cote D'Ivoire Ghana Indonesia Cameroon Nigeria Brazil Ecuador Peru Dominican Republic Colombia
Production (Tonnes) 1,472,313 858,720 656,817 291,512 236,521 213,843 177,551 107,922 81,246 56,163
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Table 2 Structural composition of cocoa pods. CPH 80.2 9.1 5.9 22.6 61.0
Epicarp 82.8 10.1 5.0 17.3 62.0
Mesocarp 64.0 4.6 1.9 29.5 80.0
ADF Nitrogen-free Crude fat (ether extract) Cellulose Hemicellulose Lignin Pectin Ca K P Mg mg/kg Na Zn Fe Cu
50.0 62.2 1.2 35.0 11.0 14.6 6.1 0.32 3.18 0.15 0.22
45.0 66.8 0.8 30.0 17.0 15.0 5.1 0.58 4.61 0.16 0.39
70.0 63.7 0.3 57.5 10.0 12.0 2.1 0.19 1.56 0.06 0.10
9.1 64.9 197.1 13.2
6.0 23.5 106.3 5.6
7.2 30.8 112.4 7.1
103.2
21.3
31.9
33.6
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Adapted from Sobamiwa & Longe (1994).
34.0 70.0 1.1 20.8 7.0 13.2 10.5 0.13 2.66 0.09 0.15
SC
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Mn
3.1 40.4 90.1 7.2
Endocarp 87.1 6.7 6.9 15.3 41.0
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Component (%) Moisture Ash Protein Crude Fiber NDF
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Table 3 Chemical composition (%) of cocoa pod husk. Reference
Moisture Protein
Ash
Lipid
Total carb
Vriesmann, Amboni 2011 Laconi & Jayanegara 2015 Esong et al., 2015 Lateef et al., 2008 Chun et al., 2016
8.5 NR 13.0 NR 10.5
8.6 8.4 8.0 8.2 2.1
6.7 NR 13.0 11.3 9.0
1.5 2.5 0.6 4.7 1.5
32.3 20.6 23.0 NR 17.5
36.6 55.7 50.0 18.3 NR
21.4 38.8 NR NR 24.24
Martínez et al., 2012
6.6
4.2
8.4
2.3
29.0
56.0
NR
Yapo et al., 2013
8.5
8.9
7.3
2.3
NR
59.0
19.4
Range
6.4-14.1
2.1-9.1
5.9-13.0
0.6-4.7
17.5-47.0
18.3-59.0
14.7-38.8
7.8
5.0
5.8
1.4
901
186
57.7
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Lignin
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TDF, total dietary fiber NR, not reported
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Variance
TDF
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0.74 0.35 0.26 NR 0.34 0.81 0.18 0.29 0.49 0.35 0.04 0.30 0.40 0.35 0.34 NR 0.18 0.40
Bean husk 1.50 0.80 0.70 NR 0.72 1.87 0.27 0.48 0.45 0.79 0.06 0.45 0.20 0.71 0.70 0.12 0.42 0.25
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CPH 0.80 0.44 0.22 0.09 0.29 0.77 0.21 0.24 0.43 0.40 0.05 0.37 0.38 0.41 0.30 0.04 0.21 0.44
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Amino Acids Aspartic acid Alanine Argininea Cystine Glycine Glutamic acid Histidinea Isoleucinea Leucinea Lysinea Methioninea Phenylalaninea Proline Serine Threoninea Tryptophan Tyrosine Valinea
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Total amino acids 6.09 5.82 10.49 EAI (%) 43.70 45.5 39.56 BCAA 1.11 1.18 1.18 AAA 0.58 0.48 0.87 Fischer Ratio 1.91 2.46 1.36 Lysine/Arginine 1.82 1.35 1.13 Arg+Glu+His 1.20 1.25 2.84 Met+Cys 0.14 0.04 0.06 a Essential amino acids EAI, essential amino acid index BCAA (Val+Leu+Ile) AAA (Phe+Tyr) Fischer ratio (BCAA/AAA) NR, not reported Data for CPH from Donkoh, Atuahene, Wilson & Adomako, 1991 Cocoa husk data adapted from Bonvehi & Coll, 1999
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Table 4 Amino acid content (g/100 g) of coca pod husk and cocoa husk.
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Table 5
a
Referencec 19 28 66 58 25 63 34 11 35 339
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Cocoa huskb 18 32 53 53 21 58 47 8 55 345
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His Ile Leu Lys Met + Cys Phe + Tyr Thr Trp Val Total
mg amino acid/g protein Cocoa pod huska 27.4 24.3 31.3 39.2 56.1 66.2 52.2 47.3 18.3 5.4 75.7 64.9 39.2 45.9 5.2 57.4 54.1 362.8 347.3
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Essential and reference amino acid pattern of cocoa pod husk and cocoa husk
Adapted from Donkoh, Atuahene, Wilson & Adomako, 1991 Adapted from Bonvehi & Coll, 1999 c FAO/WHO/Expert Consultation (1990) reference pattern for 2-5-year old child
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b
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Table 6 Cocoa pod pectin characteristics. NA-HYP 10.7 41.0 17.6 59.2 60.8 14.7 9.9 20.3 11.6 4.9 1.7 0.5 1.8
OP 9.8 56.6 17.1 66.0 69.9 3.6 3.9 16.8 10.0 2.8 2.7 0.7 tr
MOP 8.6 20.8 9.4 56.0 NR NR NR 19.7 18.0 3.0 2.5 0.8 1.0
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BW 12.6 42.6 19.2 44.6 51.9 5.5 8.3 25.4 14.4 5.7 5.0 1.9 3.1
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50W 7.5 37.0 29.0 45.1 55.8 9.6 9.8 21.7 21.4 4.5 4.6 1.9 0.9
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CA-HYP Yield 10.1 DE 40.3 DA 15.9 Uronic 65.1 Carbohydrate 64.0 Protein 13.8 Phenolic* 9.4 Gal 16.7 Rha 8.2 Glc 4.1 Ara 1.9 Xyl 1.4 Man 2.6
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Adapted from Vriesmann (2012) for CA-HYP; Vriesmann, Amboni & Petkowicz (2011) for 50W and BW and Vriesmann (2012) for OP and MOP; NR-not reported. CA-HYP and NA-HYP-citric and nitric acids high yield pectin, respectively; OP and MOP optimized and modified optimized nitric-acid extracted pectins from cocoa pod husk pectins; 50W and BW- water soluble pectins extracted from cocoa pod husk flour at 50 and 100 °C, respectively; DE-degree of esterification; DA-degree of acetylation. Values expressed as %, m/m, dry base. *Percentage of gallic acid.
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Table 7 Volatile compounds identified in cocoa pod husk (CPH), cocoa pod husk (CPHF) fermentation in solid-state (with Rhizopus stolonifera ) and cocoa.
+ +
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+ + + + +
+**
+ +
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+ + + + + + +
Cocoa
+ + + +
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+
CPHF
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CPH
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Compounds ALCOHOLS 5-Methyl-2-hexanol (2S,4S)-Pentanediol E-Linalol pyranoxide 1,2-Pentanediol 11-Methyldodecanol 1-Hexanol 1-Isopropyl-1-butanol 1-Methoxy-2-butanol 1-Nonanol 1-Octen-3-ol 2,3-Butanediol 2,6-Dimethylcyclohexanol 2-Ethyl-1-hexanol 2-Ethyl-3-pentanol 3,4-Dimethylpent-2-en-1-ol Epoxylinalol Linalool n-Tridecanol Phenylethyl alcohol (2-Phenylethanol) Trans-linalool oxide α-Hydroxytoluene α-Methylbenzyl alcohol (Benzyl alcohol) α-Terpinol ALDEHYDES (E,E)-2,4-Heptadienal Heptanal Methocycitronellal Nonanal Octanal Trans-2-hexenal Trans-2-octenal α-Toluic aldehyde (2-Phenyl acetaldehyde) β-Cyclocitral KETONES n-Amyl methyl ketone 1-Phenylethanone
+
+*, **
+ +
+ + +
+*, ** +** +*’** +**
+ + + + + + + + + + +
+ + +
+*
+
+**
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+ + + +
+
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+ + + + +
+ + + +
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+**
SC
+
+
+ +
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2-Dodecanone Pyrrole-α-methyl ketone Sulcatone trans-3-Octen-2-one β-Ionone ESTERS 1-Ethylpentyl acetate Ethyl iso-allocholate Methyl N-hydroxybenzenecarboximidoate Methyl salicylate Oxalic acid, bis(6-ethyloct-3-yl)ester β-Hydroxybutyric acid methyl ester ϒ-Hydroxybutyric acid cyclic ester ORGANIC ACIDS 10,12-Tricosadiynoic acid D,L-Mevalonic acid lactone Isovaleric acid HYDROCARBONS (-)-Aristolene 1,2,4-Trimethylcyclopentane 13-Phenylpentacosane 1-Heptadecene (+)-δ-Amorphene 2,6,10,15-Tetramethylptadecane 2-Cyclopropylidene-1,7,7-trimethylbicyclo[2,2,1]heptane 2-Ethylhexene 2-Methyl-n-hexacosane D-Limone Eicosane Heneicosane Isoledene Valerena-4,7(11)-diene α-Copane ϒ-Cadinene ϒ-Muurolene OTHERS Octanenitrile 1,4-Butanediamine Rosefuran
+
+**
+ + +
+
+ + + + + + + + + + + + +
+
+
+ + +
From Tiburcio, 2017, *From Alvarez et al., (2016), **From Aprotosoaie, Luca & Miron (2016)
Endocarp1
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Mesocarp1
Pod shell/husk2
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67-76%
Mucilage/pulp2
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Epicarp1
EP
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8.7-9.9%*
Bean shell/husk2 2.1-2.3%
Bean1 21-23% (30-40 beans/pod)
Fig 1. The cocoa fruit structures1 and wastes2 (with information of Babatope, 2005; Oddoye, AgyenteBadu & Gyedu-Akpto, 2013; Awarikabey et al., 2014; Sobowale et al., 2015; Papalexandratou, & Nielsen, 2016; *By difference).
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Fig. 2. Potential applications of cocoa pod husk.
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Fig. 3. Potential health benefits of bio-compounds found in cocoa pod husk.
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Highlights
1. CPH is an important industrial waste from which no benefit has been taken yet.
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2. The investigation is limited to produce high-value-added products from CPH. 3. Bio compounds such as dietary fiber, pectins, antioxidants and minerals can be obtained from CPH.
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4. The extraction of this bio compounds may have applications in food industry, among others, because their potential health benefit.
PII:
S0924-2244(17)30751-3
DOI:
10.1016/j.tifs.2018.09.022
Reference:
TIFS 2329
To appear in:
Trends in Food Science & Technology
Received Date: 27 November 2017 Revised Date:
20 September 2018
Accepted Date: 21 September 2018
Please cite this article as: Campos-Vega, R., Nieto-Figueroa, K.H., Oomah, B.D., Cocoa (Theobroma cacao L.) pod husk: renewable source of bioactive compounds, Trends in Food Science & Technology (2018), doi: https://doi.org/10.1016/j.tifs.2018.09.022. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Abstract Background
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Cocoa Pod Husk (CPH) is the main by-product from the coca industry constituting 67-76 % of the cocoa fruit weight. This waste represents an important, and challenging, economic, environmental renewable opportunity, since ten tons of wet CPH are generated for each ton of dry cocoa beans.
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Scope and Approach
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This review highlights the value that can be added to this industrial co-product to generate new pharmaceutical, medical, nutraceuticals or functional food products. Key Findings and Conclusions
The quality and functionality of cocoa pod husk (CPH) has being improving through processing (fermentation, enzymatic hydrolysis, and combustion, among others), guiding to
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their use as source of volatile fragrance compounds, lipase extraction, skin whitening, skin hydration and sun screening, ruminants’ food, vegetable gum, organic potash, antibacterial and nanoparticles synthesis with antioxidant and larvicidal activities. However, their
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exploration to produce high-value-added products, specially for the food industry, is limited
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as well as their potential health benefits. Cocoa pod husk, the main by-product from cacao industry (up to 76%), is an abundant, inexpensive, and renewable source of bioactive compounds like dietary fiber, pectin, antioxidant compounds, minerals and theobromine, justifying their valorization. This review highlights the value addition that can be achieved with this valuable industrial co-product to generate new pharmaceutical, medical, nutraceuticals or functional food products. Keywords: Cocoa by-product; Antioxidants; Dietary fiber; Pectin.
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Cocoa (Theobroma cacao L.) pod husk: renewable source of bioactive compounds
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Rocio Campos-Vegaa*, Karen H. Nieto-Figueroaa and B. Dave Oomahb
a
Programa de Posgrado en Alimentos del Centro de la República (PROPAC), Research and Graduate Studies in Food Science, School of Chemistry, Universidad Autónoma de Querétaro, 76010 Santiago de Querétaro, Qro, Mexico. b
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Rocio Campos-Vega: [email protected] B. Dave Oomah: [email protected] Karen H. Nieto-Figueroa: [email protected] * Corresponding author: Tel.: (55) 1921200 Ext 5590.
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Retired, formerly with the National Bioproducts and Bioprocesses Program, Pacific Agri-Food Research Centre, Agriculture and Agri-Food Canada, Summerland, BC V0H 1Z0, Canada.
1
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Abbreviations
2
50W
Pectin obtained by aqueous extraction in a water bath at 50 °C for 90 min
3
5-ASA
5-aminosalicylic acid
4
ABTS
2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid)
5
ADF
Acid detergent fiber
6
BHT
Butylated hydroxytoluene
7
CHPFR
Cocoa hull product from fermented and roasted beans
8
CKPFR
Cocoa kernel product from fermented and roasted beans
9
CLEA
Cross-linked enzyme aggregate
10
CPE
Cocoa pod extract
11
CPH
Cocoa pod husk
12
CPHE-AgNPs
Cocoa pod husk extract synthesized with silver nanoparticles
13
DA
Degree of acetylation
14
DE
Degree of esterification
15
DPPH
2,2-diphenyl-1-picrylhydrazyl
16
EE
Epicatechin equivalent
17
FRAP
18
GAE
19
HM
20
IC
21
IL
22
LDL
Low-density lipoprotein
23
LM
Low methoxy pectin
EP
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SC
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1
Ferric reducing antioxidant power
AC C
Gallic acid equivalent
High methoxy pectin Inhibitory concentration
Interleukin
2
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MCC
Microcrystalline cellulose
25
MIC
Minimum inhibitory activity
26
NA-HYP
Nitric acid extracted (pH 3.5, 100 °C, 30 min) high performance pectin
27
NDF
Neutral detergent fiber
28
NO
Nitric oxide
29
NSP
Non-starch polysaccharides
30
OP
Optimized pectin extracted with nitric acid (pH 1.5, 100 °C, 30 min)
31
ORAC
Oxygen radical absorbance capacity
32
Pas
Proanthocyanidins
33
PDMS
Polydimetylsiloxane
34
RE
Rutin equivalent
35
ROS
Reactive oxygen species
36
SPME
Solid-phase microextraction
37
TcANR
Anthocyanidin reductase
38
TcANS
Anthocyanidin synthase
39
TcLAR
Leucoanthocyanidin reductase
40
TDF
41
TE
42
TEAC
43
TNF-α
Tumor necrosis factor-α
44
TSH 565
Trinidad selection hybrid 565
45
WAP
Weeks after pollination
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Total dietary fiber
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Trolox equivalent
Trolox equivalent antioxidant capacity
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1. Introduction
48
Theobroma cacao L. is claimed to be the only commercially cultivated and most prominent in
49
the market among the 22 species of the Theobroma genus (World Agriculture, 2011). The
50
Theobroma cacao tree probably originated from divergent areas in Central and South America;
51
the Upper Amazon region (10,000 – 15,000 years ago), the upper Orinoco region of north east
52
Colombia and North West Venezuela, the Andean foothills of North West Colombia, Central
53
America from southern Mexico (Chiapas-Usumacinta) to Guatemala (Young, 1994). However,
54
Central and South American countries account for only about 14% of the current (2016, latest
55
available data) world cocoa production compared to those from African countries (⅔ of world
56
production) (FAO, 2018). Three countries, Cote D’Ivoire, Ghana and Indonesia together
57
cultivate and produce 61 and 67%, respectively of globally traded cocoa, whereas the top ten
58
countries account for ~93% of total world cocoa production (Table 1). Global cocoa production
59
is estimated at 4.59 million tonnes for 2017/2018 (ICCO, 2018). In 2016, the annual production
60
of cocoa, in decreasing order, by the eight largest cocoa producing countries were Côte D’Ivoire,
61
Ghana, Indonesia, Nigeria, Ecuador, Cameroon, Brazil and Malaysia. These countries together
62
produced about 4.23 million tonnes, representing ~ 95% of the world production (ICCO, 2015).
63
The cocoa bean constitutes one third (33%) of the fruit weight, leaving behind 67% of the fruit as
64
CPH as a waste by-product (Oddoye, Agyente-Badu & Gyedu-Akoto, 2013) (Fig.1). In other
65
words, ten tons of wet CPH are generated for each ton of dry cocoa beans, thereby representing a
66
serious disposal problem and an underexploited resource (Vriesmann, Amboni & Petkowicz,
67
2011). Pods are fully developed (100-350 mm long, 0.2-1 kg wet weight) from pollinated
68
flowers after 5-6 months. Three of the major cocoa diseases ((black pod, pod rot and cocoa pod
69
borer), also known as the disease trilogy (Evans, 2007) affect the pod specifically resulting in
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significant crop loss (Fowler & Coutel, 2017). Several initiatives have therefore been undertaken
71
to counter the severe crop loss, for example, development of varieties with thicker cuticle that
72
are more resistant to the common black pod rot and/or other pathogens (Fowler & Coutel, 2017).
73
Pod size, type and index (> 60 pods/tree, low disease incidence) are discriminating
74
morphological characteristics among cocoa genotypes and therefore variation on pod traits
75
(mainly pod size; length, width, thickness) may be associated with different morpho-geographic
76
groups (Ballesteros, Logos & Ferney, 2015).
77
The pod has been described as a natural laminated material consisting of three distinctly different
78
layers: epicarp, mesocarp and endocarp (outer, middle and inner pericarp, respectively) (Fig.1).
79
The endocarp is a soft whitish tissue protecting the delicate cocoa beans in a well-lubricated
80
inner chamber; the mesocarp displays a hard-composite structure able to hold the cocoa beans in
81
place even under high impact; and the outermost relatively soft layer is the yellow cover (when
82
ripe) that is directly exposed to sunshine, after which it turns black indicating rot due to
83
degradation (Babatope, 2005). These three distinct layers have been analyzed for their chemical
84
composition and compared to the whole CPH when incorporated as feed component in broiler
85
chick diets (Sobamiwa & Longe, 1994). High proportion of ash (47% CPH), hemicellulose
86
(50%) and minerals (K, Ca and P) (41-66%) predominated in the epicarp; fiber (crude, NDF and
87
ADF-44-48%) and cellulose (53 %) in the mesocarp; protein (50%), crude fat (50%) and pectin
88
(59%) in the endocarp (Table 2). The epicarp was the most limiting portion of CPH in the
89
feeding trial, presumably due to the antagonistic inhibitory effect of lignin and pectin on CPH
90
utilization in broiler diets (Sobamiwa & Longe, 1994).
91
Cocoa pod vary in color (from green [Forastero] to red [Criollo] or variable [Trinitario, the
92
Forastero x Criollo hybrid]) and thickness when ripe depending on their clone. Pod color is a
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reflection of the exocarp (the outer 1-3 mm layer fruit tissue of pods harvested 18 weeks after
94
pollination [18WAP]). This exocarp accumulates high levels of soluble and insoluble
95
proanthocyanidins [PAs] (170 and 8 mg/g dw, respectively) compared with flowers, leaves and
96
seeds due to highly expressed PAs synthesis genes [anthocyanidin synthase (TcANS),
97
anthocyanidin reductase (TcANR) and leucoanthocyanidin reductase (TcLAR)]. Furthermore,
98
epicatechin and catechin contents in the exocarp (18WAP) were: ~30 and 0.5 mg/g dry weight
99
(dw), respectively (Liu et al., 2013); such information is unavailable for the pericarp.
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100 2. Composition
102
The CPH constitutes 67-76% of the whole fruit by weight. It has been extensively investigated as
103
poultry and/or livestock feed because of its protein (5.9-9.1%), fiber (22.6-35.7%), crude fat
104
(1.2-10%) and mineral contents, among others (Oddoye, Agyente-Badu & Gyedu-Akpto, 2013).
105
Fresh manually chopped CPH (~ 1 cm thick) had the following percent composition: organic
106
matter 87, which includes crude protein, fiber, fat and nitrogen free extract (8.4, 55.7, 2.5 and
107
20.6%, respectively). The fiber fraction consisted of neutral and acid detergent fibers (80.7 and
108
74.6%), hemicellulose (6.0), cellulose (35.3) and lignin (38.8) with up to 40% in vitro
109
digestibility (dry and organic matter) (Laconi & Jayanegara, 2015). However, total carbohydrate,
110
total dietary fiber and lignin contents of CPH vary widely (Table 3). Moreover, chemical
111
composition depends upon the pretreatment used to process CPH, described in the next sections.
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2.1. Drying methods
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The moisture content of fresh CPH is about 90% (Vriesmann, Amboni & Petkowicz, 2011),
115
therefore quick drying is essential to prevent deterioration; this is achieved by slicing the fresh
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pods, drying to ~65% moisture, grinding into pellets and drying the pellets to 10% moisture prior
117
to storage (Oddoye, Agyente-Badu & Gyedu-Akpto, 2013). However, the type and conditions of
118
drying can affect CPH composition. For example, dry milled (< 1 mm) CPH has low lipids
119
(1.5% db), high ash (6.7%), protein (8.6%), total dietary fiber (36.6%) composed primarily of
120
insoluble fiber (74%) and soluble phenolics (4.6%) with carbohydrates and Klason lignin
121
accounting for 32.3% and 21% of CPH, respectively (Vriesmann, Amboni & Petkowicz, 2011).
122
CPH dried in a circulated air oven (80 °C, 1 day) and ground to a fine powder (22 µm) contained
123
cellulose (26.4%), lignin (24.2%), hemicellulose (8.7%), carbohydrate (17.5%), ash (9%), crude
124
protein (2.1%), fat (1.5%) and moisture (10.5%) (Chun, Husseinsyah & Yeng, 2016). Oven dried
125
(40-45 °C) milled (< 1.68 mm) CPH contained ~ 60% total dietary fiber, predominantly non-
126
starch polysaccharides (NSP) (42%), soluble sugars (13%), proteins (N x 6.25 = 9%), ash (8%),
127
fat (2%), phenolics (7% dw) and minerals (0.5-6%, mainly K, P, Ca and Mg); the reducing
128
sugars consisted mainly of fructose with some glucose (~ 64% of total soluble sugar). The ratio
129
of insoluble to soluble dietary fiber (4.2) in addition to high water retention and swelling
130
capacities (6.5 g/g and 7.3 mL/g) suggests that CPH may be suitable for improving bowel transit
131
time (Yapo et al., 2013). In contrast, air dried, ground (0.40-0.45 mm) CPH had high cellulose
132
(35.4% db), hemicellulose (37%), ash (12.3%), moisture (14%) and low lignin (14.7%) contents.
133
The high (74%) hollocellulose [cellulose + hemicellulose] of air dried CPH indicates its
134
potentially excellent quality performance for the pulp and paper industry (Daud et al., 2013).
135
Hemicellullose and lignin contents increased (27 and 14%, respectively), whereas cellulose
136
content decreased (10.7%) when boiled CPH was compared with directly dehydrated (48 °C, 48
137
h) and milled CPH (< 1 mm). For boiling, pods were washed, chopped into ~2.5 cm pieces,
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138
completely covered with water and heated (98 °C, 10 min), drained and dehydrated (48 °C, 72 h)
139
(Pérez et al., 2015).
140 2.2. Animal feed quality
142
Several studies focused on improving CPH feed quality by reducing its high fiber and increasing
143
protein contents. In this context, fresh anaerobically treated CPH (7 days, 3% molasses with
144
Phanerochaete chrysoporium inoculum that produces lignin degrading enzymes) significantly
145
increased protein content (19%, p < 0.05) concomitantly reducing crude fiber and lignin contents
146
(18%, p < 0.01). Ammoniated cocoa pod with 1.5% urea increased hemicellulose content the
147
most (2.2x) and reduced crude fiber content (8%), presumably by reducing its physical strength,
148
disrupting the silicified cuticular barrier and cleavages of some lignin-carbohydrate bonds. Cow
149
rumen exposure (3% w/w, anaerobic conditions for 7 days) reduced crude fiber, crude fat and
150
lignin contents (27.5, 92 and 12.6%, respectively) of fresh CPH (Laconi & Jayanegara, 2015).
151
Ground (< 250 µm) CPH treated with 0.1 M NaOH (solid to liquid ratio 50 g/L, room
152
temperature, 1 h) incurred 26.6% weight loss, primarily due to lignin removal and changed its
153
structure from compact and smooth to more porous and rough surface increasing its adsorption
154
capacity (Pua et al., 2013). Such alkalization generally encourages dairy feed intake, increases
155
buffering capacity and reduces the initial acid load of the diet thereby enabling more feed in the
156
ration without triggering acidosis. CPH crude protein and total soluble carbohydrates increased
157
(36%) with concomitant reduction in crude fiber, hemicellulose, cellulose, lignin and total
158
tannins (17, 21, 26, 17 and 88%, respectively) on solid-state fermentation (Pleurotus ostreatus
159
with 0.075% MnCl2 [w/w], 5 weeks) (Alemawor et al., 2009a). Solid state fermentation of CPH
160
with Rhizopus stolonifera LAU 07 increased protein (95%) and reduced fiber (7.2%) contents
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(Lateef et al., 2008). Ten days’ fermentation with Phanerochaete chrysosporium significantly (p
162
< 0.05) increased crude protein of CPH compared to unfermented CPH enabling the highest
163
lignin degradation (39%) and efficient CPH bioconversion (Suparjo & Nelson, 2017). Other
164
fermentation such as Rhizopus oryzae (28 °C, 6 days) of CPH and rice bran (1:9 ratio mixed
165
substrate, sterilized at 121 °C, 15 min) also increased CPH protein content (14-15%), and free
166
amino acids (glutamic acid 34x, aspartic acid 3.7x, alanine 4.6x and valine 1.2x) (Sriherwanto et
167
al., 2016). Furthermore, enzyme cocktails (Pentopan MonoBG + Viscozyme L; Viscozyme L +
168
Pectinex 5XL and Pentopan MonoBG + Viscozyme L + Pectinex 5XL) effectively maximized
169
sugar release (42-53%) from CPH with a corresponding reduction (7-14%) in crude fiber and
170
NSP enhancing its use in poultry feed (Alemawor et al., 2009b). In vitro natural fermentation
171
(washed pods, sun-dried for 14 days and milled into powder; anaerobic fermentation for 3 days
172
at room temperature) increased protein (22-116%), ash (11-31%), fat (68-75%), moisture (49-
173
142%) and flavonoid (29-101%) contents of pod husk depending on cocoa clones (Criollo,
174
Forastero, Trinitario). The fermentation improved CPH by reducing crude fiber (59-73%),
175
phytate (20-81%) and tannin (41-87%) with negligible changes in carbohydrate contents of CPH
176
(Shodehinde & Abike, 2017).
177
CPH was investigated to develop and transfer appropriate technologies for animal feed
178
production as part of a pilot scale production and commercialization of cocoa by-products in
179
Ghana in a ten-year project (ICCO/CFC/CRIC, September 1993-July 2003). Full annual
180
production capacity was about 5,500 tonnes of pelletized animal feed from CPH. The cost
181
benefit and financial viability analysis indicated that the production of animal feed from CPH
182
was unlikely to be economically feasible or profitable (Adomako, 2006). However, a recent
183
study showed that sun dried CPH can be used at high inclusion rate (20%) to achieve similar
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feed intake and live weight gains with rabbits fed maize-based diets. In addition, CPH was
185
economically beneficial compared to maize in feeding growing rabbits (Esong et al., 2015).
186 2.3. Biomass
188
CPH ash has been extensively investigated particularly for its potential application as partial
189
replacement of and/or combination with fertilizers, biogas and biofuels (Dias, 2014). However,
190
the high ash content can inhibit the combustion process since oxygen cannot easily penetrate the
191
ash to reach the burning biomass (Martínez-Ángel, Villamizar-Gallardo& Ortíz-Rodríguez,
192
2015). Potassium (2.5-4% db) is the predominant CPH mineral, often up to 7% (db) in CPH from
193
cocoa grown in Ghana accounting for nearly three-quarters (~70%) of the total ash weight
194
(Donkoh et al., 1991). Although the high potassium concentration has been exploited for soap
195
production, it can lead to potential fouling problems and consequently to formation of deposits
196
and agglomerations when fired in boilers for combustion or in the gasification process
197
(Martínez-Ángel, Villamizar-Gallardo& Ortíz-Rodríguez, 2015). Other important CPH minerals
198
(Ca, 0.3-0.8%; Mg, 0.02-0.06%; P, 0.04-0.12%; S, 0.02-0.05% and Si, 0.5% db) together
199
accounts for 19.9, 21.9 and 27.4% of ash weight (Dias, 2014; Martínez-Ángel, Villamizar-
200
Gallardo& Ortíz-Rodríguez, 2015). High proportion of ash and minerals (47 and 41-66% of the
201
total pod, respectively) predominate in the epicarp, the most lignified cocoa pod pericarp
202
(Sobamiwa & Longe, 1994).
203
The ten-year project (ICCO/CFC/CRIC) also investigated potash (ash) production from CPH for
204
soft soap manufacturing. The potash from CPH is high in potassium carbonate content and as
205
such makes better soap than others in the market. Potash production from CPH was deemed a
206
profitable venture based on economic analysis (Adomako, 2006).
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207 2.4. Proteins
209
The protein characteristics of CPH have not been adequately investigated. Reports on the protein
210
characteristics of CPH are scarce and limited probably due to its reduced growth, food utilization
211
efficiency and/or protein efficiency ratio observed earlier in animal diets (Donkoh, Atuahene,
212
Wilson & Adomako, 1991; Fagbenro, 1995). One hundred and forty-four proteins were
213
identified from 700 protein spots detected in CPH proteome by MALDI-TOF/TOF MS in
214
combination with 2-DE analysis (Awang, Karim & Mitsui, 2010). Almost half of the identified
215
proteins (48%) are involved in primary and energy metabolism functioning as housekeeping
216
proteins. These proteins are involved in general cellular processes related to glycolysis (enolase,
217
triose-phosphate isomerase, phosphoglycerate kinase), Calvin cycle, and Tricarboxylic acid cycle
218
(malate dehydrogenase). Six proteins including leucoanthocyanidin dioxygenase and
219
anthocyanidin reductase, cinnamyl alcohol dehydrogenase and polyphenol oxidase pertained to
220
the phenylpropanoid pathway responsible for the production of secondary metabolites (lignins,
221
flavonoids, and phytoalexins). Caffeic acid 3-O-methyltransferase and polyphenol oxidase
222
involved in lignin synthesis were differentially expressed in the two clones, LAF17 with smooth
223
and waxier pod surface and the cocoa pod borer-resistant ICS39 clone with harder and thicker
224
sclerotic layer (Awang, Karim & Mitsui, 2010).
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2.4.1 Amino acids
227
The average protein content in CPH (generally ~ 8.5%) is lower than those previously reported
228
in cocoa bean husk (15%) (Bonvehí & Coll, 1999). Data on amino acids is limited to a single
229
report (Donkoh et al., 1991) of CPH compared to an earlier (1984) study. CPH protein has
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higher (10-15%) levels of total and essential amino acids and above half (~ 56%) of the total
231
amino acid content of bean husk (Table 4 and 5) due to asparagine, alanine, arginine, glycine,
232
glutamine, lysine, serine, threonine and tyrosine concentrations. CPH had twice the acidic
233
(aspartic + glutamic acids) and basic (arginine + histidine + lysine) amino acids, proline and
234
valine content of bean husk, whereas the levels of histidine, leucine and methionine were similar
235
in both husks. CPH protein has higher Fischer ratio and lower content of aromatic amino acids
236
than those of cocoa bean husk. The lysine/arginine ratio, a determinant of the cholesterolaemic
237
and atherogenic effects of protein, is low for CPH protein, but higher than those of cocoa bean
238
husk. A soluble cocoa fiber product (0.79 PER, contributing 28 g protein/kg diet) obtained by
239
enzymatic treatment of cocoa bean husk reduced the negative effects of dietary-induced
240
hypercholesterolemia in an animal model (Ramos et al., 2008).
241
The biological value (37.6%) of bean husk (Bonvehí & Coll, 1999) corresponds to the apparent
242
digestibility of fresh and sun-dried cocoa pod husk (37.8 and 32.1%, respectively) reflected in
243
their nitrogen retention (17 and 12.2%, respectively) in sheep rations containing 53% (dm) CPH
244
(Wong & Hassan, 1988). In addition to protein, CPH also contains theobromine (~ 0.2%), a
245
commercially significant appetite stimulant for ruminants (Trout, Zoumas & Tarka, 1978).
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2.5. Pectin
248
2.5.1. Extraction methods and properties
249
Hot water extracted pectic material from CPH contains galacturonic acid (27%), galactose
250
(21%), rhamnose and arabinose, whereas ammonium oxalate (0.5%) extraction resulted in
251
depolymerized pectin (Blakemore, Dewar & Hodge, 1966). Pectin has also been extracted with
252
mild acetic acid (0.2 N, pH 2.8, 1:3 w/v) from ripe CPH with higher yield (8-11%, db) than those
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reported earlier (Dittmar, 1958 cited in Adomako, 1972) from sun dried CPH (5.3-7.1%). The
254
acetic acid extracted pectin had the following composition: ash (8.9-9.8% comprising Ca and K
255
as major components), protein (1.1%), galacturonic acid (62%), galactose (4.65%), rhamnose
256
(2.9%), arabinose (1.7%) and xylose (1.2%) with carbohydrate composition similar to apple
257
pectin (Adomako, 1972).
258
Investigation of CPH pectins intensified after a hiatus of almost four decades, particularly in a
259
series of studies by the Brazilian group (Vriesmann et al., 2011-2017). Nitric acid (pH 1.5, 100
260
°C, 30 min) extraction from CPH produced highly esterified (56.5% degree of esterification
261
[DE]) and acetylated (17.1% degree of acetylation [DA]) pectin (9.8%, db yield) with uronic
262
acid (66%), homogalacturonan and rhamnogalacturonans; this pectin is considered high methoxy
263
(HM) pectin (Vriesmann, Teófilo & Petkowicz, 2011). Hot aqueous extraction (50 °C [50W] and
264
100 °C [boiling water], 90 min, 1:25, w/v) was used in a follow up study to obtain 7.5 and 12.6%
265
pectin from milled and dried CPH (Vriesmann, Amboni & Petkowicz, 2011). The water soluble
266
pectins were highly acetylated (19.2 and 29% DA), composed of low methoxy
267
homogalacturonans (37 and 42.3% DE) with rhamnogalacturonan insertions exhibiting non-
268
Newtonian shear-thinning behavior (Vriesmann, Amboni & Petkowicz, 2011). CPH treatment
269
(aqueous citric acid pH 3, 95 min, 95 °C) yielded 10.1% (db) pectin with low moisture (2.7%),
270
high carbohydrate (64%), protein (13.8%), low phenolics (9.4%), low-methoxy, highly
271
acetylated homogalaturonan (DE, 40.3%; DA, 15.9%) containing 65% uronic acid and exerting
272
non-Newtonian shear –thinning behavior (Vriesmann, Teófilo & Petkowicz, 2011). The pectin
273
forms gels under low pH/high sucrose content, suggesting applications as additive in acidic
274
conditions. Similar highly acetylated (DA, 17%) CPH pectin was obtained by optimized nitric-
275
acid-mediated extraction of CPH and the best gel properties were observed at 1.32% galacturonic
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acid equivalent concentration, 60% sucrose and pH 2.7, demonstrating its use as a gelling and
277
thickening agent (Vriesmann & Petkowicz, 2013). High yield (10.7%, db) of CPH pectin (DE,
278
41%; DA, 17.6%) was obtained with nitric acid extraction (pH 3.5, 100 °C, 30 min) under reflux
279
with high uronic acid (59.2%), significant proportions of galactose (20.3%) and rhamnose
280
(11.6%). The large pectic molecule was purported to be primarily responsible for its gelling
281
properties; forming gels at low pH (< 2.5) and water activity (1% w/v; 60% v/v ethylene glycol)
282
(Vriesmann & Petkowicz, 2017). Pectin extracted with hot acid (nitric acid [pH 3, 100 °C, 30
283
min]) or citric acid [pH 3, 95 °C, 95 min]) had similar chemical composition (65% uronic acid,
284
8.2% rhamnose, 16.7% galactose, 40.3% DM and 15.9% DA) (Vriesmann & Petkowicz, 2017).
285
Extraction conditions greatly affect yield and chemical properties of CPH pectins (Table 6).
286
Thus, pectin yield from cocoa husks varied (3.4-7.6%) with uric acid content ranging from 31-
287
65% and degree of methylation (7-58%) depending on solvent and extraction conditions (Chan
288
& Choo, 2013). For example, the highest pectin yield (7.6%) is obtained with hot citric acid
289
(1.25, w/v; pH 2.5, 95 °C, 3h), whereas hot water under similar conditions (1.25 w/v; 95 °C, 3h)
290
extracts pectin with the highest uronic acid (65%) content. Furthermore, pectin yield increases
291
significantly (p < 0.05) with reduction in substrate –extractant ratio (from 1:25 to 1:10 w/v)
292
using citric acid at pH 2.5. Water and hydrochloric acid extracted low methylated (LM) pectin
293
(7.2-39.3% DM), whereas citric acid (1:25 w/v, pH 2.5, 1.5 h) produced wide degree of
294
methylation range and high methylated (HM) pectin (58%) (Chan & Choo, 2013). Similarly,
295
acidified (1N HNO3) water treatment (1:25 w/v, 75 °C, 90 min) differing in pH (1, 2 or 3)
296
produced CPH pectins varying in yield (3.7-8.6%), galacturonic acid content (51-75%),
297
galactose (5.2-13.9%), total neutral sugars (7.9-24.3%), calcium (48.6-155.7 µmol/g), degree of
298
methylation (37-52%), DA (3.2-9.8%), intrinsic viscosity (162-304 mL/g) and viscosity-average
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molecular weight (43-82 kDa) (Yapo & Koffi, 2013). Hydrochloric acid (1.58 M) extraction (62
300
°C, 4.8 h) produced CPH pectin (yield =11.7%) with high DE (58.5%) and galaturonate content
301
(49.9%) (Hutomo, Rahim & Kadir, 2016). The highest CPH pectin yield (23.3% db) was
302
obtained with hot water (1.05 g/mL) extraction in a water bath (50 °C); the pectin had low
303
moisture (0.2%), ash (1%), high DE (26.8%) and swelling (357, 275 and 360 swelling index in
304
0.1M HCl, phosphate buffer [pH 6.8], and distilled water, respectively) (Adi-Dako et al., 2016).
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307
The hot water soluble pectin had Hausner ratio of 1.17, compressibility index of 14.6% and angle
308
of repose ~38° indicating good flow properties. It was predominantly rich in potassium (2.27%),
309
magnesium (0.22%), phosphorous (0.096%) and sulphur (0.094%), copper (10.9% of minor
310
elements), zinc (8.3%) and nickel (3.7%). The pectin exhibited dose-dependent moderate
311
antimicrobial activity (minimum inhibitory activity-MIC) against Staphylococcus aureus and
312
Escherichia coli (MIC: 0.5-1 mg/mL); Pseudomonas aeruginosa, Bacillus subtilis, and
313
Salmonella typhi (MIC: 1-2 mg/mL); and Shigella spp (Adi-Dako et al., 2016). Freeze-dried hot
314
water extracted CPH pectin (26.8% DE) and 4% hot aqueous citric acid soluble pectin showed
315
no toxicity on the major haematological indices, bilirubin levels and the spleen when
316
administered up to 71.4 mg/kg in Sprague Dawley rats for 90 days (Adi-Dako et al., 2018). CPH
317
pectin can therefore be safely incorporated in pharmaceutical formulations as natural polymer
318
excipients.
319
The high methoxy pectin obtained by nitric acid was used to prepare microparticles containing 5-
320
aminosalicylic acid (5-ASA) for drug delivery to the colon. The microparticles (1:1, 2:1, 3:1 and
321
4:1) obtained by spray drying showed better drug retention with increased pectin content
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(Vriesman, 2012). Saponification of this high methoxy pectin promoted distinct increase in
323
biological activities; it triggered the secretion of pro-(NO, TNF-α and IL-12) and anti- (IL-10)
324
inflammatory mediators in peritoneal microphages demonstrating its application as an immune
325
modulator (Amorim et al., 2016).
326
CPH gums have been described as essentially acidic protein-polysaccharide containing 3-5%
327
protein with galactose, glucose, rhamnose, arabinose, galacturonic acid, and glucuronic acid in
328
1:0.21:1.9:0.2: 4.2:1.3 molar ratios with high (35-40%) uronic acid content. Crude gum yield
329
(9.1% of dry CPH) similar to reported polysaccharide yields (8-11%, dw) had high potassium ion
330
(9.6% dw) concentration similar to previously observed CPH ash content (9%) (Figueira, Janick
331
& BeMiller, 1994). Alcohol extracted gum (5.25%) (sun dried samples were boiled twice with
332
70% EtOH, then extracted twice with boiling 70% MeOH) from CPH exhibited pseudoplastic
333
behavior obeying the power law flow model with high viscosity (1053 mPas at 5%
334
concentration) similar to karaya gum (1160 mPas at 3% concentration) (Samuel, 2006). CPH
335
gum can potentially substitute costly karaya gum, at least partly in many formulations.
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2.6. Phenolics and antioxidants
338
2.6.1. Effect of the extraction methods
339
Total phenolic content of CPH ranged between 46 to 57 mg gallic acid equivalent (GAE)/g dry
340
matter (Karim et al., 2014b). However, lower total phenolic (2.1-3.7 mg GAE/g) has been
341
reported depending on the location of cocoa growth and solvent system used in extraction. For
342
example, methanol: acetone extracted higher total phenolic of CPH from two locations (Cone
343
and Taura) in Ecuador (3.53 and 3.65 mg GAE/g) than ethanol (2.07 and 2.27 mg GAE/g) that
344
was reflected in their antioxidant activities (38 and 44 µM TE/g ABTS, 34 µM TE/g DPPH and
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4.6 µM TE/g FRAP) compared to ethanol (24 µM TE/g ABTS, 18 and 21 µM TE/g DPPH and 2
346
µM TE/g FRAP) (Martínez et al., 2012). Acetone-water (7:3 v/v) and ethanol (70%) extracted
347
CPH phenolics (94.92 and 49.92 mg GAE/g extract, respectively) with the acetone extract
348
exhibiting antifungal activity against Fusarium oxysporum (Mu’nisa, Pagarra & Maulana, 2018).
349
Pods chopped into 1 cm2, sun dried and ground (1 mm) contained phenolic acid (19.6-49.5 mg
350
GAE/g) and flavonoid (4-22.4 mg rutin equivalent [RE]/g) depending on the clone (Karim et al.,
351
2014a). Several polyphenolic compounds were identified by LC-MS/MS in CPH extract (80%
352
aqueous ethanol, 40 °C, 30 min); phenolic acids (protocatechuic acid and its derivatives and p-
353
hydroxybenzoic acid), flavonoids (apigenin, rhamnetin, kaempferol derivatives, flavone
354
derivatives), luteolin, apigenin and linarin (Karim et al., 2014b). Some of these phenolics
355
quantified in fresh CPH consisted of catechin (36%), quercetin (21%), epicatechin (21%) and
356
gallic (11.3%), coumaric (6.5%) and protocatechuic (4.5% of total phenolic compounds) acids
357
(Valadez-Carmona et al., 2017). The total phenolics, flavonoid and flavonol contents of the fresh
358
CPH was 3.24 mg GAE/g, 0.97 mg and 0.34 mg epicatechin equivalent (EE)/g, dw respectively
359
(Valadez-Carmona et al., 2017).
360
Aqueous ethanol (80%) extract of the pod exhibited high antioxidant activity (0.26 and 0.57 EC50
361
for DPPH or 20.35 and 45.26 mg/ml) about four times lower than that of BHT. The extract (1 g)
362
also displays strong ferric reduction potential reducing 80 mol of Fe2+ from Fe3+ (Karim et al.,
363
2014a). Ethanol extract (50% aqueous ethanol) from a Colombian cocoa clone (TSH 565)
364
contained epicatechin (0.25-0.35 mg/g) and varied in antioxidant activity (FRAP 137-169 µM
365
TE/g; ABTS 116-230 µM TE/g; ORAC 252-343 µM TE/g) depending on the extraction process
366
(Sotelo, Alvis & Arrázola, 2015). Low- molecular weight soluble phenolics obtained by three
367
step sequential extraction exhibited strong antioxidant activities (85% DPPH inhibition, EC50 =
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25 g/g; 52 µM TE/g ABTS and 130 µM TE/g FRAP) significantly higher (p < 0.05) than those of
369
fermented and roasted cocoa hull and kernel products (Yapo et al., 2013). The antioxidant
370
capacity of fresh CPH was 30.6 and 15.1 µM TEAC/g (ABTs and DPPH assays, respectively)
371
similar to those reported earlier (38 and 33 µM TEAC/g for ABTS and DPPH, respectively,
372
(Martínez et al., 2012).
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373 2.6.2. Effect of processing method
375
Processing affects CPH polyphenolic content. For example, microwave drying (595 W, 11.5
376
min) increased total phenolics (3.4x), flavonoid (5.8x), flavonol (5.8x), phenolic compounds
377
(catechin 2.2x, quecetin 73%, epicatechin 3.3x, gallic acid 3.8x, coumaric acid 4.9x and
378
protocatechuic acid 5.9x) and antioxidant capacity (ABTS 2.9x and DPPH 3.9x) of CPH,
379
compared to hot air and freeze-drying (Valadez-Carmona et al., 2017). Pre-treatment to prevent
380
enzymatic (polyphenol oxidase) oxidation such as soaking (1% citric acid, 15 min) before sun
381
drying significantly (p < 0.05) increased total phenolic (24%) and flavonoid (13%) contents
382
compared to unsoaked sun dried CPH (Sartini, Asri & Ismail, 2017). Furthermore, ultrasound
383
(60 Hz, 2 h) extracted significantly (p < 0.05) higher phenolic than those obtained by agitation
384
(200 rpm, 6 h) (21-23 vs 16-19 mg GAE/g) (Sotelo, Alvis & Arrázola, 2015). Total phenolic
385
content (extracted by 50% (v/v) ethanol solution, 35 °C, 150 rpm, 2h) of dried (60 °C, 24 h) CPH
386
decreased 42% on sterilization (autoclave 120 °C, 20 min) and increased 37% after solid-state
387
fermentation with Rhizopus stolonifera NRRL 28169 ((30 °C, pH 5.5, 72 h), presumably due to
388
the generation of new bioactive molecules (Tiburcio, 2017). HPLC of aqueous ethanol extract of
389
CPH revealed the presence of 24 phenolic peaks with 3 major unidentified components
390
accounting for 72% of the total peak area compared to 14 peaks with 3 major peaks accounting
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for 81% of the total peak area (33% higher than CPH) in R. stolonifera fermented CPH.
392
Furthermore, CPH displayed higher concentrations of phenolic compounds compared to
393
fermented CPH although the latter exhibited superior antioxidant activity (DPPH and ORAC)
394
than CPH, presumably due to polyphenolic hydrolysis and/or transformation (Tiburcio, 2017).
395
Industrial fermentation such as those used for cocoa bean (6 days in wooden boxes) from
396
Mexican cocoa genotypes can be simulated (applied) to increase phenolic content of CPH similar
397
to those reported for cocoa bean husk (Hernández-Hernández et al., 2018). Total polyphenol and
398
tannin levels decreased by ≥ 11 and ≥ 89%, respectively after fungal treatment (Aspergillus niger
399
or Talaromyces verruculosus 0.15% w/w, moisture 1:3 w/v, 7 days) to eliminate theobromine
400
and reduce ochratoxin A content of CPH (Oduro-Mensah et al., 2018). Similar reduction in
401
phenolic concentration occurred on cocoa shell after solid state fermentation with Penicillium
402
roqueforti without affecting flavonol and anthocyanin contents, indicating significant
403
consumption of these metabolites during fermentation (Lessa et al., 2018). However, CPH
404
antioxidant capacity generally increased after solid state fermentation by Rhizopus stolonifera
405
LAU 07. The IC50 of the CPH substrates (fermented/unfermented – 5.5/14.7 mg/mL) increased
406
2.7 folds in DPPH-scavenging capacity (Lateef et al., 2008).
407
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2.7. Volatiles
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A recent study (Tiburcio, 2017) identified 50 volatile compounds in CPH by SPME (5/95%
410
Carboxen/Polydimethylsiloxane, 75 µm) followed by desorption and separation on 95% PDMS,
411
5% phenyl GC column (30 m x 0.25 mm, 0.25 µm film thickness). These compounds consisted
412
of 16 alcohols, 11 hydrocarbons, 8 aldehydes, 7 ketones, 5 esters, 2 amines and isovaleric acid.
413
Solid-state fermentation of CPH with Rhizopus stolonifera NRRL 28169 (incubated in a rotatory
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shaker 100 rpm, 30 °C, pH 5.5, 7 days) reduced the number (from 50 to 33) of identified volatile
415
compounds. Thirteen volatile compounds [(2S,4S)-(+)-pentanediol, 1-methoxy-2-butanol, 2,3-
416
butanediol, epoxylinalol, phenylethyl alcohol, D-limonene, α-copaene, ethyl iso-allocholate,
417
methyl N-hydroxybenzenecarboximidoate, methyl salicylate, nonanal, octanal, and α-toluic
418
aldehyde] were present in both fermented and unfermented CPH (Table 7).
419
Ten volatiles in CPH are present in cocoa, five (2,3-butanediol, phenylethyl alcohol, nonanal, α-
420
toluic aldehyde and methyl salicylate) of which have been identified in both unfermented and
421
fermented CPH. 2,3-Butanediol imparts sweet chocolate notes reminiscent of the natural odor of
422
cocoa butter, whereas α-toluic acid (2-phenylacetaldehyde) and methyl salicylate confer floral
423
and nutty sensory perceptions, respectively (Aprotosoaie, Luca & Miron, 2016). Phenylethyl
424
alcohol (2-phenylethanol) is associated with the typical “floral” volatiles of dark chocolate
425
extracts resulting from microbial actions occurring during fermentation. It is also the most odor-
426
active compound in dried and fermented cocoa (Álvarez et al., 2016). Linalool, trans-linalool
427
oxide and isovaleric acid were identified only in nonfermented CPH.
428
phenylethanol are major alcohols in roasted nibs; linalool concentrations in cocoa vary with its
429
origin: West Africa (0-0.5 mg/kg), Malaysia (0-0.2 mg/kg) and Ghana (0.2-0.8 mg/kg). The
430
linalool/benzaldehyde ratio has been proposed as a flavor index for cocoa. Isovaleric acid has
431
been identified in fermented cocoa beans as a result of sugar metabolism. Benzyl alcohol, also
432
known as α-methylbenzyl alcohol and 1-hexanol were identified in the fermented CPH; the latter
433
confers fruity and green notes in cocoa (Aprotosoaie, Luca & Miron, 2016).
Linalool and 2-
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2.8. Other compounds
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The methyl ester of linoleic acid (9,12-octadecadenoic acid) is one of the three major
437
components of CPH ethanol (70%) extract (Mu’nisa, Pagarra & Maulana, 2018). Linoleic acid in
438
turn is the predominant (53% of the total peak area) component of bio-oil obtained from sun-
439
dried (1-2 weeks) and ground (< 1 mm) cocoa pod husks by pyrolysis (550-600 °C). The bio-oil
440
also contained palmitic, carboxymethoxy succinic, linolenic, hexanoic, caproic, pyruvic, azelaic
441
and caprylic acids (13.2, 5.6, 5.3, 3.8, 3.3, 2.2, and 2.0% of total peak area, respectively) (Adjin-
442
Tetteh et al., 2018). Linoleic acid was also present in petroleum extract (Soxhlet) of sundried (10
443
days) ground CPH powder along with other fatty acids (arachidic, palmitic, pentadecanoic,
444
stearic acids) and other compounds (Adewole et al., 2013).
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445 3. Uses and applications
447
3.1. Fragrance compounds
448
CPH impregnated with nitrogen sources has been used for the synthesis of volatile fragrance
449
compounds by a Rhizopus oligosporous strain. Solid-phase microextraction (SPME) coupled
450
with GC-MS revealed maximum abundance of volatile fragrance components when CPH was
451
fermented for 96 h (1 x 105 inoculum spores/g, 24 °C, 4-5 mm substrate thickness and pH 6.5).
452
The highest numbers and abundance of volatile compounds was obtained with L-phenylalanine
453
as the nitrogen source. This demonstrates CPH potential as an inert support for secondary
454
metabolite bioconversion by fungus strain, i.e. volatile fragrance components in a solid state
455
fermentation system (Norliza, 2006). Another study by the same group (Norliza & Rozita, 2006)
456
showed the release of three compounds, benzaldehyde, phenetyl alcohol and phenyl
457
acetaldehyde when CPH (1 mm) was mixed with L-phenylalanine (2.5, 3.0 and 3.5%, w/w) and
458
fermented with Rhizopus oligosporous for 4 days.
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Furthermore, many of the compound identified by Tiburcio (2017) in both fermented and
460
unfermented CPH [1-octen-3-ol, nonanal, octanal, α-terpineol, ionones, methyl salicylate, β-
461
hydroxybutyric acid methyl ester, α-methylbenzyl alcohol and rosefuran] are well-known
462
flavor/aroma compounds (Table 5).
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463 3.2. Skin & hair treatment
465
The skin whitening effect of CPH aqueous ethanol (80%) extract was demonstrated based on in
466
vitro mushroom tyrosinase assay and sun screening effect (UV-absorbance at 200-400 nm
467
wavelength) (Karim et al., 2014b). Resveratrol and fatty acids, such as linoleic acid, in CPH has
468
been reported to have in vitro skin whitening properties without adverse effects (Ohguchi et al.,
469
2003; Parvez et al., 2006). When tyrosinase enzyme activity is inhibited, melanin production is
470
reduced, resulting in a fairer skin (Karim et al., 2014b). The extract induced human fibroblast
471
cell proliferation at low concentration and displayed UVB sunscreen potential superior than
472
commercial UV-protecting agents (avenobenzone and octylmethoxycinnamate). It was also
473
highly effective against collagenase, better than kojic acid (a skin-lightening cosmetic product),
474
although lower against elastase and tyrosinase than pine bark extract (positive control, effective
475
in maintaining cellular function which affects the skin condition) (Karim et al., 2014b).
476
Application of gel from the CPE extract reduced in vivo skin wrinkles within 3 weeks and after 5
477
weeks (6.4, and 12.4%, respectively); skin hydration also increased (3%) after 3 weeks’
478
application (Karim et al., 2016).
479
Cocoa pod ash is a commercial ingredient used as a component of African Black Soap that in
480
turn is used in environmentally-free conditioning cleansing cream (Koiteh, 2013).
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3.3. Animal appetite stimulant
483
CPH stimulates appetite in ruminants due to the presence of theobromine (0.2-0.25% weight),
484
particularly at low theobromine (0.05-0.1%) inclusion in the feed. This commercially significant
485
appetite stimulation enables weight gain in a short time to bring animals to market sooner due to
486
increased feed consumption (Trout, Zoumas & Tarka, 1978). CPH contains approximately 0.15-
487
0.4% (w/w) theobromine limiting its inclusion to 13.7% (w/w) in animal feed due to the 300
488
mg/kg theobromine content limit imposed by the European Union (Oduro-Mensah et al., 2018).
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491
The pod may be used as a texturizing agent after drying (30-60% moisture) and grinding to
492
extremely small size (0.05-0.12 inch). Juice from the pods can be separated from the
493
parenchymatous tissues (cocoa fruit flesh representing 82% of the fruit) to form a pure
494
hydrocolloid derivative that can be used as conventional vegetable gum (Drevici & Drevici,
495
1980). The juice imparts high viscosity, preserves aroma, odor and color of products and
496
improves rheological properties due to its carbohydrate composition (galacturonic acid,
497
galactose, rhamnose, arabinose and xylose with galacturonic acid) (Drevici & Drevici, 1980).
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3.5. Antibacterial
500
Some CPH extracts are effective against two major bacteria responsible for most hospital
501
infections: Gram negative Salmonella choleraesuis (1 mg/mL MIC) and Gram positive
502
Staphylococcus epidermidis (2.5 mg/mL MIC). The extract also exhibited strong inhibitory
503
activity against Pseudomonas aeruginosa (Santos et al., 2014). Fractionation (solvent partition)
504
of CPH extracts enabled identification of bioactive molecules such as phenols, steroids, or
23
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terpenes. The extract was prepared by air drying (3 months) chopped pieces of husks (1.5-2.5 cm
506
wide), pouring distilled water over the husks and collecting the aqueous extract that was dried
507
(room temperature, 72 h) and ground (< 1.5 mm). The dry crude water extract represents 0.16%
508
of the fresh CPH (Santos et al., 2014).
509
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3.6. Nanoparticles larvicidal & antioxidant
511
CPH extract synthesized with silver nanoparticles (CPHE-AgNPs) exhibited antioxidant activity
512
with potent larvicidal activity (70-100% at 10-100 µg/ml; 43.5 µg/ml LC50) against Anopheles
513
mosquito larvae at 10-100 µg/ml concentration. The CPHE-AgNPs exhibited high stability,
514
antioxidant activity (33-85% and 14-84% DPPH-free radical scavenging and ferric ion reducing
515
activities at 20-100 µg/ml; 49.7 IC50) and synergistically improved (43-100%) antibacterial
516
activities of cefuroxime and ampicillin. The nanoparticles effectively in vitro inhibited multidrug
517
resistant Klebsiella pneumonia and Escherichia coli isolates at 40 µg/ml concentration and also
518
inhibited Streptococcus pyogenes, Staphylococcus aureus, Pseudomonas aeruginosa, Aspergillus
519
flavus, Aspergillus fumigatus and Aspergillus niger growth in emulsion paint (Lateef et al.,
520
2016).
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3.7. Encapsulating agent
523
CPH has also been investigated in green chemistry for the synthesis of nanoparticles against
524
multidrug resistant clinical bacteria because of its antioxidant, antimicrobial and larvicidal
525
activities (Lateef et al., 2016). Alpha-cellulose was obtained from CPH (26% yield) with 2.74%
526
ash, high crystallinity (27%), molecular weight (63,342) and 390 degree of polymerization
527
[glucose units] (Hutomo et al., 2012). CPH pectin may also serve as a suitable encapsulating
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528
agent alone or in combination with nanolignocellulose as demonstrated for citrus pectin to
529
protect probiotics in low pH fruit juice and gastrointestinal tract (Khorasani & Shojaosadati,
530
2017).
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533
Candida boidinii XM02G, isolated from cocoa cultivation areas, has been used to produce
534
xylitol from CPH. Xylitol was obtained in concentrations of 11.34 g.L-1, corresponding to a
535
yield (Yp/s) of 0.52 g.g-1 with a fermentation efficiency (ε) of 56.6% (Santana et al., 2018).
536
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3.9. Other valuable compounds
538
Ash (average yield 7.2%) from controlled combustion of CPH contained about 75% potash as
539
potassium carbonate with 1% impurities (iron, calcium and magnesium) that renders the organic
540
potash a premium product suitable for the pharmaceutical and food industries (Woode, 2015).
541
The high potassium content of CPH enables its use as a catalyst for biodiesel production due to
542
microstructural development when calcined at 700 °C for 4 h (Betiku et al., 2017). Carbonization
543
of dried and refined CPH with activated carbon reduces free fatty acid absorption (87%) of waste
544
cooking oil. This CPH can potentially be used as a potassium carbonate catalyst in the
545
transesterification process for biodiesel production from waste cooking oil (Rachmat, Mawarani
546
& Risanti, 2018). Microcrystalline cellulose (MCC - 5-10 µm rod-like shape, 11.635 nm
547
diameter and 74% crystallinity) was obtained by subjecting CPH to alkali treatment, bleaching,
548
and hydrochloric acid hydrolysis. This MCC can be used as a reinforcing agent with starch (2:8
549
mass ratio) and glycerol (20%) to produce high tensile strength (0.637 MPa) bioplastics (Lubis et
550
al., 2018).
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CPH can be hydrolyzed (1M HCl, 4 h, 75 °C) to yield maximum glucose (30.7%, w/v) that
552
produces ethanol (17.3 %, v/v) on fermentation (26 h) with Saccharomyces cerevisiae (Samah et
553
al., 2011).
554
Lipase extraction from CPH was achieved with the highest enzyme activity using response
555
surface methodology. Cross-linked enzyme aggregate (CLEA) was produced in the presence of
556
ammonium sulphate (30 mM) and stabilized/immobilized with glutaraldehyde (70 mM) and
557
bovine serum albumin (0.23 mM) (Yusof et al., 2016). The immobilized lipase had superior
558
stability in response to temperature (25-60 °C) and pH (5-10), and hydrophilic organic solvents
559
such as methanol (60%) compared to the free enzyme. The highest yield of lipase (11.43 U/ml)
560
was obtained with 7% CPH and 50 mM sodium phosphate buffer (pH 8) (Khanahmadi et al.,
561
2015).
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562
CPH can be easily converted into valuable products similar to those proposed and commercially
564
viable for cocoa shells. In this context, the pyrolysis plant (GmbH, Germany) can industrially
565
produce 3 products: charcoal (65-75%) as the main product of low temperature (250-300 °C)
566
torrefaction/carbonization; pyrolysis fuel (30-50%) and charcoal (25-35%) at medium
567
temperature (300-550 °C) vaporization; syngas (60-80%) and charcoal (15-20%) at high
568
temperature (600-1000 °C) gasification (Voigt, 2017). The three products, charcoal, pyrolysis
569
fuel and non-condensable gas account for 38, 32 and 30% of the total output volume,
570
respectively from the pyrolysis process. Thus, charcoal (280 kg/h) and biofuel (230 L/h) are
571
produced from an infeed of CPH (1 t/h) amounting to 60,000 Euros equivalent to an annual
572
saving of 504 million Euros. Furthermore, the Bühler shell burning technology can save
573
producers up to 50% in total cocoa production costs by CPH combustion system thereby
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574
reducing their carbon footprint by up to 2800 t/a. Fig. 2 summarizes the potential applications of
575
CPH.
576 4. Potential health benefits
578
Notwithstanding the foregoing, the utilization of CPH to enhance the quality and functionality of
579
pharmaceutical and food products is limited. Moreover, no research is available on the health
580
benefits of bioactive compounds from CPH. Therefore, in this section the health benefits of these
581
compounds from other plants are presented.
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4.1. Pectins
584
The readily available CPH can be used to recover value-added compounds such as pectins.
585
Pectins extracted from several plant by-products are widely used as gelling, thickening and
586
stabilizing agents and have several positive effects on human health, including lowering
587
cholesterol and serum glucose levels, reducing cancer and stimulating the immune response
588
(Vriesmann, Amboni & Petkowicz, 2011).
589
Pectin favorably influences cholesterol levels in blood. It helps reduce blood cholesterol in a
590
wide variety of subjects and experimental conditions as comprehensively reviewed
591
(Sriamornsak, 2001). Consumption of at least 6 g/day of pectin is necessary to have a significant
592
effect in cholesterol reduction, lower amounts are ineffective (Ginter et al., 1979). Pectin acts as
593
a natural prophylactic substance against poisoning with toxic cations. It is effective in removing
594
lead and mercury from the gastrointestinal tract and respiratory organs (Kohn, 1982). Pectin’s
595
high water binding capacity provides a feeling of satiety, thus reducing food consumption.
596
Experiments showed a prolongation of the gastric emptying half-time from 23 to 50 minutes of a
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meal fortified with pectin (Holt et al., 1979). These attributes of pectin are used in the treatment
598
of overeating-related disorders (Di Lorenzo et al., 1988).
599 4.2. Minerals
601
Four minerals (K, P, Ca and Mg) detected in CPH are quantitatively important (0.5- 6.0%). The
602
presence of these CPH minerals which are required for maintaining vital functions in living
603
human cells makes it a potential source of those elements (Yapo et al., 2013).
604
Minerals are essential constituents of skeletal structures such as bones and teeth; play a key role
605
in the maintenance of osmotic pressure, and thus regulate the exchange of water and solutes
606
within the animal body; serve as structural constituents of soft tissues; they are essential for the
607
transmission of nerve impulses and muscle contraction; minerals play a vital role in the acid-base
608
equilibrium of the body, and thus regulate the pH of the blood and other body fluids; also serving
609
as essential components of many enzymes, vitamins, hormones, and respiratory pigments, or as
610
cofactors in metabolism, catalysts and enzyme activators (FAO, 2017).
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611 4.3. Dietary fiber
613
The total dietary fiber in CPH (59%) includes 11 and 48% of soluble and insoluble dietary fiber,
614
respectively (Yapo et al., 2013). CPH contains higher TDF compared to other dietary fiber
615
sources, such as citrus (35-37%), apple (51%) and banana (33-52%) by-products, but lower than
616
those of coconut fiber (63%), pea hull (75%) and yellow passion fruit rind (74-82%) by-products
617
(Yapo et al., 2013).
618
High dietary fiber intake increases stool weight and transit time which may deter large bowel
619
disorders such as constipation, diverticulitis and large bowel cancers (Elleuch et al., 2011). Most
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non-absorbed carbohydrates have laxative effects, both by increasing bacterial mass or osmotic
621
effects, and by water binding of unfermented fiber. The etiology of cancer involves both
622
inherited and environmental (dietary) factors. The overall evidence for an effect of total fiber
623
intake on the risk of colorectal cancer is not considered sufficient to serve as a basis for
624
guidelines on dietary fiber intake. However, individuals with lesser fiber intakes may have an
625
increased risk (Mudgil & Barak, 2013).
626
Recent observational studies consistently show an inverse association between dietary fiber
627
intake and the risk of coronary heart disease (Mudgil & Barak, 2013). Postulated mechanisms for
628
lower levels of total and low-density lipoprotein (LDL) cholesterol include alterations in
629
cholesterol absorption and bile acid re-absorption, and alterations in hepatic metabolism and
630
plasma clearance of lipoproteins (Theuwissen & Mensink, 2008). In some countries the evidence
631
for the cholesterol-lowering properties of certain viscous fibers, especially β-glucans from oats,
632
is considered sufficient for claims on the reduction of the risk of coronary heart disease (Mudgil
633
& Barak, 2013).
634
Some cohort studies inversely associate dietary fiber intake and the risk of developing type 2
635
diabetes (Chandalia, 2000). Furthermore, viscous fibers such as pectin and guar gum delay
636
gastric emptying, whereas slowly digested starch and resistant starch increase satiety in vivo
637
(Mudgil & Barak, 2013). Presumably other, as yet unidentified substances in such foods can
638
explain this; perhaps it is the overall combination of the dietary fiber, nutrients and bioactive
639
substances acting synergistically, that is critical to health. However, dietary fiber such as
640
resistant starch, non-digestible oligosaccharides and polydextrose may help to prevent and
641
alleviate bowel disorders, and decrease risk factors for coronary heart disease and type 2 diabetes
642
(Kaczmarczyk, Miller & Freund 2012).
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643 4.4. Antioxidants compounds
645
Polyphenols are among the other bioactive compounds found in CPH. Polyphenolic compounds
646
usually accumulate in the outer parts of plants, such as shells, skins and husks (Vriesmann,
647
Amboni & Petkowicz, 2011). The total phenolic content of CPH is estimated at ∼7% and
648
significantly higher (p < 0.05) than in cocoa hull product from fermented and roasted beans
649
(CHPFR) (∼2-3%) and cocoa kernel product (CKPFR) (Yapo et al., 2013). Therefore, CPH can
650
be a potential source of antioxidant-dietary fiber-rich food, which may be used to compensate for
651
their shortage or complete lack in refined modern diets currently associated to various free
652
radical-induced disorders (Yapo et al., 2013).
653
Free radicals, or Reactive oxygen species (ROS), play a biochemical role in the development of
654
cancer, multiple sclerosis, Parkinson's disease, rheumatoid and inflammatory diseases. Metabolic
655
disease and environmental contaminants may increase ROS in tissues, leading to increased
656
oxidative stress in cells. However; antioxidants are able to ‘scavenge’ free radicals from the
657
body, reducing oxidative stress and the potential development or progression of many disease
658
states (Zumbé, 1998). Studies with procyanidins BI and B3 and (+)-catechin show that these
659
polyphenols are capable of trapping hydrophilic peroxyl radicals in vitro, and their radical
660
scavenging activity increases with polyphenol concentration (Ariga & Hamano, 1990).
661
Furthermore, antioxidant activity of polyphenols has been demonstrated in vitro to prevent lipid
662
peroxidation, a type of ROS induced cell injury and low density lipoprotein (LDL) oxidation
663
(Zumbé, 1998).
664
The anti-genotoxic effect of catechin, epicatechin, gallocatechin and epigallocatechin have all
665
been demonstrated in vitro, protecting organisms such as S. typheriurn and E.coli against
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mutation following exposure to various known carcinogens. This protective, anti-genotoxic
667
effect of the polyphenols has also been demonstrated in in vitro studies of mammalian cells
668
exposed to such cancer inducers as nitrosamine, benzo[a]pyrene, aflatoxin BI, tobacco smoke
669
and smoked meat extracts. Furthermore, polyphenols prevent DNA damage in vitro and in vivo
670
following exposure to these types of compound (Agarwal & Mukhtar, 1996; Yang et al., 1996).
671
Bioactive compounds extracted from CPH can exert potential health benefits and may therefore
672
be an alternative source of these important components (Fig.3).
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673 Conclusions
675
There is an urgent need for practical and innovative ideas to use this low cost CPH and exploit its
676
full potential increasing the overall sustainability of the cocoa agro-industry. Since changes
677
towards better efficiency and sustainability can also involve actions to improve the valorization
678
of by-products and of food related waste, the large amounts of organic compounds (i.e. pectin,
679
antioxidants, dietary fiber and minerals) contents in the CPH justify its valorization. CPH is a
680
good source of nutraceuticals, however their use in food industry is minimal partly due to limited
681
research. CPH potential food applications include extraction of aroma compounds, vegetable
682
gums, or texturizing agents, among others.
683
This by-product can be tailored through processing for diverse functionality and bioactivity.
684
Furthermore, CPH should be explored as a functional ingredient generating healthy and
685
innovative functional food, cosmetic and medical products.
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Conflict of interest: The authors declare no competing interests.
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Acknowledgements
689
Author Karen Haydeé Nieto Figueroa, was supported by a scholarship from the Consejo
690
Nacional de Ciencia y Tecnología (CONACyT-Mexico) [grant number 854976]. The funding
691
provided by UAQ (Universidad Autónoma de Querétaro) and CONACyT-Fondos Mixtos
692
(FOMIX-QRO-279751) are appreciated.
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Sriamornsak, P. (2001). Pectin: The role in health. Journal of Silpakorn University, 21(22), 6077. Sriherwanto, C., Reksohadiwinoto, B. S., Mahsunah, A.H., Suja’i, I., Toelak, S. & Rusmiyati, M.
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Suparjo, S., & Nelson, N. (2017). Determination length of fermentation of cocoa pod husk with
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Theuwissen, E., & Mensink, R. P. (2008). Water-soluble dietary fibers and cardiovascular
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Valadez-Carmona, L., Plazola-Jacinto, C.P., Hernández-Ortega, M., Hernández-Navarro, M.D.,
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Young, A.M. (1994). The Chocolate Tree. A Natural History of Cacao. Washington, DC, Smithsonian Institution Press.
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Zumbé, A. (1998). Polyphenols in cocoa: are there health benefits? Nutrition Bulletin, 23(1), 94-
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Table 1 Cultivation and production of cocoa from top ten countries.
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%Total 27.96 16.51 16.69 7.10 8.22 7.06 4.45 1.23 1.70 1.63
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Adapted from FAO, 2018.
%Total 32.96 19.23 14.71 6.53 5.30 4.79 3.98 2.42 1.82 1.26
Cultivation (Hectares) 2,851,084 1,683,765 1,701,351 723,853 838,046 720,053 454,257 125,580 172,940 165,844
SC
Country Cote D'Ivoire Ghana Indonesia Cameroon Nigeria Brazil Ecuador Peru Dominican Republic Colombia
Production (Tonnes) 1,472,313 858,720 656,817 291,512 236,521 213,843 177,551 107,922 81,246 56,163
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Table 2 Structural composition of cocoa pods. CPH 80.2 9.1 5.9 22.6 61.0
Epicarp 82.8 10.1 5.0 17.3 62.0
Mesocarp 64.0 4.6 1.9 29.5 80.0
ADF Nitrogen-free Crude fat (ether extract) Cellulose Hemicellulose Lignin Pectin Ca K P Mg mg/kg Na Zn Fe Cu
50.0 62.2 1.2 35.0 11.0 14.6 6.1 0.32 3.18 0.15 0.22
45.0 66.8 0.8 30.0 17.0 15.0 5.1 0.58 4.61 0.16 0.39
70.0 63.7 0.3 57.5 10.0 12.0 2.1 0.19 1.56 0.06 0.10
9.1 64.9 197.1 13.2
6.0 23.5 106.3 5.6
7.2 30.8 112.4 7.1
103.2
21.3
31.9
33.6
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Adapted from Sobamiwa & Longe (1994).
34.0 70.0 1.1 20.8 7.0 13.2 10.5 0.13 2.66 0.09 0.15
SC
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Mn
3.1 40.4 90.1 7.2
Endocarp 87.1 6.7 6.9 15.3 41.0
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Component (%) Moisture Ash Protein Crude Fiber NDF
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Table 3 Chemical composition (%) of cocoa pod husk. Reference
Moisture Protein
Ash
Lipid
Total carb
Vriesmann, Amboni 2011 Laconi & Jayanegara 2015 Esong et al., 2015 Lateef et al., 2008 Chun et al., 2016
8.5 NR 13.0 NR 10.5
8.6 8.4 8.0 8.2 2.1
6.7 NR 13.0 11.3 9.0
1.5 2.5 0.6 4.7 1.5
32.3 20.6 23.0 NR 17.5
36.6 55.7 50.0 18.3 NR
21.4 38.8 NR NR 24.24
Martínez et al., 2012
6.6
4.2
8.4
2.3
29.0
56.0
NR
Yapo et al., 2013
8.5
8.9
7.3
2.3
NR
59.0
19.4
Range
6.4-14.1
2.1-9.1
5.9-13.0
0.6-4.7
17.5-47.0
18.3-59.0
14.7-38.8
7.8
5.0
5.8
1.4
901
186
57.7
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Lignin
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TDF, total dietary fiber NR, not reported
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Variance
TDF
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0.74 0.35 0.26 NR 0.34 0.81 0.18 0.29 0.49 0.35 0.04 0.30 0.40 0.35 0.34 NR 0.18 0.40
Bean husk 1.50 0.80 0.70 NR 0.72 1.87 0.27 0.48 0.45 0.79 0.06 0.45 0.20 0.71 0.70 0.12 0.42 0.25
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CPH 0.80 0.44 0.22 0.09 0.29 0.77 0.21 0.24 0.43 0.40 0.05 0.37 0.38 0.41 0.30 0.04 0.21 0.44
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Amino Acids Aspartic acid Alanine Argininea Cystine Glycine Glutamic acid Histidinea Isoleucinea Leucinea Lysinea Methioninea Phenylalaninea Proline Serine Threoninea Tryptophan Tyrosine Valinea
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Total amino acids 6.09 5.82 10.49 EAI (%) 43.70 45.5 39.56 BCAA 1.11 1.18 1.18 AAA 0.58 0.48 0.87 Fischer Ratio 1.91 2.46 1.36 Lysine/Arginine 1.82 1.35 1.13 Arg+Glu+His 1.20 1.25 2.84 Met+Cys 0.14 0.04 0.06 a Essential amino acids EAI, essential amino acid index BCAA (Val+Leu+Ile) AAA (Phe+Tyr) Fischer ratio (BCAA/AAA) NR, not reported Data for CPH from Donkoh, Atuahene, Wilson & Adomako, 1991 Cocoa husk data adapted from Bonvehi & Coll, 1999
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Table 4 Amino acid content (g/100 g) of coca pod husk and cocoa husk.
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Table 5
a
Referencec 19 28 66 58 25 63 34 11 35 339
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Cocoa huskb 18 32 53 53 21 58 47 8 55 345
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His Ile Leu Lys Met + Cys Phe + Tyr Thr Trp Val Total
mg amino acid/g protein Cocoa pod huska 27.4 24.3 31.3 39.2 56.1 66.2 52.2 47.3 18.3 5.4 75.7 64.9 39.2 45.9 5.2 57.4 54.1 362.8 347.3
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Essential and reference amino acid pattern of cocoa pod husk and cocoa husk
Adapted from Donkoh, Atuahene, Wilson & Adomako, 1991 Adapted from Bonvehi & Coll, 1999 c FAO/WHO/Expert Consultation (1990) reference pattern for 2-5-year old child
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Table 6 Cocoa pod pectin characteristics. NA-HYP 10.7 41.0 17.6 59.2 60.8 14.7 9.9 20.3 11.6 4.9 1.7 0.5 1.8
OP 9.8 56.6 17.1 66.0 69.9 3.6 3.9 16.8 10.0 2.8 2.7 0.7 tr
MOP 8.6 20.8 9.4 56.0 NR NR NR 19.7 18.0 3.0 2.5 0.8 1.0
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BW 12.6 42.6 19.2 44.6 51.9 5.5 8.3 25.4 14.4 5.7 5.0 1.9 3.1
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50W 7.5 37.0 29.0 45.1 55.8 9.6 9.8 21.7 21.4 4.5 4.6 1.9 0.9
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CA-HYP Yield 10.1 DE 40.3 DA 15.9 Uronic 65.1 Carbohydrate 64.0 Protein 13.8 Phenolic* 9.4 Gal 16.7 Rha 8.2 Glc 4.1 Ara 1.9 Xyl 1.4 Man 2.6
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Adapted from Vriesmann (2012) for CA-HYP; Vriesmann, Amboni & Petkowicz (2011) for 50W and BW and Vriesmann (2012) for OP and MOP; NR-not reported. CA-HYP and NA-HYP-citric and nitric acids high yield pectin, respectively; OP and MOP optimized and modified optimized nitric-acid extracted pectins from cocoa pod husk pectins; 50W and BW- water soluble pectins extracted from cocoa pod husk flour at 50 and 100 °C, respectively; DE-degree of esterification; DA-degree of acetylation. Values expressed as %, m/m, dry base. *Percentage of gallic acid.
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Table 7 Volatile compounds identified in cocoa pod husk (CPH), cocoa pod husk (CPHF) fermentation in solid-state (with Rhizopus stolonifera ) and cocoa.
+ +
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+ + + + +
+**
+ +
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Cocoa
+ + + +
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+
CPHF
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Compounds ALCOHOLS 5-Methyl-2-hexanol (2S,4S)-Pentanediol E-Linalol pyranoxide 1,2-Pentanediol 11-Methyldodecanol 1-Hexanol 1-Isopropyl-1-butanol 1-Methoxy-2-butanol 1-Nonanol 1-Octen-3-ol 2,3-Butanediol 2,6-Dimethylcyclohexanol 2-Ethyl-1-hexanol 2-Ethyl-3-pentanol 3,4-Dimethylpent-2-en-1-ol Epoxylinalol Linalool n-Tridecanol Phenylethyl alcohol (2-Phenylethanol) Trans-linalool oxide α-Hydroxytoluene α-Methylbenzyl alcohol (Benzyl alcohol) α-Terpinol ALDEHYDES (E,E)-2,4-Heptadienal Heptanal Methocycitronellal Nonanal Octanal Trans-2-hexenal Trans-2-octenal α-Toluic aldehyde (2-Phenyl acetaldehyde) β-Cyclocitral KETONES n-Amyl methyl ketone 1-Phenylethanone
+
+*, **
+ +
+ + +
+*, ** +** +*’** +**
+ + + + + + + + + + +
+ + +
+*
+
+**
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+ + + +
+
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+ + + + +
+ + + +
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+**
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+
+
+ +
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2-Dodecanone Pyrrole-α-methyl ketone Sulcatone trans-3-Octen-2-one β-Ionone ESTERS 1-Ethylpentyl acetate Ethyl iso-allocholate Methyl N-hydroxybenzenecarboximidoate Methyl salicylate Oxalic acid, bis(6-ethyloct-3-yl)ester β-Hydroxybutyric acid methyl ester ϒ-Hydroxybutyric acid cyclic ester ORGANIC ACIDS 10,12-Tricosadiynoic acid D,L-Mevalonic acid lactone Isovaleric acid HYDROCARBONS (-)-Aristolene 1,2,4-Trimethylcyclopentane 13-Phenylpentacosane 1-Heptadecene (+)-δ-Amorphene 2,6,10,15-Tetramethylptadecane 2-Cyclopropylidene-1,7,7-trimethylbicyclo[2,2,1]heptane 2-Ethylhexene 2-Methyl-n-hexacosane D-Limone Eicosane Heneicosane Isoledene Valerena-4,7(11)-diene α-Copane ϒ-Cadinene ϒ-Muurolene OTHERS Octanenitrile 1,4-Butanediamine Rosefuran
+
+**
+ + +
+
+ + + + + + + + + + + + +
+
+
+ + +
From Tiburcio, 2017, *From Alvarez et al., (2016), **From Aprotosoaie, Luca & Miron (2016)
Endocarp1
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Mesocarp1
Pod shell/husk2
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67-76%
Mucilage/pulp2
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Epicarp1
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8.7-9.9%*
Bean shell/husk2 2.1-2.3%
Bean1 21-23% (30-40 beans/pod)
Fig 1. The cocoa fruit structures1 and wastes2 (with information of Babatope, 2005; Oddoye, AgyenteBadu & Gyedu-Akpto, 2013; Awarikabey et al., 2014; Sobowale et al., 2015; Papalexandratou, & Nielsen, 2016; *By difference).
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Fig. 2. Potential applications of cocoa pod husk.
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Fig. 3. Potential health benefits of bio-compounds found in cocoa pod husk.
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Highlights
1. CPH is an important industrial waste from which no benefit has been taken yet.
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2. The investigation is limited to produce high-value-added products from CPH. 3. Bio compounds such as dietary fiber, pectins, antioxidants and minerals can be obtained from CPH.
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4. The extraction of this bio compounds may have applications in food industry, among others, because their potential health benefit.