Bentil, J. A. 1 *, Dzogbefia, V. P. 2 and Alemawor, F. 2 IJAMBR 3 (2015) ISSN

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1 IJAMBR () - ISSN - Enhancement of the nutritive value of cocoa (Theobroma cacao) bean shells for use as feed for animals through a two-stage solid state fermentation with Pleurotus ostreatus and Aspergillus niger Bentil, J. A. *, Dzogbefia, V. P. and Alemawor, F. Department of Animal Science, College of Agriculture and Natural Resources, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana. Department of Biochemistry and Biotechnology, College of Science, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana. Article History Received December, Received in revised form January, Accepted January, Key words: Agroindustrial byproducts, Cocoa bean shells, Solid state fermentation, Aspergillus niger, Pleurotus ostreatus, Theobromine. Article Type: Full Length Research Article ABSTRACT The study investigated the suitability of cocoa bean shell (CBS), a by-product from cocoa processing industry as alternative energy source for animal feeding when subjected to a two-stage solid state fermentation with Pleurotus ostreatus and Aspergillus niger. It involved the optimization of the fermentation period by the afore-mentioned fungi. The already dried shells were milled into mm particle size, composted and pasteurized. The pasteurized substrate was fermented with the spawn of P. ostreatus (oyster mushroom) for six weeks followed by A. niger fermentation for seven days. The fermentation of the shells with P. ostreatus significantly increased (p<.) the protein content by.% and decreased (p<.) the cellulose, hemicellulose and lignin by 9.9, 9.99 and.% respectively, at the sixth week; the optimum fermentation period. The degradation of the complex carbohydrates by the fungus increased the level of soluble carbohydrates from. to.% which resulted in a.% increase in metabolizable energy (M.E) in the fermented product. The calcium, phosphorus and potassium contents of the P. ostreatus fermented shells were also significantly enhanced (p<.) at the end of the fermentation period. The P. ostreatus fermented CBS which was subsequently fermented with A. niger had its theobromine content significantly reduced (p<.) by.9% at the end of the seven days of fermentation. The CBS fermented product hence becomes more suitable for use as animal feed material. BluePen Journals Ltd. All rights reserved INTRODUCTION The poultry industry is faced with several challenges one of which is the scarcity of conventional energy source feedstuffs particularly maize, a major energy source in animal feed. This is due to the high demand on such ingredients by humans and in the poultry and livestock *Corresponding author. profdocjab@yahoo.com. industries. The high competition for energy source ingredients between humans and animals has led to escalating prices of these ingredients hence increased the cost of feeding (Adebowale, 9). Several attempts have been made by researchers to address this pestering issue facing the poultry industry by the use of alternative and cheaper sources of ingredients to replace the less available and expensive ones (Olubamiwa et al., ; Hamzat and Babatunde, ).

2 Int. J. Appl. Microbiol. Biotechnol. Res. One such alternative source is the use of agro-industrial by-products which are produced by agro-allied industries and are abundantly found in the environment. Most of these industries spend huge sum of money for proper disposal of these wastes which also increases their production cost. However, improper disposal of these wastes by the industries that produce them may lead to environmental pollution. Cocoa bean shells (CBS) produced in vast quantities from cocoa and chocolate factories is a potential tropical feed resource therefore its utilization in animal feeding will greatly reduce the disposal problem facing the cocoa processing factories (Aina, 99). The percentage dry weight of CBS contains.-. crude protein,.9-. ether extractable components, 9.-. crude fibre,.-. ash,.9-. moisture and.-. nitrogen-free extractives dependent on the extent of processing of the cocoa beans as reported by EFSA (). Several researchers have attempted to use CBS for animal feeding especially in poultry diets (Olubamiwa et al., ; Hamzat and Babatunde, ), but at minimal inclusion levels due to the low feed conversion efficiency observed in poultry diet. This is for the reason that, CBS is lignocellulosic i.e. contains cellulose and lignin that are difficult to digest by poultry (Hamzat and Babatunde, ). Moreover, the high theobromine content of CBS (Gohl, 9) makes it impossible to feed more of the byproducts to poultry since it is highly toxic to their digestive system. Mushroom forming fungi are therefore amongst nature s most powerful decomposers, secreting strong extracellular enzymes due to their aggressive growth and biomass production (Adenipekun, 9). They are primary decomposers and saprophytic fungi, which grow easily in clusters on decomposing lignocellulosics, capable of utilizing a wide range of substrate materials with high biological efficiency and good mycelia growth rate (Achio, 9). Lignocellulose decomposers undergo extracellular digestion hence their capability of producing a wide range of enzymes such as laccases, cellulases and hemicellulases that are responsible for the breakdown of complex polysaccharides such as lignin, cellulose and hemicelluloses respectively, into simpler soluble substances which are highly absorbable for growth and development (Sharma et al., 999). Studies have also established that fungus from Basidiomycetes and Ascomycetes families are capable of not only biodegrading lignocellulosics but also antinutritional factors such as tannins, alkaloids, lectins, and others that may be present in the agricultural wastes through extracellular enzymatic hydrolysis (Adamafio et al., ; Alemawor et al., 9). This study therefore seeks to address these outlined limitations associated with the use of CBS as suitable animal feeding material by investigating the effect of a two-stage fermentation of CBS with Pleurotus ostreatus and Aspergillus niger on its nutritive and anti-nutritive values. MATERIALS AND METHODS Experimental design The completely randomized design (CRD) was used to sample substrates from the fermentation media to ascertain the effects of the fungi fermentation on the nutritive and anti-nutritive of the CBS. Source of experimental materials The already dried CBS were obtained from the Cocoa Research Station at Mpintsin in the Western region of Ghana. P. ostreatus (Oyster mushroom) spawn used for the first stage fermentation was obtained from Robart Farms, a mushroom production farm at Kenyasi, Kumasi in the Ashanti region of Ghana. The identification and isolation of A. niger for the second stage fermentation was undertaken at the Pathology Laboratory of the Faculty of Agriculture, Kwame Nkrumah University of Science and Technology, Kumasi. Fermentation of cocoa bean shells with P. ostreatus spawn Size reduction The already dried CBS used as substrate for the fermentation was milled into a uniform particle size of mm using a hammer mill. Composting The milled substrate was then heaped on black rubber sheet to a height of. m, mixed with tap water to a % moisture content (Alemawor et al., 9) and covered with the rubber sheet to protect it from direct effect of sun and rain. It was then left for four days with intermittent turning over. A temperature of C and ph. were recorded at the end of the composting period. The composted substrate was then mixed with calcium carbonate in a ratio of : grams w/w (Thulasi et al., ) to control the ph of the material during fermentation. Pasteurization The composted substrate was wrapped in aluminium foil in g portions and autoclaved at C for min.

3 Bentil et al. Inoculation After the pasteurized substrates have been cooled, g of P. ostreatus grains was introduced onto g of the substrates in already sterilized trays. A control in replicates of three was also set where substrates were not inoculated with the P. ostreatus spawn. Incubation The inoculated substrates were incubated at C for a period of eight weeks to allow the growth of the mycelia through the substrates. Optimization of P. ostreatus fermentation The fermentation process was terminated each week by drying the substrates in an oven (Wagtech) at C for two days and the moisture content in the fermentation media checked. The dried substrates were then milled to homogeneity and kept in dessicator for chemical analysis. Chemical analysis The proximate composition of the raw and treated substrates which included crude protein, crude fibre, ether extract (crude fat), ash were determined by proximate analysis (AOAC, ). The cellulose, hemicellulose and lignin contents were computed from acid detergent fibre (ADF), neutral detergent fibre (NDF) and acid detergent lignin (ADL) determined by van Soest Method of Analysis (van Soest, 99). Calcium and Potassium were determined using Flame Photometry and the Phosphorus using the spectrophotometric Vanandium Phosphomolybdate method. The anti-nutritional factor (theobromine) was determined using gravimetry method by Holmes (AOAC, ). Fermentation of P. ostreatus fermented CBS with A. niger Identification and isolation of A. niger from CBS A portion of sterilized Potato Dextrose Agar (PDA) was poured into already sterilized Petri dishes in duplicates. The PDA containing Petri dishes were inoculated with the shells in a Laminar Flow and incubated at room temperature for days. The A. niger isolated from the fungal culture was subjected to lactophenol cotton blue staining which confirmed the morphology and spore colouration characteristic of the species. Preparation of sub-cultures for fermentation A mm cork borer was used to pick spores of the A. niger from each of the pure cultures which were inoculated into already sterilized Petri dishes with PDA in replicates. The Petri dishes were then incubated at C for six days. Preparation of A. niger spore suspension At the end of the six days incubation, spores of the mould were harvested by flooding each of the -day old cultures with ml of sterilized distilled water and dislodging the spores by scraping. The spore suspensions were then decanted and pooled into a ml Erlenmeyer flask to the mark. Determination of spore concentration One () ml of spore suspension was taken from the stock into a ml measuring cylinder and made to the mark with sterile distilled water. The diluted suspension was shaken and a portion pipetted and released into the grooves of a haemacytometer and placed under a light microscope for the counting of the spores. The spores were counted within five regions of the haemacytometer and the average taken. The average value was then multiplied by number of cells/ml and a dilution factor of. The spore concentration was obtained to be. x cells/ml. Substrate for A. niger fermentation The dried and grounded P. ostreatus fermented CBS (POFCBS) for week six, the optimum period of the P. ostreatus fermentation was used as substrate for the A. niger fermentation Inoculation and incubation of substrates Forty () grams of the sterile CBS powder (substrate) was inoculated with ml of the spore suspension of spore concentration of. x cell/ml (Krishnan et al., 9). Two controls were set up having non- P. ostreatus fermented CBS (NPOFCBS) and P. ostreatus fermented CBS (POFCBS). Substrates inoculated with the A. niger spore suspension were kept in an incubation room at C for seven days.

4 %Moisture content Int. J. Appl. Microbiol. Biotechnol. Res. Figure. Effect of fermentation of CBS with P. ostreatus on moisture content. Optimization of A. niger fermentation After each day, the fermentation process was terminated by drying the substrates in an oven (Wagtech) at C for two days to inactivate the A. niger. The dried substrates were then prepared for theobromine determination by titrimetric method. Data collection and analysis Results were presented as mean standard values of three replicates each. A one-way analysis of variance (ANOVA) was carried out at p<. level of significance. Graphs were plotted using the Graph Pad Prism (Version.) software. RESULTS AND DISCUSSIONS Proximate composition of CBS fermented with P. ostreatus There was a marginal reduction of moisture content (from. to.%) observed up to the sixth week of fermentation after which there was a decline when the CBS was fermented with P. ostreatus (Figure ). This could be attributed to the mycelia growth of the mushroom which is supported by the assertion made by Bano et al. (9) that mycelia of mushrooms contain some amount of moisture. Sawyer (99) also reported that water forms about % of the composition of mushrooms. The decline of the moisture content after the sixth week may be as a result of the formation of fruiting bodies of the mushroom which utilizes nutrients as well as water for growth (Rypacek, 9). The general increase in the ash content (Figure ) could be attributed to the enrichment of minerals by mycelia of the mushroom grown on the shells. According to Sawyer (99), mushrooms have richer supply of minerals than many meat products and two times higher than in vegetables. Additionally, the fruiting bodies of mushrooms contain about % ash as on dry matter basis (Bano et al., 9). The CBS was significantly enriched (p<.) in crude protein content by the fermentation with P. ostreatus having an optimum level at the sixth week of fermentation (.%) (Figure ). The increase in the crude protein

5 % %Crude protein Protein %Ash content Bentil et al. Figure. Effect of fermentation of cocoa bean shells with P. ostreatus on ash content. Figure. Effect of fermentation of cocoa bean shells with P. ostreatus on crude protein content.

6 %Crude Fibre % Crude fibre Int. J. Appl. Microbiol. Biotechnol. Res. Figure. Effect of fermentation of cocoa bean shells with P. ostreatus on crude fibre content. level is in agreement with the report by Vijay et al. () who reported that P. ostreatus has the potential of converting cheap lignocellulosic materials into valuable protein materials at low cost. However, the growth of fruiting bodies after the optimum level resulted in a reduction due to the use-up of the crude protein by the fruiting bodies. There was a significant decline (p<.) in the fibre content observed from the st week (.%) to the th week (.%) (Figure ) when CBS was fermented with P. ostreatus. The decrease in fibre content was as a result of extracellular enzymes secreted by the fungus that was responsible for the biodegradation of the cell wall components (Chesson, 99) compared to the control (uninoculated) that had no significant change (p<.) in the fibre content throughout the fermentation period. The degradation of the complex carbohydrates into soluble components is the synergistic actions of several extracellular enzymes such as glucosidases which reduce oligosaccharides to their monomeric units (Aderemi and Nworju, ) Individual fibre components of cocoa bean shells fermented with P. ostreatus The decrease in the cellulose content of the shells was found to be significant (p<.) unlike the control which showed no significant change throughout the fermentation period. There was a 9.9% decrease in the cellulose content of the fermented shells at the sixth week, the optimum period of fermentation (Figure ). The cellulose degradation was facilitated by the synergistic action of hydrolytic enzymes (cellulases) secreted by the fungus during the fermentation (Chesson, 99; Datta and Chakravarty, ). There was a general decreasing trend of hemicellulose content observed throughout the fermentation period but peaked at the sixth week, the optimum fermentation period recording a 9.99% decrease in hemicellulose content (Figure ). The degradation of the hemicellulose was as a result of extracellular hemicellulases such as xylanases secreted by the fungus during the fermentation process (Chesson, 99). This result strongly supports a study by Brimpong et al. (9) who also observed a % decrease in hemicellulose after complete colonization of corn cobs by the mycelia of the oyster mushroom with no further reduction thereafter. The lignin content of the shells was significantly reduced (p<.) by the th week when fermented with the P. ostreatus recording.% level of degradation (Figure ). An investigation conducted by Brimpong et al. (9) on the growth of oyster mushroom on corn cobs reported a decrease of lignin content by.% after complete colonization beyond which there was no further reduction. The degradation of lignin was as a result of the production of lignin-degrading extracellular enzymes such as peroxidases and laccases that oxidize both the

7 % Hemicellulose content % % Hemicellulose content % Hemicellulose content % % Hemicellulose content % Hemicellulose content content %Cellulose content % Cellulose content Bentil et al. Figure. Effect of fermentation of cocoa bean shells with P. ostreatus on cellulose content. Fermentation Fermentation time time (Weeks) (Weeks) Fermentation Fermentation Time Time Time Time Time (Weeks) (Weeks) time Time (Weeks) (Weeks) (Weeks) Figure. Effect of fermentation of cocoa bean shells with P. ostreatus on hemicellulose content.

8 %Lignin content % Lignin content Int. J. Appl. Microbiol. Biotechnol. Res. Figure. Effect of fermentation of cocoa bean shells with P. ostreatus on lignin content. aromatic rings and the aliphatic side chains to produce compounds more absorbable by the fungi (Youri et al., ). Changes in mineral content of CBS fermented with P. ostreatus The calcium content of the raw shells was found to be. mg/kg whereas that of the control and inoculated shells recorded 9. and 9. mg/kg respectively, an increase of.% when compared to the control at the optimum period of fermentation (th week) (Figure ). The increase may be attributed to the enrichment of the shells by the mycelia growth of the mushroom known to contain appreciable amount of calcium (Sawyer, 99). The respective values of phosphorus recorded were.,. and. mg/kg for the raw, the uninoculated (control) and the inoculated shells, respectively (Figure 9). Phosphorus had the least improvement by the bioprocess as observed (an increase of.%). This result confirms earlier work done by Alemawor et al. (9) where phosphorus recorded a least value of the mineral elements after P. ostreatus fermentation of cocoa pod husk over the same period. This increase may be attributed to the enrichment of the shells by the mycelia growth of the mushroom known to be rich in phosphorus and other elements needed by the body (Sawyer, 99). The potassium content of the treated shells was significantly improved (p<.) recording. mg/kg when compared with the control at the sixth week of fermentation. The raw shells and the control recorded. and. mg/kg of potassium content which was not significantly different (p<.) (Figure ). The increase is attributed to the enrichment of the shells by the mycelia growth of the mushroom known to contain appreciable amount of minerals including potassium (Sawyer, 99). Changes in theobromine content of cocoa bean shells fermented with A. niger Though the theobromine level in the raw shells (. mg/g) was far lower than literature value (.-. g/kg theobromine) (EFSA, ), it was significantly reduced (p<.) when fermented with A. niger as compared to the raw shells and the controls (Figure ). There was therefore a.9% reduction in theobromine content after the A. niger fermentation of the shells for seven days. This result confirms a study conducted by Adamafio et al. () who reported a.% detheobromination in cocoa pod husk fermented with A. niger over the same period. The degradation of theobromine was aided by its use as a sole carbon and energy source by A. niger which occurs via the demethylase route involving the expression of enzymes such as theobromine demethylase, theobromine oxidase,

9 Bentil et al. Figure. Changes in calcium content of P. ostreatus fermented cocoa bean shells. Figure 9. Changes in phosphorus content of P. ostreatus fermented cocoa bean shells. Figure. Changes in Potassium content of P. ostreatus fermented cocoa bean shells.

10 Int. J. Appl. Microbiol. Biotechnol. Res. 9 Figure. Changes in theobromine content of A. nigerfermented cocoa bean shells. Key: PAFCBS, P. ostreatus and A. niger fermented cocoa bean shells; POFCBS, P. ostreatus fermented cocoa bean shells; NPOFCBS, Non-P. ostreatus fermented cocoa bean shells. xanthine dehydrogenase, xanthine oxidase, urease and uricase (Yamoka-Yano and Mazzafera, 999; Dash and Gummadi, ; Huq, ). It was further observed that the theobromine levels of both the P. ostreatus fermented CBS (POFCBS) and the non-p. ostreatus fermented CBS (NPOFCBS) serving as controls were not significantly different (p<.) at the end of the fermentation period. This result confirms the observation of Alemawor et al. (9) that, methylxanthines usually present in cocoa products and by-products are not affected by P. ostreatus fermentation. The huge difference observed in the theobromine levels of the raw shells (. mg/g) and the controls (9. and 9. mg/g) could be attributed to the heating methods applied such as pasteurization as heating is reported to equally reduce the theobromine level in cocoa by-products (Menon, 9). Conclusion The tremendous reduction in the lignocellulose content and the theobromine content of the CBS fermented with P. ostreatus and A. niger respectively, makes the CBS more suitable for use as feed material for animals especially monogastrics. RECOMMENDATIONS Notwithstanding the suitability of the fungi treated CBS as feed material for animals, it is recommended that: An assay should be conducted to ascertain the mycotoxicity level in the fungi treated CBS since the strain of A. niger used in the study was not specified whether or not it produced Aflatoxin during the fermentation process. An in vitro digestibility studies should be conducted to ascertain the level of fibre digestibility of the fermented CBS in terms of total sugars. REFERENCES Achio, S. (9). Understanding the biology of the oyster mushroom (Pleurotus ostreatus), as a means to increase its productivity. Proceedings of nd African Conference on Edible and Medicinal Mushrooms, Accra, Ghana, March -, Abstract, p.. Adamafio N. A., Ayombil F. & Tano-Debrah K. (). Microbial detheobromination of cocoa (Theobroma cacao) pod husk. Asian J. Biochem. :-. Adebowale E. A. (9). The maize replacement of value of fermented cassava peels (Manihot utilissma POHL) in rations for sheep. Trop. Anim. Prod. :-. Adenipekun, C.O. (9). Uses of mushrooms in bioremediation. Proceedings of nd African Conference on Edible and Medicinal Mushrooms, Accra, Ghana, March -, Abstract, p.. Aderemi F. A. & Nworju F. C. (). Nutritional status of cassava peels and root sieviate biodegraded with Aspergillus niger. American- Eurasia. J. Agric. Environ. Sci. ():-. Aina A. A. (99). Potential of cocoa by-product as livestock feeds. Proceeding of National Workshop on Alternative Formulation of Livestock Feed held at ARMTI, Ilorin. Pp. -. Alemawor F., Dzogbefia V. P., Oldham J. H. & Oddoye O. K. (9). Effect of fermentation on the composition of cocoa pod. Influence on time and Mn + supplementation on the fermentation process. Afr. J. Biotechnol. (9):9-9. Sharma, S. G., Jyoti, N. & Singh, V. K. (999). Studies on recycling Pleurotus waste. Mushroom Res. ():-.

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