Theobromine Content in Cocoa Pod Husk (
Content in Cocoa Pod Husk (
Theobroma cacao
)
Fermented by
Aspergillus spp
. in Different of Chop Sizes and
Fermentation Times
F. F. Munier1 & H. Hartadi21Graduate Student of Animal Science Institute, Gadjah Mada University, Fauna
Street No. 3, Bulaksumur, Yogyakarta, 55281
2Animal Science Faculty, Gadjah Mada University, Fauna Street No. 3,
Bulaksumur, Yogyakarta, 55281
Abstract
Theobromine is one of alkaloid in cocoa pod husk (CPH). Fresh CPH has high theobromine content reach 0.40% (400.000 ppm) which is fresh CPH portiontheobromine content reach 0.40% (400.000 ppm) which is fresh CPH portioncontent reach 0.40% (400.000 ppm) which is fresh CPH portion in ruminant ration more than 30% to be affected of health. This research aim was to know the change of theobromine content of CPH with different chop sizes weretheobromine content of CPH with different chop sizes werecontent of CPH with different chop sizes were fermented in the different time of incubation by Aspergillus spp. Three species of Aspergillus spp. were used in this research i.e. A. niger, A. oryzae and A. ficuumficuum BPT. There were 4 chop sizes i.e.; irrigular size (A1), 1x5 cm (A2), 3x5 cm (A3), and 5x5 cm (A
4). The incubation times were; 0, 5, 7, and 9 days, with three replications.
The solely parameter was theobromine content which were prepared according totheobromine content which were prepared according to content which were prepared according to AOAC (1990) and analyze using HPLC (Chen et al., 2008). The result of research showed that CPH fermented using A. niger, the lowest of theobromine content fortheobromine content for content for chop size A2 and incubated 9 days was 38.088 ppm. CPH fermented using A. oryzae had the lowest of theobromine content in Atheobromine content in Acontent in A
2 and 9 days only 36.215 ppm. CPH
fermented with A. ficuum had the lowest of theobromine content in Aficuum had the lowest of theobromine content in A had the lowest of theobromine content in Atheobromine content in A content in A2 and 9 days reach 42.914 ppm. It was concluded that the best of CPH fermented for reducing ofCPH fermented for reducing offermented for reducing ofermented for reducing of theobromine content for 1x5 cm (A content for 1x5 cm (A2) chop size with 9 days fermentation, fermentedchop size with 9 days fermentation, fermentedwith 9 days fermentation, fermented by A. oryzae.
Keywords: theobromine, cocoa pod husk, Aspergillus spp., chop sizes, incubationtheobromine, cocoa pod husk, Aspergillus spp., chop sizes, incubation, cocoa pod husk, Aspergillus spp., chop sizes, incubation times
Introducton
a long year whch s CPH abundant avalable n harvest season. Utlzaton of fresh CPHas feed supplementhas lmtng factor whch one alkalod s theobromne. CPH has hgh of theobromne contentor 3,7-dmethylxanthne(Mahyuddn and Bakre, 1993; Aregheore, 1999). The hgh concentraton theobromne affected the health ofAregheore, 1999). The hgh concentraton theobromne affected the health of). The hgh concentraton theobromne affected the health of theobromne affected the health oftheobromne affected the health ofaffected the health of anmal (Goldng, 1985). The theobromne contenttheobromne content contentofCPH about 0.17-0.20% (Wong0.17-0.20% (Wong and Hassan, 1988), whle n cocoa beans reached 1.5-3.0%, and n dry cocoa beans8), whle n cocoa beans reached 1.5-3.0%, and n dry cocoa beans), whle n cocoa beans reached 1.5-3.0%, and n dry cocoa beanswhle n cocoa beans reached 1.5-3.0%, and n dry cocoa beans1.5-3.0%, and n dry cocoa beans.5-3.0%, and n dry cocoa beans5-3.0%, and n dry cocoa beans.0%, and n dry cocoa beans%, and n dry cocoa beans reached 1.8% (Wllson, 1999)..8% (Wllson, 1999)..
Decreasng of theobromne content can be done wth fermentaton method us-theobromne content can be done wth fermentaton method us-content can be done wth fermentaton method us-ng mcrobe agent such as fuus-ng. Some researchers used A. niger as decomposeras decomposer of CPH n sold fermentaton process (Yamaoka-Yano and Mazzafera, 1999; Huq,(Yamaoka-Yano and Mazzafera, 1999; Huq, 2006) or A. oryzae. oryzae (Lubs et al., 2002). They found that CPH theobromne contenttheobromne contentcontent were reduced due to the role of enzyme actvty that secreted by Aspergillus spp.
Based on those results, n ths research we used A. niger, A. oryzae and A. fi- fi-cuum n sold fermentaton wth CPH as a substrate. The am of ths research was to decrease theobromne content of CPH n Laboratory scale that subsequently thetheobromne content of CPH n Laboratory scale that subsequently the content of CPH n Laboratory scale that subsequently the fermented CPH would used for alternatve forage, and also we studed about storage tme of fermented CPH as feed.
Materals and Methods
Ths experment was fermented CPH usng three of Aspergillusspp..e. A. ni-ger, A. oryzae and A. ficuum, n four types of chop szes .e. rregular sze (A1) as control, 1 x 5 cm (A2), 3 x 5 cm (A3), and 5 x 5 cm (A4) and four knds of ncubaton tmes .e. 0, 5, 7 and 9 days. CPH was ar dred for 8 hours after choppng, whle powder fung were used as much as 1% and then added urea 1% of CPH weght. Urea dssolved wth sterle water n rato 1 : 10 parts. Fresh CPH used 1 kg every treatment whch ths was spread n plastc box (the frst coatng). CPH sprayed wth urea and noculated of Aspergillus spp., the next wth same procedure was done for the second and thrd coatngs.
After completon of fermentaton process, CPH was removed and to be ar-fermentaton process, CPH was removed and to be ar-process, CPH was removed and to be ar-dred for 6 hours. The weghng of CPH and to be contnued wth samples dryng usng oven n temperature of 55 oC for 4 days. About 100 g of fermented CPH was
dred usng freeze dryer for 4 days. Dred CPH was ground wth the mortal whch was surrounded by ce to mantan optmal temperature and theobromne content,theobromne content,content, and then followed by analyss of theobromne content of CPH. Analyss of theobro-theobromne content of CPH. Analyss of theobro-content of CPH. Analyss of theobro- theobro-mne content was recommended by EFSA (2008) usng Hgh-Performance Lqudcontent was recommended by EFSA (2008) usng Hgh-Performance Lqud Chromatography(HPLC).
Results and Dscusson
Al-kalod s organc component from plant whch has contan of ntrogen, commonly as alkals and t has bology actvty. The alkalod n plant has functon for attact of pest (Urch, 1994). Fermentas treatment can decrease CPH theobromne content. CPHtreatment can decrease CPH theobromne content. CPHtheobromne content. CPHcontent. CPH non fermented has hgher of theobromne content (Table 1).theobromne content (Table 1).content (Table 1).(Table 1).e 1).).
Note: A1= rregular sze, A2= 1 x 5 cm, A3= 3 x 5 cm, A4= 5 x 5 cm.
Table 1. Theobromne content of cocoa pod husk fermented byTheobromne content of cocoa pod husk fermented by A. niger (ppm)
Incubaton
The fndng results showed that fermentaton process affected the decreasngfermentaton process affected the decreasng process affected the decreasng of CPH theobromne content. In A. niger fermentaton treatment, there was an effectfermentaton treatment, there was an effect on CPH theobromne content due to dfferences of chop szes and ncubaton tmes.CPH theobromne content due to dfferences of chop szes and ncubaton tmes. Reducng of CPH chop sze was parallel to reducng of theobromne content. Thetheobromne content. The content. The A
2 and A4 chop szes showed average of the lowest and the hghest of theobromne
content, respectvely. On the other sde, ncreasng of ncubaton tme was nversed wth decreasng of average of theobromne content. The lowest average of theobro-The lowest average of theobro-mne content was acheved through fermentaton at 9 days of ncubaton (reduced was acheved through fermentaton at 9 days of ncubaton (reduced9 days of ncubaton (reduced 76.91%). From ths experment, the lowest of theobromne content (38.088 ppm) was acheved n A
2 chop sze when ncubated durng 9 days.ncubated durng 9 days.
Fermentaton usngermentaton usngusng A. oryzaeoryzae n treatment of chop szes and ncbaton tmes showed effect to decreasng of CPH theobromne content that was presented n Ta-content that was presented n Ta-that was presented n Ta-ble 2. Reducng of CPH chop sze had correlaton wth reducng of theobromneReducng of CPH chop sze had correlaton wth reducng of theobromnetheobromne content. The A2 chop sze showed average of the lowest theobromne content, whle A
4 was the hghest theobromne content. The longer ncubaton tme supported
re-ducng theobromne content. The lowest average of theobromne content was occurtheobromne content. The lowest average of theobromne content was occur content. The lowest average of theobromne content was occurThe lowest average of theobromne content was occur was occur at 9 days fermentaton (reduced 23.81%). From ths experment, the lowest of theo-9 days fermentaton (reduced 23.81%). From ths experment, the lowest of theo-bromne content (36.215 ppm) was acheved n A2 chop sze when ncubated durngncubated durng 9 days.
average of theobromne content was acheved through fermentaton at 9 days of was acheved through fermentaton at 9 days of9 days of ncubaton (decreased 28.09%). From ths experment, the lowest of theobromne content (36.512 ppm) was acheved n A2 chop sze when ncubated durng 9 days.ncubated durng 9 days.
The average for theobromne content n CPH non fermentaton (0 day) was 192.556 ppm (192.556 mg/kg), kg). Ths value was lower from reported by HaryatHaryat and Harjosuwto (1984); Wong and Osman (1988) about 0.17-0.20% (1,700-2,000Wong and Osman (1988) about 0.17-0.20% (1,700-2,000 mg/kg), but EFSA (2008) reported hgher reach 0.40% (4,000 mg/kg). DfferentDfferent of theobromne content for non fermented CPH was caused by dfferent treatmentcontent for non fermented CPH was caused by dfferent treatmenton fermented CPH was caused by dfferent treatmentwas caused by dfferent treatment of chop sze. Ths research, CPH was chopped and ar dred durng 8 hours whchchop sze. Ths research, CPH was chopped and ar dred durng 8 hours whch. Ths research, CPH was chopped and ar dred durng 8 hours whch was decrease water content and followed by decreasng of theobromne content.theobromne content.. The cocoa bean had theobromne content hgher reached 1.5-3.0% (15,000-30,000content hgher reached 1.5-3.0% (15,000-30,0001.5-3.0% (15,000-30,000.5-3.0% (15,000-30,0005-3.0% (15,000-30,000.0% (15,000-30,000% (15,000-30,000 mg/kg) (Harrson, 2001), whle dry beans had theobromne content lower only 1.8%) (Harrson, 2001), whle dry beans had theobromne content lower only 1.8%Harrson, 2001), whle dry beans had theobromne content lower only 1.8%whle dry beans had theobromne content lower only 1.8%had theobromne content lower only 1.8%content lower only 1.8% (18,000 mg/kg) (Wllson, 1999).. The CPH theobromne content was the lowestcontent was the lowest among 3 varetes of Aspergillusspp. after fermentaton process that was A. niger n A2 wth ncubaton of 9 days (45.052 ppm).9 days (45.052 ppm).
Reducng of CPH theobromne content caused by decomposng actvty ofcontent caused by decomposng actvty ofactvty of
Aspergillus spp. whch was durng fermentaton process to result heat (ncreasng
Note: A1= rregular sze, A2= 1 x 5 cm, A3= 3 x 5 cm, A4= 5 x 5 cm.
Table 2. CPH theobromne content KBK nonfermented and fermented wth A. niger (ppm)
Incubaton
Table 3. CPH theobromne content KBK nonfermented and fermented wth A. ficuum (ppm)
of substrate temperature). Besde that occured hydrolyss n CPH substrate. In ths step, theobromne dssolve n cell lqud and came out together wth evaporatontheobromne dssolve n cell lqud and came out together wth evaporatondssolve n cell lqud and came out together wth evaporaton process. Rohan (1963) and Alamsyah (1991) were reported that theobromne con-theobromne con- con-tent reduce durng fermentaton process because soluble n cell lqud and dffu-son n cocoa bean nb. Dffudffu-son process wll stop f occur balance of theobromnetheobromne content n cocoa bean. Decreasng of CPH theobromne content was relatvely low. Decreasng of CPH theobromne content was relatvely lowtheobromne content was relatvely low between A. niger compared A. ficuum. Ths case had relatton wth ablty A. niger
to utlze theobromne as energy source. Accordng to Asano et al. (1993) and Hakltheobromne as energy source. Accordng to Asano et al. (1993) and Hakl et al. (1999) that A. niger niger had ablty to utlze theobromne as energy source th-theobromne as energy source th-rough methylxanthnes degradaton.
Conclusons
The chop sze of 1x5 cm and ncubaton tmes of 9 days affected decreasng of CPH theobromne content. The best of CPH fermented for reducng of theobromnetheobromne content. The best of CPH fermented for reducng of theobromne content. The best of CPH fermented for reducng of theobromneCPH fermented for reducng of theobromnefermented for reducng of theobromneermented for reducng of theobromne content for chop sze 1x5 cm (Achop sze 1x5 cm (A2) wth 9 days fermentaton, fermented by A. oryzae.
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