Effect of Inoculations of Trichoderma viride and Saccharomyces cerevisiae Mixed Culture on Chemical Composition , Fiber , Digestibility and Theobromine Cocoa Pod Fermentation

The objective of the study was to identify the effect of Trichoderma viride and Saccharomyces cerevisiae inoculant on chemical compositions, digestibility, and theobromine concentration of fermented cocoa pod. This experiment consisted of four treatments, namely cocoa pods without fermentation as control (R0); fermentation of cocoa pods with inoculant T. viride (R1); fermentation of cocoa pods with inoculant S. cerevisiae (R2); and fermentation of cocoa pods with inoculant T. viride and S. cerevisiae mixed culture (R3). Each treatment had 3 replicates, and then was fermented for 10 days. Variables observed were the chemical compositions i.e dry matter (DM), organic matter (OM), crude protein (CP), ether extract (EE), crude fiber (CF), nitrogen free extract (NFE), fiber fraction (Neutral detergent fiber and acid detergent fiber), in vitro digestibility, and theobromine concentration. Data were analysed by one-way analysis of variance and followed by Duncan’s new multiple range test (DMRT), if there were any significant difference. Results showed the inoculum affected (P<0.05) the chemical composition, fiber fraction and in vitro digestibility. However, theobromine was not detected on cocoa pod without fermentation and fermentation. Compared to group R0, inoculation with T. viride and S. cerevisiae mixed culture (P<0.05) resulted in higher DM concentration (92.78% vs 89.72% respectively), higher CP (7.43% vs 5.63% respectively), higher NDF (79.41% vs 61.18% respectively), higher ADF (73.04% vs 47.94% respectively), but was not significantly different on DM and OM digestibility (21.22% vs 22.24%, and 22.67% vs 24.31% respectively) than cocoa pod without fermentation. It is concluded that inoculant T. viride and S. cerevisiae mixed culture increased CP concentration, but had no effect on in vitro digestibility.


INTRODUTION
Limited availability of forage, force people to seek for other alternative feeds that could replace a part or total forages. Plantation wastes to be one option in dealing with the issue of the lack of forage availability in the dry season, but waste products of plantation could also pollute the environment. According to Ditjen Perkebunan (2012), there were approximately 1,745,789 ha of cocoa plantation in 2011.
Cocoa pod contain various minerals. Toharmat et al. (2006), reported that feeding cocoa pod to Etawa goat as a fiber source also provide, Mg and Zn in a higher content than those provided by grass, bark coffee husk, and rice straw. Wahyuni et al. (2008) reported that fermented cocoa pod can be used as animal feed in growing goats up to the level of 40%. Puastuti et al. (2008) reported that a ration based cocoa pod without ammoniation but with organic Zn supplementation resulted in weight gain equivalent to the daily ration of grass-based, caused by sufficient consumption rate and nutrient digestibility, N retention and good fermentation characteristics. Ginting (2004) reported that pod husks of more than 15% in the ration can reduce performances of goats.
Cocoa plantation wastes such as pod husks are commonly just piled up in the field after being harvested. This practice can cause discomfort odor or emerging which cause disease to the cocoa plants. Pod husks contained anti nutritive substances such as theobromine that limits its level of inclusion in the diets. Efforts to improve the nutritive quality of the cocoa pod including, drying, addition of urea (ammoniation), and fermentation. The objectives of this study was to investigate the effect of fermenting cocoa pod using different types of innoculant on the chemical composition, digestibility, and theobromine content.

MATERIAL AND METHODS
The experiment was performed at the Laboratory Animal Feed Technology, Laboratory Biochemistry of Nutrition, Feed Animal Nutrition Department, Faculty of Animal Science, Gadjah Mada University.
Instruments used included scales capacity 10 kg with sensitivity of 0.1 kg, sealer and vacuum, grinder with sieve diameter's 1 mm, analytic scales with sensitivity of 0.0001 g, pH meter, digital thermometer, oven, lactic analysis tools, ammonia rumen tools, in vitro tools, gas chromatography tools, and high performance liquid chromatography Knauer type UV 6000 LP. Materials used included cocoa pod, inoculant of L. plantarum, inoculant of S. cerevisiae, cassava meal, water, chemicals for proximate analysis, ruminal fermentation, theobromine analysis, and in vitro digestibility.

Substrate preparation, fermentation, and treatments
Fresh cocoa pod was collected from traditional farming Gunung Kidul Regency. Cocoa pod was cut to size of 1×5 cm and sun-dried for 10 hours to decrease the water content. Two species of fungal were used as innoculant, namely T. viride and S. cerevisiae.
Fermentation was conducted at laboratory scale in room that was sterile. The room was watered with desinfectan, the tools were sterealised with alcohol (90%). Cocoa pod was fermented in fermenter bag (as silo), and inoculated with T. viride and S.cerevisiae was performed at 1% dry matter, by mixing and spreading the innoculant evenly. Silo was made to be aerobic by making a hole.The length of fermentation was set at 10 days at room temperature (Ginting & Krisnan 2006).
Three processing treatments and one control treatment were as follows: R0: Unfermented cocoa; R1: Cocoa pod fermented using T. viride; R2: Cocoa pod fermented using S. cerevisiae; and R3: Cocoa pod fermented using T. viride and S. cerevisiae mixture.

Chemical analysis
Samples of dried silages were analyzed for chemical compositions using proximate analyses (AOAC 2005). Neutral Detergent Fiber (NDF) and Acid Detergent Fiber (ADF) analyses were performed according to the procedures of Van Soest & Robertson (1985). In vitro digestibility study was performed according to the procedures of Tilley & Terry (1963) at Biochemical Nutrition Laboratory, of Animal Science Faculty, the Gadjah Mada University. Theobromine analysis to was conducted as recommended by European Food Safety Authority using HPLC (AOAC 2005).
Data of chemical composition, fiber fraction, and in vitro digestibility were analyzed using analysis of variance (ANOVA) and followed by Duncan's new multiple range test (DMRT) if there were any significant differences (Steel & Torrie 1993). All of statistic calculation was performed using software of Statistical Product and Service Solution version 16.0 (Soleh 2005).

Proximate and fiber fraction composition of fermented cocoa pod
The fermented cocoa pods have higher dry matter content than that of the unfermented cocoa pod. The Increased dry matter contents of the fermented cocoa pod might be caused by any growth of fungal in cocoa pod. The growth of fungi was characteritically slower than that of bacterial T. viride secreted cellulose at eight days, which can be influenced dry matter content. Suparjo et al. (2009) reported that fungi would utilized the organic matter from substrate and caused losses in dry matter. Mulato & Widyatomo (2003) showed that time of fermentation can affect the dry matter content of substrates. The proximate compositions were shown in Table 1.  Crude protein content was significantly affected by the type of innoculants. Inoculation with T. viride had highest effect on crude protein level. Ginting & Krisnan (2006), showed that fermentation using Trichoderma sp. as an innoculant increased the crude protein content of the substrates. In the current experiment the crude protein content was lower when treated with T. viride and S. cerevisiae mixed culture, which could be due to any negative interactions between both of innoculant.
Fungal innoculation significantly increased crude fiber contents of the fermented cocoa pods. This higher crude fiber content could be caused by increased cell wall, of the hyfa of fungal. Utomo (2001), reported that crude fiber is organic matter which was divided to celulosa, hemicellulosa, and lignin. All of these components were found in hyfa of fungi. Cocoa pods fermentation with T. viride and S. cerevisiae mixed culture have higher free extract nitrogen content. During the stationary phase, the T. viride used the substrate free extract nitrogen before degrading the cellulose. Kamara et al. (2008) showed that glucose at early phase was used by T. viride for growing and then strated to degrade of fiber. The fiber fractions of substrates were presented in Table 2.
Neutral detergent fiber and ADF content were significantly higher in the fermented cocoa pod compared to the unfermented ones. Fungi types affected the NDF and ADF content of fermented cocoa pod. Cell wall increased during the prolonged time of fermentation. This result differed to that reported by Alemawor et al. (2009), who reported that cocoa pods fermented with Aspergillus niger have lower cell wall contents.

In vitro digestibility of cocoa pod fermentation
The effects of fermentation and type of inoculants on the in vitro digestibility were showed in Table 3. In vitro digestibility of substrate dry matter and organic matter was significantly affected by fermentation. Unfermented cocoa pod have higher DM and OM digestibility than those fermented with T. viride, but were not significantly different to than those of fermented cocoa pods using inoculant of T. viride and S. cerevisiae mixed culture. Utomo (2001), reported that ADF is hardly to be degraded so that it could become an inhibitor in feed. Yunus (1997) reported that factors that affect the in vitro digestibility are composition of feed, ruminal fluid temperature, time of incubation and analysis method.  The theobromine content of fermented and unfermented cocoa pods presented in Table 4. Theobromine content of unfermented and fermented cocoa pods were not detected by HPLC. This was caused by the pre-preparation procedures including sun dried for 10 hours, this porocess might have reduced theobromine content. Mulato & Widyatomo (2003), showed that dried and boiled can decrease theobromine content.

CONCLUSION
It is concluded that inoculation with T. viride and S. cerevisiae mixed cultures in fermenting cocoa pods affected the chemical composition and fiber fraction. However it did not affect the in vitro DM and OM digestibility of cocoa pod. The theobromine content of the fermented and unfermented was not detected in this study.