Evaluation of kepok banana corm fermented with Saccharomyces cerevisiae and Aspergillus niger as feeds

Banana (Musa spp. L.) are major staple crops, particularly in developing countries (Azhar and Harrison, 2008; Vatanasuchart et al., 2012). Indonesia produces a 3.5 million metric tons per year and a 55 million metric tons are produced globally. Bananas are generally consumed directly as dessert bananas (mainly from the Cavendish subgroup) or fried, in the instance of plantains (starchy cooking bananas) (Azhar and Harrison, 2008; Mohapatra et al., 2010).
        
The high banana production also produces an abundance of by-products. These can be used as low-cost, high-energy animal feeds, with starches and non-starch polysaccharides comprising 70% of the total carbohydrates. However, the banana and its waste also contain antinutritional ingredients, such as tannins, saponins, sterols, glycosides, quinones, terpenoids, polyphenols and alkaloids (Jamuna et al., 2011; Krishna et al., 2013).
        
Efforts to overcome problems in using such waste as feeds include fermentation by yeast (Saccharomyces cerevisiae) and fungus (Aspergillus niger), prior to use. Both microorganisms produce amylase and glucosidase, enzymes that have been known to degrade starch molecules and separate glucose from the non-reducing sugar terminal (Aiyer, 2005; Bhat et al., 2005; Englyst et al., 2007; Winarno, 2010).
        
Although banana pseudostems and peels are often used as fodder, the corms have not been used for animal feeds. The corms are readily obtainable at low-cost and their use could also decrease waste pollution. Furthermore, Pisang Kepok, or the Kepok banana, is a plantain banana produced in Indonesia that generates corms containing 79% carbohydrates (dry weight basis) and hence, fermentation of the corms may be potentially useful as feed.
        
Therefore, the purpose of this study was to evaluate the nutrient and tannin contents of fermented Kepok banana corms using S. cerevisiae and A. niger, as a function of fermentation time and microbe type.

Banana waste and nutrient fermentation
 
Fresh Kepok banana corms, were obtained from local farmers in the district of Kupang, East Nusa Tenggara, Indonesia. The microbial yeast (S. cerevisiae) used had 1.25 x 10¹³ CFU/g and A. niger had 1.03 x 10¹² CFU/g, as determined by the Laboratory of Microbiology, Faculty of Veterinary Medicine, Nusa Cendana University.
 
Research design
 
A completely randomized design (CRD) with a 3 x 3 factorial arrangement was used, with three replications. Two main factors were investigated: microbial strains (P) and incubation time (Q). Factor P comprised three subgroups, P₁: S. cerevisiae, P₂: A. niger and P₃: S. cerevisiae + A. niger. Factor Q consisted of three different incubation times, Q₁: 48 h, Q₂: 72 h and Q₃: 96 h.
        
S. cerevisiae and A. niger were cultured separately in a nutrient medium containing 40 g glucose (analytical grade), 2 g (NH₄)₂SO₄, 1.5 g KH₂PO₄ and 1 g MgSO₄ mixed in 1000 ml of distilled water, as previously described (Dhanasekaran et al., 2011; Retnoningtyas et al., 2013; Tudor et al., 2013) but with modifications. The medium was homogenized using a magnetic stirrer and then sterilized by autoclaving at 121°C for 15 min. Once the liquid culture medium was cooled, 2 g of the microorganism was added before incubation at 30°C for 96 h.
 
Kepok banana corms fermentation
 
Steamed Kepok banana corm flour (substrate) was inoculated with the liquid culture at 10% w/v, as recommended by Aziz et al., (2012). The inoculated substrates were enclosed within sealed polybags (2 kg capacity) and fermented under aerobic conditions. The fermentation product was inactivated by drying at 60°C for 24 h, as described by Ozturk et al., (2009) and Jenses et al., (2013) with modifications, and then stored in sterile plastic bags at 4°C until analysis.
 
Statistical analysis
 
All experimental factors were conducted in triplicate. Data were analyzed using analysis of variance (ANOVA). Duncan’s multiple range test was used to determine differences among treatment mean values (p<0.05). Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS) version 14.

Chemical composition of corms
 
Kepok banana corms contained 87.70% dry matter, 90.26% organic matter, 3.58% crude protein, 2.15% crude lipid, 19.33% crude fiber, 57.41 g/100 g neutral detergent fiber (NDF), 25.02 g/100 g starch, 3385 Kcal/kg energy and 909.14 mg/100 g tannins. The energy content exceeded that of rice bran (3100 Kcal/kg) and the carbohydrate content was higher compared to plantain fruits at 78.9% (Akubor, 2005). Thus, the energy derived from banana wastes can potentially be used as an alternative animal feed.

Influence of microbial fermentation on energy and protein contants
 
The highest dry matter obtained was 90.26%, (Table 1) which was achieved by P₃ (S. cerevisiae + A. niger). Thus, fermentation of Kepok banana corms using the microbial consortium (S. cerevisiae + A. niger) resulted in the most effective degradation of the complex carbohydrates into glucose, maximizing the amount of dry matter produced.
 

        
The highest organic matter (88.30%) was generated by P₁ (S. cerevisiae) (Table 1). A similar observation was made by Dhanasekaran et al. (2011) for pineapple peel fermentation, where produced maximum wet and dry biomass with S. cerevisiae used as inocula. The highest crude protein content of 5.56% was attained by P₁ (S. cerevisiae). This finding was in agreement with previous work that reported an increase in crude protein content in fermented substrates was most likely a result of the high protein contents of the yeast cells (Anonymous, 2014). Besides containing high levels of protein, yeast (S. cerevisiae) also contain mannan oligosaccharides and b-glucan (Price et al, 2010), chitin and fatty acids (Anonymous, 2014).
        
The highest energy content (3452.33 Kcal/kg), which corresponded to P₃ (S. cerevisiae + A. niger) was not significantly different (P>0.05) to P₂ (A. niger) at 3452.00 Kcal/kg. This result indicates that the microbial consortium used for fermentation was able to raise the energi contents of substrate comparing with culture alone. The increasing energy contents in fermented corms of the study was because the capability of enzyme amylase from the microbial consortium used to convert high starch into sugars. Since enzyme a-Amylase, Glukoamylase, cellulase, sacrosidase and lipase (Aiyer, 2005; Winarno, 2010) contained in Saccharomyces cerevisiae and a-Amylase, a-glucosidase, b-glukosidase, glukoamylase, cellulase, protease, lypase, mananase and pectinase enzymes produced from Aspergillus niger (Uthumporn et al., 2010) the nutrient structure bonds of the corm during fermentation was hidrolyzed and degraded then converted it into the energy.
        
The incubation times (Q1, Q2 and Q3) significantly affected (P<0.01) the nutrient contents in the fermented Kepok banana corms (Table 2). The highest crude protein content of 5.15% was occurred in Q3 (96 h incubation), in line with a previous study, which reported that incubation for 72 h using S. cerevisiae increased the protein value derived from pineapple peels (Dhanasekaran et al., 2011).
 

        
The proximat analysis showed highly significant interactions (P<0.01) between both microbes types and incubation time on the nutrient contents of Kepok banana corms fermentation.The dry matter contents varied from the highest value of 91.84% for P₁Q₁ (S. cerevisiae for 48 h) to the lowest value of 87.02% for P₁Q₂ (S. cerevisiae for 72 h). The highest organic matter of 89.93% was acquired by P₁Q₃ (S. cerevisiae for 96 h) and the lowest of 86.36% by P₃Q₂ (S. cerevisiae + A. niger for 72 h). These results are consistent with previous reports that have used enzymes to increase the nutritional content of fermentation products (Zobel and Stephen, 1995; Aiyer, 2005; Smith et al., 2005).
        
The maximum crude protein content (5.81%) was achieved by P₁Q₃ (S. cerevisiae for 96 h), whereas the lowest value of 4.40% was attained by P₃Q₂ (S. cerevisiae + A. niger for 72 h). Using the Saccharomycopsis fibuligera yeast for rice fermentation, Nicolau et al., (2011) reported an increase in the contents of sugar, protein, amino acids, phosphorus and B vitamins.The increase in crude protein value by fermentation of Kepok banana corms was due to the ability of proteolytic enzymes to convert organic and inorganic matter into proteins and the proteinaceous contributions from the microbial (S. cerevisiae and A. niger) cells (Price et al., 2010).
        
The crude fiber values of the present results are consistent with previous research on cassava waste fermentation using a microbial consortium of Trichoderma viride, S. cerevisiae and A. niger that showed a decrease in crude fiber content of the substrate (Suryani, 2013).The NDF contents varied from the highest value of 43.45 g/100 g for P₁Q₁ (S. cerevisiae for 48 h) to the lowest value of 38.29 g/100 g for P₂Q₃ (A. niger for 96 h). The highest starch content of 35.54 g/100 g was attained by P₃Q₂ (S. cerevisiae + A. niger for 72 h). Uthumporn et al. (2010) mentioned that the starch granules of corn, mung beans, cassava and sago, can be decomposed using a-amylase and glucoamylase via fermentation at 35oC for 24 h.The P₃Q₂ (S. cerevisiae + A. niger for 72 h) generated the highest energy content of 3511 Kcal/kg, while P₁Q₂ (S. cerevisiae for 72 h) provide the lowest at 3397 KCal/kg. Previous studies verified that fermentation of raw foods can reduce the tannin contents (Lakra and Sehgal, 2009; Njidda and Ikhimioya, 2012). High tannin contents in the feeds have resulted in reduced consumption, digestion and absorption of nutrients in pigs (Akande et al., 2010). Condensed tannins and non-starch polysaccharides in feeds may also decrease the growth performance of pigs (Emiola and Gous, 2011).
        
Kepok banana corm flour fermented with S. cerevisiae gave maximal nutrient contents at varied incubation times i.e. 91.84% dry matter (48 h), 89.93% organic matter (96 h), 5.81% crude protein (96 h) and decreased crude fiber to 15.61% (72 h). Using A. niger at varied incubation time resulted in a reduction of NDF to 38.29 g/100 g (96 h) and tannins to 727.35 mg/100 g (72 h). Using the microbe consortium (S. cerevisiae + A. niger) for 72 h, generated maximum in starch (35.54 g/100 g) and energy (3511 Kcal/kg) values. The increased starch and energy content  in the fermented product may be useful ingredients as feed.