| Livestock Research for Rural Development 26 (4) 2014 | Guide for preparation of papers | LRRD Newsletter | Citation of this paper |
Two studies on the effect of fermentation on nutrients contents of fermented copra meal, cellulases activity, bird performance and carcass percentage were carried out. In the first study, copra meal was fermented by Trichoderma viride with 4 levels of inoculum; L-1: without inoculum, L-2: 17.7 CFU (Coliform units) /g/kg copra meal, L-3: 35.4 CFU/g/kg copra meal, L-4: 53.1 CFU/g /kg copra meal, and 4 different incubation times; T-4: 4 days, T-6: 6 days, T-8: 8 days and T-10: 10 days. Fermented copra meal was analysed for nutrients and extracted to produce crude enzyme. Cellulase activity was analyzed by the DNS (3,5-dinitrosalicylic acid) method. In the second study, an enzyme product derived from fermented copra meal was tested in an in-vivo study. A total of 200 broiler chickens were kept for 6 weeks and given 5 different types of feeds: CM0: without copra meal; CM5-E: 5% copra meal + 0.5% crude enzyme; CM10-E: 10% copra meal + 0.5% crude enzyme; CM15-E: 15% copra meal + 0.5% crude enzyme; CM20-E: 20% copra meal + 0.5% crude enzyme). Feed and water were available at all times. The parameters observed were weight gain, feed intake, feed conversion ratio and breast meat percentage. The first study adopted a factorial randomized design (4 levels of inoculum, 4 incubation times and three replications), while the second study applied a completely randomized design with 5 treatments and 5 replicates (cages).
Inoculum and incubation times significantly improved nutritive value of fermented copra meal. Celulase activity of crude enzyme was 0.84 g glucose/l. Growth and feed conversion ratio of birds fed a diet with 15% ot enzyme-treated copra meal were the same as in birds fed the control diet without copra meal. Breast meat percentage of birds fed copra meal diets supplemented with crude enzyme was similar to that from birds fed the control diet.
Key words: bioconversion, coconut cake, fungi, nutritive value, poultry
Copra meal, the residue after oil extraction of coconut, is produced in large quantity in many Asian and Pacific countries. Problems associated with the use of copra meal for poultry are due to its high dietary fiber (Saha 2003), imbalanced amino acids and bulkiness (Sundu et al 2009). These are the reasons why the use of copra meal in poultry diet is limited, being less than 5% in commercial diets. Effort to improve the quality of this by-product through commercial enzyme supplementation comes up with limited success (Sundu et al 2004). This is due possibly to the fact that commercial enzymes were mainly designed for maize-soybean based diets.
An enzyme that is suitable for copra meal-maize based diets needs to be produced to tackle the problem of low quality of copra meal. The application of solid state fermentation (SSF) as an enzyme production method could offer environmental, economical and nutritional benefits over conventional method (submerged liquid fermentation) (Filler 2001). Microbes in the class of fungi are commonly used to produce many different type of enzymes (McCleary 1988), including cellulases.
Cellulolitic enzymes produced from filamentous fungi, such as Trichoderma sp, have gained increasing attention due to the fact that these fungi could not only secrete cellulase in large quantity but also had a potential to fully hydrolyze crystalline cellulose (Kubicek 1992). Among Trichoderma sp, Trichoderma viride is one of the celulolitic fungi that could produce cellulase with high efficacy to break down cellulose. The use of this fungus with copra meal as a substrate in enzyme production is scarcely reported. The present study was designed to determine the effects of fermentation of copra meal by Trichoderma viride on cellulase activity, nutrients content of fermented copra meal and its effect on broiler performance.
Trichoderma viride was obtained from the Laboratory of Microbiology, Faculty of Science, University of Brawijaya, Indonesia. Copra meal, purchased locally, was produced by mechanical extraction. Copra meal was finely ground to 1-2 mm particle size. A method of Ganjar (2006) was applied for solid state fermentation procedures. Solid state fermentation was performed with fine copra meal as a solid substrate. The substrate was autoclaved for 20 minutes at 20 psi and then cooled to room temperature prior to inoculation of fungi spores with different concentrations (TV0: no inoculum, TV1: 17.7 coli form unit (CFU)/g/kg copra meal, TV2: 35.4 CFU/g/kg copra meal and TV3: 53.1 CFU/g/kg copra meal). The copra meal and fungi were mixed thoroughly and added with distilled water to maintain moisture content to about 80%. The mixture was put on the tray with 2 cm thickness and kept with different incubation periods of 4, 6, 8 and 10 days. The fermented copra meal was then harvested and dried at 60oC for 24 hours. The fermented copra meal was analysed for proximate fractions. Analysis of the crude protein content, crude fat and crude fiber was carried out by the methods of AOAC (1990).
Trichoderma viride-fermented copra meal produced in this study was extracted by using a method of Jacob and Prema (2006). Fermented copra meal was diluted with distilled water (ratio of 1 kg substrate to 5 l distilled water). The mixture was then placed in a rotary shaker for 1 h with rotation speed of 200 rpm. The mixture was filtered with muslin and the supernatant was then centrifuged with speed of 2,500 rpm for 15 minutes. The fluid obtained was called crude enzyme and used for analysis of cellulase activity.
The parameters measured were crude protein, lipid and crude fiber of fermented copra meal. A completely randomized factorial structure with four different concentrations of inoculum, four different incubation periods and three replicates was adopted. Data were analyzed by analysis of variance using Minitab 14 statistical program (Minitab 2003). Differences among treatments were tested for significance by using Tukey Test (Steel and Torrie 1980).
Enzyme isolation was done by diluting 600 ml of extracted crude enzyme with distilled water up to 1000 ml (400 ml distilled water). To the mixed liquid was added 650 g zwavelzuur amonium (ZA or ammonium sulphate) and stirred until thoroughly mixed to completely dilute ZA. The solution was placed in a beaker sealed with aluminum foil and then allowed to stand for 24 hours. After 24 hours, enzymes will rise to the surface and the enzymes were taken by filtering them with filter paper. Analysis of cellulase activity was done just for the treatment with the lowest crude fiber content (treatment of 53.1 CFU/g/kg copra meal with 10 days incubation period of time). The analysis of cellulase activity was performed by measuring the reducing sugars released using the DNS Method (Omojosola 2008).
The crude enzyme used in this experiment was the enzyme produced from the fermented copra meal with the lowest crude fiber content (treatment of 53.1 CFU/g/kg copra meal with 10 days of incubation time).
A total of 200 day-old male broiler chicks of Cobb commercial strain were used in this study. The birds were randomly distributed into each treatment of 8 birds. The cages were equipped with trough feeders and drinkers. The birds were fed starter diet from day 1 to 21 and grower diet from day 22 to 42 (Table 1). Feed and water were available at all times. Feed was topped up twice a day.
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Table 1: Ingredietns and nutrient composition of the experimental diets |
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|
Ingredients |
Percentage of copra meal in the diet |
|||||||||
|
0 |
5 |
10 |
15 |
20 |
||||||
|
Starter |
Grower |
Starter |
Grower |
Starter |
Grower |
Starter |
Grower |
Starter |
Grower |
|
|
Copra meal |
0 |
0 |
5.0 |
5.0 |
10.0 |
10.0 |
15.0 |
15.0 |
20.0 |
20.0 |
|
Maize |
52.5 |
54.3 |
44.4 |
49.5 |
44.0 |
53.4 |
45.0 |
48.3 |
40.0 |
42.7 |
|
Full fat soybean |
23.0 |
23.5 |
23.5 |
23.0 |
24.5 |
23.0 |
24.5 |
24.7 |
25.3 |
25.0 |
|
Fish meal |
13.5 |
10.0 |
13.8 |
11.0 |
12.5 |
10.5 |
11.6 |
8.0 |
10.0 |
7.0 |
|
Rice bran |
8.5 |
10.0 |
10.8 |
9.0 |
7.0 |
12.0 |
1.0 |
1.0 |
1.2 |
1.4 |
|
Coconut oil |
0 |
0 |
0 |
0 |
0 |
0 |
0.5 |
0.7 |
1.0 |
1.6 |
|
DCP |
1.5 |
1.4 |
1.5 |
1.5 |
1.1 |
1.0 |
1.4 |
1.3 |
1.5 |
1.2 |
|
Premix |
0.3 |
0.2 |
0.3 |
0.3 |
0.3 |
0.3 |
0.3 |
0.3 |
0.3 |
0,3 |
|
DL- Methionine |
0.3 |
0.2 |
0.3 |
0.3 |
0.3 |
0.1 |
0.3 |
0.3 |
0.3 |
0.3 |
|
L-Lysine |
0.2 |
0.2 |
0.2 |
0.2 |
0,1 |
0.1 |
0.2 |
0.2 |
0.2 |
0,2 |
|
Celite |
0.2 |
0.2 |
0.2 |
0.2 |
0.2 |
0.2 |
0.2 |
0.2 |
0.2 |
0.2 |
|
Calculated composition, % in DM: |
|
|
|
|
|
|
|
|
||
|
Protein |
22.1 |
20.6 |
22.0 |
20.6 |
22.1 |
20.2 |
22.1 |
20.5 |
22.1 |
20.6 |
| Crude fiber | 3.5 | 3.7 | 4.3 | 4.2 | 4.7 | 5.3 | 4.8 | 5.4 | 5.4 | 5.4 |
|
Methionine |
0.7 |
0.6 |
0.7 |
0.7 |
0.7 |
0.6 |
0.7 |
0.7 |
0.7 |
0.6 |
|
Lysine |
1.4 |
1.3 |
1.4 |
1.3 |
1.3 |
1.2 |
1.4 |
1.3 |
1.3 |
1.2 |
|
Calcium |
1.2 |
1.0 |
1.2 |
1.1 |
1.1 |
0.9 |
1.1 |
0.9 |
1.1 |
0.8 |
|
Phosphorus |
0.8 |
0.7 |
0.8 |
0.7 |
0.8 |
0.6 |
0.8 |
0.7 |
0.7 |
0.6 |
|
ME (Kcal/kg) |
3088 |
3104 |
3095 |
3118 |
3074 |
3107 |
3055 |
3095 |
3031 |
3091 |
|
DCP: Dicalcium Phosphate; ME: Metabolizable energy |
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Fifty ml of crude enzyme were sprayed onto 10 kg diet to produce a dose rate of 0.5% crude enzyme using a small presure sprayer. The diets were then air dried for three days prior to feeding. The treatment diets were CM0: diet without copra meal; CM5-E: Diet with 5% copra meal plus 0.5% crude enzyme; CM10-E: Diet with 10% copra meal plus 0.5% crude enzyme, CM15-E: Diet with 15% copra meal plus 0.5% crude enzyme and CM20-E: Diet with 20% copra meal plus 0.5% crude enzyme.
On day 42, two birds were randomly taken from each cage and fasted overnight. On day 43, the birds were individually weighed and killed by cervical dislocation. The carcasses and the breast meat were weighed after plucking and eviscerating by removing internal organs, head and shank. Data were expressed in percentages.
The parameters measured were body weight, feed intake, feed conversion ratio and breast meat percentage. A completely randomized design with five different treatments (diets) and five replicate cages of 8 birds each was adopted. Data were analyzed by analysis of variance using the Minitab 14 statistical program (Minitab 2003). Differences among treatments found in the analysis of variance were tested for significance by using Tukey Test (Steel and Torrie 1980).
The inoculum increased the crude protein of Trichoderma viride-fermented copra meal by 30, 24 and 13% with 17.7, 35.4 and 53.1 g CFU/kg copra meal (Table 2).The increase in protein content of fermented copra 17.7 meal is consistent with the previous findings of Dairo and Fasuyi (2008), who found a 17% increase of protein due to fermentation of copra meal. The authors speculated that additional protein in the fermented copra meal was contributed by the microorganisms. However, it is hard to explain where and how the Trichoderma viridae got the additional nitrogen. Possibly, the increased protein of fermented copra meal is only a matter of a decrease in the other fractions (lipids and crude fiber).
|
Table 2: The effect of inoculum level on composition of Trichoderma viride-fermented copra meal (% in DM) |
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|
Parameters |
Level of T. Viride, g CFU/kg copra meal |
SEM |
p |
|||
|
0 |
17.7 |
35.4 |
53.1 |
|||
|
Crude protein, % |
17.5a |
22.7c |
21.7c |
19.7b |
0.497 |
<0.0001 |
|
Lipid, % |
21.2a |
5.80b |
2.31b |
3.08b |
1.13 |
<0.0001 |
|
Crude fiber, % |
18.1a |
13.8b |
10.3b |
10.3b |
0.467 |
<0.0001 |
|
Values with common superscript within a row are not different at p<0.05. |
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The 21% lipid in copra meal was relatively high. However, some publications indicated high concentration of lipid in copra meal. Khanongnuch et al (2006) reported that copra meal contained nearly 19% lipid. Ankrah (1998) even reported lipid content of copra meal being 4 to 21.2%. This may indicate that the copra meal used in this study was poorly extracted. The lipid and crude fiber contents of the copra meal were decreased when the copra meal was fermented. It is not difficult to rationalise this phenomenon as these two compounds were the source of energy for maintanance and production of the fungi. The energy was released to the air as an energy lost. Interestingly, incubation time led to an increase in lipid content (Table 3). The early study of Kalsum and Sjofjan (2008) indicated that lipid content of Neurospora sitopila-fermented tofu waste followed the parabolic curve. In the first three days of fermentation, lipid content of fermented tofu increased over time and started decreasing on fourth day. The same trend was observed in the present study where an increased lipid content of fermented copra meal took place up to 8 days of fermentation and then decreased on day 10. Gross energy content of the fermented copra meal increased with increased levels of inoculum and incubation time. This improvement may be partly due to an increase in either protein content or lipid content.
|
Table 3: The effect of incubation time on com[osition (5 in DM) of Trichoderma viride fermented copra meal. |
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|
Parameters |
Incubation time, days |
SEM |
p |
|||
|
4 |
6 |
8 |
10 |
|||
|
Crude protein, % |
20.4 |
20.5 |
21.7 |
22.0 |
1.1 |
0.148 |
|
Lipid, % |
8.33ab |
8.48a |
8.51a |
7.06b |
0.42 |
0.013 |
|
Crude fiber, % |
14.2a |
13.5ab |
13.3ab |
12.6b |
0.50 |
0.30 |
|
Values with common superscript within a row are not different at p<0.05 |
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It has long been believed that Trichoderma viride is a cellulolytic fungi. Mandels (1970) stated that this fungus had the potential to produce relatively large amounts of cellulases to degrade cellulose. Kubicek (1992) also stated that cellulases produced from Trichoderma sp had potential to optimally hydrolise cystalline cellulose. Accordingly, the use of Trichoderma viride to break down feedstuffs high in cellulose, such as copra meal, was recommended. The lowest levels of crude fiber found in the present study was in the inoculum level of 53.1 CFU/g with 10 days of incubation time. This may indicate that more cellulases were produced in this treatment as more crude fiber was lost. It is for this reason cellulases activity of crude enzyme produced from this particular treatment was measured and this crude enzyme was then used in the present study to improve broiler performance. Data indicated that cellulases activity obtained from 10 days-fermented copra meal with inoculum level of 53.1 CFU/g were in the range between 0.79 and 0.89 g glucose/ liter (Table 4). Since there was no information in the data base on cellulase activity of fermented copra meal, it is too early to conclude that Trichoderma viridae suited to the copra meal substrate to produce cellulases.
|
Table 4: Cellulase activity of extracted crude enzyme (g glucose/l) |
|
|
Replications |
Cellulase activity |
|
1 |
0.79 |
|
2 |
0.83 |
|
3 |
0.88 |
|
4 |
0.89 |
|
5 |
0.80 |
|
Average |
0.84 |
|
SEM |
0.02 |
Sundu et al (2004) stated that the addition of copra meal in the diet depressed feed intake due partly to increased fiber content of the diet. High fiber content leads to the diet being bulky and low in digestibility. The addition of crude enzyme produced from fermented copra meal with Trichoderma viride could maintain feed intake to the same level as on the control diet (Table 5). This indicates that the problem of bulkiness of copra meal could be overcome by the addition of 0.5% crude enzyme produced from Trichoderma viride-fermented copra meal. Results of this study showed that the cellulase enzyme present in crude enzyme apparently worked well. To test this possibility, further studies on digestibility of feed, especially the digestibility of crude fiber, are needed.
In a previous study, the use of copra meal in the diet has been reported to suppress the growth of broilers (Sundu et al 2005). In this present study, growth rate of birds fed the Trichoderma viride-fermented copra meal at the 15% level was similar to that of birds fed the control diet. This indicated that the crude enzyme may play a positive role in feeds containing copra meal.
|
Table 5: The effect of increasing levels of copra meal supplemented with crude enzyme on feed intake, body weight, feed conversion and carcass and breast meat in broilers |
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|
Parameters |
|||
|
FI (g) |
BW (g) |
FCR |
Breast meat (%) |
|
4347 |
2490a |
1.73bc |
28.1 |
|
4339 |
2318b |
1.87a |
27.2 |
|
4337 |
2357b |
1.84ab |
23.3 |
|
4306 |
2526a |
1.71c |
27.2 |
|
4336 |
2244b |
1.93a |
28.2 |
|
5.98 |
25.0 |
0.02 |
0.32 |
|
0.22 |
<0.001 |
<0.001 |
0.17 |
|
Values with common superscript within a column are not different at p<0.05. |
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Carcass quality is an important factor in assessing meat production of broilers. Breast meat can be an indicator of the quality of carcass as breast meat is the most expensive chicken flesh. The percentage of breast meat found in this current study was 23-28%.
Ankrah E K 1998 Chemical composition of cake samples of Ghanaian copra (cocos nucifera). Ghana Journal of Agricultural Science 31: 123-124.
AOAC 1990 Association of Official Analytical Chemist Official Methods of Analyses. Third Edition. AOAC. Washington DC.
Dairo F A S and Fasuyi A O 2008 Evaluation of fermented palm kernel meal and fermented copra meal protein as substitute for soybean meal protein in laying hens. Journal of Central European Agriculture 98: 35-44.
Filler K 2001 Production of enzymes for the feed industry using solid substrate fermentation. In: Proceedings of Alltech's 17th annual symposium. (Eds, T.P. Lyons and K.A. Jacques). Nottingham University Press. United Kingdom.
Gandjar I 2006 Mikologi Dasar dan Terapan.Yayasan Obor Jakarta, Indonesia.
Jacob N and Prema P 2006 Influence of mode of fermentation on production of polygalaturonase by a novel strain of streptomyces lydicus. Food Technology and Biotechnology 44: 263-267.
Kalsum U and O Sjofjan 2008 Effects of incubation time of tofu by-product and tapioca waste mixture fermented by Neurospora sitophila on nutrient contents. National Seminar on Technology of Animal Science and Veterinary, Indonesia.
Khanongnuch C, Sa-nguansok C and S Lumyong 2006 Nutritive quality of mannanase-treated copra meal in broiler diets and effectiveness on some fecal bacteria. International Journal of Poultry Science 5:1087-1091.
Kubicek C P 1992 The cellulose proteins of Trichoderma Reesei: Structure, multiplicity, mode of action and regulation of formation. Advances in Biochemical Engineering Biotechnology 45: 1-27.
Mandels M 1970 Cellulases. In: Annual Report on Fermentation Processes. (Ed, G T Tsao). Academic Press, New York.
McCleary B V 1988 Synthesis of betaD-mannopyranosides for the assay of beta N-mannosidase and exo beta D-mannanase. Methods in Enzymology 160: 515-518.
Minitab 2003 Minitab user's guide. Data analysis and quality tools. Release 14 for windows. Minitab Inc, Pennsylvania, USA.
Omojasola P F, O P Jilani and S A Ibiyemi 2008 Cellulase production by some Fungi Cultured on pineapple waste. Nature and Science 6: 64-75.
Saha B C 2003 Hemicellulose bioconversion. Journal of Industrial Microbiology and Biotechnology 30: 279-291.
Steel R G D and J H Torrie 1991 Principle and Procedure Statistics 2nd Edition. McGraw-Hill Book Co, Inc, Singapore.
Sundu B, Kumar A and Dingle J 2005 Growth pattern of broilers fed a physically or enzymatically treated copra meal diet. Australian Poultry Science Symposium 17: 291-294.
Sundu B, Kumar A and Dingle J 2004 The effects of levels of copra meal and enzymes on bird performance. Australian Poultry Science Symposium 16:52-54.
Sundu B, Kumar A and Dingle J 2009 Feeding value of copra meal for broilers. World’s Poultry Science Journal 65:481-489.
Received 7 September 2013; Accepted 10 March 2014; Published 5 April 2014