Effects of fungal treatment on the in vitro degradation of cassava

M Ramin*, H Yaakub, A R Alimon and Z A Jelan

Department of Animal Science, Faculty of Agriculture, Institute of Tropical Agriculture, Universiti Putra Malasyia,
43400 UPM Serdang, Selangor, Malaysia
* Current address: Department of Agricultural Research for Northern Sweden, Swedish University of Agricultural Sciences,
SE-901 83 Umeå, Sweden
hali@agri.upm.edu.my

Abstract

The objective of this experiment was to increase the nutrient value of cassava by the solid state fermentation technique (SSF).  Peeled cassava root was fermented for 10 days under solid state fermentation culture with two different fungi (Aspergillus niger and Rhizopus oryzae).  A follow-up in-vitro gas production experiment was conducted in order to determine the kinetics of fermentation (degradation rate) using cassava from the two SSF. .

There was an increase  in crude protein concentration due to treatment of cassava with A. niger but not with R. oryzae compared to the unfermented treatment (fresh cassava). Acid detergent fibre (ADF) and neutral detergent fibre (NDF) were greater  for both fermented treatments compared to the unfermented treatment. The rate of fermentation, in vitro organic matter digestibility (IVOMD) and metabolizable energy (ME) were greater, and gas production was higher, from the cassava fermented with R. oryzae after 10 days SSF compared to cassava fermented for 10 days with A. niger or the unfermented cassava.  It was concluded that A. niger is an effective fungus to increase the crude protein of cassava.

Keywords: Aspergillus niger, kinetics of fermentation, Rhizopus oryzae,solid state fermentation

Introduction

Cassava is a major root crop in tropical countries. It is currently a worldwide subject to develop its potential as feed for animals especially ruminants. Cassava is very low in protein, however, it contains high amount of starch (Reade and Gregory 1975). Cassava roots contain high levels of energy and have been used as a source of readily fermentable energy in ruminant rations (Khampa et al 2009). Because of the low protein of cassava root, its use in animal feeding requires supplementation of such diets (Iyayi and Losel 2001). Traditionally, foods of animal origin are relied upon to meet the protein needs. However, conventional protein sources from fish meal and soybean are expensive. Protein production from cassava in submerged fermentation by yeasts and filamentous fungi has been demonstrated (Reade and Gregory 1975). Protein enrichment of cassava by using methods which are less expensive is desirable. Solid state fermentation (SSF) and enzyme treatments are potentially beneficial. A number of studies have shown that using enzyme producing microbes in SSF increases the protein value and the bioavailability of nutrients (Fernandez et al 1989). Fungal fermentation has been recognized to be an inexpensive method for increasing the protein level under solid state conditions. Because in vivo experiments might not be the most suitable for evaluation of the feed treatment benefits (Gooselink et al 2004), an in vitro gas production technique was utilized in the present experiment. Such technique has the ability to characterize feeds not only by the quantity of digestible carbohydrates they provide, but also by the rate at which the carbohydrates are fermented (Nagadi et al 2000).

Consequently, it was the objective of the present experiment to identify a fungus with the potential to improve the degradability and protein value of cassava roots.

Materials and methods

Solid state fermentation

Two species of fungi (A. niger and R. oryzae) were obtained from the University Department of Plant Protection, sub cultured on potato dextrose agar and kept refrigerated at 4°C. Fresh cassava was obtained form the market, peeled and ground by using a sieve and oven dried at 60ºC. Dry cassava (50 g) was placed into each of twenty seven 500 ml Erlenmeyer flasks, followed by 4.5 g of (NH₄)₂SO₄, 1.35 g of urea, 2.5 g of KH₂PO₄ and 25 ml of distilled water. The flasks were covered with cotton plugs and sterilized at 120°C for 20 min. They were then divided into three groups of nine flasks each and each group was assigned one of three treatments: CN:  Cassava without fungus (control), AN: Cassava + Aspergillus niger; and RO: Cassava + Rhizopus oryzae (RO). Approximately 1 cm² of corresponding fungus was inoculated into each of nine flasks for each treatment, to prepare fresh SSF cultures. Three cultures (replicates) in each treatment were fermented for 7 and 10 days at 35ºC. After completion of fermentation, the content of each flask was removed and oven dried at 60ºC for 48 h. The contents were then ground using a blender, homogenized, and stored at 4ºC for further analysis.  The crude protein (CP) concentration in the contents was determined as Kjeldahl N × 6.25. The acid detergent fiber (ADF) and neutral detergent fiber (NDF) were analyzed as described by Van Soest et al (1991) without the addition of amylase and sodium sulfate. Hemicellulose was calculated as NDF-ADF.

In vitro gas production

Fresh cassava (control) and cassava after 10 days fermentation with A. niger and R. oryzae from the first experiment were used as substrates. The 10 day fermentation with A. niger and R. oryzae were selected because on completion they produced higher CP compared to the other fermentations. The cumulative gas production in the current in vitro gas production experiment was measured according to the method of Menke and Steingass (1988). Rumen contents were collected in pre-warmed flasks, 2 h before morning feeding from three fistulated goats fed a normal diet (60% hay and 40% concentrate). Rumen fluid was obtained by squeezing rumen contents through four layers of cheesecloth. The strained rumen fluid was mixed (1:2 v/v) with anaerobic medium (buffer) to form a suspension as described by Menke and Steingass (1988). The suspension was then kept warm with continuous stirring at 39°C for approximately 20 min under continuous bubbling of CO₂. Approximately 200 mg of each sample was weighed into a 100 ml syringe (Fortuna, Hiberle Labortechnik, Germany) and 30 ml of the suspension was added to each of the syringes. The syringes were incubated for 48 h at 39°C. The cumulative gas production was recorded in each syringe at 2, 4, 8, 12, 24, 32 and 48h of incubation.

Metabolizable energy and organic matter digestibility

Based on equations described by Menke and Steingass (1988), the  ME (MJ/kg DM) and IVOMD (%) were calculated.  The crude protein values after ten days fermentation were used to calculate ME and IVOMD.

ME (MJ/KG DM) =1.56+0.139*GP24 (ml gas/200 mg DM in 24 h) +0.0074*Crude Protein (g/kg DM) +0.0178*Crude Fat (g/kg DM)

IVOMD (%): 14.88+0.8893*GP (ml Gas/200 mg DM) + 0.0448*Crude Protein (g/kg DM) + 0.0651*Crude Ash (g/kg DM)

Kinetics of gas production

The rate of gas production was calculated by using the NEWAY program  as described by Martínez et al (2005) with the model

Y=a+b (1-exp^-ct)

Where Y is gas volume at time t,

a+b is potential gas production (ml/g DM) and

c (h^-1) is the rate at which gas is produced (Kiran and Krishnamoorthy 2007).

Statistical analysis

A 3×3 factorial design was performed and analyzed by the general linear model (GLM) procedure, using a statistical package of SAS version 9.2  (SAS Institute 2008). The model used was:

Y_ijr=µ+t_i+d_j+ (td)_ij+e_ijr

Where Y_ijr is the dependent variable, µ is the overall mean, t_i is the effect of treatment, d_j is the effect of day, (td) _ij is the interaction of day and treatment and e_ijr is the residual error.

The means were compared by Duncan multiple range test and considered significant at P < 0.05.

Results

Solid state fermentation

The chemical composition of the fresh cassava is given in Table 1. The effect of SSF on chemical composition of cassava is given in Table 2. The mean values of CP after 4, 7 and 10 days of fermentation were higher for the treatment fermented with A. niger . Interaction was significant for all parameters indicating the effect of time on fermentation (Table 2).

Time of fermentation (4, 7 or 10 days) had no effect on the concentrations of CP, NDF, ADF and hemicellulose in cassava of the control (CN) treatment (Figure 1). The CP concentration in the treatment with A. niger was almost double that of the control. The Rhizopus oryzae (RO) treatment did not increase CP concentration.

Figure 1. Effects of fungal cultivation (Aspergillus niger or Rhizopus oryzae)  under solid state condition on the concentration of crude protein (A), neutral detergent fibre (B), acid detergent fibre (C), hemicellulose (D) and ash (E) in cassava at 4, 7 and 10 days of fermentation. C: Control, AN: Aspergillus niger; RO: Rhizopus oryzae a, b, c, d, and e represent the significant differences (P < 0.05)

Changes with fermentation time in concentrations of cell wall constutuents tended to reflect the decreasing concentration of the soluble components that were used as energy by the fungi but this effect was not consistent for the RN treatment (Figure 1).

In-vitro gas production

The rate and extent of gas production from the control (CN) and RO treatments were similar and much higher than from the AN treatment (Table 3 and Figure 2). Presumably this reflected that on the AN treatment the substrate was being partially converted to protein while on the control and RO treatments the end product was mainly gas. .

Discussion

The increase in CP concentration in the cassava fermented with A.niger is similar to the findings of Iyayi and Losel (2001) and Aderimi and Nworgu (2007). However, both these groups of researchers reported increases in NDF and ADF as well as in CP. Our results of an increase in CP but a decrease in cell wall constituents appear to be more logical as increases in NDF and ADF fractions would occur as the soluble fractions would be fermented  preferentially.

Conclusions

• Solid state fermentation of cassava root with A. niger was effective in increasing the crude protein content but this was at the expense of a decrease in the energy value.

  
  

• Fermentation with R. oryzae did not appear to affect the nutritive value of the cassava root as neither the crude protein content nor the degree of gas production were changed as compared with untreated cassava root.

Acknowledgments

The authors wish to thank Mr. Saparin and Mr. Zakaria for their technical assistance and the Academic Science Grant Scheme for financial support.

References

Aderemi F A and Nworgu F C 2007 Nutritional Status of Cassava Peels and Root Sieviate Biodegraded With Aspergillus niger. American-Eurasian Journal of Agricultural and Environmental Science 2 (3): 308-311.

Fernandez M J, Roche E, Pou J, Garrrido F and Garrido D 1989 Enzyme production by solid-state cultures of aerobic fungi on lignocellulose substrates. In: Coughlan M P (Ed) Enzyme Systems for Lignocellulose. Elsevier Applied Science Publishers. London pp 177-191.

Gosselink J M J, Dulphy J P, Poncet C, Jailler M, Tamminga S and Cone J W 2004 Prediction of forage digestibility in ruminants using in situ and in vitro techniques. Animal Feed Science and Technology 115: 227-246.

Iyayi E A and Losel D M 2001 Protein enrichment of cassava by-products through solid state fermentation by fungi. Journal of Food Technology in Africa 6 (4): 116-118.

Khampa S, Chaowarat P, Singhalert R and Wanapat M 2009 Supplementation of yeast fermented cassava chip as a replacement concentrate on rumen fermentation efficiency and digestibility on nutrients in cattle. Asian Journal of Animal Science 3 (1): 18-24.

Kiran D and Krishnamoorthy U 2007 Rumen fermentation and microbial biomass synthesis indices of tropical feedstuffs determined by the in vitro gas production technique. Animal Feed Science and Technology 134: 170-179.

Menke K H and Steingass H 1988 Estimation of the energetic feed value obtained from chemical analysis and in vitro gas production using rumen fluid. Journal of Animal Research and Development: 28: 7-55.

Nagadi S Herrero M and Jessop N S 2000 The influence of diet of the donor animal on the initial bacterial concentration of ruminal fluid and in vitro gas production degradability parameters. Animal Feed Science and Technology 87: 231-239.

Reade A E and Gregory K F 1975 High-Temperature Production of Protein- Enriched Feed from Cassava by Fungi. Applied Microbiology 30 (6): 897-904.

Martínez S, Pedraza R M, González M L, González C E and Guevara G 2005 Influence of the donor animal on the in vitro gas production with the use of voided bovine faeces. Livestock Research for Rural Development. Volume 17, Article #129. Retrieved October 5, 2010, from http://www.lrrd.org/lrrd17/11/mart17129.htm

SAS institute 2008 SAS User’s guide Release 9 2. SAS Institute Inc Carry NC USA.

Van Soest P J, Robertson J B and Lewis B A 1991 Methods for dietary fibre, neutral detergent fiber and non-starch polysaccharides in relation to animal nutrition. Journal of Dairy Science 74: 3583-3597. http://download.journals.elsevierhealth.com/pdfs/journals/0022-0302/PIIS0022030291785512.pdf

Received 20 March 2011; Accepted 23 May 2011; Published 1 July 2011

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