The significant increase in demand for livestock products in recent years in developing countries has required an increase in animal feed supply. In this context the role of cassava as a cheap carbohydrate source capable of supplying adequate calories to livestock is very significant. However, due to its low protein, vitamin and mineral content and lack of the sulphur containing amino acids such as methionine, it is often considered as inferior to maize or wheat. The crude protein content of whole cassava roots is around 3.5% dry weight and 40–60% of the total nitrogen is non-protein nitrogen. Careful formulation of the cassava diet is important to nutritionally balance the feed. Fermentation has been identified as one of the less expensive means of increasing the protein quality of cassava.
The use of microorganisms to convert carbohydrates, lignocelluloses and other industrial wastes into foodstuffs rich in protein is possible due to the following characteristics of microorganisms.
Microorganisms have a very fast growth rate.
They can be easily modified genetically for growth on a particular substrate under particular cultural conditions.
Their protein content is quite high varying from 35 to 60%.
They can be grown in slurry or on solids.
Their nutritional values are as good as other conventional foods rich in protein.
NEED FOR PROTEIN ENRICHMENT OF CASSAVA USING MICROBIAL TECHNIQUES
The economic feasibility of using cassava based rations for animals depends mainly on the price of cassava in relation to alternate energy sources and the price of the supplementary protein sources to be added to balance the protein requirements of animals to be fed. Because of the very low protein content of the cassava tubers, any substitution of cassava for cereals in compounded feeds necessitates the inclusion of a considerable amount of supplementary protein. Experimental studies conducted by Gomez et al. (1976) showed that a swine feeding programme based on cassava meal required approximately 60 to 65% more protein supplement than a similar feeding programme using maize as an energy sources. Therefore, in developing countries the potential for cassava use as animal feed depends mainly on the availability of cheap protein sources. An alternate approach is to enrich cassava flour with microbial protein. The microbial enrichment process is relatively cheap and the enriched product can increase the potential of cassava as a feed.
MICROBIAL TECHNIQUES FOR PROTEIN ENRICHMENT OF CASSAVA Following the successful experiments of Brook et al.(1969), Stanton and Wallbridge (1969), and Gray and Abou-El-Seoud (1966), attempts hav been made in many laboratories to develop fermentation techniques to produce microbial proteins using either cassava flour/cassava wastes or enriching cassava flour/cassava wastes.
Submerged fermentation of cassava
In the submerged fermentation system water is always in a free state and carbon, nitrogen, phosphorous and other nutrients are in a suspended or dissolved state. Simple aseptic inoculation of microorganisms under such conditions might convert some of the non-protein nitrogen into protein, but submerged fermentation is only economically worthwhile when done on an industrial scale, using processes that require a strict control of fermentation and which take place in a sterile environment.
Gray and Abou-El-Seoud (1966) studied the protein production efficiency of several filamentous fungi by growing them on ground cassava roots supplemented with ammonium chloride and corn steep liquor. They found that Cladosporium eladosporoides gave good mycelial yield and produced products containing 13–24% crude protein. strasser et al. (1970) described a process in which the yeast Candida utilis was used to produce a product containing 35% crude protein on a dry weight basis. However, it is important to note that: 1) prior to fermentation with yeasts, enzymatic or acid treatment of starch is necessary; 2) the entire fermentation has to be carried out under aseptic conditions; and 3) since the yeast takes time to settle or remains in a suspended form in the medium recovery of cell mass from such a fermentation system can be tedious. In order to avoid this, centrifugation or ultrafiltration can be used to achieve separation.
Extensive studies on the use of cassava based submerged fermentation systems have been carried out at the University of Guelph, Ontario, Canada. These reported the advantages of using thermotolerant filamentous fungi for the production of protein rich animal feeds from cassava (Reade and Gregory, 1975; Gregory, 1977 and Gregory et al., 1977). Screening of suitable organisms was carried out using a low pH (3.5) and a temperature in excess of 45°C, since only a few thermotolerant fungi will grow under these conditions and contaminating bacteria, fungi and other organisms could be eliminated.
After rat feeding experiments to test pathogenicity, three cultures which gave protein efficiency ratios of 2.3 or more were selected. The fermentation conditions for protein production from cassava mash by Aspergillus fumigatus are given in Table 1. However, due to safety considerations, this culture was not recommended for practical application.
The use of Cephalosporium eichhorniae 512 (ATCC38255) which is capable of protein production from cassava carbohydrate for use as animal feed was also studied (Mikami et al.,1982). This strain is a true thermophile showing maximum growth at 45 to 47°C and achieving maximum protein yield at 45°C and no growth at 25°C. It has an optimum pH of about 3.8 and is an obligate acidophil, being unable to sustain growth at pH 6.0 in a liquid medium and above pH 7.0 on solid media. The optimum growth conditions for this fungus, (pH 3.8 and temperature 45°C) were strongly inhibitive to the potential contaminants. This fungus rapidly hydrolyzed cassava starch and did not utilize sucrose, but around 16% of the sucrose components of cassava were chemically hydrolyzed during the process. Fungal growth with cassava meal (50 g/1) was complete in around 20 h, yielding around 22.5 g/1 (dry biomass) containing 41% crude protein (48 to 50% crude protein in the mycelium) and 31% true protein (7.0 g/1).
|CONDITION||REASON FOR SELECTION|
|Carbohydrate concentration = 4% (approx. 15% fresh cassava)||Fermentation is completed in 20 h from a 6.7% inoculum, permitting a daily production schedule. Higher concentrations take longer, lower concentrations give lower yields.|
|Mash heated to 70°C for 10 min, immediately after grinding, in one- half final volume||Gelatinize starch, permitting complete utiliz- ation, prevents development of antifungal activity, provides desired starting tempera- ture after dilution to final volume|
|Nitrogen source = Urea (1.72 g/1)||No automatic pH control required; whereas, (NH4)2SO4 results in excess acidity|
|Mineral supplement = KH2PO4(0.25 g/1)||Assures sufficient phosphorous even with cassava roots which are low in P. All other mineral requirements except “S” are supplied by cassava roots.|
|Initial pH adjustment with sulphuric acid||Supplies sulfur requirement as well as acidity|
|pH 3.5||Optimum for protein production|
|Temperature = 45°C||Inhibits bacterial and yeast growth, thus permitting use of nonaseptic conditions (although optimum temperature for the fungus is 37–40°C)|
|Vigorous agitation and aeration during growth||Provides rapid oxygen transfer to growing cells|
Muindi and Hanssen (1981a,b) described a fermentation procedure to increase the protein content of cassava root meal (CRM) with Trichoderma harzianum. The organism was grown in a 4% CRM medium containing inorganic nitrogen. The growth medium had the following composition (g litre -1) CRM - 40.0, NH4NO3 - 1.00, KH2PO4 - 1.50, MgSO4.7H2O - 0.25, CaCl2 - 0.01, MnSO4.4H2O - 0.001,ZnSo4 - 0.001, CuSO45H2O - 0.0001. The fungal biomass with the remaining CRM was collected by filtration at the end of fermentation. Satisfactory results were obtained using a temperature of 23°C, a pH of 4.0 - 4.2 and a fermentation time of 60 h. The estimated efficiency of conversion of the CRM into CRM/biomass was shown to be 30%.
The submerged cultivation of T. harzianum resulted in a substantial improvement in the protein content and quality of CRM. However, the metabolizable energy content of enriched cassava root meal was found to be about 9.1 MJ kg-1 dry matter (DM), which was significantly lower (P<0.05) than that of non-enriched CRM (12.2 MJ Kg-1 DM). The CP content of the enriched CRM product used was 37.6% on dry matter basis, compared with the 2.4% CP of the untreated CRM. The nonprotein nitrogen content accounted for about 30% of the total CP value. The mean apparent digestibility coefficient of the total CP was about 66% whereas that of amino acid was about 81%. Data from this study indicate that fungal enriched CRM could be used in chicken diets.
Protein enrichment of cassava by solid state fermentation
Generally solid-state fermentation is characterized by water addition limited to the saturation of the solid without allowing the separation of the liquid from the solid phase. Besides the reduction in the fermenter volume the advantages include: simplicity, ease of adaptation to rural conditions, elimination of foaming and a reduced cost of a final product which contains up to 20% protein (Brook et al., 1969).
Several organisms and fermentation methods have been tried to increase the protein content of cassava and cassava wastes using solid state fermentation. Some of the techniques developed are reported here.
The effect of substituting corn with fermented cassava in broiler rations was investigated by Varghese et al. (1976). They first evaluated the role of natural nitrogenous supplements like chicken manure, pineapple bran, groundnut, etc., in enhancing the fermentation of cassava. Direct fermentation of cassava, alone, with Aspergillus, Neurospora and Rhizopus elevated the protein values by 3% using nitrogenous supplements, e.g., 25% pineapple bran increased the protein from 4 to 5%, whilst a mixture of 12.5% pineapple bran and 12.5% chicken manure increased the protein to 7%. Soyabean and groundnut were found to be even better additives to facilitate protein enrichment.
The possibility of using fungal strains isolated from Mexican, African and Oriental traditional foods to upgrade the protein content of cassava by solid state fermentation was investigated by Raimbault et al. (1985). The protein content of cooked enriched cassava varied from 10.9 to 16.5 percent and the residual sugar content from 28.2 to 45.2 percent. In this process milled dry cassava roots were supplemented with a mineral salt solution of high ionic strength and fermented with Sporotrichum pulverulentum in solid state culture. The fungus produced 30.4 g high quality protein/100 g dry cassava in 48 h at 45°C in an aerated bench scale tray fermenter.
An artisanal static process for protein enrichment of cassava by solidstate fermentation tested in pilot units in Burundi (Central Africa) produced enriched cassava containing 10.7% of dry matter protein compared with 1% before fermentation (Daubresse et al., 1987). In this process cassava chips which had been processed into pellets of 2–4 mm diameter, were moistened (40% water content) and steamed. After cooling to 40°C, the cassava was mixed with a nutrient solution containing the inoculum (Rhizopus oryzae strain MUCL 28627) and 3.4 g urea, 1.5 g KH2PO4, 0.8 g MgSO4. 7H2O and 22.7 g citric acid per 100 g dry matter. During the fermentation, the cassava, which had a moisture content of around 60% and a pH of 3.5, was spread in a thin layer (2–3 cm thick) on perforated trays and placed in an aerated humidified enclosure for 65 h incubation. The production of protein enriched cassava was 3.26 kg dry matter/m2 per tray.
Several levels of urea were used in experiments relating to this fermentation. The study showed that after the 65 h fermentation, there was little loss of nitrogen when the 2.2 and 4.5 g doses of urea were mixed with each 100 g of cassava, though acidity increased to pH 5 during fermentation. Using 6.8 g urea, a rapid increase in pH to 5.5 was observed accompanied by a loss of nitrogen. When the dose of urea was doubled from 2.2 to 4.5 g/100 g cassava the protein content of the final product increased by 29% from 8.81 to 11.37 of total real nitrogenous dry matter. However, the non—protein fraction of the fermentate was higher (38.41%) with the incorportion of 4.5 g urea than 2.2 g (24.25%). Tripling the urea dose gave poor results.
Balagopalan and Padmaja (1988) developed a solid state fermentation process for the protein enrichment of cassava flour and cassava starch factory wastes using the fungus Trichoderma pseudokoningii Rifai. The various treatments studied and results obtained are given in Table 2. The results showed that using this process it was possible to convert the substrate, using the minimum of nutrients [0.15% (NH4)2SO4] to a protein enriched animal feed. The highest increase in protein content was observed, i.e., 14.32 g/100 g dry matter (DM) from an initial 1.28 g/100 g dry matter, where cassava flour was the sole ingredient. Overall, although protein production took place using both substrates, it was greatest where cassava flour was present and reduced as the level of cassava starch factory wastes increased. Increased amylolytic activity and the subsequent reduction in starch content during the solid state fermentation also indicated the ability of the organism to carry out bioconversion of starch to protein.
|Cassava mixture flour/wastes||Fermentation period (days)|
Source: Balagopalan and Padmaja, 1988
The laboratory technology was evaluated with regard to its potential use in the large scale production of SCP enriched poultry feed at the Central Tuber Crops Research Institute, Trivandrum. In this study a mixture of cassava flour and starch factory wastes were prepared in the ratio 50:50. The moisture content was adjusted to 15 percent and 0.2 percent urea was included. The mixture was then boiled using steam for 60 min. and then allowed to cool to ambient temperature. A one week old inoculum of Trichoderma pseudokoningii, prepared using a cassava flour-waste mixture, in the ratio of one Kg of inoculum to 10 kg substrate, was thoroughly mixed with the substrate. The inoculated substrate was then spread over a cemente floor at a thickness of 2–3". The substrate was frequently turned to release heat generated during the fermentation. Since moisture was lost from the substrate, water was sprinkled ocasionally over the fermentate. The solid state fermentation, which was developed to suit village conditions, was terminated at the end of six days incubation and the protein content estimated.
Contamination only occurred in cases where excess moisture and high temperatures develop. In order to obtain continuity in the production of the enriched feed, paralles batches were maintained. The enriched feed was then dried and stored in gunny bags for further feeding trials with poultry. The results obtained are discussed later in the section on animal experiments.
PROTEINS FROM WASTES
A study was undertaken by Manilal et al. (1987) to utilize starch factory wastes by means of a solid state fermentation process. The waste consisted of a concentrated, dried primary effluent collected from a starch factory. Experiments were carried out on samples with and without nutrient enrichment. In both cases the moisture content was adjusted to 60%. Spores of a one week old culture of Aspergillus niger were used in the studies and the samples were incubated at 30°C for 120 h.
Initial biomass protein in the material was 1.60% (w/w). A stepped increase in the protein content was observed during the first three days, so that the material contained 7.0% protein by the third day. During the next two days of incubation the protein content only increased by and additional 0.7%. In the case of non-enriched samples the initial protein content was 1.0% which increased to a maximum level of 3.7% on the third day of incubation. No further increase in the protein content occurred during the fourth and fifth day of incubation.
Feeding experiments carried out with pigs and sheep fed microbial protein enriched cassava root meal showed promising results (Paraksa and Saeow, 1987 and Adeyanju, 1979).
Padmaja and Balagopalan (1990) studied Trichoderma pseudokoningii enriched cassava waste: flour mix (50:50) as an energy source in broiler rations, using three levels of feed inclusion viz. 50, 55 and 60 percent. The calculated metabolizable energy values for the various test diets ranged from 2360 to 2450 Kcal/Kg and the crude protein (g/Kg) ranged from 200 to 233.
Growth performance and percentage carcass yields showed that use of up to 60% of the enriched material did not adversely affect the birds performance. A further evaluation trial was conducted using 60% enriched feed in a starter rations and 65% in finisher rations. The performance of the birds was compared with those fed on a non-enriched mix (50:50). The performance of birds fed the non-enriched ration was similar to those fed with SCP feed. However, the cumulative feed intake was less for the test birds which led to a reduction in overall feed use. This study clearly shows the potential that exists for commercial broiler farming to switch over to a cassava waste based feed, from the conventional feeds (Table 3).
|Treatment/Body Wt. (g)||Age in weeks (Starter Stage)||Finisher stage|
|T1 (50% SCP)||233||420||894||1118||1420||1720||1850|
|T2 (55% SCP)||254||430||895||1120||1380||1690||1990|
|T3 (60% SCP)||248||415||912||1163||1395||1705||1915|
In view of anticipated world protein shortages, microorganisms offer a variety of possibilities for increased protein production. However, microbial proteins have a high nucleic acid content, though from the studies presented here it would suggest that their use in animal feeds will not cause problems. Single cell proteins by virtue of their rich amino acids composition, high vitamin B and digestible nutrient content can be used to totally or partially replace conventional vegetable and animal proteinaceous feedstuffs. Screening of safe microorganisms for high protein production and the use of asporogenous mutants is a positive development in the utilization of cassava for SCP production. The application of biotechnological techniques to produce better strains of microorganisms is also suggested as a future approach.
In the application of in situ utilization of cassava as an animal feed, the already developed cassava fermentation techniques offer the advantage of being simple and low cost and can be transferred to rural areas through trained extension services. In particular the solid state fermentation technology seems to be the most appropriate in rural areas since it requires little environmental control and equipment.
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