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Department of Animal Sciences, University Pertanian Malaysia, Serdang, Selangor, Malaysia
The dwindling food and feed reserves in the world have increased interest in the exploitation of carbohydrate residues that at present largely go to waste and are a pollution hazard. Within the past decade fresh impetus has been given to the serious study of these carbohydrate residues as substrates for the production of protein enriched foods or feeds through microbial fermentation. Part of this impetus has stemmed from wider recognition of malnutrition in the developing countries and efforts to combat it. At the same time, with the ever-increasing seriousness of the waste problems from the processing of food and natural carbohydrate sources, the production of microbial protein from these wastes and by-products could be a profitable way of overcoming this difficulty.
Carbohydrate residues are available in large quantities in many parts of South-East Asia. Some of these residues have been used as substrates to grow micro-organisms, and their nutritive value has been documented (1).
In Malaysia, as in many of her neighbouring countries, there are increasing needs for protein sources. Protein consumption has been reported to be about 45 g/day/person and to consist of not more than 17 g of animal protein. Efforts have been made to increase animal protein sources, such as meat from poultry and beef. However, with the high cost of imported concentrated feeds, especially of protein, meat production will eventually become economically unattractive.
Realizing these facts, considerable research has been conducted in Malaysia within the past ten years - and is currently being intensified - to maximize the use of various agro-industrial wastes, including those of carbohydrate residues, for useful animal feed and thus, indirectly, for food.
This paper presents the broad outline of the current work done in Malaysia on the use of carbohydrate residues, and the advances made toward the realization of finished products for large-scale application.
The major agro-based industries producing residues or wastes are rubber and oil palm, and, to a lesser extent, coconut, pineapple, sugar cane, cassava, and sago factories. Some of these wastes contain a high proportion of carbohydrates, especially those from cassava, pineapple, sago, and sugar cane.
The oil palm industry is one of the major revenue-earning industries in Malaysia in addition to rubber and cocoa. Palm oil production has increased rapidly so that its foreign exchange earning was nearly as high as that of rubber in 1978 compared to its second place behind rubber before that year. Today, oil palm cultivation covers about 650,000 ha, producing about 2 million tons of palm oil annually. The waste from palm oil processing, generally known as palm oil mill effluent or palm oil sludge, is obtained from the sterilization and clarification processes. Assuming an extraction rate of 20 per cent for palm oil, about 107 tons per year of fresh fruit bunches are processed, resulting in an estimated 6.8 million tons of palm oil sludge discharged each year.
The sludge contains about 10 per cent crude protein, 12 per cent crude fat, 12 per cent crude fibre, and 54 per cent soluble carbohydrates on a dry matter basis, making it a useful substrate for the production of single-cell protein for feeding farm animals. Because of its high biological oxygen demand (BOD), the disposal of this effluent into the streams causes ecological and pollution hazards. Several attempts have been made to utilize these effluents, among which are: (a) direct use of the sludge as land fertilizer, and (b) utilization of the effluent as animal feed. The methods attempted for the conversion of sludge into animal feeds include moisture reduction through dehydration, filtration, centrifugal solids recovery, biodegradation, and the use of effluent as substrate for the production of single-cell protein and cultivation of Chlorella algae.
The filtration process for palm oil sludge involves the separation of the solids from the sludge using muslin-coverd filters or a metal-mesh screen.
The centrifugal solids recovery method entails three phases: concentration by centrifugal means, fermentation, and extraction. The centrifugal sediment is subjected to anaerobic fermentation to improve the protein content of the solids. In the extraction phase, the raw, concentrated, or fermented slurries are mixed with locally available agricultural by-products before complete drying. The details of this procedure and subsequent feeding trials in animals have been published (2-7).
Biodegradation of palm oil sludge using selected micro-organisms on continuous fermentation has been shown to improve the quality of the effluent and at the same time to reduce its BOD load. This process was jointly initiated by Dunlop Estates Malaysia, Ltd., and Dunlop Bioprocesses, Ltd., England, to produce a feed designated "Prolima." The solid fraction, separated by filtration, contains 35 per cent protein. Preliminary findings from nutritional and safety evaluation in rats and poultry showed that the product has a biological value of approximately 80 per cent of that in soybean meal and does not contain carcinogenic substances or toxins.
Sludge in its raw form is an emulsion from which solids and oil wastewater are obtained The residual wastewater has recently been shown to be suitable as a substrate for algae (Chlorella sp.) production, thereby reducing the BOD (8). Algae are known to be valuable products for humans and animals.
The effective use of palm oil sludge as animal feed will be of economic significance, especially in view of the rising cost of imported feedstuffs, and at the same time will reduce pollution.
Rubber (Hevea brasiliensis) is one of the major plantation crops as a source of income and employment in Malaysia, representing about 20 per cent of foreign exchange revenue. The plantation covers about 1.7 million ha of land, producing about 1.5 million tons of rubber annually. On the basis of an averagesized rubber factory producing about 20 tons of rubber and 410,000 litres of effluent per day, there are approximately 80 million litres of effluent discharged from factories daily into nearby streams and rivers, mostly without any treatment. The effluent consists mainly of process water and a small amount of uncoagulated latex and latex serum, The latex serum contains large amounts of carbohydrates (sugars), proteins, fats, and inorganic and organic salts.
Reports have indicated that this effluent waste serum provides a good growth medium for a variety of micro-organisms, such as Candida utilis and Saccharomyces cerevisiae, giving a yield of about 10 to 15 g/litre waste serum (9,10). Harvested C. utilis has been claimed to contain 50 per cent crude protein on a dry matter basis. Other findings have also shown that green algae, particularly Chlorella sp., could grow well in waste serum, growth being facilitated by prior anaerobic treatment of the waste (11-13).
Fresh-water fish such as Tilapia sp., Ophiocephalus sp., and Tricopodus sp. were found to grow well in the stabilization treatment pond containing rubber effluent so that algal protein and fish protein can be obtained simultaneously (14). Effluent has also been used as a liquid fertilizer to provide N, P. and Mg for grasses (giant, rapier, star, Brachiaria mutica, Axonopus compressus) and significantly increased the yield and protein content of the grasses (15-17).
It is evident from these results that rubber effluent has the potential to be used as a source of nutrients for culture media for micro-organisms or as fertilizer in which the end-products can be used either as proteinaceous foods or animal feeds.
Coconut ranks as the third largest plantation crop in terms of area after rubber and palm oil, covering about 283,000 ha of land and producing about 90,000 tons of coconut oil. The copra industry produces a large quantity of coconut water, at the rate of 136 litres per 1,000 nuts; the volume of coconut water produced annually is about 11 million litres. Coconut water contains about 2 per cent (W/V) carbohydrates (mainly sugars), and other substances capable of inducing rapid growth of beneficial micro-organisms (18).
Jayatissa and co-workers (19) have shown that inoculating coconut water with a local strain of Saccharomyces cerevisiae resulted in the production of coconut toddy (potable spirit). It has also been used as a growth medium for large-scale production of acid-producing bacteria (20). A combination of coconut water and sugar cane juice has been shown to produce vinegar using S. cerevisiae (for alcoholization) and Acetobacter rancens (for acidification) (18). Attempts have been made in Malaysia to investigate the suitability of coconut water as a growth medium for the production of single-cell protein and certain amino acids (H.K. Choke, personal communication, 1979).
There are approximately 17,000 ha of land under pineapple cultivation, producing about 0.25 million tons of fruit. Because of stiff export competition with Hawaii and Taiwan, its production has declined sharply within the last few years.
Of the whole fruit, only about 20 per cent is canned, while the remainder, in the from of peeled skin, core, base, crown, is discharged as waste (21). The liquid portion of the waste contains a high concentration of carbohydrates in the form of sugars that can be concentrated as a syrup for alcohol production. The solid portion can be used for bran or silage for feeding farm animals (22; 23). The juice, which contains about 7 per cent total sugar, has been used as a growth medium for single-cell protein production, yielding up to 15 g/litre of Candida utilis (14), and for the production of vinegar (24).
Sugar cane cultivation in Malaysia is rather new compared to that of oil palm and rubber, covering about 20,000 ha of land with an estimated annual production of about 1 million tons. The plantations are located in Perlis, Perak, and Negeri Sembilan states of Peninsular Malaysia, where two out of three of the original sugar mills are in operation. Major by-products obtained from the sugar manufacturing process are molasses (40,000 tons per year) and bagasse. Molasses, containing about 50 per cent carbohydrates (total sugar), has been used as raw material in many fermentation industries for the production of alcohol, monosodium glutamate, and vinegar as a substrate to facilitate latex coagulation, and as a source of energy for feeding farm animals (7; 25; 26).
The wastes obtained from the sugar mills include condensed water, cane wash-water, flood washings, and boiler blowdown, and these have contributed to the pollution problem at the mills. There have been some suggestions to use oxidation or stabilization ponds and recycle the wastewater to reduce the BOD content of these wastes. The bagasse is used as fuel or as raw material for manuacturing paper and soft boards.
Another promising source of carbohydrate is the sago palm (Metroxylon sagu). This palm tree grows wild in the swampy areas of Malaysia, Indonesia, and New Guinea. Sago palms cover about 14,000 ha of land in Malaysia. Each tree can produce about 300 kg of sago. Sago starch and its by-products are obtained from the trunk (pith) when the tree is about 12 to 15 years old. In areas where the sago palm grows, sago pith is used for human consumption and livestock feeding. The total amount of sago pith used for livestock feed in Indonesia and Malaysia is estimated to be about 15,000 and 25,000 tons per year (7; 27). By-products from sago palm include unprocessed sago pith, sago meal, and sago refuse. Sago is very low in protein but is exceptionally high in carbohydrates. Results have shown that soluble carbohydrates in sago meal ranged from 51 to 92.5 per cent (28; 29). Based on the chemical composition of sago and cassava, sago residues should be as effective as those of cassava as a growth medium for microorganisms.
Studies have shown the technical feasibility and nutritional desirability of converting carbohydrates and their residues into products containing a large amount of protein by means of micro-organisms. The use of carbohydrate substrates such as cassava and sugar cane for microbial protein production can be of significance in many developing countries where there is a surplus of carbohydrates and an inadequate supply of proteins,
Cassava has been the subject of worldwide efforts to develop its potential as a food, a feed, and an industrial commodity (30). The root is very low in protein (less than 3 per cent), but it yields the highest amount of starch per unit of land of any crop known, in that about 90 per cent of its carbohydrate content is fermentable (31). The residues from cassava processing include the solid wastes (pulps and sand-drum waste) and liquid wastes. The liquid wastes are obtained from the wash tank (root washwater) and from the extraction and separators during the starch extraction process (separator wastewater).
Yeast was among the micro-organisms considered as potential protein sources using wastewaters from cassava starch factories. A process for growing Torula yeast (Torula utilis) on separator wastewaters has been developed. Large numbers of yeast cells have been successfully recovered from this yeast propagation, amounting to about 40 per cent of the total solids in the wastewater. This process could contribute to greater use of organic nutrients in waste effluents abundantly available in Malaysia, thus providing an economical means for minimizing their pollution hazards.
Efforts have been made to improve the nutritional value of cassava root as animal feed by various supplementations, e.g., protein and amino acid concentrates, fats, minerals, vitamins, and feed additives. However, the supplementation approach has not been fully satisfactory as this is dictated by economic factors (7). An alternative approach to improve its protein value through microbial fermentation has been investigated because the tradition of fermenting cassava for human consumption e.g., "tapeh" (peuyeum) in Indonesia and "gari" in Africa was the original concept (25; 32). Largescale fermentation of cassava with micro-organisms for protein enrichment or microbial protein production has also been studied (6; 33).
The use of micro-organisms for the production of dietary protein from carbohydrates has been extensively reviewed (34-38). The ability of micro-organisms to grow on cassava in submerged fermentation has been studied, and some potentially useful yeasts and fungi identified (32; 39-43). Reade and Gregory (44) described a process for producing microbial protein from cassava using thermotolerant, filamentous fungi (Aspergillus fumigates, Sporotrichum thermophile, Paecilomyces sp.) by fermentation at a low pH. The biomass showed a high protein value (36.9 per cent), but had a low level of sulphur-containing amino acids (33). Rats fed these fungi (dried biomass) had a low weight gain, reduced protein efficiency ratios (PER), and net protein ratio values (NPR) (45). Methionine supplementation at 0.6 per cent was found to improve these values. In their subsequent trial, Khor and co-workers (46) found that rats fed a thermo-tolerant fungus (A. fumigatus) - grown on a cassava carbohydrate and salts medium had heavier kidneys than those on the conventional diet. A slight increase in blood urea nitrogen was observed at the 40 per cent level of feeding. Determination of the nutritive value of the biomass produced from the pilot plant is currently under way (Gomez, personal communication, 1979).
Stanton and Wallbridge (1) reported a different approach in cassava fermentation in that moist-solid fermentation was employed by incorporating fungal protein into an extruded paste made from cassava flour. The resulting product showed a marked increase in protein content over that of the original cassava substrate. It was claimed that the strain of Rhizopus or other moulds selected could be considered dominant, thus making careful and expensive sterilization unnecessary. Based on this process, a cassava microbial enrichment project was conducted to develop a technique that could be adapted for operation on a village or larger industrial scale for the production of enriched cassava as animal feed.
Micro-organisms capable of using cassava as a substrate for growth and synthesis were isolated from cassava tubers, chips, and residues at cassava-processing centres in Malaysia. These organisms were screened for their relative efficiency in protein enrichment of cassava, based on the level of protein nitrogen and non-protein nitrogen before and after the fermentation. Among the strains of fungi investigated, Rhizopus, Aspergillus, and Neurospora were found capable of increasing the protein of cassava. It was, however, recognized that, while cassava could be enriched by moist-solid fermentation, the protein levels attained were inadequate for these materials to serve as substitutes for cereal grains.
Further isolations were therefore undertaken, and, in addition, the role of nitrogenous supplements such as poultry excrete, pineapple waste, groundnut, soybean, and urea was assessed. Poultry excrete were added to supply nitrogen in the form of uric acid for conversion into microbial protein as well as the necessary nutrients for fungal growth. The addition of small quantities of soybean and groundnut meals was found essential to stimulate microbial activity during fermentation and thereby increase the rate of protein conversion.
Optimum conditions for the growth of micro-organisms were at a temperature of 28°C ± 2°C and a relative humidity of 80 per cent. On the laboratory scale, optimal fermentation was found when the cassava was spread out in thin layers about 6 cm thick. The final mixture contained about 50 per cent moisture following autoclaving or steaming, and the substrate was then subjected to fermentation for about 48 hours. A schematic presentation of the process is shown in figure 1.
Chemical analyses and amino acid composition of the final products are shown in tables 1 and 2. Rhizopus-fermented cassava contained about 90 per cent dry matter, 12 per cent crude protein, consisting of 75 per cent true protein (9 per cent protein nitrogen), 2 per cent fat, 7 per cent crude fibre, 16 per cent ash, 51 per cent total carbohydrates, 2.5 per cent calcium, and 0.8 per cent phosphorus. The amino acid composition shows that the products are too low in Iysine and methionine to meet the requirements for pigs and poultry. The metabolizable energy ranged from 2.2 to 2.4 kcal per gram of dry matter. Preliminary findings on the microbiological aspects and feeding experiments have been reported (6).
FIG. 1. Flow Chart of Cassava Fermentation Process
TABLE 1. Proximate Analysis of Rhizopus and Aspergillus Mycelia and Their Respective Fermented Cassava Products
|CP (N x 6.25) (%)||12.00||12.20||13.75||13.60|
|True protein (%)||8.50||9.00||9.83||10.77|
|Ether extract (%)||1.99||2.05||2.04||1.88|
|Crude fibre (%)||8.19||7.87||7.76||7.25|
|N-free extract (%)||51.41||51.36||52.29||53.37|
|Gross energy (kcal/g)||4.08||4.12||3.94||3.87|
a. Substrates composed of 65 per cent cassava
chips and 35 per cent poultry excrete, inoculated with either
Rhizopus (IR-35) or Aspergillus (IA-35).
b. Substrates composed of 65 per cent cassava chips, 25 per cent poultry excrete, and 10 per cent supplements, inoculated with either Rhizopus (IR-25S) or Aspergillus (IA-25S),
The efficacy of fungi in making use of a wide range of mineral nitrogen sources (e.g., ammonium salts, urea, biuret) and oilseeds has been demonstrated (47-49), but apparently there has been little work done to compare the preferences of fungi for these nitrogen sources.
Fungi have been found to grow more slowly than bacteria or yeasts. A protein doubling time of two hours has been reported for Neurospora, and the same rate of protein production has been maintained by the growth of fungal mycelium in a 3,000-litre fermentor (36; 37). Based on the calculation of protein nitrogen at pre- and post-fermentive stages, a gain of 15 per cent true protein over that of the original substrate was recorded. The conversion efficiency was estimated to give a yield of 15.8 9 dry mycelium per 100 g carbohydrates metabolized (50).
Results from the two-year nutritional and safety evaluation of the microbially enriched cassava in poultry and pigs showed that substitution of maize by fermented cassava up to 60 per cent (30 per cent of the total diet) produced performance equally as good as that of maize-based diets. However, substitution beyond the 30 per cent dietary level resulted in increased feed consumption, reduced weight gain, poor feed efficiency and carcass quality, and low egg production. A progressive depigmentation of the egg yolk, fat, and skin tissues was observed, suggesting that supplementation of pigment is required on enriched cassava-based diets for poultry.
TABLE 2. Levels of Essential Amino Acids (Grams per 16 9 N) in Rhizopus Mycelium, Substrates, and Fermented Cassava and Their Recommended Levels in Poultry Feeding
|Valine||4.90||3.00||2.50||5.00||4.30||3.80||3.30||4 30||5 00|
TABLE 3. Recommended Feeding Levels (Percentages of Diet) of \/arious Fermented Cassava Products for Broilers, Laying Hens, and Pigs
Feeding up to 60 per cent of fermented cassava to broilers for ten weeks, layers for forty weeks, and pigs for six months did not affect their general health or mortality rate. Haematology, blood chemistry, and histo-pathological examinations of tissue samples failed to reveal any abnormalities attributable to the inclusion of these products in the diets. Recommended feeding levels of fermented cassava for broilers, layers, and pigs are shown in table 3.
The successful production of microbially enriched cassava with a protein content superior to maize on the laboratory scale prompted the construction of a pilot plant of sufficient capacity to produce enough feeding material for about 200 pigs, 3,000 broilers, and 2,500 layers. The cost of fermented cassava in 1977 was estimated to be in the range of M$290 to M$390 per ton, depending on the composition of the substrates. The detailed operation of this pilot plant has been described (50).
There exists a great potential in the use of micro-organisms such as fungi for the production of highquality feedstuffs from the abundantly available agro-industrial wastes, particularly carbohydrate residues. Processes of traditional fermented food preparation in Asia and Africa can be modified, improved, and adopted for this purpose. Residues from cassava, sago, and sugar cane processing, by virtue of their relatively low cost and abundance, are considered to be suitable substrates for microbial fermentation.
The use of moist-solid fermentation for the enrichment of carbohydrate residues has several advantages over the submerged liquid-type fermentation adopted elsewhere.
This process is relatively simple, does not involve sophisticated equipment, and is, therefore, more suited to village-level operation. It is hoped that a group of dominant micro-organisms can be discovered that will grow under a highly specific set of environments so that sterilization of the substrates before fermentation is not required. It is also recognized that, since the product is prepared in moist-solid form, there is no necessity for the tedious and expensive step of extracting the final product from the liquid mixture as required in submerged fermentation.
To minimize the cost further, the drying step could be omitted by mixing the end-product with compounded feed before feeding farm animals.
Screening of micro-organisms with high conversion efficiency of protein from carbohydrate residues should be worthy of further investigation. From the safety and nutritional point of view, microbially enriched carbohydrate residues such as fermented cassava have great potential for feeding livestock. However, whether it is practical or not to produce microbially enriched carbohydrate residues appears to be primarily a question of economics.
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