Materials and methods
Jacques C. Senez
Laboratoire de Chimie Bacterienne, C.N.R.S., Marseille, France
In spite of a combination of currently unattractive economics and political opposition in some quarters, large-scale production of single-cell protein (SCP) will undoubtedly develop soon in industrialized countries in Western Europe and in Japan and the USSR, where the development of new protein sources is becoming an absolute, urgent necessity. A priori, it could also be expected that the SCP industry would provide a contribution to the problem of hunger in the Third World. In this regard, however, there are several major obstacles.
To be economically viable, an SCP production unit should have a minimal capacity of at least 100,000 tons per year, corresponding to a capital cost of US$50 - 70 million. On the other hand, a plant producing 100,000 tons of SCP from paraffins would require an equal supply of substrate and should thus be associated with an oil refinery having a minimal capacity of about 3 to 5 million tons of crude oil per year. Similar considerations apply to the production of SCP from natural gas or from methanol. Such facilities are obviously absent in most non-oil producing countries of Asia, Africa, and Latin America. Moreover, these countries may have neither a potential market nor a transportation and distribution network for the commercialization of 100,000 tons of SCP per year.
Clearly, those countries that cannot currently import food or feeds because of currency shortages will also be unable to import industrial SCPs. Consequently, it is of utmost importance for them to develop their own protein resources. In addition to hydrocarbons and methanol, a wide variety of raw materials potentially utilizable for SCP production might be considered. However, most of them are available at too high a cost to be economically competitive, or exist in quantities too low for protein production on a significant scale. Among the substrates suitable in cost and supply, special emphasis is usually put on cellulosic materials, but, at the moment, the many attempts made in this direction have had little success, the main difficulty being the lack of cellulolytic organisms with an adequate growth rate.
In contrast, starchy materials, more specifically cassava in the tropical regions or potatoes in temperate climates, are of obvious interest because of both high productivity per hectare and excellent rate of conversion to biomass by a great number of fast-growing micro-organisms.
In order to be economically competitive, the production of protein from starch should not be undertaken by classical fermentation in liquid medium, under aseptic conditions, followed by biomass separation and drying. As in the case of SCP production from paraffins or methanol, the optimization of such sophisticated technology would require a minimal production of well over the potential market of most developing countries, and would result in high investment and operation costs. Moreover, in the developing countries, the collection, transportation, and storage of large quantities of raw materials would result in major difficulties. Considering these factors, a more practical approach would be to enrich starchy materials with protein by means of a simplified technology that can be applied at the farm or village level, and that would allow the combination of cultivation of raw material, its conversion into protein, and its direct utilization for animal feeds. Economically, a great and decisive advantage of such an integrated procedure is to prevent intermediary profits and speculation that would inevitably take place if either the raw material or the final product were commercialized.
To be workable at the farm level, a protein enrichment process should not require aseptic conditions, and should be performed in a single operation. Additionally, the product must be sufficiently rich in protein to be utilizable as such, without a secondary fortification step. This last requisite creates a biotechnological difficulty that has been responsible for the failure of many previous attempts to achieve direct protein enrichment of starchy materials. In a mash of raw material dense enough to be directly utilized for animal feeding, the major problem is to maintain aerobic conditions and oxygen transfer efficiency so as to prevent anaerobic contamination of the culture.
A new procedure of solid state fermentation (1) fulfilling the above specifications was developed in collaboration with Drs. M. Raimbault and F. Deschamps at the French Office de la Recherche Scientifique et Technique d'Outre Mer (ORSTOM), and the Institut de Recherche en Chimie Applique (IRCHA), respectively. A preliminary report on this procedure has already been presented (2) at the 5th International Conference on the Global Impacts of Applied Microbiology, held in Bangkok in November 1977.
Tempeh and many other food preparations obtained by solid state fermentation of soybeans or other materials with filamentous fungi (3 - 5) are traditionally used in various parts of Asia and Africa. On the other hand, procedures for direct protein enrichment of cassava by liquid (6, 7) or solid state fermentation have been described. However, protein enrichment by these methods does not exceed 3 - 4 per cent, and therefore is insufficient for use as a complete feedstuff. The principle of the new procedure devised by ORSTOM and IRCHA for protein enrichment of cassava and other starchy materials is summarized in Table 1.
TABLE 1. Protein Enrichment of Cassava by Solid State Fermentation
|Initial substrate (g)|
|water||100 - 120|
|Optimal growth conditions|
|T: 35 - 40C; initial pH: 3.5|
|Inoculum: 2 x 107 spores/g flour|
|Incubation time: 30 hours|
|Composition of the product|
|Protein**||18 - 20% in dry matter|
|Residual sugar***||25 - 30% in dry matter|
* Carbohydrates: so per cent; protein: 1 per cent; water: 30 - 35 per
** Determined by the Lowry method.
*** Determined by enzymatic hydrolysis (amyloglucosidase) and Somogyi-Nelson titration
All of the operations are conducted in a commercial dough mixer of ten kg capacity, modified for that purpose. The coarsely ground raw material, with 30 - 35 per cent moisture content, is gently steamed for 15 - 20 minutes to break the starch granules. After cooling to 40C, the preparation is mixed with water containing the inoculum (spores), the nitrogen sources (ammonium sulphate and urea), and mineral salts, to 55 per cent final moisture content. After mechanical stirring, the inoculated substrate spontaneously takes the form of well separated and uniform granules of about 1 mm diameter.
Aeration is performed by passing humidified air through the perforated bottom of the tank. Conventional probes are used to monitor, after mixing and water spraying, the temperature, pH, and moisture content. To date, all experiments have been performed with a selected strain of Aspergillus niger having high amylolytic activity and suitable amino acid composition However, other filamentous fungi could be utilized as well.
With the organism currently utilized, the optimal temperature is +40 C, but growth still takes place at temperatures from +30 to +45 C without significant changes in the final protein yield. The initial moisture content is critical, with an optimum of 55 per cent. In the course of fermentation, the water content is progressively increased to a final value of 70 75 per cent.
This method of protein enrichment has already been worked out with a variety of starchy materials, namely cassava, whole potatoes, potato waste from industrial starch works, and banana refuse. The results are summarized in Table 2, showing that, after 30 hours of incubation, a product is obtained that contains, on average, 20 per cent true proteins, measured by the Lowry method, and 25 per cent residual reducing sugars. The rate of conversion of carbohydrates to protein is 20 to 25 per cent, corresponding to 40 to 50 per cent conversion into dry weight biomass.
TABLE 2. Protein Enrichment of Various Raw Materials
|Initial composition||Final product|
The kinetics of a fermentation on potato waste are shown in Figure 1, illustrating the production of protein, reducing sugars, water content, and the pH of the preparation. The curve, marked by crosses, is of special interest. It shows that, during a total incubation time of 30 hours, the monitoring devices for mechanical stirring and water spraying had to operate for only five hours, thus demonstrating the excellent efficiency of the cooling device. Additionally, it requires a remarkably low expenditure of power, a fact of obvious importance in regard to the production cost of solid state fermentation and to its economic feasibility at the farm level in tropical regions.
Figure.1. Solid State Fermentation of Potato Waste
It has been pointed out that, owing to perfectly aerobic and highly selective conditions, no aseptic precautions have to be taken, and sporulation of the mould is totally inhibited. Nutritional and toxicological tests on rats and chickens are in progress, and the preliminary results are quite satisfactory, showing a nutritional value similar to that of soybean meal.
Currently, the studies on solid state fermentation are being actively developed in France by ORSTOM and IRCHA in close collaboration with the Applied Scientific Research Corporation of Thailand. The scaling-up of the process to a fermenter unit of 1 m has been undertaken and is in progress. This equipment, which is expected to be operative in the coming months, will be utilized for large-scale nutritional and toxicological testing on target animals (pigs and poultry), for further optimization of substrate preparation and growth conditions, and for determination of the actual investment and operation costs. It is intended that the experimentation will be extended to the setting up of experimental production units in tropical Asia and Africa, in order to adapt the procedure to local climatic and agro-economic conditions.
As already pointed out, the two main sources of starch potentially available for protein enrichment are cassava in tropical regions and potatoes in temperate climates. Protein enrichment of cassava is of special interest in those semi-arid regions of Latin America and Africa where climatic conditions are not suitable for the cultivation of soybeans or other protein-rich feeds.
The productivity of cassava per hectare varies widely from one region to another, depending on climatic and agro-technological conditions. From about 16 tons (harvested weight) per hectare in northeastern Brazil, the yield can be easily increased by the use of fertilizers and by improved cultural practice to 40 and even 60 tons per hectare. Other advantages of cassava are low production costs, easy storage in the soil for several months, and the fact that cassava is also an excellent source of calories.
On the basis of a productivity of 40 tons per hectare and 20 per cent protein enrichment via solid state fermentation, cassava or potatoes may provide 2.4 tons of protein per hectare, i.e., the supply required for the feeding of 65 pigs (Table 3). This is about four times the quantity of protein per hectare provided by soybean cultivation in the United States. The crop yield and protein productivity per hectare of other protein sources conventionally utilized for animal feeding are reported in Table 4.
TABLE 3. Agro-Economic Prospects of Cassava Enrichment
|A. Productivity of raw material and of protein||Cassava||Soybeans*|
|Raw material (tons/ha)||40||1.8**|
|Moisture content (%)||70||-|
|Protein (tons/ha)||2.4*** 0.6|
B. Conversion into animal product (pork)****
|Alimentary conversion rate (units of feed per unit liveweight gain)||3:1|
|birth to weaning||11.3 kg|
|- weaning to slaughtered||25.5 kg|
|- total||36.8 kg|
C. Overall agro-economic prospect
(1) Protein productivity per hectare: protein-enriched cassava versus soybeans:
(2) One hectare of cassava can produce, via solid state fermentation, enough protein for feeding:
c.a. 65 pigs
* 34 per cent protein.
** data from U.S. Department of Agriculture.
*** for 20 per cent protein enrichment.
**** From: C.A. Shacklady, in G. de Pontanel (ed.), Proteins from Hydrocarbons, pp. 115 - 128, Academic Press, New York, 1972.
Birth to weaning: 70 days; + 25 kg; diet with 15 per cent protein.
Weaning to slaughter: 130 days; + 85 kg; diet with 10 per cent protein.
Total: 200 days; 110 kg.
TABLE 4. Optimal Productivity of Protein-Rich Feeds
* 40 tons per hectare of cassava with 70 per cent moisture content.
Based on prices in October 1978 and on the average yields of agricultural products, a comparison can be made of the gross product per hectare of corn, wheat, soybeans, and protein-enriched cassava. The figures in Table 5 strikingly demonstrate the economic advantage of protein enrichment by solid state fermentation. In the case of cassava, the value of the residual sugars (25 per cent dry weight) should increase the gross product figure by about 10 per cent. On the other hand, for a rural community combining the production of raw material with protein enrichment and direct utilization for animal feeding, the actual gross product should be estimated, not from the commercial value of protein, but from the value of the feedstock produced. Moreover, as already pointed out, one of the major agro-economic advantages of proteinenriched cassava is the possibility of feedstock production in regions where no other suitable source of conventional feed protein is available.
It is obvious that the economic competitiveness of protein enrichment by solid state fermentation depends ultimately on the investment and production costs of the process. It would be premature to propose a really accurate estimate in this regard, and information must be obtained from pilot operations at the farm level. However, based on its present state of technological development, and on the data reported in Table 5, the conclusion is that the process will prove valuable.
TABLE 5. Comparison of Productivity and Gross Product per Hectare
* On 29 September 1978.
** Cassava: 40 tons per hectare, with 70 per cent moisture, dry product containing 20 per cent protein.
*** Estimated from current price (US$213.6, Rotterdam, cif) of soybean meal with 44 per cent protein.
Protein enrichment of starchy materials was achieved by a simple, inexpensive process of solid state fermentation not requiring aseptic conditions and workable at the farm or village level for direct animal feeding. From cassava, banana refuse, potatoes, and other substrates potentially available in tropical or temperate climates, the process provides foodstuffs containing up to 20 per cent protein and 35 per cent residual sugars. On the basis of 40 tons productivity (harvest weight) per hectare, cassava and potatoes could thus provide four times more protein per hectare than is obtained by soybean cultivation. Hence, its agro-economic prospects compete favourably with the cultivation of corn, wheat, and soybeans.
1. M. Raimbault and J.C. Germon, Procds d'enrichissement en protines de produits comestibles solides, Patent B.F. no. 76.06. 677, 9 March 1976.
2. M. Raimbault, F. Deschamps, F. Meyer, and J.C. Senez, "Direct Protein Enrichment of Starchy Products by Fungal Solid State Fermentation," paper presented at the 5th International Conference on Global Impacts of Applied Microbiology, Bangkok, November 1977.
3. C.W. Hesseltine, "A Millennium of Fungi, Food, and Fermentation," Mycologia 57: 1 49 - 1 97 (1 965).
4. A. Martinelli and C.W. Hesseltine, "Tempeh Fermentation,'' Food Technol. 18: 167 - 171 (1964).
5. W.D. Gray, "The Use of Fungi in Food and in Food Processing,'' Chemical Rubber Co. Critical Review in Food Technology 1: 225 - 329 (1970).
6. A.E. Reade and K.F. Gregory, "High Temperature Production of Protein-Enriched Feed from Cassava by Fungi," Applied Microbiol. 30: 897 - 90411975).
7. K.F. Gregory, A.E. Reade, G.L. Khor, J.C. Alexander, J.H. Lumsden, and G. Losos, "Conversion of Carbohydrates to Protein by High Temperature Fungi," Food Technol. 30 (3): 30 - 35 (1976).
Regarding the yield of biomass from fungal treatment of cassava, approximately 50 per cent of the starch is converted, in the course of which the percentage of protein content in the starting material is increased between ten- and twenty -fold.