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Protein enrichment of starchy substrates by solid-state fermentation

J.C. Senez
Laboratoire de Chimie Bactérienne - CNRS, Marseilles, France

M. Raimbault and F. Deschamps
Centre de Recherche IRCHA, Vert-le-Petit, France

Introduction
Technical aspects
Summary
References

Introduction

In spite of current economic constraints, large-scale industrial production of single-cell proteins (SCP) will undoubtedly soon develop in the industrialized countries of Western Europe, Japan, and the USSR, for whom new protein sources are becoming an absolute and urgent necessity. A priori, one would expect the SCP industry to provide a decisive 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 to 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 methanol. Such facilities are obviously absent in most non-oil-producing developing countries of Asia, Africa, and Latin America. Moreover, these countries may not have a potential market or an appropriate transportation and distribution network for the commercialization of 100,000 tons of SCP per year.

Clearly, those countries that cannot now import food or feeds because of currency shortage will also not be able to import industrial SCP from abroad. 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 usable for SCP production might be considered. However, most of them are too high in cost to be economically competitive or are available in quantities too low for protein production on a really significant scale. Among the substrates suitable with respect to cost and supply, special emphasis is usually given to cellulosic materials, but, at the moment, the many attempts made in this direction have not been notably successful, 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 more temperate climates - are of obvious interest, both because of their high productivity per hectare, and their 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 and methanol, optimal use of such sophisticated technology would require a minimal production 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 lead to major difficulties.

Given these considerations, a quite different approach is suggested, consisting of protein enrichment of starchy material by a simplified technology that can be applied at the farm or village level, and that will thus simultaneously combine the cultivation of raw material, its conversion into protein, and its direct use for animal feeds. Economically, the decisive advantage of such an integrated approach is that it prevents intermediary profit-taking and speculation that would inevitably develop if either the raw material or the product were commercialized.

To be workable at the rural level, a protein enrichment process should not require aseptic conditions and should be performed in a single operation. Additionally, the final product must be sufficiently rich in protein to be utilizable as such, without a secondary concentration step. This last requisite entails 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 used directly for animal feeding, the major problem is to maintain aerobic conditions and oxygen transfer efficiency so as to prevent anaerobic contamination of the culture.

Tempeh and many other food preparations obtained by solid-state fermentation of soybeans or other materials with filamentous fungi (1-3) are traditionally used in various parts of Asia and Africa, but they do not increase the protein content of the initial materials. On the other hand, procedures for direct protein enrichment of cassava by liquid (4; 5) or solid-state (6) fermentation have been described. However, protein enrichment by the solid technique did not exceed 3 to 4 per cent, and therefore was insufficient for use as a complete feedstuff. The liquid process with fungi presented technical or sanitary problems.

A new procedure for solid-state fermentation (7) fulfilling the above specifications was developed in France. A preliminary report of this technique was presented at the Fifth International Conference on the Global Impacts of Applied Microbiology (81.

Technical aspects

Laboratory Investigations

The principle of this new procedure is based on the homogeneous distribution of spores and mineral salts in the mass of starchy substrate. The preparation of a porous, granulated material with adequate pH, temperature, and moisture content is essential to ensure good aeration and rapid growth of mycelium within the mass.

TABLE 1. Protein Enrichment of Cassava by Solid-State Fermentation

a. Carbohydrates, 90 per cent; protein, 2 per cent; water 8-9 per cent.
b. Percentage of the dried product, determined by the Lowry method.
c. Percentage of the dried product, determined by enzymatic hydrolysis (lamyloglucosidase) and Somogyi-Nelson titration.
d. Percentage of the wet product.

TABLE 2. Protein Enrichment of Various Raw Materials

All results in percentage of the dried material.

Thus, the coarsely ground raw material, with 30 to 35 per cent moisture, is maintained at 70 to 80 for 10 to 15 minutes by gently steaming to gelatinize the starch granules. After cooling to 40 C, the preparation is mixed with water containing the inoculum (spores), the nitrogen sources (ammonium sulphate and urea), and potassium phosphate, to a 55 per cent moisture content. By means of mechanical stirring, the inoculated substrate spontaneously takes the form of well-separated and uniform granules of about 2 to 3 mm in diameter.

General conditions for protein enrichment of cassava or other starchy materials are summarized in table 1. This method has already been worked out with a variety of starchy materials, namely cassava, whole potatoes, potato wastes from industrial fecula works, and banana refuse. The results are reported in table 2, showing that, after 30 hours of incubation, one obtains a product containing an average of 20 per cent true protein, measured by the Lowry methods, and 25 per cent residual reducing sugars. The rate of conversion of carbohydrates to protein is 20 to 25 per cent.

Up to now, experiments have been performed with a selected strain of Aspergillus niger having high amylolytic activity and suitable amino acid composition. However, it should be pointed out that many other filamentous fungi, particularly among strains traditionally used in Asia for producing fermented foods for human consumption, were successfully tested by this technique. This method does not require aseptic conditions, because selective growth of the mould results from acidic pH, low moisture content, and heavy spore inoculation. Microscopic examination of the products indicates that all spores germinate after six to eight hours, and during the growing phase all the mycelia develop. At the end of the fermentation no spores could be observed. Bacteriological controls of fermented products indicate neither pathogens nor significant development of anaerobic bacteria. The aerobic microflora remain at the same level during the first 20 hours, at which time the number of aerobic bacteria quickly decreases.

Experimental Pilot-Scale Studies

The laboratory results led to the design of new equipment for this solid-state fermentation process (9). All the operations were conducted in a commercial bread making blender modified for that purpose.

Steaming or aeration was done by passing steam or air through the perforated bottom of the tank. A control system using conventional probes was designed to keep suitable pH, moisture, and temperature by stirring the product and spraying it with water or mineral solutions. This control system was monitored by a temperature sensor; as soon as the temperature reached the desired point, pH, temperature, and regulation time could be monitored by a simple check of growth rate and harvest-time.

With the organism now used, the optimal temperature is 40 C, but the same growth takes place at temperatures from 30 to 45 C without a significant change in the final protein yield. The initial moisture content is critical, the optimum being 55 per cent. During the course of fermentation, the water content is progressively increased to a final value of 70 to 75 per cent. The kinetics of a fermentation using potato waste are reported in figure 1, showing the production of protein, reducing sugars, and water content as well as the pH of the preparation. The curve marked by crosses is of special interest, since it shows that during a total incubation time of 30 hours the monitored devices for mechanical stirring and spraying had to operate for only five hours, thus demonstrating the excellent efficiency of the cooling device. Additionally, it corresponds to a remarkably low expenditure of power, a fact of obvious importance with regard to the production cost of solid-state fermentation, thus making it economically feasible at the village level in tropical regions.

Currently, the studies on this solid fermentation process are being actively developed in France by the Office de la Recherche Scientifique et Technique Outre-Mer and the Institut National de Recherche Chimique Appliquée in close collaboration with industry for utilization of potato wastes. The scaling-up of the process to a fermentor unit of 1,200-litre capacity (see photograph) is in progress. This equipment, which is expected to be operative in the coming months, will be used for large-scale nutritional and toxicological testing on target animals (pigs and poultry), for further improvements in substrate preparation and growth conditions, and, finally, for the determination of actual investment and operation costs. It is intended that the experiment will be extended to the setting up of trial production units in tropical Asia and Africa, in order to adapt the procedure to local climatic and agroeconomic conditions.

Agro-economic Perspectives

FIG. 1. Solid-State Fermentation of Potato Waste

As already pointed out, the two main sources of starch potentially available for protein enrichment are cassava in tropical countries 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 proteinrich 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 north-eastern Brazil, the yield can be easily increased by the use of fertilizers and improved cultivation practices to 40 and even 60 tons per hectare. Other advantages of cassava are low production costs, easy storage in the ground for several months, and high calorie content for animal feeding.

TABLE 3. Agro-economic Prospects of Cassava Compared with Soybeans

a. 34 per cent protein.
b. Data from US Department of Agriculture.
c. Based on 20 per cent protein enrichment, with 25 per cent loss of dry matter during fermentation.
d. From ref. 10.
e. 70 days; + 25 kg diet with 15 per cent protein.
f. 130 days; + 85 kg; diet with 10 per cent protein.
g. 200 days; 110 kg.

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 1.8 tons of protein per hectare, i.e., the supply required for feeding 50 pigs (table 3). This is about three 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 used for animal feeding are shown in table 4.

From October 1978 prices and from data on average yields of agricultural products, one can compare the gross product per hectare of corn, wheat, soybeans, and protein-enriched cassava. Actually, in the case of cassava, the value of the residual sugars (35 per cent, dry weight) should increase the gross product figure. On the other hand, for a rural community combining the production of raw material with protein enrichment and direct use for animal feeding, the real 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 protein-enriched cassava is that it allows feedstock production in regions where no other suitable source of conventional feed protein is available.

TABLE 4. Optimal Productivity of Protein-Rich Feeds

a. 40 tons per hectare of cassava, with 70 per cent moisture content, 25 per cent lost during fermentation, dry weight

Obviously, to be economically competitive, the process of protein enrichment by solid-state fermentation depends ultimately on the investment and production cost of the process. It would be premature to give a truly accurate estimate in this regard until information is obtained from pilot operations at the farm level. However, in the present state of technological development it can be assumed that the process will prove to be valuable.

Summary

Protein enrichment of starchy materials destined for direct animal feeding was achieved by a simple, cheap, and non-aseptic process of solid-state fermentation applicable at the farm or village level. The process provides feedstuffs containing up to 20 per cent protein and 35 per cent residual sugars derived from cassava, banana refuse, potatoes, and other substrates potentially available in tropical or temperate climates. On the basis of 40 tons productivity (harvest weight) per hectare, cassava and potatoes could thus provide three times more protein than soybeans and compete favourably with the cultivation of corn, wheat, and soybeans.

References

1. C.W. Hesseltine, "A Millennium of Fungi, Food and Fermentation," Mycologia, 57: 149-197 11965).

2. A. Martinelli and C.W. Hesseltine, "Tempeh Fermentation," Food Technol., 18: 167-171 (1964).

3. W.D. Gray, "The Use of Fungi in Food and in Food Processing," Critical Review in Food Technology (Chemical Rubber Co.), 1: 225-329 (1970).

4. A.E. Reade and K.F, Gregory, "High Temperature Production of Protein-Enriched Feed from Cassava by Fungi," Appl. Microbiol., 30: 897-90411975).

5. 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: 30-35 1 1 976).

6. E.J. Brook, W.R. Stanton, and A. Wallbridge, "Fermentation Methods for Protein Enrichment of Cassava," Biotechnol. Bioeng., 11: 1271-1284 (1969).

7. M. Raimbault and J.C. Germon, "Procédé d'Enrichissement en Protéines de Produits Comestibles Solides," Pat. B.F. No. 76.06.677, 9 Mar. 1976.

8. M. Raimbault, F. Deschamps, F. Meyer, and J.C. Senez, "Direct Protein Enrichement of Starchy Products by Fungai Solid Fermentation" (paper presented to the 5th international Conference on the Global Impacts of Applied Microbiology, Bangkok, Thailand, 21-26 Nov. 1 977),

9. F. Deschamps and F. Meyer, "Nouveau Fermenteur pour Milieux Solides," Pat B.F. No. 79.02.625,1 Feb. 1979.

10. C.A. Shacklady, in H. Goonelle de Pontanel, ea., Proteins from Hydrocarbons (Academic Press, New York, 1973), pp. 115-128.

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