14 Solid-State Fermentation of Manioc to Increase Protein Content

Nguyen Ngoc Thao and Nguyen Hoai Huong

Manioc (cassava) is grown extensively in Vietnam and other tropical countries for its high yields in infertile soil. Although manioc is high in carbohydrates, its use is limited by its low protein content (1 to 4 percent). Manioc has been used at levels of 10 to 15 percent in poultry feed and 35 to 50 percent in pig feed. Powdered dried fish debris (gills, scale, tail, etc., from the fish processing industry), oil cake (from coconut or peanut oil production), or soybean flour have been used to raise protein levels in such feeds, but these products raise the price of feed significantly.

To upgrade the protein content in manioc, yeast cells or fungi can be inoculated in a manioc-containing medium along with nutrients containing nitrogen, phosphorus, and potassium. The use of mycelial fungi has the following advantages:

· The protein content in fermented product can increase to 30 percent.

· Fungal protein can be substituted completely for animal protein.

· The product has a low nucleic acid content.

· The product contains a favorable spectrum of amino acids.

Solid-state fermentation and liquid-state fermentation are two methods used for cultivation of fungus. Liquid-state fermentation processes are well developed in industrialized countries but are not suitable for rural farms in developing countries. Solid-state fermentation is a simple process that does not require modern equipment, power supply, or sterile conditions. In addition, the capital investment is low, permitting countryside operation and the use of available manual labor.

Many studies of solid-state fermentation of manioc have been conducted. The cultivation of Aspergillus niger in a manioc medium at 35° to 40°C for 30 hours has resulted in protein content increases of 5 to 18 percent; carbohydrate content decreased from 65 to 28 percent (1). The protein from this fermentation can be competitive with soybean protein.

In addition to A. niger, other fungi such as A. awamori, A. hennebegii, A. fugamitus, Rhizopus chinensis, and Sephalo sporium lichlorniae can be grown in acid medium at high temperatures. The protein content of the fermented product can reach 48 percent.

In Vietnam, A. niger and A. hennebegii were cultivated on a maltolized-manioc medium or a mixture of manioc and rice flour. This research comes from the demand of the husbandry industry and is designed to develop a fermentation process for on-farm use.

MATERIALS

Dried manioc pieces were ground to the size of 5 to 10 mm. Spores of A. niger were cultivated by surface fermentation on a medium containing rice hulls, rice bran, or manioc flour as carbohydrate, and urea (2 percent), ammonium sulfate (8 percent), and potassium phosphate (4 percent) at pH 4.5. Spores were collected after 7 days of cultivation.

METHODS

After a defined period of fermentation, the product was dried at 65° to 70°C, ground, and analyzed. The moisture content was determined by drying at 105°C to constant weight. The protein content was determined by precipitating with a solution of CaSO4 (6 percent) and NaOH (1.25 percent); the precipitate was analyzed by the Kjeldahl method. The starch content was determined by hydrolyzing the preparation with HCl and using the Bertrand method. The reduced glucose content was determined by the Bertrand method.

Table I shows that A. niger could not grow in medium containing urea as the only nitrogen at a concentration of 4.5 percent because of the resultant alkalinity. With (NH4)2SO4 as the N source, the pH was maintained at 4 to 5 during the fermentation. The maximum protein content was attained in medium containing urea (4 percent) and ammonium sulfate (5.8 percent). The content of protein can reach 17 percent in comparison with the one cultivated in only urea-containing medium. However, 1.55 percent N protein was achieved in the culture medium containing 3.1 percent N with the transformation efficiency of 49 percent.

TABLE 1 Effect of Nitrogen Sources on Protein Formation

N Protein

Percent Percent N Protein

N in Culture in Fermented in Fermented

N°	Source	Medium	Product	Preparation
1 Urea	2.0	0.93	5.2	0.83
	3.3	1.54	8.0	1.28
	4.0	1.86	8.2	1.3
	4.5	2.10	no growth	--
	5.0	2.33	no growth	--
2 Ammonium sulfate
(AS)	7.4	1.54	4.4	0.70
Urea 2% + AS 10%		3.05	7.2	152
Urea 3.3% + AS 4%		3.10	9.36	1 49
Urea 4.0% + AS 5.8%		3.1	9.7	1.55
Urea 4.5% + AS 7.4%		3.1	no growth

The transformation efficiency was 70 percent in medium containing only urea (4 percent).

The P and K elements (Table 2) were added to the medium containing urea 3.3 percent and ammonium sulfate 4.4 percent (1-5) or urea 3.3 percent (6-9), respectively. The results suggested that the P and K sources had no clear effect on protein formation.

In Table 3, the effect of humidity on the protein content is shown. Table 4 illustrates the effect of sterilizing conditions on the yield of protein. Table 5 shows the effect of the amount of inoculum culture on protein synthesis.

RESULTS

Manioc flour cannot be used as a carbohydrate source in the culture medium because it agglomerates and excludes air necessary for the growth of the fungal mycelium.

Manioc pieces of 0.5 to 1.0 centimeters are best for this solid fermentation method. The protein content of fermented preparation decreased 50 percent when using manioc pieces that were 1.0 to 2.0 centimeters in size.

The analysis of a fermented preparation after 2 days of fermentation, drying at 65° to 70°C, and grinding is shown in Table 6.

TABLE 2 Effects of Nutrients on Biosynthesis of Protein (The P and K elements were from chemical fertilizers)

Chemical Fertilizer	P Percent + K Percent	Protein Percent
1	3.3 + 1.0	10.21
2	2.3 + 0.5	11.34
3	2.3 + 1.5	10.41
4	4.3 + 0.5	11.46
5	4.3 + 0.5	9.3
6	3.3 + 1.0	10.0
7	2.3 + 0.5	10.0
8	1.3 + 0.5	11.25
9	0.3 + 0.5	9.62

TABLE 3 Effect of Initial Humidity on Protein Content

	Humidity of	Protein
N°	Culture Medium(a)	Percent	Notes
1	45	7.9	The change of humidity from
2	50	9.36	60 to 70 percent occurred
3	55	9.64	depending on the atmospheric
4	60	11.08	temperature and humidity.
5	65	11.23
6	70	11.37
7	75	Poor growth

a The medium for this experiment contained urea (4 percent), P (1.3 percent), and K (0.5 percent).

TABLE 4 Effect of Sterilization Conditions on Protein Production

, ,

N°	Temperature °C	Time Minutes	Percent Protein	Notes
1	100	45	8.6	The culture medium
2	100	90	7.35	can be sterilized
3	120	30	7.6	at 100°C in
4	120	45	7.50	45 minutes.
5	120	60	6.30

 

TABLE 5 Effect of the Amount of Inoculum Culture on Protein Synthesis

	Percent of Inoculum	Percent	Notes
N°	Culture	Protein
1	0.5	6.0	The maximum percent of
2	1.0	8.6	protein was achieved in 2 percent
3	1.5	10.0	of inoculum culture. There was
4	2.0	12.5	formation of black spore in
5	3.0	12.0	fermented preparation when using more than 2 percent of inoculum culture.

TABLE 6 Product Analysis

			Fermented
		Manioc Pieces	Preparation,
N°	Index	Culture-Medium, Percent	Percent
1	Protein	1.5-2.0	10.0-13.3
	Starch	33	11
	Reduce sugar	4.4	8.11
	Total sugar	8.4	13.00

CONCLUSION

This solid-state fermentation method can be used to upgrade by six to seven times the protein content in manioc pieces. The resulting fermented product contains 10 to 13 percent protein, which is suitable for use as a feed additive.

REFERENCE

1. Raimbault, M.J. 1985. Fermentation Technology 63(4):395-399.