Protein enrichment of cassava by-products using Aspergillus niger and feeding the product to pigs

Nguyen Van Phong, Nguyen The Hoa Ly, Nguyen Van Nhac and Du Thanh Hang

Hue University of Agriculture and Forestry, Vietnam
hangduthanh@yahoo.com.vn

Abstract

A fermentation procedure to increase the protein content of cassava bran with Aspergillus niger is described. The organism was grown in a cassava bran medium with different inorganic nitrogen (ammonium sulphate) levels (0,  0.5,  1.0 and 1.5% N in DM). The fermentation time was 144h.

The true protein contents were 3.3, 5.2,  6.4 and 5.1% in DM, for the four levels of ammonium sulphate, respectively. The enriched cassava bran supported higher growth rates and better feed conversion  than the non-enriched product when included in a fattening diet for pigs, both on-station and on-farm. The results indicate that the fungal-enriched cassava bran can be safely used in diets for fattening pigs.

Key words: ammonium sulphate, cassava bran, conversion, fermentation, growth, starch processing

Introduction

About 200 tonnes per month of cassava roots are processed into starch by artisan methods in the Hue city area. Two by-products from this process are bran and fines with about 100 tonnes  produced monthly. Most of the by-products are utilized for feeding live stock. However, they are low in protein content,  so that  large quantities of protein-rich oilseed and fish meals have to be added when these feeds are used as the basis of diets for non-ruminant live stock diets, in order to raise the concentration and the quality of the protein. 

The protein-enrichment  of cassava bran using microbial techniques was therefore investigated. The aims were to  determine whether Aspergillus niger, in the presence of added inorganic nitrogen,  could be used to increase  the protein content of cassava bran and thus improve it's feeding value for pigs.

Materials and methods

Propagation of the fungus

The strain of Aspergillus niger used in the investigation was obtained from the Department of Microbiology, University of  Danang. It had been isolated originally from cassava bran. The cassava bran used as the fermentation medium was taken  from a small starch-processing mill in the Hue area and had been dried. Three different carbohydrate sources were compared in the following fermentation media (g/100g) in  order to propagate the culture:

• MM:  60g cassava bran + 30g maize meal + 10g Aspergillus-niger

• RB: 60g cassava bran + 30g rice bran + 10g Aspergillus-niger

• CR:  60g cassava bran + 30g cassava root meal + 10g Aspergillus-niger

The cassava bran, rice bran, maize meal, and cassava root meal were steamed for 25 minutes and then cooled to ambient temperature (30ºC) before incubating with Aspergillus-niger spores,  keeping the moisture content of the mixture at 60%. The media were spread in a thin layer (2-3 cm) on a tray and were fermented aerobically. Test samples were also fermented in petri dishes to observe the development of the fungal cultures. Samples were taken after 6 days of incubation and analysed for dry matter (DM), crude protein (CP), ether extract and ash. On the basis of the analysis the medium containing rice bran was selected  for  propagating the culture for the production experiment.

Production experiment

The following experiment was designed to determine the optimum level of addition of ammonium sulphate. The selected levels (as % of DM) were: 0, 0.5, 1.0 and 1.5 g (NH₄)₂SO_{4 .}

Cassava bran was adjusted to a moisture content of 50 to 55%.  The inoculum of Aspergillus-niger was one week old and was added to the cassava bran in the ratio 1: 9 (fresh basis),  mixed well, and placed in an aerated area.  Samples were taken for chemical analysis at 0, 3, 6 and 9 days after incubation and analysed for DM and nitrogen (AOAC 1990).

Feeding trial on-station

The experiment was carried out from September to November, 2001 on the University farm. Three treatments were compared using 12 pigs (Large White x Mong Cai) with average weight of  26.5 kg. The treatments were:

• Control: Traditional diet based on rice bran

• ECB:  Enriched cassava bran replacing 30% of rice bran  in the traditional diet

• CB: Non-enriched cassava bran replacing 30% of rice  bran in the traditional diet.

The composition of the control diet was (%): rice bran 40, maize meal 40, groundnut cake 10, fish meal 10. The pigs were weighed on the initial and final day of the experiment. Refusals were collected the following morning before the first meal for calculating feed intake and feed conversion.

Feeding trial on-farm

The experiment was carried out from February to May, 2002 in Phu Thuorng village. There were  two treatments applied to 12 pigs (Large White x Mong Cai), six castrated males and six females, of average initial weight of 28 kg. One group was  fed a diet of 30% enriched cassava bran + 70% basal  diet; the other group was fed a diet of 30% non-enriched cassava + 70% of basal diet. The diet was divided equally into 3 meals per day at 7.00, 12.00 and 17.00. The pigs were weighed at the beginning and at the end of the experiment which lasted 70 days.

Chemical analysis

Samples of feeds were analysed for DM, N, crude fibre, ether extract and ash according to AOAC (1990) procedures.  True protein was determined by the Stutzer method (AOAC 1990). The samples were  boiled with water to dissolve the amides. True protein was then precipitated with copper hydroxide and the aqueous mixture was filtered to separate the amides from the protein. The true protein which remained in the residue on the filter paper was analysed for N by the standard Kjeldahl procedure.

Statistical analysis

Statistical analysis was performed using the general linear model procedure of the ANOVA option in the Minitab software (Minitab 2000). Sources of variation were treatments and error.

Results and discussion

Incubation experiments

The rice bran appeared to be the best medium for propagating the Apergillus niger, as measured by the increase in protein after 6 days of incubation (Table 1).

Cassava enrichment experiment

The true protein of the media increased rapidly during the first 6 days of incubation with no further increase up to 9 days (Table 2; Figure 1). The optimum level of ammonium sulphate was 1.0% of substrate DM.

Table 2: Mean values for concentrations of crude and true protein (as % in DM)after incubation for 0, 3, 6 and 9 days with increasing levels of ammonium sulphate
	Level of ammonium sulphate, % in DM				SEM	p
	0	0.5	1.0	1.5
0 days
Crude protein	1.7	2.0	2.4	2.6
3 days
True protein	3.1	3.4	5.4	4.1	0.09	<0.001
Crude protein	4.2	5.8	7.0	6.8
6 days
True protein	3.3	5.2	6.4	5.1	0.09	<0.001
Crude protein	4.5	7.1	9.8	8.9
9 days
True protein	3.2	5.3	6.4	5.5	0.07	<0.001
Crude protein	4.6	7.3	8.1	9.5

Figure 1: True protein production in cassava by-product incubated with Aspergillus niger in presence of ammonium sulphate at levels of 0 to 1.5% in the DM of the medium (values at 0 days are for crude protein; it was assumed this would be mainly as true protein)

Similar findings were reported by by Balagopalan et al (1988) who developed a solid state fermentation process for the protein enrichment of cassava flour and cassava starch factory wastes using the fungus Trichoderma pseudokonigii rifai. Our results show that using this fermentation process it was possible to convert the substrate to a protein-enriched animal feed, using the minimum of additional nutrients [0.15% (NH₄)₂SO₄].

There appeared to be a small increase in the lipid concentration of the media after incubation with Aspergillus, but this was not affected by addition of ammonium sulphate (Table 3).

On-station feeding trial

The overall changes in the cassava bran after incubation with Aspergillus are shown in Table 4.

The pigs fed the diet with enriched cassava bran grew faster, and had better feed conversion, than those fed the non-enriched product (Table 5), the overall performance being similar to that on the traditional diet. The implication is that the nutritive value (mainly the protein content) of the enriched cassava bran diet had been improved to a level comparable with the rice bran. 

On-farm trial

The results of the on-farm trial (Table 6) were similar to those on-station. Growth rates and feed conversion were improved on the enriched cassava bran diet compared to the non-enriched product.

Conclusions

• Incubating cassava bran with Aspergillus-niger in the presence of 1% ammonium sulphate increased the true protein from 2 to 6% in DM

• Pigs grew faster with better feed conversion when the traditional diet was partially replaced (30%) with the enriched cassava bran compared with the non-enriched product.

Acknowledgements

This research was partially financed by the MEKARN project, supported by Sida (Sweden).

References

AOAC 1990 Official Methods of Analysis 13^th Edition. Association of Official Analytical Chemists, Washington DC

Balagopalan C, Padmaja G and George M 1995 Improving the nutritional value of cassava products using microbial techniques. In:  Roots, tubers, plantains and bananas in animal feeding (Editor: D Machin). FAO, Rome AHPP 95 http://www.fao.org/ag/aga/agap/frg/ahpp95/95-127.pdf

Minitab 2000 Minitab reference Manual release 13.31. User’s guide to statistics. Minitab Inc., USA

Received 7 May 2013; Accepted 20 June 2013; Published 1 July 2013

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