The protein content of cassava residue, soybean waste and rice bran is increased through fermentation with Aspergillus oryzae

Tran Thi Thu Hong and Le Thanh Ca

Faculty of Animal Science and Veterinary, Hue University of Agriculture and Forestry,
102 Phung Hung Street, Hue City
hongnbh@yahoo.com

Abstract

Cassava root waste, soybean waste and rice bran were fermented with  Aspegillus oryzae alone or together with Saccharomyces cerevicae,and Lactobacillus fermentum with the aim of improving the protein content.

After 21 days of fermentation, there were increases in the content of crude protein, and decreases in NDF,  in all the substrates with the effect being most pronounced for cassava root waste, followed by soybean waste and rice bran. There were no apparent benefits from including Saccharomyces cerevisiae or Lactobacillus fermentum  together with Aspergillus oryzae as agents in the fermentation..

Key words: agro-industrial by-products, fungi, incubation, Lactobacillus fermentum, Saccharomyces cerevisiae

Introduction

In animal raising, the feed price occupies up 75-80% of the production cost. There is therefore a strong incentive to find cheaper ingredients to replace the soybean and fish meal that are the main protein sources on diets for pigs and poultry. The waste from processing of cassava for starch extraction is widely available in Vietnam as is the residue from production of Tofu. Both these products have relatively high levels of cell wall carbohydrates and cassava waste is very low in protein. The presence of anti-nutritional factors (cyanogenic glucosides in cassava waste and anti-trypsin compounds in soybean waste) are other constraints that have to be considered.

Recently, fermentation with fungi, yeast, and bacteria has been studied with the aim of reducing non-nutritional components and increasing the nutritive value of agro-industrial by-products (Aro 2008).

The objective of this study was to evaluate the use of a combination of micro-organisms - Aspergillus oryzae, Saccharomyces cerevicae and Lactobacillus fermentum - to improve the nutritive value of cassava root waste, soybean waste and rice bran.

Materials and methods

By-products

Cassava residue was collected directly from a cassava starch processing factory, in Phong An Commune, Phong Dien District, Thua Thien Hue Province. Soybean waste was collected from a factory processing soybeans for Tofu. Rice bran was purchased from the market.  

Microorganisms

Aspergillus oryzae was provided by Tokyo University, Japan.  Saccharomyces cerevisiae was purchased in the market as it is used locally for production of rice wine. Lactobacillus fermentum was isolated from brewery grain waste which was obtained from the HUDA beer factory. It was stored at -70^oC (Trần Thị Thu Hồng et al 2009).

Media for growth of micro-organisms

For production of Aspergillus oryzae, the media was PDA (Potato dextrose agar) prepared as follows. The skin was removed from 200 – 250 g fresh potatoes, which were then cut into small pieces, 1 liter of water added and cooked for 2 – 3 hours. Pure water was then  added to make a total volume of  1 liter.   The suspension was filtered and to the filtrate was added 10 g of glucose, and the solution put into flasks and autoclaved at 121^oC for 15 minutes. Aspergillus oryzae was added to the flasks that contained the PDA media, which were then incubated 2 days at 28^oC for collecting spores. The flasks were kept at 4^oC and using as starter for the fermentation of the three substrates.

The MRS (De Man Rogosa Sharpe) media was used for culturing Lactobacillus fermentum. The composition of MRS media was: Proteose pentone no.3  10g, Beef Extract 10g, Yeast Extract 5g, Dextrose 20g, Polysorbate 80 1g, Ammonium citrate 2g, Sodium  Acetate  5g, Magnesium sulfate 0.1g, Manganese sulfate 0.05g, Dipotassium phosphate 2g, Agar 15g.

To 70 g MRS media, was added 1 liter of water. The solution was autoclaved, distributed into Petri dishes, inoculated with Lactobacillus fermentum and then incubated at 37^oC, for 72 hours.  After that the cultures of Lactobacillus fermentum were kept in eppendoft typs containing glycerin at 4^oC for use in the fermentation studies.     

Fermentation of the substrates

The treatments were:

• AO: Aspergillus oryzae

• AS: Aspergillus oryzae + Saccharomyces cerevisiae

• ASL: Aspergillus oryzae + Saccharomyces cerevisiae + Lactobacillus fermentum

The cultures (about 2ml of each) were mixed thoroughly with 1000 g of the substrates together with 0.5 % NaCl and 0.5 % NaNO₃ (% DM basis). The substrates after mixing with the micro-organisms were put in thick polyethylene bags (20 x 30 cm). These were laid on the floor and the contents spread evenly to a depth of 1 cm. Water was previously added to the rice bran to give it a moisture content of about 65%.  Samples were taken from the bags before fermentation and then after 7, 14 and 21 days of fermentation.

Chemical analysis 

Analyses were made of DM, ash, crude protein and crude fibre according to Vietnam standards (TCVN 4326 – 86, TCVN 4327 – 86, TCVN 4328 – 8, TCVN 4329 – 86, respectively. Neutral detergent fibre and acid detergent fibre were determined according to Van Soest et al (1991). Total energy (GE) was determined by Bomb Calorimetry (Bomb Calorimeter 6300, Parr Instrument Company). HCN was analysed by the method of Easley et al (1970). 

Statistical analysis

The data were analyzed using the GLM option in the ANOVA Program of the Minitab Software, version 13.31 (Minitab 2000). Treatment means which showed significant differences at the probability level of P<0.05 were compared using Tukey’s pair-wise comparison procedures.

Results and discussion

Effect of fermentation time and different micro-organisms on the composition of the substrates

There appeared to be no advantage from adding Saccharomyces cerevisiae or Lactobacillus fermentum  to the substrates (Table 1;  Figures 1-3). When there were differences, these were small. The discussion of the effect of time of fermentation is therefore presented only for the treatment with Aspergillus (Table 2; Figures 4 to 9).  After 21 days of fermentation, there were increases in the content of crude protein in all the substrates with the effect being most pronounced for cassava waste (300% increase in CP content), followed by soybean waste (185% increase) and rice bran (136% increase).  As was to be expected, the changes in NDF content were negative, with the greatest reduction observed for cassava waste (44.5%) with smaller effects for soybean waste (13.1%) and rice bran (14%).

Figure 1. Effect of micro-organism and fermentation time on crude protein content of cassava root processing waste	Figure 2. Effect of micro-organism and fermentation time on crude protein content of rice bran	Figure 3. Effect of micro-organism and fermentation time on crude protein content of soybean waste

Figure 4. Effect of fermentation with Aspergillus oryzae on crude protein content of cassava root processing waste	Figure 5. Effect of fermentation with Aspergillus oryzae on crude protein content of rice bran	Figure 6. Effect of fermentation with Aspergillus oryzae on crude protein content of soybean processing waste

Figure 7. Effect of fermentation with Aspergillus oryzae on NDF content of cassava root processing waste	Figure 8. Effect of fermentation with Aspergillus oryzae on NDF content of rice bran	Figure 9. Effect of fermentation with Aspergillus oryzae on NDF content of soybean processing waste

The data show clearly that fermentation by Aspergillus oryzae led to increases in the concentration of crude protein n the substrates, especially in the cassava root waste. However, in the absence of final weights of the substrates at the end of each stage of the fermentation it is difficult to decide what proportion of the change was due to de facto protein synthesis and what was due to oxidation of the substrate. An indication of the possible extent of the changes can be made by assuming different degrees of substrate oxidation (Table 3).

Table 3. Predicted increases in total amount of protein synthesized in cassava root waste due to fermentation by Aspergillus oryae, assuming different amounts of substrate were oxidized (eg: 25%, 50% or 70%)
Substrate, g	1000*	750#	500#	300#
CP, g	35.1	87.8	58.5	35.1
Increase in CP, g	0	52.7	23.4	0.0
* Substrate at the beginning # Assumed amount of substrate at the end of fermentation

If it is assumed that between 25 and 50% of the substrate was oxidized during the fermentation then the net gain in protein would vary from 70 to 150%. Subsequent studies will be directed to measuring directly the relationship between amounts of substrate oxidized and the net gains in protein as a result of fermentation by Aspergillus oryzae.

Supporting evidence for a net improvement in protein level and quality through fermentation of cassava root waste by fungi can be seen in the report by Nguyen Van Phong et al (2013). These authors recorded increases from 4.6 to 9.5% in crude protein and 3.2 to 5.5% in true protein (% in DM), after incubation of cassava root waste with Aspergillus niger. Furthermore, the protein-enriched cassava root waste supported faster growth rates and better feed conversion in pigs than the non-enriched cassava root waste.

References

Aro S O 2008 Improvement in the nutritive quality of cassava and its by-products through microbial fermentation. African Journal of Biotechnology Volume 7 (25), pp. 4789-4797.

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

Nguyen Van Phong, Nguyen Thi Hoa Ly, Nguyen Van Nhac, and Du Thanh Hang 2013 Protein enrichment of cassava by-products using  Aspergillus niger and feeding the product to pigs. http://www.lrrd.rg/lrrd15/7/hang25130.htm

Tran Thi Thu Hong, Pham Hong Son, Tran Quang Vui, Do Thi Loi and Hoang Anh Tuan 2009  The influence of inclusion of Lactobacillus fermentum isolated from brewery grain in the diet on the performance of post-weaning piglets. Journal of Science, Hue University, Volume 55, 2009.

Received 26 April 2013; Accepted 21 June 2013; Published 1 July 2013

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