Protein enrichment of cassava (Manihot esculenta Crantz) root by fermentation with yeast, urea and di-ammonium phosphate

Nouphone Manivanh, T R Preston¹ and Nguyen Thi Thuy²

Faculty of Agriculture and Forest Resource, Souphanouvong University, Luang Prabang, Lao PDR
nouphone2007@yahoo.com
¹ Centro para la Investigación en Sistemas Sostenibles de Producción Agropecuaria (CIPAV), Carrera 25 No 6-62 Cali, Colombia
² Cantho University, Cantho, Vietnam

Abstract

Cassava root was fermented with yeast, urea and di-ammonium phosphate (DAP) to determine the degree of conversion of crude to true protein. A completely randomized design (CRD) was used with 6 treatments arranged as a 2*3 factorial with four replications. The treatments were: root processing: steamed and not steamed; DAP: 0, 1 and 2% of the substrate DM. The fermentation was over 14 days with samples taken for determination of true and crude protein at 0, 3, 7 and 14 days.

The true protein in cassava root increased with a curvilinear trend (R² = 0.99) from 2.4 to 6.51% in DM as the fermentation time increased from zero to 14 days; the ratio of true to crude protein increased from 0.22 to 0.63 over the same period. Increasing the proportion of DAP from zero to 2% of the substrate DM increased the true protein from 5.57 to 7.29% in DM after 14 days of fermentation. Steaming the cassava root prior to fermentation improved slightly (p=0.17) the conversion of crude to true protein.

Key words: aerobic, crude protein, feed resources, pigs, steaming, true protein

Introduction

The major problems of small-holder pig production in upland areas of Lao PDR are high piglet mortality and low growth rates. Almost all pigs are of local breed (Mou Lat), managed in scavenging systems and suffer feed inadequacy in both quality and quantity. According to the survey by Phonepaseuth et al (2010) most piglets in upland areas had a low growth rate (20-50 g/day) and high mortality (30-50%). Weaned pigs required from 5 to 8 months to reach live weights of 20 to 30 kg.

Cassava (Manihot esculenta Crantz) is the third most important crop in Lao PDR and has increased dramatically following the development of industrial production of starch for export. The root is composed almost entirely of carbohydrate and has very low protein content which limits its value as livestock feed. Solid state fermentation of the root is a promising technology as this has the potential to raise the protein content to levels required to balance the carbohydrate thus presenting the opportuitg to make an almost complete feed for monogastric animals such as pigs and poultry (Boonnop et al 2009; Kaewwongsa et al 2011).

In previous research (Manavanh and Preston 2016), we showed that the true protein content in cassava root could be increased to 14% in DM by fermenting it aerobically with yeast, supplemented with urea and di-ammonium phosphate.

The aim of the research described in this paper was to test the effect of different levels of phosphorus derived from di-ammonium phosphate (DAP), with or without prior steaming of the root before the fermentation.

Materials and methods

Location

The experiment was carried out in the Laboratory of the Animal Science Department in the Faculty of Agriculture and Forest Resource in Souphanouvong University. The site is located 7 km from Luang Prabang City, Lao PDR. The mean daily temperature in this area at the time of the experiment was 27 ^oC (range 22-32°C).

Experimental design

The experiment was arranged as a 2*3 factorial in a completely randomized design (CRD) with 4 replications.

The treatments were:

Root processing

Steamed (ST) and not steamed (NST)

DAP:

0, 1 or 2% of root DM

Procedure:

Steaming

Cassava roots were peeled and chopped by hand into small pieces (1-2 cm). One portion was steamed for 30 minutes in a bamboo basket placed above a pan containing boiling water (Photo 1). It was then cooled for 15 minutes prior to being mixed with the yeast, urea and DAP. The other portion of the cassava root was mixed directly with yeast, urea and DAP without prior steaming.

Photo 1. The steaming of the cassava root	Photo 2. Aerobic fermentation of the cassava root.

The steamed cassava root was removed from the bamboo basket allowed to cool for 15 minutes. The steamed and un-steamed cassava root were then mixed with urea, DAP and yeast (Table 1). The proportions of urea were varied according to the level of DAP so that the substrates were iso-nitrogenous. The mixed substrates were then transferred to bamboo baskets covered with plastic netting to allow free entrance of air (Photo 2) and allowed to ferment for 14 days.

Measurements

On days 0, 3, 7 and 14 samples were taken from each treatment/replicate and analyzed for DM, N, ash and true protein. The fresh weight of the substrates in each treatment were weighed at each time interval to determine the relative amounts of substrate DM utilized in the fermentation process.

Chemical analysis

DM, N and ash were analyzed according to AOAC (1990) methods. For estimation of true protein, 2 g of the fresh sample were put in a 125ml Erlenmeyer flask with 50 ml of distilled water, allowed to stand for 30 minute, after which 10ml of 10% TCA (trichloracetic acid ) were added and allowed to stand for a further 20-30 minutes. The suspension was then filtered through Whatman #4 paper by gravity. The filtrate was discarded and the remaining filter paper and suspended substrate transferred to a kjeldahl flask for standard estimation of total N. The measurements of crude and true protein were done on the fresh sample.

Statistical analysis

The data were analysed with the General Linear Model option of the ANOVA program in the MINITAB software (Minitab 2000). Sources of variation were: days, steaming, DAP level, interactions day*DAP, day*steaming and error.

Results and discussion

The level of true protein in the substrate increased with a curvilinear trend (R² = 0.99) from 2.4 to 6.51% in DM as the fermentation time increased from zero to 14 days; the ratio of true protein to crude protein increased from 0.22 to 0.63 over the same period (Table 2;  Figures 1 and 2).

Steaming the cassava root prior to fermentation appeared to have a slightly beneficial effect (p=0.17) on conversion of crude to true protein (Table 2).

Figure 1. Effect of length of fermenttion on concentrations of true and crude protein	Figure 2. Curvilinear response in the true:crude protein ratio with increasing length of fermentation

Increasing the proportion of DAP from zero to 2% of the substrate DM increased the average level of true protein from 4.28 to 5.6% in DM (Table 3), the final values after 14 days of fermentation being increased from 5.57 to 7.29% (Table 4; Figure 3).

Figure 3. Increasing the percentage of DAP in the fermentation substrate increased the true:crude protein ratio

Changes in the mass of substrate during fermentation

About 30% of the original DM in the substrate had been fermented by the end of 14 days, the rate of loss showing a curvilinear trend with time, with the major change taking place in the first 3 days (Table 5; Figures 4 and 5).

Figure 4. Changes in the mass of substrate during the fermentation	Figure 5. Proportion of the original substrate fermented during different stages of the fermentation

Discussion

The increase in the true protein content of the cassava root by fermentation with yeast, urea and DAP is supported by reports from several researchers.  Fermentation of cassava peels by a pure culture of S. cerevisiae increased the protein content from 2.4% to 14.1%, according to Antai and Mbongo (1994). Oboh and Kindahunsi (2005) reported that the fermentation of cassava flour with S. cerevisiae increased the protein level from 4.4% to 10.9% in DM. Krisada et al (2009) carried out a similar fermentation with fresh cassava root using urea and yeast. The crude protein was increased from 3.2 to 21.1% in DM with 90% of the crude protein in the form of true protein. Phiny et al (2012) fermented broken rice in an anaerobic system simulating the “farmer” production of rice wine.  The difference in procedure was the addition of urea (1% of the rice) as well as yeast and no distillation. The crude protein content was raised from 7% in DM in the broken rice to 23% in DM after fermentation for 3 days. The proportion of “crude” to “true” protein was not measured but the 37% increase in pig growth rate when the protein-enriched rice was included in the diet, compared with 16.5% improvement in growth rate, for supplementation with fish meal, indicated that much of the increase in “crude” protein was as “true” protein. Manivanh and Preston (2016) used cassava root as the carbohydrate source, with incorporation of DAP as a source of phosphorus to supplement the yeast and urea. True protein content of the cassava root was raised to 14% in DM (from 2.5% in unfermented root) and growth rate of Moo Laat pigs was increased by 46% compared with the control diet in which the protein was from ensiled Taro  foliage (leaves and petioles).  

Phosphorus is required for the growth of all biological entities, including yeast,  thus the 30% increase in true protein in the fermented cassava pulp by raising the level of DAP (20% phosphorus) from cero to 2% (in DM) was to be expected. There appear to be no comparable studies on effects of phosphorus levels in protein-enrichment of carbohydrate with yeast and urea.

Cost of producing true protein by solid state fermentation of cassva root

The true protein present in the original 300g of substrate DM  was 300*2.38/100 = 7.4g. After 14 days the true protein had increased to 210*7.3/100 = 15.2g. Thus 15.2 - 7.4 = true protein produced at a cost 90 g substrate fermented = a protein yield of 16.5 g from 100 g substrate DM fermented  Assuming the factory delivered price of fresh cassava root is USD 0.05/kg then the cost of 1 kg of protein in PECRwould be approximately USD1.00.

A simpler comparison is by  calculating the cost of 1000 kg of DM of PECR (with 7.5% true protein  in DM) compared with a ration of similar protein content derived from a mixture of casava root and soybean meal (See Table 6). It appears the PECR with 7.5% true protein DM could be of lower cost that a similar feed producing by combiningensiked cassava root  with soybean meal

Conclusions

• The true protein in cassava root increased with a curvilinear trend (R² 0.99) from 2.4 to 6.51% in DM as the fermentation time increased from zero to 14 days, the proportion of true protein in crude protein increasing from 0.22 to 0.63 over the same period.
  
  
• Increasing the proportion of DAP from zero to 2% of the substrate DM increased the true protein from 5.57 to 7.29% after 14 days of fermentation.
  
  
• 30% of the original root DM was fermented in the partial conversion of root carbohydrate to true protein after 14 days of fermentation.
  
  
• Steaming the cassava root prior to fermentation appeared to have a slightly positive effect on conversion of crude to true protein.

Acknowledgments

This research is part of the requirement by the senior author for the degree of PhD at Nong Lam University. The support from the MEKARN II project, financed by Sida, is gratefully acknowledged, as is the help received from the Animal Science Department, Faculty of Agriculture and Forest Resource, Souphanouvong University, Lao PDR.

References

Antai S P and Mbongo P N 1994 Utilization of cassava peels as substrate for crude protein formation. Plant Foods for Human Nutrition, v.46, p.345-351.

AOAC 1990 Official methods of analysis.Association of Official Analytical Chemists, Arlington, Virginia, 15^th edition, 1298 pp.

Boonnop K, Wanapat M, Nontaso N and Wanapat S 2009 Enriching nutritive value of cassava root by yeast fermentation. Scientia Agricola Volume 66 no.5 Piracicaba http://dx.doi.org/10.1590/S0103-90162009000500007

Manivanh N and Preston T R 2016 Replacing taro (Colocasia esculenta) silage with protein-enriched cassava root improved the nutritive value of a banana stem (Musa spp) based diet and supported better growth in local pigs (Moo Laat breed).Livestock Research for Rural Development. Volume 28, Article #97. Retrieved July 14, 2016, from http://www.lrrd.org/lrrd28/5/noup28097.html

Minitab 2000 Minitab Software Release 13.

Oboh G and Akindahunsi A A 2005 Nutritional and toxicological evaluation of Saccharomyces cerevisiae fermented cassava flour. Journal of Food Composition and Analysis, v.18, p.731-738,

Phiny C, Preston T R, Borin K and Thona M 2012: Effect on growth performance of crossbred pigs fed basal diet of cassava root meal and ensiled taro foliage supplemented with protein-enriched rice or fish meal. Livestock Research for Rural Development. Volume 24, Article #65.  http://www.lrrd.org/lrrd24/4/phin24065.htm

Phonepaseuth P, Somchanh K and Phouthone P 2010 Survey on smallholder pig production system in Xieng Ngeun and Pak Ou district, Louangprabang province. Livestock Research Center, National Agriculture and Forestry Research Institute, Vientiane Lao PDR.

Received 24 September 2016; Accepted 2 November 2016; Published 1 December 2016

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