Improving nutritive value of cassava root (Manihot esculenta Crantz) by fermentation with yeast (Saccharomyces cerevisiae), urea and di-ammonium phosphate

Nouphone Manivanh, T R Preston¹, Le Van An² and Tran Thi Thu Hong²

Faculty of Agriculture and Forest Resource Souphanouvong University, LuangPrabang, Lao PDR
noumanivanh@gmail.com
¹ Centro para la Investigación en Sistemas Sostenibles de Producción Agropecuaria (CIPAV), Carrera 25 No 6-62 Cali, Colombia
² Faculty of Animal Science and Veterinary Medicine, Hue University of Agriculture and Forestry, Hue University, 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, pH and ammonia. A completely randomized design (CRD) was used with 2 treatments with four replications. The treatments were anaerobic and aerobic fermentation. The substrate was cassava root 93.6% + di-ammonium phosphate (DAP) 2% + urea 1.4% + yeast 3​% (DM basis). The fermentation was over 7 days with samples taken for determination of true and crude protein, and ammonia, at 0 and 7 days. pH was measured at 0 and 3h after preparing the substrates and every 24h until end of day 7.

The pH decreased with fermentation time, according to an almost linear trend, from 5.8-5.9 immediately after mixing the substrate, to 5.3-5.6 in 3h and to 3.3-3.5 after 7 days. The level of crude protein after mixing the substrate and additives was 10.8% in DM and did not change over the 7 days of fermentation. True protein in the substrate increased from 2.5 to 6.6% in DM as the fermentation time increased from zero to 7 days. There were no differences in all these criteria as between the aerobic and anaerobic condition, other than a tendency for the pH to fall slightly more quickly in the first 4 days in the anaerobic condition followed by a slower rate of fall to reach almost the same final value after 7 days, as for the aerobic condition. It is suggested that the incomplete conversion of urea-N and ammonia-N to yeast protein was because of incomplete hydrolysis of urea to ammonia due to action of urease being inhibited by the fall in pH during the fermentation.

Key words: aerobic, ammonia, anaerobic, pH, true protein

Introduction

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 of 1-3% in DM (Stupak et al 2006) which limits its nutritive value as livestock feed.

Solid state fermentation of the root with urea and di-ammonium phosphate (DAP) is a promising technology as this has the potential to raise the protein content to levels required to balance the carbohydrate, thus presenting the opportunity to make an almost complete feed for monogastric animals such as pigs and poultry (Boonnop et al 2009).

The problem, in the studies reported so far, is that not all the added nitrogenous compounds (urea and DAP) were converted to “true” protein, the levels of which never exceeded some 50 to 70% of the “crude “ protein in experiments with yeast-fermented cassava root (Vanhnasin and Preston 2016a) and cassava root pulp (Sengxayalth et al 2017a). A related problem was that when the protein-enriched cassava root (or cassava pulp) was included in feeding systems for pigs, the growth rate was reduced markedly when more than 30% of the diet crude protein, as replacement for soybean meal, was from the protein-enriched root (Vanhnasin et al 2016b) or pulp (Sengxayalth et al (2017b). In the experiment by Sengxayalth et al (2017b), pig growth rate was close to zero when the protein-enriched pulp replaced 100% of the soybean meal. Similar reductions in growth rate of pigs. When protein-enriched cassava root pulp exceeded 30% of the dietary crude protein, were reported by Hong et al (2017).

In an attempt to solve this problem, Manivanh and Preston (2016) increased the level of DAP to 2% of the substrate, reducing the level of urea to 1.2%. This had the effect of increasing the phosphorus level in the substrate with a related increase in the proportion of the added N as ammonia, replacing urea-N. The linear increase in the proportion of true to crude protein that resulted was attributed to the increased level of phosphorus, but another explanation could have been the partial change in the origin of the added NPN – from urea to ammonia. Yeast cannot directly use urea which must first be hydrolysed to ammonia by urease. However, the activity of urease is inhibited at low pH (Kay and Reid 1934), which falls rapidly when the cassava root is fermented.

It was therefore hypothesized that the limiting factor to the synthesis of true protein from crude protein in the fermentation of cassava root could be the decrease in pH in the fermentation substrate preventing the hydrolysis of urea to ammonia and thus decreasing the availability of nitrogen for growth of the yeast.

The following experiment was set up to test this hypothesis.

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°C (range 22-32°C).

Experimental design

Mixtures (DM basis) of 93.6% cassava root, 3% yeast, 1.4% urea and 2% diammonium phosphate (DAP) were fermented for 7 days under aerobic or anaerobic conditions. There were 4 replications of each of these treatments in a completely randomized design (CRD) with 4 replications.

Procedure

Cassava roots were peeled and chopped into small pieces (1-2 cm) and steamed for 30 minutes in a bamboo basket placed above a pan containing boiling water. It was then cooled for 15 minutes prior to being mixed with the yeast, urea and DAP. One half of the mixed substrate was transferred to bamboo baskets covered with plastic netting to allow free entrance of air and allowed to ferment for 7 days (aerobic condition). The other portion of the substrate was packed tightly into 0.5 liter plastic bags, which were closed (anaerobic condition), and in which it was stored for 7 days.

Measurements

Samples were taken from each treatment/replicate on day 0 (3 h after mixing the components of the substrate), and then every 24h until end of day 7 of the fermentation for measurement of pH, crude protein, true protein and ammonia.

Chemical analysis

The pH of each sample was measured with a digital pH meter, prior to addition of sodium hydroxide for subsequent analysis for ammonia by steam distillation (AOAC 1990). Crude protein was analyzed by kjeldahl digestion with sulphuric acid followed by distillation 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 minutes, after which 10ml of 10% TCA (trichloroacetic 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. Urea-N was estimated by subtraction of true protein-N and ammonia-N from the crude protein-N. The measurements of crude and true protein and ammonia were done on the fresh samples.

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 of fermentation, interaction system*fermentation time and error.

Results and discussion

Chemical composition of substrates

The pH decreased with fermentation time, according to an almost linear trend, from 5.8-5.9 immediately after mixing the substrate, to 5.3-5.6 in 3h and to 3.3-3.5 after 7 days (Table 1; Figure 1). The level of crude protein after mixing the substrate and additives was 10.8% in DM and, as expected, did not change over the 7 days of fermentation. The level of true protein in the substrate increased from 2.5 to 6.6% in DM as the fermentation time increased from zero to 7 days, such that the ratio of true to crude protein increased from 0.23 to 0.63 over the same period (Table 1; Figure 2). There were no differences in all these criteria as between the aerobic and anaerobic condition, other than a tendency for the pH to fall slightly more quickly in the first 4 days in the anaerobic condition followed by a slower rate of fall to reach almost the same final value after 7 days, as for the aerobic condition (Figure 1).

Figure 1. Effect of fermentation time on pH of cassava root fermented with yeast, urea and DAP, under anaerobic and aerobic condition

Figure 2. Effect of fermentation on true and crude protein content of cassava root supplemented with urea, DAP and yeast

The proportion of true protein in the substrate after the 7-day fermentation was doubled from 34 to 62% of the total nitrogen, and appeared to be derived almost equally from the nitrogen present originally as ammonia (from DAP) and from urea (Figure 3). However, urea was not determined directly but was assumed to be the source of the N remaining after accounting for the protein-N and ammonia-N at the end of the fermentation. Two questions to be answered are: i) Why all the ammonia was not used for yeast growth?; and ii) why was the urea not completely hydrolyzed to ammonia? The latter question could perhaps be explained as being the consequence of the rapid fall in the substrate pH inhibiting the action of urease, the action of which is decreased at low pH (Kay and Reid 1934).

Figure 3. Distribution of the nitrogen as urea, ammonia and true protein at the beginning and after 7 days of fermentation

Conclusions

• The pH decreased with fermentation time, according to an almost linear trend, from 5.8-5.9 immediately after mixing the substrate, to 5.3-5.6 in 3h and to 3.3-3.5 after 7 days.
  
  
• The level of crude protein after mixing the substrate and additives was 10.8% in DM and did not change over the 7 days of fermentation.
  
  
• The true protein in the substrate increased from 2.5 to 6.6% in DM as the fermentation time increased from zero to 7 days, such that the ratio of true to crude protein increased from 0.23 to 0.63 over the same period.
  
  
• There were no differences between the aerobic and anaerobic condition, other than a tendency for the pH to fall slightly more quickly in the first 4 days in the anaerobic condition followed by a slower rate of fall to reach almost the same final value after 7 days, as for the aerobic condition.
  
  
• It is suggested that the incomplete conversion of urea-N and ammonia-N to yeast protein is because of incomplete hydrolysis of urea to ammonia due to action of urease being inhibited by the fall in pH during the fermentation.

Acknowledgments

This research is part of the requirement by the senior author for the degree of PhD at HUE 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

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

Hong T T T, Lien P T B, Hai D T, Hang P T and Quan N H 2017 Protein-enriched cassava root pulp as partial replacement for fish meal in diets for growing pigs. Livestock Research for Rural Development. Volume 29, Article #184. Retrieved April 10, 2018, from http://www.lrrd.org/lrrd29/9/hong29184.html

Kay W W and Reid M A H 1934 The optimum buffer p_H for hydrolysis of urea by urease, and the preparation of stable urease powder. Biochemistry Journal 28(5): 1798–1801. William Whittle Kayand Muriel Anne Hislop Reid¹

Manivanh N, Preston T R and Thuy N T 2016 Protein enrichment of cassava (Manihot esculenta Crantz) root by fermentation with yeast, urea and di-ammonium phosphate.Livestock Research for Rural Development. Volume 28, Article #222. Retrieved December 18, 2016, from http://www.lrrd.org/lrrd28/12/noup28222.html

Minitab 2000 Minitab Software Release 13.

Sengxayalth Phoutnapha and Preston T R 2017a Fermentation of cassava pulp with yeast, urea and diammonium phosphate (DAP). Livestock Research for Rural Development. Volume 29, Article #177. http://www.lrrd.org/lrrd29/9/pom29177.html

Sengxayalth Phoutnapha and Preston T R 2017b Effect of protein-enriched cassava pulp on growth and feed conversion in Moo Laat pigs. Livestock Research for Rural Development. Volume 29, Article #178. http://www.lrrd.org/lrrd29/9/pom29178.html

Stupak M, Vanderschuren H, Gruissem W and Zhang P 2006 Biotechnological approaches to cassava protein improvement. Trends in Food Science & Technology, v.17, p.634-641.

Vanhnasin P, Manivanh N and Preston T R 2016a Effect of fermentation system on protein enrichment of cassava (Manihot esculenta) root.Livestock Research for Rural Development. Volume 28, Article #175. Retrieved April 10, 2018, from http://www.lrrd.org/lrrd28/10/vanh28175.html

Vanhnasin P and Preston T R 2016b Protein-enriched cassava (Manihot esculenta Crantz) root as replacement for ensiled taro (Colocasia esculenta) foliage as source of protein for growing Moo Lat pigs fed ensiled cassava root as basal diet. Livestock Research for Rural Development. Volume 28, Article #177. Retrieved April 10, 2018, from http://www.lrrd.org/lrrd28/10/vanh28177.html

Received 28 March 2018; Accepted 22 April 2018; Published 1 May 2018

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