Effect of diammonium phosphate (DAP) on conversion of crude to true protein content in a solid-state fermentation of mixed cassava pulp and maize grain

Du Thanh Hang and Ho Le Quynh Chau

Faculty of Animal Science and Veterinary Medicine, Hue University, Vietnam
duthanhhang@huaf.edu.vn

Abstract

In a solid-state fermentation over 21 days of a mixture of cassava pulp and maize grain (70:30) , supplemented with urea (2%) and DAP (0, 0.5, 1.0, 1.5 and 2%), the conversion of crude to true protein was better when the inoculum was Bacillus subtilis rather than Aspergillus nigeror a combination of the two micro-organisms.

Increasing the level of DAP to 1.5% of the substrate increased the conversion of crude to true protein, but there were no benefits from the higher level of 2%. Increasing the length of the fermentation time resulted in a curvilinear response in the conversion of crude to true protein, which reached a maximum after 14 days, and then declined. Some 24% of the crude protein was not accounted for as true protein at the end of the fermentation.

Key words: Aspergillus niger, Bacillus subtilis, fiber, non-protein-nitrogen

Introduction

Vietnam currently has 560.000 ha of cassava, with the total production of 9.7 million tonnes of tubers. There are more than 100 factories for processing cassava roots to extract starch. The byproducts - cassava pulp and cassava root peel - account for about 45% of the original cassava tubers. The problem is that much of the cassava byproducts from factories are non-marketable and cause environmental pollution.

Cassava pulp has a high potential as an energy source for livestock as it contains appreciable amounts of starch and other non-fibrous carbohydrates. 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 (Okpako et al 2008; Aderemi et al 2007; Tran Thi Thu Hong and Nguyen Van Ca 2013). Additional phosphate results in increased biomass growth of yeast and bacteria (Papagianni et al 1999).

The objective of this study was to determine the optimal levels of a combined source of phosphorus and ammonia [DAP (NH₄)₂ HPO₄] when added to a mixture of cassava root pulp, maize grain and urea fermented with either Aspergillus niger, Bacillus subtilis or a mixture of the two micro-organisms.

Materials and methods

Location and time

The experiment was carried out in the Analysis Center of Hue University, Viet Nam from Jun to September of 2017

Treatments and design

In a solid-state fermentation of a mixture of dried cassava pulp and maize grain (70:30 DM basis) with 2% urea, the treatments in a 5*3*5 factorial random block design with 4 replicates were 5 levels of DAP (0, 0.5, 1.0, 1,5 and 2.0%), 3 sources of inoculum (Aspergillus niger, Bacillus subtilis and a combination of both) and 5 periods of fermentation (0, 3, 7, 14 and 21 days).

Materials

Cassava pulp in dried meal form was collected from the cassava starch processing factory in Phong Dien district, Thua Thien Hue province. DAP was bought from the Medical Equipment Store in Hue city.Aspergillus niger (A.niger) and Bacillus subtillis ( B.sub.) were obtained from Lan Oanh Scientific Instrument Company, Ltd in Ho Chi Minh city.

Microorganisms and inoculum preparation

A. niger was cultured on yeast extract, peptone dextrose and glucose at 37^oC in bottles placed on a reciprocal shaker operated at 150 rpm for 48 h, after which it was stored at 4^oC. This suspension, containing approximately 10⁹CFU/g B.sub was transferred to a solution containing 5g peptone, 5g NaCl and 2g yeast extract peptone dextrose. The suspension was incubated at room temperature, with shaking at 150 rpm during 48h after which it contained approximately 1.1*10 ⁴/g,

Fermentation procedure

The DAP, urea and carbohydrate substrate were added to the suspensions of the microorganisms (Table 1) and placed in trays at a depth of approximately 5 cm for fermentation at room temperature during periods of 0, 3, 7, 14 and 21 days (total number of trays was 3*5*3*4=180).

Chemical analysis

DM, crude protein and true protein (following treatment of the samples with trichloroacetic acid to precipitate the protein) and crude fiber were determined on samples taken from each of the substrates following AOAC (1997) procedures.

Statistical analysis

The data were analyzed by the GLM option in the ANOVA program of the Minitab (2016) software. Sources of variation were levels of DAP, source of inoculum, periods of fermentation, interaction DAP level * fermentation period and error. Polynomial regressions were fitted to the data relating crude and true protein percentages, ratio of true to crude protein and concentrations of DM and crude fiber with period of fermentation. For this analysis the data were restricted to the DAP level of 1.5% in substrate as.

Results

The conversion of crude to true protein was better for B. sub. than for A. niger or the combination of the two micro-organisms (Table 2). As was to be expected, increasing the level of DAP led to an increase in the crude protein content of the substrate (Figure 1), but this was not reflected in the rate of conversion of crude to true protein (Figure 2), except when the DAP level was 1.5% in DM.

Figure 1. Effect of level of DAP on concentration of crude (red shading) and true (blue shading) protein after 21 days of fermentation	Figure 2. Effect of level of DAP on proportion of crude protein converted to true protein after 21 days fermentation

Figure 3. Effect of level of DAP on content of DM (blue symbols) and crude fiber (red symbols) in the substrate after 21 days of fermentation

The content of crude and true protein in the substrate DM, and the ratio of true to crude protein, increased with curvilinear trends as the fermentation was extended to 21 days. The level of true protein, and ratio of true to crude protein, reached peak values at 14 days followed by a decline at 21 days (Table 2; Figures 3 and 4). The DM in the substrate and the crude fiber in the DM both decreased linearly with length of fermentation (Figure 5).

Figure 4. Effect of fermentation time on concentration of crude and true protein with 1.5% DAP in the substrate DM	Figure 5. Effect of fermentation time on the proportion of true to crude protein with 1.5% DAP in the substrate DM

Figure 6. Effect of fermentation time on crude fiber and DM content of the substrate with DAP content of 1.5% in DM

Discussion

The increase in crude protein content of the substrate as the fermentation proceeded was probably a consequence of the loss of substrate DM as the carbohydrate was metabolized by the microorganisms for their own growth with accompanied loss of carbon dioxide. The reduction in the crude fiber content of the DM as the fermentation proceeded confirms the known capacity of both Aspergillus niger and Bacillus subtillis to use fiber as an energy source (Wizna 2009; Jahromi 2010). Manavanh et al (2017) reported that 25% of substrate DM was metabolized when cassava root was fermented with yeast (Saccharomyces cerevisiae), urea and DAP.

The apparent benefit from providing 1.5% of the diet DM as DAP was probably because of the additional phosphorus provided by this supplement. Although both nitrogen and phosphorus are confounded in the DAP, support for the positive role of the phosphorus can be found in the results of a similar experiment (Malavanh and Preston 2016) in which cassava root was fermented with isonitrogenous levels of nitrogen from urea DAP. In this case, the conversion of crude to true protein was linearly related to the level of DAP in the range 0 to 2% in diet DM. Benefits from supplementary phosphorus in solid-state fermentations with A. niger were reported by Papagianni et (1999).

The curvilinear response in the conversion of crude to true protein, which reached a maximum after 14 days fermentation, and then declined, implies that some factor other than ammonia was limiting microbial growth. Some 24% of the crude protein was not accounted for as true protein at the end of the fermentation, a finding corroborated by several researchers who have studied solid-state fermentation of cassava root and cassava root pulp with yeast, urea and DAP (Hong et al 2017; Manivanh and Preston 2016; Sengxayalth Phoutnapha and Preston 2017a; Vanhnasin et al 2016). The nature of this residual non-protein-nitrogen fraction has not been identified.

Manavanh et al (2018) reported that only a small fraction (about 5%) was in the form of ammonia and hypothesized that the remainder was probably in the form of undissociated urea due to the reduction in pH (less than 4 after 7 days fermentation) inhibiting the action of urease. Whatever may be the nature of this non-protein fraction, it would appear to be non-nutritional, and even sub-toxic, at least to monogastric animals, as several studies reported major decreases in growth rate of pigs when the proportion of yeast-fermented cassava root or root pulp exceeded some 30% of the diet DM (Vanhnasin et al 2016; Hong et al 2017; Manivanh and Preston 2016; Sengxayalth Phoutnapha and Preston 2017b).

Identification of the exact nature of the residual non-protein-nitrogen fraction in urea/DAP fermented carbohydrate is the logical next step in this area of research.

Conclusions

In a solid-state fermentation of cassava pulp and maize grain, supplemented with urea and DAP, the conversion of crude to true protein was better forBacillus subtilis than for Aspergillus nigeror for the combination of the two micro-organisms.

Increasing the level of DAP to 1.5% of the substrate increased the conversion of crude to true protein, but there were no benefits from the higher level of 2%.

Increasing the fermentation time resulted in a curvilinear response in the conversion of crude to true protein, which reached a maximum after 14 days fermentation, and then declined. Some 24% of the crude protein was not accounted for as true protein at the end of the fermentation.

Acknowledgments

This research was supported by the Mekong Basin Animal Research Network (MEKARN II) financed by Sida.

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Received 8 May 2018; Accepted 10 June 2018; Published 3 July 2018

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