in substrates with length of fermentation
| Livestock Research for Rural Development 31 (5) 2019 | Guide for preparation of papers | LRRD Newsletter | Citation of this paper |
In a solid-state fermentation of a mixture of cassava pulp-maize grain (70:30), with urea (2%) and diammonium phosphate (1.5%), the conversion of crude to true protein was better when the inoculum was Bacillus subtilis rather than Aspergillus niger and when the fermentation was for 14 rather than 21 days. The rate of conversion of substrate to true protein was 7.2 g DM per 1 g of true protein over the 14-day fermentation.
When the basal diet of growing pigs was replaced with protein-enriched cassava pulp responses in live weight gain and feed conversion were curvilinear with no improvements (live weight gain) and slight improvement (feed conversion) as the enriched pulp was increased to 25% of the diet DM, followed by deterioration in response with higher levels of enriched pulp.
We suggest that promoting studies on high biomass-producing plants such as Colocasia esculenta (L) Schott is a more appropriate way forward to produce high quality protein for animal feeding than solid state fermentation of carbohydrate that can best be used directly by the animals.
Key words: Aspergillus niger, Bacillus siubtilis, digestibility, greenhouse gas, nitrogen retention, true-protein
Cassava plays an important socio-economic role as a secondary crop in Viet Nam. The cropping area of 570.000 ha produces some10 million tonnes of tubers that after processing for starch yield byproducts - cassava pulp and cassava root peel – accounting for about 45% of the original weight of tubers. Solid-state fermentation of these byproducts with fungi, yeast, and bacteria has been shown to improve the true protein content (Hong and Ca 2013; Manivanh et al 2016; Vanhnasin and Preston 2016a; Sengxayalth et al 2017a). However, the overall conversion rate of substrate to true protein has not been reported and attempts to produce a balanced diet for pigs by this process have not been successful (Vanhnasin and Preston 2016b; Sengxayalth et al 2017b; Manivanh et al 2018b; Hong et al 2917).
The objective of this study was to develop a feeding system for growing pigs by fermenting a mixture of 70% cassava pulp and 30% maize with two micro-organisms - Aspergillus niger, Bacillus subtilis - in combination or separately, with supplementary nitrogen and phosphorus from urea and diammonium phosphate.
Two experiments were carried out in Hue University in Viet Nam from October 2017 to March 2018.
Aspergillus niger and Bacillus subtilis were used to ferment a mixture of cassava pulp and maize grain (70:30 DM basis), with 2% urea and 1.5% diammonium phosphate (DAP), with 3 replications of each organism employed separately or in combination. The fermentation was for 21 days with measurements taken at 0, 3, 7, 14 and 21 days. The inoculum of microorganisms was prepared by the procedure described by Hang and Quynh Chau (2018).
The DAP, urea and carbohydrate substrate were added to the suspensions of the microorganisms 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. After each period of fermentation, measurements were made of crude and true protein according to AOAC (2016). The data were analysed as a 3*6 factorial using the GLM program in the Minitab (2016) software.
For individual and combined micro-organisms there were no advantages in extending the fermentation beyond 14 days (Table 1). Fermentation with B. subtilis supported a higher rate of conversion of crude to true protein than A. niger with intermediate results from the combination of the two micro-organisms.
|
Table 1.
Effect of source of fermenting micro-organism and duration
of fermentation on true |
|||||
|
TP |
CP |
TP/CP |
DM |
CF |
|
|
A.niger |
8.95b |
13.5b |
65.8b |
75.4a |
26.2a |
|
A+B |
10.a |
14.4a |
71.87a |
76.8a |
23.4c |
|
B.sub |
11.7a |
14.3a |
74.56a |
77.1a |
25.1b |
|
SEM |
0.13 |
0.09 |
1.123 |
0.697 |
0.089 |
|
p |
<0.001 |
<0.001 |
<0.001 |
0.22 |
<0.001 |
|
Fermentation, days |
|||||
|
0 |
7.33d |
12.87d |
56.98d |
86.4a |
26.7a |
|
3 |
9.35c |
13.52c |
68.98c |
82.6b |
26.0a |
|
7 |
10.5b |
14.21b |
73.75b |
74.90c |
24.8b |
|
14 |
11.7a |
14.81a |
79.15a |
72.2c |
23.5c |
|
21 |
11.2ab |
14.92a |
74.9b |
65.9d |
21.8d |
|
SEM |
0.11 |
0.111 |
1.45 |
0.627 |
0.116 |
|
p |
<0.001 |
<0.001 |
<0.001 |
<0.001 |
<0.001 |
|
ab Means within main treatments without common superscript differ at p<0.05 |
|||||
|
Table 2.
Changes in the composition of the substrates during 21
days |
|||||
|
Days |
DM, g |
TP, g |
CP, g |
TP/CP |
CF |
|
0 |
86.4 |
6.33 |
11.1 |
57.0 |
23.1 |
|
3 |
82.6 |
7.72 |
11.2 |
69.2 |
21.5 |
|
7 |
74.9 |
7.86 |
10.6 |
73.9 |
18.6 |
|
14 |
72.2 |
8.45 |
10.7 |
79.0 |
17.0 |
|
21 |
65.9 |
7.38 |
9.83 |
75.1 |
14.4 |
After 14 days of fermentation, 14.2 g of substrate had been used to produce an additional 2.12 g of true protein (Table 3; Figure 1), a conversion of 6.7 g substrate per 1g of true protein. It would appear that the fermenting organisms used almost equally the fiber and the non-fiber components in the substrate for protein synthesis.
|
Table 3.
Balance of DM, crude protein and true protein before |
|||
|
DM, g |
CP, g |
TP, g |
|
|
Begin |
86.4 |
11.1 |
6.33 |
|
After 14d |
72.2 |
10.7 |
8.45 |
|
Gain/loss |
-14.2 |
-0.4 |
2.12 |
|
| Figure 1. Changes in concentration of dry matter and true protein in substrates with length of fermentation |
The negative feature of the fermentation is that the other end-product will have been carbon dioxide, resulting in an overall negative carbon footprint for the process.
The combination of Aspergillus niger and Bacillus subtilis with14 days of fermentation was chosen to produce the protein-enriched feed for the pig growth experiment (Table 4).
|
Table 4.
Ingredients in the fermentation of |
||
|
Cassava pulp |
75 |
|
|
Maize |
11.5 |
|
|
Rice bran |
10 |
|
|
Mixture B. subtilis and A. niger |
5 |
|
|
DAP |
1.5 |
|
|
Urea |
2 |
|
The fermentation was done daily in quantities sufficient to ensure the availability of the protein-enriched feed throughout the feeding trial.
The four treatments in a random block design with 4 replicates were:
PEC0: Control diet with no protein-enriched supplement
PEC12.5: The control diet with 12.5% of PEC (DM basis)
PEC25: The control diet with 25% of PEC
PEC50: The control diet with 50% pf PEC
Sixteen castrated male (Mong Cai x Large White) pigs (24.5± 0.5 kg) were housed individually in metabolism cages. After 45d (pigs were 54±0.6 kg), feces and urine were collected over 5 days for measurement of digestibility and N retention. The growth experiment was continued to 80 days. Feed offered and refused and liveweights were recorded.
|
Table 5.
Chemical composition of the ingredients of
feeds |
|||
|
Feed |
DM |
CP |
CF |
|
Rice bran |
86.0 |
11.0 |
5.96 |
|
Maize |
87.0 |
8.5 |
2.6 |
|
Fish meal |
87.0 |
45.0 |
3.2 |
|
PEC |
39.0 |
13.6 |
17.6 |
|
PEC : Protein-enriched cassava pulp/maize/rice bran |
|||
|
Table 6. Composition of experimental diets (% in DM) |
||||
|
PEC0 |
PEC12.5 |
PEC25 |
PEC50 |
|
|
Maize |
39.6 |
35.2 |
28.7 |
18.6 |
|
Rice bran |
39.9 |
34.8 |
29.8 |
17.9 |
|
Fish meal |
20.0 |
17.0 |
16.0 |
13.0 |
|
PEC |
0.0 |
12.5 |
25 |
50 |
|
Premix Min-Vit |
0.5 |
0.5 |
0.5 |
0.5 |
|
Compositión, % in DM |
||||
|
CP |
16.4 |
16.1 |
16.1 |
16.0 |
|
TP# |
16.4 |
15.8 |
15.6 |
15.0 |
|
CF |
4.8 |
6.2 |
7.3 |
9.1 |
|
# Based on 80% of protein in PEC is true protein (experiment 1) |
||||
DM, crude and true protein and crude fiber were determined following AOAC (1997) procedures.
The data were analyzed according to the GLM option in the ANOVA program of the Minitab (2016) software. Sources of variation were diets and error.
Responses in live weight gain and feed conversion to inclusion of the protein-enriched feed were curvilinear with no improvement (LW gain) or slight improvement (feed conversion ) as the protein-enriched feed provided up to 25% of the diet DM followed by deterioration in response, for both criteria, with 50% replacement by PEC (Table 7: Figures 1 and 2).
|
Table 7.
Mean values for feed intake, live weight gain and feed
conversion in pigs fed |
||||||
|
PEC0 |
PEC12.5 |
PEC25 |
PEC50 |
SEM |
p |
|
|
Live weight, kg |
||||||
|
Initial |
24.8 |
24.1 |
24.6 |
24.1 |
0.486 |
0.63 |
|
Final |
78.6a |
79.1a |
79.2a |
72.9b |
0.011 |
0.01 |
|
Daily gain |
0.67a |
0.6a |
0.6a |
0.61b |
0.08 |
0.02 |
|
DM intake, kg/d |
1.74ab |
1.72b |
1.69b |
1.79a |
0.33 |
0.04 |
|
FCR# |
2.6b |
2.50b |
2.47b |
2.95a |
0.05 |
0.001 |
|
#DM intake /LW gain ab Means without common superscript differ at p<0.05 |
||||||
 |
 |
| Figure 2. Curvilinear response in growth rate of pigs fed protein- enriched cassava pulp replacing up to 50% of the basal diet |
Figure 3. Curvilinear response in feed conversion of pigs fed protein- enriched cassava pulp replacing up to 50% of the basal diet |
Apparent digestibility of organic matter and crude protein did not differ among diets, but that for crude fiber showed a linear decrease as the protein-enriched feed in the diet was increased (Table 8).
|
Table 8.
Mean values for DM intake and digestibility coefficients in
pigs fed increasing |
||||||
|
PEC0 |
PEC12.5 |
PEC25 |
PEC50 |
SEM |
p |
|
|
DMI, kg/d |
1.63 |
1.5 |
1.74 |
1.7 |
0.08 |
0.172 |
|
Digestibility, % |
||||||
|
CP |
67.2 |
74 |
71 |
68 |
2.37 |
0.158 |
|
OM |
64 |
63 |
64 |
66 |
2.39 |
0.82 |
|
CF |
28a |
26a |
23b |
19c |
2.15 |
0.01 |
|
ab Means without common superscript differ at p<0.05 |
||||||
N retention, expressed on daily basis, or as percent of N digested, decreased linearly as the proportion of protein-enriched feed in the diet was increased (Table 9; Figures 4 and 5).
|
Table 9.
Mean values for N-balance in pigs fed creasing levels of
protein-enriched cassava |
||||||
|
PEC0 |
PEC12.5 |
PEC25 |
PEC50 |
SEM |
p |
|
|
N balance, g/d |
||||||
|
Intake |
43.2 |
36.8 |
38.4 |
38.4 |
1.76 |
0.242 |
|
Feces |
14.6 |
10.1 |
11.2 |
12 |
1.28 |
0.098 |
|
Urine |
5.92 |
6.88 |
7.68 |
8.48 |
0.8 |
0.07 |
|
Retention |
22.3a |
20.5ab |
19.7ab |
17.6b |
0.956 |
0.004 |
|
Ret%digN |
77.9a |
76.7ab |
72.4ab |
66.7b |
1.69 |
0.001 |
|
ab Means without common superscript differ at p<0.05 |
||||||
 |
 |
| Figure 4. Linear decrease in N retention in pigs fed protein-enriched cassava pulp replacing up to 50% of the basal diet |
Figure 5. Linear decrease in N retention as percent of N digested in pigs fed protein-enriched cassava pulp replacing up to 50% of the basal diet |
Developing alternatives to imported soybean meal for the pig industry is a laudable activity in tropical countries. The ready availability of carbohydrate-rich by-products from cassava roots processed for starch production has been the incentive to develop solid-state fermentation systems to promote growth and reproduction of micro-organisms such as yeast and the fungal strains studied in the present paper than can use ammonia-nitrogen for this purpose. The results of the experiments described in this paper, together with those from many other researchers, prove that the process is feasible. The problem has been to devise a feeding system in which the “protein-enriched” feeds, developed with this process, can satisfactorily replace conventional protein sources such as fish and soybean meals.
The results from the feeding trial described in Experiment 2 confirm those from other sources -- that protein-enriched feeds produced by solid-state fermentation can replace no more than 20-30% of the dietary protein supply, beyond which performance deteriorates to a point that is not economical. In most cases the decline in performance is caused by a reduction in feed intake. As most observations indicate that the maximum degree of conversion-of added NPN sources to true protein is of the order of 70-80%, the obvious conclusion is that the residual NPN is the factor leading to reduced intake and therefore growth rate. This point has not been adequately studied. Manivanh et al (2018a) found no residual ammonia after yeast-fermentation of cassava root meal. They 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.
The other issue raised by the present research is the efficiency of conversion of substrate carbohydrate to microbial protein. The results from experiment 1 indicate that it requires about 7 g of carbohydrate to produce 1 g of microbial protein using the mixed fungal strains of B. subtilis and A. niger. Manivanh et al (2016) reported a similar ratio using yeast as the fermentative organism. The related issue is that the byproduct of the fermentation process is carbon dioxide the presence of which in the atmosphere causes global warming. This leads to the question of the choice of system to produce the required protein. The alternative way is by growth of protein-rich plants which absorb atmospheric carbon dioxide as their source of energy and carbon-rich structure, as opposed to producing it as a byproduct of metabolism as in the case of protein produced by microbial growth. We suggest that promoting studies on high biomass-producing plants such as Colocasia esculenta (L) Schott (Hang et al 2018) is a more appropriate way forward to produce high quality protein for animal feeding than solid state fermentation of carbohydrate that can best be used directly by the animals.
This research was supported by the Mekong Basin Animal Research Network (MEKARN II) financed by Sida.
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Received 1 January 2019; Accepted 7 April 2019; Published 1 May 2019