Partial protein replacement with processed Bambara ... · PARTIAL PROTEIN REPLACEMENT WITH...

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FUNAI Journal of Science and Technology

3 (2), 2017, 1-13

PARTIAL PROTEIN REPLACEMENT WITH PROCESSED BAMBARA GROUNDNUT

MEAL: EFFECT ON SELECTED TISSUE ENZYME ACTIVITIES IN BROILER

CHICKENS (STARTER PHASE)

Ogunbode, S.M.

1, Raji, A.O.

1, Usman, M.D.

1, Sulyman, F.O.

1, Salau, A.K

1., Agboola, A.F.

2,

Olaogun, O.O.3, and Majolagbe, O.H.

2

1Department of Chemical Sciences, Biochemistry and Nutrition Unit, Fountain University,

Osogbo, Nigeria 2Department of Animal Science, University of Ibadan, Ibadan, Nigeria

3Premier Feedmills, Ibadan.

(Received 4 March, 2017; Revised 9 July, 2017; Accepted:12 July, 2017)

Abstract

This study was conducted to investigate the effect of partial replacement (20% inclusion) of soya

bean meal with Bambara groundnut meal (using different processing methods) on vital tissue

enzymes activities of broiler chicks. Eighty four 2 weeks unsexed commercial Arbor Acre broiler

chicks were placed on seven experimental dietary treatment (iso-energetic and iso-nitrogenous)

consisting of three replicates of four birds per replicate. Soya bean meal based diet without

Bambara groundnut seed served as the control diet (T1). The various processing methods

explored included raw Bambara groundnut seed meal based diet (RBG T2) boiled Bambara

groundnut (BBG-T3), soaked Bambara groundnut (SBG-T4), fermented Bambara groundnut

without decantation (FBG (WO)-T5), fermented Bambara groundnut with decantation (FBG (W) –

T6) and roasted Bambara groundnut (ROBG –T7). Weight gain was monitored as an index of

performance study. The various enzymes studied included aspartate and alanine transaminases

(AST and ALT), gamma glutamate transferase (GGT), alkaline phosphatase (ALP) and total

protein. Inclusion of the experimental meals at 20% had no statistical significant difference

(p>0.05) on the various tissues studied compared with the control. The study thus concluded that

replacement of Bambara groundnut meal at 20% against soya bean meal had no deleterious

effect on performance and vital tissues of broiler chickens at the starter phase.

Keywords: Bambara groundnut meal, enzyme study, weight gain, fermentation.

Partial protein replacement with processed Bambara... Ogunbode, et. al.,

FUNAI Journal of Science and Technology, 3(2), 2017

1. Introduction

Human populace is growing at a geometric

progression; whereas the resources used in

satisfying the populace is growing at an

arithmetic progression especially in the

developing countries (Frauke Voosholz,

2014). One of such resources is the demand

for quality protein that is very vital for growth

and repair of worn out tissues. Animal protein

such as poultry is a complete protein because

it contains virtually all the essential amino

acids needed for survival. Unfortunately, the

cost of getting these animal products is

exorbitant as a result of high cost of

production, especially feeding cost, due to

heavy competition for leguminous grains

between man and animal. This major problem

has necessitated the search for alternative,

cheap, non-conventional ingredients with less

competetion. One of such legumes is Bambara

groundnuts, a protein-rich crop (Obizoba,

1998), which is cheap and readily available.

Legumes are important sources of proteins,

lipids, minerals and vitamins required for the

proper growth of chicks (Balogun and Fetuga,

1986). However, their use is limited by the

presence of antinutritional factors (Nwokolo

and Sim, 1987). The presence of

antinutritional factors such as trypsin and

chymotrypsin inhibitors in legumes lowers

digestibility of legume protein (Elegbede,

1998). It is known however that processing

techniques like soaking, toasting, boiling and

fermentation can improve nutritional quality

and bioavailability of nutrients present in

legumes such as Bambara groundnut. The

various processing techniques are effective

ways of achieving desirable changes,

especially in poultry production, removal of

undesirable components and effective

utilization of the full potential of legumes as

feed stuff (Gloria et al., 1995).

The present study therefore aimed at studying

the effect of partial replacement of soya bean

meal with Bambara groundnut meal in broiler

chicken diets and determining its effect on

selected biochemical parameters of broiler

chickens.

2.0 Materials and methods

Chemicals and reagents

Assay kits for alkaline phosphatase (ALP),

aspartate aminotransferase (AST), alanine

aminotransferase (ALT) and gamma-

glutamyltransferase (GGT) were products of

Randox Laboratories, County Antrim, UK. All

other reagents were of analytical grade and

prepared with distilled water.

2.1 Sample collection

The experiment was conducted at the pullet

section of Biochemistry and Nutrition



Laboratory, Department of Chemical

Sciences, Fountain University, Osogbo,

Nigeria. The Bambara groundnut seed was

purchased from Wadata market, Makurdi,

Nigeria.

2.2. Sample preparation

Five different processing methods were

carried out on the seed.

2.3. Boiling: This was carried out by pouring

1 kg of the Bambara groundnut in 8 litres of

distilled water at 1000C for 60 minutes until it

was well cooked.

2.4. Soaking: This was done by soaking 1 kg

of the seeds in 8 litres of distilled water for 12

hours.

2.5. Fermentation without decantation: This

was done by soaking 1 kg of the seeds in 8

litres of distilled water in an air-tight container

for 60 hours without decantation.

2.6. Fermentation with decantation: This

was done by soaking 1 kg of the seeds in 8

litres of distilled water in an air-tight container

for 12 hours. This was thereafter decanted and

replaced with another 8 litres of distilled water

for another 48 hours.

The water from the aforementioned processing

methods were sieved out and the nuts were

sun dried for 4 days before milling

(Omoikhoje, 2008).

2.7. Roasting: Roasting of the Bambara

groundnut was done by heating 1 kg of the

seeds in a frying pan containing fine sand with

continous stirring until brownish. Excessive

burning was avoided so as not to completely

destroy the essential amino acids. The toasted

nuts were then milled (Oloyede et al., 2010).

2.8. Raw Bambara groundnut: The seeds

were rinsed with tap water, sun-dried and

milled into powdery form.

Proximate analyses of the processed seeds

were carried out and the obtained results

served as a guide into formulating diets used

to feed the birds as shown in the gross

composition of the experimental diets used for

the study (Table 1).

2.9 Management of experimental birds

Eighty-four 1-day old broiler chicks (Arbor

Acre strain, RTO Farms, Osogbo Nigeria) of

both sexes were randomly distributed into

seven dietary treatments of three replicates.

The treatment groups were fed as shown in

Table 1. The cages were cleaned and

disinfected prior to the arrival of the birds.

The birds were brooded for two weeks with

feed and water ad libitum. The individual

weights of the birds were taken prior to the

commencement of the experiment and

repeated weekly to allow for calculation of

weight gain. The chicks were vaccinated

against Newcastle disease virus (lasota

vaccine) on day 8 when the maternal antibody

had subsided (Ogunbode et al., 2014), and



Table 1: Gross composition (g/100gDM) of experimental diets

SBM = Soya Bean Meal, GNC = Groundnut Cake, BBG = Boiled Bambara groundnut, SBG = Soaked Bambara

groundnut, FBG(wo)= Fermentation without decantation, FBG(w) = Fermentation with decantation, ROBG = Roasted

Bambara Groundnut, *Premix supplied the following information kg of diet: Vitamin A (12,500,000 I. U), vitamin D3

(2,500,000 I. U), vitamin E (40,000mg) vitamin K3 (2,000 mg), vitamin B1 (3,000 mg), vitamin B2 (5,500 mg), Naicin

(55,000 mg), calcium panthothenate (11,500 mg) vitamin B6 (5000 mg) vitamin B12 (25 mg), choline chloride

(500,000 mg), folic acid (1,000 mg), biotin (80 mg), Mn (120,000 mg), Fe (100,000 mg), Zn (80,000 mg), Cu (8,500

mg), I (1,500 mg), Co (300 mg), Se (120 mg)

against Infectious Bursal Disease (IBD) virus

on day 10 via drinking water. The experiment

lasted for two weeks.

2.10. Blood collection and preparation of

tissue homogenates

At the end of the feeding trial, the broilers

were starved overnight so as to empty the

crop. The birds were weighed, sacrificed and

blood samples were collected via jugular vein.

Serum was separated by centrifugation for

five minutes at 3000 revolutions per minute.

The serum was collected using Pasteur pipette

and kept frozen until needed. The de-feathered

broilers were dissected and the liver and

kidneys were then removed, cleaned with

tissue paper and weighed. The organs were

homogenized in ice-cold 0.25 M sucrose

solution (1:4 w/v) as described by Akanji and

Ngaha (1989).

2.11. Measurement of enzyme activities

Protein concentration was determined by

Biuret method as described by Gornall et al.

(1949). The activities of enzymes were also

determined by standard procedures as

described for ALP, Wright et al. (1972);

aminotransferases, Reitman and Frankel

(1957); and GGT, Rosalki and Tarlow (1974).

Ingredients Control Raw BBG

FBG (wo)

FBG (w) ROBG SBG

Maize 60 58.6 58.6 58.6 58.6 58.6 58.6

Bambara 0 5 5 5 5 5 5

SBM 24.41 19.41 19.41 19.41 19.41 19.41 19.41

Fish meal 70% 1 3 3 3 3 3 3

GNC 5 5 5 5 5 5 5

Wheat offal 4 4 4 4 4 4 4

Oil 1.8 1.2 1.2 1.2 1.2 1.2 1.2

Limestone 1.5 1.5 1.5 1.5 1.5 1.5 1.5

Bone meal 1.27 1.27 1.27 1.27 1.27 1.27 1.27

Methionine 0.15 0.15 0.15 0.15 0.15 0.15 0.15

Lysine 0.23 0.23 0.23 0.23 0.23 0.23 0.23

Salt 0.39 0.39 0.39 0.39 0.39 0.39 0.39

Premix 0.25 0.25 0.25 0.25 0.25 0.25 0.25

Total 100 100 100 100 100 100 100



2.12. Statistical analysis

All experimental values were represented as

means ± SEM (n = 3). Analysis of variance

(ANOVA) was used to test for differences

between means followed by Duncan’s and

Tukey-Kramer’s tests for significant

differences (P<0.05) between the variables

used in this study.

3. Results

Bambara groundnut seed meal was used to

replace soybean meal at 20 % inclusion level

across all processing methods used for this

study. Table 2 shows the calculated nutrients

composition of the various diets used for the

study. The energy level was between 3002 and

3009 kcal/g while the crude protein inclusion

level was between 19.96 and 20.28%. Table 3

shows the proximate composition (g/100

gDM) of the Bambara groundnut seeds

processed using different methods, the crude

protein value ranged from 18.2% in raw seed

to 24.54% in fermented seeds without

decantation. The crude protein value was

within the range obtained in literature,

Oloyede et al. (2007). Table 4 shows the

proximate composition (g/100 gDM) of the

formulated diets using the various processed

seeds.

The crude protein value ranged from 19.86 to

20.30%, while the crude fibre was within the

range of 7.73 and 10.06 %. Chart 1 shows the

daily weight gain of broiler chickens fed with

the different experimental diets. Statistically,

there were no significant differences (P<0.05)

between the various diets used. Chart 2 shows

the specific activities of alkaline phosphatase

(ALP) in the serum, liver and kidney of broiler

chickens fed with the various processed

experimental diets. The results reveal no

significant (P>0.05) changes in the enzyme

activities in the serum and organs when

compared with the control diet. Chart 3 and 4

showed the specific activities of alanine and

aspartate aminotransferases in the liver and

serum of broiler chickens fed with

experimental diets. The results revealed no

significant (P>0.05) differences in the enzyme

activities in the serum and liver when

compared with the control diet. Chart 5 shows

the specific activities of gamma glutamyl

transferase in the liver and serum of broiler

chickens fed with the various experimental

diets. The results revealed no significant

(P>0.05) changes in the enzyme activities in

the serum and liver when compared with the

control diet.



Table 2: Calculated Nutrient Composition of Experimental Diets (%)

Control

BBG

SBG

FBG(wo)

FBG(w)

ROBG

RBG

Energy (kcal/g) 3002 3002 3004 3004 3005 3004 3009

C.P 20.03 20.19 20.19 20.1 20.28 20.11 19.96

Methionine 0.48 0.54 0.54 0.54 0.54 0.54 0.54

Methionine +

Cystine 0.81 0.78 0.78 0.78 0.78 0.78 0.78

Lysine 1.11 1.41 1.41 1.41 1.41 1.41 1.41

Calcium 0.95 1.12 1.12 1.12 1.12 1.12 1.12

Phosphorus 0.38 0.43 0.43 0.43 0.43 0.43 0.43

Sodium 0.18 0.2 0.2 0.2 0.2 0.2 0.2

Linoleic acid 2.13 1.85 1.85 1.85 1.85 1.85 1.85

Crude fibre 3.49 3.67 3.62 3.62 3.62 3.6 3.63

Chloride 0.22 0.23 0.23 0.23 0.23 0.23 0.23

Fat 4.95 4.76 4.77 4.76 4.76 4.76 4.88

Total phosphorus 0.43 0.45 0.45 0.45 0.45 0.45 0.45

Threonine 0.72 0.85 0.85 0.85 0.85 0.85 0.85

Tryptophan 0.25 0.22 0.22 0.22 0.22 0.22 0.22 SBM=Soya Bean Meal, CP= crude protein Av=average, BBG=Boiled Bambara groundnut, SBG=Soaked Bambara groundnut,

FBG (wo)=Fermentation without Bambara Groundnut, FBG(w)=Fermentation with decantation, ROBG=Roasted Bambara

Groundnut, RBG= Raw Bambara Groundnut.

Table 3: Proximate composition (g/100 gDM) of the different processed Bambara Groundnut

seed meal

Parameter (%) BBG SBG FBG(wo) FBG(w) ROBG RBG

Crude protein 22.75 22.81 21.06 24.54 21.11 18.2

Dry matter 92.75 91.35 91.4 91.05 94.5 91.4

Crude fibre 8.43 7.67 7.45 7.57 7.2 8.52

Ash 2.52 2.99 2.38 2.31 2.42 2.49

Ether extract 7.42 7.67 7.39 7.43 7.2 9.78

NFE 58.88 58.86 61.72 58.15 62.07 61.01

BBG=Boiled Bambara groundnut, SBG=Soaked Bambara groundnut, FBG (wo)=Fermentation without Bambara Groundnut,

FBG(w)=Fermentation with decantation, ROBG=Roasted Bambara Gr oundnut, RBG= Raw Bambara Groundnut, NFE=Nitrogen Free Energy.


7

Table 4: Proximate composition (g/100 gDM) of the formulated diets with Bambara Groundnut

seed meal

Parameter

(%) Control BBG SBG FBG(wo) FBG(w) ROBG RBG

Dry matter 92.2 92.75 91.35 91.4 91.05 94.5 91.4

Crude

protein 20.05 20.14 20.16 19.99 20.30 20.09 19.86

Crude fibre 10.06 8.95 8.46 7.91 8.01 7.73 9.42

Ash 3.78 2.68 2.99 2.56 2.5 2.71 2.5

Ether extract 7.9 7.89 8.01 7.89 7.35 7.85 9.93

NFE 58.21 60.34 60.38 61.65 61.97 64.60 58.39

BBG=Boiled Bambara groundnut, SBG=Soaked Bambara groundnut, FBG (wo)=Fermentation without Bambara Groundnut,

FBG(w)=Fermentation with decantation, ROBG=Roasted Bambara Groundnut, RBG= Raw Bambara Groundnut, NFE=Nitrogen Free Energy.

Chart 1: Weight gain of the broiler chickens fed with the different processed Bambara

groundnut seed meal replaced diets

The results are mean ± SEM of 3 determinations. The weight gained are expressed as g/d (gram per day).

BBG=Boiled Bambara groundnut

SBG=Soaked Bambara groundnut

FBG(wo)=Fermentation without decantation

FBG(w)=Fermentation with decantation

ROBG=Roasted Bambara Groundnut

RBG=Raw Bambara Groundnut.


8

Chart 2: Specific activities of alkaline phosphatase in the serum, liver and kidneys of broiler

chickens fed with the different processed Bambara groundnut seed meal replaced diets.

The results are mean ± SEM of 3 determinations. Enzyme activities are expressed as nmol min-

1mg

-1protein.






RBG=Raw Bambara Groundnut


9

Chart 3: Specific activities of alanine aminotransferase in the serum and liver of broiler


The results are mean ± SEM of 3 determinations. Enzyme activities are expressed as nmol min-

1mg

-1protein.






RBG=Raw Bambara Groundnut

Chart 4: Specific activities of aspartate aminotransferase in the liver and serum of Broiler

chickens fed with the different processed Bambara seed meal replaced diets.

The results are mean ± SEM of 3 determinations. Enzyme activities are expressed as nmol min-1mg-1protein.








10

Chart 5: Specific activities of Gamma glutamyl transferase in the liver and serum of broiler


The results are mean ± SEM of 3determinations. Enzyme activities are expressed as nmol min-

1mg

-1protein.







4. Discussion

Carbohydrates are an important source of

energy. The nitrogen-free extract (NFE)

represents the soluble carbohydrates of the feed,

such as starch and sugar. Crude fiber represents

insoluble carbohydrates (AOAC, 1995).Proteins

are important constituents of foods, they are a

major source of energy, as well as containing

essential amino-acids, such as lysine,

tryptophan, methionine, leucine,isoleucine and

valine, which are

essential to human health, but which the body

cannot synthesize. Many food proteins are

enzymes which are capable of enhancing the

rate of certain biochemical reactions (AOAC,

1995).Crude fiber is a traditional measure of

fiber content in feeds; it gives an estimate of

indigestible fiber in foods. The basis of many

fiber analysis techniques is to develop a

procedure that mimics the processes that occur

in the human digestive system (AOAC, 1995).


11

Cooking and soaking methods are good

processing methods and the stability of anti-

nutritional factors could be reduced by up to

15% (Bressani, 2002). Palatability had also

been shown to influence feed intake and overall

performance of animals (Jurgens, 2002;

Holness, 2005). The various processing

methods as per weight gain compete favourably

with the control diets. This could imply that the

various processing methods had improved the

palatability of the seed. It could also be that

20% inclusion level of Bambara groundnut seed

meal in the diet had no deleterious effect, since

birds on the raw seed inclusion diet did not give

a poor performance effect as depicted in the

weight gain (Table 5). Legumes, including

Bambara seeds, contain phytochemical

compounds, some of which are antinutrients.

Their presence may confer favourable and/or

unfavourable effects on the feed stock used in

this study. There is therefore, the need to assess

the safety of the feed stock. Activities of

enzymes have been commonly used to assess

the safety or otherwise of plant materials in the

cellular system (Akanji et al., 2013).

Alkaline phosphatase (ALP) has been used as a

marker of plasma membrane integrity and

endoplasmic reticulum (Akanji et al., 1993). It

catalyses the hydrolysis of phosphate esters and

also transports ions and metabolites across the

cellular membrane. Aminotransferases

(ALT/AST) are the most commonly used

indicators of cellular necrosis and high levels in

serum may indicate liver malfunctioning

(Akanji, 1993). They occupy a central position

in amino acid Metabolism. They are present in

the liver, heart, kidney, skeletal muscle and

other tissues (Tiez, 1987). Both enzymes are

markers of liver damage caused by exposure to

chemicals (Nelson and Cox, 2000) with alanine

aminotransferase more liver specific (Tiez,

2008). Gamma glutamyl transferase (GGT) is

present in renal and hepatic tissue with renal

tissue having the highest level of activity of the

enzyme (Tietz 1995; 1999). Its presence in the

serum originates primarily from the

hepatobiliary system (Tietz, 1987). GGT

activity is activated in all forms of liver disease

with the highest level in cases of intrahepatic or

post-hepatic biliary obstruction. It is more

sensitive than alkaline phosphatase and

transaminases in detecting jaundice, cholangitis

and cholecystitis (Tietz, 1999). The observed

consistency, without significant differences, in

the activities of these enzymes in the liver as

well as serum may indicate that the Bambara

seeds have no observable effects on the liver

cells and may be relatively safe for

consumption. Since ALP is a membrane

enzyme, the membranes might not have been

affected. This contradicts the findings of


12

Oloyede et al. (2007) that reported higher ALP

and aminotransferase activities in the serum.

This could be due to the fact that Oloyede et al.

(2007) used the processed seeds as the sole

source of protein. This study, on the other hand

did a partial replacement of the soybean meal

for groundnut meal at 20%.

5. Conclusion

The results obtained from this study showed

that activities of liver and serum enzymes of

broiler chickens at the starter phase were not

affected when Bambara groundnut seed meal

was used to replace soybean meal at 20%

inclusion level. Further research need be carried

out to investigate indices of liver function and

other organs at the finishing stage of broiler

rearing.

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