Proximate Composition Analysis this refers to the determination of the major constituents of

feed and it is used to assess if a feed is within its normal compositional parameters or somehow

been adulterated. This method partitioned nutrients in feed into 6 components: water, ash, crude

protein, ether extract, crude fibre and NFE.

Proximate analysis was carried out in the Central Laboratory, Faculty of Agriculture, University

of Benin, Benin City Edo State. With little modification by (E. M. Isikhuemen and I. U. Efenudu

et al, 2020)

Moisture Determination

Moisture is determined by the loss in weight that occurs when a sample is dried to a constant

weight in an oven. About 2g of a feed sample is weighed into a silica dish previously dried and

weighed. The sample is then dried in an oven for 650C for 36 hours, cool in a desiccator and

weigh. The drying and weighing continues until a constant weight is achieved.

wt of sample+ dishbefore drying−wt of sample + dishafter drying

%Moisture= X 100
Wt of sample taken

Since the water content of feed varied very widely, ingredients and feed are usually compared for

their nutrient content on moisture free or dry matter (DM) basis.

%DM = 100 - %Moisture.

Ash
Ash is the inorganic residue obtained by burning off the organic matter of feedstuff at 400-600 C

in muffle furnace for 4hrs. 2g of the sample is weighed into a pre-heated crucible. The crucible is

placed into muffle furnace at 400-6000C for 4hrs or until whitish-grey ash is obtained. The

crucible is then placed in the desiccator and weighed

wt of crucible +ash – wt of crucible

%Ash= X 100
wt of sample

Ether Extract

The ether extract of a feed represents the fat and oil in the feed. Soxhlet apparatus is the

equipment used for the determination of ether extract. It consist of 3 major components

1. An extractor: comprising the thimble which holds the sample

2. Condenser: for cooling and condensing the ether vapour

3. 250 ml flask

Procedure: about 150ml of an anhydrous diethyl ether (petroleum ether) of boiling point of 40-

600C is placed in the flask. 2-5 g of the sample is weighed into a thimble and the thimble is

plugged with cotton wool. The thimble with content is placed into the extractor; the ether in the

flask is then heated. As the ether vapour reaches the condenser through the side arm of the

extractor, it condenses to liquid form and drop back into the sample in the thimble, the ether

soluble substances are dissolved and are carried into solution through the siphon tube back into

the flask. The extraction continues for at least 4 hrs. The thimble is removed and most of the

solvent is distilled from the flask into the extractor. The flask is then disconnected and placed in

an oven at 650C for 4 hrs, cool in desiccator and weighed.

wt of flask + extract – tare wt of flask

%Ether extract = X 100
wt of sample
Crude Fibre

The organic residue left after sequential extraction of feed with ether can be used to determine

the crude fibre, however if a fresh sample is used, the fat in it could be extracted by adding

petroleum ether, stir, allow it to settle and decant. Do this three times. The fat-free material is

then transferred into a flask/beaker and 200 mls of pre-heated 1.25 % H 2SO4 is added and the

solution is gently boiled for about 30 mins, maintaining constant volume of acid by the addition

of hot water. The Buckner flask funnel fitted with whatman filter is pre-heated by pouring hot

water into the funnel. The boiled acid sample mixture is then filtered hot through the funnel

under sufficient suction. The residue is then washed several times with boiling water (until the

residue is neutral to litmus paper) and transferred back into the beaker. Then 200 mls of pre-

heated 1.25 % Na2SO4 is added and boiled for another 30mins. Filter under suction and wash

thoroughly with hot water and twice with ethanol. The residue is dried at 650 C for about 24 hrs

and weighed. The residue is transferred into a crucible and placed in muffle furnace (400-6000 C)

and ash for 4hrs, then cool in desiccator and weigh.

Dry wt of residue before ashing−wt of residue after ashing

% Crude fibre= X 100
wt of sample

Crude Protein

Crude protein is determined by measuring the nitrogen content of the feed and multiplying it by a

factor of 6.25. This factor is based on the fact that most protein contains 16 % nitrogen. Crude

protein is determined by kjeldahl method. The method involves: Digestion, Distillation and

Titration.
Digestion: weigh about 2 g of the sample into kjeldahl flask and add 25 mls of concentrated

sulphuric acid, 0.5 g of copper sulphate, 5 g of sodium sulphate and a speck of selenium tablet.

Apply heat in a fume cupboard slowly at first to prevent undue frothing, continue to digest for 45

mins until the digesta become clear pale green. Leave until completely cool and rapidly add 100

mls of distilled water. Rinse the digestion flask 2-3 times and add the rinsing to the bulk.

Distillation: Markham distillation apparatus is used for distillation. Steam up the distillation

apparatus and add about 10 mls of the digest into the apparatus via a funnel and allow it to boil.

Add 10 mls of sodium hydroxide from the measuring cylinder so that ammonia is not lost. Distil

into 50 mls of 2 % boric acid containing screened methyl red indicator.

Titration: the alkaline ammonium borate formed is titrated directly with 0.1 N HCl. The titre

value which is the volume of acid used is recorded. The volume of acid used is fitted into the

formula which becomes

14 x VA x 0.1 x X w x 100
%N =
1000 x 100

VA = volume of acid used w= weight of sample

% crude protein = % N x 6.25

Nitrogen Free Extract (NFE)

NFE is determined by mathematical calculation. It is obtained by subtracting the sum of

percentages of all the nutrients already determined from 100.

% NFE = 100 - (% Moisture + % CF + % CP + % EE + % Ash)

NFE represents soluble carbohydrates and other digestible and easily utilizable non-nitrogenous

substances in the sample.

Reference:

Association of Official Analytical Chemists (AOAC). Official Methods of Analysis of AOAC

International, 17th ed.; AOAC International: Gaithersburg, MD, USA, 2000.

Noureddini, H.; Byun, J. Dilute-acid pretreatment of distillers’ grains and corn fiber. Bioresource

Technol. 2010, 101, 1060–1067.

E.M. Isikhuemen, B.O Ogbomwan, and I.U Efenudu (2020) Evaluation of Phytochemicaland

Mineral Constituents of Piper guineense Schum. And Thonn. and Piper UmbellatumLinn:

Implications for Ethnomedicine. Europran Journal of Medicinal Plants, 31(1): 84-97,

2020

Mineral element determination

The determination of mineral contents was carried out using Atomic Absorption

Spectrophotometer (AAS model: SOLAAR 968 Unicam Series) for Ca, Fe, Mg and Zn; Flame

Photometer for Na, K and Spectrophotometer (model: Spectronic 20D+) for P followed methods

described by AOAC (2003) For wet digestion of samples, 1.0 g of the powdered sample was

weighed in digestion flask. 12 ml of concentrated HNO 3 was added and kept overnight at room
temperature. 4.0 ml of HClO4 was added to the mixture and heated in digestion block; starting at

50°C and gradually increasing to 250°C. The appearance of fumes at 70 – 80 min signaled

completion of digestion. The mixture was allowed to cool before transferring into 100 ml

volumetric flask and thereafter made to mark with distilled water. The wet digested solution was

stored in plastic reagent bottle for use in determination of minerals following the principles and

procedures expounded by Gul and Safdar (2009).

Reference:

AOAC. Official methods of analysis of the association of officials’ analytical chemists. 17th

Edition. Association of official analytical chemists. Arlington, Virginia; 2003.

Gul S, Safdar M. Proximate composition and mineral analysis of Cinnamon. Pakistan Journal of

Nutrition. 2009;8(9): 1456–1460.