Proximate Analysis is the partitioning of compounds in a feed into six groups based on the chemical properties of the compounds. The six categories are:
(i) Moisture (ii) Ash
(iii)Crude protein (or Kjeldahl protein) (iv) Crude lipid
(v) Crude fibre
(vi) Nitrogen-free extracts (digestible carbohydrates)
All the analysis above were carried out using satandard Methods of Analysis by the Association of Analytical Chemists (AOAC, 2000).
3.11.1. Determination of Percentage Moisture Content
Moisture content determination is an important factor that critical in food quality, preservation, and resistance to deterioration. Moisture content of foods can be determined by a variety of methods.
Procedure
Apparatus and Instrument needed
Analytical balance
Desiccator
Crucible with lid
Thermosetting Oven
Tongs
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Washed crucibles were oven dried at 105 ⁰C for an hour to ensure total dryness. They were then transferred into the desiccator to cool for about 30 minutes. The crucibles were weighed on an electronic balance and the weight recorded as (W1). 5 g of seed sample were weighed into the dried preweighed crucible (W2). The crucibles and the content were oven-dried at 105
0C for 4 hours. The samples were removed from the oven and dried until a constant weight was obtained. After drying, the crucible was transferred into the desiccator to cool for about 45 minutes and weighed (W3). This analysis was carried out in triplicate and the average value was recorded as moisture content.
Calculations
(%) Moisture content = Loss in weight due to drying Weight of sample before drying (%) Moisture content = 𝑊2−𝑊3𝑊2−𝑊1 * 100
Where,
W₁ = Weight of empty Crucible
W₂ = Weight of empty crucible + sample before drying
W₃ = Weight of crucible+ sample after drying (constant weight).
% Total Solid = 𝑊3−𝑊1𝑊2−𝑊1 * 100
Or % Total solid = (100 - % Moisture content) Totals Solid is the part that is not water.
3.11.2 Determination of Total Percentage Ash Content
Ash is the inorganic residue after the water and organic matter have been removed from a substance by heating in the presence of certain oxidizing agents. The ash is not usually the same as the inorganic matter present in the original material since there may be losses due to
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the volatilization or chemical interaction between the constituents. The importance of Ash content is that it gives an idea of amount of mineral elements present and the content of organic matter in the sample. The organic matter account for quantitative constituents of protein, lipids or fat, carbohydrate, plus nucleic acid. The measure of ash content provides a measure of the total amount of minerals within a food. The principle of this method is based on the fact that minerals in a food substance are not destroyed on heating and also have low volatility compared to other components of the food. Analysis of ash content can be done either by dry ashing, wet ashing or plasma dry ashing. The method that is chosen for the analysis depends on the reason for the analysis, the type of food analyzed, and also the equipment available. Ashing is also a preparation step in analysis of specific minerals either by atomic spectroscopy or other traditional methods. Dry ashing for the majority of the samples,
Dry ashing is incineration at high temperature (525 °C or higher). It is accomplished in a muffle furnace. There are many different types of crucibles made of different materials such as quartz, porcelain, steel or platinum crucibles. The crucible selection is based on cost, reactivity of the crucible to the sample and resistance of the crucible to very high temperatures Dry ashing has the advantages of not needing external reagents and requiring little attention.
Apparatus
Crucible or porcelain or metal dishes can be used Muffled furnace, Desiccator, Tong
Procedure
A clean crucible was pre-dried in an oven for 30 minutes at 100 0C to assure total dryness of the crucible. It was then transferred into the desiccator to cool for 30 minutes and weighed on an electronic weighing balance as W1. 5 g of sample was weighed into it and weighed as W2. It was placed in a muffle furnace for 4 hours and the temperature was slowly increased to 450
˚C to avoid incomplete ashing. Samples were ash until it becomes whitish in colour. It was
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removed into the desiccator with a tong and cooled to room temperature for an hour. Sample was reweighed as W3. The percentage ash was calculated as followed and average taken:
Calculation: =
% Ash content = 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑎𝑠ℎ
𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 (𝑎𝑓𝑡𝑒𝑟 𝑑𝑟𝑦𝑖𝑛𝑔)∗ 100
% Ash content = 𝑊3−𝑊1
𝑊2−𝑊1 * 100
% Organic matter = 100 - % Ash
3.11.3. Determination of Percentage Crude Fat Content
Fats are defined as mixtures of various glyceride of fatty acids, which are sparingly soluble in water and some certain organic solvents e.g ethyl ether, petroleum ether, acetone, ethanol, methanol, benzene. Extraction is carried out with Soxhlet apparatus with ether or petroleum ether. The fat content may be determined by either the Soxhlet method, the Mojonnier method or the Soxtherm automated method. The most conventional of these three is the Soxhlet method. The usual procedure is to continuously extract the fat content using 40/60 petroleum ether in a Soxhlet extractor. The ether extraction method is based on the principle that non-polar components of sample are easily extracted into organic solvents.
Apparatus
Thimble
Reflux condenser
Round bottomed flask
Heating mantle
Appropriate solvent
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Previously dried, fat free thimble was weighed as W1. 5 g of sample was weighed into the thimble and weighed as W2. The thimble and the sample was carefully wrapped and tied.
Washed and dried 500 ml round bottom flask was weighed as W3. The flask was half filled with 40/60 petroleum ether and the sample was dropped into the sample holder of the soxhlet extraction apparatus. The Soxhlet extractor was fitted with reflux condenser as shown figure 3.7. The flask was then placed on a heating mantle and the heat source was adjusted to allow it to boil gently at 34 0C. It was allowed to siphon over 5 hours. The condenser was detached and the thimble removed. Petroleum ether was distilled from the flask. The distilling flask containing the oil was air dried at 100 0C for exactly 5 minutes to remove the solvent residues in oil. This was put inside a desiccator to cool and the weight was taken as W4. The percentage fat contained was determined thus:
% Crude fat = 𝑊𝑒𝑖𝑔ℎ𝑡 of Flask + oil – Weight of empty flask 𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑆𝑎𝑚𝑝𝑙𝑒 * 100 (%) Crude Fat = 𝑊4−𝑊3
𝑊2−𝑊1*100
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Figure 3.7 A set up of Soxhlet apparatus for the extraction of crude fat
3.11.4 Determination of Percentage Crude Fibre
Crude fibre is the portion of the plant material which is not ash. Fibre was originally thought to be bad and the indigestible portion of foodstuffs. However, the benefits of a diet high in fibre have been discovered. Foods with high fibre content can absorb cholesterol and toxic agents in food. It also raises the excretion of bile and sterols. It is known however that fibre consists of cellulose which can be digested to a considerable extent by both ruminants and non-ruminants.
The interest in fibre in food and feed has increased, based on the noticed number of serious illness associated with diet low in fibre. Fibre swells and form gelatinous mass with high water retention capacity with the digestive system. Diseases such as constipation (the intestine absorbs water and volume of the feaces will decrease), appendicitis, Hemorrhoid and Diabetes mellitus have been related to low-fibre diet in humans.
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The starch and the protein part of food were dissolved by boiling with acid and then with a very strong base (NaOH). The residue, which comprises of cellulose and lignin was washed and dried and weighed. The residue is ashed and the weight is subtracted from the weight of the residue.
3 g of defatted sample was weighed (W1) into 250 ml beaker containing 200 ml of 0.125 M or 1.25 % tetraoxosulphate (iv) acid (Sulphuric acid). The mixture was heated in a steam bath at 70 – 90 0C for 2 hours, it was then allowed to cool. The cooled mixture was filtered using a muslin cloth over a Buckner funnels. The residue was washed three times with hot distilled water to remove the acid and then put in a beaker containing 200 ml of potassium hydroxide.
The mixture was heated as before over a steam bath for 2 hours. The solution was filtered and the residue washed three times with hot distilled water, then with petroleum ether and water.
The final residue obtained was put in clean preweighed (W2) crucible and dried at 120 0C to a constant weight. The crucible with the oven dried sample as put in a muffle furnace and ashed at 550 0C for 30 minutes such that the sample became ash white. The crucible and its contents were removed from the furnace, cooled in a desiccator and reweighed (W3). Percentage fibre was calculated as followed:
% Crude fibre = weight of oven dried sample – weight of ash X 100 Initial weight of sample
% Crude fibre = 𝑊2−𝑊3
𝑊1 ∗ 100
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3.11.5. Determination of Percentage Crude Protein
Proteins are necessary nutrients for the human body. They are one of the building blocks of body tissue, and also serve as a fuel source. Protein is a nutrient that is important for growth and maintenance of the body. Crude protein is calculated on the basis that protein is the only food composition that has Nitrogen. Average Nitrogen content of protein is about 16 %, the amount of nitrogen is multiplied by 6.25 to calculate the crude protein, and this is used only for Kjedahl method of analysis of protein.
The analysis of Crude protein was determined using Kjeldahl method. This process involved 3 different stages namely; digestion, distillation and titration.
A chemical mixture of 150 g of K2SO4 and 10 g of CuSO4 was made. 1 g of sample and 10 g of chemical mixture was weighed into a 250 ml digesting tube. 12 ml of concentrated H2SO4
was carefully added to the mixture. The digesting tubes containing samples were kept in a rack and digested for 30 minutes at 420 0C in a fume cupboard. After digestion, the samples were allowed to cool to room temperature for about 1 hr. 80 ml of distilled water was then added to the digested samples. 25 ml of diluted digested sample as well as 25 ml of NaOH were measured into a distillation tube. Distillation was carried out using 5 ml of Boric Acid and methyl red indicator. This process was stopped when the conical flask containing boric acid-indicator solution reached 100 ml mark. The distillate was titrated using HCl until end point was reached.
At this stage, the purple colour obtained during distillation changed to dark yellow. The formula used for protein calculation is:
% Nitrogen = (V2/V1) × C ×0.0140 ×(S−B)
Z ∗ 100
% Protein = % Nitrogen x 6.25
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3.11.6 Carbohydrate Content Determination
The carbohydrate content of the sample was obtained by difference, that is, as the difference between the total summations of percentage moisture, fat, fibre, protein and ash.
% Carbohydrate= 100 – (% moisture + % fat + % protein + % fibre + % ash)