Identificación de metabolitos secundarios.

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b(yellow-blue) axis – positive values are yellow; negative values are blue and 0 is neutral

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and also 12ml of concentrated H₂SO₄ added. Digestion was done for one hour at 420⁰C. The distillation was done using 2200 FOSS distillation unit with addition of 80mls of water, 40 ml NaOH (40%). The distillate was collected in 4% boric acid prepared with bromocresol green and melthyl red indicators. The distillate was titrated with 0.1 M HCl. Equation 7 was applied.

Nitrogen, (%) = Titre −Blank x 14.007 x 0.1 x 100 1000 x sample weight (mg ) (7)

Crude Protein, % = %N multiplied by 5.95 3.4.3 Determination of fat content

The fat contents were determined using the method described by AOAC (2000). The principle of Soxhlet fat extraction method was used but with a modern fat extractor with automated control unit (Foss Soxtec 2055, Foss Tecator Analytical AB Hoganas, Sweden). The equipment has the advantage of analysing the fat contents of six samples in 1h: 15min compared to the conventional Soxhlet which takes up to 8 h.The equipment has six extraction units with each unit having thimble for loading the samples and aluminium cups where the extracted fat are collected. The differences in the weight of the pre-weighed cups and after extraction are estimated as the percentage of fat in the samples. 1 g of the sample is weighed into the thimble and the mouth of the thimble is plugged with defatted cotton wool, and inserted into the extraction unit.

The extraction cups were cleaned, dried, weighed and 80ml of petroleum ether were measured into each cups and the heating temperature adjusted to 135°C which is the extraction temperature for fat using petroleum ether as stated in the manual. The cups were set into the Soxtec unit with each cup aligning with its respective thimble.

There are three stages involved; the boiling/extraction, rinsing and the drying. The extraction stage was for 30minutes in „boiling‟ position and 30 min in „rinsing‟

position after which it was aerated for 15 min. The cups were removed, cooled in a dessicator and weighed. Equation 8 was used for the calculation of crude fat.

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W 1 x100(8)

Weight of the cup with the extracted oil = W3

Weight of the empty cup = W2

Weight of sample =W1

3.4.4 Determination of ash content

The ash content was determined using the method described by AOAC (2000).

Crucibles were washed and dried in the laboratory hot air oven (Surgifriend Medicals, England. SM9053) maintained at 105⁰C for 30 min. It was allowed to cool in a dessicator and weighed. 2.5 g of the samples were then weighed into the crucible and charred on a heater inside a fume cupboard to drive off most of the smoke. The samples were transferred into a pre-heated muffle furnace (Surgifriend Medicals, England. SM9080) maintained at 550⁰C until a light grey ash was observed. The crucibles were transferred directly into a dessiccator, cooled and weighed immediately.The ash content was calculated using equation 9.

Ash content,%= weight of crucible +ash −(weight of empty crucible

weight of sample x 100

(9) 3.4.5 Determination of crude fibre

Crude fibre was determined using the method described by AOAC (2000). Two grams of sample was weighed into 250 ml conical flask. 100 ml of 1.25 % H2SO4 was added and boiled under reflux for 30 min. The sample was filtered and completely rinsed with distilled water. The residue was transferred with spatula back into the conical flask, 100 ml of 1.25 % NaOH added and again boiled under reflux for another 30 min. The residue was filtered and rinsed thoroughly after which the residue was transferred into a crucible and dried in oven at 180 ⁰C for 3 h, then cooled and

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weighed. The sample was returned to muffle furnace and ash for 2 h at 550 ⁰C until completely ashed. It was cooled and weighed. The calculation of crude fibre was done using equation 10.

Crude fibre, (%) =^{W 2−W 3}

W 1 (10) W1 = weight of the sample

W2 = weight of the crucible + sample after oven drying W3 = weight of the crucible after ashing

3.4.6. Determination of carbohydrate

Carbohydrate was estimated by difference using equation 10:

Carbohydrate= [100 - %(moisture+ protein+ fat+ ash+ crudefibre) (11) 3.4.7. Determination of free fatty acid

The general titrimetric method for the determination of free fatty acid (FFA) as described by AOAC, (2000); Akinoso and Adeyanju,(2010) were used. The principle involvespreparation of a mixture of 25ml diethyl ether and 25ml alcohol and 1ml phenolphthalein solution (1 %), and carefully neutralised with 0.1 M sodium hydroxide. 1 g of oil sample was put in the mixed neutral solvent and titrated with aqueous 0.1 M sodium hydroxide with the warm solution shaken continuously until a pink colour which persists for 15 s is obtained. The FFA was calculated as oleic acid (1ml 0.1M sodium hydroxide equals 0.0282g oleic acid).

3.4.8 Determination of amylose content

Amylose content of the rice was estimated as described by Adeyemi, (2009).

This involves digestion of 100mg of rice flour with 1ml of 95% ethanol and 9ml of 1N NaOH, the sample was heated for 10 min in a boiling water bath, and diluting the gelatinized starch with distilled water, 5ml of the starch solution was pipetted into a

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100 ml volumetric flask, 1ml of 1N acetic acid and 2ml of iodine solution added, and made up to volume and absorbance was read at 620nm. Amylose content was determined by reference to a standard curve.

3.4.9 Estimation of metabolizable energy

The metabolizable energy of the rice and flakes were calculated from the three basal nutrients in the samples (protein, fat, and carbohydrate) using Atwater factor as described by FAO (2002). To calculate the ME, the percentage of fat, protein, and carbohydrate eelier estimated in the rice and flakes are then multiplied by their respective Atwater factors, added together, and multiplied by 10. Equation 12 was applied in the calculation of metabolizable energy

ME (kcal/kg) = 10 (3.82 x CP) + (8.37 x CF) + (4.16 x CHO) (12) Where ME= Metabolizable energy

CP= % crude protein CF=% crude fat

CHO= % carbohydrates 3.4.10 Determination of phytate

The method for evaluation of phytic acid was carried out as described by Garcia-Estepa et al., 1999. The ground samples (5.0±0.5 g) were extracted under magnetic agitation with 40.0 ml of extraction solution (10 g/100 g Na₂SO₄ in 0.4 mol/l HCl) for 3 h at room temperature. The suspension was centrifuged at 5000 rpm for 30 min and the supernatant was filtered using whatman no. 1. 10ml of supernatant (containing between 3.3 and 9.0 mg of phytic acid) waspipetted into a 100 ml centrifuge tube together with 10.0 ml of 0.4 mol/l HCl, 10.0 ml of 0.02 mol/l FeCl₃ and 10.0 ml of 20 g/100 g sulphosalicylic acid, shaked gently and the tube used was

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sealed with a rubber cork through which passes a narrow 30 cm long glass tube, to prevent evaporation.

The tube was placed in a boiling water bath for 15 min, and then allowed to cool. The sample was centrifuged at 5000 rpm for 10 min, decanted, filtered and the residue was washed several times with small volumes of distilled water. The supernatant and washed fractions were diluted (100.0 ml). One aliquot (20.0 ml) adjusted to pH 2.5.0.5 by addition of glycine was diluted to 200 ml. The solution was heated at 70 °C and, whilst still warm, titrated with 50 mmol/l EDTA solution. The 4:6 Fe/P atomic ratios was used to calculate phytate content.