Essential oil optimizes the susceptibility of Callosobruchus maculatus and enhances the nutritional qualities of stored cowpea Vigna unguiculata

The intensive use of synthetic pesticides in cowpea storage has led to the development of resistance by Callosobruchus maculatus and subsequent degradation of grain quality. In an attempt to circumvent these constraints, the susceptibility of C. maculatus to 2,2-dichlorovinyldimethyl phosphate (DDVP) and Lippia adoensis essential oil (EO) was investigated and variations in the proportions of nutritional values of treated grains 150 days after storage were assessed. The survival rate was recorded after five generations. The resistance index and biochemical parameters of grains were determined. The results from this study revealed that the survival rate and resistance index significantly increased proportionally with damage in DDVP treatments (r = 0.889; p = 0.018) while in EO treatments, those values remained low without significant variations (p = 0.0764) throughout the generations. DDVP stored grains yielded higher crude protein values, but lower carbohydrates, tannins, phenolics and minerals compared to EO. Eighteen amino acids were detected in EO treated grains and 14 in DDVP which was devoid of albumin and prolamin. Lippia adoensis EO could therefore represent a safe alternative bio-pesticide to cope with insect resistance and enhance the nutritional qualities of stored cowpea seeds.

3. The Chemical Score (CS) was calculated as previously described by Block and Mitchell [2] and later reported by Elhardallou et al. [3]. It is defined as the ratio of a gram of the limiting amino acid in a tested sample (AA s ) to the same amount of the corresponding amino acid in a reference diet (whole-egg protein) (AA ref ) multiplied by 100: Where: : Chemical Score of a given amino acid (i); : Amount of a given amino acid (i) in a sample; : Corresponding amount of a given amino acid (i) in the reference sample.
4. Essential amino acid index (EAAI) was calculated according to Oser [4] with slight modifications. It is defined as a geometric mean of the ratios of the essential amino acids in the test protein (EAAs) relative to their corresponding amounts in the whole egg protein (EAA ref ).

Where:
: Essential Amino Acid Index of a given sample; : Amount of given essential amino acid (i) in a sample; : Amount of essential amino acid (i) in the reference sample; : Number of a given amino acid (i) in a sample. 5. The Net Protein Value (NPV) was also evaluated as follows: 6. The biological value (BV) of cowpea seeds in each treatment was calculated using the method of Oser (1951) [4] as the follows: BV = 1.09 (EAA) -11.7 7. Protein efficiency ratio (PER) was estimated according to the regression equation reported by Alsmeyer et al. [5] and reported by Ilesanmi and Gungula [6]. Predicted protein efficiency ratio (P-PER) was computed as follows:

Total Carbohydrate determination
2g of cowpea flour was homogenized in 100ml of 95% ethanol. The homogenate was centrifuged at 1100 rpm for 10 min and used for the estimation of total sugar by anthrone method [7] as reported by Parthiban et al. [8].
Principle: Carbohydrate was first hydrolysed into simple sugars using dilute hydrochloric acid. In hot acidic medium, glucose is dehydrated to hydroxmethyl furfural. This compound forms with anthrone a green coloured product with absorption maximum at 630 nm.

Reagents:
 Standard solution of glucose: 100 mg of glucose was dissolved in 100 ml of water in a standard flask;  Working standard: 10 ml of the stock was diluted to 100 ml. 1ml of this solution contains 100µg of glucose;  Anthrone reagent: 0.2% anthrone was dissolved in ice cold concentrated sulphuric acid. Prepared fresh before use;  2.5 N HCl.

S3. Experimental procedure
100mg of the sample was weighted and poured into a boiling tube, hydrolysed by keeping it in a boiling water bath for 3 hours with 5 ml of 2.5 N HCl and cooled to room temperature. It was neutralized with solid sodium carbonate until the effervescence ceases and the volume adjusted to 100 ml before centrifuging at 1000 rpm for 10 minutes. The supernatant was collected and 0.2 to 1 ml were taken for analysis. Prepare the standards by taking 0.2-1ml of the working standards. 1 ml of water serves as a blank made up the volume to 1ml in all the tubes with distilled water, and then added 4.0 ml of anthrone reagent, heated for 8 minutes in a boiling water bath, cooled rapidly and read the green to dark green colour at 630 nm using spectrophotometer (Eppendorf AG, Germany).

Calculation
A standard graph was drawn by taking the concentration of glucose on X axis and spectrophotometer reading on Y axis. From the graph the concentration of glucose in the sample was calculated.

Moisture Determination
5g of cowpea flour was placed in a crucible and dried at 110°C to a constant weight after the initial weighing. Moisture content of the grains was calculated by the difference of initial and the final weight of the seed samples.

Ash Determination
5g of ground cowpea seeds was placed in a crucible, dried in oven at 100°C and then burnt to ashes at 600°C for 8hours in carbolite muffle furnace. This was cooled to room temperature until a white ash was obtained. The ash content was determined by the difference in weight of the crucible before and after cooling.

Fat determination
Soxhlet ether extraction was used for fat extraction and quantification. Petroleum ether was added to 5g of cowpea seed flour and placed in an extraction apparatus (a thimble). Extraction was carried out for 18 hours. Soon after, ether was allowed to evaporate to dryness and only fat remained in the flask and was recovered. The amount of fat was determined by the difference in the weight of the flask before and after ether dryness [9].
Five grams of ash samples were dissolved in 10mL of 2M HNO 3 and oven-dried. An extra volume of 5 mL of 2M HNO 3 was then added, boiled and filtered through a Whatman filter paper into a 100ml volumetric flask and the filtrate was made up with distilled water. The Digital Flame Photometry (model PFP7) was used to determine the sodium, potassium and calcium.
Total magnesium, iron, zinc, copper, sulfur and elements in traces (cobalt, boron, selenium, and manganese) were determined by atomic absorption spectrophotometer (BUCK 210VGP model) [10]. The Vanadomolybdate reagent at 400nm was used to determine and quantify Phosphorus from the sample filtrate by colorimetric method (Colorimeter SP 20, Bausch and Lamb).