3.0                                           MATERIAL AND METHODS
3.1       Source of African yam bean (AYB)
The African yam bean (Brown colour) will be purchased from Meat market, Abakiliki, Ebonyi State. The bean will packaged in polyethylene bags, transported to the laboratory and kept at ambient temperature until required.
3.2   Preparation of sample
         Prior to processing of milk and moi moi from African Yam Bean. First, the beans will be sorted manually to remove extraneous materials like dirt residue and diseased seeds to obtain healthy ones.

3.2       Production of African Yam Bean Milk and Moi Moi
            African yam bean seeds will be divided into four portions. Two portions will be soaked for 12 hours dehulled and dried under the sun. The dried seeds will be milled with locally fabricated attrition mill to obtain fine flour. African Yam Bean milk will be prepared from the flour as described by (Anminigo et al; 2007). The bean flour will be blended with 850C hot water (1:4 seed: water) in blender for 3 mins. The resulting slurry will be filtered through two layers of double folded cheese cloth and coarse particles will be removed by allowing the filtrate to settle for 10 mins. The moimoi will also be prepared from the flour by addition other local condiments and wrapped in aluminum foil and placed in boiling water in a pot with lid and allowed to steam for 2hours.
 Another two portion will be soaked for 12hours, dehulled and wet milled. One portion will be heated for 5 mins at 100oC. The slurry will be filtered through two layers of double cheese cloth and coarse particles were removed by allowing the filtrate to settle for 10 mins. The moimoi  will also be prepared form the wet bean and other local condiments will be added and wrapped in aluminum foil and placed in boiling water in a pot with lid and allowed to steam for 2 hours.
3.3       Proximate Analysis
            The crude protein, fat, moisture, and ash of the four processing methods were determined by AOAC (1990), while carbohydrate was ruined by difference.
            The moisture content will be determined by AOAC method (1990) using dry oven method. Five grains of the sample were weighted separately into crucible of a known weight (W1) the sample in the crucible (W2) were place in an oven for 6 hours at 1050C and were cooled in desicator and were reweighed (W3). The moisture content will be calculated using the equation below.
%         Moisture        =          W2-W3            x          100
                                                W2-W1                           1
Where W1      =          Weight of empty crucible
            W2       =          Weight of the empty crucible +weight of the sample
before drying
            W3       =          Weight of crucible + weight of sample after drying
3.3.2               Ash Determination
            The ash content of the sample will be determined by the method described by AOAC (1990) by putting about 5g in curable of known weight and will be dried in an Oven for about 4 hours at 1050C. The sample in the curable will be ashes in a muttle furnace at 5500C until white or grey ash were gotten. It will be cooled in a desolator and reweighed (W3) the percentage ash content will be calculated using the equation below;
% Ash =         
Where  W1     =          Weight of empty crucible
            W2       =          Weight of sample + weight of crucible before ashing
            W3       =          Weight of sample + weight of curable attar ashing,
3.3.3   Fat Determination
            The fat will be determined by AOAC method (1990) by weighing five grams of the sample into a labeled thimble, and thimble weighed. The bathing flask will be filled with about 100 millimeter to n-hexane, (Boiling pt 40-60). The extraction thimble will be assembled and allowed to reflux for about six times hour. The thimble will be removed and then hexane collection from the solution of fat. The fat left behind in the flask will be placed in the oven to dry at 1050C for one hour. The round will be cooled in the desicator and weighed. The percentage of fat in the sample will be calculated using the formula below.
% Fat  =         
3.3.4   Protein Determination
            The AOAC (1990) described method will be used to determined the protein by Kjedahl procedure using a protein factor of 6.25. Two grams of the sample will be weighed into a digestion tube and (H2SO4) 98% will be added using a dispenser. The tube will be placed in a pre-heated digester, cooled and diluted with water and will be placed in the distillation. A conical flask contain 35ml of boric acid with indicator will be placed under the condenser output. About 25ml of 40% sodium hydroxide (NaoH) will be filtrated with 0.1m tetramosulphate vi acid to purplish-greg end point.
            Percentage nitrogen (% N2) will be calculated using the formula below
% Nitrogen   
% crude protein = % N x 6.25
3.3.5   Carbohydrate Determination
The AOAC (1990) described method will be used to determined the carbohydrate content by difference, the percentage of moisture, protein, fat and ash were subtracted from 100 to get the carbohydrate value.
%Carbohydrate=100 - (moisture 1% protein + % fat+% ash)

3.4       Function properties
3.4.1 Water/oil absorption capacity (WAC/OAC)
The WAC/OAC will be determined using the method described by Onwuka (2005). One gram of the sample will be weighed into a conical centrifuge tube where a warring whirl mixer will be used to mix the sample thoroughly and 10ml distilled water/oil for 30 seconds. The sample will be allowed to stand for 30 minutes at room temperature and centrifuge at 5,000xg for 30minutes. The volume of the free water/oil the supernatant will be read directly from the graduated centrifuge tube.
            The amount of oil or water absorbed (total minus free) in multiplied by its density for conversion to grams.
3.4.2               Determination of Emulsification for capacity (EC)
The method of Onwuka (2005) will be adopted and use 2 gram of the sample will be blended with 25ml of distilled water at room temperature after complete dispersion, 5ml of oil (groundnut oil) will be added and blended continuously for another 30 second transferred into a centrifuge which will be finally directly from the tube.
            The emulsion capacity is expressed as the amount of oil emulsified and held per gram of same.
Emulsion capacity   =          x          x          100
                                                Y                      1
Where x         =         height of emulified layer
              Y       =          height of whole solution in the centrifuge tube.

3.4.3               Bulk density
          Bulk density will be calculated using the method, of Okaka and Potter (1979). Fifty gram of the sample will be placed in a graduated cylinder and the volume will be taped against a table until the flour be tightly packed. The volume of 1the sample will be noted and the bulk density will be calculated as weight per unit volume of the sample.
Bulk density (g/ml)=              Weight of sample(g)
                                                  Volume of sample (ml)

3.5.1               Tannins
The method of Onwuka (2005) will be adopted. One gram of the sample will be dispersed in 10 ml distilled water and agitated, left to stand for 30mins at room temperature being shaken every 5 min. At the end of the 30 mins, it was centrifuged and the extract gotten. 2.5 ml of the supernatant (extract) was dispersed into a 50 ml volumetric flask. Similarly 12.5ml of standard tannic acid solution was dispersed into a separate 50ml flask. A 1.0 ml Folic-Denis reagent was measured into each flask, followed by 2.5 ml of saturated Na2co3 solution. The mixture was diluted to mark in the flask (50ml) and incubated for 90min at room temperature. The absorbance was measured at 250nm a Grenway model 600 electronic spectrophotometer. Reading was taking with the reagent blank at zero. The tannin content was given as follows
%   Tannin     An   x    C    x  100    x   5             
               As                       w
Where            An       =         Absorbance of text sample
                        As       =         Absorbance of standard solution
                        C         =         Concentration of standard solution
                        W        =          weight of sample used
            The method of Onwuka (2005) will be adopted and use. fifty gram of the sample and dispensed into 50ml of 10% acetic acid solution in ethanol, shake the mixture well and allowed to stand for 4hours before filtering. The filtrate will be evaporated to one quarter (1/4) of its original volume. A drop of concentrated NH40H to precipitate the alkaloids. The precipitate will be filtered with a weighed filter paper and wash with 1% NH40H solution. The filtering should do with a weighed filter paper. The precipitate in the filter paper is dried in the oven at 60oc for 30minis and reweigh. The weight of alkaloid is determined and expressed as a percentage of the sample weight.
%Alkaloid     =         
Where            W        =          Weight of sample
                        W1       =          Weight of empty filter paper
                        W2       =          Weight of paper plus precipitate

3.5.3   OXALATES
            The titration method will be used as describe by Onwuka (2005). It involves three steps digestion, Oxalate precipitation and permanganate titration. 2gram of the sample is suspended in 190ml 4 distilled water in a 250ml volumetric flask. 10ml of 6m Hcl is added and the suspension digested at 100oC for 1 hour. Cooled, and then make up to 250ml mark before titration. Duplicate portions of 125ml of the filtrate are measured into beakers and four drops of methyl red indicator added. Followed by the addition of conc NH4OH solution until the test solution changes from salmon pink colour to a faint yellow colour (pH-4-5). Each portion is then heated to 900c, cooled and filtered to remove precipitate containing ferrous ion. The filtrate is again heated to 900c and 10 ml of 5% of CaCl2 solution is added while being stirred constantly. After heating, it is cooled and left overnight at 50C. The solution is then centrifuged at 2500 rpm for 5 minutes. The supernatant is decanted and the precipitate completely dissolved in 10ml of 20% (v/v) H2So4 solution. At this point, the total filtrate resulting from digestion of 2 gram of the sample is made up to 300ml. Aliquots  of 125ml of the filtrate is heated until near boiling and then titrated against O.o5M standardized KMnO4 solution to a faint pink colour which persist for 30 seconds.
            The calcium oxalate content is calculated content is calculated using the formula.
T  x   (Vme) (DF) x 105        (mg/100g)
               (ME)  x Mf

Where T         =          The titer of KMn04 (ml)
Vme                =          The volume – mass equivalent (ie1cm3 of 0.05M KMn04
solution is equivalent to 0.000225g anhydrous oxalic acid)
 DF                  =          Is the dilution of tractor VT /A (2.4 where
                                    ÑT is the total value of titrate (300ml) and
            A  Is the aliquot used (125 ML),
            ME      =          is the molar equivalent of KMn04 in
                                    Oxalate (KMnO4 redox reaction).
            Mf       =          is the mass of the sample used.
3.6       Sensory Evaluation
Sensory evaluation of the sample will be carried out using twenty semi-trained panelist consisting of students from Food Science and Technology Department, Ebonyi State University, CAS Campus Abakaliki. The panel will be instructed to evaluate appearance, taste/flavor, texture, colour and general acceptability using 9-point hedonic scale as described by Ihekoronye and Ngoddy (1985) with 1-dislike extremely, 5= neither like or dislike and 9=like extremely. Sample will be coded and presented in random sequence to the panelists. 
3.7       Statistical analysis
Data will be analyzed by analysis of variance (steel and Torie,1980).The difference between mean values will be determined by Least significant difference. Significance will be accepted at 5% probability.
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