MATERIALS AND METHODS
2.1 Materials
2.1.1 Plant Materials
The fresh fruits used for this study were apple (Malus domestica), orange (Citrus sinensis), pineapple (Ananas comosus) and red grape (Vitis vinifera).
2.1.2 Collection of samples
Industrially processed fruit juices (100% and 50%) from four different companies (A, B, C and
were purchased from shoprite and various other local grocery stores in Enugu State of Nigeria. Some of the fresh and ripe fruits used in this study were purchased from Ogige market in Nsukka Local Government, and others from Shoprite, both located in Enugu State of Nigeria. The industrially processed fruit juices purchased were within expiry date. The fresh fruits used were identified in the Department of Plant Science and Biotechnology, University of Nigeria, Nsukka.
2.1.3 Instrument/Equipment
The equipment used were those obtained in the Department of Biochemistry as well as other laboratories in the University of Nigeria, Nsukka. They include: atomic absorption spectrophotometer (Jenway), beaker (Pyrex, England), blender and grinder PM-B999 (Pyramid), EDTA bottles (Axiom, United Kingdom), electronic weighing balance (HX-T), flame photometer, freezer (Haier thermocool), hot air oven (Universal), microsyringes (Lifescan), mortar and pestle (Lifescan), refractometer, VIS spectrophotometer (Spectronic 20D), water bath and weighing boat.
2.1.4 Chemicals
All chemicals used for this study were of analytical grade and were obtained from reputable sources. The chemicals and their manufacturers include: absolute ethanol (JHD Ltd, China), AgNO3 (JHD, China), ammonia solution (JHD, China), ammonium chloride (Burgoyne, India), ammonium molybdate (BDH, England), ascorbic acid (CVL, India), calcine (BDH, England), carbon tetrachloride (JHD, China), copper sulphate (Vickers Ltd, England), concentrated hydrochloric acid (JHD, China), concentrated sulphuric acid (JHD, China), D- glucose
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(Burgoyne, India), distilled water (Lavans, Nigeria), ethylene diamine tetra acetic acid (JHD, China), erichrome black T-indicator (BDH, England), glacial acetic acid (JHD, China), hydrogen peroxide (JHD, China), hydroquinone (Kermel, China), iodine (Burgoyne, India), nitric acid (BDH, England), oscinol (Chemlight, India), phosphomolybdic acid (May and Baker, England), potassium cyanide (Burgoyne, India), potassium iodide (Biochemicals IIC, France), potassium hydroxide (May and Baker, England), resorcinol (Vickers Ltd, England), starch (Naafco, London), sodium acetate (May and Baker, England), sodium ethyl ethiocarbonate ( Hopkins and Williams, England), sodium sulphate (BDH Analar, England), sucrose (Burgoyne, India) and tartaric acid (Kermel, China).
2.1.5 Experimental Design
A total of 20 samples (juices) were used for the study; four (4) of which were freshly extracted juices of apple, orange, pineapple and red grape (designated 1). Eight (8) were industrially processed 100% fruit juices (designated 2) and the other 8 were industrially processed 50% fruit juices of apple, orange, pineapple and red grape origin (designated 3). There were four groups (apple, orange, pineapple and red grape juice). Each group had five fruit juice samples (of the same fruit origin) comprising one freshly prepared fruit juice, two industrially processed 100% fruit juices from companies A and B, and two industrially processed 50% fruit juices from companies C and D.
2.2 Methods
2.2.1 Extraction of Juice from Fresh Fruits
Extraction of juice from the fresh fruits was carried out using the method of Nzeagwu and Onimawo (2010). The apple and red grapes were properly washed and crushed with a blender after which they were filtered using a cheese cloth and the filtrates were collected as the freshly extracted apple and red grape juices, respectively. Pineapple and orange were properly washed and peeled after which the pineapple was crushed with a blender and the orange was squeezed to extract the juice. They were filtered using a cheese cloth and the filtrates were collected as the pineapple and orange juices, respectively.
2.2.2 Determination of Total Soluble Solid (TSS) Content
The total soluble solid content of the fruit juice samples was determined using a refractometer as described by Jasmine (2012) and Hossain et al. (2012). The refractometer was calibrated to 0%
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mark using water. The TSS content of each of the fruit juices was determined using the refractometer by placing a drop of the sample on its prism. The percentage of TSS was obtained from direct reading of the refractometer.
2.2.3 Determination of Sugar Contents of Fruit Juice Samples
Fructose, glucose and sucrose levels of each of the fruit juice samples (both industrially processed and freshly prepared) were determined.
2.2.3.1 Determination of Fructose Content
The fructose content of each of the fruit juice samples was determined by resorcinol reagent method as described by Buba et al. (2013). A quantity, 0.1 ml of fruit juice was pipetted into appropriately labelled test tube and made up to 1ml with distilled water after which resorcinol reagent (1 ml) was added. One mililitre (1 ml) of fructose solution B (contains 50 ml concentrated HCl in 10 ml distilled water) was added to each of the test tubes and the mixture was boiled in a water bath at 80oC for 8 min. The preparation was cooled to room temperature and the absorbance was read at 530 nm against a blank composed of water and the reagents. Fructose was used as the standard and the fructose content of each of the samples was calculated from the readings obtained.
/100 = ×
=
2.2.3.2 Determination of Glucose Content
The glucose content of the fruit juice samples was determined by Folin-Wu method as described by Plummer (1987). A quantity, 0.1 ml of fruit juice was pipetted into appropriately labelled test tubes and made up to 1 ml with distilled water after which alkaline copper reagent (1 ml) was added. The mixture was boiled in a water bath for 5 min and cooled to room temperature after which phosphomolybdic acid reagent (1 ml) was added. The preparation was diluted to 10 ml with distilled water and the absorbance was read at 420 nm against a blank composed of water and the reagents. Glucose was used as the standard and the glucose content of each of the samples was calculated from the readings obtained.
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/100 = ×
2.2.3.3 Determination of Sucrose Content
The concentration of sucrose in each of the samples was determined by the method described by Buba et al. (2013). A quantity, 0.1 ml of fruit juice was pipetted into appropriately labelled test tubes and made up to 1ml with distilled water after which 2% w/v oscinol (5 ml) was added. The mixture was boiled in a water bath for 12 min and cooled to room temperature. The absorbance of each of the preparation was read at 620 nm against a blank composed of water and oscinol reagent. Sucrose was used as the standard and the sucrose content of each of the samples was calculated from the readings obtained.
/100 = ×
2.2.4 Determination of the Mineral Contents of Fruit Juice Samples
The fruit juice samples were digested after which the mineral contents (including heavy metals) of each of the samples were determined.
2.2.4.1 Wet Digestion of Fruit Juice Samples
Fruit juice samples were digested prior to mineral determination using the method described by AOAC (2000). A quantity, 5 ml of each of the fruit juice samples was accurately measured into appropriately labelled conical flasks after which concentrated HNO3 (20 ml) and H2O2 (20 ml) were added. The preparations were heated in a furnace at a temperature of 500oC until the HNO3 and H2O2 had evaporated. The digested samples were afterwards tested for mineral contents.
2.2.4.2 Determination of Sodium (Na) and Potassium (K) Contents
Sodium and potassium contents of digested samples were determined by flame photometric method as described by AOAC (2000) and Ndife et al. (2013). The samples were run in a flame photometer with standards of sodium and potassium, respectively, and the sodium and potassium contents of the samples were calculated from the readings obtained.
/100 = ×
= ⁄
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2.2.4.3 Determination of Magnesium (Mg) Content
Magnesium content of digested samples was determined by complexometric titration using EDTA as described by Okoye and Ibeto (2009). Ten (10) ml of each of the digested samples was added into appropriately labelled conical flasks after which distilled water (25 ml) and ammonia-ammonium chloride buffer (25 ml) were added. Erichrome black T-indicator was added to each of the conical flasks. A blank was prepared with water and other reagents after which the preparations were titrated with 0.01 N EDTA. The Mg content of each of the samples was calculated from the titre readings obtained.
× × ×
/100 =
×
Where; TV = Titre value, NE= Normality of EDTA, AM= Atomic mass of magnesium, DF= Dilution factor, WD= Weight of the digested sample and AT= Aliquot (ml) for titration.
2.2.4.4 Determination of Calcium (Ca) Content
Calcium content of digested samples was determined by complexometric titration using EDTA as described by Okoye and Ibeto (2009). Ten (10) ml of each of the digested samples was added into appropriately labeled conical flasks after which distilled water (25 ml) and 10% w/v KOH (25 ml) were added. A pinch of calcine indicator was added to each of the conical flasks. A blank was prepared with water and other reagents without the samples. The preparations were titrated with 0.01 N EDTA. The Ca content of each of the samples was calculated from the titre readings obtained.
× × ×
/100 =
×
Where; TV= Titre value, NE= Normality of EDTA, AM= Atomic mass of calcium, DF= Dilution factor, WD= Weight of the digested sample and AT= aliquot (ml) for titration.
2.2.4.5 Determination of Phosphorus (P) content
Phosphorus content of digested samples was determined by spectrophotometric method as described by AOAC (2000). A quantity, 5 ml of each of the digested samples was added into appropriately labeled test tubes after which ammonium molybdate (1 ml) and ascorbic acid (1 ml) were added. The solution was diluted with 3 ml of distilled water after which the absorbance
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was read at 882 nm against the blank. The phosphorus content of each of the samples was calculated from the readings obtained.
/100 = ×
2.2.4.6 Determination of Iron (Fe) Content
Iron contents of digested samples were determined as described by AOAC (2000). Five (5) ml of each of the digested samples was added into appropriately labeled test tubes after which 2% hydroquinone (1 ml), acetate buffer (5 ml) and 0.1% 2, 2 dipyridyl (1 ml) were added. The solution was shaken vigorously and a change of colour to pink was closely observed after which the absorbance was read at 530 nm against the blank. The Fe content of each of the samples was calculated from the readings obtained.
/100 = ×
2.2.5 Determination of Heavy Metal Contents of Fruit Juice Samples
Each of the digested samples was screened for contaminations with heavy metals such as copper, zinc, arsenic and lead. Atomic absorption spectrophotometer model 9100 with cathode lamps specific for Cu, Zn, As and Pb was used to determine the Cu, Zn, As and Pb contents respectively of each of the digested samples (Ndife et al., 2013).
2.2.6 Statistical Analysis
Data obtained from this study were analyzed using statistical product and service solutions (SPSS) version 18. Least Significant Difference (LSD) and Duncan were used to compare mean values. Data were reported as mean±standard deviation. Mean values were considered statistically significant at p < 0.05.