Investigation of Essential Metals in White Lupin Grain in Some

Research Article Volume 9 Issue No.12 Investigation of Essential Metals in White Lupin Grain in Some Selected Amhara Region, Ethiopia Zemenay Zewdu Department of Chemistry College of Natural and Computinal Science, University of Gondar, Ethiopia

Abstract: Back ground: Plants are the most important sources of food for human beings. Among plant sources, legumes products are important sources of protein and minerals in the diets of millions of people in the world. White lupin (Lupinus albus), the local name of “Gibto” is one of the traditional pulse crops in Ethiopia especially in Amhara regional state. Objective: This study focuses on the determination of essential metal in white lupin. Methods: In this study, the concentration of selected metals (Ca, Fe, Zn and Cd) in raw lupin cultivated in different part of Amhara Region, Ethiopia particularly in Debretabor, Merawi and Kossober were determined using FAAS. Result: The observed metal concentration in three cultivars of raw lupin was in the range of Ca (87.71±0.638 to101.14±0.42 mg/100g), Fe (3.83±0.22 to 10.3±0.1g/100g), Zn (15.91±0.865 to 28.22±0.14 mg/100g) and Cd (1.32 ± 0.04 to 1.38 ± 0.02 mg/100g). The difference in metal concentration between samples based on the difference in geographical location were determined by using one way ANOVA Duncan’s multiple range test at a significant level of P < 0.05. Conclusion: The result shows that the ability of white lupin seeds to accumulate relatively higher amount of Ca, Fe, and Zn. Toxic metal Cd was found to be comparatively at lower level. The content metals in all sample of lupin were similar trained across the verities that could be arranged in the following order: Ca > Fe > Zn > Cd. Further study should be carried out to analyze the soil metal content on which white lupin is grown and to study correlation of metals between soil and lupin plants.

Key Words: White lupin, essential metals, FAAS, concentration.

I. BACKGROUND and minerals. They are used in many parts of the world for both animal and human nutrition3. The other legume from the same Plants are the most important sources of food for human beings. family Leguminosae called lupin, it is one of the traditional Among plant sources, legumes products are important sources of pulse crops in Ethiopia especially in Amhara regional state. It is protein and minerals in the diets of millions of people in the used as human food and animal feed since early Roman times. It world1,2. The legumes used by humans are commonly called is an economically and agriculturally valuable Plant and its seeds food legumes or grain legumes. The food legumes can be are employed as a protein source for animal and human nutrition divided into two groups, the pulses and the oilseeds. Pulses in various parts of the world, not only for their nutritional value, group consists of dried seeds of cultivated legumes, which have but also for their adaptability to soils and climates4. The plant is been eaten for a long time. Grain legume is important source of characterized by having various flowering spikes in large range significant amounts of proteins, carbohydrates, fibber, vitamins of colours5.

Figure.1. Flower of different lupin species

Lupin seeds have a high protein and fat content but their use for of α-galactosides from Lupinus albus L. Var. maltoilupa on the human nutrition is very limited due to the presence of high chemical composition of prepared flour and the daily availability alkaloid content requires large processing steps3. Different of N, total P, Ca, Mg, Fe, Zn and Mn was studied. From those studies have been under taken regarding the metal contents of study the content of Mn, Cu, Fe, Zn, P, Mg and K were 63.9 ± different cultivars of white lupin. In Spain, the effect of removal 1.30, 6.62 ± 0.28, 63.3 ± 0.14, 55.0 ± 1.11 (µg/g), 565.8 ± 0.90,

IJESC, December 2019 24228 http:// ijesc.org/ 189.61 ± 5.81 and 600 ± 23.7 mg/100 g of dry matter Sample collection and processing respectively6. White lupin (Lupinus albus) sample from each sampling site were collected randomly from the mentioned market and Similarly in Portugal the lead and copper amount also studied in processed. The raw lupin sample was washed with tap water and white lupin root and leaves using anodic striping voltametry. as well with distilled water. Finally it is rinsed with deionized From these study toxicity symptom of lead is observed in leaves water and air dried. The dried whole seed was powdered and and roots of white lupin7. Likewise in Europe, the amount of sieved. The powder was collected and stored with clean and some macronutrients of lupin was analyzed. These study dried plastic bottles in a refrigerator7. established that the presence of K, P, Ca, Mg, Mn, and low level of Na. The manganese level of white lupin was found 896 mg/kg Reagents and Chemicals which can approaches toxicity concentration and the level of Ca, All reagents and chemicals used in the study were analytical P, Mg, K, Na, and Mn was found 2.1 to 4.66, 4.29 to 7.20, 1.20 grade. HNO3 (69-72%) HClO4 (6062%) both from (SD Fine 7 to 2.25, 8.6 to 11.1 and 0.1 to 0.2 g/kg respectively . Chem Industries Mumbai, India), and H2O2 (30%, Scharlau, European Union), Lanthanum nitratetrihydrate (99.9%,Aldrich, Tizazu et al (2010) studies that chemical composition, physical USA) and standard stock solutions containing 1000 mg/L, in 2 and chemical properties of Gibto seed grown in Ethiopia. Up on % HNO3, of the metals Ca, Fe, Zn and Cd (BUCK SCIENTIFIC his study the metal content of lupin in two cultivars of white GRAPHIC) were used. lupin (Debretabor and Dembecha) has been reported. From his observation the P and Ca level of Debretabor lupin are 248.77 Digestion of white lupin sample and 97.98 mg/100 g respectively and in Dembecha lupin the P Sample digestion was carried out under optimum conditions of and Ca level are 249.02 and 67.13 mg/100 g respectively. In Nitric acid-perchloric acid mixture (7:3 v/v), digestion general based on his study the content of P, Fe, Zn and Ca were temperature (250 °C) and a total digestion time of 3:30 hours. 248.90, 12.51, 4.68 and 82.56 mg/100 g respectively1. Getachew Through applying the optimized procedure, 7 mL concentrated (2009) in Ethiopia studied that the chemical composition and the HNO3 (69-72 %) acid and 3 mL of HClO4 (60-62 %) (7:3) was effect of traditional processing on nutritional composition of added to 0.5 g powdered samples. The mixture was digested Gibto (Lupinus albus L.) grown in Gojam area. His observation under a hot plate by covering the beaker by watch glass until the conducted in two areas of Dangla and Tilili. The mineral entire sample was digested and 1-2 mL colorless solution was composition of the two cultivars was reported that, the Dangla remained. After digestion the sample was allowed to cool for 10 sample has 6.00, 2.11, 58.43, and 8.93 mg/100 gm contents of minutes at room temperature followed by addition of 10 mL Fe, Zn, Mn and Mg respectively. The values of the same types of deionized. The solution was filtered and diluted with deionized minerals for the Tilili sample were 6.72, 1.81, 63.54, 59.14 and water. On the same way the blank solution was prepared from 10 2 9.46 mg/100 gm respectively . mL of reagents (i.e. a mixture of 7 mL of HNO3 and 3 mL of HClO4), boil the mixture as the same time and temperature used The concentration of mineral in white lupin is determined by the for the sample digestion procedure8. For Ca analysis lanthanum choice of cultivars and environmentally determined by soil type nitrate hydrated was added to the sample then placed in a freezer and climate conditions. Appropriate conditions together with to avoid interference until analysis9. selection of lupin cultivars that have a high metal concentration can be used to promote high metal bioavailability. For this Instruments and Apparatus reason, it is necessary to determine the level of selected metals in Polyethylene bags were used for handling and transporting the white lupin seed collected from markets of production site of collected samples, mixer grinder (BRILLIANT B×1002, AN 150 south Gondar, west Gojam and Awi zone (Debretabor, Merawi 9001:2008 CRTIFIED CAMPANY) for grind and powdered the and Kossober) and to compare the result. Since trace metals have dried sample, digital analytical balance for weigh samples, role in disturbing at high levels in human tissue and body fluids. beaker, used to digest the lupin powder, hot plate(C-MAG HS 4 Therefore; knowing the content of metals in white lupin grain is 525, IKA) to digest the dried and powdered lupin samples, fume a universal interest from a nutritional point of view. There are no hood, refrigerator for keeping the digested sample till analysis, other studies carried out in determining the level of metals in pipette, filter paper, 50 mL of volumetric flask. Finally BUCK Ethiopian white lupin commonly known as “Gibto”. Therefore; SCINTIFIC 210 VGP flame atomic absorption spectroscopy the main objective of this study is to determine the (FAAS) for analysis of metals using air-C2H2 flame concentrations of metals in white lupin (Lupinus albus) grain grown in different parts of Ethiopia. Analysis of metals in white lupin sample Before analysis blank solutions were prepared for calibrating the II. MATERIAL AND METHOD instrument, it was prepared by digesting the mixture of reagents following the same digestion procedure as the samples and Study area diluted to 50 mL with deionized water10. Measurements were made using the hollow cathode lamps for Ca, Fe, Zn and Cd at Samples of white lupin were collected from open markets of the proper wavelength and the slit width was adjusted using air sampling area. The sampling sites are Debretabor, Merawi and acetylene flam. The concentration of each element in the sample Kossober. The geographic locations of the sampling sites are is equal to the quotient of the intercept and the regression found in Amhara region. Debretabor is found in South Gondar, coefficient of the calibration curve. Then each metal content was Merawi is found in west Gojam (Bahirdar zuria) and Kossober is calculated (using Microsoft excel- 2007 and SPSS soft ware found in Awi zone. The sampling techniques were random version 20) and the results were reported as mean standard sampling. ± IJESC, December 2019 24229 http:// ijesc.org/ deviation10. The metal contents of each sample were calculated Method detection limit: A method’s detection limit is the using the following formula. smallest amount or concentration of analyte that can be detected with statistical confidence11. To determine the detection limit a b  v triplicate analysis of the four blank samples were performed and Metalcontent(mg/100g)  (1) the pooled standard deviation of the four reagents blank was 10W calculated. The detection limit was obtained by multiplying the Where; W = weight in gm of the sample pooled standard deviation of the blank reagent by three. a = concentration in ppm of sample V = volume in ml of the extract Method Limits of Quantitation (MQL) b = concentration in ppm of blank Limit of quantification (LOQ) is the smallest concentration or absolute amount of analyte in a sample that can be determined III. EVALUATION OF ANALYTICAL RESULTS with suitable precision and accuracy. According to American Chemical Society’s Committee on Environmental Analytical Precision Chemistry, the quantification limit of each element was To ensure the precision of the analysis, each sample was calculated as ten times the standard deviation of the blank If the digested in triplicate and triplicate reading was carried out. The analyte concentration is below the LOQ value, results are precision of the results were evaluated by relative standard reported as non-detectable. As a result LOQ is a very important deviation of each metals analyzed for each sample. For Ca, Fe, parameter in risk measurement11. The method detection limit, Zn, and Cd analysis the relative standard deviations of all method limit of quantitation and the detection limit of the measurement (from results shown in table 5) were found in the instrument of each metal have shown in table 3. acceptable range.

Table.1. List of working standards for determination of metals in all Lupin samples using flame atomic absorption spectrometer. No Element Concentration of standards, in Correlation coefficient(R) 1 Ca 2 6 10 12 0.99862

2 Fe 0.5 1 2 4 0.9984 3 Zn 0.5 1 2 4 0.99972 5 Cd 0.5 1 1.5 2 0.99835

Table.2. Instrumental operating conditions for determination of metals in white lupin samples using FAAS. No Element Flame Type Wave Detection Slit width length(nm) limit (mg/L) (nm)

1 Ca Acetylene gas 422.7 0.05 0.7 2 Fe Acetylene gas 372.0 0.05 0.7

3 Zn Acetylene gas 213.9 0.005 0.7

5 Cd Acetylene gas 228.9 0.01 0.7

Table.3. Method Instrumental detection limit (IDL), Method detection limit (MDL) = 3Sblank and Quantitation limit (MQL) =10Sblank in mg/L for all metals determined in white lupin samples (n=3). Metals MDL MQL IDL (mg/L) (mg/L) (mg/L)

Ca 0.611 2.038 0.05

Fe 0.397 1.326 0.05

Zn 0.055 0.185 0.005

Cd 0.00867 0.0289 0.001

1. *Values are mean of 3×standard deviation of the four reagent 2. *Values are mean of 10×standard deviation of the four reagent blank determination measured three times; MDL= method blank determination measured three times; IDL = Instrumental detection limit. detection limit; MQL = Method Limits of Quantitation

IJESC, December 2019 24230 http:// ijesc.org/ 3. *Values are detection limit of FAAS instrument; IDL= Instrumental detection limit

IV. RESULT AND DISCUTION

0.020 0.010 0.018

0.016 0.008

0.014

0.012 0.006

0.010 Y = 0.00158X-0.000322 R = 0.99862 0.008 0.004 Y = 0.0023X-0.000305 Absorbance R = 0.9984 Absorbance 0.006

0.004 0.002

0.002 0.000 2 4 6 8 10 12 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 Concentration (ppm) Concentration (ppm)

(a) Calibration graph of Ca standard (b) Calibration graph of Fe standard solution

0.45

0.40

0.35

0.30

0.25 Y = 0.23972X-0.04765 0.20 R = 0.99835 Absorbance 0.15

0.10

0.05

0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 Concentration (ppm)

(e) Calibration graph of Cadimium standard

Figure.2. Calibration graph of Ca, Fe, Zn, and Cd standard solution as shown below (2a to 2d):

The calibration graph for the four elements was determined in correlation coefficient between 0.99835 and 0.99972 for all four the concentration range of shown above in table 1. The elements. calibration curves were linear over the entire range with

Table.4. Atomic absorption spectrophotometer results of metal contents (mean ± SD, N = 3) (mg/100 g) of raw white lupin that are cultivated in some selected Amhara region of Ethiopia. Ca Fe Zn Cd Metals (mg/100 g) (mg/100 g) (mg/100 g) (mg/100 g)

Debretabor 99.29±0.66a 3.83±0.22d 17.36±0.26e 1.38 ± 0.02g

Merawi 101.14±0.42a 10.3±0.1c 28.22±0.14f 1.34 ± 0.01g

Kossober 87.71±0.638b 5.43±0.0561d 15.91±0.865e 1.32 ± 0.04g

*Values in the same column that are followed by a different from place to place. Because, the geographical location, litters (a-g) are significantly different at P < 0.05 by using one environmental condition, chemical and physical property of the way ANOVA Duncan’s multiple-range test soil, type of fertilizer used and PH of the soil are the main Level of metals in raw white lupin: When plants grow under parameters affecting different in chemical composition of all different climatic condition, its chemical composition is different plants12. As a result the entire above mentioned factor can

IJESC, December 2019 24231 http:// ijesc.org/ influence on the content of metals in lupine grains. As table 4 VI. REFERENCE shows, Ca was found in maximum concentration compared with other metals. The Ca content of Merawi lupin was higher [1]. Volume 10 No. 8 August 2010 CHEMICAL COMPOSI (101.14±0.42 mg/100 g) than Debretabor lupin (99.29± TION, PHYSICOCHEMICAL AND FUNCTIONAL PROPE 0.66mg/100 g) and kossober lupin (87.71±0.638b mg/100 g). The RTIES OF LUPIN (. 2010;10(8):3029-3046. profile of the metal content of whit lupin seed were found to be in the order Ca > Zn > Fe> Cd. The higher level of Ca in Lupin [2]. P.Getachew, chemical composition and the effect of Seed is obvious since Ca is usually abundant in ground water traditional processing on nutritional composition of Gibto and surface water which can be easily absorbed by plants. From (Lupinus albus L.) grown in, Gojam area, M.SC Thesis in Food this result no significant different was observed on Ca content of Science and Nutrition, 2009, Addis Ababa university , Addis Debretabor and Merawi lupin, while kossober raw lupin has Ababa, Ethiopia. significantly different (P < 0.05). This suggests that due to the variation in geographical location and environmental condition. [3]. Kohajdová Z, Karovičová J, Schmidt Š. Lupin Composition Similarly the micronutrient, Fe and Zn were found in relatively and Possible Use in Bakery – A Review. 2011;29(3):203-211. higher amount in lupin grain and they are found in the range from 10.3±0.1mg/100g to 3.83±0.22 mg/100 g and 28.22±0.14 [4]. Yeheyis L. White lupin ( Lupinus albus L .), the neglected to 15.91±0.865 mg/100 g respectively. Since the major soil type multipurpose crop : Its production and utilization in the mixed of lupin growing area is Nitosoil and Acrisoil with soil pH crop-livestock farming system of Ethiopia. 2010;(May 2014). ranging between 4 and 54.So, it has better accumulation of micronutrients like Fe and Zn. Also, toxic metals Cd were found [5]. Z.Nigusie, Contribution of white lupin ( Lupinus albus L.) in the range 1.320 ± 0.0442 to 1.38 ± 0.015 mg/100g. The for food security in North-Western Ethiopia, Asian journal of presence of toxic metals Cd might be due to several factors some plant science, 11,5 ( 2012) 200-205 of these are contamination of the plant during growth, atmospheric input and the use of phosphate fertilizer. But in [6]. Orres JEMP, Randa PIA, Ari M, Urado ÄPEZ, Rbano GLU. Ethiopia, there is no enough industry to cause atmospheric Nutritional Potential of Raw and Free r -Galactosides Lupin ( pollution. So the major source of contamination of the plant with Lupinus albus Var . multolupa ) Seed Flours . Effect of Phytase toxic metals might be comes from agricultural activities, such as Treatment on Nitrogen and Mineral Dialyzability. 2005:3088- the use of animal wastes, fertilizers and pesticides and the use of 3094. irrigation with a contaminated sludge. And also storage place of the seed after harvesting, use of contaminated material for [7]. Republic C. Nutritional Value of Lupine in the Diets for transportation of the seed might be the main source of Pigs (a Review) Bohumila Písaříková, Zdeněk Zralý. 2009:399- contamination of lupin seed with toxic metals. 409. doi:10.2754/avb200978030399.

V. CONCLUSION AND RECOMMENDATION [8]. Bhowmik S, Datta BK, Saha AK. Determination of mineral content and heavy metal content of some traditionally important This study determined level of metals Ca, Fe, Zn and Cd in aquatic plants of tripura , India using atomic absorption white lupin in three major farming areas of Ethiopia using spectroscopy. 2012;8(4):1467-1476. FAAS. The result shows that the ability of white lupin seeds to accumulate relatively higher amount of Ca, Fe, and Zn. Toxic [9]. Length F. Determination of some trace metal levels in onion metal Cd was found to be comparatively at lower level. The leaves from irrigated farmlands on the bank of River. content metals in all sample of lupin were similar trained across 2008;7(10):1526-1529. the verities that could be arranged in the following order:

Ca > Fe > Zn > Cd [10]. Nawaz MS, Sheikh SA, Nizamani SM, Bhanger MI, Afridi I. Determination of mineral elements in Jamun fruit ( Eugenia Furthermore, in order to aware users about the metal jambolana ) products. 2010;20(1):1-7. composition and in order to keep users safe from health risk due to lack of minerals used, further study should be carried out [11]. Jinadasa BKKK. Preparation of a standard operation which will have collect a large number of samples from all procedure for validation of laboratory methods for trace metal major lupin growing area of the country and also it is analysis in seafood for national aquatic resources research and recommended to analyze the soil metal content on which white development agency ( NARA ), SRI LANKA. 2010. lupin is grown and to study correlation of metals between soil and lupin plants. Secondly, it is recommended that to validate [12]. Aminzadeh M, Amiri F, Abadi EA, Mahdevi K. Factors the method and to analyze each metal using another instrument Affecting on Essential Chemical Composition of Thymus rather than FAAS specially using modern instrument like, ICP- kotschyanus in Iran Former MSc Student of Natural Resources , MS, XRF etc to know the exact concentration range of metals in University of Tehran , Iran. 2010;8(7):847-856. white lupin.

ACKNOWLEDGEMENT: I am grateful to university of Gondar for the financial support and for allowing undertaking the laboratory activities. Conflict of Interest The author declare that they have no conflict of interests

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