MINERAL CONTENTS OF BARLEY GRAINS AND ITS PROCESSED FOODS (KOLO, PORRIDGE, BREAD AND INJERA) CONSUMED IN ETHIOPIA

Barley (Hordeum vulgare L.) is one of the most widely cultivated stable food crops in the world. Barley grain samples were collected from four selected areas (Bahir Dar, Bure, Finote Selam and Debre Markos) of Ethiopia and four types of processed food (kolo, porridge, bread and injera) were prepared from it. The levels of essential and non-essential metals in barley grains and its processed food were determined by microwave plasmaatomic emission spectrometry after wet digestion with a mixture of HNO3 and HClO4 (5:1, v/v). The concentration (mg/kg dry weight) in the barley grains were in the ranges K (5482-6516), Mg (546-643), Ca (445-684), Mn (7.319.80), Fe (127-439), Cu (0.88-1.86), Zn (42.8-56.8), Pb (0.39-2.73), Cd (3.01-4.66). The concentrations of all the metals in the four types of processed barley foods showed variation among each other. The results indicate that Ethiopian barley grains and its processed foods are good source of essential metals.


INTRODUCTION
Cereals are common food crops of the world. They provide food calories and proteins to human. They are staple foods for most of the population. Barley (Hordeum vulgare L.) is a cereal crop. It belongs to the tribe Triticeae of family Poaceae. It is an important cereal crop and cultivated over broad environmental conditions in the world [1][2][3]. It ranks fourth in the world in production after wheat, maize and rice [4].
Barley is used commercially for animal feed, to produce malt, and for human food applications as it is rich in protein, carbohydrates, dietary fibers, minerals, vitamins and antioxidants [5,6]. Barley is also an important crop in Ethiopia [3]. It is ranked fifth among the cereals on the basis of area of cultivation while third on the basis of production per unit area in Ethiopia. It covers 7.56% of the land under grain crop cultivation with a yield of 1.96 tones/ha [7].
Ethiopia is second largest producer of barley in Africa next to Morocco, accounting for about 26% of the total barley production in the continent [8,9]. It serves as a component of various foods. It is used in soups and stews, and the grain is used locally in bread, biscuits, and the traditional beremeal bannock. Barley is used commercially for animal feed, to manufacture malt, which is primarily used in beer production, and for human food applications [10,11]. It is used for the preparation of Ethiopian fermented bread injera, porridge, roasted snack, and in homemade beer. Barley provides basic necessities of life (food, feed, and beverages) for many people in the Ethiopian highlands [2]. Barley is used in the preparation of different recipes in Ethiopia. It is deep rooted in the culture of people's diets. It has also been used for the preparation of various types of traditional foods such as kolo (roasted grains), kita (fried bread), dabo (baked bread), beso (snack), genfo (porridge), chuko (barley conserved with butter), tihlo (dough balls), shorba (soup), kinche (cooked broken grains), and injera (pan cake bread) in Ethiopia [12,13].

Description of processed foods from barely
In Ethiopia, barley is a dependable source of food in the highlands areas. Its grain is used for the preparation of different foodstuffs, such as kolo, porridge, bread and injera, and local drinks, such as tella, borde and beer [9]. This study is concerned on four types of barley foods such as kolo, porridge, bread and injera. Kolo is a dehulled, well-roasted barley whole grain that is eaten as a snack, alone or mixed with roasted seeds such as ground nut, chick pea, sunflower or safflower seeds. Kolo is commonly eaten both at home and at various social gatherings, such as after funerals ceremonies, during many official meetings or during traveling. Kolo is also eaten by people having gastritis problems [34].

Porridge
Porridge is widely eaten breakfast foods in Ethiopia. It is most commonly consumed during a special celebration such as birthdays and weddings. Traditionally, in many parts of Ethiopia, there is a common practice to prepare barley porridge for an expectant mother. A postnatal mother eats porridge with spiced butter for breakfast and her guests are also served porridge. Traditionally porridge is also considered as a supplementary food for children aged between 6 months and 24 months to make the baby grow faster and stay healthy [12].

Bread
Bread is the most widely eaten food. It has desirability to all population rich and poor, rural and urban. It is a good source of nutrients, such as macronutrients (carbohydrates, protein and fat) and micronutrients (minerals and vitamins) that are all essential for human health. The widespread consumption of bread in the world has necessitated the study of the composition of the bread to improve its nutritive value [35].

Injera
Injera is a fermented, pancake-like soft, circular flat bread with small bubbly structures or eyes (honey-comb-like holes) on its top surface, which are produced due to the production and escape of CO2 during fermentation and baking, respectively. It is prepared from various cereals depending on availability such as teff, barley, sorghum, maize, wheat and rice or a combination of some of these cereals [3].

Sample preparation for metal determination
Samples were washed with tap water to remove the adsorbed soil and other particulate matters and the barley grains were placed in the hot water for 10 min. The clean grains were crushed in the traditional mortar to remove kernel and dried in the air. The dried grains were separated from the kernel by winnowing and rinsed with deionized water. The samples were exposed to sunlight in open air for drying.
A 200 g portion of the dried barley grains were ground using electronic blending device and stored in sample bottles. The flour was used to prepare porridge, bread and Injera.
A 200 g barley grain samples were roasted using metal pans, cooled and ground using the electronic blending device. The roasted grain is called "kolo" in Ethiopia. The powdered kolo sample was stored sample bottles.
The porridge was prepared according to Ethiopian traditional procedure. A 200 g barley flour was added to the 100 mL of boiling distilled water and homogenized with wooden spoon for 5-10 min. The prepared porridge was put in to oven at 200 o C for 3 h to complete dryness. The dried porridge sample was ground to powder and stored sample bottle.
The bread was prepared according to the traditional procedure used in Ethiopia. A 200 g barley flour and 0.5 g starter (yeast) was mixed well with 200 mL distilled water and after 1 h the dough was fermented. The metal pan was heated and the dough was placed on the pan. The pan was covered with lid, and after 10 min the bread was inverted upside down for uniform heating. The bread was taken off from the pan and allowed to cool. It was cut in to pieces and exposed to oven at 150 o C for 3 h to complete dryness until a constant weight was obtained. The dried bread sample was crushed in to powder form and stored in the sample bottle.
Injera was made by mixing 200 g barley flour with 200 mL distilled water to make a dough, and triggering a fermentation process by inoculating the dough with yeast, a starter culture, left over from a previous fermentation. The fermentation lasted 3 days, then 200 mL boiled distilled water was added after which the dough was thinned into a batter before baking on an open platter. A clay plate was heated and the dough was placed on the plate. The plate was covered with lid, and after 5 min the injera was taken off from the plate and allowed to cool. The injera was cut in to pieces and exposed to oven at 150 o C for 3 h to complete dryness. The dried injera sample was crushed in to powder form and stored in the sample bottle.
It should be noted that traditionally the heated metal pan is used for preparing bread and kolo. Therefore, the heated metal pan was used for preparing bread and kolo in the present study. Normally contamination occurs when liquid sample is in contact with the metal pan. However, the contamination will be negligible when a solid sample is in contact with the metal. Therefore, we expect negligible contamination of metals from the metal pan during preparation of bread and kolo.

Optimization of digestion procedure
Wet acid digestion is one of the methods commonly used for samples of organic matrix. It is based on changing digestion parameters like temperature, volume ratio of reagent and time. Kjeldahl apparatus is one of the wet acid digestion apparatus by which organic component are assumed to be decomposed in the form of different gases leaving metallic elements in the residue. Several trials for the optimization of digestion procedure for sample preparation were made as shown in Table 2. 30 min without removing the condenser. After removing the condenser about 10 mL distilled water was added to the solution by rinsing the neck of the round bottom flask and the tip of the condenser which was in contact with the flask to dissolve the precipitate formed on cooling and to reduce dissolution of filter paper by digest residue during filtration using Whatman filter paper in to 50 mL volumetric flask. The solution was diluted to 50 mL with deionized water.
A 0.5 g of the four types of processed foods from barley were also digested separately in the same manner as the barley grains.

Instrument calibration
MP-AES was calibrated using four series of working standards for each metal of inters. The working standard solution of each metal was prepared by diluting the standard solutions. Concentrations of the working standards, correlation coefficients of the calibration curves for each metal are listed in Table 3. From the correlation coefficients in Table 3 for each metal it is possible to conclude that the change in the emission intensity with concentration is in good positive correlation and are linearly fit.

Method detection and quantification limits
The method detection limit (MDL) and method quantification limit (MQL) were determined as three and ten times the standard deviation of blank solutions, respectively [36]. The results are given in Table 4 which clearly indicated that the studied metals can be determined at trace levels in the barley grain and its processed foods.

Validation of optimized procedure
Spiking experiments were used to validate the optimized procedure. The samples were spiked with known concentration of each metal and digested and analyzed in similar conditions using optimized procedure for sample analysis. The percentage recoveries were obtained within the range 93.4-107% which is within the acceptable range for all metals.

Statistical analysis
The statistical software (SPSS Version 22) was used for the analysis of variance (ANOVA) and Pearson correlation. The graphical expression was done using Microsoft Excel 7 in addition to data analysis.

Levels of metals
Each sample was analyzed in triplicate. The mean values were determined from the results of triplicate analysis of each sample for each metal and the results are reported in terms of mean values ± SD (Table 5 and 6).

Distribution of metals in barley grain sample
The plants uptake metals by different and complex biochemical processes. The accumulation of metals depends on the ability of particular plant to absorb metals from the soil and the availability of the minerals in the soluble forms in the particular areas. The variation in the level of metals in soil depends on the degree of pollution of the biosphere from the rapid industrialization and modern large scale agricultural activities [34][35][36]. The use of sewage sludge, pesticides, herbicides and fertilizers on agricultural lands highly affect the quality of food products for humans and animals. The distribution and accumulation of metals in barely grain are the reflections of the mineral composition of the soil and the degree of mineral pollution of the environment in which the barely plant grows. The metal concentrations of barley grain vary considerably at different locations due to differences in composition of chemicals and fertilizers and also due to variation in the soil composition and climatic conditions [34][35][36].

Levels macro essential metals in barley grains
There was a wide variation in the level of macro-essential metals among the samples from the four different sites ( The major metals in barley grains showed variations among the sample sites. This might be due to the differences in the availability of the minerals in the soluble and usable forms, differences in the natural occurrence of these minerals in the areas, and differences in the degree of contamination of the soil by these metals. The use of different fertilizers, soil acidity and water for irrigation could also be the causes for the differences [34][35][36].

Levels of essential trace metals in barely grains
It can be clearly seen from Tables 5 that Fe is the highest accumulated trace essential metal. This may be due to the nature of the soil produced either from fertilizer or animal and plant decomposition followed by Zn, Mn and Cu with concentration ranges 42.8-56.8, 7.31-9.8 and 0.88-2.73 mg/kg, respectively. The overall concentration of trace essential metals in barely grain was in the order Fe > Zn > Mn > Cu. The metals in the barley grains were in the same order in all the four sites. The lower concentration of trace minerals in the barley grain may be due to soil characteristics. There was a higher degree of variation in the level of Fe by sample sites. It was also at the highest level among the micro-essential metals. Cu exhibited small variation among the four sample sites as shown in Table 5.

Levels of toxic metals in barley grains
The levels of Pb and Cd found in the present work are presented in Table 5. The amount of Pb and Cd determined in the barley grain from Bahir Dar was higher than from Bure, Fenote Selam and Debre Markos. The amount of Pb and Cd in the barley grain from Bahir Dar may be due to use of different fertilizers and pesticides containing Pb and Cd as an ingredient. Exposure to contamination during storage and transportation by cultivators could be the other causes for the higher values. The dietary exposure to Cd and Pb were estimated to be about 0.8 µg/kg of body weight daily and 0.02-3 µg/kg of body weight daily, respectively by the World Health Organization [4]. Accordingly the dietary intake of Cd and Pb should be less than 0.007 mg/kg of body weight, per week and 25 µg/kg of body weight daily, respectively [4].

Concentration of metals in barley food samples
The levels of metals in barley foods from the four sample sites were determined and are given in Table 6. There is a wide variation in the levels of metals in the four types of barley processed foods. Among the four types of barley foods kolo contains lowest amounts of all the metals. This is expected because during the preparation of kolo the barley grains are roasted which results in opening of the grains and subsequent loss of metals. In contrast to kolo, barley injera contains highest amounts of almost all the metals. This is because injera is baked at relatively lower temperature than the roasting of barley grains during kolo preparation. Barley porridge contains slightly lower amounts of metals than the injera but relatively higher levels of metals than the kolo and bread. Barley bread contains relatively higher levels of metals than the kolo but lower levels of metals than the injera and porridge.
There is no general trend in the levels of toxic metal Cd and Pb in the four types of barley processed foods. The highest amount of Cd (4.66 mg/kg) was obtained in the Bahir Dar injera while the lowest amount of Cd (1.6 mg/kg) was found in the Debre Markos bread. The highest amount of Pb (5.32 mg/kg) was observed in the Bahir Dar bread and lowest amount of Pb (1.50 mg/kg) was found in the Bure kolo sample. The variation in the levels of Cd and Pb in the four types of barley processed food from four different areas may be due to use of different types and amounts of fertilizers and pesticides and differences in the nature and sources of irrigation water.
The variation in the levels of metals in the barley grain and its processed food depends up on the nature of individual metal, differences in the food processing, soil properties, environmental conditions and use of different fertilizers and pesticides. The variation in the levels of metal in the four types of barley foods may be also due to differences in the nature and sources of irrigation water and overall in the nature of soil and climatic conditions in the four different areas of barley cultivation.
It should be noted that there is wide variation in the mineral contents of barley grains collected from the four different sampling sites. There is also a wide differences in the preparation of four types of barley foods. The variation in the mineral contents of the barley grains from the four sampling sites is due to variations in the geographical locations and environmental conditions of the four sampling sites. While the variations in the mineral contents of the barley grains and the processed foods from the four sampling sites are due to variations in the geographical locations and environmental conditions of the four sampling sites as well as the variations in the processing of four types of barley foods. These two factors resulted in the random variations in mineral contents in the barley grains and its processed foods.

Comparison of metal levels of present study with literature values
Several studies have been done on barley grain by different investigators in different countries. However, there is no detailed study conducted on the levels of metal contents in barley grains cultivated in Ethiopia. Therefore, the results of present study have been compared with the results reported from other countries in the literature as summarized in Table 7. The comparison shows that the levels of metals found in the present study are within the ranges of levels of metals reported in the literatures except Mg, Mn and Cu which are lower than the literature values. The level of toxic metal Cd in this study is above the literature cited. However, there are variations in the levels of individual metal in the barley grain from different countries. The variations are expected due to differences in the soil properties, geographical locations, climatic conditions and agricultural practices in different countries.

Analysis of variance (ANOVA)
One-way ANOVA was applied to assess the sources of variations observed in the level of metals as to whether it came from experimental procedure or due to heterogeneity among the samples. The results showed that there is a significant difference in the mean values of all the metals in the barley grain collected from four sampling area of Ethiopia. The differences are most likely due to heterogeneity among the samples because of the differences in the geographical locations and climatic conditions of the sampling sites.

Pearson correlation of metals in barley grain
Pearson correlation coefficients were employed to correlate the level one metal over the other metal. Pearson correlation coefficients revealed that there is negative, weak and/or moderate positive correlation between metals with each other. It was found that there was a strong positive correlation of Cu with Zn and Pb in the barley grain. The results are given in Table 8. These strong correlations may be due to their common natural sources as well as from similarity in their chemical properties.

Daily intake of minerals from barley
Metal concentrations in barely (this study), the amount that a person can get from 200 g barley per day, RDI and upper limit values of metals recommended by experts and agencies for a normal adult man is given in Table 9. The amount of mineral intake by a person who consumes 200 g barley per day is shown in Table 9. The data shows that amount of major metals (K, Mg and Ca) a person can get from barley is lower than the daily recommended values. Therefore the person needs K, Mg and Ca from other sources. The amount of trace metals (Mn and Cu) that the man can get from barley is also below the required amount. Hence other diet is required for to get these metals up to the recommended values. The amount of Fe supply from barley for all the sample sites is very sufficient. The amount of Zn a person can get from barley is within the range of daily recommended intake from the Bahir Dar but not from other three sites. The values for Pb and Cd are above the allowable limits. The man must not consume large quantity of foods from barley regularly.

CONCLUSION
The levels of metals in barley grain and four types of barley processed foods were determined. The order of metals contents in the raw grain and processed foods were different from each other. This is due to the differences in food processing practices. Barley grain from the selected sites accumulated relatively higher levels of K (5482-6516 mg/kg) and Fe (128-439 mg/kg) among major and trace metals, respectively, and lower level of Cu (0.88-1.86 mg/kg). The ANOVA result at 95% confidence level suggested significant differences in the level of metals among the barley grain samples collected from the four different regions of Ethiopia. These differences may be due to the difference in the soil chemical composition and environmental conditions which control the degree of mineral absorption by barley plants. The barley grain and its processed foods are good sources of essential metals. However, the Pb and Cd contents in the barley grains are above the allowable limits. Therefore people should not consume large quantity of foods from barley regularly.