ASSESSMENT OF ESSENTIAL AND NON-ESSENTIAL METALS IN ETHIOPIAN TRADITIONAL FERMENTED ALCOHOLIC BEVERAGES

Evaluation of the levels of essential and non-essential metal concentrations in Ethiopian traditional fermented alcoholic beverages (Tella, Tej, Birz, Korefe, Keribo and Borde) was conducted using atomic absorption spectrophotometer (AAS) after HNO3/H2O2 digestion process. A total of 10 bulk samples were collected randomly from vending houses at five different sub-cities of Addis Ababa, the capital city of Ethiopia (Lideta-Kolfe, Bole-Kirkose, Yeka-Gullele, Arada-Addis Ketema and Akaki-Kaliti-Lafto) and from five nearby towns (Sebeta, Dukem, Sululta, Sendafa, and Burayu) of Oromia Regional State. The considered metals include: Ca, Mg, Fe, Zn, Cd, Cr, Cu, Ni, Pb, Co and Mn to ascertain their levels. With 88–96% recovery Ca, Mg, Fe, Zn, Cu, Cd, Pb and Mn were detected at varying concentrations in the beverages, while Co, Ni and Cr were below detection limits. All the beverages were rich in Ca, Mg and Zn. Among the trace metals found Zn was the first. The levels of the trace metals were generally low, and within statutory safe limits set by World Health Organization (WHO). Mineral concentrations were found to be significantly different among the beverages studied (p < 0.05). The levels of metals in the Ethiopian fermented beverages were found comparable with the reported ones.


INTRODUCTION
Traditional alcoholic beverages are indigenous to a particular area and are prepared by the local people using old age techniques and locally available raw materials [1,2]; accordingly, different countries, including Ethiopia have various indigenous alcoholic beverages [3][4][5][6][7]. The most familiar traditional fermented beverages are: high alcoholic beers such as Tella and Korefe; low alcoholic beers such as Keribo, Buqri, Shameta, Borde and wine such as Tej made from honey [8,9]. Similarly, the most popular distilled beverage is Areke [8,9].
Living organisms (including plants, animals and microorganisms) store and transport metals so as to get appropriate concentration for later uses in physiological reactions as well as a means of protection against the toxic effects of the metals [10]. Mineral elements play a critical role in building body tissues and regulating numerous physiological processes. They are thus essential constituents of enzymes and hormones; regulate a variety of physiological processes, and are required for the growth and maintenance of tissues and bones [10]. In contrast, essential elements at excessive levels and toxic metals may lead to severe effects that include enhance premature aging and cause oxidative damage [11], reduced growth and development, cancer, organ damage, nervous system damage, and in extreme cases, death [12][13][14][15]. Toxic metals such as Cd and Hg are known to impair kidney functions and reproductive capacity, cause hypertension, tumor and hepatic dysfunction; whereas Pb can cause renal failure, liver damage, impaired hearing or cause mental retardation and in women, a shortened gestation period [11]. However, unlike in commercial alcoholic products, the role of metals in promoting or degrading the quality of traditional alcoholic beverages is not well documented.
containing 1000 mg L -1 in 2% HNO 3 of the metals (Ca, Mg, Fe, Zn, Mn, Cu, Cr, Co, Ni, Cd, and Pb) (Buck Scientific Puro-Graphic tm ) were used for preparation of calibration standards and in the spiking experiments. Working standard solutions were obtained by suitable dilution from stock solution. Distilled-deionized water was used throughout the experiment for sample preparation and dilution, and rinsing of apparatus prior to analysis.

Apparatus and instruments
A refrigerator (Samsung Hitachi, Japan) was used to keep the fermented alcoholic beverage samples till the analysis. 250 mL round bottomed flasks fitted with reflux condensers were used on Kjeldahl apparatus hot plate to digest the fermented alcoholic beverages samples. Flame atomic absorption spectrophotometers (Analytik Jena ZEEnit 700P, Germany) equipped with deuterium arc background connectors and hollow cathode lamps with air-acetylene flame was used for the analysis of the analyte metals (Ca, Mg, Cr, Ni, Cu, Zn, Cd, Pb, Mn, Co and Fe) in the samples. A micropipette (100-1000 μL) was used for the volume measurement of reagents and standards.

Sampling
The most popular Ethiopian traditional fermented beverages, Tej (honey wine), Tella (a malt beverage like beer), Borde, Birz (honey water drink), Korefe and Keribo were selected. A total of 10 bulk samples were collected randomly from vending houses at five different sub-cities (Lideta-Kolfe, Bole-Kirkose, Yeka-Gullele, Arada-Addis Ketema and Akaki-Kaliti-Lafto) of Addis Ababa, the capital city of Ethiopia and from five nearby towns (Sebeta, Dukem, Sululta, Sendafa, and Burayu) of Oromia Regional State. 150 mL of the beverages were taken from the three sites of the towns and woredas of Addis Ababa to prepare 450 mL bulk samples of each type. All the samples were collected using glass amber bottles. The prepared bulk samples of the beverages were kept in a refrigerator at 4 ºC until the analysis time.

Digestion of samples
The digestion procedures were optimized by varying: (i) the ratio and amount of HNO 3 and H 2 O 2 added to the sample, (ii) the digestion time and (iii) the temperature required for the digestion. The procedures which consumed least amount of the reagents and lower time and temperature for the digestion, giving clear solution with no precipitate were considered as the optimum procedure. Based on these parameters, different optimization results for different beverages were obtained. Smaller amounts of reagents and lower temperature and time than the optimum parameters resulted in incomplete digestion of samples which were indicated by colored and unclear digest with precipitates. The optimized digestion procedures are indicated in Table 1.

Method performance evaluation
Method validation is the process used to confirm that the analytical procedure employed for a specific test is suitable for its intended use. Results from method validation can be used to judge the quality, reliability and consistency of analytical results; it is an integral part of any good analytical practice. The parameters: accuracy, precision, specificity, limit of detection and limit of quantification are used for method validation. In this study, precision of the results was evaluated by standard deviation of the results of the nine measurements, triplicate digestion and triplicate readings. The method detection limits for each metal were calculated from the standard deviation(s) of the replicate analysis of six blanks prepared by the optimized procedure: MDL = 3×S of blank [42,43].

Recovery test
The accuracy of the analytical procedure was investigated by spiking a suitable known amount of the analyte metals into a test portion of the sample having a known concentration of the analyte, and analyzing the spiked test portion along with the original sample. The recovery test for all samples was performed in triplicates.

Determination of the metals in alcoholic beverages samples by FAAS
Stock standard solutions containing 1000 mg L -1 in 2% HNO 3 of the metals, Ca, Mg, Mn, Fe, Cu, Zn, Ni, Co, Pb, Cd, and Cr were used for the preparation of calibration standards and in the spiking experiments. All the analyses were carried out using flame atomic absorption spectrophotometer at the wavelengths specific for each metal. For the preparation of calibration curve, a series of four working standards were prepared for each metal. The working standards were prepared daily from the intermediate standards, that were earlier prepared from the stock solutions (1000 mg L -1 ).

Statistical analysis
Mean values obtained for the metals studied in the alcoholic beverage samples were compared by One-Way ANOVA at 95% level using IBM SPSS statistics version 20; assuming that there were significant differences among them when the statistical comparison gives p < 0.05.

Figures of merit
The analytical wavelengths, the correlation coefficients, and the correlation equations of the calibration curves for the determination of metals in fermented alcoholic beverage samples by FAAS are given in Table 2. The correlation coefficients of all the calibration curves were > 0.999 and these correlation coefficients showed that there was a very good correlation (relationship) between concentration and absorbance.

Method validation
To determine method detection limit, replicate analyses for 6 blank samples were performed and the pooled standard deviation of the six reagents blank was calculated. The detection limits were obtained by multiplying the pooled standard deviation of the reagent blank by three. The method detection limits of each metal for each sample type are given in Table 3. The method detection limits were generally comparable with that of the instrument for all samples.

Recovery
The accuracy of the analytical procedure was investigated by spiking 25% amount of the analyte metals recorded in the samples into a test portion of the sample having a known concentration of the analyte, and analyzing the spiked test portion along with the original sample. For elements having a result below detection limit, 0.25 mg L -1 was the amount added. The recovery results are presented in Table 3.

Determination of metals in Ethiopian traditional fermented alcoholic beverages samples by FAAS
The concentrations of Ca, Mg, Cu, Zn, Ni, Pb, Cd, Cr, Mn, Fe and Co in the samples were determined by AAS using an air/acetylene flame at the wavelengths specific for each metal. To bring the concentration within the linearity range further dilution was done for Ca, Mg and Zn. All the analyses were carried out in triplicate. The summary of mean concentrations (mean  SD) for the metals in different types of traditional alcoholic beverages are presented in Tables 4  -6.

Comparisons of the levels of the metals among Ethiopian fermented alcoholic beverages
Among the beverages, Ca concentration was maximum in the Tella (161 mg L -1 ) and minimum in the Borde (1.17 mg L -1 ) and Mg content was also maximum in the Tella (40.4 mg L -1 ) and minimum in the Borde (0.39 mg L -1 ). Zn content was maximum in the Tella (10.9 mg L -1 ) and minimum in the Tej (1.32 mg L -1 ) while Fe content was maximum in the Korefe (0.028 mg L -1 ) and Borde (0.028 mg L -1 ) and minimum in the Tella (0.008 mg L -1 ). Cu content was maximum in the Borde (1.15 mg L -1 ) and minimum in the Birz (0.038 mg L -1 ), whereas it was below the detection limit in the Tella and Tej. Cd concentration was maximum in the Keribo (2.37 mg L -1 ) and minimum in the Birz (1.54 mg L -1 ), while it was below the detection limit in the Tej, Borde and Korefe. Pb content was maximum in the Keribo (0.48 mg L -1 ) and minimum in the Korefe (0.13 mg L -1 ) while it was below the detection limit in the Tej, Borde and Birz. Mn content was maximum in the Borde (0.018 mg L -1 ) and minimum in the Tej (0.003 mg L -1 ). Cr, Ni and Co contents were found below the detection limit in all the beverages except Cr in Tej (0.10 mg L -1 ) and Ni in Tella (4.73 mg L -1 ).

Distribution pattern of the metals in Ethiopian fermented alcoholic beverages
In general, the concentration of the metals in Tella samples was in the order: Ca > Mg > Zn > Ni > Pb > Cd > Fe. Metals such as Cu, Co, Mn and Cr in all samples were found below detection limits. In Tej (Table 5) like in Tella Ca (2.16 -11.8 mg L -1 ) was the leading followed by Mg (1.08 -7.4 mg L -1 ) and Zn (0.35 -3.42 mg L -1 ), respectively. In general, except Ni, Cu, Cd, Co and Pb which were found below detection limits, the decreasing order of metals with average concentrations was, Ca > Mg > Zn > Cr > Fe > Mn. Likewise, the decreasing orders of metals in terms of their average concentrations were: for Birz Ca > Mg > Cd > Zn > Fe; for Keribo Ca > Mg > Zn > Cd > Cu > Pb > Fe; for Korefe Ca > Zn > Mg > Cu > Pb > Fe and for Borde Zn > Ca > Cu > Fe > Mn (Table 6).
Among the Ethiopian fermented alcoholic beverages, Tella was found higher in Ca, Mg, Zn concentrations and was followed by Tej, Birz, Keribo ̴ Korefe and Borde, respectively. This is mostly by variation in composition of the raw materials. In most fermented alcoholic beverages, especially Ni, Cr, Co and Mn were below detection limits. This has an agreement with the ultra micro-elements composition (< 10 μg L -1 ) of fermented beverages such as wine [44]. In Tej samples most of the metals especially heavy metals were below detection limits. This is mostly from its preparation pattern, especially hops or (Rhamnus prenoides (Gesho)) boiling with portion of must [45]. Toxic metals such as Pb and Cd were found in Tella, Korefe, and Keribo samples; on the other hand, they were not detected in Tej, Birz and Borde samples. Korefe was rich in Fe and Cu than Tella. This is because Korefe is semi-liquid; as a result, it has additional Fe and Cu which are found in the solid part. Likewise, Birz was rich in Zn, Cu, Fe and Mn than Tej. Since Birz has less fermentation time than Tej, therefore, the probability of these metals to be diminished by yeast is not significant. In addition, in the preparation of Birz, there is no treatment of the wort with hops-boiling. Birz, Korefe, Keribo and Borde were found rich in Cu whereas it was absent in Tella and Tej. This is mostly related with the action of yeast and the fermentation time required by the beverages. In general, the total metal content of Ethiopian fermented alcoholic beverages varied widely. This is because the metallic content in fermented alcoholic beverages are depending upon, the metallic content of raw materials, the effect of processes such as hop-boiling, fermentation and filtration [45].
In general, though, there was a difference with the reported ones for few exceptional, the levels of metals in Ethiopian traditional fermented alcoholic beverages were almost comparable with reported metal contents of different alcoholic beverages like wine, beer, and others. In addition, most of the beverages were found below the limits set by WHO, but for toxic metals, Cd and Pb some few samples have levels that exceeded the WHO limits.

Statistical analysis
Statistical analysis of data was made to verify whether there was a significant difference in metal contents between the six Ethiopian traditional fermented alcoholic beverage varieties analyzed. In the present study, the significance of variation within sample and between samples has been studied using one-way ANOVA and calculations were made using SPSS software.
No significant difference (p > 0.05) at 95% confidence level was observed for Ca, Mg, Cu and Mn concentrations in all the beverages varieties analyzed. On the other hand, significance difference (p ≤ 0.05) at 95% confidence level was observed for Cd, Fe, Pb and Zn concentrations in all the beverages varieties analyzed. For some metals, there was significant variation (p ≤ 0.05) at 95% confidence level among some beverage varieties while no significant variation was observed among the remaining beverage varieties when pair-wise comparison was performed. Table 7 summarizes pair-wise comparison of each metal in each sample with the remaining sample types. Absence of significant difference in some mineral nutrients in beverage varieties indicates the presence of similarities in certain factors or variables such as composition of the raw materials. Similarly, presence of significant difference in concentration for some minerals indicates the differences in the sources of metals in the beverages. This is because the metallic content in fermented alcoholic beverages depends upon the metallic content of raw materials, the effect of processes such as hop-boiling, fermentation and filtration.

CONCLUSIONS
Traditional fermented alcoholic beverages of Ethiopia were investigated for their major, trace and toxic metallic contents. The results confirmed that metals concentrations in Ethiopian fermented beverages differ among samples of different types and also among samples of the same types because metals in beverages are derived from various raw materials, equipment and brewing processes. In terms of metallic content, the Ethiopian fermented alcoholic beverages were found comparable with beer, wine and African traditional beverages. Most metals in these beverages were below the WHO limits. However, for toxic metals Cd and Pb some samples have higher values than the limit set by WHO.