Organic Contaminants and Microbial Load of Native Beers Locally Prepared Within Jos Metropolis

collaboration between all authors. Author SYG designed the work and analyzed the samples. Authors KHJ and SGM collected and processed the samples. Each author proofread and edited the draft manuscript before submission for publication in this Journal. ABSTRACT Introduction: Ethyl alcohol toxicity is implicated in impaired immune system, disease conditions such as hepatitis, cirrhosis, and cancer. The incidence of these ailments among drinkers of native beers: burukutu , pito and goskolo Thin layer chromatography culture Gram staining, catalase and biochemical tests applied to the aim of the work. Results: Results indicated the presence of not only ethyl alcohol but of ethanal, Streptococci, Candida krusei, Candida pseudotropicalis, Candida tropicalis and Lactobacilli in burukutu and pito samples. No microbial growth was observed for the control. There were no spots on TLC plates for goskolo . There were other spots on the TLC plates for burukutu and pito samples which did not match the R F values of ethanol, acetate and ethanal standards implying the presence of other contaminants in the samples. Candida and Streptococci species are pathogenic, ethanal is toxic, and presence of other spots on TLC plates could be for other toxicants in the samples. Conclusion: Contaminants in the native beer samples may contribute significantly to the observed complications of disease conditions associated with chronic consumption of native beers.


INTRODUCTION
Foods, including native beers, become hazardous when contaminated by biological agents such as bacteria, fungi and their toxins. For example, they may contain mycotoxin contaminants such as zearolenone, citreoviridin, moniliformin and ochratoxins, which are secondary metabolites of Aspergillus flavus, Fusarium graminaerum, Pennicillium citreoviride, Fusarium moniliforme and Aspergillus ochraceus respectively, or bacterial toxins, produced by Staphylococcus aureus and Clostridium botilinum respectively [1,2,3].

Experimental Design
Five samples each of burukutu and pito were purchased fresh from the field randomly in Jos metropolis. Qualitative analysis of Burukutu, Pito and Goskolo (banned liquor in the area) samples for acetate, ethanal and ethyl alcohol presence was done against the respective standards by thin layer chromatography.

Microbial Profile of Samples of Burukutu and Pito
Five (5) samples each of burukutu and pito were analysed for the presence of microorganisms.

Nutrient agar medium
14 g was dissolved in 500 ml of distilled water in a 1000 ml bottle. The bottle was made airtight and autoclaved at 121°C/in 2 for 15 minutes. Thereafter, it was removed and kept at room temperature to cool. The molten agar was then poured into ready-to-use sterile Petri dishes and allowed to solidify. The plates were subjected to sterility check by incubating them un-inoculated in an upside down position in an incubator at 37°C for 24 hours. They were deemed not contaminated if no growth was observed the following day. The plates were then inoculated with native beer samples using sterile wire loop and incubated at 37°C for 24 hours in oven.

Universal beer agar
Beer samples were diluted as follows: 1 ml aliquot of a beer sample was pipetted into a sterile 20 ml sample bottle followed by addition of 9ml sterile distilled water which was aseptically transferred, mixed thoroughly and allowed to settle. 1 ml of the diluted sample was measured using a 2 ml sterile pipette and transferred into labeled sterile Petri dishes; subsequently, 15 ml of sterilised molten UBA medium was poured and mixed gently with the diluted sample and the agar medium to ensure homogeneity and then allowed to solidify. The prepared Petri dishes were then incubated in an incubator at 37°C in inverted position for 72 hours.

Gram Staining
Beer samples were stained both with primary stain (Gram stain) and secondary stain (safranin). Using immersion oil, slides were viewed under microscope (at×100 magnification).

Catalase Test
This was carried out on colonies thus: 3 drops of hydrogen peroxide (H 2 O 2 ) were added onto a sterilised glass slide. With the aid of a sterile glass rod, some colonies of the organism were picked from the grown culture in the Petri dish and added on to the H 2 O 2 solution on the slide. Observations were made and results recorded.
Biochemical tests of samples were performed by inoculating them in glucose, xylose, sucrose, galactose, urea slant, raffinose, dolcitol and inositol; thereafter, they were incubated at 37°C for 48 hours in an incubator.

Determination of Volatile Organic Compounds in Burukutu and Pito
This was done by distillation followed by thin layer chromatographic method. Filtered samples were distilled at 78°C over a period of 4 hours. Resultant distillates and residues were kept in labeled capped test tubes and refrigerated at 0°C until needed for TLC analysis.
For the TLC analysis, glass plates were used as inert supports to each of which silica gel slurry of 0.25 mm thickness was layered having been prepared by mixing 50 g of the silica powder in 95 mls of distilled water. Plates were activated at 105°C for 1 hour. Samples were spotted on the TLC plates using capillary tubes. Mobile phase used contained n-hexane/petroleum ether in the ratio 50:70 v/v respectively. Spotted TLC plates were placed vertically in an air tight chromatographic tank saturated with the mobile phase with level just below the origin. The set up ran for 55 minutes after which the solvent front was marked off immediately using pencil. To identify the spots on the TLC plates, 50% H 2 SO 4 in water (v/v), 10% H 2 SO 4 in methanol (v/v) and iodine crystals were used. Tables 1 and 2 bear results for TLC analysis of ethanol metabolites in burukutu, pito and goskolo respectively. Numerous spots were obtained for both distillates and residues. Among these spots, the relative fronts (R F ) values of some matched those of acetate, ethanal and ethyl alcohol standards. Burukutu had more spots than Pito.

Organic Metabolites
No spots were observed for Goskolo samples.
The result for pito indicated spots which were fewer than in burukutu; these spots meant, in addition to ethyl alcohol, that there were other components in the samples; residue of pito gave rise to three spots whose R F values were 0.7, 0.5, and 0.1 respectively.
The R F value for ethyl alcohol standard and that of residue number two spot were the same--hence pito contains ethyl alcohol even though there were other spots on the plate that did not correspond with those of the standards used indicating the likely presence of other components whose standards may not have been used during the experiment.  Table 3 bears results for goskolo samples where no spots were obtained. Perhaps an appropriate or suitable mobile phase was not used and therefore the seeming 'zero' spot.

Microbial Profile
The results of the screening for microorganisms in the samples are summarised in Tables 4 and 5 where different species of microorganisms were isolated in burukutu and pito samples; the most prevalent microorganisms in the samples were Saccharomyces cerevisiae, Candida krusei, Lactobacilli strains, and Streptococci strains in burukutu.
In pito samples, Candida pseudotropicalis, Candida tropicalis were the dominant. Goskolo was not subjected to microbial analysis.

Saccharomyces cerevisiae, Candida krusei, Streptococci species, and Candida tropicalis
were isolated in all the test groups but control. The control used was star® (a brand of factorybased lager beer). They were confirmed to be present using biochemical tests. Observations were compared with standard references in literature. Gram staining native beer samples indicated the presence of Streptococci species (gram positive), yeast cells, and Lactobacillus.

DISCUSSION
The aim of this work was to analyse for the presence organic contaminants including microorganisms present in native beer samples. Contaminants present in native beers could themselves be toxic. [6] reported contamination of native beers by iron; also, [7] reported contamination of traditional alcoholic beverages by both zinc and manganese. Alcoholic solutions of salts of cadmium, lead, iron and zinc caused adverse effects on some hepatic and nephrotic parameters [8].
The results of TLC of burukutu samples indicated several spots some of which had identical relative fronts (R F ) values compared with those of standards of acetate (ethanoic acid), ethyl alcohol and ethanal (acetaldehyde)--hence acetate (ethanoic acid), ethyl alcohol and ethanal were present in burukutu. The presence of ethanal (acetaldehyde) in burukutu samples suggests that drinkers are at risks of its toxic effects. Acetaldehyde is a metabolite of ethyl alcohol oxidation which is toxic and could cause damage to, especially the brain inducing behavioural abnormalities, impaired memory, and sedative effects; it is also a carcinogen [9]. Acetaldehyde, the major alcohol-reactive metabolite, has been detected in the intestine of Wistar rats after alcohol exposure [10,11]. Therefore, when drinkers of burukutu exhibit these abnormal behaviours and signs, it may be the result of synergistic action between acetaldehyde and ethyl alcohol or that of acetaldehyde. However, [12] reported that acetaldehyde modulates rather than mediates some of ethyl alcohol toxic effects whereas [13] reported that acetaldehyde does not contribute at all to the pharmacological effects of ethyl alcohol contending that in vivo concentration of acetaldehyde in target organs are insufficient to induce significant pharmacological actions. It readily reacts with the amino moiety of polypeptides/proteins and amino acids forming adduct thereby causing mutation and hence impaired function of the protein. It induces the deficiency of vitamin B1 which is critical to the function of the brain; furthermore, acetaldehyde induces deficiency of NAD + and niacin, the consequences of which is necrotic! Another metabolite of ethyl alcohol oxidation present in burukutu samples is acetic acid generated due to the catalytic action of aldehyde dehydrogenase, the enzyme that catalyses the oxidation of acetaldehyde (ethanal) to acetate. By its catalytic action, aldehyde dehydrogenase generates acetic acid which is a slow process. NAD + is the coenzyme required to activate both alcohol dehydrogenase and aldehyde dehydrogenase. In the conversion of acetate to acetylCoA, the equilibrium favours non-formation of acetylCoA and therefore acetate appears in blood whose accumulation causes acidosis.

Plate 1. Control; no growth after 24 hours of incubation at 37°C
That there were other spots with their corresponding R F values suggest that burukutu and pito contain components other than ethyl alcohol alone; drinkers therefore ingest ethyl alcohol along with other undesirable components. The extent of contamination was higher in the residues than in the corresponding distillates. In reality, sellers stir the product well before serving and hence the crude, not the distillate, is the one actually ingested. spoilage microbes, food utensils, packaging materials, and domestic water are sources of potentially toxic chemicals in foods [14].

Plate 3. Grown colonies on nutrient and blood agar after 24 hours of inoculation
In most cases, the catalase tests results for samples were positive (white effervescence on the slides) following addition of hydrogen peroxide solution [15].
Streptococci are pathogenic gram positive species which are implicated in meningitis, pneumonia, tonsillitis, septic arthritis, otitis media, and scarlet fever [16]; also cause colonisation of mucosal surfaces of the host naso-pharynx and upper airways [17,18,19]. Therefore, such conditions in drinkers of native beers might be due to this microbe or in synergy with alcohol since alcohol is known to suppress immune system [20]. 6 higher in the residues than in the corresponding sellers stir the product well before serving and hence the crude, not the distillate, is the one actually ingested. Food spoilage microbes, food utensils, packaging materials, and domestic water are sources of potentially toxic chemicals in foods [14].

nutrient, MRS and agar after 24 hours of inoculation nutrient and blood after 24 hours of inoculation
In most cases, the catalase tests results for positive (white effervescence on the slides) following addition of hydrogen are pathogenic gram positive species which are implicated in meningitis, pneumonia, tonsillitis, septic arthritis, otitis ever [16]; also cause colonisation of mucosal surfaces of the host pharynx and upper airways [17,18,19]. Therefore, such conditions in drinkers of native beers might be due to this microbe or in synergy with alcohol since alcohol is known to suppress

CONCLUSION
From the foregoing, organic toxicants such as ethanal as well as Streptococci species, which are pathogenic, are present in native beers. Hence, consuming native beers predisposes the drinker to these pathogens more so that the sanitary culture of the 'brewers' of the native beers is generally suspect which favours microbial growth.

Plate 5. Microbial catalase test results after inoculation; effervescence formation solution
From the foregoing, organic toxicants such as and Candida species, which are pathogenic, are present in native beers. Hence, consuming native beers predisposes the drinker to these pathogens more of the 'brewers' of the native beers is generally suspect which favours Authors have declared that no competing Okoye ZSC. Fusarium mycotoxins in maize harvested from farms in Jos Bioscience Research -28.