MICROBIOLOGICAL ANALYSIS OF KUNUN-ZAKI: A FERMENTED MILLET DRINK IN BENIN CITY, EDO STATE, NIGERIA

Microbiological and physicochemical analyses were carried out on samples of fermented millet drink Kunun Zaki. Fifteen samples were obtained from Ikpoba Hill Market and Aduwawa Quarters. The microbiological analysis was carried out using the standard plate count technique to determine the total microbial population. The mean count of bacteria and fungi was 2.57x10 cfu/ml and 0.98x10cfu/ml respectively. Microorganisms identified were Lactobacillus sp, Bacillius sp, Staphylococcus aureus, Streptococcus sp, Escherichia coli, Pseudomonas sp, Mucor sp and Fusarium sp. The mean value of the pH and titrable acidity was 4.26±0.09 and 2.73±0.08ml 0.1M NaOH respectively. The moisture content was high with a mean of 85.90±0.95 and mean solid content of 14.1±0.95. This study has shown that kunun-zaki sold in Ikpoba Hill Market and Aduwawa Quarters is highly contaminated with microorganisms. Practices of good hygiene are therefore necessary in an environment where kunun-zaki is produced, stored, prepared and packaged.


INTRODUCTION
Food borne intoxication and poisoning is common in man and local beverage and drinks may be common sources of infection (WHO, 1996). The consumption of these local drinks is of public health significance. Hence, local drinks may serve as vehicles for food-borne pathogens such as Staphylococcus, Salmonella, Shigella, Listeria, Escherichia etc. Millet is one of the main cereal staples of West Africa. There are various species of millets found and they include Bulrush millet (Nigeria millet), Pennisetum typhoideun in America, popularly known as Pearl millet, Pennisetum mericanum, foxtail millet (Setaria italic) and finger millet (Eleusine coracana). The grain had its origin in Central America and West Asia (Efiuvwevwere and Akoma, 1995). They are widely grown in Ghana, Cameroon and throughout the Savannah zone of Nigeria such as Bauchi, Sokoto, Katsina and Kano States (Ònuorah et al., 1987). The quality of millet determines its use, if the grain is to be used as seed for planting, it should be pure, have a good yield and be free from disease and insect pest. If the grains are to be eaten by man or livestock, a high protein content is desirable and it must taste good (Oranusi et al., 2003). Kunun-zaki is an indigenous fermented non-alcoholic beverage that is widely consumed for its thirst quenching properties. Though consumed throughout the year, it is extensively consumed during the dry season. The drink can also be produced Food Scientech Journal 3(2) 2021 from fermented sorghum, guinea-corn and maize (Amusa and Odunbaku, 2008).
It is a popular drink with characteristic sweet-sour taste and fermented cereal drink, it is consumed both in rural and urban areas of Northern Nigeria and enhances lactation in nursing mothers, increase libido, sustain erection and increase sperm count (Amusa and Ashaye, 2009). Other food products derived from these cereals include; malted alcohol known as 'Oyokpo' 'pito' or 'burukutu' (Ekanem et al., 2018;Innocent et al., 2011). Like other grains, maize and millet contain essential nutrients such as vitamins A, B and C, minerals like potassium, zinc, anti-diabetic, anti-diuretic and anti-cancerous compound which are useful in treatment of diseases like diabetics, cancer and urogenital tract infections (Amusa and Odunbaku, 2008).
Organisms usually associated with millets grains include Aspergillus sp, Penicillim sp., as well bacteria like Bacillus sp, Staphylococus aureus and Lactobacillus sp. (Elmahmood and Doughari, 2007) which predispose consumers to food borne infection and diseases. In a study on safety and quality evaluation of street foods sold in Zaria, Nigeria, Umoh and Odoba (1999) found the mean aerobic counts for kunu ranged from 3.67±0.67 to 4.29 ± 1.14log10/g with those sold by mobile food sellers having a significantly higher mean (4.29±1.14 log10/g) than that sold by stationary food sellers (3.67±0.4 log10/g) with Bacillus cereus and Staphylococcus aureus being the major bacteria isolated. Oranusi et al. (2003) also worked on the hazards and critical control points of kunu-zaki, in Northern Nigeria and discovered that S. aureus contamination in all the samples after pitching increased to 2.90 log10cfu ml -1 while Bacillus counts increased from 1.69 to 4.36 log10cfu ml -1 .
The current food safety challenges rises slowly over the years and requires strategic efforts to be controlled (USFDA, 2008). These products are being produced on daily basis for sales in markets, offices, schools, motor parks and as drinks during festivities, weddings and naming ceremonies. The production procedures and sales of these products are carried out under unhygienic conditions which may predisposed them to many pathogens of public health importance. This study seeks to give an in-depth focus into the sources of microorganisms that could contaminate Kunun-zaki and also identify practices that would aim at reducing the microbial load of the beverage.

MATERIALS AND METHODS Sample Collection
Fifteen samples of kunun zaki drinks were purchased from different sales outlets in Ikpoba hill market and Aduwawa quarters in Benin City, Edo State during May-June 2011. These samples were placed in sterile bags and transported in a cold pack to the laboratory for analysis within one hour of collection. Statistical analysis of the sample results were carried out using descriptive statistics (mean and standard deviation).

Preparation of Media and Samples
1 ml of every Kunun zaki beverage was placed into 9 ml of distilled water and serial dilution was carried out up to 10 -10 dilution. The culture media used for microbiological analyses which include nutrient agar, MacConkey agar, potato dextrose agar, eosin methylene blue agar and mannitol salt agar, were prepared according to manufacturers' instruction (Gadage et al., 2004).

Isolation of Microorganisms
Using pour plate method, 1 ml from the dilutions were inoculated on Nutrient agar, MacConkey agar, potato dextrose agar for the enumeration of total microorganism, coliform count and enumeration of fungi respectively. After inoculation, Petri dishes containing Nutrient agar, MacConkey agar and Mannitol salt agar were incubated at 37 o C for 24 h (Mannitol salt agar was used for the isolation of Staphylococcus), while inoculated plates containing Potato dextrose agar was incubated at 28 o C for 5-7 days (Gadage et al., 2004).

Identification and Characterization of isolates
The isolated organisms were characterized and identified based on their cultural, morphological, and biochemical tests (Buchanan and Gibbson, 1974;Gadage et al., 2004). The colonies were sub-cultured, Gram stained and subjected to biochemical tests such as oxidase, catalase, coagulase, urease, citrate, indole and sugar fermentation tests. These results were also checked on PIBWIN (Probable identification of microorganisms).

Physicochemical analysis
pH of the Kunun zaki drinks was measured by dipping the pH electrode into 10 ml of the beverage placed in a beaker, and the reading were recorded. Total titrable acidity was measured as percentage lactic acid by adding 3 drops of phenolphthalein indicator into 10ml of the drink placed in a conical flask and thoroughly shaken. The mixture was then titrated against 0.1 M NaOH (Sodiun hydroxide) to a pink color end point and the titre value was calculated. Moisture and solid content were determined by methods described by Ceese (1995).

RESULTS AND DISCUSSION
The results of this study showed that the mean total viable bacterial and fungal count was 2.51x10 7 cfu/ml and 0.98x10 7 cfu/ml respectively as shown in Table 1.
These results revealed that samples gotten from Aduwawa market (B2) had highest bacterial count while Sample from Ikpoba hill market (C1) has the lowest bacterial count. It also showed that samples gotten from Aduwawa market (A1) had highest fungal count while Sample from Ikpoba hill market (C3) has the lowest fungal count. This showed that kunun drinks sold in Aduwawa are heavily contaminated.
In many earlier reports, the pH was 4.3 (Ekanem et al., 2018 ); 3.80 and 3.99 reported by Innocent et al. (2011), 2.42 to 3.83 recorded by Otaru et al. (2013), 5.25 to 5.65 reported by Amusa and Ashaye, (2009). The acidity of the kunu drinks may be due to the presence of some bacteria which help in acid fermentation of the kunu products (Ekanem et al., 2018). The results of the investigation showed that the samples of Kunun-zaki contained a fairly large microbial population. The high microbial densities could be related to the fact that usually a heterogeneous population of microorganism are usually involved in the fermentation process and also that foodstuffs are also susceptible to microbial contamination during the processing and storage.  Amusa and Odunbaku (2008); Oyenuga et al., (2003); Elmahomood and Doughari (2007). Sources of the organisms may be traced to the cereals as had been reported by Food Scientech Journal 3(2) 2021 Efiuvwevwere and Akoma (1995). The presence of Lactobacillus indicates that kunun-zaki is a lactic acid bacteria fermented beverage and it is not unexpected because they help in fermentation process. Lactobacillus in food samples tend to dominate by preventing other pathogenic microorganisms from surviving in the beverage, and their ability to produce lactic acid reduces the pH of food medium. Most food poisoning organisms cannot tolerate low pH, therefore the isolation of Lactobacillus favors this fact. Efiuvwevwere and Akoma (1995) reported the presence of some bacteria including Lactobacillus spp in kunun drink. The presence of Staphylococus aureus indicate contamination from handlers as Staphylococcus aureus is a normal flora of the skin, nose, throat, palms, hairs and mucus membrane and a common etiological agent of septic arthritis (Charles et al., 2005; Emmanuel-Akerele and Uchendu, 2021). The contamination could have been contracted by sneezing or by picking of the nostrils by food handlers. Staphylococus aureus can also produce Staphylococcal bacteraemia and abscesses in cell during food infection. Escherichia coli in food is an indication of faecal contamination of product. However, E. coli is a normal floral of the intestinal tract of man, presence of it in excess could lead to gastroenteritis and bacterial diarrhea disease (Emmanuel-Akerele and Uchendu, 2021). This fail to agree with the WHO standards which suggested that water that contain >10coliforms/100ml or one E. coli/100ml with or without other coliforms is unsatisfactory for human consumption. Streptococcus sp. may also have been enumerated from the beverage as a result of the handlers, since it is also normal flora of the throat and the buccal activity. The presence of Bacillus could render a beverage unsuitable for human consumption (Innocent et al., 2011). It is possible that the Bacillus is a spore former and as such the spores were easily distributed and was able to withstand high temperature and pH to fully germinate (Otaru et al., 2013). The organism has the potential of causing an array of infections. The presence of Pseudomonas is not of great significance due to their low population. They could have occurred due to environmental contamination. The pathogenic microorganisms isolated exceed permissible limit (Efiuvwevwere and Akoma, 1995;USFDA, 2008). Production of kunun drinks should be done under hygienic conditions to avoid proliferation and spread of these pathogenic organisms.
The fungal isolates present could be traced right to when the grains were either being harvested or stored. The presence of Aspergillus, Penicillium, and Fusarium in the kunun-zaki samples might not be too surprising as they are known as common spoilage organism of carbohydrate foods as   well as storage microflora of many cereals including sorghum (Ekanem et al., 2018;Omonigho and Osubor, 2002). The fungal may produce spores attached to the grains and overcome adverse condition during the preparation and finally germinate in the finished product. The presence of these fungi such as Aspergillus, Mucor, Fusarium and Rhizopus is associated with spoilage of the beverage (Oyenuga et al., 2003). Some of   Efiuvwevwere and Akoma (1995) and Amusa and Ashaye (2009), who attributed these to the presence of lactic acid bacteria. The acidity tends to increase with increase in fermentation period resulting into spoilage. Consequently, the low pH value may have encouraged the growth of fungi and this could be responsible for the species of microorganism isolated. The pH brought about a corresponding increase in the titratable acidity and sour taste flavour of the kunun-zaki drink. The moisture and solid content of the analyzed Kunun-Zaki had overall mean of 85.90±0.95 and 14.1± 0.95 respectively. The low moisture contents could be due to the ease of moisture loss during production (drying resulting from hydrolytic enzyme activities during malting and incubation). The relatively high solid content indicates that the kunun can develop off flavor and colour if stored at room temperature for few days. The presence of all these organisms indicated in this report are of great public health concern as it has passed the permissible limit by WHO (WHO, 1996;Ekanem et al., 2018), and in situations where the beverage is contaminated, quick medical