DOI: https://doi.org/10.4316/fens.2021.041 392 Journal homepage: http://fens.usv.ro/index.php/FENS Journal of Faculty of Food Engineering, Ştefan cel Mare University of Suceava, Romania Volume XX, Issue 4 - 2021, pag. 392 - 402 MICROBIOLOGICAL, PHYSICOCHEMICAL, NUTRITIONAL AND ANTI- NUTRITIONAL EVALUATION OF LOCALLY MADE NON-ALCOHOLIC KUNUN ZAKI BEVERAGE SOLD IN LAGOS STATE, NIGERIA *Bukola Caroline OGUNYEMI 1 , Adewale Kayode OGUNYEMI 2 , Simeon Kolawole ODETUNDE 3 , Emmanuel Olukayode OLUMUYIWA 4 , Adesola Oluwafunmilayo OLALEKAN 5 , Kabir Olusegun AKINYEMI 6 , Bamidele Abiodun IWALOKUN 7 1Department of Chemical Sciences, Yaba College of Technology, Yaba, Lagos State, Nigeria, bukolaalagbada@gmail.com 2Department of Microbiology, University of Lagos, Akoka, Lagos State, Nigeria. 3Department of Biological Sciences, School of Pure and Applied Sciences, Lagos State Polytechnic, Ikorodu, Lagos State,, Nigeria. 4Department of Biological Sciences, College of Natural and Applied Sciences, Bells University of Technology, Ota, Ogun State, Nigeria. 5Molecular Biology and Biotechnology Unit, Department of Medical Laboratory Science, College of Medicine, University of Lagos. Lagos State, Nigeria. 6Department of Microbiology, Lagos State University, Lagos State, Nigeria. 7Molecular Biology Division, Nigerian Institute of Medical Research (NIMR), Yaba, Lagos State, Nigeria. *Corresponding author Received 3rd August 2021, accepted 23rd December 2021 Abstract: Kunun zaki is a traditionally fermented cereal-based beverage produced locally and generally consumed throughout Nigeria. This study aimed to evaluate the microbiological, physicochemical, nutritional and anti-nutritional content of kunun zaki samples sold in various markets in Lagos, Nigeria using standard methods. The total bacteria and coliform count in kunun zaki ranged from 12 x 106 - 39 x 106 cfu/ml for bacteria and 3 x 105 - 17 x 105 cfu/ml for coliform respectively, while mould and yeast had total counts of 5 x 104 - 19 x104 cfu/ml and 1.0 x 104 - 9 x 104 cfu/ml respectively. The predominant bacterial species isolated were Staphylococcus aureus, E.coli, Klebisella sp., Bacillus sp. Streptococcus sp., Citrobacter feundii and Pseudomonas aeruginosa. The predominant fungal species isolated from the samples were Fusarium sp., Aspergillus parasiticus, Aspergillus niger, Candida albicans, Penicillium sp. and Saccharomyces cerevisiae. The pH, titratable acidity and specific gravity of the samples ranged from 3.87-4.28, 0.18-0.43 g/L and 0.736-0.75 respectively. The proximate analysis revealed that kunu samples contained 0.3-0.6% protein, 0.4-1.1% fat, 1.3-1.62 ash, 12.8-20.4% carbohydrate and moisture content ranging from 76.75-84.45%. The results for the mineral content analysis showed the presence of Phosphorus, Calcium, Potassium, Copper, Manganese and Magnesium. This study revealed that kunun zaki obtained from different markets in Lagos, Nigeria contained pathogenic microorganisms that pose threats to public health. Awareness campaign, training of local producers, and monitoring of compliance with safety standards are required to reduce risk of contamination. Keywords: Kunun; Coliform; proximate; bacteria; fungi; mineral. 1. Introduction Fermented cereal-based non-alcoholic beverages like kunun zaki have formed an essential part of the dietary lifestyle of many adult and children in developing countries [1]. Kunun zaki, is a highly nutritious, refreshing and filling cereal- based beverage which is widely consumed in Nigeria due to its sweet, creamy, mailto:bukolaalagbada@gmail.com Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XX, Issue 4 – 2021 B.C. OGUNYEMI, A.K. OGUNYEMI, S.K. ODETUNDE, E.O. OLUMUYIWA, A.O. OLALEKAN, K.O. AKINYEMI, B.A. IWALOKUN, Microbiological, Physicochemical, Nutritional and Anti-Nutritional evaluation of locally made non-alcoholic Kunun zaki Beverage Sold in Lagos State, Nigeria, Food and Environment Safety, Volume XX, Issue 4 – 2021, pag. 392 – 402 393 refreshing taste and consistency [2]. Kunun zaki has been reported to contain a variety of nutrients including carbohydrates, proteins, vitamins and several amino acids [3]. The traditional production of kunun zaki majorly requires the use of various grains, which include sorghum (Sorghum bicolor), pearl millet (Pennisetum glaucum), rice (Oryza sativa), fonio (Digitaria exilis) and maize (Zea mays) [4]. Spicy ingredients such as chili pepper, ginger and cloves are also used in addition to sweet potatoes, malted cereals as well as saccharifying agents to improve aroma, taste, nutritional quality and medicinal values of kunun zaki [5,6]. According to Adeleke and Abiodun [7], the appearance of kunun zaki is milky cream and is usually consumed few hours after its production. The method of processing kunun zaki generally varies based on the producer's household, personal interests, taste and cultural preferences. This results in a difference in the presentation, appearance, taste, flavor, consistency and overall quality of kunun zaki [8]. Due to the traditional method of preparation of kunun zaki, the ingredients used are neither quantified nor standardized, therefore the risk of contamination is very high [9]. Kunun zaki and other cereal-based products have been reported to contain probiotic bacterial communities including Bifidobacterium spp., Lactobacillus brevis, Weissella confusa, Streptococcus lutetiensis, Streptococcus gallolyticus [10,11]. Several scientific studies have evaluated the microbial and nutritional composition of kunun zaki in various parts of Nigeria with different results. According to Osuntogun and Aboabo [12], “kunun zaki contains lactic acid bacteria (LAB) such as Lactobacillus spp., Streptococcus spp. and Leuconostoc spp. which can cause food borne illnesses”. Findings have shown that non-alcoholic beverages consumed in different parts of Nigeria contained anti-nutritional factors such as phytate and trypsin inhibitors, which may negatively affect their nutritional values [13,14]. Presently, there is a high intake of non-alcoholic beverages in different parts of Lagos, Nigeria, and some of these beverages are produced under unhygienic conditions [15]. This suggests that safety and nutritional values associated with the consumption of kunun zaki may be compromised. Hence, the present study aimed to evaluate the microbiological, physico-chemical, nutritional and anti- nutritional qualities of kunun zaki sold in Lagos, Nigeria. 2. Materials and Methods Sample collection Collection of kunun zaki samples were collected randomly at thirteen (13) major markets in Lagos State, Nigeria. A total of one hundred (100) samples of kunun zaki were collected, stored in sterile bottles and transferred to the laboratory for further evaluation. However, the samples were stored at 4º C and analyzed within 6 h after collection on sampling days twice per week for 1 month. Figure 1 shows the satellite image of sampling points in Lagos State, Nigeria. Microbiological Analysis One milliliter (1.0 ml) of kunun zaki sample aseptically taken and introduced into 9 ml sterile water previously autoclaved at 121ºC and 15psi for 15 min to make 1:10 dilution. This was followed by 6 serial 10-fold dilutions (10-1 – 10-7) with 1 ml of each dilution mixed with molten agar (45ºC) to determine microbial count. Nutrient agar, MacConkey agar, Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XX, Issue 4 – 2021 B.C. OGUNYEMI, A.K. OGUNYEMI, S.K. ODETUNDE, E.O. OLUMUYIWA, A.O. OLALEKAN, K.O. AKINYEMI, B.A. IWALOKUN, Microbiological, Physicochemical, Nutritional and Anti-Nutritional evaluation of locally made non-alcoholic Kunun zaki Beverage Sold in Lagos State, Nigeria, Food and Environment Safety, Volume XX, Issue 4 – 2021, pag. 392 – 402 394 potato dextrose agar were used for total bacterial count, total coliform count and total fungal count respectively. Malt extract agar supplemented with streptomycin was used for total yeast count. Plates for total and coliform count determination were incubated at 37°C for 48 h, while those for total and yeast count determination were incubated at 28 ºC for 72 h [16]. All experiments were performed in triplicates with mean count calculated and measured as mean colony forming unit (cfu/ml) of kunun zaki [17]. Colonial, morphological and biochemical characterization of bacteria isolates were carried out using standard microbiological techniques according to Bergey’s manual of Determinative Bacteriology [18]. Fungal identification and enumeration was based on their colony elevation, colour, texture, shape and arrangement of conidia (spherical or elliptical, unicellular or multicellular), branched or unbranched mycelia, presence or absence of cross walls (whether septate or non-septate) and others. They were enumerated according to ‘illustrated manual on identification of some seed borne fungi’ [19] and ‘illustrated genera of imperfect fungi’ [20]. Determination of pH, Titratable Acidity and Specific gravity The pH, titratable acidity and specific gravity of the kunun zaki samples were determined according to the method of Association of Official Analytical Chemists (AOAC) [21]. The titratable acidity was expressed as % lactic acid present in the sample. Determinations were done in triplicates and mean values were calculated. Proximate and Mineral Analysis Moisture content, crude fat, protein and carbohydrate content were determined according to AOAC methods [21]. ‘Atomic Absorption Spectrophotometer’ (AAS) was used to determine three mineral contents (Calcium, Copper, Iron, and Magnesium) of the samples, while other minerals (Potassium, Phosphorus) were determined using‘flame photometry’according to AOAC [21]. Determinations were performed in triplicates and mean values were calculated. Anti-nutritional factors determination The levels of phytate and trypsin inhibitor in the samples were determined using the colorimetric method [22,23]. Determinations were done in triplicates and mean values were calculated and expressed as mg per 100g (mg/100g) sample. Statistical Analysis Data obtained in this study were subjected to one-way analysis of variance (ANOVA) Graph pad prism® software package, version 5.0 (Graph- Pad Software Inc., San Diego, CA, USA). 3. Results and Discussion The microbial counts in kunun zaki are presented in Table 1. The total bacteria and coliform count in kunun zaki ranged from 12 x 106 - 39 x 106 cfu/ml for bacteria and 3.0 x 105 - 17 x 105 cfu/ml for coliform respectively. Total count of mould ranged from 5.0 x 104 - 19 x104 cfu/ml while total yeast count ranged from 1.0 x 104 - 9 x 104 cfu/ml.The results showed that Ojuelegba had the highest bacteria count (39 x 106 cfu/ml) followed by Oyingbo (35 x 106 cfu//ml) while Yabatech had the least count (12 x 106 cfu/ml). The samples from Oyingbo had the highest fungi count (19 x 104 cfu/ml) followed by Orile (17 x 104 Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XX, Issue 4 – 2021 B.C. OGUNYEMI, A.K. OGUNYEMI, S.K. ODETUNDE, E.O. OLUMUYIWA, A.O. OLALEKAN, K.O. AKINYEMI, B.A. IWALOKUN, Microbiological, Physicochemical, Nutritional and Anti-Nutritional evaluation of locally made non-alcoholic Kunun zaki Beverage Sold in Lagos State, Nigeria, Food and Environment Safety, Volume XX, Issue 4 – 2021, pag. 392 – 402 395 cfu/ml) while Unilag had the least fungi count (5 x 104 cfu/ml). Bariga had highest yeast count (9 x 104 cfu/ml) while Surulere had the least yeast count (1.0 x 104 cfu/ml). The occurrences of bacteria and fungi species were presented in Figures 2 and 3. Bacillus sp. had the highest occurrence (27.14%) followed by E.coli (22.86%) while Citrobacter feundii had the least occurrence (5.71%). Whereas Saccharomyces cerevisiae had the highest occurrence (61.72%) followed by Candida albicans (25.31%). Penicillium sp. (1.23%) had the least occurrence. From this present study, the results indicate that kunun zaki presented high microbial counts. Coliforms and E. coli are known as indicators of faecal contamination in food products. According to WHO guidelines for drinking water [25], indicator organisms must not be present (=0 cfu/100ml). The coliform counts obtained in this study are far higher than the 104 cfu/g taken as tolerable in foods in developed countries [26]. Studies have shown that Escherichia coli, Clostridium, Staphylococcus, Campylobacter, and Vibrio are some of the common bacteria that cause food-related illness with severe implications [27]. The presence of coliform and E. coli in the kunun zaki indicates fecal contamination that may be due to unhygienic practices during or after processing the beverage [28]. Poor water quality, an unsanitary environment, low quality of materials, unclean equipment, stock containers, and improper handling by vendors could be the cause of microbial contamination in all kunun zaki samples [29, 30]. Ekanem et al. [31] previously reported that “samples of kunnu had total colony counts ranging from 0.5×105 to 3.2 ×105, 0.5×105 to 3.2 ×105, 5.2×105 to 8.0×105 (cfu/ml) for coliform bacteria, heterotrophic bacteria and heterotrophic fungi respectively”. Efiuwevwere and Akoma [32] also reported the occurrence of high microbial communities in kunu-zaki samples produced in Jos metropolis. Anumudu and Anumudu [33] found the following occurrence (%) for nine genera of bacteria in kunnu: “Staphylococcus sp. (16.66%), E. coli (13.33%), Citrobacter sp. (13.33%), Proteus sp. (10.00%), Serratia sp. (10.00%), Lactobacillus sp. (10.00%), Salmonella sp. (10.00%), Streptococcus sp. (10.00%) and Enterobacter sp. (6.66%)”. Table 1 Microbial counts in the kunun zaki samples. Sampling site Bacterial counts (x 106 cfu/ml) Coliform counts (x 105 cfu/ml) Fungi counts (yeast) (x 104 cfu/ml) Fungi counts (mould) (x 104 cfu/ml) Jibowu 18 3 2 7 Surulere 15 6 1 9 Tejuosho 20 8 2 11 Orile 34 11 3 17 Obalende 19 9 NYG 10 Iyana- Ipaja 42 12 4 15 Ojuelegba 39 16 6 13 Unilag 16 5 NYG 5 Yabatech 12 4 NYG 6 Sabo- Yaba 26 7 5 16 Oyingbo 35 17 7 19 Lagos Island 17 8 8 8 Bariga 31 16 9 15 NYG-No yeast growth Seven (7) bacteria species were isolated and identified (E. coli (3 strains), Klebisella sp. (5 strains), Staphylococcus aureus (5 strains), Bacillus sp. (3 strains), Streptococcus sp. (3 strains), Citrobacter feundii (3 strains), and Pseudomonas aeruginosa (3 strains)). Six (6) fungal species were isolated and identified as Aspergillus parasiticus, Aspergillus niger, Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XX, Issue 4 – 2021 B.C. OGUNYEMI, A.K. OGUNYEMI, S.K. ODETUNDE, E.O. OLUMUYIWA, A.O. OLALEKAN, K.O. AKINYEMI, B.A. IWALOKUN, Microbiological, Physicochemical, Nutritional and Anti-Nutritional evaluation of locally made non-alcoholic Kunun zaki Beverage Sold in Lagos State, Nigeria, Food and Environment Safety, Volume XX, Issue 4 – 2021, pag. 392 – 402 396 Fusarium sp., Penicillium sp., Candida albicans and Saccharomyces cervisae (Table 2 and 3). The result from this study is consistent with previous findings [34], which reported the isolation of Staphylococcus aureus, Shigella sp., Streptococcus sp., Salmonella sp., E. coli, Lactobacillus sp., Klebsiella sp., Citrobacter sp. in samples of kunu zaki produced in Kwara State, Nigeria. Adeyemi and Umar [1] had earlier reported the presence of Lactobacillus plantarum, Leuconostoc mesenteriodes, Mucor spp., Rhizopus spp. and Saccharomyces cerevisiae in kunun zaki prepared with a combination of sorghum and millet; while Osuntogun and Aboaba [12] isolated Lactobacillus, Streptococcus, Aspergillus and Penicillium. However, findings from Olasupo et al. [35], have confirmed the presence of lactic acid bacteria as the only test organism (including Lactobacillus salivarius, Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus jensenii, Lactobacillus cellobiosus and Lactobacillus plantarum) found in kunun zaki. Adebayo et al. [3] isolated ‘Aspergillus niger, Rhizopus stolonifer, Aspergillus nidulans and Aspergillus flavus from kunun zaki. The results from this study indicates the presence of contaminants, which may be due to unhygienic practices or low production quality [3, 33, 36]. The consumption of such contaminated beverage may pose serious health risks to the consumers. Proximate Analysis As presented in Table 4, the result of the proximate analysis in this study showed that kunun zaki from different locations of Lagos metropolis contained 0.3-0.6% protein, 0.4- 1.65% fat, 1.3-1.62% ash and 12.8-20.4% carbohydrate with moisture content ranging from 76.75-84.45%. The result of the proximate analysis in this present study are similar to a previous study by Onyeleke and Shittu [37] who reported that kunun zaki sold in Minna contain 0.33% protein, 1.0% fat, 1.52 ash and 12.2% carbohydrate. Previous report by Essien et al. [38] described that the loss of protein during processing may be responsible for the low protein content observed in the drinks. Fig.2: Occurrence of Bacteria species in the kunun zaki beverage obtained from different markets Fig.3: Occurrence of Fungi species in the kunun zaki beverage obtained from different markets Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XX, Issue 4 – 2021 B.C. OGUNYEMI, A.K. OGUNYEMI, S.K. ODETUNDE, E.O. OLUMUYIWA, A.O. OLALEKAN, K.O. AKINYEMI, B.A. IWALOKUN, Microbiological, Physicochemical, Nutritional and Anti-Nutritional evaluation of locally made non-alcoholic Kunun zaki Beverage Sold in Lagos State, Nigeria, Food and Environment Safety, Volume XX, Issue 4 – 2021, pag. 392 – 402 397 Table 2 Morphology and biochemical tests of isolates from kunun zaki products Isolates 1 2 3 4 5 6 7 8 9 10 Identification BC1 rod - + + - - + + + + E.coli BC2 rod - + + + -- + + - + Klebsiella sp. BC3 coccus + + + + - + + - + Staphylococcus aureus BC4 rod + + + - + + - + + Bacillus sp. BC5 coccus + + + + - + + - + Staphylococcus aureus BC6 rod - + + + -- + + - + Klebsiella sp. BC7 rod - + + - - + + + + Citrobacter freundii BC8 rod - + + - + + - - - Pseudomonas aeruginosa BC9 coccus + + - - - + + + + Streptococcus sp. BC10 rod - + + - - + + + + Citrobacter freundii BC11 rod + + + - + + - + + Bacillus sp. BC12 coccus + + - - - + + + + Streptococcus sp. BC13 rod - + + - - + + + + E.coli BC14 rod - + + - + + - - - Pseudomonas aeruginosa BC15 coccus + + + + - + + - + Staphylococcus aureus BC16 rod - + + + -- + + - + Klebsiella sp. BC17 rod + + + - + + - + + Bacillus sp. BC18 coccus + + + + - + + - + Staphylococcus aureus BC19 rod - + + + -- + + - + Klebsiella sp. BC20 rod - + + - - + + + + Citrobacter freundii BC21 coccus + + + + - + + - + Staphylococcus aureus BC22 rod - + + - - + + + + E.coli BC23 rod - + + - + + - - - Pseudomonas aeruginosa BC24 rod - + + + -- + + - + Klebsiella sp. BC25 coccus + + - - - + + + + Streptococcus sp. 1-Shape; 2-Gram status; 3-Growth in Air; 4-Catalase; 5-Urease; 6-Oxidase; 7-Glucose; 8-Maltose; 9-Arabinose; 10- Sorbitol, +=Positive, -=Negative Table 3 Microscopic and macroscopic characteristics of fungi isolates Colony colour Colony Diameter (mm) Conidia colour Conidiophore colour Conidiophore surface texture Phialides Identification black 33±1.4 Brown to black brown very rough, globose Biseriate Aspergillus niger dark green 28±2.2 brown colourless distinctly rough, globose Uniseriate Aspergillus parasiticus pinkish white 32±0.9 light brownish orange rough-walled, with an apical swelling biseriate Fusarium sp. black 25±1.3 green blue fuzzy, smooth uniseriate Penicillium sp. smooth creamy colonies 40±2.2 brush-like white white biseriate Candida albicans cream 18±1.2 white white white Biseriate Saccharomyces cerevisiae Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XX, Issue 4 – 2021 B.C. OGUNYEMI, A.K. OGUNYEMI, S.K. ODETUNDE, E.O. OLUMUYIWA, A.O. OLALEKAN, K.O. AKINYEMI, B.A. IWALOKUN, Microbiological, Physicochemical, Nutritional and Anti-Nutritional evaluation of locally made non-alcoholic Kunun zaki Beverage Sold in Lagos State, Nigeria, Food and Environment Safety, Volume XX, Issue 4 – 2021, pag. 392 – 402 398 Table 4 Proximate analysis of kunun zaki products obtained from various Lagos markets Proximate contents Moisture content Fat Ash Protein CHO Jibowu 76.75±1.3 1.63±0.02 1.5±0.05 0.25±0.05 12.8±0.10 Surulere 79.47±0.36 1.42±0.01 1.3±0.25 0.6±0.10 17.2±0.10 Tejuosho 81.72±0.32 1.59±0.01 1.38±0.03 0.37±0.01 20.2±0.05 Orile 79.31±0.39 1.48±0.03 1.62±0.03 0.45±0.01 18.3±0.10 Obalende 82.38±0.72 1.57±0.06 1.38±0.010 0.33±0.07 17.6±0.50 Iyana-Ipaja 78.63±1.30 1.65±0.03 1.6±0.10 0.5±0.10 19.4±0.10 Ojuelegba 78.52±0.49 1.63±0.06 1.48±0.09 0.45±0.05 18.4±0.10 Unilag 81.16±0.02 1.57±0.06 1.6±0.10 0.37±0.03 17.3±0.05 Yabatech 82.13±0.33 1.54±0.01 1.5±0.10 0.42±0.02 18.8±0.40 Sabo-Yaba 84.45±0.21 1.61±0.02 1.3±0.10 0.6±0.10 19.3±0.40 Oyingbo 83.29±0.81 1.54±0.02 1.55±0.11 0.3±0.01 20.4±0.70 Lagos Island 78.24±0.03 1.43±0.02 1.5±0.10 0.4±0.05 19.8±0.25 Bariga 83.77±0.06 1.61±0.20 1.4±0.10 0.5±0.05 20.3±0.10 CHO-Carbohydrate; S.I units for all parameters are in % Physicochemical analysis The physicochemical properties of kunun zaki are presented in Table 5. The pH value of the kunun zaki samples ranged from 3.87- 4.28 which shows that the samples are acidic. Studies have shown that the pH value of a food is considered a measure of microbial spoilage. Table 5 Physicochemical characteristics of kunun zaki samples sold in Lagos, Nigeria Sampling site pH titratable acidity Specific gravity Jibowu 3.87±0.05 0.24±0.05 0.745±0.01 Surulere 4.03±0.01 0.18±0.01 0.738±0.02 Tejuosho 4.1±0.05 0.36±0.03 0.75±0.01 Orile 4.16±0.02 0.32±0.02 0.742±0.02 Obalende 3.97±0.05 0.43±0.01 0.745±0.015 Iyana-Ipaja 4.23±0.18 0.28±0.01 0.741±0.00 Ojuelegba 4.27±0.05 0.31±0.06 0.739±0.00 Unilag 4.12±0.05 0.18±0.01 0.738±0.00 Yabatech 4.28±0.03 0.24±0.01 0.745±0.00 Sabo-Yaba 4.05±0.25 0.32±0.01 0.743±0.00 Oyingbo 4.2±0.06 0.37±0.01 0.748±0.00 Lagos Island 4.15±0.03 0.41±0.02 0.736±0.00 Bariga 4.27±0.01 0.28±0.05 0.739±0.00 Microorganisms have been reported to have optimum pH requirements and so the higher the pH of a food, the more prone it is to microbial spoilage. This indicates kunun zaki as a food with high acid content (<4.6) may therefore be more resistant to microbial spoilage [39]. The pH values recorded in this study were lower than the previously reported values of 4.70-5.75 [40], but comparable to 3.3- 4.3 reported by Elmahmood and Doughari [41]. In this study, the titratable acidity (TTA) of kunun zaki ranged from 0.18 to 0.43 of Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XX, Issue 4 – 2021 B.C. OGUNYEMI, A.K. OGUNYEMI, S.K. ODETUNDE, E.O. OLUMUYIWA, A.O. OLALEKAN, K.O. AKINYEMI, B.A. IWALOKUN, Microbiological, Physicochemical, Nutritional and Anti-Nutritional evaluation of locally made non-alcoholic Kunun zaki Beverage Sold in Lagos State, Nigeria, Food and Environment Safety, Volume XX, Issue 4 – 2021, pag. 392 – 402 399 lactic acid. This result is consistent with previous studies by Adebayo et al. [3] and Essien et al. [38]. Since high acidity and low pH can be attributed to the absorption of amino acids and the release of organic acid, the acidity of kunun zaki may be as a result of lactic acid production during fermentation [42]. Microorganisms utilize carbohydrates and this contributes to the production of more hydrogen ions, which signify increased production of acid in the beverage [43]. In this study, the specific gravity of the kunun zaki samples ranged from 0.736-0.75. This is lower than the values (1.02-1.06) reported by Omowaye- Taiwo and Oluwamukomi [44]. Mineral Composition The results obtained for the mineral content of kunun zaki are presented in Table 6. In this study, the kunun zaki samples contained P (176.4-278mg/100g), Ca (290.7-427.2mg/100g), K (155- 203mg/100g), Cu (0.1-0.9mg/100g), Mn (0.4-1.5mg/100g) and Mg (90.2- 112.8mg/100g). The mineral content of kunun zaki recorded in this study is similar to a previous study by Nkama et al. [45]. Minerals are nutritionally important components in food and they are essential for health and cellular functions in the body [12]. In this study, calcium had the highest value followed by phosphorus, potassium, magnesium and manganese while copper was the least abundant in kunun zaki samples. This is consistent with previous findings by Ofudje et al. [33] who observed that kunun zaki has high content of calcium and low content of copper and manganese. The results for potassium, magnesium and copper are similar to results from previous study by Omowaye- Taiwo and Oluwamukomi [44]. Table 6 Mineral Composition ofkunun zaki products obtained from various Lagos markets Sampling site P (mg/100g) Ca (mg/100g) K (mg/100g) Cu (mg/100g) Mn (mg/100g) Mg (mg/100g) Jibowu 180.4±0.10 320.7±0.20 155±2.0 0.5±0.10 0.7±0.10 90.5±0.02 Surulere 176.4±0.20 290.7±0.10 160±2.0 0.3±0.20 1.2±0.15 107.2±0.10 Tejuosho 205.1±0.00 308.5±0.10 172±1.0 0.2±0.1 1±0.11 98.2±0.20 Orile 240±1.0 407.5±0.02 159±2.0 0.6±0.1 1.1±0.16 91.5±0.40 Obalende 195±2.0 370.3±0.3 163±3.0 0.1±0.01 0.9±0.01 93.7±0.10 Iyana-Ipaja 187.4±0.20 350.2±0.2 174±1.0 0.35±0.12 1.25±0.06 90.2±0.20 Ojuelegba 195±2.0 410.7±0.1 180±0.58 0.51±0.06 1.3±0.06 95.1±0.05 Unilag 230.5±0.10 345.1±0.03 203±1.0 0.27±0.07 0.8±0.10 108.4±0.10 Yabatech 240±2.0 290.7±0.2 165±1.0 0.4±0.03 0.7±0.11 112.8±0.10 Sabo-Yaba 278±2.0 350.2±0.1 190±1.0 0.22±0.01 1.2±0.20 102.9±0.79 Oyingbo 251.8±0.10 380±2.0 185±1.0 0.3±0.10 1.5±0.10 94.2±1.2 Lagos Island 270.4±0.10 345±1.0 175±2.0 0.4±0.09 0.7±0.20 90.8±1.6 Bariga 265±2.0 427.2±0.10 200±2.0 0.9±0.20 0.4±0.10 91.4±0.3 P-Phosphorus; Ca-Calcuim; K-Potassium; Cu-Copper; Mn-Manganese; Mg-Magnesium Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XX, Issue 4 – 2021 B.C. OGUNYEMI, A.K. OGUNYEMI, S.K. ODETUNDE, E.O. OLUMUYIWA, A.O. OLALEKAN, K.O. AKINYEMI, B.A. IWALOKUN, Microbiological, Physicochemical, Nutritional and Anti-Nutritional evaluation of locally made non-alcoholic Kunun zaki Beverage Sold in Lagos State, Nigeria, Food and Environment Safety, Volume XX, Issue 4 – 2021, pag. 392 – 402 400 Anti-nutritional contents of kunun zaki samples The result of the anti-nutritional contents (phytate and trypsin) of kunun zaki samples are presented in Table 7. The phytate content of the samples ranged from 0.093mg/100g to 0.27mg/100g, while a range of 0.058mg/100g to 0.081mg/100g was obtained for trypsin inhibitor. According to Food and Agriculture Organization (FAO) [46], anti-nutritional factors are generally defined as naturally occurring substances present in grains and other substances present in grain as a result of fungal or other environmental contamination. The reductions in the concentration of anti-nutritional factors in several grains have been attributed to the use of different processing techniques including germination, fermentation, soaking and cooking [47]. Adelekan et al. [12] reported a reduction in the concentration of trypsin inhibitor from 0.067 mg/100 g in unmalted kunun zaki to 0.057 mg/100 g in malted kunun zaki and similar occurrence was found in the result of the phytic acid. Studies have shown that some anti-nutritional factors affect the nutritional value of grains, while others have significantly serious consequences [46]. Table 7 Anti-nutritional contents of kunun zaki samples sold in Lagos metropolis. Sampling site Phytate (mg/100g) Trypsin (mg/100g) Jibowu 0.15±0.01 0.068±0.01 Surulere 0.17±0.01 0.072±0.01 Tejuosho 0.093±0.03 0.061±0.001 Orile 0.14±0.04 0.069±0.003 Obalende 0.27±0.05 0.08±0.01 Iyana-Ipaja 0.22±0.03 0.065±0.005 Ojuelegba 0.18±0.01 0.073±0.002 Unilag 0.14±0.02 0.072±0.002 Yabatech 0.12±0.01 0.074±0.002 Sabo-Yaba 0.2±0.01 0.062±0.001 Oyingbo 0.18±0.01 0.058±0.005 Lagos Island 0.17±0.01 0.073±0.001 Bariga 0.13±0.02 0.081±0007 4. Conclusion The study showed that kunun zaki contains many essential nutrients, such as carbohydrates, protein, and fat. In addition, kunun zaki also contains essential mineral elements necessary for growth and development. The presence of contaminants in the kunun zaki samples, shows that the vended kunun zaki had been prepared under low hygienic condition and may pose serious health risk to consumers. Hence, improving the nutritional value and microbial quality of locally produced kunun zaki has become imperative. 5. References [1]. ADEYEMI I.A, UMAR S. Effect of method of manufacture on quality characteristics of kunun zaki; A millet-based beverage. Nigeria Food Journal. 12: 944-947. (1994). [2]. OBADINA A.O., OYEWOLE O.B., AWOJOBI T.M. (2008). Effect of steeping time of milled grains on the quality of kunnu-Zaki (A Nigerian beverage). AJFS 2: 033-036. (2008) [3]. ADEBAYO G.B., OTUNOLA G.A., AJAO T.A. Physicochemical, microbiological and sensory characteristics of kunu prepared from millet, maize and guinea corn and stored at selected temperature. Adv J Food Sci Technol. 2(1): 41-46, (2010) [4]. AHMED E.U., MUSA N., NGODDY P.O. Sensory attributes of extruded cereal legume blends of instant kunnu-zaki beverage analogue proceedings on the Second Annual NIFSI Conference. pp: 5-6, (2003). Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XX, Issue 4 – 2021 B.C. OGUNYEMI, A.K. OGUNYEMI, S.K. ODETUNDE, E.O. OLUMUYIWA, A.O. OLALEKAN, K.O. AKINYEMI, B.A. IWALOKUN, Microbiological, Physicochemical, Nutritional and Anti-Nutritional evaluation of locally made non-alcoholic Kunun zaki Beverage Sold in Lagos State, Nigeria, Food and Environment Safety, Volume XX, Issue 4 – 2021, pag. 392 – 402 401 [5]. MAKINDE F., OYELEKE O. Effect of sesame seed addition on the chemical and sensory qualities of sorghum based kunun- zaki drink. Afr J Food Sci Technol. 3:204–212, (2012). [6]. OLAOYE O.A., UBBOR S.C., UDUMA E.A. Determination of vitamins, minerals, and microbial loads of fortified nonalcoholic beverage (kunun zaki) produced from millet. Food Sci Nutr. 4(1):96–102. https://doi.org/10.1002/fsn3.267, (2015) [7]. ADELEKE R.O., ABIODUN O.A. Physico- chemical Properties of Commercial Local Beverages in Osun State, Nigeria. Pakistan J Nutr. 9 (9):853-855, (2010) [8]. ADELEKAN A.O., ALAMU A.E., ARIS N.U., ADEBAYO Y.O., DOSA A.S. Nutritional, Microbiological and Sensory Characteristics of Malted Soy-Kunu Zaki: An Improved Traditional Beverage. Adv Microbiol. 3:389-397, (2013). [9]. ABOH M.I., OLADOSU .P. Microbiological assessment of kunnu-zaki marketed in Abuja Municipal area council (AMAC) in the Federal Capital Territory (FCT). Nigeria. African J Microbiol Res. 8 (15):1633-1637, (2014). [10]. CRITTENDEN .R., KARPPINEN S., OJANEN S., TENKANEN M., FAGERSTRÖM R., MÄTTÖ J., SAARELA M., TIINA S., MATTILA‐SANDHOLM T., POUTANEN K. (2002). In vitro fermentation of cereal dietary fibre carbohydrates by probiotic and intestinal bacteria. J Sci Food Agric. 82:781-789, (2002). [11]. OGUNTOYINBO F.A., TOURLOMOUSIS P., GASSON M.J., NARBAD A. Analysis of bacterial communities of traditional fermented West African cereal foods using culture independent methods. Int J Food Microbiol. 145: 205-210, (2011). [12]. OSUNTOGUN B., ABOABO O.O. Microbiological and physiochemical evaluation of some non-alcoholic beverages. Pakistan J Nutr. 2(3):21-33, (2006). [13]. OMEMU A.M., OKAFOR U.I., OBADINA A.O., BANKOLE M.O., ADEYEYE S.A.O. Microbiological assessment of maize ogi cofermented with pigeon pea. Food Sci Nutr. 6. 10.1002/fsn3.651, (2018). [14]. AJAYEOBA T.A., DULA S., IJABADENIYI O.A. Properties of Poly-γ-Glutamic Acid Producing-Bacillus Species Isolated From Ogi Liquor and Lemon-Ogi Liquor. Front microbiol. 10:771, (2019) [15]. GBADAMOSI A., IWALOYE O., BAMBER D. An exploratory study of students' consumption of non-alcoholic beverages in Nigeria: A qualitative perspective. Nutr Food Sci. 39:609- 618, (2009). [16]. FAWOLE M.O., OSO B.A. Laboratory manual of Microbiology. 5th Edition, Spectrum Books Limited, Ibadan, pp.22-23, (2007). [17]. HARRIGAN W.F., MCCANCE M.E. Laboratory Methods in Food and Dairy Microbiol. 8th (Edn.), Academic Press Inc, London, pp. 7-23, (1990). [18]. HOLT J.G., KREIG N.R., SNEATH, P.H.A., STANLEY J.T., WILLIAM S.T. Bergey’s manual of Determinative Bacteriology. Baltimore, USA: William and Wikins 213pp, (1994). [19]. KULWANT S.J.C. An illustrated manual on identification of some Seed borne Aspergilli, Fusaria, Penicillia and their Mycotoxins. Hellerup, Denmark. Danish Government Institute of Seed Pathology for Developing Countries and Department of Biotechnology, The technical university of Denmark, (1991). [20]. BARNETT H.L., HUNTER B.B. Illustrated Genera of Imperfect Fungi. 3rd Edition, Burgess Publishing Co., Minneapolis, 241 pp, (1972). [21]. Association of official Analytical Chemists (AOAC). Official Methods of Analysis 20th Edition. Washington D.C, (2005). [22]. HAUG W., LANTZSCH H.J. Sensitive method for the rapid determination of phtytate in cereals and cereal products. J Sci Food Agric. 34:1423–1426. doi:10.1002/jsfa.2740341217, (1983) [23]. HOU H.J., CHANG K.C. (2004). Storage conditions affect soybean color, chemical composition and tofu qualities. Journal of Food Processing and Preservation, 28:473-488, (2004). [24]. CENTER FOR FOOD SAFETY (CFS). Microbiological Guidelines for Food (for ready-to- eat-food in general and specific food items). Center for food safety, 1-46, (2014). [25]. WORLD HEALTH ORGANIZATION (WHO). Guidelines for Drinking-Water Quality; Fourth Edition Incorporating the First Addendum: Geneva, Switzerland, 2017; p. 149 (2017). [26]. COOKE E.M., GIBSON G.L. Essential clinical microbiology. New York, NY: John Wiley and Sons Ltd. Intestinal diseases; pp. 16–21, (1990). [27]. AMOAH D. Microbial risk assessment of mixed vegetable salads from selected canteens in the Kumasi metropolis, Ghana. Pp 41-56, (2014). Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XX, Issue 4 – 2021 B.C. OGUNYEMI, A.K. OGUNYEMI, S.K. ODETUNDE, E.O. OLUMUYIWA, A.O. OLALEKAN, K.O. AKINYEMI, B.A. IWALOKUN, Microbiological, Physicochemical, Nutritional and Anti-Nutritional evaluation of locally made non-alcoholic Kunun zaki Beverage Sold in Lagos State, Nigeria, Food and Environment Safety, Volume XX, Issue 4 – 2021, pag. 392 – 402 402 [28]. KHAN M.M., ISLAM M.T., CHOWDHURY M.M.H., ALIM S.R. Assessment of microbiological quality of some drinks sold in the streets of Dhaka University Campus in Bangladesh. Int. J. Food Contam. 2, 4, (2015). [29]. AUAD L.I., GINANI V.C., STEDEFELDT E., NAKANO E.Y., NUNES A.C.S., ZANDONADI R.P (2019). Food safety knowledge, attitudes and practices of Brazilian food truck food handlers. Nutrients, 11, 1784, (2019). [30]. NEMO R., BACHA K., KETEMA T. Microbiological quality and safety of some-street vended foods in Jimma Town, Southwestern Ethiopia. Afr. J. Microbiol. Res. 11, 574–585, (2015). [31]. EKANEM J.O., MENSAH B.J., MARCUS N.S., UKPE B.A. (2018). Microbial Quality and Proximate Composition of Kunu Drinks Produced and Sold in Ikot Ekpene Metropolis, Akwa Ibom State, Nigeria. J Appl Sci Environ Mgt. 22 (11):1713–1718, (2018). [32]. EFIUWEVWERE B.J.O., AKOMA O. (1995). The Microbiology of Kunu- Zaki a cereal Beverage from northern Nigeria during the fermentation (production) process. J Microbial Biotechnol.11:491-493. (1995). [33]. ANUMUDU I.C., ANUMUDU C.K. Bacteriological quality of kunu-zaki sold on the streets of Owerri metropolis, Nigeria. Af J Bio Sc.1(1): 18-22. (2019). [34]. EDEM V.E., ELIJAH A.I., UMOH S.H. (2017). Microbial Quality of kunun-zaki sold in Eiyenkorin, Kwara State, Nigeria. Nigerian J Agric Food Environ.13 (2), 112-116, (2017). [35]. OLASUPO N.A., OLUKOYA D.K., ODUNFA S.A. Identification of Lactobacillus species associated with selected African fermented foods. Zeitschrift fur Naturforschung C-A J Biosci. 5 (2):105-108, (1997). [36]. OFUDJE E.A., OKON U.E., ODULEYE O.S., WILLIAMS O.D. Proximate, Mineral Contents and Microbial Analysis of Kunu-Zaki (A Non-Alcoholic Local Beverage) in Ogun State, NigeriaJ Adv Biol Biotechnol. 7(1): 1-8, (2016). [37]. ONYELEKE S.B., SHITTU A. (2005). Quality Food Assessment of kunnu-zaki sold in Minna. Proceedings Science and Technology” Handbook of Cereal of the Biotechnology Society of Nigeria (10 Annul Science and Technology (CRC Press) 99 2 edition th Conference) Minna, Niger State (2005). [38]. ESSIEN E., MONAGO C., EDOR E.A. Evaluation of the nutritional and microbiological quality of Kunnu-zakin (a cereal based non- alcoholic beverage) in Rivers State, Nigeria. The Internet Journal of Nutrition and Wellness 10: 1-10, (2011). [39]. BABAJIDE J.M., OLALUWOYE A.A., TAOFIK SHITTU T.A., ADEBISI M.A. Physicochemical properties and phytochemical components of spiced cucumber-pineapple fruit drink. Nigerian Food J. 31: 40– 52, (2013). [40]. AMUSA N.A., ASHAYE O.A. Effects of processing on nutritional, microbiological and sensory properties of kunnu-zaki (A sorghum based nonalcoholic beverage) widely consumed in Nigeria. Pakistan J Nutr. 8(3):288-292, (2009). [41]. ELMAHMOOD A.M., DOUGHARI J.H. Microbial quality assessment of kunu-zaki beverages sold in Griei Town of Adamawa State, Nigeria. African J Food Sci. 20 (2): 011-015, (2017). [42]. ASHIRU A.W., OLALEYE O.O., EGBENNI P.O. Occurrence of Pathogenic organisms in kunun Drink Sold within Lagos Metropolis, Proceedings of the 27th Annual NIFST Conference. pp. 84, (2003). [43]. FASOYIRO S.B., BABALOLA S.O., OWOSIBO S.B. Chemical composition and sensory qualities of fruit flavoured roselle (Hibiscus sabdariffa) World J Agric Res. 1 (2):161-164, (2005). [44]. OMOWAYE-TAIWO O.A., OLUWAMUKOMI M.O. Physical and nutritional composition of instant kunun-zaki powder obtained by three drying methods. FJRS 1:114-122, (2015). [45]. NKAMA I., AGARRY O.O., AKOMA O. Sensory and nutritional quality characteristics of powdered ‘kunun zaki’: A Nigerian fermented cereal beverage. African J Food Sci. 4(6):364 –370, (2010). [46]. FOOD AND AGRICULTURAL ORGANIZATION (FAO) of the United Nations. Food and Agricultural Organization (FAO) of the United Nations, Sorghum and Millets in Human Nutrition (FAO Food and Nutrition Series, No. 27), Rome. ISBN 92-5-103381-1, (1995). [47]. ADEYEMO S.M., ONILUDE A.A., OLUGBOGI D.O. Reduction of Anti-nutritional factors of sorghum by lactic acid bacteria isolated from Abacha - an African fermented staple. Front Sci. 6(1): 25-30, (2016). 1. Introduction