MATERIALS AND METHODS

Sample collection: Biradon, kesham and kindrimo samples were purchased from Fulani Farm and four markets: Bulunkutu, Gwange, Monday and Bama in Maiduguri and transported to the Microbiology Laboratory in sterile corked plastic tubes packed in iced container. Twenty samples were purchased from each place for each of the milk product. Purchases were done between 8.00 and 11.00 a.m. each day lasting from March 2010 to July 2010.

Microbiological analyses: Biradon, kesham and kindrimo samples about 40 mL each contained in sterile screw-capped plastic bottles were used for the isolation and enumeration of the microorganisms. Ten milliliters of each sample was aseptically introduced into 90 mL of sterile normal saline and was homogenized by hand-shaking for 5 min followed by further decimal dilutions to up to 10^-6 concentrations. A 0.1 mL quantity of appropriately diluted sample was used to inoculate freshly prepared media as described previously (Okonkwo, 2011). Media employed for the isolation and enumeration of the organisms included: Nutrient Agar (NA) (Biotech Lab. Ipswich, UK) for Total viable counts; MacConkey agar (Fluka Biochemika Steinhein, Germany) for Coliform; Baird Parker Medium (BPM) (Lab M. Ltd, Bury Lancashire BL9 6As, United Kingdom) for S. aureus; Eosine Methylene Blue Agar (EMBA) (Himedia Laboratories Pot Ltd, India) for E. coli and Desoxycholate Citrate Agar (DCA) (Park Scientific Limited, Moultan Park, Northampton) for Salmonella and Shigella sp. All media except DCA were sterilized by autoclaving at 121°C for 15 min. DCA was sterilized by boiling over gauze as recommended by the manufacturer. Emergent colonies following incubation were counted using digital colony counter (Labtech, New Delhi, India).

Isolation and enumeration of E. coli: Isolation and enumeration of E. coli was done on EMBA plates inoculated as described above and incubated at 37°C for 48 h after which typical colonies with greenish metallic sheen were subjected to further biochemical testing for E. coli.

Isolation and enumeration of S. aureus: BPM were inoculated as above, incubated at 37°C for 48 h and subsequently examined for characteristics typical of S. aureus. Greyish-black or black colonies with or without a halo were presumptively identified as Staphylococci as recommended by Macfaddin (1977) and coagulase test was carried out to further characterize S. aureus.

Isolation and enumeration of Salmonella and Shigella sp.: Macfaddin (1977) procedure was used for the isolation and enumeration of Salmonella and Shigella sp. Typical colonies with black centres were identified as Salmonella sp. whereas, pinkish colonies were identified presumptively as Shigella and each subjected to further biochemical testing.

Coliform count (CC): Sterile MacConkey agar plates (MAP) were inoculated as above, incubated at 37°C for 48 h and subsequently examined for typical pinkish colonies.

Total viable count (TVC): TVC was performed by inoculating sterile Nutrient Agar plates as above, incubating at 37°C for 48 h and subsequently examining for emergence of colonies.

Biochemical identification of the isolates: The biochemical tests for the identification of the isolates were the IMVC; namely: indole, methyl-red, Voges-proskauer and citrate utilization and Triple Sugar Iron (TSI), urease, oxidase, coagulase and catalase tests described previously (Okonkwo, 2011). Confirmation of test results were based on Cowan and Steel (1965) and Cheesbrough (2004) s’ procedures.

Statistical analysis: Data were analyzed by Multiple-Sample Comparison using STATGRAPHICS Centurion XVI Version 16.1.05 (32-bit). A one-way Analysis of Variance (ANOVA) was performed and when the F-test in the ANOVA was significantly (p≤0.05) different between the means, Multiple Range Tests were conducted to tell which means were significantly different from others.

RESULTS

E. coli count (log₁₀ cfu mL^-1): The mean E. coli counts (log₁₀ cfu mL^-1) of biradon, kesham and kindrimo were between 1.24 and 3.30, 2.40 and 4.82 and 1.02 and 3.66, respectively from the different markets (Table 1) with an overall mean of 2.43, 3.31 and 2.82 log₁₀ cfu mL^-1, respectively. The mean E. coli count of Gwange (kesham) was significantly higher (p≤0.05) than that of the other markets.

Table 1:	Mean counts of different microorganisms isolated from biradon, kesham and kindrimo from different markets in Maiduguri (counts were expressed as log 10 cfu mL-1)
NB: BI: Biradon, KE: Kesham, KI: Kindrimo, TVC: Total viable counts. There is statistically significant difference *(p<0.05) between organisms in the column with asterisks and other organisms in the same row

Table 2:	Minimum and maximum values of count of microorganism isolated from different markets from biradon, kesham and kindrimo in Maiduguri (counts were expressed as log 10 cfu mL-1)
NB: BI: Biradon, KE: Kesham and KI: kindrimo, TVC: Total viable counts

E. coli counts (log₁₀ cfu mL^-1) of biradon, kesham and kindrimo from different markets ranged from 0.0 to 6.22, 0.0 to 6.30 and 0.0 to 5.48, respectively (Table 2) with distribution and percentage frequency of occurrence between 10 and 40, 10 and 30 and 10 and 36%, respectively. The overall mean percentage E. coli were 21.6, 23.2 and 26.4%, respectively (Table 3).

S. aureus count (log₁₀ cfu mL^-1): The mean S. aureus counts of biradon, kesham and kindrimo were between 1.00 and 4.12, 0.30 and 1.68 and 1.33 and 4.31, respectively from the different markets (Table 1) with an overall mean of 2.65, 1.16 and 2.41 log₁₀ cfu mL^-1, respectively. The mean S. aureus count of Gwage market was significantly lower (p≤0.05) than that got from other markets. The S. aureus counts (log₁₀ cfu mL^-1) of biradon, kesham and kindrimo ranged from 0.0 to 6.79, 0.0 to 3.87 and 0.0 to 6.39, respectively (Table 2) whereas, the distribution and percentage frequency of occurrence was between 15 and 48, 10 and 40 and 10 and 30, respectively (Table 3).

Shigella sp. count (log₁₀ cfu mL^-1): The mean Shigella count of biradon, kesham and kindrimo were between 0.0 and 0. 30, 0.10 and 1.00 and 0.12 and 0.40, respectively from the different markets (Table 1) with an overall mean of 0.15, 0.40 and 0.24 log₁₀ cfu mL^-1, respectively. There was no significant (p≤0.05) difference between the means of Shigella from the markets. However, Shigella was significantly (p≤0.05) lower than other organisms in the same row except in Fulani farms. The range values of count of Shigella (log₁₀ cfu mL^-1) for biradon, kesham and kindrimo fell between 0.0 and 0.48, 0.0 and 2.48 and 0.0 and 0.90, respectively (Table 2) and percentage distribution between 0.0 and 4, 2 and 10 and 2 and 8%, respectively (Table 3).

Table 3:	Distribution and % frequency of occurrence of different microorganisms isolated from biradon, kesham and kindrimo samples in Maiduguri
NB: BI: Biradon, KE: Kesham and KI: Kindrimo, TVC: Total viable

Salmonella count (log₁₀ cfu mL^-1): The mean Salmonella count of biradon, kesham and kindrimo were between 1.00 and 2.00, 1.45 and 2.36 and 0.45 and 1.85, respectively from the different markets (Table 1) with an overall mean of 1.44, 1.83 and 1.23 log₁₀ cfu mL^-1, respectively. Results obtained for Salmonella had no significant difference (p≤0.05) from results of Shigella from the different milk products. The different market means were statistically (p≤0.05) homogeneous. The range value of counts of Salmonella (log₁₀ cfu mL^-1) for biradon, kesham and kindrimo were between 0.0 and 4.02, 0.0 and 4.76 and 0.0 and 3.30, respectively (Table 2) and the distribution and percentage frequency of occurrence between 2 and 15, 5 and 20 and 2 and 20%, respectively (Table 3).

Coliform count (log₁₀ cfu mL^-1): The mean coliform count of biradon, kesham and kindrimo were between 1.88 and 4.30, 3.12 and 5.80 and 2.24 and 4.55, respectively from the different markets (Table 1) with an overall mean of 2.94, 4.12 and 3.72 log₁₀ cfu mL^-1, respectively. Biradon from Bama market was significantly higher than other biradon from other markets. Similarly, mean coliform of kesham and kindrimo were significantly higher than other organisms in the same row except the TVC. The range value of counts of coliform (log₁₀ cfu mL^-1) for biradon, kesham and kindrimo were between 0.0 and 6.48, 0.0 and 7.51 and 0.0 and 6.70, respectively (Table 2). The distribution and percentage frequency of occurrence of coliform ranged between 20 and 40, 30 and 48 and 26 and 44%, respectively. The overall mean percentage coliform was 30.8, 38.6 and 35.2%, respectively (Table 3).

Total viable count (TVC) (log₁₀ cfu mL^-1): The mean TVC of biradon, kesham and kindrimo were between 4.75 and 6.22, 6.10 and 8.60 and 4.65 and 8.20, respectively from the different markets (Table 1) with an overall mean of 5.34, 7.27 and 6.58 log₁₀ cfu mL^-1, respectively. TVC was significantly higher than mean values obtained for other groups of organisms in almost all of the samples except some coliform results that were statistically homogeneous with TVC. The range value of counts of TVC (log₁₀ cfu mL^-1) for biradon, kesham and kindrimo were between 0.0 and 8.48, 0.0 and 9.85 and 0.0 and 9.90, respectively (Table 2).

DISCUSSION

Biradon, kesham and kindrimo samples were analysed microbiologically for the presence of E. coli, S. aureus, Salmonella and Shigella sp. frequently implicated in food safety problems. Coliform was used as a measure of sanitary quality and TVC was used to indicate the overall microbial quality and shelf-life of the product. While E. coli was used as an index of faecal contamination, S. aureus, Salmonella and Shigella sp. were used as measure of organisms frequently associated with foodborne diseases.

The mean E. coli content of kesham is greater than kindrimo greater than biradon in almost all of the samples except in Bulunkutu and Bama where kindrimo had slightly higher level of E. coli than kesham. Mean E. coli content of biradon of between 1.24 and 3.30 log₁₀ cfu mL^-1 of the present study (Table 1) perhaps supports 1.85 and 2.31 obtained by Abdalla and El-Zubeir (2006). For kesham and kindrimo, E. coli mean values of between 2.40 and 4.80 log₁₀ cfu mL^-1, respectively (Table 1) supports 2.92 and 4.76 obtained by Gran et al. (2002) but contradicts lower mean values of other fermented milk products (yoghurt, roub and mish) obtained by Abdalla and El-Zubeir (2006). The range of E. coli of between 0 and 6.20 for the raw milk biradon also supports that obtained by Adesiyun et al. (1997) and Abdalla and El-Zubeir (2006). The range of E. coli in Kesham and kindrimo also supports previously published data on fermented milk by Abdalla and El-Zubeir (2006). Similarly, results obtained for percentage E. coli content of the samples supports previously recorded data by Soomro et al. (2002); Fadel and Ismail (2009) and Okonkwo (2011) but disagrees with lower values of not detectable to 20% reported by Ekici et al. (2004); Tambekar and Bhutda (2006) and El-Zubeir et al. (2008). Using E. coli as an index of faecal contamination, the samples with E. coli content up to 1 (log₁₀ cfu mL^-1) are feared to have recent faecal contamination and capable of triggering some negative health consequences.

The mean S. aureus contents of the biradon (2.65 log₁₀ cfu mL^-1) (Table 1) relates to that of raw milk obtained by Abdalla and El-Zubeir (2006). The result is however, significantly lower than that obtained by El-Zubeir and Mahboba (2007); Mohamed and El-Zubeir (2007) and Gran et al. (2002). Mean S. aureus contents of kesham of 1.16 supports the mean obtained previously for fermented milk by Abdalla and El-Zubeir (2006) mish and (Okonkwo, 2011) (nono). Mean S. aureus contents of kindrimo (2.41) is higher than values obtained by Abdalla and El-Zubeir (2006) (mish) and (Okonkwo, 2011) (nono) which are also fermented milk products. The range value of count of S. aureus between 0.0 to 6.7 (Table 2) of biradon supports 0.0-4.90 obtained by Abdalla and El-Zubeir (2006) for whole and skim milk but disagrees with high values obtained by El-Zubeir and Mahboba (2007) in Sudan. The range value of count of S. aureus between 0.0 to 3.8 and 0.0 to 6.3, respectively for kesham and kindrimo (Table 2) support 0.0 and 6.60 of (Okonkwo, 2011) but contradicts Abdalla and El-Zubeir (2006) results of 0.00-2.90 log cfu mL^-1. If S. aureus is found in foods or drinks handled by man, it is no surprise since Jablonski and Bohach (1997) noted that S. aureus could be spread from humans to food by direct contact, indirectly by skin fragments or through respiratory tract droplet nuclei and also since it could be normal on healthy human skin. Similarly, Tormo et al. (2011) also noted that Staphylococcus was the dominant bacterial sp of milk and inferior health condition of the animal increased the contamination of milk with Staphylococci. The percentage distribution Staphylococci in samples of between 15 and 48, 10 and 40 and 10 and 30, respectively for biradon, kesham and kindrimo, was lower than 75% obtained by Ekici et al. (2004) from cow milk and 60% from goat milk Tormo et al. (2011). The values however supported that obtained by Tambekar and Bhutda (2006) and El-Zubeir et al. (2008).

Low Shigella count throughout the sampling supports the previous finding of Okonkwo (2011) where no Shigella sp. was detectable in some areas studied. Similarly, mean Shigella counts of biradon, kesham and kindrimo of 0.15, 0.40 and 0.24 log₁₀ cfu mL^-1, respectively (Table 1) are statistically homogeneous with 0.30 recently obtained (Okonkwo, 2011). The range value obtained for Shigella also corroborates the earlier result of Okonkwo (2011). The mean Salmonella sp. count of biradon, kesham and kindrimo of the order 1.0 to 2.0, 1.45 to 2.36 and 0.45 to 1.85 log₁₀ cfu mL^-1, respectively (Table 1) support the value obtained by Abdalla and El-Zubeir (2006) for both fermented and raw milk and 1.17 previously obtained by Okonkwo (2011). The results however, contradict not detectable obtained by Gran et al. (2003). In public health view point, 10⁶ cells of Salmonella sp. are needed to initiate infection in healthy individuals. The percentage distribution of Salmonella in samples of between 2 and 15, 5 and 20 and 2 and 20%, respectively for biradon, kesham and kindrimo, supports 16.30% obtained by Tambekar and Bhutda (2006) and 24% previously obtained by Okonkwo (2011) but contrasts not detectable reported by Ekici et al. (2004). The percentage distribution of Shigella was low and also supported low values previously recorded by Tambekar and Bhutda (2006), Akabanda et al. (2010) and Okonkwo (2011).

The mean coliform count of 2.94, 4.12 and 3.72 log₁₀ cfu mL^-1 obtained, respectively for biradon, kesham and kindrimo (Table 1) were significant in the sense it indicates an overall poor hygiene quality of the products. Mean coliform content of biradon of between 1.88 and 4.30 supports 3.96 previously obtained by Samolada et al. (1998) but disagree with higher value of 3.32x10⁶ cfu mL obtained by Mohamed and El-Zubeir (2007). Similarly, mean coliform contents of kesham and kindrimo of between 3.12 and 5.80 and 2.24 and 4.45 log₁₀ cfu mL^-1 agrees partly or wholly with values obtained by Gran et al. (2002) and El-Bakri and El-Zubeir (2009) but disagrees with lower values obtained by Lore et al. (2005) and Nahar et al. (2007). Inadequate cleaning of milk utensils is noted to be the most common source of coliform in raw milk (Murphy and Boor, 2000).

The mean TVC of between 4.75 and 6.22 log₁₀ cfu mL^-1 obtained for biradon (Table 1) agrees with 5.70 obtained by Samolada et al. (1998) but disagree with higher value of 7.9x10¹⁰, 1.4x10⁸ and 3.8x10⁸ cfumL^-1 obtained by El-Zubeir and Mahboba (2007) and lower means of 2.1x10² cfu mL^-1 obtained by Obi and Ikenebomeh (2007) and 3.6 log₁₀ cfu mL^-1 average bacterial counts of Tormo et al. (2011). Similarly, mean TVC of kesham and kindrimo between 6.10 and 8.20, respectively agrees with values obtained Rihab et al. (2010) and Nahar et al. (2007) and partly with El-Bakri and El-Zubeir (2009) since these authors recorded higher values in plain yoghurt. The mean TVC contradicts lower mean value of 3.76 log₁₀ cfu mL^-1 obtained by Okonkwo (2011) for aerobic mesophilic count. The range value of TVC 0.0 to 8.48; 0.0 to 9.85 and 0.0 to 9.90 log₁₀ cfu mL^-1, for biradon, kesham and kindrimo, respectively (Table 2) supports values obtained by Savadogo et al. (2004), El-Zubeir et al. (2008), El-Bakri and El-Zubeir (2009) and Okonkwo (2011).

CONCLUSION

All the milk products both raw and fermented studied harbour some degree of microorganisms which are capable of endangering human health when consumed in sufficient dosage. There is no significant difference between results obtained for the different milk products. The sources through which these organisms gain access into the milk products are: via the milking animals themselves, the process line, process environment and distribution channel. Proper pasteurization and safety distribution of the products in a hygienic environment is recommended.