Microbiological Analyses and Safety Evaluation of Nono: A Fermented Milk Product Consumed in Most Parts of Northern Nigeria

Okonkwo, Ogbonna Innocent

INTRODUCTION

‘ Nono’ (Hausa) is an opaque white to milky coloured liquid food drink got from fermented raw milk. It is a healthful food whose consumption transverses the Saharan tribes of West African Sub-region extending to the inhabitants of the Mediterranean region and also the Middle East. In the Middle East, it is called ‘dahi’ or ‘lassi’ (Nahar et al., 2007). Nono also called nunu by some tribes in Nigeria contains good quantities of amino acids, calcium, phosphorous and vitamins A, C, E and the B complex (Nebedum and Obiakor, 2007).

Predominantly, nono is being prepared and hawked by the nomadic Hausa/Fulani cattle herdsmen, who control over 80% of Nigerian’s cattle production. Consumption of nono was limited to Fulani/Hausa indigenous (Obi and Ikenebomeh, 2007; Adesokan et al., 2011) since most non indigenous see their preparation as apparently unhygienic and since it has poor shelf life (Yahuza, 2001) it is gaining wider acceptance nowadays.

Raw milk has low keeping quality and at room temperature, spontaneous microbial spoilage occurs turning the product sour few some days. This is brought about by the activity of lactic acid bacteria (Wouters et al., 2002). Depending on its preservation and process-line, microorganisms other than lactic acid bacteria could be found in the nono. Milk’s nutritional composition makes it not only suitable for human nutrition but also ideal for microbial life. The growth of microorganisms in food could make the food grossly unwholesome and harmful to consumers. Outbreaks of milk-borne diseases have occurred despite pasteurisation, as a result of either improper pasteurisation or product recontamination (Altekruse et al., 1998; Da Silva et al., 1998; Hartman, 1997; Nebedum and Obiakor, 2007).

It is noticed that raw milk often contains microorganisms which may likely cause foodborne diseases (Adesiyun et al., 1995; Headrick et al., 1998; Steele et al., 1997). Even when the milk is fermented, the fermentation process with the attendant drop in pH may not rid the product of these organisms and may be carried to consumers. Salmonellae and other microorganisms have been known to underscore the importance of milk and milk products as vehicle for human infections. For these reasons, McEwes et al. (1988) reasoned that the presence of Salmonellae and other human pathogens in unpasteurized milk is a public health hazard. Food safety guideline for food banks recommends that milk and milk products including cream and cream products, ice cream, frozen desserts, yoghurt and similar foods must be pasteurized, held and distributed in their original unopened containers.

Different categories of microorganisms including fungi, bacteria, rickettsia and viruses could be found in milk since the udder of the animal could harbour organisms while others come as contaminants due to poor handling. Most handlers or sellers of nono are street peddlers. Often, not all nono sent to the market by peddlers is sold the same day and in most developing countries, such unsold ones have to go back to market with no special attention to preservation or safety. Thus pathogenic organisms that might have gained access into the product have enough time to multiply and/or produce harmful metabolites. The present work was aimed at investigating the microbiological quality and safety of nono consumed in Maiduguri, Nigeria.

MATERIALS AND METHODS

Sample collection: Nono samples were purchased from five different markets namely: Bulunkutu, Gwange, Monday, Bama Road Motor Park and Unimaid Motor Park Markets all within Maiduguri metropolis. Forty samples were randomly purchased from each of the markets. Purchased samples were transported to the University of Maiduguri Microbiology Laboratory in sterile corked plastic tubes packed in iced container. Purchases were done between 9.00 am and 11.00 am each day lasting from September 2009 to February 2010. Our laboratory prepared nono got from fresh cow milk (purchased at Fulani Farm in Maiduguri) was also analysed.

Nono preparation and consumption: It is produced from cow milk collected in calabash and allowed to ferment naturally for 24h (Eka and Ohaba, 1977; Olasupo et al., 1996).

Microbiological analyses: Nono samples approximately 40 mL each contained in pre-autoclaved containers were used for the isolation and enumeration of the microorganisms. In each isolation protocol, nono sample was shaken and 10 mL of the sample was aseptically introduced into 90 mL of sterile normal saline solution and homogenized by shaking followed by further decimal dilutions to up to 10^-6concentrations. A 0.1 mL quantity of appropriately diluted sample was used to inoculate freshly prepared media and surface-plated. Media employed for the isolation and enumeration of the organisms included: 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 spp. Nutrient Agar (NA) (Biotech Lab. Ipswich, UK) was used for aerobic mesophilic, anaerobic mesophilic and psychrotrophic microorganisms’ counts. Yeasts and mould counts were carried out on a Potato Dextrose Agar (PDA) (Lab M. Ltd, Bury Lancashire BL9 6As, United Kingdom) plate. Media were sterilized by autoclaving at 121°C for 15 min except DCA which involved only boiling over gauze. In all cases of colony counts, the resulting colonies following inoculation and incubation were counted using digital colony counter (Labtech, New Delhi, India).

Aerobic mesophilic counts (AM): Inoculated NA plates were incubated at 30°C for 48 h.

Anaerobic mesophilic counts (AAM): Inoculated NA plates were incubated in anaerobic jars (Equtron) containing heated iron nail and candle as recommended by Cheesbrough (2004) at 30°C for 48 h.

Psychrotrophic microorganisms’ counts: Inoculated NA plates were incubated at 6.5°C for 24-48 h.

Yeasts and moulds counts: PDA plates containing Chloramphenicol were inoculated as above. Incubation was at 28°C for 5 days. Colonies were thereafter counted.

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

Isolation and enumeration of S. aureus: BPM were inoculated as above and incubated at 37°C for 48 h. 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: Isolation and enumeration of Salmonella and Shigella sp. was as recommended by Macfaddin (1977).

Biochemical identification of the isolates: The biochemical tests for the identification of the isolates were the citrate utilization, indole, methyl-red, Voges-proskauer, Triple Sugar Iron (TSI), urease, oxidase, coagulase and catalase tests. Cowan and Steel (1965) and Cheesbrough (2004) procedures were used for these biochemical tests.

The sample pH: The pH of various samples was measured using a pH meter (WPA pH Meter, India) after standardization with pH 4, 10 and 7 buffers (BDH England).

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 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.

Table 1:	Mean microbial counts (counts were expressed as log₁₀cfu mL^-1) and mean pH of samples of nono from different markets in Maiduguri

NB: Ec: E. coli, Sa: S. aureus, Sg: Shigella, Ss: Salmonella counts, Am: Aerobic mesophilic, Aam: Anaerobic mesophilic counts, Pm: Psychrotrophic microorganism, Ym: Yeast and mould counts. *Indicate statistically significant difference (p<0.05) between organisms in the column and other organisms in the same row

Table 2:	Minimum and maximum values of count of microorganism isolated from different markets in Maiduguri

Counts were expressed as log₁₀ cfu mL^-1. Total No. of samples = 40 from each market. NB: Ec: E. coli, Sa: S. aureus, Sg: Shigella, Ss: Salmonella counts, Am: Aerobic mesophilic, Aam: Anaerobic mesophilic counts, Pm: Psychrotrophic microorganism, Ym: Yeast and mould counts

RESULTS

E. coli count (log₁₀ cfu mL^-1): The mean E. coli counts (log₁₀ cfu mL^-1) were between 1.37 and 3.29 from the different markets (Table1) with an overall mean of 2.21 log₁₀ cfu mL^-1. The mean E. coli count of Unimaid was significantly higher (p≤0.05) than Monday, Gwange and Bama but homogeneous with Bulunkutu market. E. coli count from different markets ranged from 0.0 to 7.89 log₁₀ cfu mL^-1 (Table 2).

S. aureus count (log₁₀ cfu mL^-1): The mean S. aureus counts fell between 2.98 and 0.76 with an overall mean of 1.51 log₁₀ cfu mL^-1(Table 1). The mean S. aureus count of Bulunkutu market was significantly higher (p≤0.05) than that got from other markets. The S. aureus count of the samples ranged from 0.0 to 6.60 log₁₀ cfu mL^-1(Table 2).

Shigella sp count (log₁₀ cfu mL^-1): The mean Shigella count was between 0.0 and 0.92 and had an overall mean of 0.30 log₁₀ cfu mL^-1. There was no significant (p≤0.05) difference between the means of Shigella from the markets. The range values of count of Shigella fell between 0.0 and 6.0 and percentage distribution between 0.0 and 12.0. Shigella was not isolated from three markets under study (Table 1-3).

Salmonella count (log₁₀ cfu mL^-1): The mean Salmonella count was between 0.43 and 2.37 and the overall mean was 1.17 log₁₀ cfu mL^-1(Table 1). Bulunkutu market value was significantly higher (p = 0.05) than that of other markets. The range value of counts of Salmonella was between 0.0 and 6.5 and the distribution and percentage frequency of occurrence between 4.0 and 22.0 (Table 3).

Table 3:	Distribution and (%) frequency of occurrence of different microorganisms isolated from nono samples

Values in parenthesis are individual market (%) frequency. In each market, 40 nono samples were analysed (n = 40)

Aerobic mesophilic count (log₁₀ cfu mL^-1) (AMC): The mean AMC from the different markets fell between 2.53 and 4.89. The overall mean AMC was 3.76 log₁₀ cfu mL^-1 (Table 1). There was no significant difference between the means of AMC (Table 2). The range values of AMC fell between 0.0 and 8.90.

Anaerobic mesophilic counts (log₁₀ cfu mL^-1) (AAMC): The mean AAMC was between 0.69 and 4.09 with an overall mean of 1.76 log₁₀ cfu mL^-1(Table 1). There was a significant (p≤0.05) difference between the means obtained from Bulunkutu and other markets analysed. The range values of AAMC were between 0.0-6.30 (Table 1).

Psychrotrophic microbial count (PMC) (log₁₀ cfu mL^-1): The mean PMC (log₁₀ cfu mL^-1) was between 0.86 and 6.45 from the different markets. The overall market mean for PMC was 0.76 log₁₀ cfu mL^-1(Table 1). There was no significant difference between the means obtained from the markets. The range values of PMC fell between 0.0 and 4.3 (Table 2).

Yeast and mould count (YMC) (log₁₀ cfu mL^-1): The mean YMC fell between 0.81 and 1.61. The overall mean YMC was 1.23 log₁₀ cfu mL^-1(Table 1). There was no significant difference between the mean values obtained from the markets. The range values of YMC from the markets were between 0.0 and 5.00 (Table 2).

The sample pH: The pH range of the nono samples from the various markets was between 3.8 and 5.4 (Table 1).

DISCUSSION

The presence of E. coli (43%), S. aureus (28%), Shigella (8%) and Salmonella (24%) in nono sold within Maiduguri Metropolis is quite high to attract public health attention. From the results, E. coli was the most frequent isolate (Table 1-3). Results obtained for % E. coli content of the samples fell within the range of 15-65 which supports that obtained by Soomro et al. (2002) but lower than not detectable to 20% reported by Ekici et al. (2004) and 2.5% by Mohamed and El-Zubeir (2007). The mean E. coli count of log 2.21 cfu mL^-1 supports that obtained by Abdalla and El-Zubeir (2006). While originally E. coli had been associated with the contamination of milk and milk products (Kulshrestha, 1990), researches have linked the presence of E. coli with the presence of other enteric pathogens (Adesiyun et al., 1995; Norwegian Food Control Authority, 1994; Smoot and Pierson, 1997; Van den Berg, 1988).

The mean S. aureus counts (log₁₀ cfu mL^-1) of 1.51 contradicts a lower mean of 0.48 obtained by Abdalla and El-Zubeir (2006) from yoghurt samples and the % distribution in samples of 28 was lower than 75% obtained by Ekici et al. (2004) from cow milk and 60% from goat milk Tormo et al. (2011). The range value of count of S. aureus between 0.0 and 6.60 also contradicts Abdalla and El-Zubeir (2006) results of 0.00-2.90 log cfu mL^-1. Processing operations and fermentation parameters could have brought about the discrepancy. The presence of S. aureus in the milk samples corroborates the finding of Tormo et al. (2011) that Staphylococcus was the dominant bacterial sp. of milk and inferior health condition of the animal increased the contamination of milk with Staphylococci. 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. Mastitis udder also harbours S. aureus (Wellenberg et al., 2002). Consequently, milk from mastitis cows could be another reservoir for S. aureus (Gran et al., 2002). The range of S. aureus in this study exceeded the estimated minimum for the organism to produce intoxication and also, the pH (3.8-5.4), temperature (30-45°C) and the holding time conditions agreed with the requirement for S. aureus to produce sufficient enterotoxin (Rorvik and Granum, 1999).

Shigella sp. was not detectable in three areas under study. This organism is not an intrinsic flora of the animal; therefore, contamination of the fermented milk with the organism could have arisen from handling. Twenty-four percent of the assayed samples showed the presence of Salmonella spp. The mean Salmonella counts (log cfu mL^-1) of 5.9 contradicts 1.12 obtained by Abdalla and El-Zubeir, (2006) and % distribution of 24 contrasts not detectable reported by Ekici et al. (2004) and Mohamed and El-Zubeir, (2007). Salmonella are pathogens that could originate from the animals themselves. Thus, presence of Salmonella in fermented milk is not surprising since they could either be transmitted from the animal before preparation or could have come via cross contamination. Since, these organisms including E. coli and S. aureus were not isolated from our laboratory prepared nono, it is possible to get pathogen-free nono via general hygiene of the process line thereby saving consumers from the potential health risks.

The aerobic mesophilic count (AM) ranged from not detectable 0 to 8.9 log₁₀ cfu mL^-1 and the mean AM counts was 3.76 log₁₀ cfu mL^-1 (Table 2). The mean aerobic mesophilic count (log cfu mL^-1) of 3.76 agreed closely with 3.6 average bacterial counts of Tormo et al. (2011). Aerobic colony count is useful for indicating the overall microbial quality of food product. It generally does not relate to food safety hazards but acts as an indicator for food quality and shelf-life duration (Pianetti et al., 2008). Except for Bulunkutu, anaerobic organisms were generally very low throughout the study. The reason for this could be their absence during nono preparation and distribution. The mean Psychrotrophic microbial counts (log cfu mL^-1) of nono were low throughout the counts with ranges between 0 and 4.3 log₁₀ cfu mL^-1. It stands to reason that storage of the product at such temperature could increase its shelf life.

The mean YMC of 1.23 log₁₀ cfu mL^-1 was higher than not detectable recorded by Ukwuru and Ogbodo (2011) from fermented tigernut- a milk product but however contradicts higher value of 3.48 reported by Awad et al. (2006) from similar product. Range yeast and mould count of 0.81-1.61 was partly in support of 1.44-2.06 obtained by Nahar et al. (2007) but lower than 1.83x10³-3.7x10⁶ obtained by Savadogo et al. (2004). The reason for the contradictions could be linked to the chance fermentation practiced by many local producers as differences in YMC of the same product from different manufacturers had been documented (El-Bakri and El-Zubeir, 2009). Yeasts are known to cause spoilage in products such as yoghurt and sour milk; however they are also important because they produce desirable favours as in cheese ripening (Fleet, 1990; Rohm et al., 1992; Jakobsen and Narvhus, 1996). The mean YMC of 1.23 log₁₀ cfu mL^-1 (Table 1) was lower than 10⁵-10⁶ cells g^-1reported to cause pronounced spoilage (Fleet, 1990).

The pH range of samples between 3.8 and 5.4 supports that obtained by El-Bakri and El-Zubeir (2009) but however contradicts 5.51-6.29 reported by Adesokan et al. (2011) and 5.7 by Obi and Ikenebomeh (2007). The differences in pH could be as a result of some factors including; length of fermentation and the starter culture. If the keeping time of nono increases prior to consumption, the acidity increases and his determines the number and kind of contaminating organisms.

CONCLUSION

The significant (p≤0.05) difference between the means of the microbial groups in this study could be due to lack of standardized method of nono preparation, or the health status of the milk producing animal or other environmental variables. The recommendation of the food safety guideline for the pasteurization of food like milk and distribution in unopened form for food safety could account for the many microorganisms isolated from the nono. The practice of preparation and distribution of nono in open calabash contravenes such principles. Proper hygiene in the process-line of nono is recommended since the markets selected were the major nono selling points in Maiduguri and since the laboratory prepared nono had no detectable pathogen.