2. Materials and Methods
2.1. Collection of and Transportation of Samples
Six samples of full cream typical 8 h old of typical indigenous traditional African fermented milk were collected in duplicates from a Kalenjin farm in (Kenya) labelled as KE and two farms in Uganda; the Karamojong, labelled (UG 1) and Acholi in Gulu (UG 2). The samples were collected during the rainy season (July - September) in sterile plastic milk bottles. The samples were immediately put on ice in an ice box and transported to the laboratory. On arrival in the laboratory, pH, titratable acidity, and microbiological analyses of the samples were taken within four hours then after 24, 48 and 72 h to check the microbiological growth during storage. Broth dilution and pour plate methods were used for the microbial analyses (11). The remaining samples were then stored in a fridge (4oC). The yoghurt samples were prepared according to the Official Methods of Analysis Chemist (AOAC )(12).
2.2. pH Measurement
The pH of the samples was measured with a Mettler Toledo Delta 320 pH meter, at room temperature (20oC ± 2). The pH electrode was firstly calibrated at pH 4 and 7 with standard buffer solutions. The calibrated pH electrode was inserted into a 10 ml sample. The readings were recorded accordingly. All measurements were carried out in triplicate.
2.3. Titratable Acidity of Fermented Milk Sample
20 g of well-shaken yoghurt or un-fermented milk was weighed accurately into a 250-mL Erlenmeyer flask, 40 mL of boiled and cooled distilled water was added to it. With a sterile pipette, 2-3 drops of the indicator (phenolphthalein) were added to the milk as an indicator of the endpoint. The content of the flask was titrated against 0.1N sodium hydroxide (NaOH) until the sample changed colour to persistent light pink. The initial and final readings on the meniscus burette were recorded, prior to starting the titration and at the endpoint, respectively. The amount (mL) of 0.1N NaOH titrated was calculated by subtracting the initial volume from the final volume to give the amount of NaOH used to reach the endpoint. This was performed at least three times per sample. The per cent lactic acid was then calculated using the equation Eq [
1] below:
where:
Vt= Volume of titrant (ml NaOH)
N = Normality of titrant
90 = Equivalent weight for lactic acid
Vs = Volume of sample used (ml yoghurt/milk)
2.4. Sample Preparation for Analysis
10 millilitres (ml) of each sample were aseptically weighed and homogenised with 90 ml of sterile quarter-strength Ringer’s Solution (pH 7.2) using a Stomacher lab-blender (Seward Medical, London, UK) for 2 minutes. Serial dilutions (10−1 to 10−8) were prepared in the same diluent and duplicate counting plates were prepared. For pour plating, one millilitre of the sample was taken from the chosen dilution to obtain an expected count of 30 to 300 for Aerobic Mesophilic Bacterial Count, 15 to 150 for Coliform count, and 10 to 200 for Yeast and Mould count (13). The media and sample dilutions were gently mixed and allowed to set. All counts were made in duplicate plates. For surface plating, 0.1 ml of the dilutions were spread on the surface of dried media plates.
All media were prepared according to the manufacturers’ instructions. Sterile quarter-strength Ringer’s Solution (BR 0052, Thermo Fischer Scientific, Loughborough, UK) was used as an isotonic diluent for the microorganisms. The quarter Ringer solution was sterilized by autoclaving at 121˚C for 15 minutes. All media were prepared with deionized water. Glassware such as Petri dishes, test tubes, pipettes and flasks were sterilized in a hot oven at 160˚ C for one hour.
2.5. Microbial Analysis
The yoghurt samples were examined for Total Aerobic Mesophilic Bacterial Count. This estimates the number of viable aerobic bacteria per gram or millilitre of the product measured in colony-forming unit per ml (cfu/ml) according to the procedures of Abebe et al., (14). Samples were prepared as above (section 2.4). Aerobic mesophilic bacteria were counted on pour plates of Plate Count Agar (PCA), (Oxoid M325, Basingstoke, Hampshire, UK) incubated in an inverted position at 30oC for 48±1h (15).
Lactobacilli were enumerated on pour plates of de Man Rogosa and Sharpe agar (MRS, LAB098) at pH 5.5 (16) incubated in an inverted position incubated anaerobically in an anaerobic jar at 42±1°C for 48±2 h. A further analysis was carried out on MRS agar + Vancomycin for the enumeration of leuconostocs incubated anaerobically at 32oC for 48±2 h in Anaerobic jars (Biolab and Oxoid) with gas generating kits (Oxoid BR 38B). Streptococci were enumerated on M17 Agar (LAB092) and M17 broth (CM0817, pH 6.5), incubated aerobically for 48 ±2 h at 37±1°C (17).
For Salmonella identification, 25 ml of the sample was pre-enriched with 225 ml of Buffered Peptone Water (BPW) and incubated for 24 h at 37oC. A portion (0.1 ml) of the pre-enriched culture was transferred to 9.9 ml of Rappaport-Vassiliadis (RV) broth and incubated at 42oC for 24 h. A loopful of the enrichment broth was then transferred to Xylose Lysine Deoxycholate (XLD) agar and incubated at 37oC for 24 h. Characteristic Salmonella colonies having a slightly transparent zone of reddish colour and black centre were sub-cultured on nutrient agar and confirmed biochemically using Triple Sugar Iron (TSI) and Simon citrate agar according to the procedures of Gebeheyu et al. (18) with some modification.
Escherichia coli and coliform bacteria were enumerated on Violet Red Bile Agar (VRBA, Oxoid CM 107B Ltd Basingstoke, Hans UK and Violet red bile agar (Oxoid CM 107 with added MUG supplement BRO 71 E), Thermo Fischer Scientific, Loughborough, UK) (19) incubated aerobically for 24±2h at 37±1°C. The supplement containing 4-methylumbelliferyl-B-d-glucuronide (MUG) allowed the separate enumeration of E. coli which contain glucuronidase activity. The presence of E. coli was further tested using indole production in tryptone water (Oxoid, UK) with Kovac’s reagent (Biolife), as previously reported by Moushumi and Prabir (20).
For the general enumeration of Salmonella and Shigella spp., the sample (25 ml) was pre-enriched with 225 ml of Buffered Peptone Water (BPW) and incubated for 24h at 37oC. A portion (0.1 ml) of the pre-enriched culture was transferred to 10 ml Rappaport-Vassiliadis (RV) broth and incubated at 42oC for 24h. A loopful of the enrichment broth culture was then transferred to Xylose Lysine Deoxycholate (XLD) agar and incubated at 37oC for 24h. Characteristic Salmonella colonies having a slightly transparent zone of reddish colour and black centre were sub-cultured on nutrient agar and confirmed biochemically using Triple Sugar Iron (TSI) and Simon citrate agar (21). Red colonies only, were regarded to be Shigella.
Most Probable Number technique was used for the enumeration of Bacillus cereus using selective media mannitol yolk Polymyxin (MYP) B agar and polymyxin pyruvate egg mannitol bromothymol blue agar (PEMBA).(22)
For S. aureus counts were enumerated on Baird–Parker’s medium (Oxoid CM 0275 + SR054C) Staphylococcus aureus was detected using the reference method of the International Dairy Federation (23).
Listeria monocytogenes was enumerated in a well-mixed sample (25 ml), homogenized in 225 ml of Listeria Enrichment Broth A and B then incubated for 24h at 37oC (24) and on Listeria selective medium (Oxford formulation CM856, Oxoid UK) adjunct with Oxoid™ Listeria selective supplement (SR0140, Oxoid, UK). The latter was then incubated for 48 h at 30 °C. A loop full of the enrichment culture broth was streaked in duplicate onto Polymyxin-Acriflavine-Lithium Chloride-Ceftazidime-Aesculin-Mannitol (PALCAM) selective agar (Oxoid, CM877) and incubated for 48h at 37oC. Suspected Listeria monocytogenes colonies were further characterized using Gram staining and catalase test. The color of Listeria spp. colonies typically ranged from greyish green to brownish green with black zones of 1–3 mm diameter of aesculin hydrolysis. Five presumptive Listeria monocytogenes colonies were selected from each Petri dish of selective agar and cultivated on trypticase soy agar medium (CM0131, Oxoid, UK) supplemented with 0.6% yeast extract and subsequently placed into an incubator for 24 h at 30oC to perform further analyses, including examination of non-spore Gram-positive coccobacilli strains for catalase, umbrella growth in motility, nitrate reduction, MR/VP, β-hemolysis production biochemical tests (acid formation from glucose, rhamnose, xylose, and mannitol fermentation) and a further characterised using Gram stain and catalase test were carried out (24).
Yeast and mould counts were enumerated on Malt Extract Agar (MEA) (1.5% Agar No 2) (Oxoid) and Potato Dextrose Agar (+0.005 g/L chloramphenicol). The plates were incubated at 20 and 25 ± 1°C for 5 days. Yeast and mould colonies were counted separately (25).
Analytical Profile Index (API) Biochemical Test
The analytical profile index or API is a biological classification of bacteria based on biological tests, allowing fast identification. This system is developed for quick identification of clinically relevant bacteria and because of this, only known bacteria could be identified. The Biochemical and Physiological tests were carried out with the appropriate API strips to identify the presumptive bacteria.
Table 1.
Summary of culture and media used for the isolation of microorganisms in traditional African fermented milk (cfu/ml).
Table 1.
Summary of culture and media used for the isolation of microorganisms in traditional African fermented milk (cfu/ml).
Medium for growth |
Microorganisms |
Time (Hours) |
Growth condition and incubation Temperature |
Growth condition and incubation Temperature |
Plate Count Agar (Oxoid M325) |
Total aerobic mesophilic aerobic bacteria |
48±2h |
aerobic 30±1oC |
aerobic 30±1oC |
MRS agar, LAB098) |
Mesophilic Lactobacilli
|
48±2h |
aerobic 35±1oC |
aerobic 35±1oC |
MRS agar (LAB 098 + Vancomycin) |
Leuconostoc |
48±2h |
anaerobic 30±1oC |
anaerobic 30±1oC |
MRS agar (pH 5.5) LAB098 |
Thermophilic Lactobacilli
|
48±2h |
anaerobic 42±1oC |
anaerobic 42±1oC |
MRS agar (pH 6.) LAB098 |
Thermophilic Lactococci |
48±2h |
anaerobic 42±1oC |
anaerobic 42±1oC |
M17 agar (LAB 092) |
Mesophilic Streptococci
|
48±2h |
anaerobic 30±1oC |
aerobic 35±1oC |
Violet Red Bile Lactose agar with MUG supplement BRO 71 E), |
Non-Sorbitol E. coli
|
24 ±2h |
aerobic 37±1oC |
aerobic 37±1oC |
Violet Red Bile Agar (VRBA) |
Total coliform |
24 ±2h |
aerobic 30±1oC |
aerobic 30±1oC |
XLD |
Salmonella and Shigella spp. |
24 ±2h |
aerobic 37±1oC |
aerobic 37±1oC |
Baird–Parker’s medium (Oxoid CM 0275 + SR054C) |
Staphylococcus aureus |
24 ±2h |
aerobic 37±1oC |
aerobic 37±1oC |
Listeria Enrichment Broth A and B |
Listeria. Monocytogenes |
24 ±2h |
aerobic 30±1oC |
aerobic 30±1oC |
B. cereus agar |
B. cereus |
|
aerobic 30±1oC |
aerobic 30±1oC |
1.5% Malt Extract and Agar No. 2 |
Yeast and mould |
|
aerobic 25±1oC |
aerobic 25±1oC |
PDA + chloramphenicol |
Mould |
|
aerobic 30±1oC |
aerobic 30±1oC |
4. Discussion
In this study, the physicochemical, and microbiological attributes of typical traditional African yoghurt from Northern Uganda and western Kenya, were assessed to establish the status of microbial risks associated with the traditional fermented milk. Sour milk is processed at the household level by leaving the fresh raw milk to ferment naturally for 1 -3 days at ambient temperature. Fermentations are carried out spontaneously in gourds or earthenware pots. Sometimes sour milk from previous batches is added to speed up the fermentation process (26).
In the three days from production to analysis), the pH of the tested traditional fermented milk was low (2.9 -3.6). Makut et al. (27), Mathara et al. (28), Ifeanyi et al. (29) and Digbabul et al. (31) reported pH results ranging from pH 3.5- - 5.11 for traditionally fermented yoghurt. The low pH in this study was reflected in the titratable acidity which was 1.26 ± 0.1, 0.71 ± 0.1; 0.92 ± 0.1% for UG 1, UG 2 and KE respectively.
The Aerobic Mesophilic Bacterial count (AMBC) in fermented milk indicates the sanitary conditions during the production and handling of raw milk or post-fermentation contamination (32). The average AMBC obtained in the current study was very high (x 109 cfu/ml). This number failed to comply with the Health Protection Agency guidelines (33) for acceptable microbial limit (x 106 cfu/ml) in fermented milk products. In regards to the microbial quality of the tested samples, the AMBC was not significantly different (p>0.05) from each other.
The mean counts for mesophilic lactobacilli were highest in UG 1 (x 108) followed by KE (107), and lower in UG 2 (106 cfu/ml). However, the thermophilic lactobacilli were 107 cfu/ml in UG 1 but higher in UG 2 samples (109 cfu/ml) although lower (106 cfu/ml) and 3 logs lower in KE samples and UG 1 respectively. A high level of thermophilic lactobacilli was recovered in UG 2 sample with counts of 109 cfu/ml. The high AMBC (106 - 109 cfu/mL) could come from the already high numbers of bacteria in raw milk as observed by other researchers in raw milk taken from different areas of Africa (5, 8, 10, 12, 13, 16). Hot weather at the production areas also enhances the growth of microorganisms in the milk if contaminated before or during processing (33). Besides having high counts of AMBC, the yoghurt samples had a rich diversity of microorganisms, predominated by lactic acid bacteria and yeasts.
In Africa, fermentation is spontaneous with back slopping using the previously fermented milk as starters rather than specific starter cultures as elsewhere in the world. Thus it comes as no surprise that this typical African fermented milk harboured such a rich and diverse type of microbes, especially lactic acid bacteria. The level of the bacteria recovered in the samples is in agreement with those reported for Zambia by Yambayamba and Zulu, (5). Similarly, high bacterial counts (5.6 -7.5 log cfu/ml) were reported by Abdalla and Abdel Nabi (34) in zabadi (x 108 cfu/ml) of Sudan and Egypt; (34); in the traditional fermented milk of Zimbabwe (x 108 cfu/ml) (35); in the traditional fermented milk of Morocco (36). In South Africa, a high number of microorganisms (x 108 cfu/ml) was reported too (37, 38). This high number of mesophilic bacteria could be due to the warm ambient temperature (28-35oC) of the natural fermentation of the milk at the time. The presence of microorganisms in traditional fermented milk depends on the nature of the fermented milk and the temperature of the regions where they were obtained from (39). It also follows the level of contamination at the production site. Contamination can occur during milking, especially where hygiene practices such as pre-milking udder washings are poor (40). It is therefore important to remove both visible dirt and bacteria from the outer surface of the udder which are likely to contribute to the contamination of the raw milk. Most of the traditional herders in the region of study do not practice pre-udder washing (14).
Furthermore, other workers (6, 41, 42) noted that mesophilic bacteria such as Leuconostoc spp. are observed in traditional fermented milk products in regions with cold climates. Whereas, in warm regions, thermophilic bacteria such as Lactobacillus and Streptococcus dominate (43). This could explain the high numbers of mesophilic bacteria in these samples because they were fermented and collected during the rainy season and cooler months (25-35°C) in both Kenya and Uganda.
Lactic acid bacteria were in the range of 108 log cfu/ml. The counts of thermophilic lactobacilli and lactococcal were 2.87 x 107 in UG 1 and 1.54 x 109 in UG 2 and 1.74 x 108 in KE samples. Obadai and Dodd (44) reported counts of LAB in the range of x 108 to x 1010 in nyarmie, the traditional fermented milk of Ghana. This agrees with those reported by Owusu-Kwarteng et al. (45) and in nunu, of Ghana’s traditional fermented milk product and by Mathara et al., (46) of kule naoto in Kenyan traditional milk. In this report, the most dominant streptococci were S. thermophilus. The abundance of Lactobacillaceae and Streptococcaceae over other families suggested the dominance of LAB during the fermentation process, and this was equally reported in other studies (43, 46, 47). In addition, this high number of lactic acid bacteria could be due to the natural selection and/or temperature of fermentation. Fewer leuconostocs suggest that this group are unable to compete with other lactic acid bacteria in mixed cultures (48). This gives them a selective disadvantage over other lactic acid bacteria (48) and a selective advantage over thermophilic bacteria. Lactic-acid bacteria are generally recognized as safe (GRAS) as well as being part of the natural microbiota of various foods and are often used as starter cultures. Many LAB such as Lactococcus, Leuconostoc, Pediococcus, and Lactobacillus species demonstrate success in inhibiting microorganisms and other pathogens in yoghurt (49, 50).
Coliforms were high in UG 1 sample (x 105 cfu/ml) but lower in UG 2 and KE (x 103 cfu/ml) samples in this study. Counts of coliform in UG 1 samples suggested poor handling and processing conditions of the milk (51). Other pathogens such as E. coli, Salmonella species, Bacillus cereus and S. aureus were also recovered with counts between 103 and 104 cfu/ml. Hamama (52) reported similar results in ‘Lben’ and ‘Jben’ the Moroccan traditional fermented dairy products. Salmonella species are known pathogens that can cause food poisoning if contaminated milk or milk products are consumed. In the present study, Salmonella species were recovered in UG 1 samples irrespective of the low pH (pH 2.9). Salmonella, as enteric pathogens, encounter a low pH value in the environment, especially during its transit in the host. According to Foster (53), Salmonella species such as Salmonella typhimurium periodically confront acid environments during its life. In an experiment, Liyuwork et al. (54) observed antimicrobial resistance in Salmonella species isolates from dairy products in Addis Ababa. Chatti et al., (56) reported acid-resistant Salmonella isolated from food and waste water in Tunisia. Although Salmonella is supposed to be destroyed or inactivated during fermentation of highly acidic products such as yoghurt in which the pH value is less than 4.55, this is not the case in this study where the acidity is low, yet the pathogen was still detected in some of the samples. This could be due to the fact that Salmonella can survive in various environmental niches for long periods of time (53).
Many diseases are transmissible via milk products and pathogenic and acid-tolerant bacteria in acidic foods have recently been a cause of public health concern. Unpasteurised milk has been a major vehicle for the transmission of pathogens such as E. coli, L. monocytogenes and Salmonella (57). It can be assumed that other sources of contamination by microorganisms are unclean teats, milkers’ hands and the use of the same milking and fermentation vessels (58). The presence of coliforms has long been thought to indicate faecal contamination (57, 58), however, recent reports regarding this diverse group of bacteria indicate that only a fraction are faecal in origin, while the majority are environmental contaminants (59). Low counts of coliforms might be due to the high acidity of the products. However, coliforms were still recovered even in such high acidity product
Yeast and mould can build up on equipment surfaces and under the surface of the package lid which often contaminate the fermenting milk (59). The presence of yeast and mould in milk and its product is undesirable as they can cause changes in the product with reduced shelf life rendering it unacceptable for consumption (60). In this study, yeasts and moulds formed a high number of the components of the microbial population. The high number of yeasts and fungus in the products suggests a high presence of yeasts in the environment where the milk was fermented.
In addition, it indicates that yeasts are a significant part of the microflora of these naturally fermented milk products in these areas. Yeasts could be a common part of the flora of the milking parlour (25) and milk containers or fermentation vessels and could impact the overall quality of these products. Yeasts and mould can produce toxic metabolites which are not destroyed during fermentation. This finding agrees with the reports of Akabanda et al. (61) and Savova and Nikolova (62). In this study, several yeasts and mould species were also recovered in the traditional yoghurt similar to the report of Savova and Nikolova (62). Growth of yeasts is mostly undesirable in milk and dairy products because these microorganisms harbour a high risk of spoilage. However, yeasts play an important role in foodstuffs, as they are able to grow in a broad range of pH environments and usually adapt to coexistence with LAB in acidic environments (63). Saccharomyces cerevisiae, a lactose fermenting yeasts present in the yoghurt might have contributed to lowering the acidity of the products. Yeasts also contribute to the flavour of the product (25).
Getachew et al. (64) commented that the variety of microorganisms present in naturally fermented milk products creates rich and full flavours that are hard to imitate. However, the use of appropriate traditional equipment is crucial to pathogen control. Additionally, the equipment must be easy to clean and sanitize, to prevent the formation of niches where microorganisms can grow and settle, forming biofilms (65). Furthermore, lack of pasteurisation, inadequate storage and maturation conditions, the temperature of water used for cow udder washing, the practice of mixing milk lots, the type of milk container, use of refrigeration, and milk filtration are some of the major risk-enhancing factors in traditional milk fermentation (56).
To minimize contamination during milking, effective hygienic practices need to be applied to the hands of the milkers and udder of the animals, and the general environment such as reducing faecal sources of contamination, (66) as well as the milking equipment (67). Washing hands without detergent may not improve the hygienic conditions of milk and milk products (68). Poor drying practices following hand washing and the use of old and unclean clothes for other farm activities is a risk factor for milk contamination (69). Traditional knowledge plays a role in awareness creation in the community to manage their day-to-day activities in livestock management (70). The main advantage of spontaneous fermentation processes is that they are appropriate to rural situations, since they were, in fact, created by it.
Several reports on the microbiological quality of fermented milk of Africa from different countries give knowledge of the various microorganisms in yoghurt and other traditional fermented milk (70). However, there are still gaps that need to be filled regarding pathogen control in traditional milk fermentation environments as microorganisms in traditional dairy products continue to be identified. Although many countries have milk safety regulations and surveillance systems for monitoring foodborne pathogens to ensure food safety, such surveillance of milk and milk products is not conducted on a routine basis in most African countries. Consistency in the day-to-day implementation of milking procedures is an important part of good dairy farming practices for milking. The need to use the guide developed by Food and Agriculture Organisation (71) would help to improve the standard of milk quality at traditional farms and farming practices.