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Article

Microbiological Quality of Typical Traditional Fermented Milk from Northern Uganda and Western Kenya

by
Betty A. Ogwaro
1,2,*,
Hazel Gibson
1,2,
Dave J. Hill
1,2 and
Elizabeth A. O’Gara
1,2
1
School of Life Sciences, University of Wolverhampton, Wulfruna Street, Wolverhampton WV1 1LY, UK
2
Faculty of Science and Engineering, Research Institute for Healthcare Science, University of Wolverhampton, Wulfruna Street, Wolverhampton WV1 1LY, UK
*
Author to whom correspondence should be addressed.
Dairy 2023, 4(3), 445-461; https://doi.org/10.3390/dairy4030030
Submission received: 18 May 2023 / Revised: 3 July 2023 / Accepted: 7 July 2023 / Published: 18 July 2023
(This article belongs to the Section Milk and Human Health)
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Abstract

:
In this study, the microbiological quality of traditionally fermented milk from Northern Uganda and Western Kenya was analysed. Six samples of typical traditionally fermented milk were collected randomly from traditional cattle keepers in Karamojong (UG 1) and Acholi (UG 2) in Northern Uganda and Kalenjin in Western Kenya (KE). The microbial quality of the collected samples was assessed through the use of conventional methods for total aerobic mesophilic bacteria, total coliform, lactic acid bacteria, Staphylococcus aureus, Listeria monocytogenes, yeasts, and mould counts. The mean aerobic mesophilic bacterial counts were 5.14 × 109 coliform forming units (cfu)/mL. The mean counts for mesophilic lactobacilli ranged from 106 to 108 cfu/mL. The mean thermophilic lactobacilli count ranged from 107 to 109 cfu/mL, while the mean thermophilic lactococci counts ranged from 106 to 109 cfu/mL. On the other hand, the Streptococci counts were between 106 and 108 cfu/mL. The mean count for the non-sorbitol E. coli was 3.87 × 103 cfu/mL. These results suggest that although the pH of the traditional yoghurt in this study was low, the acidity was not sufficient to inhibit growth of microorganisms in the product. Although it is difficult to avoid the microbial contamination of milk during milking, it is of the utmost importance to maintain a very high level of hygiene in dairy farming practices, and the correct cleaning of teats during milking is very important for good udder health and optimum milk quality, and to ensure safety. The results regarding microbial contamination pose public health concerns, and therefore the appropriate government agencies must pay attention to ensure that the environment in which yoghurt is produced is in the best condition to reduce contamination.

1. Introduction

Dairy products, especially traditionally fermented milk, are important foods among cattle keepers in Africa. They are integral to rural families’ diets and play a central role in the enhancement of food security and income generation [1]. Traditional African herders ferment raw milk to preserve it as they lack refrigeration facilities, but also because fermented or acidified milk products are used in weaning and as a drink or complementary food, especially among nomads.
In the traditional sector, milking is performed by hand in the open air or in a shelter, generally under poor hygiene conditions [2]. The udder is rarely washed before milking; if this is performed, the water is derived from various sources other than tap water, which contributes to the contamination of the raw milk [3], as do other sources of contamination during milking [4]. Hygiene practices regarding the hands of the milker or the udder, the milking equipment and the surrounding environment are inadequate [5]. Traditional farmers mitigate the impact of a lack of refrigeration, poor handling, and environmental contamination by fermenting the milk into several products. The microbiological aim of fermentation is to achieve a pH fall that prevents or reduces the growth of pathogens. Milk is fermented at household/village level through the application of traditional fermentation technology. The milk ferments spontaneously due to acidification of the raw milk by indigenous microflora in the milk [6]. Some of these products have demonstrated therapeutic and probiotic effects; although they show inconsistent product quality and limited shelf life.
The fermentation period varies from community to community. The main process parameters are fermentation time and temperature, type of fermentation container, the presence/absence of back slopping, removal of whey, and addition of raw milk. In Ghana, Mashini ghakushika, a traditional fermented milk, is fermented for three to four days at ambient temperatures before consumption. Mabisi is fermented for about 48 h, after which the product is stirred and ready for consumption. Âudaï is left to stand overnight to allow fermentation at room temperature [7]. The fermented milk is consumed the following day. In Southern Africa, milk is fermented for between 24 h and 48 h before it is ready for consumption [8]. Kule naoto of Kenya is fermented for two to three days before consumption [9]. In Uganda, among the Karamojong and Acholi, milk is fermented for 2448 h in a warm part of the house, after which time it is ready for consumption. The practice of boiling milk before use is increasingly used in Africa; however, people still more frequently consume and even prefer raw milk and/or products derived from unpasteurised milk.
From a food safety perspective, the important pathogens that are related to raw milk and fermented milk products are bacteria such as Staphylococcus aureus, Salmonella spp. Escherichia coli, Listeria monocytogenes, and Bacillus cereus.
Salmonella belongs to the Enterobacteriaceae family. It is a rod-shaped, Gram-negative, facultative anaerobic bacterium that can use glucose for fermentation. It causes enteric (typhoid) fever, gastroenteritis, and a localised type of disease with loci in one or more organs and which is accompanied by septicaemia [10,11]. Salmonella has been reported to survive in acidic environments [12].
E. coli are a large and diverse group of bacteria. Although most strains are harmless, some can cause diarrhoea, urinary tract infections, respiratory illness, pneumonia, etc. A particular strain of E. coli known as E. coli O157:H7 causes a severe intestinal infection in humans. According to Massa et al. [13], Conner and Kotrola [14] and Ogwaro et al. [15], E. coli strains (including E. coli O157:H7) can survive milk fermentation and have been found to be present in yoghurt and traditionally fermented milk after 48–72 h of fermentation.
L. monocytogenes is a Gram-positive, pathogenic bacterium that is widely found in both agricultural and food processing environments. It is known to secrete a wide variety of virulence factors, which make the microorganism responsible for different infections, both localised and systematic [16,17]. It is a possible cause of subclinical mastitis and thus can be released into milk. L. monocytogenes has been isolated in raw milk from dairy farms. The main concern in the dairy industry is the ability of L. monocytogenes to withstand extreme conditions. It can grow in a wide range of pHs, between 4.3 and 10 [18].
S. aureus is a common contaminant in the dairy industry and one of the most common causative agents of food poisoning [19]. It is an opportunistic pathogen that can cause infections in warm-blooded animals and is a leading cause of nosocomial infections in humans and bovines with mastitis [20,21,22].
B. cereus group members are among the main organisms responsible for the spoilage of dairy products. At the farm, the presence of B. cereus is associated with the contamination of soil, fodder and bedding that are in contact with cow udders. At the time of milking, spores are conveyed in the milk [23].
Several researchers [24,25,26,27] have reported the presence of microorganisms in some Kenyan or Ugandan spontaneously fermented milk products. There is little modernisation of the production of traditional fermented products through the use of starter cultures under controlled conditions or exploitation of their probiotic and antimicrobial potential. Some data regarding milk quality and the incidence of pathogens in milk from large commercial dairy farms is well documented, but there is limited or no data in the literature regarding microbiological quality or pathogen prevalence in Northern Uganda. It is necessary to understand which microorganisms are present in traditionally fermented, ready-to-drink milk since their presence is directly connected to the health of consumers. Assessment of the microbiological quality of traditionally fermented milk is required to protect consumers against exposure to any health hazard and to ensure that the product will not deteriorate microbiologically during its anticipated shelf life [27].
This assessment aimed to determine the microbiological quality of ready-to-drink fermented milk from Western Kenya and Northern Uganda. Few research studies have been focused on the microbiological quality of fermented milk from these areas, especially in Northern Uganda.

2. Materials and Methods

2.1. Collection of Samples

Six samples of full cream, 18-h-old, typical traditional African fermented milk were collected randomly in duplicate from two farms in each location: from the Kalenjin farm (Kenya), labelled as KE, and from the Karamojong farm, labelled UG 1, and the Acholi farm in Gulu (UG 2), both in Uganda. The samples were collected during the rainy season (July–September) in sterile plastic milk bottles. All the samples were produced in gourds. After collection, the samples were transported immediately at 4–5 °C in a cold box to the laboratory for analysis.

2.2. Sample Analysis

The fermented milk was 36 h old when it arrived in the laboratory. Within four hours of arrival, the pH, titratable acidity (TA) and microbiological content of the samples were analysed, and these tests were repeated after 24, 48, and 72 h at 25 °C to check the microbiological growth during storage. Broth-dilution and pour-plate methods were used for the microbial analyses [28]. The samples were prepared according to the Official Methods of Analysis published by the Association of Official Analytical Chemists (AOAC) [29] and were analysed in duplicate.

2.2.1. pH Measurement

The pH of the samples was measured with a Mettler Toledo Delta 320 pH meter, at room temperature (20 ± 2 °C). The pH electrode was calibrated before the measurements at pHs 4 and 7 with standard buffer solutions. The calibrated pH electrode was inserted into a 10 mL sample and the readings were recorded. All measurements were performed out in triplicate.

2.2.2. Titratable Acidity of Fermented Milk Sample

A 20 g sample of well-shaken yoghurt or unfermented milk was weighed accurately into a 250 mL Erlenmeyer flask, and 40 mL of boiled, cooled, distilled water was added to it. With a sterile pipette, two-to-three drops of phenolphthalein were added to the milk to act as an indicator of the endpoint. The flask contents were titrated against 0.1 N 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 0.1 N NaOH that had been titrated was calculated by subtracting the final volume from the initial volume to give the amount of NaOH used to reach the endpoint. This was performed at least three times per sample. The percentage of lactic acid was then calculated through the use of Equation (1):
TA (%) = Vt × N × 90 × 100/Vs × 1000
in which:
  • Vt = volume of titrant (ml NaOH).
  • N = normality of titrant.
  • 90 = equivalent weight for lactic acid.
  • Vs = volume of sample used (ml yoghurt/milk).

2.3. Isolation of Microorganisms

2.3.1. Sample Preparations

Ten millilitres of each sample were aseptically pipetted into 90 mL of sterile quarter-strength Ringer’s solution (pH 7.2) and blended through the use of a Stomacher lab blender (Seward Medical, London, UK) for 2 min, then mixed thoroughly. Serial dilutions (10−1 to 10−8) were prepared in sterile quarter-strength Ringer’s solution.

2.3.2. Isolation of Microorganisms

Portions (1 mL) of the chosen dilutions were plated on the following media (Table 1).
(i)
Plate count agar (Oxoid M325, Basingstoke, Hampshire, UK), incubated in an inverted position at 30 ± 1 °C for 48–72 ± 1 h [28], for enumeration of total aerobic plate counts. This estimates the number of viable aerobic bacteria per gramme, or millilitre of the product measured, in colony-forming units per ml (cfu/mL) [29]. Plates that contained 30 to 300 colonies were counted.
(ii)
de Man Rogosa and Sharpe agar [30] (MRS, LAB098 pH 5.5) incubated anaerobically for 48 ± 2 h at 42 ± 1 °C in anaerobic jars (Biolab and Oxoid) with gas generating kits (Oxoid BR 38B) for selective enumeration of thermophilic lactobacilli, particularly Lactobacillus delbrueckii subsp. Bulgaricus.
(iii)
Rogosa agar [31] (E. Merck, D-61 Darmstadt, Germany), incubated anaerobically for 48 ± 2 h at 35 ± 1 °C, for enumeration of mesophilic lactobacilli. A further analysis was performed on MRS agar + vancomycin for the enumeration of Leuconostocs incubated anaerobically at 32 °C for 48 ± 2 h.
(iv)
M17 agar [32] (Oxoid CM 0817, pH 6.5), incubated aerobically for 48 ± 2 h at 30 ± 1 °C for the enumeration of lactococci, especially Streptococcus thermophilus. Ten isolates were obtained randomly from the countable plates of MRS, M17, and Rogosa agars, incubated at 42 °C for thermophilic bacteria and at 35 °C for mesophilic bacteria. Isolates were cultivated in MRS broth (CM359) pH 5.5 and pH 6.5 at 37 °C for 48–72 h for selective enumeration of Streptococcus thermophilus, Lactobacillus delbrueckii subsp. Bulgaricus, Lactobacillus acidophilus, Lactobacillus casei, and Bifidobacterium.
(v)
Xylose lysine deoxycholate (XLD) agar for the enumeration of Salmonella and Shigella spp. A 25 mL portion of the sample was pre-enriched with 225 mL of buffered peptone water and incubated for 24 h at 37 °C. A portion (0.1 mL) of the pre-enriched culture was transferred to 9.9 mL of Rappaport-Vassiliadis broth and incubated at 42 °C for 24 h. A loopful of the enrichment broth was then transferred to XLD agar and incubated at 37 °C for 24 h. Characteristic Salmonella colonies that each showed a slightly transparent zone of reddish colour and black centre were sub-cultured on nutrient agar and confirmed biochemically using triple sugar iron and Simon citrate agar according to the procedures of Gebeheyu et al. [11], with some modification.
(vi)
Violet red bile agar (Oxoid CM 107 with added 4-methylumbelliferyl-β-D-glucuronide (MUG) supplement BRO 71 E), incubated aerobically for 24 ± 2 h at 37 ± 1 °C for enumeration of non-sorbitol E. coli. The use of the supplement that contained MUG enabled the separate enumeration of E. coli that show glucuronidase activity. Plates were examined under long-wave UV light (366 nm) for the presence of fluorescing colonies. The presence of E. coli was tested further using indole production in tryptone water (Oxoid, UK) with Kovac’s reagent (Biolife, Sarasota, FL, USA), according to AOAC methods [29], and as previously reported by Moushumi and Prabir [33]. Further analysis of total coliform was performed using violet, red bile lactose agar incubated aerobically for 24 + 2 h at 30 ± 1 °C. Only plates containing 15 to 150 colonies for coliform were counted.
(vii)
Bacillus cereus was enumerated through the use of the most probable number technique with the selective agar mannitol yolk polymyxin B and polymyxin pyruvate egg mannitol bromothymol blue [29].
(viii)
Baird-Parker’s medium (Oxoid CM 0275 + SR054C) was used to enumerate S. aureus according to the reference method of the International Dairy Federation [34] and ISO 6888, 1:2021 [35].
(ix)
A listeria-selective medium was used for the enumeration of L. monocytogenes. The International Dairy Federation method [17] was used. A 25 mL sample was homogenised in 225 mL of listeria enrichment broths A and B, and then incubated for 24 h at 37 °C on listeria-selective medium (Oxford formulation CM856, Oxoid™, UK) adjunct with listeria-selective supplement (SR0140, Oxoid™, UK). The latter was then incubated for 48 h at 30 °C. A loopful of the enrichment culture broth was streaked in duplicate onto polymyxin acriflavine lithium chloride ceftazidime aesculin mannitol agar (Oxoid, CM877) and incubated for 48 h at 37 °C. Suspected L. monocytogenes colonies were characterised through the use of the Gram staining and catalase test. Five presumptive L. monocytogenes colonies were selected from each Petri dish of selective agar and cultivated on trypticase soy agar medium (CM0131, Oxoid, UK) that had been supplemented with 0.6% yeast extract. They were placed subsequently into an incubator for 24 h at 30 °C. Listeria spp. Colonies typically ranged from greyish green to brownish green with black zones of 1–3 mm diameter of aesculin hydrolysis.
(x)
Malt extract agar (1.5% agar no 2) (Oxoid) and potato dextrose agar (+0.005 g/L chloramphenicol) were used to enumerate yeast and mould counts. The plates were incubated at 20 and 25 ± 1 °C for five days. Yeasts and mould colonies were counted separately. Plates that contained 10 to 200 yeast and mould colonies were counted [36].
Further analyses were performed, which included examination of non-spore Gram-positive coccobacilli strains for catalase positivity [37], umbrella growth in motility, nitrate reduction, performance in the methyl red/Voges Proskauer test, and β-haemolysis production. The cultures were characterised based on the microscopic appearances of 24-h-old cultures through the use of Gram-stained preparations and analytical profile index (API) biochemical tests (acid formation from glucose, rhamnose, xylose, and mannitol fermentation). All media were prepared with deionised water. Glassware, such as Petri dishes, test tubes, pipettes, and flasks, was sterilised in a hot oven at 160 °C for one hour.

2.4. Analytical Profile Index Biochemical Test

API was used to identify the Gram-positive and Gram-negative bacteria and yeast to the species level. API strips are used usually to detect enzymatic activity, mostly related to the fermentation of carbohydrates or the catabolism of proteins or amino acids by the inoculated organisms. Identification of the lactic acid bacteria (LAB) was performed using API 50 CH and API 50 CHL media (bioMérieux sa). For the enteric and non-enteric bacteria, API 20E and API 20NE were used, respectively, according to the manufacturer’s instructions.
Ten representative isolates of each microorganism were selected. From each plate, a single isolated colony (from pure culture) was suspended in sterile distilled water. The bacterial suspension was placed in each compartment by use of a Pasteur pipette, the strips were incubated, and the procedure was performed according to the manufacturer’s instructions. The galleries were incubated at 30 or 37 °C and reactions were observed after 24 h and 48 h. All positive and negative test results were compiled to obtain a profile number, which was then compared with profile numbers in a commercial codebook (the APILAB PLUS database (bioMérieux sa) or online to identify the bacterial species.

2.5. Data Analysis

Microbial counts were transformed to logarithmic values (log cfu/mL) and the transformed values were analysed through the application of one-way analysis of variance in Prism Graph Pad (San Diego, CA, USA), version 9.0. All samples were collected in duplicate. The data were expressed as the mean ± standard deviation. Comparison tests were conducted to determine whether or not there were significant differences (p ≤ 0.05) between and within the different fermented milk samples from the three regions.

3. Results

3.1. pH and TA

Table 2 represents means of the pH and TA values of the fermented milk samples. There were no differences in pH or TA readings in each location. The mean pH of the samples was 2.9 ± 0.1% in the UG 1 sample, 3.4 ± 0.1% in the UG 2 and 3.6 ± 0.1% in the KE. There was a significant difference (p < 0.05) among means. The mean TAs of the samples were 1.26 ± 0.1% in UG 1, 0.92 ± 0.1% in UG 2, and 0.7± 0.1% in KE. A highly significant difference (p < 0.001) among the means of the TA measurements was observed. The UG 1 fermented milk sample had a significantly lower pH (2.9 ± 0.01) and higher TA (1.26 ± 0.1%) than the others. The KE sample showed a higher pH (3.6) but a higher TA (0.92%) than did the UG 2, which had a pH of 3.4 but a TA of 0.71%. There was no significant difference (p > 0.05) among the acidities of samples from each region. During the three days of storage at 25 °C, the pHs of the samples did not change much from the pHs or TAs at the time of initial analysis (36 h) after collection (data not shown).

3.2. Microbial Counts

Table 3 shows summaries of the microbial counts that were obtained from the traditional fermented milk samples that were 36 h old (considered ready for consumption). Values obtained in the counts were transformed into log cfu/mL 36 h old (considered ready for consumption). The total aerobic mesophilic bacteria count (AMBC) is an indicator of the sanitary conditions of the handling of raw milk and good-quality milk products [38]. The results show that the mean total AMBC in the samples was between 9.4 and 9.9 log cfu/mL. The mean counts of mesophilic anaerobic lactobacilli on MRS (35 ± 1 °C) were lower (6.33 log cfu/mL) in UG 2 and higher in UG 1 (7.8 log cfu/mL) in KE (Table 3). The counts of LAB in UG 2 were close to the total AMBCs, which suggested that most of the bacteria in UG 2 were LAB. The highest coliform counts were obtained from UG 1 samples (5.33 log cfu/mL). E. coli was detected in all the samples, and the highest count was obtained from UG 1 samples (4.08 log cfu/mL), while the same level of contamination was noted in UG 2 and KE (3.34 and 3.34 log cfu/mL), respectively. From the results, it can be assumed that E. coli was the major component of the coliform.

3.3. Microbial Analysis

A total of 22 different types of microorganisms were grouped according to their colony phenotypes and Gram stain reactions. The prevalence of each group of microorganisms is presented in a pie chart (Figure 1) expressed as percentage of the total number of groups of the isolates (n) obtained from the samples. Aerobic mesophilic bacteria were the largest group of microorganisms, comprising 34% of the total count (Figure 1). Mesophilic lactobacilli and the Leuconostoc group comprised 18%, while 8% of the isolates were thermophilic lactobacilli spp. And 9% were streptococci species. 11% of the total count was coliforms, whilst yeasts and moulds and other (unidentified) represented 9% and 11% respectively.

3.3.1. Identification of the Isolates with API Biochemical Analysis

The presence of multiple and closely related species in these products makes the differential or selective enumeration of bacteria difficult due to the similarity in growth requirements and overlapping biochemical characteristics of the species. However, after the Gram stain, the isolates were subjected to API biochemical analysis. Table 4 shows the presumed predominant microorganisms that were identified through the use of API biochemical analysis. Mesophilic aerobes grown on M17 and MRS agars at 35 °C dominated the samples. Of the pathogens, Bacillus cereus had the highest number of microorganisms in the group (Table 4).
LAB were the dominant groups of bacteria in all the samples, and these could be identified by their phenotypic and microscopic appearance. They were grouped under Lactobacillus, Streptococcus, and Lactococcus spp.
The phenotypical characteristics of the presumed Lactobacillus bulgaricus and Streptococcus thermophilus were compared with those of the laboratory collections of Lactobacillus bulgaricus NCIMB 11778 and Streptococcus thermophilus NCIMB 10378, when Gram-stained and in API biochemical analysis. Ten isolates which were Gram positive, coccoid or spherical shaped were grouped under Streptococci spp., and another 10 under the lactobacilli, of which three isolates were identified preliminarily as beta-bacteria (heterofermentative Lactobacillus). Others included Leuconostoc and Enterococcus spp.
From the UG 1 samples, twelve different colonies were identified and grouped according to their Gram stain reactions. Three of the ten isolates were Gram positive, very tapered rod-shaped and according to the API 50 CHL biochemical test, they were identified as Lactobacillus spp. Ten of the twelve isolates were Gram-positive and coccoid or spherical in shape. They were grouped under the Streptococcus spp. Of the mesophilic lactobacilli, API tests indicated L. cremoris, L. mesenteroides, Lactococcus spp., and L. lactis spp.
Many of the Gram-positive mesophilic groups with large sporulated ends were identified with API 20E preliminarily as belonging to the Bacillus. The other group, which were also Gram-positive with round or coccoid in groups or chains, were identified as belonging to the Staphylococcus and Enterococcus spp., which are common in the cattle environment. S. caprae, which colonise healthy human skin, nails, and nasal mucosa, were identified in the UG 2 sample. The Gram-positive diplococci or pairs of short-chained isolates were grouped into the Enterococcus family. The coliforms were dominated by E. coli. With API 20E, presumed E. faecalis, E. agglumeritus and E. durans were preliminarily identified.
From the KE sample, 17 different isolates were preliminary identified. API analyses indicated that four of the isolates were of lactobacilli spp. Others were less distinct but identified as Staphylococci species. Table 4 shows the phenotypical and morphological characteristics of some of the yeasts that were isolated from the fermented milk.

3.3.2. Yeast and Mould

Many fermented dairy products such as yoghurt and sour milk are contaminated with yeast species that become the major cause of spoilage. This is because the low pH of these products offers a favourable environment for their growth. Yeasts are known to be the major contaminants of products such as fermented or acidified milk. This can be seen in the results observed in this study, in which the isolated yeasts were identified but not to the species level. Identification was achieved mainly due to the phenotypical and morphological characteristics of the isolated yeasts and moulds. These characteristics showed that the most common yeasts and moulds present in the samples of the fermented milk from Northern Uganda were Saccharomyces and Kluyvermnyces spp., and in the samples from Western Kenya, Kluyvermnyces; Penicillium spp. and Aspergillus species. Although yeasts were isolated from UG 2 samples, Aspergillus flavus was not detected. A. flavus is associated with corn and groundnuts and produces toxins. In a traditional setting, each farmer produces corn and groundnuts for home consumption. A. flavus can be transferred from the farmer’s hands or a cow’s skin to contaminate the milk. This contaminant requires monitoring in dairy farming. However, the API system that was used did not identify most of the yeast and mould isolates.

3.4. Survival of Pathogens during Storage

The aim of this study was to assess the microbial quality of typical traditional African fermented milk that is ready for consumption. However, we also studied the survival of pathogens in milk that was stored for three days. Samples of UG 1, UG 2, and KE were stored at 25 °C for a further three days after the initial assessment of the fermented milk had been performed when it was 36-h-old and viable cell counts for general coliforms, E. coli, L. monocytogenes, S. Typhimurium, S. aureus, and B. cereus had been found (Table 3). Viable counts were performed very 24 h to assess the survival of these organisms. It is of note that traditional farmers in the areas of study consume the milk within 24–48 h of production. The milk storage aimed to observe the survival pattern of the pathogens for possible improvement.
Figure 2 shows that coliforms were still recovered after three days of storage (104 h after production). The viable counts declined by approximately two log units in the UG 1 and UG 2 samples and by approximately 1.5 log units in the sample (KE). E. coli counts declined after 36 h by three to four log units in all the samples during the three days of storage by 2.8 log units were still recovered in all the samples after 104 h of fermentation, which indicated some resistance even in the low pH environment of the products. S. typhimurium was not detected in KE samples after two days of storage (60 h old) but it survived after this time in UG 2 samples (Figure 2). L. monocytogenes was not detected after 60 h of production and storage. B. cereus survived for up to 60 h after production and storage and was not recovered after three days of storage. S. aureus survived in all the samples up to 84 h and were not detected after 104 h in the UG samples but were present in the KE samples.
Coliform bacteria are often used as indicator organisms for the hygiene status of milk on farms. Reports state increasingly that the total number of Gram-negative bacteria reflect accurately the hygiene status of processing environments on farms, yet this report shows that even Gram-positive bacteria such as S. aureus, which colonise healthy skin, nails and nasal mucosa of humans, and B. cereus, which is usually associated with soil, also reflect the hygiene status of farms and especially of the farmers.

4. Discussion

In this study, the physicochemical and microbiological attributes of traditional African yoghurt produced in Northern Uganda and Western Kenya were assessed to establish the status of the microbial risks that are associated with this product. Milk is processed at the household level to become sour; this is achieved by leaving fresh, raw milk to ferment naturally for one to three days at ambient temperature. Fermentation occurs spontaneously in gourds or earthenware pots. Sometimes sour milk from previous batches is added to speed up the fermentation process.
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. [39], Nduko et al. [40], Ifeanyi et al. [41], and Digbabul et al. [42] reported that the pH of traditionally fermented yoghurt ranged from pH 3.5 to 5.11. The low pH in this study was reflected in the TA level, which was 1.26 ± 0.1, 0.71 ± 0.1, and 0.92 ± 0.1% for the UG 1, UG 2, and KE samples, respectively.
The AMBCs in fermented milk indicate the sanitary or quality conditions that prevailed during the production and handling of raw milk, or post-fermentation contamination. The average AMBC obtained in the current study was very high (109 cfu/mL), which indicated poor quality of the products. This number is over the microbial limit that is acceptable (106 cfu/mL) for such products according to the Health Protection Agency (HPA) guidelines [43]. Regarding the microbial quality of the tested samples, the AMBCs were not significantly different (p > 0.05) from each other. The mean counts for mesophilic lactobacilli were highest in the UG 1 sample (108 cfu/mL) followed by the KE (107 cfu/mL), and lowest in the UG 2 (106 cfu/mL). The thermophilic lactobacilli were 107 cfu/mL in UG 1, higher in UG 2 (109 cfu/mL), but lower (106 cfu/mL) in KE samples. The high AMBCs (106–109 cfu/mL) may have been due to the already high numbers of bacteria in the raw milk. This has been observed by other researchers who have studied raw milk taken from different areas of Africa [44]. Hot weather in the production areas also enhances the growth of microorganisms in milk if it is contaminated before or during processing [45]. Besides the high AMBC counts, the yoghurt samples had a rich diversity of microorganisms, predominantly LAB and yeasts.
In Africa, fermentation is spontaneous and back slopping is used, in which the previously fermented milk is used as a starter culture. Elsewhere in the world, specific milk starter cultures (Lactobacillus bulgaricus and Streptococcus thermophilus) are used to ferment milk into yoghurt under a controlled environment. Thus, it came as no surprise that these typical African fermented milk products harboured rich and diverse types of microbes, especially LAB. The amounts of the bacteria recovered from the samples were similar to those reported from Zambian milk by Yambayamba and Zulu [5]. Similarly, high bacteria count (5.6–7.5 log cfu/mL) were reported in the traditional yoghurt (108 cfu/mL) of Sudan and Egypt [46]; in the traditional fermented milk of Zimbabwe (108 cfu/mL) [47]; and in traditional fermented milk from Morocco [48]. In South Africa, a high number of microorganisms (108 cfu/mL) were also reported [49,50]. These high numbers of mesophilic bacteria could have been due to the warm ambient temperature (28–35 °C) at which the milk fermented at the time. The presence of microorganisms in traditional fermented milk depends on the nature of the milk and the temperatures of the regions from which they have been obtained [46]. It also depends on the level of contamination at the production site. Contamination can occur during milking, especially in cases in which hygiene practices such as washing the udders pre-milking are poor [51]. It is therefore important to remove visible dirt from the outer surface of the udder because this is likely to contribute to the contamination of the raw milk. Most traditional herders in the region of study do not practise pre-milking udder washing.
Other researchers [52] have noted that mesophilic bacteria such as Leuconostoc spp. are found in traditional fermented milk products in regions with cold climates, whereas, in warm regions, thermophilic bacteria such as Lactobacillus and Streptococcus dominate [53]. This could explain the high numbers of mesophilic bacteria that were found in these samples, because they were fermented and collected during the rainy season and cooler months (25–35 °C) in Kenya and Uganda.
The amounts of LAB were in the range of 108 log cfu/mL. The counts of thermophilic lactobacilli and Lactococcus were 2.87 × 107 in the UG 1, 1.54 × 109 in the UG 2 and 1.74 × 108 in the KE samples. Obadai and Dodd [54] reported counts of LAB in the range 108 to ×1010 in nyarmie, in the traditional fermented milk of Ghana. These results agree with those reported by Owusu-Kwarteng et al. [55] in nunu, Ghana’s traditional fermented milk product, and by Mathara et al. [56] in kule naoto, Kenyan traditional milk. In this report, the 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 [57,58]. In addition, this high number of LAB may have been due to natural selection and/or temperature of fermentation. The presence of fewer Leuconostocs suggests that this group is unable to compete with other LAB in mixed cultures [57]. This gives them a selective disadvantage over other LAB and a selective advantage over thermophilic bacteria. LAB are generally recognised as safe; they are part of the natural microbiota of various foods and are often used as starter cultures. Some lactic acid bacteria such as Lactococcus, Leuconostoc, Pediococcus, and Lactobacillus species are found to inhibit the growth of microorganisms and other pathogens in yoghurt which could be the same effect in traditionally fermented milk.
The numbers of coliforms were unsatisfactorily high in the UG 1 sample (105 cfu/mL) but at a borderline of acceptance in the UG 2 and KE samples (103 cfu/mL) in this study. Counts of coliform in UG 1 samples suggested poor handling and processing conditions of the milk. Levels of other pathogens such as E. coli, Salmonella species, Bacillus cereus, and S. aureus were also unsatisfactory according to the HPA guidelines, with counts of between 103 and 104 cfu/mL. However, B. cereus did not maintain its contamination after 72 h of storage, probably due to the acidity of the products. Hamama [59] reported similar results in ‘lben’ and ‘jben’, 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 low pH values in the environment, especially during their transit in the host. According to Foster [60], Salmonella species such as Salmonella Typhimurium periodically confront acid environments during their lives. In an experiment, Liyuwork et al. [61] observed antimicrobial resistance in Salmonella species isolated from dairy products in Addis Ababa. Chatti et al. [12] reported the isolation of acid-resistant Salmonella from food and wastewater in Tunisia. Although it is generally thought that Salmonella is destroyed or inactivated during the fermentation of highly acidic products, such as yoghurt in which the pH value is less than 4.55, this was not the case in this study. This could be because Salmonella can survive in various environmental niches for long periods.
Many diseases are transmissible via milk products, and pathogenic and acid-tolerant bacteria in acidic foods have recently been the causes of public health concern. Unpasteurised milk has been found to be a major vehicle for the transmission of pathogens such as E. coli, L. monocytogenes, and Salmonella. It can be assumed that sources of contamination by microorganisms include dirty teats and milkers’ hands, and the repeated use of milking and fermentation vessels [62]. The presence of coliforms has long been thought to indicate faecal contamination; however, recent reports regarding this diverse group of bacteria indicate that only a fraction are faecal in origin, while the majority are environmental contaminants [63]. Low counts of coliforms might be due to the high acidity of the products, which restricted the growth of the microorganisms. However, coliforms were still recovered even in such high acidity conditions, suggesting adaptation to the acidic environment.
Yeasts and mould can build up on equipment surfaces and under the surface of package lids. These often contaminate fermenting milk [64]. The presence of yeasts and mould in milk, and their products, is undesirable as they can cause changes in the product that reduce the shelf life and render it unacceptable for consumption. In this study, many of the components of the microbial population were yeasts. The high number of yeasts in the products suggests a high presence of yeasts in the environment where the milk was fermented. In addition, it indicates that yeasts form a significant part of the microflora of these naturally fermented milk products in these areas. When milk is fermented according to good manufacturing practice, it should contain less than 10 yeast cells per gramme, and a shelf life of three to four weeks is expected if they are correctly refrigerated. However, milk that is contaminated with an initial load of 100 or more yeast cells per gramme will quickly spoil as the yeast cells multiply.
Yeasts may be a common part of the flora of the milking parlour and equipment and may impact the overall quality of the products. Yeasts and mould can produce toxic metabolites that are not destroyed during fermentation [65]. In this study, several yeast and mould species were recovered from the fermented milk, similar to the report of Savova and Nikolova [66]. Despite their undesirability due to spoilage effects, yeasts play an important role in foodstuffs. Getachew et al. [67] commented that the variety of microorganisms present in naturally fermented milk products created rich and full flavours that are hard to imitate. Yeasts can grow in a broad range of pH environments and usually adapt to coexistence with LAB in acidic environments [62]. Saccharomyces cerevisiae, a lactose fermenting yeast present in the yoghurt, may have contributed to lowering the acidity of the products [68].
The use of appropriate traditional equipment is crucial to pathogen control. A complex relationship exists between the microbiology of milk and milk products and their processing environment. The equipment must be easy to clean and sanitise, to prevent the formation of niches in which microorganisms can grow and settle, forming biofilms [64]. Only a few pathogens of concern associated with milk and milk products typically originate from the production environment: Listeria monocytogenes, Salmonella ssp., and most recently Cronobacter sakazakii. These pathogens have been widely reported to persist in processing plant niches for years and in some cases decades. 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 [69].
To minimise contamination during milking, effective hygiene practices must be applied to the hands of the milkers, the udders of the animals and the milking equipment, and the general environment; for example, faecal contamination must be prevented [61]. Washing hands without detergent is insufficient to improve the cleanliness of milk or milk products. Poor drying practices after hand washing and the use of old, dirty clothes for other farm activities is a risk factor for milk contamination [67]. Traditional knowledge plays a role in the creation of awareness in the community to manage day-to-day activities in livestock management [69]. The main advantage of spontaneous fermentation processes is that they are appropriate to rural situations, where they were created.
Several reports on the microbiological quality of fermented milk in Africa provide knowledge of the various microorganisms in yoghurt and other traditional fermented milk. However, gaps remain regarding the knowledge of pathogen control in traditional milk fermentation environments as microorganisms continue to be identified in traditional dairy products. Although many countries have milk safety regulations and surveillance systems which monitor foodborne pathogens to ensure food safety, such surveillance of milk and milk products is not conducted routinely in most African countries. Consistency in the day-to-day implementation of milking procedures is an important part of good dairy farming practices. Use of the guide developed by the US Food and Agriculture Organization [70] would help to improve the standard of milk quality and farming practices at traditional farms. The period of incubation and temperature of storage of the products must also be considered during assessment for improvement of fermented milk as well as the safety of traditional fermented milk. Some researchers [7,8,9,13,14,15] have reported that the numbers of pathogens decrease when the fermented milk is stored for more than seven days at 22 °C or they are eliminated when the milk is stored at 25 °C or atmospheric temperature for 15 days. This period must be considered together with pH or acidity of the product. The organoleptic characteristics of the product may become unsatisfactory at a pH below 5.0 because the increased acidity makes the product unacceptable to the consumer.

5. Conclusions

This study of traditional fermented milk of Northern Uganda and Western Kenya has shown that although the products are of low pH, they harbour high and variable loads of bacteria and therefore could pose health risks to consumers. In respect to food safety, the consumption of unpasteurised milk and dairy products poses significant health risks to consumers due to the persistence in the dairy chain in Africa of microbial pathogens of significant safety concern. The presence of microorganisms such as E. coli, Salmonella spp., Bacillus spp., and Staphylococcus aureus indicates the need to improve hygiene in traditional fermented milk production among farmers. The cross-contamination of milk products with microorganisms is an ongoing risk throughout traditional milk production.
To improve the quality of traditionally fermented milk, training must be conducted, and awareness raised regarding hygiene practices on the farm. Such practices include the cleaning and sanitisation of hands before and after milking, udder washing, drying the udder with clean dry cloths, and correct washing of milk equipment. Instruction in ways to avoid cross-product contamination from the environment and equipment should be stepped up. This should include everyone in the household who is involved in milking and processing, especially women. Rural people’s preference for fermented milk with raw milk over boiled or pasteurised milk, and the fact that smallholder dairying and informal markets dominate dairying in Africa, must be recognised. Additionally, clear messages must be promulgated regarding the dangers of consuming dairy products made from raw milk.

Author Contributions

Conceptualisation, B.A.O. and D.J.H.; formal analysis, B.A.O., D.J.H. and H.G.; data Collection, B.A.O.; data analysis, B.A.O., D.J.H., H.G. and E.A.O.; writing—original draft, B.A.O., D.J.H., H.G. and E.A.O.; writing—review B.A.O., D.J.H., H.G. and E.A.O.; methodology B.A.O., D.J.H., H.G. and E.A.O.; supervision; H.G. and E.A.O.; editing, H.G., D.J.H. and E.A.O. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding, but research fees were paid by Betty Ogwaro through the support of the Government of the Republic of South Sudan through salary payments to Betty Ogwaro during the study period.

Data Availability Statement

The data presented in this study are openly available.

Acknowledgments

We appreciate sincerely the support and collaboration of Lensio Onek Angole from the University of Juba in facilitating the sample collection.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. A pie chart of the diverse microorganisms from the traditional African yoghurt (n = 22). n = number of total groups identified.
Figure 1. A pie chart of the diverse microorganisms from the traditional African yoghurt (n = 22). n = number of total groups identified.
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Figure 2. Survival of the coliform, E. coli, L. monocytogenes, S. typhimurium, S. aureus and B. cereus in traditionally fermented milk during storage for 104 h at 25 °C in UG 1 [◼], UG 2 [◼], and KE [◼] samples.
Figure 2. Survival of the coliform, E. coli, L. monocytogenes, S. typhimurium, S. aureus and B. cereus in traditionally fermented milk during storage for 104 h at 25 °C in UG 1 [◼], UG 2 [◼], and KE [◼] samples.
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Table
Table 1. Summary of cultures and media used for the isolation of microorganisms in the samples.
Table 1. Summary of cultures and media used for the isolation of microorganisms in the samples.
No.Medium for GrowthMicroorganismsTime (hour)Growth Condition and Incubation Temperature (°C)
iPlate count agar
(Oxoid M325)		Total aerobic mesophilic bacteria48 ± 2aerobic 30 ± 1
iiMRS agar (LAB098) (pH 6.5)Mesophilic lactobacilli48 ± 2aerobic 35 ± 1
iiiRogosa agarMesophilic lactobacilli48 ± 2anaerobic 35 ± 1
ivMRS agar (LAB 098 + vancomycin)Leuconostoc48 ± 2anaerobic 30 ± 1
VMRS agar (pH 5.5) (LAB098)Thermophilic lactobacilli48 ± 2anaerobic 42 ± 1
viiMRS agar (pH 6.0) (LAB098)Thermophilic lactococci48 ± 2anaerobic 42 ± 1
viiiM17 agar (LAB 092)Streptococci48 ± 2anaerobic 30 ± 1
viiiViolet red bile lactose agar with MUG supplement BRO 71 E),Non-sorbitol E. coli24 ± 2aerobic 37 ± 1
ixViolet red bile agar Coliform24 ± 2aerobic 30 ± 1
XXLDSalmonella and Shigella spp.24 ± 2aerobic 37 ± 1
xiBaird-Parker’s medium (Oxoid CM 0275 + SR054C)Staphylococcus aureus24 ± 2aerobic 37 ± 1
xiiListeria enrichment broths A and BListeria monocytogenes24 ± 2aerobic 30 ± 1
xiiiBacillus cereus agarBacillus cereus24 ± 2aerobic 30 ± 1
xiv1.5% malt extract and agar no. 2Yeasts 120 ± 2aerobic 25 ± 1
xvPDA + chloramphenicolMould120 ± 2aerobic 30 ± 1
Key: MRS =Man Rogosa Sharpe; XLD = xylose lysine deoxycholate.
Table
Table 2. Mean pH and TA of the fermented milk (±SD, n = 6).
Table 2. Mean pH and TA of the fermented milk (±SD, n = 6).
SamplepHTA (%)
UG 12.9 ± 0.011.26 ± 0.1
UG 23.4 ± 0.010.71 ± 0.1
KE3.6 ± 0.010.92 ± 0.1
Table
Table 3. Mean microbial counts of the probable microorganisms in the fermented milk samples (log cfu/mL).
Table 3. Mean microbial counts of the probable microorganisms in the fermented milk samples (log cfu/mL).
No.MicroorganismUG 1UG 2KE
iTotal aerobic mesophilic bacterial counts9.359.689.69
iiMesophilic lactobacilli (aerobic)7.247.287.28
iiiMesophilic lactobacilli (anaerobic)7.86.337.28
ivLeuconostoc5.775.685.70
vMesophilic lactococci7.948.208.32
viThermophilic lactobacilli.8.238.298.32
viiThermophilic lactococci7.857.767.76
viiiStreptococci8.228.028.02
ixNon-sorbitol E. coli4.083.343.34
xColiform5.334.334.24
xiSalmonella spp.2.972.872.93
xiiShigella spp.0.000.000.00
xiiiStaphylococcus aureus4.414.354.11
xivListeria monocytogenes1.181.131.12
xvBacillus cereus2.922.862.82
xviYeasts8.338.368.52
xviiMould8.048.178.21
Key: UG1, UG2, Uganda samples 1 and 2. KE: Kenya yoghurt samples. n = 6 (samples analysed in duplicates). Values are means ± SD.
Table
Table 4. Phenotypic and morphological characteristics of yeasts and mould isolated from the samples.
Table 4. Phenotypic and morphological characteristics of yeasts and mould isolated from the samples.
IsolateMacro-Colony Morphology (Margin, Colour, Elevation, Cell Appearance)UG1UG2KE
1Cream, smooth, oval shape entire and ellipsoidal cell√√√
2Undulating, white top with green base, slightly convex, spheroidal to short ellipsoidal. (Blue colony on Kluveymyces differential medium)√√√
3Yellow-green, powdery and pale yellowish on reverse Aspergillus flavus√ND√
4Dirty white with yellow spores at the centre, base orange, slightly radially furrowed (Microsporum spp.).√√√
5Cream-yellow, powdery, and pale yellowish on reverse, capsulate margin, slightly raised centre, filamentous cells√√√
6White to cream, yellowish, wrinkled, nearly flat elevation, oval cells and ellipsoidal √√ND
7White to cream coloured, flat with aerial mycelium (Aspergillus spp.)√√√
8Green with a red base√√√
9White at the base and black spores at the top√√√
10White pin head, clear zones around the colony√√√
11Black, yellow to pale cream in the centre (Aspergillus) NDNDND
12White measuring 1–4 mm, opaque and flat. Ropy to the touch√√√
13Straw cream at the centre, base orange, slightly radially furrowed√√ND
14Well-formed white colonies (Aspergillus spp.)√√√
15Green and pale yellow on reverse (Penicillium)NDNDND
16White base with black conidiophores √ND√
17Greenish black, white mycelia at the margin, white in the centre (Rhizopus sp.)NDNDND
18Greenish surrounded by creamy-white ring at the margin (Penicillium)ND√ND
19White to cream, smooth, glaucous dark green on the obverse and pale yellow on the reverseNDNDND
20Cotton white to cream on the obverse and yellow to orange on the reverse with dark brown exudate√√√
21White colony, opaque and flat√√√
22Bright red coloniesNDNDND
Key: UG1, UG2, Uganda samples 1 and 2. KE: Kenya yoghurt samples. √ = Detected ND = Not Detected.
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Ogwaro, B.A.; Gibson, H.; Hill, D.J.; O’Gara, E.A. Microbiological Quality of Typical Traditional Fermented Milk from Northern Uganda and Western Kenya. Dairy 2023, 4, 445-461. https://doi.org/10.3390/dairy4030030

AMA Style

Ogwaro BA, Gibson H, Hill DJ, O’Gara EA. Microbiological Quality of Typical Traditional Fermented Milk from Northern Uganda and Western Kenya. Dairy. 2023; 4(3):445-461. https://doi.org/10.3390/dairy4030030

Chicago/Turabian Style

Ogwaro, Betty A., Hazel Gibson, Dave J. Hill, and Elizabeth A. O’Gara. 2023. "Microbiological Quality of Typical Traditional Fermented Milk from Northern Uganda and Western Kenya" Dairy 4, no. 3: 445-461. https://doi.org/10.3390/dairy4030030

APA Style

Ogwaro, B. A., Gibson, H., Hill, D. J., & O’Gara, E. A. (2023). Microbiological Quality of Typical Traditional Fermented Milk from Northern Uganda and Western Kenya. Dairy, 4(3), 445-461. https://doi.org/10.3390/dairy4030030

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