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Brazilian Journal of Microbiology logoLink to Brazilian Journal of Microbiology
. 2021 Mar 11;52(2):869–881. doi: 10.1007/s42770-021-00461-y

Identification and characterisation of the lactic acid bacteria associated with the traditional fermentation of dairy fermented product

Promiselynda Ijeoma Obioha 1,✉, L Irene I Ouoba 1,2, Amarachukwu Anyogu 3, Brigitte Awamaria 1, Sarah Atchia 1, Philippa C Ojimelukwe 4, Jane P Sutherland 1, Hamid B Ghoddusi 1
PMCID: PMC8105451  PMID: 33694058

Abstract

The aim of this research was to identify the key lactic acid bacteria associated with the fermentation of dairy traditional fermented products for developing starter cultures for controlled fermentation. A total of 100 lactic acid bacteria (LAB) were isolated from dairy traditional fermented products. Samples were obtained from eight producers in the South East of Nigeria. Isolates were identified by phenotypic and genotypic techniques including rep-PCR genotyping and sequencing of the 16S rRNA, pheS and rpoA genes. Isolates were characterised for antimicrobial activity against foodborne pathogens, exopolysaccharide (EPS) production and survival at low pH and in the presence of bile salts. All isolates clustered into 11 distinct rep-PCR groups and were identified as Lactobacillus fermentum (40%), Lactobacillus delbrueckii (23%), Streptococcus thermophilus (22%), Streptococcus infantarius (10%), Lactobacillus senioris (2%), Leuconostoc pseudomesenteriodes (2%) and Enterococcus thailandicus (1%). Lactobacillus fermentum showed a broad spectrum antimicrobial activity and survival at low pH, while Lactobacillus delbrueckii was able to tolerate low pH and produce EPS. All isolates survived in vitro exposure to 1% (w/v) bile salts over a 3-h period. L. fermentum, L. delbrueckii and S. thermophilus could be used to simulate the fermentation of dairy traditional fermented products.

Keywords: Dairy traditional fermented product, Traditional milk products, Lactic acid bacteria, Potential starter cultures, Phenotypic and genotypic identification, Traditionally fermented foods

Introduction

Traditional fermented dairy products have been consumed for thousands of years and remain popular globally and across the African continent. In areas with limited access to electricity and cold storage facilities, fermentation is an important processing technique to extend the shelf life of milk, a highly perishable food. Fermentation also makes a significant contribution to improving the organoleptic and nutritional characteristics of the final fermented product [1–3].

Dairy traditional fermented product is a naturally fermented, yoghurt-like, milk product popular amongst many cattle-owning and cattle-rearing pastoral communities in West Africa. The dairy traditional fermented product is made from cow’s milk and is drunk as a refreshing nutritional drink or served as an accompaniment to fura, a millet-based dough. Like other traditional fermented foods, dairy traditional fermented products play a significant role in the diet as it is a relatively cheap source of nutrients as well as a source of income, particularly amongst the women [4, 5]. It is also of cultural significance as traditional fermentation techniques are passed down generations, using modifications to obtain desired organoleptic characteristics.

Traditional processing of cow’s milk for dairy traditional fermented production varies across West Africa. It has been previously reported that dairy traditional fermented product is produced by fermenting raw milk fermented for ca. 24 h [6, 7] without any heat treatment. However, during this field study, it was observed that amongst the Fulani who reside in the southeastern region of Nigeria, cow’s milk is heat-treated, though not pasteurised, prior to fermentation.

Despite this important difference in fermentation practice, dairy traditional fermented product production has similar characteristics to other naturally fermented African milk products such as Amasi, Rob, Amabere and Amaruranu [8–10] with respect to; use of backslopping, non-utilisation of starter cultures and small-scale, household production. Reliance on spontaneous fermentation of milk leads to variability in the microbial consortium present in the milk and, subsequently, the quality of the final fermented product. Poor hygiene during processing and handling can contribute to the contamination of the final product [11]. Historically, dairy traditional fermented products have low acceptability outside pastoral communities due to their short shelf life and perceived low hygienic quality. It has been suggested that processing modifications such as using pasteurised milk and controlled fermentations with well-characterised lactic acid bacteria can support improving marketability to a broader range of urban consumers [7].

Recently, there has been an increased focus on studying the microbiological and physicochemical properties of African fermented milk products. However, research on dairy traditional fermented product from Nigeria has mainly concentrated on using phenotypic methods to assess microbiological quality [12–14] and less on the detailed identification of microflora associated with the fermentation. Information about fermentation temperature, time and pH change during dairy traditional fermented product production is also limited. An important first step towards improving and standardising the fermentation process for dairy traditional fermented products is the development of functional starter cultures. To achieve this, an accurate understanding of the lactic acid bacteria involved during the fermentation is required.

The purpose of this study was to enumerate, isolate and identify the predominant lactic acid bacteria (LAB) involved in the fermentation of cow milk for the production of dairy traditional fermented products in Nigeria using a combination of phenotypic and genotypic methods. Potential technological properties including tolerance to acidic pH and bile salt, exopolysaccharide production and antimicrobial activity of LAB isolates against common foodborne pathogens were also investigated.

Material and methods

Sampling

Dairy traditional fermented products were sampled in different areas of Abia State located in the southeastern region of Nigeria, West Africa. A total of eight samples were collected from eight different producers: two from producers at Eket Islamic Mosque, Umuahia, and six other samples from a farm settlement at Lokpa-Nta Fulani village. All samples were collected in sterile containers and kept on ice prior to microbiological analysis. The pH of the samples was measured with a calibrated pH metre (Whatman PHA 2000, Portugal).

Microbiological analysis

Enumeration and isolation of presumptive lactic acid bacteria

Enumeration and isolation of LAB from dairy traditional fermented product were carried out using three different media including deMan, Rogosa and Sharpe (MRS) Agar (Oxoid, CM0361 Basingstoke, UK); MRS + 0.5% l-cysteine (MRSL) (C1276, Sigma-Aldrich, UK) and M17 Agar (Oxoid, CM0785). Plates were incubated anaerobically in an anaerobic jar (Oxoid, AG0025) with gas kit (Oxoid, BR0038) added to create an anaerobic condition. Both MRSL and MRS agar plates were incubated at 37 °C for 48 h, while M17 plates were incubated at 45 °C for 48 h. After incubation, morphological characteristics of the colonies recovered from each agar were examined, and representative colonies were selected from appropriate dilutions. Bacteria were separately isolated and purified by streaking several times on the same media as appropriate. A single pure colony was picked aseptically and stored in a Microbank cryovial (Pro-Lab Diagnostics, Birkenhead, UK) at −20 °C until required for further analysis.

Phenotypic characterisation of the isolated LAB

After growth on appropriate media, colony morphological characteristics such as size, shape and colour were examined. Cell morphology was examined by microscopy using a phase contrast microscope (0.90 Dry Japan Nikon Eclipse E400). Bacteria were tested for Gram reaction using KOH (3% w/v) as described by [15, 16]. Isolates were also screened for the catalase enzyme reaction using 3% (v/v) hydrogen peroxide (H3410, Sigma) and for the oxidase reaction using an oxidase reagent (Biomerieux® ,55635), on a strip of filter paper (Whatman No. 4, Whatman Plc., Kent, UK).

Genotypic characterisation of the isolates

  1. Characterisation of the isolate by rep-PCR

A pure colony of each isolate was sub-cultured on tryptone soya agar (TSA, Oxoid, CM0131) and incubated for 24 h anaerobically at 37 °C. Bacterial DNA was extracted using Instagene matrix (Bio-Red 732-6030, Hercules, CA, USA) according to the manufacturer’s instructions. Repetitive sequence–based PCR (rep-PCR) using the GTG5 (5′-GTG GTG GTGGTG GTG-3′; 5 pmol ml1) primer as described by [17] was used to characterise isolates at the subspecies level. For the amplification, the following programme was applied: 4 min at 94 °C for initial denaturation, then 30 cycles of denaturation at 94 °C for 30 s, annealing at 45 °C for 1 min and elongation at 65 °C for 8 min. The amplification ended with a final extension at 65 °C for 16 min.

The DNA fragments were separated by electrophoresis on 1.5% (w/v) agarose gel (Bio-Rad, Hemel Hempstead UK) for 2 h in 1× Tris Borate-EDTA buffer (TBE, Sigma, UK) at 130 V. Gels were stained with ethidium bromide and DNA profiles visualised and recorded using a UV transilluminator gel documentation system (M-26X, UVP, Cambridge UK). Fingerprint patterns were analysed and clustered using the Bionumerics system (Dice’s Coefficient of similarity, UPGMA; Applied Maths, Saint-Martens-Latem, Belgium).

  • (b)

    Identification of bacteria by 16S rRNA, pheS and rpoA gene sequencing

To identify bacteria, the method described by [17] was used to sequence the 16S rRNA gene using primers pA (5′-AGAGTTTGATCCTGGCTCAG-3′) (100 mmol L−1) and pE (5′-CCGTCAATTCCTTTGAGTTT-3′). The amplification was carried out under the following conditions: 5 min at 95 °C for initial denaturation followed by 35 cycles of denaturation at 94 °C for 1min, annealing at 55 °C for 1 min, and elongation at 72 °C for 1 min. A final extension was performed for 5 min at 72 °C. PCR products were purified using the QIAquick PCR Purification kit (Qiagen GmbH, Hilden, Germany 28104) following the manufacturer’s instructions. A sequencing PCR using primer pD (5′-GTATTACCGCGGCTGCTG-3′) was carried out under the following conditions: 95 °C for 2 min, then 35 cycles at 96 °C for 15 s, 40 °C for 1 s and 60 °C for 4 min before running on a gel (Source: Bioscience, Cambridge, UK).

Where closely related species could not be clearly differentiated by 16S rRNA gene sequencing, further identification was carried out by sequencing the pheS and rpoA genes according to the method described by [18]. Primers pheS-21-F (5′-CAY-CCNGCH-CGY-GAY-ATG-C-3′) and pheS-23-R (5′-GGRTGR-ACC-ATV-CCN-GCH-CC-3′) were used to direct the amplification of the pheS gene and rpoA-21-F (5′-CAY-CCNGCH-CGY-GAY-ATG-C-3′) and rpoA-23-R (5′-GGRTGR-ACC-ATV-CCN-GCH-CC-3′) the rpoA gene. The amplification programme consisted of (i) 5 min at 95 °C, (ii) 3 cycles of 1 min at 95 °C + 2 min 15 s at 46°C + 1 min 15 s at 72 °C, (iii) 30 cycles of 35 s at 95 °C + 1 min 15 s at 46 °C + 1 min 15 s at 72 °C and (iv) a final 7 min at 72 °C. Isolates were identified to genus and species level by comparing sequences with those contained in the GenBank sequence database (NCBI, MD, USA). All 16S rRNA gene sequences were analysed using the EzBiocloud database [19].

Investigation of LAB technological properties

The isolates investigated included representative isolates of each rep-PCR group (Table 1). This included Enterococcus thailandicus, Streptococcus infantarius, Lactobacillus senioris, Lactobacillus fermentum, Lactobacillus delbrueckii subsp. indicus, Leuconostoc pseudomesenteroides and Streptococcus thermophilus.

Table 1.

Identity of the LAB isolated from dairy traditional fermented product

Isolate code *Sample location Rep-PCR group Identification by 16S rDNA, pheS and rpoA gene sequencing
1 LO1 A Lactobacillus fermentum
2 LO1 B Lactobacillus fermentum
33 LO1 B Lactobacillus fermentum
35 LO1 B Lactobacillus fermentum
54 LO1 B Lactobacillus fermentum
36 LO1 D Lactobacillus fermentum
37 LO1 D Lactobacillus fermentum
69 LO1 G Streptococcus thermophillus
71 LO1 G Streptococcus thermophillus
74 LO1 G Streptococcus thermophillus
70 LO1 H Streptococcus thermophillus
73 LO1 H Streptococcus thermophillus
72 LO1 H Streptococcus thermophillus
34 LO1 J Lactobacillus delbrueckii subsp. indicus
53 LO1 J Lactobacillus delbrueckii subsp. indicus
17 LO1 J Lactobacillus delbrueckii subsp. indicus
5 LO2 A Lactobacillus fermentum
19 LO2 A Lactobacillus fermentum
3 LO2 B Lactobacillus fermentum
38 LO2 B Lactobacillus fermentum
55 LO2 B Lactobacillus fermentum
56 LO2 D Lactobacillus fermentum
57 LO2 D Lactobacillus fermentum
75 LO2 G Streptococcus thermophillus
76 LO2 H Streptococcus thermophillus
4 LO2 J Lactobacillus delbrueckii subsp. indicus
18 LO2 J Lactobacillus delbrueckii subsp. indicus
39 LO2 J Lactobacillus delbrueckii subsp. indicus
58 LO3 A Lactobacillus fermentum
7 LO3 A Lactobacillus fermentum
20 LO3 A Lactobacillus fermentum
40 LO3 A Lactobacillus fermentum
41 LO3 A Lactobacillus fermentum
59 LO3 A Lactobacillus fermentum
6 LO3 J Lactobacillus delbrueckii subsp. indicus
8 LO3 J Lactobacillus delbrueckii subsp. indicus
21 LO4 B Lactobacillus fermentum
61 LO4 B Lactobacillus fermentum
9 LO4 E Leuconostoc pseudomesenteroides
42 LO4 E Leuconostoc pseudomesenteroides
43 LO4 F Lactobacillus senioris
79 LO4 I Streptococcus infantarius
80 LO4 I Streptococcus infantarius
77 LO4 I Streptococcus infantarius
78 LO4 I Streptococcus infantarius
10 LO4 I Streptococcus infantarius
44 LO4 I Streptococcus infantarius
22 LO4 J Lactobacillus delbrueckii subsp. indicus
60 LO4 J Lactobacillus delbrueckii subsp. indicus
46 LO5 A Lactobacillus fermentum
47 LO5 A Lactobacillus fermentum
12 LO5 A Lactobacillus fermentum
82 LO5 G Streptococcus thermophillus
88 LO5 G Streptococcus thermophillus
86 LO5 H Streptococcus thermophillus
81 LO5 H Streptococcus thermophillus
87 LO5 H Streptococcus thermophillus
83 LO5 I Streptococcus infantarius
84 LO5 I Streptococcus infantarius
85 LO5 I Streptococcus infantarius
11 LO5 J Lactobacillus delbrueckii subsp. indicus
23 LO5 J Lactobacillus delbrueckii subsp. indicus
24 LO5 J Lactobacillus delbrueckii subsp. indicus
45 LO5 J Lactobacillus delbrueckii subsp. indicus
14 LO6 A Lactobacillus fermentum
63 LO6 A Lactobacillus fermentum
13 LO6 C Lactobacillus fermentum
25 LO6 C Lactobacillus fermentum
26 LO6 C Lactobacillus fermentum
48 LO6 C Lactobacillus fermentum
50 LO6 C Lactobacillus fermentum
62 LO6 C Lactobacillus fermentum
92 LO6 G Streptococcus thermophillus
89 LO6 G Streptococcus thermophillus
91 LO6 G Streptococcus thermophillus
90 LO6 H Streptococcus thermophillus
49 LO6 J Lactobacillus delbrueckii subsp. indicus
28 EO1 A Lactobacillus fermentum
29 EO1 A Lactobacillus fermentum
51 EO1 A Lactobacillus fermentum
64 EO1 A Lactobacillus fermentum
95 EO1 I Streptococcus infantarius
30 EO1 J Lactobacillus delbrueckii subsp. indicus
65 EO1 J Lactobacillus delbrueckii subsp. indicus
27 EO1 J Lactobacillus delbrueckii subsp. indicus
94 EO1 J Lactobacillus delbrueckii subsp. indicus
93 EO1 J Lactobacillus delbrueckii subsp. indicus
15 EO1 J Lactobacillus delbrueckii subsp. indicus
31 EO2 A Lactobacillus fermentum
32 EO2 A Lactobacillus fermentum
68 EO2 A Lactobacillus fermentum
67 EO2 F Lactobacillus senioris
97 EO2 G Streptococcus thermophillus
96 EO2 G Streptococcus thermophillus
99 EO2 G Streptococcus thermophillus
100 EO2 G Streptococcus thermophillus
98 EO2 G Streptococcus thermophillus
16 EO2 J Lactobacillus delbrueckii subsp. indicus
66 EO2 J Lactobacillus delbrueckii subsp. indicus
52 EO2 K Enterococcus thailandicus
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*LO1, LO2, LO3, LO4, LO5 and LO6: Production sites from Lokpa; EO1 and EO2: Production sites from Eke

Accession number: MT956953–MT956959

Screening of LAB for tolerance to acidic conditions

Isolates were sub-cultured on MRS or M17 agar, and a single pure colony was suspended in 1 ml of sterile maximum recovery diluent (MRD) (Oxoid, Basingstoke, UK). The suspension was used to prepare an inoculum (with a final cell concentration of 107–108 CFU/ml (equivalent to 0.5 McFarland standard) using a SensitreTM nephelometer (TREK Diagnostic Systems, West Sussex, UK). An acid resistance test was performed according to the method of [20]. One millilitre of each microbial suspension was inoculated into 9 ml of phosphate buffer solution (PBS) and adjusted to pH 3 and 7 using 2 M HCl and 2 M NaOH respectively. Cultures were incubated at 37 °C for 3 h under anaerobic conditions. The survival of the bacteria was determined every 30 min using a plate-counting method on MRS and M17 agars. Plates were incubated anaerobically for 48 h at 37 °C, and viable cell counts were expressed as log10 CFU/ml.

Screening of the LAB for tolerance to a different percent bile salt concentration

Bile salt tolerance of the isolates was ascertained in sterile PBS containing either no or 1.0% (w/v) bile salts, according to [20]. The processes of inoculum preparation, medium inoculation, sampling and viable counts were carried out as described above.

Screening of LAB for exopolysaccharide production

This experiment was performed according to [21]. Skimmed milk agar plates containing 10% (w/v) skimmed milk, 1% (w/v) sucrose (10020440, Fisher Scientific, UK), 0.5% (w/v) yeast extract (10225203, Fisher Scientific, UK), 1.5% (w/v) agar and 0.08 g/L ruthenium red (11103-72-3, Fisher Scientific, UK) were prepared. Both LAB cultures from 48-h incubation and the control (Enterococcus casseliflavus, Microbiology Research Unit, London Metropolitan University) were streaked out on separate plates which were incubated anaerobically at 37 °C for 48 h. Isolates unable to produce exopolysaccharide (EPS) compared with the control appear as non-ropy, pink-coloured colonies, while EPS producers exhibit a ropy, whitish appearance [21].

Screening LAB for antimicrobial activities against the indicator of pathogenic bacteria

  1. Inhibition of indicator of pathogenic bacteria using the spot test

The spot test described by [18] was first used to evaluate the antimicrobial activity of the LAB isolates. The activity of the LAB was tested against five indicator bacteria obtained from the culture collection of the Microbiology Research Unit, School of Human Sciences, London Metropolitan University (London, UK). These include Samonella enteritidis serovar Typhimurium variant DT124, Escherichia coli NCTC 12900, Listeria monocytogenes NCTC 11994, Staphylococcus aureus CMCC 1930 and Bacillus cereus LMG 1356. An inoculum (2 μl) of each isolate was spotted on the surface of an MRS agar plate and allowed to dry at room temperature for 30 min. All cultures were incubated anaerobically at 37 °C for 24 h. After the incubation time, 100 μl of each stock solution of an indicator organism was inoculated into 10 ml tryptone soy broth (TSB) + 0.8% (w/v) agar and overlaid on the grown spotted cultures of the LAB isolates. The overlaid plates were left to dry for 1 h at ambient temperature. Control plates were set up by pouring the soft agar + indicator overlay on MRS agars without any test isolate spots. All plates were incubated aerobically for 24–48 h at 37 °C which is the optimum growth condition for the indicator bacteria. The diameter of the zone of inhibition was measured and recorded in millimetre.

  • (b)

    Inhibition potential of cell-free supernatants (CFS) of LAB cultures against indicator bacteria using a spectrophotometric method

Antimicrobial activity resulting from direct antagonism between the CFS of LAB isolates and indicator bacteria in liquid media was tested using the method described by [22] with some modifications. The CFS of LAB isolates was added 10% (v/v) to an inoculum of indicator bacteria in TSB. In the negative control, LAB CFS was substituted with 2 ml of MRS broth. The inhibitory activity of the CFS of the LAB isolates was determined by separately transferring (2 ml) of CFS of each test isolate into a universal bottle containing a mixture of 2 ml of each indicator bacterium culture and 16 ml of TSB. In the negative control, CFS was substituted with 2 ml of MRS broth. Cultures were then incubated aerobically for 24 h at 37 °C, and the optical density (OD) measured at 540 nm (JENWAY 7315, Staffordshire, UK) by comparing the OD of the mixtures containing the indicator bacteria and with that of the control mixture but before measuring the OD. The spectrophotometer was zeroed using a mixture of 2.6 ml TSB and 0.4 ml MRSB. Furthermore, to eliminate acid production as the sole antimicrobial property, the acid neutralisation test was conducted. The CFS of LAB isolates were prepared as previously described and neutralised with filter sterilised 0.1M NaOH (Sigma, S8045) to increase the pH to 6.95 ± 0.1. The inhibitory effect of the neutralised CFS on the indicator bacteria was investigated using the spectrophotometric method as described above.

  • (c)

    Screening potential of LAB isolates for production of antimicrobial peptides against indicator bacteria

Further characterisation of antimicrobial activity examined the possibility that the LAB isolates investigated could produce antimicrobial peptides with broad-spectrum activity against the indicator bacteria. Each neutralised CFS was separately treated with Proteinase K (P2308, Sigma) to a final concentration of 1 mg/ml. The treated CFS were incubated at 37 °C for 2 h according to manufacturers’ instructions. A negative control was set up using non-treated, neutralised CFS for comparison. Inhibitory activities were determined using the spectrophotometric method as described above.

Data analysis

Each experiment was conducted at least two times and data were analysed using Microsoft Excel to determine the mean and standard deviation of the number of the viable colonies also, statistical significant differences set at P ≤ 0.05 to compare the means using 1-way ANOVA. The results were expressed as mean ± standard deviation in log10 CFU/ml.

Results

Bacteria identification

After a 48 h fermentation period, all samples yielded bacterial counts of 107 CFU/ml. This corresponded with a decrease in pH from about 6.8 to 4.3 ± 0.23. A total of 100 bacteria were isolated from the eight samples of dairy traditional fermented product investigated. All isolates exhibited the primary features of LAB, i.e. Gram-positive, catalase-negative and oxidase-negative. Microscopic observations revealed that a majority of the cells were rods arranged as chains, single and diplobacilli. There were also some cocci arranged in chains, single and diplococci, while some were V-shaped and are coccobacillus.

All isolates were selected for molecular identification on the basis of their phenotypic characteristics. Rep-PCR allowed differentiation of the isolates at interspecies and intraspecies levels into 11 different groups (Fig. 1). The relatedness of the different group is variable (30–80%) as shown in Fig. 1. The combination of the 16S rRNA, pheS and rpoA gene sequencing allowed the identification of four genera of LAB including Lactobacillus, Streptococcus, Leuconostoc and Enterococcus and seven species including Lactobacillus fermentum (40%), Lactobacillus senioris (2%), Lactobacillus delbrueckii (23%), Streptococcus thermophilus (22%), Streptococcus infantarius (10%), Leuconostoc pseudomesenteriodes (2%) and Enterococcus thailandicus (1%) (Table 1). Nucleotide sequence data reported are available in the GenBank database under the accession number MT956953 to MT956959. Lactobacillus fermentum and Lactobacillus delbruckii were observed in all samples irrespective of the location and production site and were the most diverse species with 4 and 2 rep groups respectively. With regard to production sites within the same location, it was observed that from Lokpa, in addition to the two common species, Streptococcus thermophillus was recovered from LO1, LO2, LO5 and LO6; Streptococcus infantarius from LO4 and LO5 and Leuconostoc pseudomesenteroides and Lactobacillus senioris from LO4 only. In Eket, additional species recovered were Streptococcus infantarius from E01, whereas Enterococcus thailandicus, Lactobacillus senioris and Streptococcus thermophillus were noticed in the sample from E02. The main difference between the two locations was the presence of Leuconostoc pseudomesenteroides in LO4 and Enterococcus thailandicus in EO1 (Table 1).

Fig. 1.

Fig. 1

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Unweighted pair-group arithmetic mean method (UPGMA) dendogram of rep-PCR fingerprints from lactic acid bacteria isolated from traditional fermented dairy product

Using 16S rRNA gene sequencing, some bacteria could not be clearly discriminated from closely related species. This was the case for isolates identified as to Enterococcus thailandicus which could not be differentiated from Enterococcus seguinicola; Leuconostoc pseudomesenteriodes which could not be differentiated from Leuconostoc mesenteriodes, and Streptococcus infantarius, which was not separated from Streptococcus lutetiensis. Such bacteria were all clearly identified by rpoA and pheS gene sequencing (Table 1). All bacteria were identified with a percentage similarity of 98–100%.

Technological properties of LAB from dairy traditional fermented product

The effect of pH on the viability of the test isolates indicated that their tolerance to acid pH varied according to the isolate screened (Table 2). At pH 3, there was no different variation in viable cell count (about 107 CFU/ml) of Lactobacillus fermentum over the 3 h test period, while the viability of other isolates varied within the same test time (Table 2) compared to their numbers in the control at pH 7. Streptococcus infantarius, Leuconostoc pseudomesenteroides and Streptococcus thermophilus cultures lost their viability after 3 h of incubation, while the viability of Enterococcus thailandicus, Lactobacillus senioris and Lactobacillus delbrueckii subsp. indicus was reduced respectively to 102 CFU/ml, 105 CFU/ml and 104 CFU/ml. Generally, Lactobacillus fermentum exhibited the highest viability count (107 CFU/ml) after a 3 h incubation, while the least viability count (104 CFU/ml) was displayed by Streptococcus thermophilus. With the exception of Streptococcus thermophilus, all isolates showed good tolerance to bile, with no significant decrease in viable counts over the 3 h test period. Exposure to bile salts led to a 3 log decrease in S. thermophilus (Table 2).

Table 2.

Survival of the LAB from dairy traditional fermented product in low pH and their tolerance to bile salt

Isolates code Species Viable count (CFU/ml)
pH 7 pH 3 0% bile salt 1% bile salt
0 h 3 h 0 h 3 h 0 h 3 h 0 h 3 h
52 Enterococcus thailandicus 6.33 ± 0.08bc 6.18 ± 0.20bc 6.31 ± 0.04bc 2.90± 0.26e 7.68 ± 0.07ab 7.75 ± 0.09ab 7.55 ± 0.10ab 7.61 ±0.11ab
11 Lactobacillus delbrueckii subsp. indicus 6.68 ± 0.04b 5.35 ± 0.06c 5.87 ± 0.07bc 4.26 ± 0.03d 5.60 ± 0.07bc 5.73 ± 0.14bc 5.56 ± 0.14bc 5.23 ± 0.09c
13 Lactobacillus fermentum 7.85 ± 0.00ab 7.48 ± 0.02ab 7.47 ± 0.01ab 7.32 ± 0.00b 8.29 ± 0.05a 8.29 ± 0.35a 8.25 ± 0.10a 7.18 ± 0.81b
43 Lactobacillus senioris 7.78 ± 0.13ab 7.68 ± 0.06ab 7.62 ± 0.02ab 5.98 ± 0.19bc 7.59 ± 0.12ab 7.65 ± 0.05ab 7.66 ± 0.03ab 7.69 ± 0.02ab
9 Leuconostoc pseudomesenteroides 6.56 ± 0.15b 6.12 ± 0.02bc 6.33 ± 0.16bc - 7.94 ± 0.14ab 7.29 ± 0.16b 7.96 ± 0.17ab 6.39 ± 0.16bc
10 Streptococcus infantarius 6.86 ± 0.04b 6.46 ± 0.11b 6.38 ± 0.21bc - 8.18 ± 0.47a 7.84 ± 0.01ab 7.70 ± 0.07ab 7.47 ± 0.03ab
73 Streptococcus thermophilus 5.94 ± 0.03bc 4.51 ± 0.03c 4.37 ± 0.18d - 6.32 ± 0.06bc 5.26 ± 0.07c 5.40 ± 0.08bc 2.86 ± 0.10e
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Data represent the mean of number of viable colonies in two experiments expressed as mean ± standard deviation in log10 CFU/ml. Data were considered significantly different (rows/columns) when p < 0.05

Exopolysaccharides production also varied according to species. For instance, some LAB isolates exhibited long, ropy strands, while others exhibited less ropy strand formation. Amongst the seven isolates screened, Enterococcus thailandicus and Lactobacillus delbrueckii subsp. indicus exhibited characteristics ascribed to EPS production by indicating the ropy whitish colonies similar to that of the control (Table 3), while other isolates showed no indication of EPS production except Streptococcus thermophilus which showed less whitish colonies.

Table 3.

Antimicrobial activity of the LAB isolates against indicator bacteria and their exopolysaccharide (EPS) production potential

Isolate code Species Antimicrobial activity (spot test) EPS production
Indicator bacteria
Salmonella enteritidis sp. DT124 Escherichia coli NCIMB 555 Staphylococcus aureus CMCC1930 Listeria monocytogenes NCTC 11994 Bacillus cereus LMG1356
52 Enterococcus thailandicus *++ ++ ++ ++ ++++ +
11 Lactobacillus delbrueckii subsp. indicus ++ ++ ++ ++ +++ +
13 Lactobacillus fermentum +++ ++ ++ ++ ++++ -
43 Lactobacillus senioris ++ +++ ++ ++ +++ -
9 Leuconostoc psudomesenteroides ++ ++ ++ ++ +++ -
10 Streptococcus infantarius ++ +++ ++ ++ +++ -
73 Streptococcus thermophilus - - - - - -
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The diameter of the zone of inhibition was measured and recorded in millimetre

*Absence of inhibition: -, 0–10 mm: +, 11–20 mm: ++, 21–30 mm: +++, 31–40 mm: ++++; absence of ESP production = -, EPS production = +

Lactic acid bacteria isolated from dairy traditional fermented product exhibited varying levels of inhibition against common Gram-positive and Gram-negative foodborne pathogens. It was observed that Streptococcus thermophilus did not inhibit any of the indicators screened. On the other hand, Lactobacillus fermentum exhibited, in general, a broad spectrum of inhibition against both Gram-positive and Gram-negative indicator bacteria (Table 3) with inhibition zones between 11 and 40 mm according to the indicator screened. Taking specific indicators into account, Lactobacillus fermentum exhibited the most potent inhibitory effect (21–30-mm inhibition zone) against Salmonella enteritidis, while Streptococcus infantarius and Lactobacillus senioris exhibited the most substantial inhibitory effect (21–30-mm inhibition zone) against Escherichia coli. All LAB except Streptococcus thermophilus showed the same degree of inhibition (11–20-mm inhibition zone) against Staphylococcus aureus and Listeria monocytogenes. In addition, Bacillus cereus was the most susceptible indicator with the largest clear inhibition zones on average of (21–40 mm, Table 3).

All LAB showed varying degrees of antimicrobial activity due to direct antagonism between the CFS and indicator bacteria in liquid media. The pH of the CFS dropped from 6.0 (MRS broth) and 6.8 (M17 broth) to 3.97, 4.14, 4.16, 4.26, 4.29, 4.29 and 6.07 for the CFS of broth cultures of Lactobacillus delbrueckii subsp. indicus, Lactobacillus fermentum, Streptococcus infantarius, Lactobacillus senioris, Enterococcus thailandicus, Leuconostoc pseudomesenteroides and Streptococcus thermophilus respectively. The CFS of Lactobacillus fermentum exhibited the highest antimicrobial effect against all indicators screened followed by that of Lactobacillus senioris (Fig. 2). Furthermore, Streptococcus thermophilus exhibited the least effect on the growth of the indicator bacteria particularly against Escherichia coli, Bacillus cereus and Staphylococcus aureus.

Fig. 2.

Fig. 2

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Antimicrobial activity of the treated and non-treated CFS of LAB from dairy traditional fermented product against different pathogenic bacteria

Overall, inhibitory activities observed from the CFS of test isolates were removed after neutralisation. For example, neutralised CFS of Lactobacillus senioris lost its effect on the growth of all the indicator bacteria screened (Fig. 2), while other test isolates when their CFS were neutralised retained their inhibition effect only against Bacillus cereus compared to non-neutralised CFS (Fig. 2b). The antimicrobial effect of Streptococcus thermophilus against Salmonella enteritidis was also not observed to be influenced by acid neutralisation (Fig. 2c).

Further characterisation to determine the potential of isolates to produce antimicrobial peptides against the indicators screened showed that the inhibitory activities observed from the neutralised CFS against Bacillus cereus were lost after proteolytic enzyme (proteinase K) treatment. All treated CFS exhibited antimicrobial effect against Staphylococcus aureus (Fig. 2d).

Generally, for the effect on Listeria monocytogenes, Streptococcus infantarius exhibited more antilisterial activity compared to other test isolates. Lactobacillus senioris and Streptococcus thermophilus did not show inhibition potential against Listeria monocytogenes.

Discussion

The isolation and identification of LAB from dairy traditional fermented product, a traditional fermented milk product, was evaluated. Microbial counts of LAB in dairy traditional fermented product ranged between 1.34 × 107 and 8.76 × 107 and are similar to those reported for other African fermented milk products with counts of 106–108 [6, 9, 23]. The reduction in pH observed in fermented milk products like dairy traditional fermented product is associated with the production of lactic acid and other types of organic acids by fermenting lactic acid bacteria. These observations are similar to other studies on traditional African fermented milks which have been reported to range from 3.2 to 4.8 [1]. A study on kule naoto, a Maasai traditional fermented milk from Kenya, reported a final pH between of 4.17 and 5.16 [24]. In nunu, a fermented milk from Ghana, a much lower pH value of 3.1 was reported [6]. These differences may be related to factors such as the consortium of lactic acid bacteria involved in the fermentation and their particular technological properties such as acid production and fermentation time [25].

Rep-PCR was shown to be effective for the differentiation of LAB isolates at interspecies and intraspecies levels and enabled the diversity of the lactic acid bacteria responsible for the fermentation to be explored. A combination of 16S rRNA and other housekeeping genes is necessary to provide accurate bacterial identity and has been demonstrated in the identification of LAB from other fermented food materials [18, 26]. The current study demonstrated that different genera, species and subspecies of LAB including Lactobacillus fermentum, Lactobacillus senioris, Lactobacillus delbrueckii subsp. indicus, Streptococcus thermophilus, Streptococcus infantarius, Leuconostoc pseudomesenteriodes and Enterococcus thailandicus are involved in the fermentation of cow milk for dairy traditional fermented pr. Results also indicated that location may have some influence on microbial profile as Leuconostoc pseudomesenteroides and Enterococcus thailandicus were observed only in Lokpa and Eket respectively.

The predominance of Lactobacillus fermentum in traditional African fermented milk products is in agreement with other reports [6, 24, 27]. Unlike other authors who have noted Lactobacillus plantarum as a dominant LAB species in African traditional fermented cow milk products [6, 9, 28, 29], this was not our observation. Lactobacillus plantarum is usually associated with the fermentation of vegetables and root crops [30], and it has been suggested that its presence in milk may be due to contamination [1]. This observed difference could be due in part to the variation in methods used to isolate and identify LAB from fermented milk products. For example, MRS agar was shown to be a suitable medium for the enumeration and recovery of Lactobacillus spp. [4, 31–35], while M17 agar is more selective for Streptococcus species such as Streptococcus thermophilus and lactococci [36, 37]. Also, many of these studies rely on phenotyping alone for identification which can be unreliable in providing accurate identification of bacterial species. To our knowledge, this is the first study using molecular techniques to characterise the dairy traditional fermented product consumed in Nigeria.

Generally, during the production and consumption of fermented milk, bacteria involved are exposed to different environmental conditions such as acids, bile, oxygen, and oxygen-derived radicals, heat and cold stress, which could negatively affect their viability and functionality [38]. In this study, Lactobacillus fermentum followed by Lactobacillus senioris showed better viability at pH 3 for a 3 h incubation compared to other tested strains supporting the results of the study of [39] which demonstrated that Lactobacillus species are more tolerant to acid environment than the other genera of LAB. Hence, this property makes Lactobacillus species abundant in the final phases of many food fermentations.

Tolerance to bile is considered one of the essential properties required for probiotic bacteria to survive in the small intestine [40]. In this study, all the tested strains showed good tolerance to bile. Other similar studies have assessed this at different concentrations from 0.5% (w/v) up to 2% (w/v). For instance, [41] observed the higher tolerance of LAB isolated from fish intestine at 2% bile concentrations, while [42] explained that Lactobacillus strains of dairy origin survived exposure to 0.3% w/v bile salts for 4 h when screening their probiotic potential.

Extracellular polysaccharide-forming strains have some advantages of improving texture, avoiding syneresis and increasing the viscosity of the yoghurt. In addition, EPS-forming LAB have been used to improve the rheological characteristics of dairy products. In this study, Enterococcus thailandicus and Lactobacillus delbrueckii subsp. indicus showed higher EPS production, while Streptococcus thermophilus showed less EPS production. The current observation is similar to the observations from Patil et al. [43]. The authors observed that EPS production from dairy isolates varies amongst species. Also, the presence of additional metabolites in milk can influence EPS production. For instance, the addition of glucose or sucrose to milk and milk ultrafiltrate increased EPS production by ropy strains of L. lactis subsp. lactis, L. lactis subsp. cremoris and L. casei subsp. casei. [44] also observed enhanced growth and EPS production by Strep. thermophilus strains in enriched milk medium supplemented with 1.0% peptone and 0.5% yeast extract, and these observations are similar to observations in this study in regard to medium for EPS production.

In Africa, the production of traditional fermented products such as dairy traditional fermented products remains a cottage-level industry. Due to limited training, awareness and practice of Hazard Analysis and Critical Control Point (HACCP) and Good Manufacturing Practice (GMP) by producers and food handlers, the presence of pathogenic bacteria cannot be ruled out [45]. Recent reports indicate that traditional fermented products available for retail sale can serve as vehicles for pathogenic bacteria [1, 46]; therefore, antimicrobial activity is an important technological aspect when selecting for LAB starter cultures for the controlled production of fermented dairy products. Lactic acid bacteria from dairy traditional fermented product were characterised on the basis of their antimicrobial properties against three Gram-positive (Staphylococcus aureus, Listeria monocytogenes, Bacillus cereus) and two Gram-negative (Salmonella enteritidis, Escherichia coli) indicators of foodborne pathogens. The study also aimed to accurately attribute antimicrobial properties as being due to one or a combination of competition for nutrients, acid production and production of antimicrobial peptides. Results from the spot test showed that six out of seven LAB isolates exhibited varying levels of inhibition against common Gram-positive and Gram-negative foodborne pathogens. Particularly, Lactobacillus fermentum exhibited a broad spectrum of inhibition against both types of indicator bacteria. The ability of Lactobacillus fermentum strains isolated from fermented milk products to show broad spectrum inhibitory activity has been reported by other authors [47, 48]. Bacillus cereus was the most sensitive indicator when tested against all LAB isolates that showed antimicrobial activity in the spot test. This strong antagonistic activity of LAB isolates from fermented milk products against strains of Bacillus cereus has been reported by other authors [49–51]. This result is promising as it is important for any potential starter to be able to inhibit the growth of spore-forming bacteria thereby improving the safety and quality of the product.

In this study, a more sensitive assay based on a spectrophotometric method showed that most CFS of the LAB isolates from dairy traditional fermented product inhibited the growth of the indicator bacteria in broth cultures. The growth of Listeria monocytogenes was notably impeded in the presence of CFS. This is of particular interest as starter cultures for fermented milk products that show antilisterial activity are important in the food and dairy industries. Listeria spp. are commonly associated with dairy products [49] with corresponding safety issues. The ability of the CFS to inhibit the growth of the indicators shows that the antimicrobial effect cannot be solely attributed to competition for nutrients. Thus, the exact mechanism of inhibitory activities was further evaluated to ascertain if this was due to factors such as acid production or antimicrobial peptides. In general, the bacteriostatic effect of the test isolates on the indicator organisms was removed after neutralisation of the CFS indicating that acid production was most likely the main antimicrobial effect. This observation has been reported in other studies [52, 53]. Owusu-Kwarteng et al. [54] reported that none of the neutralised CFS from LAB strains studied showed antimicrobial activity against any of the Gram-negative pathogens tested. This is the case in the current study for example for Streptococcus infantarius, Lactobacillus senioris and Lactobacillus fermentum against Bacillus cereus.

The removal or reduction of inhibition after treatment with proteolytic enzymes in many cases suggests that some of the antimicrobial activities observed are likely due to the action of antimicrobial peptides such as bacteriocins or bacteriocin-like inhibitory substances (BLIS) [23, 55, 56]. Although in this study, the addition of proteolytic enzymes to the neutralised CFS was associated with a decrease in the inhibitory effect of LAB isolates, both Lactobacillus senioris and Leuconostoc pseudomesenteroides maintained inhibitory activities against E. coli after neutralisation.

Conclusion

Traditional dairy fermented products are produced by spontaneous fermentation with related issues of inconsistency in quality, safety, nutritional and organoleptic properties. Selection of multifunctional starter culture for the development of controlled fermentation could address these problems and contribute to improved food security in Africa by increasing the availability of animal products and providing a source of income for producers. Potential lactic acid bacteria for use as multifunctional starter cultures, for this milk product, include L. fermentum, L. delbrueckii and S. thermophilus. Further investigation should be carried out to develop appropriate conditions for upgrading this traditionally fermented milk product.

Declarations

Conflict of interest

On behalf of all the authors: P. Obioha, I. Ouoba, A. Anyogu, B. Awamaria, S. Atchia, P. Ojimelukwe, J. Sutherland, P. Ghoddusi, The corresponding author Promiselynda Obioha states that there is no conflict of interest

Footnotes

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