Isolation and Molecular Characterization of Some Lactobacillus Species Isolated from Three Locally Fermented Foods

Isolation and Molecular Characterization of Some Lactobacillus Species Isolated from Three Locally Fermented Foods

*Ogunsona S. B., and Oladipo I. C

Department of Science Laboratory Technonology, Ladoke Akintola University of Technology, Ogbomoso, Oyo State Nigeria

*Corresponding Author

DOI: https://doi.org/10.51584/IJRIAS.2025.100600123

Received: 22 June 2025; Accepted: 26 June 2025; Published: 19 July 2025

ABSTRACT

This study investigated the isolation and molecular characterization of lactic acid bacteria (LAB) from three locally fermented Nigerian foods; West African soft cheese (Wara), fermented yellow maize cereal porridge (Ogi solo), and fermented white maize cereal porridge (Ogi funfun). Samples were obtained from Waso market in Ogbomosho, Oyo State, and processed according to standard microbiological techniques on MRS medium under anaerobic conditions. Total LAB counts ranged from 6.0 × 10⁶ to 11.0 × 10⁶ CFU/g, with the highest count in the yellow maize cereal porridge. Five distinct LAB isolates were obtained and molecularly characterized via 16S rRNA gene sequencing. From the BLAST analysis, with a similarity of 99-100% to the reference strains, the isolates were identified as Lactococcus lactis FYM1, Lactobacillus acidophilus FYM2, Lactobacillus paracasei WAC1, Lactobacillus delbrueckii WAC2, and Lactobacillus iners FWM. All the isolates have been deposited in NCBI GenBank under accession numbers PQ758598, PQ555634, PQ346814, PQ555635, and PQ555636, respectively. The phylogenetic analysis showed that the isolates clustered within the Lactobacillaceae family and had a close relationship to other known probiotic strains. This shows the very high diversity that exists among the beneficial LAB in traditional Nigerian fermented foods and their availability for further studies in food biotechnology and probiotic development.

Keywords: Fermented food, Lactobacillus, Lactic acid bacteria, probiotics, molecular characterization.

INTRODUCTION

Fermented foods have been reported to be highly nutritious and therapeutic not only because of the macro/micronutrients present in them but also due to the presence of microorganisms that have functionalized the food through their metabolism and secondary metabolites.  These beneficial microorganisms confer nutritional and medicinal benefits on the food in which they are found (Adesemoye et al., 2025).  The metabolic activities of the beneficial microorganisms found in foods can help improve the bioavailability of phytochemicals, macro and micro nutrients in a biochemical process called fermentation.  Through the presence or inoculation of microorganisms like bacteria or yeasts into food, fermentation occurs by breaking down carbohydrate into organic acids and alcohols in anaerobic environments (Punia et al., 2022).

In Nigeria, some fermented foods can be classified based on their raw materials, these fermented food categories include sorghum-based fermented foods e.g. brown pap locally called ogi baba by the Yoruba tribe in southwestern part of Nigeria, maize-based fermented foods e.g. yellow corn meal also locally called “Ogi” by the Yoruba tribe.  Also, millet-based fermented foods locally called “burukutu”  “kunu-zaki, “masa”, and pito.  Cassava-based fermented foods e.g. fufu, gari, and lafun also belong to fermented foods classified on the bases of their raw materials which are highly consumed in Nigeria and other parts of the world (Adesulu-Dahunsi et al., 2020).   Furthermore, West African soft cheese locally called Wara is another fermented food which is locally processed using fresh cow milk curdled with juice extract of Calostropis procera leaves which can be consumed raw after fermentation or as snacks when fried. The unripe soft cheese curd is commonly consumed in the western and northern part of Nigeria.  And due to its nutritional composition, a plethora of beneficial microorganisms are found in it (Adesemoye et al., 2025)

Among the beneficial microorganisms found in fermented foods are lactic acid bacteria (LAB) (Wang et al., 2021).  Lactic acid bacteria are rod or cocci, non-spore forming, Gram-positive bacteria with strong tolerance to pH, capable of carbohydrate fermentation to produce lactic acid primarily as the end product (Oladipo et al., 2025a; 2025b); they are also known to biodegrade proteins and lipids.   Lactic acid bacteria are also capable of producing alcohols, esters, aldehydes, and some compounds like sulfur compounds to produce flavors in various fermented foods (Bintsis, 2018; Oladipo and Oyewumi, 2024).  Lactic acid bacteria constitute more than 61 genera, but the genera frequently found in fermented foods are Lactococcus, Lactobacillus, Leuconostoc, Streptococcus and Pediococcus (Wang et al., 2021).  Recently Leuconostocaceae and Lactobacillaceae have been merged into family Lactobacillaceae while the Lactobacillus genus was reclassified into 25 genera (Zheng et al., 2020; Wang et al., 2021).

Lactic acid bacteria that are beneficial and edible are called probiotics (Oladipo and Ogunleke, 2023), although not only LAB are found in this category, but LAB constitute the larger part of it.   Lactic acid bacteria are highly beneficial to man in many ways as reported by Oladipo et al. (2013).  This researched work focused on the isolation and molecular characterization of lactic acid bacteria isolated from West African cheese (Wara), yellow corn meal (Ogi solo) and white corn meal (Ogi funfun) collected from a local market within Ogbomosho metropolis.

MATERIALS AND METHODS

Sample Collections and preparation

Fermented yellow maize cereal porridge (locally called Ogi solo), fermented white maize cereal porridge (locally called Ogi funfun) and West African soft cheese (locally called wara) samples were collected from Waso market (8° 8′ 31.7940” N and 4° 14′ 42.6696” E) in Ogbomosho, Oyo State.  The samples were collected in sterile screw cap bottle and conveyed to the laboratory in ice packs to retain the microbial integrity of the samples.  One gram of the sample was aseptically added to 9 mL of sterile MRS (De-Man, Rogossa and Sharpe) broth, carefully shaken and were left to homogenize for 30 minutes. Ten-fold serial dilutions were prepared from the sample homogenate, and were streaked onto duplicate sterile petri dishes of MRS (De-Man, Rogossa and Sharpe). The streaked plates were incubated anaerobically at 30˚C for 2 – 3 days in an anaerobic incubator.  The incubated isolate were checked after 72 hours for visible colonies and the colonies were further subjected to sub-culturing to acquire a pure culture.  Fresh medium were prepared and sterilized at 121⁰C for 15 minutes and allowed to cool and then pour aseptically in the sterilized petri dishes.  The isolates inoculums were sub cultured on new plate by streaking in other to get a pure distinct colony.  The plates were incubated at 37⁰C for another 72 hours.  The plates were observed for pure and distinct colony after 72 hours

Molecular Characterization of the Isolates

Fresh culture of the isolates was subjected to DNA extraction using the EZ-10 Spin Column Bacterial Genomic DNA Miniprep Kit (Bio Basic, Markham, Canada). The DNA integrity and concentration of the isolates were determined by taking optical density measurements using a Nano Drop spectrophotometer (Thermo Fisher Scientific, Waltham, USA) (Romero-Luna et al., 2020).

Amplification and Sequencing of 16S rRNA Gene

The amplification of the desired DNA template was carried out using PCR with universal primer sets: forward primer 3′-AGAGTTTGATCCTGGCTCAG-5′ and reverse primer 5′-GGCTGCTGGCACGTAGTTAG-3′. The thermal cycler was programmed with a pre-incubation period of 2 min at 94 °C and then 35 cycles, each consisting of denaturation for 30 s at 94 °C, annealing for 30 s at 55 °C, and elongation for 1 min at 72 °C. A final elongation of 10 min at 72 °C followed the 35 cycles. The amplicons were visualized through 1% agarose gel electrophoresis with reference to a 1 kbp DNA ladder. The PCR product was quantified using a Nano Drop spectrophotometer and purified using the QIAquick (Qiagen, Germantown, USA) purification kit (Romero-Luna et al., 2020).

RESULTS AND DISCUSSION

Isolation and Molecular Identification of the Isolates

The total Lactobacillus counts of the fermented yellow maize cereal porridge (Ogi solo), fermented white maize cereal porridge and West African soft cheese (Wara) samples used in the study (Figure 1) are shown in Table 1, it was observed that the highest colony count was observed on fermented yellow maize cereal porridge plate with colony forming unit of 11.0 x 10⁶, while West African Soft cheese and fermented white maize cereal porridge had the lowest colony count of 6.0 x 10⁶ respectively. The result is in agreement with the report of David et al. (2019) who reported the colony count within the range of 6.0 x 10² CFU/ml to 2.0 x 10⁴CFU/ml in fermented foods. Also, Eji et al. (2023) reported the isolation of lactic acid bacteria from 20 locally fermented foods with CFU/g ranges from 2.0 to 2.5 x 10⁴ in all the samples.  According to Wang et al. (2021), lactic acid bacteria are regarded to be hetero-lactic fermentative because they are capable of fermenting carbohydrate to produce lactic acid and thus abundant and highly resourceful in fermentation, which lend credence to the presence of lactic acid bacteria in fermented foods used in this study.

The isolates were molecularly characterized and were identified using targeted unique sequence (1500 bp length) that were aligned using BioEdit sequence alignment editor version 7.2.5.0. The 16S rRNA gene sequences obtained showed 99%–100% similarity to reference strains in the NCBI database after the BLAST search, the isolates were identified as Lactococcus lactis FYM1, Lactobacillus acidophilus FYM2, Lactobacillus paracasei WAC1, Lactobacillus delbruecki WAC2, and Lactobacillus iners FWM (Table 2), the gel (1500bp) picture of the amplicons are illustrated in Figure 2.  After the BLAST search in NCBI data base, it was observed that Lactococcus lactis FYM1 has 99% homology with L. lactis IPLA1064 (CP184693) isolated from raw milk cheese in Denmark (Lopez-Gonzales et al., 2025).  Lactobacillus acidohilus FYM2 has 9.15% homology with L. acidophilus ATCC 4356 (CP139575) isolated from sour milk in Bioscience, Los Alamos National Laboratory, USA (Kumar, 2023).  Lactobacillus paracasei WAC1 has 100% homology with L. paracasei 77FHE (MT383743) isolated from raw cow milk sample in Faculty Of Women For Arts, Science and Education, Asmaa Fahmy, Naser City 11757, Egypt (Abo-Safe et al., 2020).  Lactobacillus delbrueckii WAC2 has 99.5% homology with Lactobacillus delbrueckii A281 (MN437943) isolated from diary product in College of Food Science and Nutritional Engineering, China Agricultural University (Ren, 2019).  Lactobacillus iners FWM has 99% homology with L. iners C025C1 (CP049231) isolated from human vagina in Institute for Genome Sciences, University of Maryland Baltimore, USA (France et al., 2020).

The isolates: Lactococcus lactis FYM1, Lactobacillus acidophilus FYM2, Lactobacillus paracasei WAC1, Lactobacillus delbruecki WAC2, and Lactobacillus iners FWM were registered in NCBI Genbank and were given accession numbers PQ758598, PQ555634, PQ346814, PQ555635 and PQ555636 respectively. Based on molecular characterization of the isolates, the result obtained highlighted Lactobacillus sp as the highest occurring organisms and this is in agreement with the report of Eji et al. (2023) who also reported the isolation and characterization of Lactobacillus plantarum, Lactobacillus acidiophilus, Lactobacillus fermentum and Lactobacillus casei from traditionally fermented food such as ogi, and soy milk.  Furthermore, Hamad et al. (2022) and Latif et al. (2023) reported the isolation and molecular identification of Lactobacillus sp. from milk based fermented foods. Oladipo et al. (2025b) also reported the isolation and molecular identification of Lacticaseibacillus casei, Lactobacillus delbrueckii, Lactobacillus johnsonii, Lactobacillus brevis and Lactobacillus jensenii from fermented African oil bean seeds, fermented Citrullus colocynthis seeds, fermented maize cereal porridge, and West African soft cheese respectively.

Figure 1: Fermented Foods Used in the Study

Table 1: The Microbial Load of the Samples

Key: CFU/ml: colony forming unit per gram.

Figure 1: Polymerase chain reaction amplification of the isolates DNA

Key: A: FYMI, B: FYM2, C: WAC1, D: WAC2, E: FWM. FYMI: fermented yellow maize cereal porridge, FWM: fermented white maize cereal porridge, WAC: West African soft cheese.

Table 2: Molecular Identity and Accession Numbers of the Isolates

Key: FYM1 and FYM2: isolates from fermented yellow maize cereal porridge, FWM: isolate from fermented white maize cereal porridge, WAC and WAC2: isolates from West African soft cheese.

The phylogenetic relationship among the isolates is shown in Figure 3; the isolates principally cluster with the Lactobacillaceae family, related to Lactobacilus delbrueckii (AB008207.1), Lactobacillus acidophilus (AB680529), Lactobacilus iners (NR036982.1), Lactobacillus paracasei (LC638743.1), Lactococcus lactis (NR113960).  The isolates from the fermented food samples form a well-supported clade that is distinct but closely related to known reference strains obtained from NCBI.  According to the phylogenic analysis, FYM1 showed 99% homology with Lactococcus lactis NBRC strain, FYM2 showed 99.6% homology with Lactobacillus acidophilus NBRC 13951 strain (Igbal et al., 2009), WAC1 showed 99% homology with Lactobacillus paracasei R094 strain (Mulaw et al., 2019), WAC2 showed 99% homology with Lactobacillus delbrueckii YIT 0080 strain (Igbal et al., 2009) and FWM showed 99% homology with Lactobacillus iners DSM 13335 (Mulaw et al., 2019).  Previous studies have highlighted the significance of fermented food as probable vehicles that play essential role in the continuous isolation of Lactic acid bacteria due to their capacity to not only generate tremendously elevated levels of lactic acid but also thrive consistently in extreme acidic state (David et al., 2019).

Figure 2: The phylogenetic tree showing the relationship between the isolates

CONCLUSION

The isolation and molecular characterization of 5 different LAB species from 3 Nigerian fermented foods commonly consumed were achieved. The characterized isolates, which have been deposited in NCBI GenBank, constitute valuable genetic resources demonstrating the immense microbial biodiversity existing in traditional Nigerian fermented foods. The results demonstrate the importance of indigenous fermented foods as sources of beneficial microorganisms and therefore form the basis for further benefits from their therapeutic and commercial exploitation.  This work contributes to food security through the isolation and molecular identification of probiotic lactic acid bacteria from locally fermented foods, which are crucial for nutritional enhancement, and shelf life extension. Understanding these LAB can help improve food availability and safety thus addressing SDG 2 (Zero hunger). The study also contributes to SDG 3 (Good health and well-being) as probiotic lactic acid bacteria are known for their health benefits which include antimicrobial properties, digestive health, and immune system support. Isolating and characterizing local strains of LAB could lead to innovative health-promoting applications.

REFERENCES

1. Abo-Safe, F., Elsagher, H. and Sakr, E. (2020) Lacticaseibacillus paracasei strain 77FHE 16S ribosomal RNA gene, partial sequence. https://www.ncbi.nlm.nih.gov/nucleotide/1?report=genbank&log$=nucltop&blast_rank=4&RID=3K6P5RX3016
2. Adesemoye, E.T., Sanni, A.I., Spano, G., Capozzi, V., Fragasso, M. (2025) Lactic Acid Bacteria Diversity in Fermented Foods as Potential Bio-Resources Contributing to Alleviate Malnutrition in Developing Countries: Nigeria as a Case Study. Fermentation 2025, 11(2), 103; https://doi.org/10.3390/fermentation11020103
3. Adesulu-Dahunsi, A.T.; Dahunsi, S.O.; Olayanju, A. Synergistic Microbial Interactions between Lactic Acid Bacteria and Yeasts during Production of Nigerian Indigenous Fermented Foods and Beverages. Food Control. 2020, 110, 106963.
4. Bintsis T. Lactic acid bacteria: their applications in foods. J Bacteriol Mycol Open Access. 2018;6(2):89–94. DOI: 15406/jbmoa.2018.06.00182
5. David, A.A.D., Orukotan A.A. and Mohammed S.S.D (2019) Conventional and molecular characterization of selected lactic acid bacteria from fermented corn gruel (ogi) and fermented milk (NONO). Science World Journal Vol. 14(No 4)
6. Eji, C.A., Okwuokei, G. C., Okafor, J.G., Akinboyewa, I., Ononye, O.D., John, C., Okoro, N., Ochigbabo, J., Chidi, P.C., Orji, N.O. (2023) Antibacterial activity of lactic acid bacteria isolated from traditionally fermented food against food pathogen. GSC Advanced Research and Reviews, 15(02), 020–031
7. France, M.T., Rutt, L., Narina, S., Arbaugh, S., Humphrys, M.S., Ma, B., Hayward, M.R., Relman, D., Kwon, D.S. and Ravel, J. (2020) Complete genome sequences of six Lactobacillus iners strains isolated from the human vagina. https://www.ncbi.nlm.nih.gov/nucleotide/ CP049231.1?report=genbank&log$=nucltop&blast rank=11&RID=3KBMYY0P013
8. Hamad, G. M., Amer, A., el-Nogoumy, B., Ibrahim, M., Hassan, S., Siddiqui, S. A., et al. (2022). Evaluation of the Effectiveness of Charcoal, Lactobacillus rhamnosus, and Saccharomyces cerevisiae as Aflatoxin Adsorbents in Chocolate. Toxins 15:21. doi: 10.3390/ toxins15010021
9. Kumar, A. (2023). The Complete Genome Sequence of Probiotic Lactobacillus acidophilus ATCC 9224 isolated from sour milk. https://www.ncbi.nlm.nih.gov/nucleotide/1?report=genbank&log$=nucltop&blast rank=2&RID=3K8595R9013
10. Latif, A., Shehzad, A., Niazi, S., Zahid, A., Ashraf, W., Iqbal, M.W., Rehman, A., Riaz, T., Aadil, R.M., Khan, I.M., Özogul, F., Rocha, J.M., Esatbeyoglu, T. and Korma, S.A. (2023) Probiotics: Mechanism of Action, Health Benefits and their Application in Food Industries. Front. Microbiol. 14:1216674. doi: 10.3389/fmicb.2023.1216674
11. Mulaw, G., Tessema, T., Muleta, D., Tesfaye, A. (2019) In Vitro Evaluation of Probiotic Properties of Lactic Acid Bacteria Isolated from Some Traditionally Fermented Ethiopian Food Products. Int J Microbiol.; (11):7179514.
12. Oladipo I. C., Sanni A. and Swarnakar S. (2013). Phenotypic and genomic characterization of Enterococcus Species from some Nigerian fermented foods. Food Biotechnology, 27 (1): 39-53. http://dx.doi.org/10.1080/08905436.2012.755627
13. Oladipo I. C. and Ogunleke O. B. (2023). Probiotics and Human Health: An Overview. Journal of Nutrition Food Science and Technology 4(2): 1-8.
14. Oladipo I. C. and Oyewumi M. M. (2024). The Role of Lactic Acid Bacteria in Food Processing, Nutrition and Human Health. International Journal of Current Microbiology and Applied Sciences 13 (10): 288 – 296 https://doi.org/10.20546/ijcmas.2024.1310.033
15. Oladipo I.C. (2025a). Safety of lactic acid bacteria in foods: A perspective.  Food and Health 7(1):1. https://doi.org/10.53388/FH2025001
16. Oladipo I. C., Ogunsona S. B., Ogunleke O. B. and Elusakin O. T. (2025b). Therapeutic Potentials of Some Lactobacillus Species Isolated from Fermented Foods and Condiments. International Journal of Current Microbiology and Applied Sciences 14(05): 59-71
17. Punia, S.; Suri, S.; Trif, M.; Ozogul, F. Organic Acids Production from Lactic Acid Bacteria: A Preservation Approach. Food Biosci. 2022, 46, 101615.
18. Ren, F. (2019) Lactobacillus delbrueckii subsp. bulgaricus strain A281 16S ribosomal RNA gene, partialsequence.https://www.ncbi.nlm.nih.gov/nucleotide/MN437943.1?report=gen bank & log$=nucltop&blast rank=2 & RID=3 K73 UCJP016
19. Romero-Luna, H.E., Peredo-Lovillo, A., Hernández-Mendoza, A. et al. Probiotic Potential of Lactobacillus paracasei CT12 Isolated from Water Kefir Grains (Tibicos). Curr Microbiol 77, 2584–2592 (2020). https://doi.org/10.1007/s00284-020-02016-0
20. Wang Y, Wu J, Lv M, Shao Z, Hungwe M, Wang J, Bai X, Xie J, Wang Y, Geng W. Metabolism Characteristics of Lactic Acid Bacteria and the Expanding Applications in Food Industry. Front Bioeng Biotechnol. 2021 May 12;9:612285. doi: 10.3389/fbioe.2021.612285. PMID: 34055755; PMCID: PMC8149962.
21. Zheng J., Wittouck S., Salvetti E., Franz C., Harris H., Mattarelli P., et al. (2020). A taxonomic note on the genus Lactobacillus: description of 23 novel genera, emended description of the genus Lactobacillus beijerinck 1901, and union of Lactobacillaceae and Leuconostocaceae. Int. J. Syst. Evol. Microbiol. 70 2782–2858. 10.1099/ijsem.0.004107.