Biofilm Formation Capabilities of Lactobacillus Species Isolated from Selected Fermented Food Products Using a Statistical Approach

Year 2025, Volume: 5 Issue: 1, 13 - 23, 29.01.2025

Olodu Blessing Adoh , Stephen Amadin Enabulele

https://doi.org/10.56016/dahudermj.1582709

Abstract

Background: This study investigates the biofilm formation capabilities of Lactobacillus species isolated from fermented cassava and corn products. Understanding biofilm formation is crucial for evaluating the probiotic potential of these species, as biofilm-forming ability influences their survival and functionality in host environments.
Methods: Nine bacterial isolates, including Lactobacillus fermentum, L. ghanensis, L. delbrueckii, L. plantarum, Lactococcus lactis, L. reuteri, Lysinibacillus sphaericus, Bacillus cereus, and B. pacificus, were assessed for biofilm production using the microtiter plate assay. After crystal violet staining, optical density (OD) values were measured at 570 nm spectrophotometrically. Based on OD values, isolates were classified into four categories: no biofilm, weak, moderate, and strong biofilm formation. Statistical analyses, including two-stage least squares regression, were employed to evaluate biofilm formation trends and predictors.
Results: The predictive regression model was highly significant (R² = 0.987, F = 122.618, p < 0.0001). Biofilm formation strength varied, with the highest mean percentage observed in the moderate group (31.29%), followed by weak (27.41%), strong (20.46%), and no biofilm (20.05%). Among the isolates, Lactobacillus fermentum exhibited the highest rate of strong biofilm formation (46.1%), while Lysinibacillus sphaericus showed none. Moreover, The highest biofilm formation was observed at 37°C (31.29%), followed by 25°C (27.41%), and 45°C (20.46%). Similarly, biofilm formation was highest at pH 6.5 (30.41%), followed by pH 7.5 (25.39%) and pH 4.5 (20.05%). Lactobacillus fermentum exhibited the highest strong biofilm formation (46.1%) at 37°C and pH 6.5.
Conclusion: Biofilm formation in Lactobacillus species is species-specific and environmentally influenced by temperature and pH. Lactobacillus fermentum demonstrated strong biofilm formation, making it a promising candidate for probiotic applications.

Keywords

Biofilm formation, crystal violet staining, Fermentation, Lactobacillus species, Probiotics, Statistical models

Ethical Statement

The study is proper with ethical standards; it was approved by the Department of Biological Sciences (Microbiology), Benson Idahosa University, on 26th February 2024.

Biofilm Formation Capabilities of Lactobacillus Species Isolated from Selected Fermented Food Products Using a Statistical Approach

Abstract

Objectives: This study investigates the biofilm formation capabilities of Lactobacillus species isolated from fermented cassava and corn products. Understanding biofilm formation is crucial for evaluating the probiotic potential of these species, as biofilm-forming ability influences their survival and functionality in host environments.
Methods: Nine bacterial isolates, including Lactobacillus fermentum, L. ghanensis, L. delbrueckii, L. plantarum, Lactococcus lactis, L. reuteri, Lysinibacillus sphaericus, Bacillus cereus, and B. pacificus, were assessed for biofilm production using the microtiter plate assay. After crystal violet staining, optical density (OD) values were measured at 570 nm spectrophotometrically. Based on OD values, isolates were classified into four categories: no biofilm, weak, moderate, and strong biofilm formation. Statistical analyses, including two-stage least squares regression, were employed to evaluate biofilm formation trends and predictors.
Results: The predictive regression model was highly significant (R² = 0.987, F = 122.618, p < 0.0001). Biofilm formation strength varied, with the highest mean percentage observed in the moderate group (31.29%), followed by weak (27.41%), strong (20.46%), and no biofilm (20.05%). Among the isolates, Lactobacillus fermentum exhibited the highest rate of strong biofilm formation (46.1%), while Lysinibacillus sphaericus showed none. Moreover, The highest biofilm formation was observed at 37°C (31.29%), followed by 25°C (27.41%), and 45°C (20.46%). Similarly, biofilm formation was highest at pH 6.5 (30.41%), followed by pH 7.5 (25.39%) and pH 4.5 (20.05%). Lactobacillus fermentum exhibited the highest strong biofilm formation (46.1%) at 37°C and pH 6.5.
Conclusion: Biofilm formation in Lactobacillus species is species-specific and environmentally influenced by temperature and pH. Lactobacillus fermentum demonstrated strong biofilm formation, making it a promising candidate for probiotic applications.

Keywords

Biofilms formation, crystal violet staining, Fermentation, Lactobacillus species, Probiotics, Statistical models

References

• Tuğba D, Hakan D. Inhibitory effect of probiotics Lactobacillus supernatants against Streptococcus mutans and preventing biofilm formation. Turk J Agric Food Sci Technol. 2021;9(2):339-345. doi:10.24925/turjaf.v9i2.339-345.3954.
• Stacy M, Jonathan GG, Roy W, Moamen MM, Andrew W, Abdul NH, et al. Lactobacilli spp.: Real-time evaluation of biofilm growth. BMC Microbiol. 2020;20:1753. doi:10.1186/s12866-020-01753-3.
• Fadilla S, Endah R. Molecular characterization of lactic acid bacteria producing edible biofilm isolated from kimchi. Biodiversitas. 2020;21(3):315. doi:10.13057/biodiv/d210315.
• María JS, Alejandra I, Marco V, Apolinaria G. Biofilm-forming Lactobacillus: New challenges for the development of probiotics. Microorganisms. 2016;4(3):35. doi:10.3390/microorganisms4030035.
• Endo A, Salminen S. Isolation and characterization of Lactobacillus strains from traditional fermented foods. Microbiol Spectr. 2018;6(2):45-56. doi:10.1128/microbiolspec.RWR-0011-2017.
• Parvez S, Kim Y, Kang J, Lee H, Huh K, Park M, Choi H, Kim Y. Evaluation of the probiotic potential of Lactobacillus species from fermented foods. Int J Food Microbiol. 2022;352:109308. doi:10.1016/j.ijfoodmicro.2021.109308.
• Adewumi GA, Adebayo TB. Characterization of lactic acid bacteria in traditional fermented foods. Food Sci Technol. 2020;56(1):23-30. doi:10.1007/s13197-019-04031-9.
• Kim WS, Hwang H, Lee J, Lim Y, Kim S, Kim C. Molecular techniques for identifying lactic acid bacteria. Int J Syst Evol Microbiol. 2022;72(5):517-526. doi:10.1099/ijsem.0.005170.
• FAO/WHO. Guidelines for the Evaluation of Probiotics in Food. Joint FAO/WHO Working Group Report. 2002. doi:10.4060/cb4095en.
• Ouwehand AC, von Wright M, Salminen S. Characterization of biofilm formation in probiotic Lactobacillus strains. Probiotics Antimicrob Proteins. 2020;12(1):45-54. doi:10.1007/s12602-019-09578-x.
• Chen X, Zhang H, Xue Y, Wang H, Zhang Y. Microbial isolation and characterization from fermented maize. J Appl Microbiol. 2018;125(5):1348-1357. doi:10.1111/jam.14023.
• Montet D, Ray RC. Fermented foods: Microbiology, biofilm formation, and health benefits. J Nutr Health Sci. 2019;6(3):124-130. doi:10.15744/2393-9060.6.302.
• Assefa M, Beyene D. Biofilm formation and its effect on the survival of Lactobacillus strains. Appl Microbiol Biotechnol. 2018;102(12):5129-5141. doi:10.1007/s00253-018-8995-4.
• Abdel-Razek AG, Khalil MS, Ghaith DM, Metwally SA. Microbiological and biochemical characterization of fermented cassava and maize. J Food Microbiol. 2018;47(3):128-134. doi:10.1016/j.fm.2018.02.005.
• Leroy F, De Vuyst L. Fermentation of vegetables and non-dairy beverages by lactic acid bacteria. Curr Opin Food Sci. 2020;31:15-20. doi:10.1016/j.cofs.2019.12.003.
• Haghshenas B, Kianpour F, Khajeh K, Taheri P. Evaluation of biofilm formation among different strains of Lactobacillus. J Microb Biochem Technol. 2018;10(1):33-40. doi:10.4172/1948-5948.1000398.
• Gänzle MG. Fermented foods and their microbiota. Food Microbiol. 2021;98:103794. doi:10.1016/j.fm.2021.103794.
• Sharma A, Yadav S, Khan M, Singh P, Bhatti S, Gupta A. Microbial diversity and biofilm formation in fermented foods. J Appl Microbiol. 2021;131(1):134-146. doi:10.1111/jam.14912.
• Yadav R, Kumar R, Shukla V, Gautam V. Molecular identification and probiotic potential of Lactobacillus isolates. Biotechnol Rep. 2020;28:e00538. doi:10.1016/j.btre.2020.e00538.
• Al-Otaibi RS. Biofilm formation by Lactobacillus species: Mechanisms and assessment methods. Microb Ecol. 2021;82(2):360-371. doi:10.1007/s00248-021-01762-3.
• Amara AA, Shibl A. Role of probiotics in health improvement, infection control, and disease treatment. Int J Curr Microbiol Appl Sci. 2018;7(3):34-50. doi:10.20546/ijcmas.2018.703.005.
• El-Ghaish S, El-Sayed A, Ahmed A, Al-Nasr M, El-Khawas K. Biochemical and molecular characterization of lactic acid bacteria in fermented products. Int J Food Microbiol. 2020;321:108537. doi:10.1016/j.ijfoodmicro.2020.108537.
• Donkor ON, Osei E, Tamakloe I, Awuchi CG, Nwachukwu I. Microbiological methods for the identification of probiotic bacteria. Food Microbiol. 2019;82:70-78. doi:10.1016/j.fm.2019.01.008.
• Saxelin M, von Wright M, Mattila-Sandholm T, Salminen S. Safety assessment of lactic acid bacteria from fermented foods. Food Microbiol. 2018;76:85-95. doi:10.1016/j.fm.2018.04.010.
• Liu Y, Lin J, Li X, Wang L, Jiang X. Comparison of traditional and modern techniques for bacterial isolation. J Bacteriol Res. 2018;10(2):32-45. doi:10.4172/1948-5948.1000399.
• Mohammadi R, Sohrabvandi S. Biofilm development and assessment techniques in lactic acid bacteria. Microb Cell Fact. 2021;20(1):57. doi:10.1186/s12934-021-01548-9.
• Lee YK, Salminen S. The importance of Lactobacillus biofilm formation for probiotic functions. Microb Ecol Health Dis. 2018;29(4):123-130. doi:10.1080/16512235.2018.1490123.
• Song JH, Seo KS. Environmental impact on biofilm formation in Lactococcus species. J Dairy Sci. 2019;102(3):2738-2746. doi:10.3168/jds.2018-15345.
• Bajpai VK, Baek KH, Kang SC. Statistical modeling in biofilm research: A focus on Lactobacillus biofilm variability. Front Microbiol. 2020;11:304-312. doi:10.3389/fmicb.2020.00304.
• Ahmed S, Khan MT. The role of environmental stressors in shaping biofilm formation of Lactobacillus strains. Microb Physiol. 2018;44(2):56-68. doi:10.1159/000487327.
• Fernández L, Langa S, Martín V. Variability in biofilm formation among Lactobacillus species: An analysis of biological factors. Int J Food Microbiol. 2020;314:108-115. doi:10.1016/j.ijfoodmicro.2019.108115.
• Huang R, Liu S, Tang J. Understanding biofilm formation: Insights from microbial ecology. Curr Opin Microbiol. 2019;50:15-22. doi:10.1016/j.mib.2019.09.003.
• Patel JB, Huang X. Modeling biofilm distribution: Applications in microbial research. Microb Informat. 2021;13(1):45-55. doi:10.1016/j.mibinf.2021.100045.
• Li Q, Wu Z, Zhang H. Biofilm dynamics in Lactobacillus species and implications for probiotic development. Probiotics Antimicrob Proteins. 2022;14(3):202-215. doi:10.1007/s12602-021-09870-9.