Endüstriyel Kefir Orijinli, Ekzopolisakkarit Üreten Lacticaseibacillus rhamnosus ve Lacticaseibacillus paracasei'ye ait Postbiyotiklerin Anti-Candidal Biyofilm Ajanları Olarak Değerlendirilmesi

Abstract

Candidal biyofilm hem tıbbi hem de endüstriyel alanlarda ciddi sorunlara neden olmaktadır. Bu çalışmada, ekzopolisakkarit üreten endüstriyel kefir izolatlarına ait postbiyotiklerinin Candida albicans'ın biyofilm ilgili genlerinin transkripsiyon düzeyine etkisinin ortaya konulması ve biyofilm önleme ve tedavi özelliklerinin belirlenmesi amaçlanmıştır. Çalışmada, süpermarketlerden alınan sekiz farklı markanın kefir örneklerinden elde edilen laktik asit bakterilerinin ekzopolisakkarit üretim kapasitesi belirlenmiştir. Ekzopolisakkarit üreten izolatların postbiyotiklerinin C. albicans biyofilmle ilişkili genleri üzerindeki transkripsiyonel etkisi Ters Transkripsiyon-Polimeraz Zincir Reaksiyonu ile gerçekleştirilmiştir. Postbiyotiklerin biyofilm önleme ve biyofilm tedavi etkileri abiyotik ortamda (polistiren mikroplak) belirlenmiştir. Lacticaseibacillus rhamnosus ve Lacticaseibacillus paracasei (ekzopolisakkarit üreten izolatlar) postbiyotiklerinin minimum inhibitör konsantrasyon (MİK) değerleri sırasıyla %12.5 ve %25 olarak belirlenmiştir. L. rhamnosus ve L. paracasei postbiyotiklerinin 2xMİK ve üzeri dozları önlemede etkili iken, MİK ve üzeri dozları biyofilm tedavisinde etkili bulunmuştur. L. rhamnosus postbiyotiği 2xMİK ve MİK dozlarında als1, als3 ve bcr genlerini aşağı regüle etmiştir. L. paracasei postbiyotiği 2xMİK dozuna maruziyette incelenen tüm genleri aşağı regüle etmiştir. L. rhamnosus postbiyotiğinin 2xMİK dozunda 24. saatte als1, als3 ve bcr genlerinde ortalama olarak sırasıyla 0.03, 0.15 ve 0.79 kat aşağı regülasyon belirlenirken; MİK dozlarında 0.31, 0.55 ve 0.77 kat aşağı regülasyon belirlenmiştir. L. paracasei postbiyotiğinde 24. saatte 2xMİK dozunda als1, als3, bcr ve hwp genlerinde sırasıyla 0.39, 0.05, 0.91 ve 0.04 kat aşağı regülasyon belirlenmiştir. Sonuç olarak, biyofilmleri önleyebilen veya tedavi edebilen yeni yaklaşımların keşfi, yeni biyolojik kontrol ajanlarının ortaya çıkmasını teşvik edecektir.

Keywords

• Biyofilm
• Candida
• Ekzopolisakkarit
• Kefir
• Lacticaseibacillus
• Postbiyotikler

Evaluation of Postbiotics Belonging to Industrial Kefir-Derived Exopolysaccharide-Producing Lacticaseibacillus rhamnosus and Lacticaseibacillus paracasei as Anti-Candidal Biofilm Agents

Abstract

Candidal biofilm is a concern in both medical and industrial fields. This study aims to reveal the effect of postbiotics on the transcriptional level of biofilm-associated genes of Candida albicans in exopolysaccharide-producing industrial kefir-derived isolates and to determine their biofilm prevention and treatment properties. In the study, the exopolysaccharide production capacity of lactic acid bacteria obtained from kefir samples of eight different brands from supermarkets was determined. The transcriptional effect of postbiotics of exopolysaccharide-producing isolates on C. albicans biofilm-related genes was carried out via reverse transcription-polymerase chain reaction. The biofilm prevention and biofilm treatment effects of postbiotics were determined in abiotic media (polystyrene microplate). The minimum inhibitory concentration (MIC) values of Lacticaseibacillus rhamnosus and Lacticaseibacillus paracasei (exopolysaccharide-producing isolates) postbiotics were determined as 12.5% and 25%, respectively. While 2xMIC and above doses of L. rhamnosus and L. paracasei postbiotics were effective in prevention, MIC and above doses were effective in biofilm treatment. als1, als3, and bcr genes were down-regulated at 2xMIC and MIC doses of L. rhamnosus postbiotic. At exposure to a 2xMIC dose of L. paracasei postbiotic, all genes examined were down-regulated. On average, 0.03, 0.15, and 0.79-fold downregulation of als1, als3 and bcr genes was determined at 24 hours at 2xMIC dose of L. rhamnosus postbiotic; 0.31, 0.55, and 0.77-fold downregulation was determined at 24 hours at MIC doses. A 0.39, 0.05, 0.91, and 0.04-fold downregulation of L. paracasei postbiotic at 2xMIC dose at 24 hours was determined in als1, als3, bcr and hwp genes, respectively. In conclusion, the discovery of new approaches that can prevent or treat biofilms could stimulate the emergence of novel bio-control agents.

Keywords

• Biofilm
• Candida
• Exopolysaccharide
• Kefir
• Lacticaseibacillus
• Postbiotics

References

1. Azami, S., Arefian, E., & Kashef, N. (2022). Postbiotics of Lactobacillus casei target virulence and biofilm formation of Pseudomonas aeruginosa by modulating quorum sensing. Archives of Microbiology, 204(2), 157.
2. Banakar, M., Pourhajibagher, M., Etemad-Moghadam, S., Mehran, M., Yazdi, M. H., Haghgoo, R., ... & Frankenberger, R. (2023). Antimicrobial effects of postbiotic mediators derived from Lactobacillus rhamnosus GG and Lactobacillus reuteri on Streptococcus mutans. Frontiers in Bioscience-Landmark, 28(5), 88.
3. Barros, P. P., Ribeiro, F. C., Rossoni, R. D., Junqueira, J. C., & Jorge, A. O. C. (2016). Influence of Candida krusei and Candida glabrata on Candida albicans gene expression in in vitro biofilms. Archives of Oral Biology, 64, 92-101.
4. Behbahani, B. A., Jooyandeh, H., Taki, M., & Falah, F. (2024). Evaluation of the probiotic, anti-bacterial, anti-biofilm, and safety properties of Lacticaseibacillus paracasei B31-2. Lwt, 207, 116676.
5. Bengoa, A. A., Dardis, C., Garrote, G. L., & Abraham, A. G. (2021). Health-promoting properties of Lacticaseibacillus paracasei: A focus on kefir isolates and exopolysaccharide-producing strains. Foods, 10(10), 2239.
6. Bengoa, A. A., Llamas, M. G., Iraporda, C., Dueñas, M. T., Abraham, A. G., & Garrote, G. L. (2018). Impact of growth temperature on exopolysaccharide production and probiotic properties of Lactobacillus paracasei strains isolated from kefir grains. Food microbiology, 69, 212-218.
7. Bertsch, A., Roy, D., & LaPointe, G. (2019). Enhanced exopolysaccharide production by Lactobacillus rhamnosus in co-culture with Saccharomyces cerevisiae. Applied Sciences, 9(19), 4026.
8. Brian-Jaisson, F., Molmeret, M., Fahs, A., Guentas-Dombrowsky, L., Culioli, G., Blache, Y., ... & Ortalo-Magné, A. (2016). Characterization and anti-biofilm activity of extracellular polymeric substances produced by the marine biofilm-forming bacterium Pseudoalteromonas ulvae strain TC14. Biofouling, 32(5), 547-560.

9. Butrungrod, W., Chaiyasut, C., Makhamrueang, N., Peerajan, S., Chaiyana, W., & Sirilun, S. (2023). Postbiotic metabolite of Lactiplantibacillus plantarum PD18 against periodontal pathogens and their virulence markers in biofilm formation. Pharmaceutics, 15(5), 1419.
10. Castro-Bravo, N., Wells, J. M., Margolles, A., & Ruas-Madiedo, P. (2018). Interactions of surface exopolysaccharides from Bifidobacterium and Lactobacillus within the intestinal environment. Frontiers in Microbiology, 9, 2426.
11. Chen, Y. S., Liu, Y. H., Teng, S. H., Liao, C. H., Hung, C. C., Sheng, W. H., ... & Hsueh, P. R. (2015). Evaluation of the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry Bruker Biotyper for identification of Penicillium marneffei, Paecilomyces species, Fusarium solani, Rhizopus species, and Pseudallescheria boydii. Frontiers in microbiology, 6, 679.
12. Dailin, D. J., Selvamani, S., Michelle, K., Jusoh, Y. M. M., Chuah, L. F., Bokhari, A., ... & Show, P. L. (2022). Production of high-value added exopolysaccharide by biotherapeutic potential Lactobacillus reuteri strain. Biochemical Engineering Journal, 188, 108691.
13. Dishan, A., Gönülalan, Z., & Dokuzcu, D. (2022). Mevcut Postbiyotik Sınıfları ve Sağlık Etkileşimleri. Beslenme ve Diyet Dergisi, 50(1), 83-91.
14. Dishan, A., Ozkaya, Y., Temizkan, M. C., Barel, M., & Gonulalan, Z. (2025). Candida species covered from traditional cheeses: Characterization of C. albicans regarding virulence factors, biofilm formation, caseinase activity, antifungal resistance and phylogeny. Food Microbiology, 127, 104679.
15. Fanning, S., Xu, W., Solis, N., Woolford, C. A., Filler, S. G., & Mitchell, A. P. (2012). Divergent targets of Candida albicans biofilm regulator Bcr1 in vitro and in vivo. Eukaryotic cell, 11(7), 896-904.
16. García-Gamboa, R., Perfecto-Avalos, Y., Gonzalez-Garcia, J., Alvarez-Calderon, M. J., Gutierrez-Vilchis, A., & Garcia-Gonzalez, A. (2024). In vitro analysis of postbiotic antimicrobial activity against Candida Species in a minimal synthetic model simulating the gut mycobiota in obesity. Scientific Reports, 14(1), 16760.
17. He, Y., Cao, Y., Xiang, Y., Hu, F., Tang, F., Zhang, Y., ... & Zhang, K. (2020). An evaluation of norspermidine on anti-fungal effect on mature Candida albicans biofilms and angiogenesis potential of dental pulp stem cells. Frontiers in Bioengineering and Biotechnology, 8, 948.
18. Hossain, M. I., Mizan, M. F. R., Roy, P. K., Nahar, S., Toushik, S. H., Ashrafudoulla, M., ... & Ha, S. D. (2021). Listeria monocytogenes biofilm inhibition on food contact surfaces by application of postbiotics from Lactobacillus curvatus B. 67 and Lactobacillus plantarum M. 2. Food Research International, 148, 110595.
19. James, K. M., MacDonald, K. W., Chanyi, R. M., Cadieux, P. A., & Burton, J. P. (2016). Inhibition of Candida albicans biofilm formation and modulation of gene expression by probiotic cells and supernatant. Journal of medical microbiology, 65(4), 328-336.
20. Jeon, S., Kim, H., Choi, Y., Cho, S., Seo, M., & Kim, H. (2021). Complete genome sequence of the newly developed Lactobacillus acidophilus strain with improved thermal adaptability. Frontiers in Microbiology, 12, 697351.
21. Kabir, M. A., Hussain, M. A., & Ahmad, Z. (2012). Candida albicans: a model organism for studying fungal pathogens. International Scholarly Research Notices, 2012(1), 538694
22. Keller, M. K., Hasslöf, P., Stecksén-Blicks, C., & Twetman, S. (2011). Co-aggregation and growth inhibition of probiotic lactobacilli and clinical isolates of mutans streptococci: an in vitro study. Acta Odontologica Scandinavica, 69(5), 263-268.
23. Khani, N., Abedi Soleimani, R., Chadorshabi, S., Moutab, B. P., Milani, P. G., & Rad, A. H. (2024). Postbiotics as candidates in biofilm inhibition in food industries. Letters in Applied Microbiology, 77(4), ovad069.
24. Kim, H., & Kang, S. S. (2019). Antifungal activities against Candida albicans, of cell-free supernatants obtained from probiotic Pediococcus acidilactici HW01. Archives of oral biology, 99, 113-119.
25. Liu, C., Xue, W. J., Ding, H., An, C., Ma, S. J., & Liu, Y. (2022). Probiotic potential of Lactobacillus strains isolated from fermented vegetables in Shaanxi, China. Frontiers in microbiology, 12, 774903.
26. Livak, K. J., & Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. methods, 25(4), 402-408.
27. Lynch, D., Hill, C., Field, D., & Begley, M. (2021). Inhibition of Listeria monocytogenes by the Staphylococcus capitis-derived bacteriocin capidermicin. Food Microbiology, 94, 103661. Mayer, F. L., Wilson, D., & Hube, B. (2013). Candida albicans pathogenicity mechanisms. Virulence, 4(2), 119-128.
28. Mohammadi, F., Hemmat, N., Bajalan, Z., & Javadi, A. (2021). Analysis of Biofilm‐Related Genes and Antifungal Susceptibility Pattern of Vaginal Candida albicans and Non‐Candida albicans Species. BioMed Research International, 2021(1), 5598907.
29. Murciano, C., Moyes, D. L., Runglall, M., Tobouti, P., Islam, A., Hoyer, L. L., & Naglik, J. R. (2012). Evaluation of the role of Candida albicans agglutinin-like sequence (Als) proteins in human oral epithelial cell interactions. PloS one, 7(3), e33362.
30. Nacef, M., Chevalier, M., Chollet, S., Drider, D., & Flahaut, C. (2017). MALDI-TOF mass spectrometry for the identification of lactic acid bacteria isolated from a French cheese: The Maroilles. International journal of food microbiology, 247, 2-8.
31. Nailis, H., Coenye, T., Van Nieuwerburgh, F., Deforce, D., & Nelis, H. J. (2006). Development and evaluation of different normalization strategies for gene expression studies in Candida albicans biofilms by real-time PCR. BMC Molecular Biology, 7, 1-9.
32. Nikoomanesh, F., Roudbarmohammadi, S., Roudbary, M., Bayat, M., & Heidari, G. (2016). Investigation of bcr1 gene expression in Candida albicans isolates by RTPCR technique and its impact on biofilm formation.
33. Pfaller, M. A., Sheehan, D. J., & Rex, J. H. (2004). Determination of fungicidal activities against yeasts and molds: lessons learned from bactericidal testing and the need for standardization. Clinical microbiology reviews, 17(2), 268-280.
34. Pokhrel, S., Boonmee, N., Tulyaprawat, O., Pharkjaksu, S., Thaipisutikul, I., Chairatana, P., ... & Mitrpant, C. (2022). Assessment of biofilm formation by Candida albicans strains isolated from hemocultures and their role in pathogenesis in the zebrafish model. Journal of Fungi, 8(10), 1014.
35. Richardson, J. P., Ho, J., & Naglik, J. R. (2018). Candida–epithelial interactions. Journal of fungi, 4(1), 22.
36. Rossoni, R. D., de Barros, P. P., de Alvarenga, J. A., Ribeiro, F. D. C., Velloso, M. D. S., Fuchs, B. B., ... & Junqueira, J. C. (2018). Antifungal activity of clinical Lactobacillus strains against Candida albicans biofilms: identification of potential probiotic candidates to prevent oral candidiasis. Biofouling, 34(2), 212-225.
37. Rossoni, R. D., De Barros, P. P., Mendonça, I. D. C., Medina, R. P., Silva, D. H. S., Fuchs, B. B., ... & Mylonakis, E. (2020). The postbiotic activity of Lactobacillus paracasei 28.4 against Candida auris. Frontiers in cellular and infection microbiology, 10, 397
38. Sarikaya, H., Aslim, B., & Yuksekdag, Z. (2017). Assessment of anti-biofilm activity and bifidogenic growth stimulator (BGS) effect of lyophilized exopolysaccharides (l-EPSs) from Lactobacilli strains. International Journal of Food Properties, 20(2), 362-371.
39. Satala, D., Gonzalez-Gonzalez, M., Smolarz, M., Surowiec, M., Kulig, K., Wronowska, E., ... & Karkowska-Kuleta, J. (2022). The role of Candida albicans virulence factors in the formation of multispecies biofilms with bacterial periodontal pathogens. Frontiers in Cellular and Infection Microbiology, 11, 765942.
40. Song, M. W., Chung, Y., Kim, K. T., Hong, W. S., Chang, H. J., & Paik, H. D. (2020). Probiotic characteristics of Lactobacillus brevis B13-2 isolated from kimchi and investigation of antioxidant and immune-modulating abilities of its heat-killed cells. Lwt, 128, 109452.
41. Song, Y. G., & Lee, S. H. (2017). Inhibitory effects of Lactobacillus rhamnosus and Lactobacillus casei on Candida biofilm of denture surface. Archives of oral biology, 76, 1-6.
42. Stepanović, S., Vuković, D., Dakić, I., Savić, B., & Švabić-Vlahović, M. (2000). A modified microtiter-plate test for quantification of staphylococcal biofilm formation. Journal of microbiological methods, 40(2), 175-179.
43. Sun, Z., Zhao, Z., Fang, B., Hung, W., Gao, H., Zhao, W., ... & Zhang, M. (2023). Effect of thermal inactivation on antioxidant, anti-inflammatory activities and chemical profile of postbiotics. Foods, 12(19), 3579.
44. Tiwari, S., Kavitake, D., Suryavanshi, M. V., Shah, I. A., Devi, P. B., Reddy, G. B., & Shetty, P. H. (2024). An expanding frontier in prebiotic research and synbiotic functional food development through exopolysaccharides from probiotic bacteria. Food and Humanity, 3, 100436.
45. Tsui, C., Kong, E. F., & Jabra-Rizk, M. A. (2016). Pathogenesis of Candida albicans biofilm. FEMS Pathogens and Disease, 74(4), ftw018.
46. Xu, X., Peng, Q., Zhang, Y., Tian, D., Zhang, P., Huang, Y., ... & Shi, B. (2020). A novel exopolysaccharide produced by Lactobacillus coryniformis NA-3 exhibits antioxidant and biofilm-inhibiting properties in vitro. Food & Nutrition Research, 64, 10-29219.
47. Yadav, M. K., Song, J. H., Vasquez, R., Lee, J. S., Kim, I. H., & Kang, D. K. (2024). Methods for Detection, Extraction, Purification, and Characterization of Exopolysaccharides of Lactic Acid Bacteria—A Systematic Review. Foods, 13(22), 3687.
48. Yocheva, L., Tserovska, L., Danguleva-Cholakova, A., Todorova, T., Zhelezova, G., Karaivanova, E., & Georgieva, R. (2024). In Vitro Inhibitory Effects and Co-Aggregation Activity of Lactobacilli on Candida albicans. Microbiology Research, 15(3), 1576-1589.
49. Zhang, K., Liu, S., Liang, S., Xiang, F., Wang, X., Lian, H., ... & Liu, F. (2024). Exopolysaccharides of lactic acid bacteria: Structure, biological activity, structure-activity relationship, and application in the food industry: A review. International Journal of Biological Macromolecules, 257, 128733.
50. Zhao, Z., Wu, J., Sun, Z., Fan, J., Liu, F., Zhao, W., ... & Hung, W. L. (2023). Postbiotics derived from L. paracasei ET-22 inhibit the formation of S. mutans biofilms and bioactive substances: An analysis. Molecules, 28(3), 1236.
51. Zordan, R., & Cormack, B. (2011). Adhesins in opportunistic fungal pathogens. Candida and candidiasis, 243-P2.