Exploring the Multifunctional Potential of Fructophilic Enterococcus faecium as a Probiotic and Extracellular Folate Producer Isolated from Wild Apis dorsata Honeybee Hives in East Nusa Tenggara
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Abstract
Folate or vitamin B9 is an essential nutrient that supports a variety of biological functions. Folate deficiency can lead to a variety of health problems, including megaloblastic anemia, digestive problems, and impaired neurodevelopment in the fetus during pregnancy. Currently, folic acid used for fortification is generally synthesized chemically, which causes unwanted side effects. Bacteria are able to produce folate, but folate production from probiotics has not been widely reported. In fact, folate-producing probiotics can provide an additional source of folate for the body through its absorption in the intestine. This study aimed to select, isolate and identify fructophilic LAB strains from wild giant honeybee hives that had probiotic potential and the ability to produce folate. The results showed that the fructophilic strains Enterococcus faecium MD05, Enterococcus sp. MD23, and E. faecium MD29, identified based on the 16S rRNA gene, are lactic acid bacteria with Gram-positive and catalase-negative properties. All three have probiotic potential, indicated by in vitro tolerance tests to pH 2.5 and 0.3% bile salt, have antimicrobial activity, are able to form aggregates against pathogens, and are sensitive to antibiotics. Enterococcus faecium MD29 was successfully proven to produce extracellular folate up to 5 ng/mL. The multi-functional characteristics of fructophilic LAB have not been widely reported. The unique characteristics of fructophilic LABs, as demonstrated by this strain, remain underexplored. Genetically, E. faecium MD29 is closely related to commensal bacteria of the digestive tract, enhancing its potential as a functional food ingredient. These findings provide a strong basis for conducting in vivo studies to evaluate the strain’s safety and validate its probiotic claims. Further research is also needed to determine the specific type of folate synthesized from fructose and its biofunctional properties.
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References
Ali, M. S., Lee, E.-B., Lim, S.-K., Suk, K., & Park, S.-C. (2023). Isolation and identification of Limosilactobacillus reuteri PSC102 and evaluation of its potential probiotic, antioxidant, and antibacterial properties. Antioxidants, 12(2), Article 238. https://doi.org/10.3390/antiox12020238
Ajibola, A., Chamunorwa, J. P., & Erlwanger, K. H. (2012). Nutraceutical values of natural honey and its contribution to human health and wealth. Nutrition and Metabolism, 9, Article 61. https://doi.org/10.1186/1743-7075-9-61
Alakomi, H. L., Skytta, E., Saarela, M., Mattila-Sandholm, T., Latva-Kala, K. & Helander, I. M. (2000) Lactic acid permeabilizes gram-negative bacteria by disrupting the outer membrane. Applied and Environmental Microbiology, 66(5), 2001-2005. https://doi.org/10.1128/AEM.66.5.2001-2005.2000
Capella-Gutiérrez, S., Silla-Martínez, J. M., & Gabaldón, T. (2009). trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics, 25(15), 1972-1973. https://doi.org/10.1093/bioinformatics/btp348
Cheng, Y., Ling, Z., & Li, L. (2020). The intestinal microbiota and colorectal cancer. Frontiers in Immunology, 11, Article 615056. https://doi.org/10.3389/fimmu.2020.615056
Corcoran, B. M., Stanton, C., Fitzgerald, G. F., & Ross, R. P. (2005). Survival of probiotic lactobacilli in acidic environments is enhanced in the presence of metabolizable sugars. Applied and Environmental Microbiology, 71(6), 3060-3067. https://doi.org/10.1128/AEM.71.6.3060-3067.2005
Czeizel, A. E., Dudás, I., Vereczkey, A., & Bánhidy, F. (2013). Folate deficiency and folic acid supplementation: The prevention of neural-tube defects and congenital heart defects. Nutrients, 5(11), 4760-4775. https://doi.org/10.3390/nu5114760
D’Aimmo, M. R., Satti, M., Scarafile, D., Modesto, M., Pascarelli, S., Biagini, S. A., Luiselli, D., Mattarelli, P., & Andlid, T. (2023). Folate-producing bifidobacteria: metabolism, genetics, and relevance. Microbiome Research Reports, 3(1), Article 11. https://doi.org/10.20517/mrr.2023.59
Darbandi, A., Asadi, A., Ari, M. M., Ohadi, E., Talebi, M., Zadeh, M. H., Emamie, A. D., Ghanavati, R., & Kakanj, M. (2022). Bacteriocins: Properties and potential use as antimicrobials. Journal of Clinical Laboratory Analysis, 36(1), Article e24093. https://doi.org/10.1002/jcla.24093
de Giori, G. S., & LeBlanc, J. G. (2018). Folate production by lactic acid bacteria. In R. R. Watson, V. R. Preedy, & S. Zibadi (Eds.). Polyphenols: Prevention and Treatment of Human Disease (2nd Ed., pp. 15-29). Academic Press. https://doi.org/10.1016/B978-0-12-813008-7.00002-3
de Melo Pereira, G. V., de Oliveira Coelho, B., Júnior, A. I. M., Thomaz-Soccol, V., & Soccol, C. R. (2018). How to select a probiotic? A review and update of methods and criteria. Biotechnology Advances, 36(8), 2060-2076. https://doi.org/10.1016/j.biotechadv.2018.09.003
Endo, A., & Salminen, S. (2013). Honeybees and beehives are rich sources for fructophilic lactic acid bacteria. Systematic and Applied Microbiology, 36(6), 444-448. https://doi.org/10.1016/j.syapm.2013.06.002
Gangadharan, D., Sivaramakrishnan, S., Pandey, A., & Nampoothiri, K. M. (2010). Folate-producing lactic acid bacteria from cow’s milk with probiotic characteristics. International Journal of Dairy Technology, 63(3), 339-348. https://doi.org/10.1111/j.1471-0307.2010.00590.x
Greppi, A., Saubade, F., Botta, C., Humblot, C., Guyot, J. P., & Cocolin, L. (2017). Potential probiotic Pichia kudriavzevii strains and their ability to enhance folate content of traditional cereal-based African fermented food. Food Microbiology, 62, 169-177. https://doi.org/10.1016/j.fm.2016.09.016
Ibrahim, S. A., Ayivi, R. D., Zimmerman, T., Siddiqui, S. A., Altemimi, A. B., Fidan, H., Esatbeyoglu, T., & Bakhshayesh, R. V. (2021). Lactic acid bacteria as antimicrobial agents: food safety and microbial food spoilage prevention. Foods, 10, Article 3131. https://doi.org/ 10.3390/foods10123131
Iorizzo, M., Letizia, F., Ganassi, S., Testa, B., Petrarca, S., Albanese, G., Di Criscio, D., & De Cristofaro, A. (2022). Functional properties and antimicrobial activity from lactic acid bacteria as resources to improve the health and welfare of honey bees. Insects, 13(3), Article 308. https://doi.org/10.3390/insects13030308
Karyawati, A. T., Nuraida, L., Lestari, Y., & Meryandini, A. (2018). Characterization of abundance and diversity of lactic acid bacteria from Apis dorsata hives and flowers in East Nusa Tenggara, Indonesia. Biodiversitas, 19(3), 845-851. https://doi.org/10.13057/biodiv/d190319
Katoh, K., & Standley, D. M. (2013). MAFFT multiple sequence alignment software version 7: Improvements in performance and usability. Molecular Biology and Evolution, 30(4), 772-780. https://doi.org/10.1093/molbev/mst010
Kos, B., Šušković, J., Vuković, S., Šimpraga, M., Frece, J., & Matošić, S. (2003). Adhesion and aggregation ability of probiotic strain Lactobacillus acidophilus M92. Journal of Applied Microbiology, 94(6), 981-987. https://doi.org/10.1046/j.1365-2672.2003.01915.x
Krawczyk, B., Wityk, P., Gałęcka, M., & Michalik, M. (2021). The many faces of Enterococcus spp.-commensal, probiotic and opportunistic pathogen. Microorganisms, 9(9), Article 1900. https://doi.org/10.3390/microorganisms9091900
Leblanc, J. G., Laiño, J. E., del Valle, M. J., Vannini, V., van Sinderen, D., Taranto, M. P., de Valdez, G. F., de Giori, G. S., & Sesma, F. (2011). B-Group vitamin production by lactic acid bacteria - current knowledge and potential applications. Journal of Applied Microbiology, 111(6), 1297-1309. https://doi.org/10.1111/j.1365-2672.2011.05157.x
Leska, A., Nowak, A., & Motyl, I. (2022). Isolation and some basic characteristics of lactic acid bacteria from honeybee (Apis mellifera L.) environment—A preliminary study. Agriculture, 12(10), Article 1562. https://doi.org/10.3390/agriculture12101562
Liu, M., Chen, Q., Sun, Y., Zeng, L., Wu, H., Gu, Q., & Li, P. (2022). Probiotic potential of a folate-producing strain Latilactobacillus sakei LZ217 and its modulation effects on human gut microbiota. Foods, 11(2), Article 234. https://doi.org/10.3390/foods11020234
Mahara, F. A., Nuraida, L., & Lioe, H. N. (2021). Folate in milk fermented by lactic acid bacteria from different food sources. Preventive Nutrition and Food Science, 26(2), 230-240. https://doi.org/10.3746/pnf.2021.26.2.230
Mahara, F. A., Nuraida, L., Lioe, H. N., & Nurjanah, S. (2023). Hypothetical regulation of folate biosynthesis and strategies for folate overproduction in lactic acid bacteria. Preventive Nutrition and Food Science, 28(4), 386-400. https://doi.org/10.3746/pnf.2023.28.4.386
Monika, K., Malik, T., Gehlot, R., Rekha, K., Kumari, A., Sindhu, R., & Priyanka, R. (2021). Antimicrobial properties of probiotics. Environment Conservation Journal, 22, 33-48. https://doi.org/10.36953/ECJ.2021.SE.2204
Moreno, M. A., Holder, M. T., & Sukumaran, J. (2024). DendroPy 5 : a mature Python library for phylogenetic computing. Journal of Open Source Software, 9(101), Article 6943. https://doi.org/10.21105/joss.06943
Ruiz, L., Margolles, A., & Sánchez, B. (2013). Bile resistance mechanisms in Lactobacillus and Bifidobacterium. Frontiers in Microbiology, 4, Article 396. https://doi.org/10.3389/fmicb.2013.00396
Saini, R. K., Nile, S. H., & Keum, Y. S. (2016). Folates: Chemistry, analysis, occurrence, biofortification and bioavailability. Food Research International, 89(Pt 1), 1-13. https://doi.org/10.1016/j.foodres.2016.07.013
Schwarz, J., Schumacher, K., Brameyer, S., & Jung, K. (2022). Bacterial battle against acidity. FEMS Microbiology Reviews, 46(6), Article fuac037. https://doi.org/10.1093/femsre/fuac037
Sensoy, I. (2021). A review on the food digestion in the digestive tract and the used in vitro models. Current Research in Food Science, 4, 308-319. https://doi.org/10.1016/j.crfs.2021.04.004
Sharma, P., Tomar, S. K., Goswami, P., Sangwan, V., & Singh, R. (2014). Antibiotic resistance among commercially available probiotics. Food Research International, 57, 176-195. https://doi.org/10.1016/j.foodres.2014.01.025
Viaud, S., Saccheri, F., Mignot, G., Yamazaki, T., Daillère, R., Hannani, D., Enot, D. P., Pfirschke, C., Engblom, C., Pittet, M. J., Schlitzer, A., Ginhoux, F., Apetoh, L., Chachaty, E., Woerther, P.-L., Eberl, G., Bérard, M., Ecobichon, C., Clermont, D., Bizet, C., Gaboriau-Routhiau, V., . . . Zitvogel, L. (2013). The intestinal microbiota modulates the anticancer immune effects of cyclophosphamide. Science, 342(6161), 971-976. https://doi.org/10.1126/science.1240537
Wang, Y., Wu, J., Lv, M., Shao, Z., Hungwe, M., Wang, J., Bai, X., Xie, J., Wang, Y., & Geng, W. (2021). Metabolism characteristics of lactic acid bacteria and the expanding applications in food industry. Frontiers in Bioengineering and Biotechnology, 9, Article 612285. https://doi.org/10.3389/fbioe.2021.612285
Yusuf, D., Nuraida, L., Dewanti-Hariyadi, R., & Hunaefi, D. (2020). In vitro characterization of lactic acid bacteria from Indonesian kefir grains as probiotics with cholesterol-lowering effect. Journal of Microbiology and Biotechnology, 30(5), 726-732. https://doi.org/10.4014/jmb.1910.10028
Zheng, C., Wang, Z., Wang, Q., Wang, S., Lao, S., He, J., & Chen, Z. (2021). Efficient preparation of the chiral intermediate of luliconazole with Lactobacillus kefir alcohol dehydrogenase through rational rearrangement of the substrate binding pocket. Molecular Catalysis, 509, Article 111639. https://doi.org/10.1016/j.mcat.2021.111639