Isolation and In-Vitro Probiotic Characterization of Fructophilic Lactic Acid Bacteria from Different Plants and The Digestive System of Bees

Year 2024, , 1013 - 1030, 01.09.2024

Mehmet Bal , Harun Önlü , Özlem Osmanağaoğlu

https://doi.org/10.21597/jist.1441350

Abstract

The aim of this study was to investigate the characteristics of fructophilic lactic acid bacteria isolated from fructose-rich niches (bee digestive tract, yam fruit, hazelnut leaf, grape, tomato, white mulberry, pear, kiwi) for their use as probiotics. Among a large number of isolates from these sources, 10 catalase-negative and Gram-positive bacterial strains were initially selected. Cell morphology and genotypic characteristics (RAPD-PZR, 16S rRNA sequencing) of the selected strains were determined in the first stage. The fructophilic isolates included in the study were identified as Enterococcus faecalis. Phylogenetic analyses of the related isolates were performed and isolates isolated from the bee gastrointestinal tract and isolates isolated from different plant sources were grouped into separate clades. Isolates whose phenotypic and genotypic characteristics were defined were then tested for their potential probiotic properties. In this context, acid, pepsin and pancreatin resistance profiles of the isolates were determined. In addition, isolates coded A6, 7, 9 and 10 were proven to be potential bacteriocin producers. In this study, it was shown that fructophilic lactic acid bacteria can be isolated from different plant sources and may have probiotic potential.

Keywords

Fructophilic lactic acid bacteria, Probiotic, Microbial typing, 16S rRNA

Thanks

We would like to thank Prof. Dr. İrfan KANDEMİR for providing honeybees for the study

Farklı Bitkilerden ve Arıların Sindirim Sisteminden Fruktofilik Laktik Asit Bakterilerinin İzolasyonu ve İn-Vitro Probiyotik Karakterizasyonu

Abstract

Bu çalışmanın amacı, genellikle fruktoz bakımından zengin nişlerden (arı sindirim sistemi, yer elması meyvesi, fındık yaprağı, üzüm, domates, beyaz dut, armut, kivi) izole edilen fruktofilik laktik asit bakterilerinin probiyotik olarak kullanılabilmelerine yönelik niteliklerinin araştırılmasıdır. Söz konusu kaynaklardan izole edilen çok sayıda izolat içerisinden ilk etapta katalaz negatif ve Gram-pozitif reaksiyon veren 10 adet bakteriyel suş seçilmiştir. Seçilen suşların ilk aşamada hücre morfolojileri, genotipik özellikleri (RAPD-PZR, 16S rRNA dizileme) belirlenmiştir. Çalışma kapsamına dahil edilmiş fruktofilik özellikteki izolatlar Enterococcus faecalis olarak tanımlanmıştır. İlgili izolatların filogenetik analizleri gerçekleştirilmiş ve arı gastrointestinal sisteminden izole edilenler ile farklı bitkisel kaynaklardan izole edilen izolatlar birbirinden ayrı kladlar içerisinde toplanmıştır. Fenotipik ve genotipik karakteristikleri tanımlanan izolatlar daha sonrasında probiyotik özellikleri itibariyle test edilmiştir. Bu bağlamda izolatların, asit, pepsin ve pankreatin direnç profilleri çıkartılmıştır. Ayrıca A6, 7, 9 ve 10 kodlu izolatların potansiyel birer bakteriyosin üreticisi oldukları kanıtlanmıştır. İlgili bu çalışmada farklı bitkisel kaynaklardan da fruktofilik özellikli laktik asit bakterilerinin izole edilebileceği ve probiyotik potansiyel içerebilecekleri gösterilmiştir.

Keywords

Fruktofilik Laktik Asit Bakterileri, Probiyotik, Mikrobiyal tiplendirme, Antimikrobiyal, 16S rRNA

References

• Ahn, Y.T., Kim, G.B., Lim, K.S., Baek, Y.J., Kim, H.U. (2003). Deconjugation of bile salts by Lactobacillus acidophilus isolates, International Dairy Journal, 13 (4), 303-311.
• Al Atya AK, Drider-Hadiouche K, Ravallec R, Silvain A, Vachee A and Drider D. (2015). Probiotic potential of Enterococcus faecalis strains isolated from meconium. Front. Microbiol. 6:227. doi: 10.3389/fmicb.2015.00227
• Ali, A.H., Bamigbade, G., Tarique, M., Esposito, G., Obaid, R., Abu-Jdayil, B., Ayyash, M. (2023). Physicochemical, rheological, and bioactive properties of exopolysaccharide produced by a potential probiotic Enterococcus faecalis 84B, International Journal of Biological Macromolecules, 240, 124425.
• Al-S'adoon, R. N. H., & Al-Rawi, A. M. (2023). A Novel in Vitro Evidence on Anticancer Effect of Local Isolate Enterococcus faecalis Bacteriocin. Journal of Global Scientific Research, 8(2), 2976-2986.
• Altschul, S.F., Madden, T.L., Schäffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs, Nucleic Acids Research, 25 (17), 3389-3402.
• Andrade-Velásquez A, Hernández Sánchez H, Dorantes-Álvarez L, Palmeros-Sánchez B, Torres-Moreno R, Hernández-Rodríguez D and Melgar-Lalanne G (2023) Honey characterization and identification of fructophilic lactic acid bacteria of fresh samples from Melipona beecheii, Scaptotrigona pectoralis, Plebeia llorentei, and Plebeia jatiformis hives. Front. Sustain. Food Syst. 7:1113920. doi: 10.3389/fsufs.2023.1113920
• Authority, E.F.S., (2010), Scientific Opinion on the maintenance of the list of QPS biological agents intentionally added to food and feed (2010 update): FSA Panel on Biological Hazards (BIOHAZ). http://www.efsa.europa. eu/fr/scdocs/doc/1944.pdf (Erişim Tarihi:06.02.2024)
• Bhunia, A., Johnson, M., Ray, B. (1988). Purification, characterization and antimicrobial spectrum of a bacteriocin produced by Pediococcus acidilactici, Journal of Applied Bacteriology, 65 (4), 261-268.
• Bujnakova, D., Strakova, E. (2017). Safety, probiotic and technological properties of Lactobacilli isolated from unpasteurised ovine and caprine cheeses, Annals of Microbiology, 67, 813-826.
• Casaus, P., Nilsen, T., Cintas, L.M., Nes, I.F., Hernández, P.E., Holo, H. (1997). Enterocin B, a new bacteriocin from Enterococcus faecium T136 which can act synergistically with enterocin A, Microbiology, 143 (7), 2287-2294.
• Cebrián, R., Baños A., Valdivia, E., Pérez-Pulido, R., Martínez-Bueno, M., Maqueda, M. (2012) "Characterization of functional, safety, and probiotic properties of Enterococcus faecalis UGRA10, a new AS-48-producer strain." Food microbiology 30.1: 59-67.
• CLSI (2015) Performance standards for antimicrobial susceptibility testing: twentysecond informational supplement. In: CLSI document M100S22. Clinical Laboratory Standard Institute, Wayne.
• Colaninno, P.M., Goldman, E., Green, L. (2021). Identification of Gram-Positive‎ Organisms, Practical Handbook of‎ Microbiology, 51-58.
• Çetin, B., & Aktaş, H. (2024). Monitoring probiotic properties and safety evaluation of antilisterial Enterococcus faecium strains with cholesterol-lowering potential from raw Cow's milk. Food Bioscience, 61, 104532.
• Daca, A., & Jarzembowski, T. (2024). From the Friend to the Foe—Enterococcus faecalis Diverse Impact on the Human Immune System. International journal of molecular sciences, 25(4), 2422.
• Dikbaş, N., Orman, Y. C., Alım, Ş., Uçar, S., & Tülek, A. (2024). Evaluating Enterococcus faecium 9 N-2 as a probiotic candidate from traditional village white cheese. Food Science & Nutrition, 12, 1847–1856. https://doi.org/10.1002/fsn3.3878
• Dunne, C., O'Mahony, L., Murphy, L., Thornton, G., Morrissey, D., O'Halloran, S., Feeney, M., Flynn, S., Fitzgerald, G., Daly, C. (2001). In vitro selection criteria for probiotic bacteria of human origin: correlation with in vivo findings, The American Journal of Clinical Nutrition, 73 (2), 386s-392s.
• Endo, A., and Sanae, O. (2008). "Reclassification of the genus Leuconostoc and proposals of Fructobacillus fructosus gen. nov., comb. nov., Fructobacillus durionis comb. nov., Fructobacillus ficulneus comb. nov. and Fructobacillus pseudoficulneus comb. nov." International journal of systematic and evolutionary microbiology 58.9: 2195-2205.
• Endo, A., Futagawa-Endo, Y., Dicks, L.M. (2009). Isolation and characterization of fructophilic lactic acid bacteria from fructose-rich niches, Systematic and Applied Microbiology, 32 (8), 593-600.
• Endo, A., Irisawa, T., Futagawa-Endo, Y., Sonomoto, K., Itoh, K., Takano, K., Okada, S., Dicks, L.M. (2011).
• Fructobacillus tropaeoli sp. nov., a fructophilic lactic acid bacterium isolated from a flower, International Journal of Systematic and Evolutionary Microbiology, 61 (4), 898-902.
• Endo, A., Salminen, S. (2013). Honeybees and beehives are rich sources for fructophilic lactic acid bacteria, Systematic and Applied Microbiology, 36 (6), 444-448.
• Endo, A., Tanaka, N., Oikawa, Y., Okada, S., Dicks, L. (2014). Fructophilic characteristics of Fructobacillus spp. may be due to the absence of an alcohol/acetaldehyde dehydrogenase gene (adhE), Current Microbiology, 68, 531-535.
• Endo, A., Tanizawa, Y., Tanaka, N., Maeno, S., Kumar, H., Shiwa, Y., Okada, S., Yoshikawa, H., Dicks, L., Nakagawa, J. (2015). Comparative genomics of Fructobacillus spp. and Leuconostoc spp. reveals niche-specific evolution of Fructobacillus spp, BMC Genomics, 16, 1-13.
• Filannino, P., Di Cagno, R., Tlais, A.Z.A., Cantatore, V., Gobbetti, M. (2019). Fructose-rich niches traced the evolution of lactic acid bacteria toward fructophilic species, Critical Reviews in Microbiology, 45 (1), 65-81.
• Fisher, K., Phillips, C. (2009). The ecology, epidemiology and virulence of Enterococcus, Microbiology, 155 (6), 1749-1757.
• Franz, C.M., Stiles, M.E., Schleifer, K.H., Holzapfel, W.H. (2003). Enterococci in foods—a conundrum for food safety, International Journal of Food Microbiology, 88 (2-3), 105-122.
• Fuller, R., Gibson, G.R. (1998). Probiotics and prebiotics: microflora management for improved gut health, Clinical Microbiology and Infection, 4 (9), 477-480.
• Gilliland, S., Staley, T., Bush, L. (1984). Importance of bile tolerance of Lactobacillus acidophilus used as a dietary adjunct, Journal of Dairy Science, 67 (12), 3045-3051.
• Harrigan, W.F., (1998), Laboratory methods in food microbiology, Gulf professional publishing, He, H., Chen, Y., Zhang, Y., Wei, C. (2011). Bacteria associated with gut lumen of Camponotus japonicus Mayr, Environmental Entomology, 40 (6), 1405-1409.
• Hotel, A.C.P., Cordoba, A. (2001). Health and nutritional properties of probiotics in food including powder milk with live lactic acid bacteria, Prevention, 5 (1), 1-10.
• Huang, Y., Adams, M.C. (2004). In vitro assessment of the upper gastrointestinal tolerance of potential probiotic dairy propionibacteria, International Journal of Food Microbiology, 91 (3), 253-260.
• Im, E. J., Lee, H. H. Y., Kim, M., & Kim, M. K. (2023). Evaluation of enterococcal probiotic usage and review of potential health benefits, safety, and risk of antibiotic-resistant strain emergence. Antibiotics, 12(8), 1327.
• Iraporda C., Irene A. Rubel, Guillermo D. Manrique et al. Lactic acid bacteria strains isolated from Jerusalem artichoke (Helianthus tuberosus L.) tubers as potential probiotic candidates, 26 February 2024, PREPRINT (Version 1) available at Research Square [https://doi.org/10.21203/rs.3.rs-3976150/v1
• Janashia, I., Carminati, D., Rossetti, L., Zago, M., Fornasari, M.E., Haertlé, T., Chanishvili, N., Giraffa, G. (2016). Characterization of fructophilic lactic microbiota of Apis mellifera from the Caucasus Mountains, Annals of Microbiology, 66 (4), 1387-1395.
• Joint, F. (2002). Guidelines for the evaluation of probiotics in food, London, Ontario, Canada, April 30 and May 1, 2002, http://www. who.int/foodsafety/publications/fs_management/probiotics2/en/index. html.
• Johnston LM, Jaykus LA. Antimicrobial resistance of Enterococcus species isolated from produce. Appl Environ Microbiol. 2004 May;70(5):3133-7. doi: 10.1128/AEM.70.5.3133-3137.2004. PMID: 15128577; PMCID: PMC404399.
• Kiessling, G., Schneider, J., Jahreis, G. (2002). Long-term consumption of fermented dairy products over 6 months increases HDL cholesterol, European Journal of Clinical Nutrition, 56 (9), 843-849.
• Kim, E.B., Tyler, C.A., Kopit, L.M., Marco, M.L. (2013). Draft genome sequence of fructophilic Lactobacillus florum, Genome announcements, 1 (1), 10.1128/genomea. 00025-12.
• Kiymaci, M., Simsek, D., Tok, K., Dirican, D., & Gumustas, M. (2023). Probiotic potential and anti-quorum sensing activity of Enterococcus faecalis and Lactobacillus kunkeei isolates from Apis mellifera. Journal of the Hellenic Veterinary Medical Society, 73(4), 4717–4728. https://doi.org/10.12681/jhvms.26743
• Knoll, C., Divol, B., Du Toit, M. (2008). Genetic screening of lactic acid bacteria of oenological origin for bacteriocin-encoding genes, Food microbiology, 25 (8), 983-991.
• Koch, H., Schmid-Hempel, P. (2011). Bacterial communities in central European bumblebees: low diversity and high specificity, Microbial Ecology, 62, 121-133.
• Kouya T, Ishiyama Y, Ohashi S, Kumakubo R, Yamazaki T, Otaki T. (2023). Philodulcilactobacillus myokoensis gen. nov., sp. nov., a fructophilic, acidophilic, and agar-phobic lactic acid bacterium isolated from fermented vegetable extracts. PLoS ONE 18(6): e0286677. https://doi.org/10.1371/journal.pone.0286677
• Kwong, W.K., Moran, N.A. (2016). Gut microbial communities of social bees, Nature Reviews Microbiology, 14 (6), 374-384.
• Leavis, H., Top, J., Shankar, N., Borgen, K., Bonten, M., van Embden, J., Willems, R.J. (2004). A novel putative enterococcal pathogenicity island linked to the esp virulence gene of Enterococcus faecium and associated with epidemicity, Journal of Bacteriology, 186 (3), 672-682.
• Luenglusontigit, P., Sathapondecha, P., Saengsuwan, P., Surachat, K., Boonserm, P., & Singkhamanan, K. (2023). Effects of postbiotic from bacteriocin-like inhibitory substance producing Enterococcus faecalis on toxigenic Clostridioides difficile. Journal of Health Science and Medical Research, 41(4), 2023918.
• Luo, Y., Ma, B.-C., Zou, L.-K., Cheng, J.-G., Cai, Y.-H., Kang, J.-P., Li, B., Gao, X.-H., Wang, P., Xiao, J.-J. (2012). Identification and characterization of lactic acid bacteria from forest musk deer feces, African Journal of Microbiol Research, 6 (29), 5871-5881.
• Lou, H., Wang, J., Wang, Y., Gao, Y., & Wang, W. (2024). Comprehensive assessment of Enterococcus faecalis SN21-3: Probiotic features and safety evaluation for potential animal use. Food Bioscience, 103688.
• Macwana, S.J., Muriana, P.M. (2012). A ‘bacteriocin PCR array’for identification of bacteriocin-related structural genes in lactic acid bacteria, Journal of Microbiological Methods, 88 (2), 197-204.
• Maeno, S., Tanizawa, Y., Kanesaki, Y., Kubota, E., Kumar, H., Dicks, L., Salminen, S., Nakagawa, J., Arita, M., Endo, A. (2016). Genomic characterization of a fructophilic bee symbiont Lactobacillus kunkeei reveals its niche-specific adaptation, Systematic and Applied Microbiology, 39 (8), 516-526.
• Mangia, N.P., Saliba, L., Deiana, P. (2019). Functional and safety characterization of autochthonous Lactobacillus paracasei FS103 isolated from sheep cheese and its survival in sheep and cow fermented milks during cold storage, Annals of Microbiology, 69 (2), 161-170.
• Maragkoudakis, P.A., Zoumpopoulou, G., Miaris, C., Kalantzopoulos, G., Pot, B., Tsakalidou, E. (2006). Probiotic potential of Lactobacillus strains isolated from dairy products, International Dairy Journal, 16 (3), 189-199.
• Mathara, J.M., Schillinger, U., Kutima, P.M., Mbugua, S.K., Guigas, C., Franz, C., Holzapfel, W.H. (2008). Functional properties of Lactobacillus plantarum strains isolated from Maasai traditional fermented milk products in Kenya, Current Microbiology, 56, 315-321.
• Nami, Y., Haghshenas, B., Bakhshayesh, R.V., Jalaly, H.M., Lotfi, H., Eslami, S., Hejazi, M.A. (2018). Novel autochthonous lactobacilli with probiotic aptitudes as a main starter culture for probiotic fermented milk, Lwt, 98, 85-93.
• Nami, Y., Vaseghi Bakhshayesh, R., Mohammadzadeh Jalaly, H., Lotfi, H., Eslami, S., Hejazi, M.A. (2019). Probiotic properties of Enterococcus isolated from artisanal dairy products, Frontiers in Microbiology, 10, 300.
• Neveling, D.P., Endo, A., Dicks, L.M. (2012). Fructophilic Lactobacillus kunkeei and Lactobacillus brevis isolated from fresh flowers, bees and bee-hives, Current Microbiology, 65, 507-515.
• Nueno-Palop C, and Narbad A. (2011). Probiotic assessment of Enterococcus faecalis CP58 isolated from human gut. Int J Food Microbiol. 28;145(2-3):390-4. doi: 10.1016/j.ijfoodmicro.2010.12.029. Epub 2011 Jan 8. PMID: 21315470.
• ONUR, M., ONLU, H. (2021). Farklı Gıda Ürünlerinden İzole Edilen Laktik Asit Bakterilerinin Bazı Probiyotik Özelliklerinin Belirlenmesi, Avrupa Bilim ve Teknoloji Dergisi, (32), 562-572.
• Onur, M., Önlü, H. (2023). Isolation, characterization of Weissella confusa and Lactococcus lactis from different milk sources and determination of probiotic features, Brazilian Journal of Microbiology, 1-17.
• Özkan, E. R., Demirci, T., & Akın, N. (2021). In vitro assessment of probiotic and virulence potential of Enterococcus faecium strains derived from artisanal goatskin casing Tulum cheeses produced in central Taurus Mountains of Turkey. Lwt, 141, 110908.
• Öztürk, H., Geniş, B., Özden Tuncer, B., & Tuncer, Y. (2023). Bacteriocin production and technological properties of Enterococcus mundtii and Enterococcus faecium strains isolated from sheep and goat colostrum. Veterinary Research Communications, 47(3), 1321-1345.
• Paramithiotis, S., Gioulatos, S., Tsakalidou, E., Kalantzopoulos, G. (2006). Interactions between Saccharomyces cerevisiae and lactic acid bacteria in sourdough, Process Biochemistry, 41 (12), 2429-2433.
• Resta-Lenert, S., Barrett, K. (2003). Live probiotics protect intestinal epithelial cells from the effects of infection with enteroinvasive Escherichia coli (EIEC), Gut, 52 (7), 988.
• Salek, F., Mirzaei, H., Khandaghi, J., Javadi, A., and Nami, Y. (2023). Apoptosis induction in cancer cell lines and anti-inflammatory and anti-pathogenic properties of proteinaceous metabolites secreted from potential probiotic Enterococcus faecalis KUMS-T48. Scientific Reports, 13(1), 7813.
• Smits, H.H., Engering, A., van der Kleij, D., de Jong, E.C., Schipper, K., van Capel, T.M., Zaat, B.A., Yazdanbakhsh, M., Wierenga, E.A., van Kooyk, Y. (2005). Selective probiotic bacteria induce IL-10–producing regulatory T cells in vitro by modulating dendritic cell function through dendritic cell–specific intercellular adhesion molecule 3–grabbing nonintegrin, Journal of Allergy and Clinical Immunology, 115 (6), 1260-1267.
• Sonei, Azin, Mohammad Reza Edalatian Dovom, and Masoud Yavarmanesh. (2024). "Evaluation of probiotic, safety, and technological properties of bacteriocinogenic Enterococcus faecium and Enterococcus faecalis strains isolated from lighvan and koozeh cheeses." International Dairy Journal 148: 105807.
• Tang, Y.-W., Ellis, N.M., Hopkins, M.K., Smith, D.H., Dodge, D.E., Persing, D.H. (1998). Comparison of phenotypic and genotypic techniques for identification of unusual aerobic pathogenic gram-negative bacilli, Journal of Clinical Microbiology, 36 (12), 3674-3679.
• Thaochan, N., Drew, R., Hughes, J., Vijaysegaran, S., Chinajariyawong, A. (2010). Alimentary tract bacteria isolated and identified with API-20E and molecular cloning techniques from Australian tropical fruit flies, Bactrocera cacuminata and B. tryoni, Journal of Insect Science, 10 (1), 131.
• Tran, T.T.; Munita, J.M.; Arias, C.A. (2015). Mechanisms of Drug Resistance: Daptomycin Resistance: Daptomycin Resistance. Ann. N. Y. Acad. Sci., 1354, 32–53.
• Todorov, S., Onno, B., Sorokine, O., Chobert, J., Ivanova, I., Dousset, X. (1999). Detection and characterization of a novel antibacterial substance produced by Lactobacillus plantarum ST 31 isolated from sourdough, International Journal of Food Microbiology, 48 (3), 167-177.
• Vásquez, A., Olofsson, T.C., Sammataro, D. (2009). A scientific note on the lactic acid bacterial flora in honeybees in the USA–A comparison with bees from Sweden, Apidologie, 40 (1), 26-28.
• Vinderola, C.G., Reinheimer, J.A. (2003). Lactic acid starter and probiotic bacteria: a comparative “in vitro” study of probiotic characteristics and biological barrier resistance, Food Research International, 36 (9-10), 895-904.
• Wilkins, T., Holdeman, L.V., Abramson, I., Moore, W. (1972). Standardized single-disc method for antibiotic susceptibility testing of anaerobic bacteria, Antimicrobial Agents and Chemotherapy, 1 (6), 451-459.
• Willems, R.J., Homan, W., Top, J., van Santen-Verheuvel, M., Tribe, D., Manzioros, X., Gaillard, C., Vandenbroucke-Grauls, C.M., Mascini, E.M., van Kregten, E. (2001). Variant esp gene as a marker of a distinct genetic lineage of vancomycinresistant Enterococcus faecium spreading in hospitals, The Lancet, 357 (9259), 853-855
• Zommiti, M., Cambronel, M., Maillot, O., Barreau, M., Sebei, K., Feuilloley, M., ... & Connil, N. (2018). Evaluation of probiotic properties and safety of Enterococcus faecium isolated from artisanal Tunisian meat “Dried Ossban”. Frontiers in Microbiology, 9, 1685.