Yığılca (Türkiye) bal arılarının bal midelerinden bakteri izolasyonu, tanımlanması ve probiyotik özelliklerinin karakterizasyonu

Yıl 2017, Cilt: 41 Sayı: 3, 253 - 261, 02.08.2017

Serpil Uğraş

https://doi.org/10.16970/ted.74860

Cited By: 8

Öz

Balarıları, dünyadaki hemen hemen bütün canlıların idamesi için hayati değer taşıyan anahtar türler olarak nitelendirilmektedir. Ancak, tüm dünyada bal arısı stoklarının büyük oranda kitlesel ölümlerine yol açan koloni çöküşü, uluslararası endişeye sebep olmaktadır. Bu kayıpları önlemek için yeni yaklaşımların ortaya çıkarılması gerekmektedir. Yapılan çalışmalarda, arıların vücutlarında bulunan probiyotik özellik taşıyan bakterilerin, arıların patojenlere karşı direnç sağlamasında aktif bir rol üstlenecekleri düşünülmektedir. Bu bağlamda, bu çalışmada sağlıklı arıların bal midesinden probiyotik özellikli laktik asit bakterilerinin izole edilmesi, bu bakterilerin arılarda hastalık etmeni patojenlere karşı etkisinin incelenmesi ve bu bakterilerin arıların bağışıklık sistemini güçlendirmek maksadıyla kullanılması amaçlanmıştır. Bu amaç doğrultusunda, 2015-2016 yılları arasında, DAGEM (Düzce Üniversitesi, Arıcılık Araştırma, Geliştirme ve Uygulama Merkezi, Yığılca, DÜZCE)’ den temin edilen arıların bal midesinden probiyotik özellikli bakteri taraması yapılmıştır. Elde edilen bakterilerin arı patojeni Melissococcus plutonius (Trüper and de' Clari, 1998) (Enterococcaceae)’ a karşı inhibisyon aktivitesi in vitro agar kuyu difüzyon metodu ile belirlenmiştir. Ardından istenen özelliklere sahip bakteri biyokimyasal, fizyolojik ve 16s rDNA analizi ile moleküler olarak Lactobacillus kunkeei (Edwards, 1998) (Lactobacillaceae) olarak tanımlanmış ve probiyotik doğası incelenmiştir. Sonuçlar değerlendirildiğinde, elde edilen izolatın gelecekte preparatlarının hazırlanarak, arıların bağışıklık sistemlerini destekleyeceği ve sonuçta, antibiyotikler ile kimyasal tedavi yöntemlerine başvurulmadan dirençli arıların üretileceği düşünülmektedir.

Anahtar Kelimeler

Bakteri, balarısı, izolasyon, Lactobacillus kunkeei, probiyotikler

Isolation, identification and characterization of probiotic properties of bacterium from the honey stomachs of Yigilca honeybees in Turkey

Öz

Honeybees are considered as a key species in nature for their vital role in the maintenance of almost all life on earth. However, the massive death of honeybee stocks worldwide, largely due to colony collapse disorder, is causing international concern. In order to avoid these losses, new approaches must be sought. In previous studies, the probiotic properties of the bacteria found in the bodies of honeybees are thought to have an active role in providing resistance against pathogens. Consequently, in this study, it is aimed to isolate probiotic lactic acid bacteria from honey stomachs of the healthy honeybee, to examine the effect of these bacteria against pathogenic bacteria and to use these bacteria to boost the immune system of bees. For this purpose, between 2015 and 2016, probiotic bacteria were screened from honey bees that provided by DAGEM (Düzce University, Beekeeping Research, Development and Application Center, Yığılca, DÜZCE). The inhibitory activity of the obtained bacteria against the bee pathogen Melissococcus plutonius (Trüper and de 'Clari, 1998) (Enterococcaceae) was determined by in vitro agar well diffusion. The bacterium with the desired characteristics were identified by biochemical, physiological and 16s rDNA analysis as Lactobacillus kunkeei (Edwards, 1998) (Lactobacillaceae) and its probiotic nature was investigated. With the evaluation of these findings, future preparations of the isolate are expected to support the bee immune system and, as a result, to produce resistant honeybees without resorting to treatment with antibiotics.

Anahtar Kelimeler

Bacterium, honeybee, isolation, Lactobacillus kunkeei, probiotics

Kaynakça

• Asenjo, F., A. Olmos, P. Henríquez-Piskulich, V. Polanco, P. Aldea, J. A. Ugalde & A. N. Trombert, 2016. Genome sequencing and analysis of the first complete genome of Lactobacillus kunkeei strain MP2, an Apis mellifera gut isolate. PeerJ, 4: e1950, DOI:10.7717/peerj.1950.
• Barganska, Z., M. Slebioda & J. Namiesnik, 2011. Determination of antibiotic residues in honey. Trends in Analytical Chemistry, 30 (7): 1035–1041.
• Corby-Harris, V., P. Maes & K. E. Anderson, 2014. The bacterial communities associated with honey bee (Apis mellifera) foragers. PLoS ONE, 9 (4): e95056, DOI:10.1371/journal.pone.0095056.
• Cornman, R. S., D. R. Tarpy, Y. Chen, L. Jeffreys, D. Lopez, J. S. Pettis, D. Van Engelsdorp & J. D. Evans, 2012. Pathogen webs in collapsing honey bee colonies. PLoS ONE 7(8): e43562, DOI:10.1371/journal.pone.0043562.
• Cox-Foster, D. L., S. Conlan, Holmes, E. C. Palacios, G. Evans, J. D. Moran, N. A. Quan, P. L. Briese, T. Hornig, M. Geiser, D. M. V. Martinson, D. Van Engelsdorp, A. L. Kalkstein, A. Drysdale, J. Hui, J. Zhai, L. Cui, S. K. Hutchison, J. F. Simons, M. Egholm, J. S. Pettis & W. I. Lipkin, 2007. A metagenomic survey of microbes in honey bee colony collapse disorder. Science, 318: 283-287.
• Crane, E. & P. Walker, 1984. Pollination Directory for World Crops, International Bee Research Association, London,183 pp.
• Doğaroğlu, M. & T. Samancı, 2006. Balda Yörelere Göre Kalıntı Hile ve Orijin Tespit Projesi. Teknoloji ve Yenilik Destek Programları Başkanlığı (TEYDEB) Arıcılık Raporu, Ankara, Türkiye.
• Endo, A., 2012. Fructophilic lactic acid bacteria inhabit fructose-rich niches in nature. Microbial Ecology in Health and Disease, 23: 10.3402/mehd.v23i0.18567, DOI:10.3402/mehd.v23i0.18563.
• Evans, J. & D. Lopez, 2004. Bacterial probiotics induce an immune response in the honey bee (Hymenoptera: Apidae). Journal of Economic Entomology, 97: 752-756.
• Forsgren, E., T. C. Olofsson, A. Vasquez & I. Fries, 2010. Novel lactic acid bacteria inhibiting Paenibacillus larvae in honey bee larvae. Apidologie, 41: 99–108.
• Free, J. B., 1993. Insect Pollination of Crops, 2. Edition, Academic press, London, 684 pp.
• Fujisawa, T. & T. Mitsuoka, 1996 Homofermentative Lactobacillus species predominantly isolated from canine feces. The Journal of Veterinary Medical Science, 58 (6): 591-593.
• Gilliam, M., 1997. Identification and roles of non-pathogenic microflora associated with honey bees. FEMS Microbiology Letters, 155: 1-10.
• Hawaz, E., 2014. Isolation and identification of probiotic lactic acid bacteria from curd and in vitro evaluation of its growth inhibition activities against pathogenic bacteria. African Journal of Microbiology Research, 8 (13): 1419-1425.
• Haynes, E, T. Helgason, J. P. W. Young, R. Thwaites & G. E. Budge, 2013. A typing scheme for the honeybee pathogen Melissococcus plutonius allows detection of disease transmission events and a study of the distribution of variants. Environmental Microbiology Reports, 5: 525-529.
• Krieg N. R. & J. G. Holt, 1984 Bergy's Manual of Systematic Bacteriology. Volume 1, Baltimore, The Williams and Wilkins Co.,964 pp.
• Maragkoudakis, P. A., G. Zoumpopoulou, C. Miaris, G. Kalantzopoulos, B. Pot & E. Tsakalidou, 2006. Probiotic Potential of Lactobacillus Strains Isolated from Dairy Products. International Dairy Journal, 16: 189-199.
• McFrederick, Q. S., W. T. Wcislo, M. C. Hout & U. G. Mueller, 2014. Host species and developmental stage, but not host social structure, affects bacterial community structure in socially polymorphic bees. FEMS Microbiology Ecology, 88: 398-406.
• Millette, M., F. M. Luquet, M. T. Ruiz & M. Lacroix, 2008. Characterization of probiotic properties of Lactobacillus strains. Dairy Science and Technology, 88 (6): 695-705.
• Mutinelli, F., 2003. Practical application of antibacterial drugs for the control of honey bee diseases. Apiacta, 38: 149-155.
• Olofsson, T. & A. Vasquez, 2008 Detection and identification of a novel lactic acid bacterial flora within the honey stomach of the honeybee Apis mellifera. Current Microbiology, 57: 356-363.
• Olofsson, T. C., M. Alsterfjord, B. Nilson, E. Butler & A. Vásquez, 2014. Lactobacillus apinorum sp. nov., Lactobacillus mellifer sp. nov., Lactobacillus mellis sp. nov., Lactobacillus melliventris sp. nov., Lactobacillus kimbladii sp. nov., Lactobacillus helsingborgensis sp. nov. and Lactobacillus kullabergensis sp. nov., isolated from the honey stomach of the honeybee Apis mellifera. International Journal of Systematic and Evolutionary Microbiology, 64: 3109–311.
• Özbek, H., 2002. Bees and Nature. Apicultural Research, 22-25.
• Padilla, C., P. Brevis, O. Lobos & E. Hubert, 1996. Bacteriocin activity of Pseudomonas sp. on enteropathogenic bacteria in an artificial aquatic system. Letters in Applied Microbiology, 23: 371–374.
• Rangberg, A., G. Mathiesen, G. V. Amdam & D. B. Diep, 2015. The paratransgenic potential of Lactobacillus kunkeei in the honey bee Apis mellifera. Beneficial Microbes, 6 (4): 513-523.
• Rezvani, M., Mendoza, M., Koci, M. D., Daron, C., J. Levy & H. M. Hassan, 2016. Draft genome sequence of Lactobacillus crispatus C25 isolated from chicken cecum. Genome Announcements, 4 (6): e01223-16.
• Salminen, S., C. Boyley, M. C. Boutron-Ruault & J. H. Cummings, 1998. Functional food science and gastrointestinal physiology and function. British Journal of Nutrition, 80: 147–171.
• Sambrook, J., E. F. Fritsch & T. Maniatis, 1989. Molecular Cloning: A Laboratory Manual. (Cold Spring Harbor Laboratory Press, 1659 pp.
• Schrezenmeir, J. & M. de Vrese, 2001. Probiotics, prebiotics, and synbiotics-approaching a definition. The American Journal of Clinical Nutrition, 73: 361-364.
• Tajabadi, N., M. Makhdzir, S. Nazamid, M. Shuhaimi, B. Rasoul & M. M. Y. Abdul, 2013. Identification of Lactobacillus plantarum, Lactobacillus pentosus and Lactobacillus fermentum from honey stomach of honeybee. Brazilian Journal of Microbiology, 44 (3): 717-722.
• Tajabadi, N., M. Mardan, M. Y. Abdul Manap, M. Shuhaimi, A. Meimandipour & L. Nateghi, 2011. Detection and identification of Lactobacillus bacteria found in the honey stomach of the giant honeybee Apis dorsata. Apidologie, 42: 642-649.
• Tamura, K., M. Nei & S. Kumar, 2004. Prospects for inferring very large phylogenies by using the neighbor-joining method. Proceedings of the National Academy of Sciences, 101 (30): 11030-11035.
• Tannock, G., 2004. A special fondness for lactobacilli. Applied and Environmental Microbiology, 70: 3189-3194.
• Turhan Eryılmaz, F., 2011. Vajinal Sekresyondan İzole Edilen Laktik Asit Bakterilerine Ait Bazı Suşların Potansiyel Probiyotik Özelliklerin Belirlenmesi. Ankara Üniversitesi, Doktora Tezi, Ankara, 95 s.
• Wilkins, T. D., L. V. Holdeman, I. J. Abramson & W. E. C. Moore, 1972. Standardized single-disc method for antibiotic susceptibility testing of anaerobic bacteria. Antimicrobial Agents and Chemotherapy, 1 (6): 451-459.
• Vásquez, A., E. Forsgren, I. Fries, R. J. Paxton, E. Flaberg, L. Szekely & T. C. Olofsson, 2012. Symbionts as major modulators of insect health: Lactic acid bacteria and honeybees. PLoS ONE 7 (3): e33188, DOI:10.1371/journal.pone.0033188.
• Vásquez, A., T. Olofsson, D. Sammataro & K. Hartfelder, 2009. A scientific note on the lactic acid bacterial flora in honeybees in the USA-A comparison with bees from Sweden. Apidologie, 40: 417-417.