FARKLI BİTKİ KAYNAKLI (YONCA, ÇAYIR OTU, MISIR SİLAJI) PREFERMENTE SIVI (PFJ) ÖRNEKLERİNDE AKTİF BAKTERİYEL POPÜLASYONUN RNA TEMELLİ 16S RRNA REAL-TİME PCR ANALİZİ İLE DEĞERLENDİRİLMESİ
Keywords:
Pre-fermente sıvı, RNA, 16S rRNA, aktif mikrobiyota, silaj fermantasyonu, RT-qPCRAbstract
Pre-fermente sıvılar (PFJ), silaj fermantasyonunu optimize etmek amacıyla yaygın biçimde kullanılan doğal biyolojik inokulantlardır. Bu çalışmada, yonca, çayır otu ve mısır silajından üretilen PFJ örneklerindeki aktif bakteriyel popülasyonlar, hücresel metabolik aktiviteyi doğrudan yansıtan RNA temelli 16S rRNA Real-Time PCR (RT-qPCR) yöntemiyle değerlendirilmiştir. Çalışma kapsamında hazırlanan bitki ekstrelerine fermantasyonun uyarılması için melas ve glikoz ilave edilerek, karışımlar 30 °C'de 5 gün boyunca anaerobik inkübasyona bırakılmıştır. TRIzol reaktifi ve bead-beating tekniğiyle gerçekleştirilen RNA izolasyonunu takiben cDNA sentezlenmiş ve SYBR Green tabanlı RT-qPCR analizleri yürütülmüştür. Elde edilen moleküler bulgular, tüm PFJ varyantlarında sigmoid karakterli amplifikasyon eğrileri ve hedef genin özgüllüğünü doğrulayan tekil erime (melting curve) pikleri oluştuğunu göstermiştir. Substrat kompozisyonlarına göre değişkenlik sergileyen eşik döngüsü (Ct) verileri incelendiğinde; özellikle zengin fermente edilebilir karbonhidrat profiline sahip mısır silajından üretilen PFJ örneklerinde laktik asit bakterileri kaynaklı en güçlü mikrobiyal aktivite tespit edilmiştir. Yoncadan elde edilen sıvılarda gözlemlenen nispeten yüksek Ct değerleri ise ilgili materyalin doğal hücresel tamponlama kapasitesiyle ilişkilendirilmiştir. Sonuç olarak, inaktif veya ölü hücrelerden kaynaklanabilecek analitik yanılgıları bertaraf eden RNA hedefli bu yaklaşımın, fermentatif sistemlerdeki fonksiyonel mikrobiyatanın karakterizasyonunda son derece özgül, duyarlı ve güvenilir bir yöntem olduğu kanıtlanmıştır.
References
Abdulamir, A. S., Yoke, T. S., Nordin, N., & Bakar, F. A. (2010). Detection and quantification of probiotic bacteria using optimized DNA extraction, traditional and real-time PCR methods in complex microbial communities. African Journal of Biotechnology, 9(10), 1481-1492.
Anwar, S., Lamaudiere, M., Hassall, J., Dehinsilu, J., Bhuller, R. K., Hold, G. L., Vázquez- Campos, X., Mahnert, A., Moissl-Eichinger, C., Gallé, B., Kainz, G., Pjevac, P., Hausmann, B., Schwarz, J., Kohl, G., Berry, D., Vancuren, S. J., Allen-Vercoe, E., Nielsen, N., … Sergaki, C. (2025). DNA reference reagents isolate biases in microbiome profiling: A global multi-lab study. mSystems, 10(11), e00466-25.
Aydın, S. S. (2023). The effect of the addition of fermented natural lactic acid bacterial liquid and some lactic acid bacterial inoculants on alfalfa silage quality, in vitro digestibility and gas production. Journal of Agricultural Sciences, 29(3), 811-820.
Aydin, S. S., & Denek, N. (2022). Amount of lactic acid bacteria in fermented natural lactic acid bacteria liquids prepared with varying sucrose inclusion at different incubation periods. Medycyna Weterynaryjna, 78(9), 456-460.
Chen, H., Kang, L., Li, K., Feng, T., Ni, Z., Gao, E., & Fang, Z. (2023). Accurate quantification on the change of some live pathogenic microbial flora in fermented feed. International Journal of Food Science and Technology, 58(7), 3552-3559.
Cocolin, L., Alessandria, V., Dolci, P., Gorra, R., & Rantsiou, K. (2013). Culture independent methods to assess the diversity and dynamics of microbiota during food fermentation. International journal of food microbiology, 167(1), 29-43.
Comtet-Marre, S., Parisot, N., Lepercq, P., Chaucheyras-Durand, F., Mosoni, P., Peyretaillade, E., ... & Forano, E. (2017). Metatranscriptomics reveals the active bacterial and eukaryotic fibrolytic communities in the rumen of dairy cow fed a mixed diet. Frontiers in microbiology, 8, 67. Daliu, P., Souto, E. B., & Santini, A. (2025). Novel applications of Lactobacillus in the food industry. Journal of Asian Scientific Research, 15(1), 111.
Denek, N., Can, A., Avci, M., & Aksu, T. (2012). The effect of fresh and frozen pre-fermented juice on the fermentation quality of alfalfa silage. Kafkas Üniversitesi Veteriner Fakültesi Dergisi, 18(5).
Dias, C. O., Scariot, M. C., de Mello Castanho Amboni, R. D., & Arisi, A. C. M. (2020). Application of propidium monoazide coupled with quantitative PCR to evaluate cell viability of Bifidobacterium animalis subsp. lactis in a non-dairy probiotic beverage. Annals of Microbiology, 70(1), 22.
Fey, A., Eichler, S., Flavier, S., Christen, R., Höfle, M. G., & Guzmán, C. A. (2004). Establishment of a real-time PCR-based approach for accurate quantification of bacterial RNA targets in water, using Salmonella as a model organism. Applied and environmental microbiology, 70(6), 3618-3623.
Filya, I. (2003). The effect of Lactobacillus buchneri and Lactobacillus plantarum on the fermentation, aerobic stability, and ruminal degradability of low dry matter corn and sorghum silages. Journal of dairy science, 86(11), 3575-3581.
Herbel, S. R., Vahjen, W., Wieler, L. H., & Guenther, S. (2013). Timely approaches to identify probiotic species of the genus Lactobacillus. Gut Pathogens, 5(1), 27.
Ji, Y., Park, S., Park, H., Hwang, E., Shin, H., Pot, B., & Holzapfel, W. H. (2018). Modulation of active gut microbiota by Lactobacillus rhamnosus GG in a diet induced obesity murine model. Frontiers in microbiology, 9, 710.
Jin, M., Zhao, Z. G., Qiu, Z. G., Wang, J. F., Chen, Z. L., Shen, Z. Q., ... & Li, J. W. (2010). Rapid method to extract high-quality RNA from activated sludge. Huan Jing ke Xue= Huanjing Kexue, 31(1), 260-265.
Jones, D. J. C. (1991). The Biochemistry of Silage (2nd edn), by P. McDonald, AR Henderson & SJE Heron. 340 pp. Kingston, Kent: Chalcombe Publications (1991).£ 49.50 (UK)£ 55.00 (elsewhere)(hardback). ISBN 0 948617 22 5. The Journal of Agricultural Science, 117(3), 386-386.
Kiraz, A. B., & Kutlu, H. R. (2016). Bakteriyel inokulant kullanımının silajlarda fermantasyon özellikleri üzerine etkileri. Harran Tarım ve Gıda Bilimleri Dergisi, 20(3), 230-238.
Koç, F., Karapinar, B., Okuyucu, B., & Korucu Erdem, D. (2020). The effects of kefir addition on the fermentation characteristics and aerobic stability of alfalfa silages.
Kung Jr, L., Shaver, R. D., Grant, R. J., & Schmidt, R. J. (2018). Silage review: Interpretation of chemical, microbial, and organoleptic components of silages. Journal of dairy Science, 101(5), 4020-4033.
Marshall, N. T., Vanderploeg, H. A., & Chaganti, S. R. (2021). Environmental (e) RNA advances the reliability of eDNA by predicting its age. Scientific reports, 11(1), 2769.
Martínez, N., Martín, M. C., Herrero, A., Fernandez, M., Alvarez, M. A., & Ladero, V. (2011). qPCR as a powerful tool for microbial food spoilage quantification: Significance for food quality. Trends in Food Science & Technology, 22(7), 367-376.
Mira Miralles, M., Maestre-Carballa, L., Lluesma-Gomez, M., & Martinez-Garcia, M. (2019). High-throughput 16S rRNA sequencing to assess potentially active bacteria and foodborne pathogens: A case example in ready-to-eat food. Foods, 8(10), 480.
Mu, L., Wang, Q., Cao, X., Li, H., & Zhang, Z. (2022). The potential of pre-fermented juice or Lactobacillus inoculants to improve the fermentation quality of mixed silage of agroresidue and lucerne. Frontiers in Microbiology, 13, 858546.
Muck, R. E., Nadeau, E. M. G., McAllister, T. A., Contreras-Govea, F. E., Santos, M. C., & Kung Jr, L. (2018). Silage review: Recent advances and future uses of silage additives. Journal of dairy science, 101(5), 3980-4000.
Nascimento Agarussi, M. C., Gomes Pereira, O., Paula, R. A., Silva, V. P., Santos Roseira, J. P., Fonseca e Silva, F., & Ribeiro, K. G. (2019). Novel lactic acid bacteria strains as inoculants on alfalfa silage fermentation. Scientific Reports, 9, 8007. https://doi.org/10.1038/s41598-019-44520-9
Ndoye, B., Rasolofo, E. A., LaPointe, G., & Roy, D. (2011). A review of the molecular approaches to investigate the diversity and activity of cheese microbiota. Dairy Science & Technology, 91(5), 495-524.
Nyanzi, R., Jooste, P. J., & Buys, E. M. (2021). Invited review: Probiotic yogurt quality criteria, regulatory framework, clinical evidence, and analytical aspects. Journal of Dairy Science, 104(1), 1-19.
Ohshima M, Kimura E and Yokota H.1997. A method of making good quality silage from direct cut alfalfa by spraying previously fermented juice. Animal Feed Science and Technology 66: 129–37. DOI: https://doi.org/10.1016/S0377-8401(96)01111-X
Oruç, A., & Avcı, M. (2024a). Şanlıurfa koşullarında ikinci ürün olarak yetiştirilen yem bezelyesi (Pisum arvense L.) + arpa (Hordeum vulgare L.) karışımı hasıllarına farklı oranlarda melas ilavesinin silaj kalitesi üzerine etkisinin araştırılması. MAS Journal of Applied Sciences, 9(3), 486–500. https://doi.org/10.5281/zenodo.13253371
Oruç, A., & Avcı, M. (2024b). Pamuk ekiminde ara ürün olarak yetiştirilen macar fiği (Vicia pannonica Crantz) + tritikale (X Triticosecale Wittm) karışımı hasıllarına farklı seviyelerde melas ilavesinin silaj kalitesi üzerine etkisinin araştırılması. MAS Journal of Applied Sciences, 9(2), 400–413. https://doi.org/10.5281/zenodo.11904700
Pahlow, G., Muck, R.E., Driehuis, F., Elferink, S.J.W.H.O. and Spoelstra, S.F. (2003). Microbiology of Ensiling. In Silage Science and Technology (eds D.R. Buxton, R.E. Muck and J.H. Harrison). https://doi.org/10.2134/agronmonogr42.c2
Pega, J., Rizzo, S., Rossetti, L., Pérez, C. D., Díaz, G., Descalzo, A. M., & Nanni, M. (2017). Impact of extracellular nucleic acids from lactic acid bacteria on qPCR and RT-qPCR results in dairy matrices: implications for defining molecular markers of cell integrity. Lwt, 80, 416-422.
Sohier, D., Pavan, S., Riou, A., Combrisson, J., & Postollec, F. (2014). Evolution of microbiological analytical methods for dairy industry needs. Frontiers in Microbiology, 5, 16. Wasfi, R., Abd El‐Rahman, O. A., Zafer, M. M., & Ashour, H. M. (2018). Probiotic Lactobacillus sp. inhibit growth, biofilm formation and gene expression of cariesinducing Streptococcus mutans. Journal of cellular and molecular medicine, 22(3), 1972- 1983.
Wang, Y., Thompson, K. N., Yan, Y., Short, M. I., Zhang, Y., Franzosa, E. A., ... & Huttenhower, C. (2023). RNA-based amplicon sequencing is ineffective in measuring metabolic activity in environmental microbial communities. Microbiome, 11(1), 131.
Yap, M., O’Sullivan, O., O’Toole, P. W., & Cotter, P. D. (2022). Development of sequencingbased methodologies to distinguish viable from non-viable cells in a bovine milk matrix: A pilot study. Frontiers in Microbiology, 13, 1036643.
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