EVALUATING THE NARINGINASE PRODUCTION POTENTIAL OF PENICILLIUM ROQUEFORTI UNDER DIFFERENT CULTURE CONDITIONS

Öz

Naringin is a major flavanone glycoside responsible for bitterness in citrus juices, particularly grapefruit juice, which often limits consumer acceptance. Enzyme-mediated hydrolysis represents a promising approach to mitigate this bitterness. Naringinase, an enzyme complex capable of catalyzing the hydrolysis of naringin, was the focus of the present study, which investigated the production potential of Penicillium roqueforti. Several culture media compositions were investigated to enhance naringinase production, and extracellular enzyme activities and pH dynamics were monitored. Based on prior studies, the initial pH values of the media were set at 4.5 and 6.5, identified as optimal for naringinase activity. The highest naringinase activity (1.47 EU/mL) was obtained in Medium 1 under different initial pH conditions. whereas variations in pH did not significantly alter the peak enzyme activity achieved in this medium. Moreover, P. roqueforti altered the pH of all media, with pH values in A1 and A2 rising to 7.86 and 8.12, respectively, by day five. A preliminary partial purification of the enzyme was performed, revealing precipitation during ammonium sulfate elution for reasons yet to be elucidated.

Anahtar Kelimeler

• Naringinase
• Penicillium roqueforti
• naringin
• microbial enzyme

PENICILLIUM ROQUEFORTI'NİN FARKLI KÜLTÜR KOŞULLARI ALTINDA NARİNGİNAZ ÜRETİM POTANSİYELİNİN DEĞERLENDİRİLMESİ

Öz

Naringin, özellikle greyfurt suyu olmak üzere turunçgil sularındaki acılıktan sorumlu olan ve tüketici kabulünü sıklıkla sınırlayan önemli bir flavonon glikozididir. Enzim aracılı hidroliz, bu acılığı hafifletmek için umut verici bir yaklaşımdır. Naringinin hidrolizini katalize edebilen bir enzim kompleksi olan naringinaz, Penicillium roqueforti'nin üretim potansiyelini araştıran bu çalışmanın odak noktasıydı. Bu bağlamda öncü bir yaklaşımı temsil eden P. roqueforti için bir dizi yeni kültür ortamı geliştirildi ve pH dinamikleriyle birlikte hücre dışı enzim aktiviteleri izlendi. Önceki çalışmalara dayanarak, ortamların başlangıç pH değerleri, naringinaz aktivitesi için optimum olarak belirlenen 4,5 ve 6,5 olarak belirlendi. Sonuçlar, her iki pH koşulunda en yüksek enzim aktivitesinin, kültürün beşinci gününde A1 ve A2 ortamlarında 1,47 EU/mL'ye ulaştığını göstermiştir. Dahası, P. roqueforti tüm ortamların pH'ını değiştirdi ve beşinci günde A1 ve A2'deki pH değerleri sırasıyla 7,86 ve 8,12'ye yükseldi. Enzimin ön kısmi saflaştırılması gerçekleştirildi ve henüz açıklığa kavuşturulamayan nedenlerle amonyum sülfat elüsyonu sırasında çökelme olduğu ortaya çıktı.

Anahtar Kelimeler

• Penicillium roqueforti
• naringin
• microbial enzyme
• naringinaz

Kaynakça

1. Bago, B., Vierheilig, H., Piché, Y., Azcón‐Aguilar, C. (1996). Nitrate depletion and pH changes induced by the extraradical mycelium of the arbuscular mycorrhizal fungus Glomus intraradices grown in monoxenic culture. New phytologist, 133(2), 273-280. https://doi.org/10.1111/j.1469-8137.1996.tb01894.x
2. Baker, S. L., Munasinghe, A., Kaupbayeva, B., Rebecca Kang, N., Certiat, M., Murata, H., & Russell, A. J. (2019). Transforming protein-polymer conjugate purification by tuning protein solubility. Nature Communications, 10(1), 4718. https://doi. org/10.1038/s41467-019-12612-9
3. Bhunia, B., Basak, B., Mandal, T., Bhattacharya, P., Dey, A. (2013). Effect of pH and temperature on stability and kinetics of novel extracellular serine alkaline protease (70 kDa). International journal of biological macromolecules, 54, 1-8. https://doi.org/10.1016/j.ijbiomac.2012.11.024
4. Bignell, E. (2012). The molecular basis of pH sensing, signaling, and homeostasis in fungi. Advances in applied microbiology, 79, 1-18. https://doi.org/10.1016/B978-0-12-394318-7.00001-2
5. Bodakowska-Boczniewicz, J., Garncarek, Z. (2022). Naringinase biosynthesis by Aspergillus niger on an optimized medium containing red grapefruit albedo. Molecules, 27(24), 8763. https://doi.org/10. 3390/molecules27248763
6. Busto, M. D., Meza, V., Ortega, N., Perez-Mateos, M. (2007). Immobilization of naringinase from Aspergillus niger CECT 2088 in poly (vinyl alcohol) cryogels for the debittering of juices. Food chemistry, 104(3), 1177-1182. https://doi.org/10.1016/j. foodchem.2007.01.033
7. Cavia-Saiz, M., Mu ̃niz, P., Ortega, N., Busto, M. D. (2011). Effect of enzymatic debittering on antioxidant capacity and protective role against oxidative stress of grapefruit juice in comparison with adsorption on exchange resin. Food Chemistry, 125, 158–163. https://doi.org/10.1016/j.foodchem.2010.08.054
8. Coton, E., Coton, M., Hymery, N., Mounier, J., Jany, J. L. (2020). Penicillium roqueforti: an overview of its genetics, physiology, metabolism and biotechnological applications. Fungal biology reviews, 34(2), 59-73. https://doi.org/10.1016/j.fbr.2020.03.001

9. Gillot, G., Jany, J. L., Coton, M., Le Floch, G., Debaets, S., Ropars, J., & Coton, E. (2015). Insights into Penicillium roqueforti morphological and genetic diversity. PLoS One, 10(6), e0129849. http://doi.org/10.1371/journal.pone.0129849
10. Golgeri M, D. B., Mulla, S. I., Bagewadi, Z. K., Faniband, B., Mishra, P., Bankole, P. O., & Romanholo Ferreira, L. F. (2025). Microbial naringinase: from microbial source to its current applications in various fields. Biologia, 1-15. https://doi. org/10.1007/s11756-025-01888-6
11. Goettig, P. (2016). Effects of glycosylation on the enzymatic activity and mechanisms of proteases. International journal of molecular sciences, 17(12), 1969. https://doi.org/10.3390/ijms17121969
12. Hasan, F., Shah, A. A., Hameed, A. (2006). Industrial applications of microbial lipases. Enzyme and Microbial Technology, 39, 235–251. https://doi.org/10.1016/j.enzmictec.2005.10.016
13. Huang, W., Zhan, Y., Shi, X., Chen, J., Deng, H., Du, Y. (2017). Controllable immobilization of naringinase on electrospun cellulose acetate nanofibers and their application to juice debittering. International journal of biological macromolecules, 98, 630-636. https://doi.org/10.1016/j.ijbiomac.2017.02.018
14. Hu, D., Zhao, R., Lin, Y., Jiang, C. (2025). Evolution and Functional Diversity of GATA Transcription Factors in Filamentous Fungi: Structural Characteristics, Metabolic Regulation and Environmental Response. Microbiology Research, 16(6), 120. https://doi.org/10.3390/microbiolres16060120
15. İmece, A., Şengül, M., Çetin, B., Aktaş, H. (2024). Effect of probiotic Lactiplantibacillus plantarum strains on some properties of grapefruit juice and naringin. Journal of Stored Products Research, 108, 102359. https://doi.org/10.1016/j.jspr.2024.102359
16. İmece, A., Şengül, M., Çetin, B., Küfrevioğlu, Ö.İ., Öztürk Kesebir, A. (2025). Comprehensive characterization of purified naringinase: effect on naringin content, antioxidant activity, phenolic profile, and grapefruit juice quality. Turkish Journal of Agriculture and Forestry, 49(3), 516-529. http://doi. org/10.55730/1300-011X.3283
17. Kachlishvili, E., Penninckx, M. J., Tsiklauri, N., Elisashvili, V. (2006). Effect of nitrogen source on lignocellulolytic enzyme production by white-rot basidiomycetes under solid-state cultivation. World Journal of Microbiology and Biotechnology, 22(4), 391-397. http://doi.org/10.1007/s11274-005-9046-8
18. Kırtıl, H. E., Metin, B., Arıcı, M. (2020). Peynir küfü olarak Penicillium roquefortı'nin taksonomisi, morfolojik, genetik ve metabolik özellikleri. GIDA/The Journal of FOOD, 45(6), 1188-1200. http://doi.org/10.15237/gida.GD20091
19. Kirk, O., Borchert, T. V., Fuglsang, C. C. (2002). Industrial enzyme applications. Current Opinion in Biotechnology, 13, 345–351. https://doi.org/10.1016/S0958-1669(02)00328-2
20. Kramer, R. M., Shende, V. R., Motl, N., Pace, C. N., Scholtz, J. M. (2012). Toward a molecular understanding of protein solubility: increased negative surface charge correlates with increased solubility. Biophysical journal, 102(8), 1907-1915. http://doi.org/10.1016/j.bpj.2012.01.060
21. Mamma, D., Kalogeris, E., Hatzinikolaou, D. G., Lekanidou, A., Kekos, D., Macris, B. J., & Christakopoulos, P. (2004). Biochemical characterization of the multi-enzyme system produced by Penicillium decumbent grown on rutin. Food Biotechnology, 18(1), 1-18. https://doi.org/10.1081/FBT-120030382
22. Mendoza-Cal, A., Cuevas-Glory, L., Lizama-Uc, G., Ortiz-Vázquez, E. (2010). Naringinase production from filamentous fungi using grapefruit rind in solid state fermentation. African Journal of Microbiology Research, 4(19),1964-1968. http://www. academicjournals.org/ajmr
23. Miguel, A. M., Martins-Meyer, T. S., Figueiredo, E. V. D. C., Lobo, B. W. P., & Dellamora-Ortiz, G. M. (2013). Enzymes in bakery: current and future trends. Food industry, 287-321. http://dx.doi.org/10.5772/54182
24. Morris, C. W. (2019). Protein Precipitation for the Purification of Therapeutic Protein (Doctoral dissertation, UCL (University College London)).
25. Müller, W. E., Schröder, H. C., Wang, X. (2019). Inorganic polyphosphates as storage for and generator of metabolic energy in the extracellular matrix. Chemical reviews, 119(24), 12337-12374. https://doi.org/10.1021/acs.chemrev.9b00460
26. Ni, H., Xiao, A. F., Cai, H. N., Chen, F., You, Q., Lu, Y. Z. (2012). Purification and characterization of Aspergillus niger α-L-rhamnosidase for the biotransformation of naringin to prunin. African journal of microbiology research, 6(24). https://doi. org/10.5897/AJMR12.1229
27. Norouzian, D., Hosseinzadeh, A., Inanlou, D. N., Moazami, N. (2000). Production and partial purification of naringinase by Penicillium decumbens PTCC 5248. World Journal of Microbiology and Biotechnology, 16(5), 471-473. https://doi. org/10.1023/A:1008962131271
28. Puri, M., Banerjee, A., Banerjee, U. C. (2005). Optimization of process parameters for the production of naringinase by Aspergillus niger MTCC 1344. Process Biochemistry, 40(1), 195-201. https://doi.org/10.1016/j.procbio.2003.12.009
29. Puri, M., Kalra, S. (2005). Purification and characterization of naringinase from a newly isolated strain of Aspergillus niger 1344 for the transformation of flavonoids. World Journal of Microbiology and Biotechnology, 21(5), 753-758. https://doi. org/10.1007/s11274-004-5488-7
30. Puri, M., Kaur, A., Singh, R. S., Singh, A. (2010). Response surface optimization of medium components for naringinase production from Staphylococcus xylosus MAK2. Applied biochemistry and biotechnology, 162(1), 181-191. http://doi.org/10.1007/s12010-009-8765-y
31. Radhakrishnan, I., Sampath, S., Kumar, S. (2013). Isolation and characterization of enzyme naringinase from Aspergillus flavus. International journal of advanced biotechnology and research, 4(2), 208-212.
32. Ribeiro, M. H. (2011). Naringinases: occurrence, characteristics, and applications. Applied microbiology and biotechnology, 90(6), 1883-1895. http://doi.org/10.1007/s00253-011-3176-8
33. Sadana, A. (1997). Bioseparations of proteins: unfolding/folding and validations (Vol. 1). Elsevier.). Samson, R. A., Houbraken, J., Thrane, U., Frisvad, J. C., Andersen, B. (2019). Food and indoor fungi. Westerdijk Fungal Biodiversity Institute.
34. Shehata, A. N., Abd El Aty, A. A. (2014). Optimization of process parameters by statistical experimental designs for the production of naringinase enzyme by marine fungi. International journal of chemical engineering, 2014(1), 273523. https://doi. org/10.1155/2014/273523
35. Silao, F. G. S., Ljungdahl, P. O. (2021). Amino acid sensing and assimilation by the fungal pathogen Candida albicans in the human host. Pathogens, 11(1), 5. https://doi.org/10.3390/pathogens11010005
36. Sindhe, A., Lingappa, K. (2023). Isolation and Molecular Characterization of the Naringinase Producing Micro-organisms for the Bio-transformation of Flavonoid. Journal of Pure & Applied Microbiology, 17(1). https://doi.org/10.22207/JPAM.17.1.38
37. Soares, I., Távora, Z., Barcelos, R. P., Baroni, S. (2012). Microorganism-produced enzymes in the food industry. Food industry, scientific, health and social aspects of the food industry, 83-94.
38. Tao, Z., Yuan, H., Liu, M., Liu, Q., Zhang, S., Liu, H., & Wang, T. (2022). Yeast extract: characteristics, production, applications and future perspectives. Journal of microbiology and biotechnology, 33(2), 151. https://doi.org/10.4014/jmb.2207.07057
39. Thammawat, K., Pongtanya, P., Juntharasri, V., Wongvithoonyaporn, P. (2008). Isolation, preliminary enzyme characterization and optimization of culture parameters for production of naringinase isolated from Aspergillus niger BCC 25166. Agriculture and Natural Resources, 42(1), 61-72.
40. Tokmakov, A. A., Kurotani, A., Sato, K. I. (2021). Protein pI and intracellular localization. Frontiers in Molecular Biosciences, 8, 775736. https://doi.org/10.3389/fmolb.2021.775736
41. Tudzynski, B. (2014). Nitrogen regulation of fungal secondary metabolism in fungi. Frontiers in microbiology, 5, 656. https://doi. org/10.3389/fmicb.2014.00656
42. Van Oort, M. (2010). Enzymes in food technology–introduction. Enzymes in food technology, 2. http://doi. org/10.1002/9781444309935
43. VinothKumar, V., Kayambu, P., RevathiBabu, S. (2010). Optimization of fermentation parameters for enhanced production of naringinase by soil isolate Aspergillus niger VB07. Food science and biotechnology, 19(3), 827-829. https://doi. org/10.1007/s10068-010-0116-9
44. Wingfield, P. T. (Ed.). (2016). Protein precipitation using ammonium sulfate. Current protocols in protein science, 84(1), A-3F. https://doi.org/10.1002/0471140864.psa03fs84
45. Yadav, M., Sehrawat, N., Sharma, A. K., Kumar, V., & Kumar, A. (2018). Naringinase: microbial sources, production and applications in food processing industry. J. Microbiol. Biotechnol. Food Sci, 8, 717-720. https://doi.org/10.15414/jmbfs.2018.8.1.717-720
46. Zhang, Y. H., Ru, Y., Jiang, C., Yang, Q. M., Weng, H. F., Xiao, A. F. (2020). Naringinase-catalyzed hydrolysis of naringin adsorbed on macroporous resin. Process Biochemistry, 93(August 2019), 48–54. https://doi.org/10.1016/j.procbio.2020.03.014