Atık Çay Ekstraktında Üretilen Aspergillus niger Misellerinden Elde Edilen Kitosanın Özellikleri

Yıl 2022, Cilt: 20 Sayı: 4, 386 - 397, 27.12.2022

Rukiye Avcı Arzu Çağrı Mehmetoğlu

https://doi.org/10.24323/akademik-gida.1224354

Öz

Bu çalışmada, atık çay ekstraktından üretilen Aspergillus niger’den kimyasal yöntemlerle kitosan özütlenerek, bu kitosan örneklerinin belirli özellikleri araştırılmıştır. Kurutulmuş atık çaylardan %5, 10 ve 20 konsantrasyonlarda hazırlanan karışımlar sterilize edilip süzüldükten sonra A. niger aşılanmış ve 25°C'de 5 gün inkübe edilmiştir. Elde edilen biyokütleden kimyasal yöntem kullanılarak kitosan ekstrakte edilmiştir. Elde edilen kitosanın yapısal özellikleri FT-IR, FESEM ve NMR ile belirlenmiştir. Üretilen kitosanın antimikrobiyal özellikleri Gram pozitif ve Gram negatif bakterilere, mayalara ve küflere karşı disk difüzyon testi kullanılarak analiz edilmiştir. Sonuçlar, %5, 10 ve 20 konsantrasyonlarda hazırlanan çay ekstraktlarında üretilen A. niger misellerinin kitosan ekstraksiyon veriminin sırasıyla %13.98, 15.71 ve 17.57 olduğunu göstermiştir. Elde edilen FT-IR, FESEM ve NMR spektrumlarının benzer çalışmalarda rapor edilen A. niger‘den üretilen kitosan ve ticari kitosan ile uyumlu olduğu görülmüştür. Gram pozitif ve negatif bakterilere karşı %1 ve 2-4 kitosan solüsyonları sırasıyla 7 ile 8 mm inhibisyon zonu oluşturmuştur. Saccharomyces cerevisiae kitosana karşı en hassas kültür olarak belirlenmiştir. Ticari kitosan, fungal kitosana göre test edilen mikroorganizmalara karşı daha fazla antimikrobiyal aktivite göstermiştir. Fungal kitosanın deasetilasyon derecesi %92 olarak bulunmuştur. Araştırmada elde edilen bulgulara göre, çevre açısından önem kazanan atık maddelerin değerlendirilmesi amacıyla atık çaylarda geliştirilen A. niger küfünden kitosan üretimi ilk defa gerçekleştirilmiş olup, çalışma bu konudaki araştırmalar için yol gösterici niteliktedir.

Anahtar Kelimeler

Aspergillus niger, Kitosan, Atık çay

Characteristics of Chitosan from Aspergillus niger Micelles Produced in Waste Tea Extract

Öz

This study investigated specific properties of chitosan extracted by chemical methods from the micelles of Aspergillus niger grown in tea waste extract. Mixtures prepared from dried tea wastes at 5, 10 and 20% concentrations were sterilized and filtered, then A. niger was inolulated and incubated at 25°C for 5 days. Chitosan was extracted from the biomass by using a chemical method. The results of FT-IR, FESEM, and NMR analyses were used to determine the structural properties of chitosan extracted. In addition, antimicrobial properties of chitosan against Gram positive and Gram negative bacteria, yeasts, and molds were determined by using the disk diffusion test. Results showed that chitosan extraction efficiencies were 13.98, 15.71 and 17.57% for A. niger micelles produced in tea extracts prepared at 5, 10 and 20% concentrations, respectively. It was observed that FT-IR and NMR spectra were compatible with both chitosan from A. niger reported by similar studies and commercial chitosan. It was observed that 1 and 2-4% chitosan solution formed inhibition zones of 7 and 8 mm in diameter against Gram positive and Gram negative bacteria, respectively. Saccharomyces cerevisiae was the most susceptible culture to chitosan. Commercial chitosan showed better antimicrobial activity against the tested microorganisms than fungal chitosan. The deacetylation degree of fungal chitosan was 92%. According to the results of this study, the production of chitosan from A. niger mold grown in waste teas was carried out for the first time in order to evaluate the waste materials that have gained importance in terms of the environment, and this study could be used as a guide for future studies on this subject.

Anahtar Kelimeler

Aspergillus niger, Tea waste, Chitosan

Kaynakça

• [1] Bartnicki-Garcia, S. (1968). Cell wall chemistry morphogenesis and taxonomy of fungi. Annual Review of Microbiology, 22, 87-108.
• [2] Aiba, S. (1992). Studies on chitosan: 4. Lysozymic hydrolysis of partially n-acetylated chitosans. International Journal of Biological Macromolecules, 14, 225-228.
• [3] Tokatlı, K., Demirdöven, A. (2015). Kitosan ve kitosan bazlı yenilebilir film uygulamaları. Akademik Gıda, 13, 348-353.
• [4] Dutta, P.K., Dutta, J., Tripathi, V. (2004). Chitin and chitosan: Chemistry, properties and applications. Journal of Scientific and Industrial Research, 63, 20-31.
• [5] Aranaz, I., Mengibar, M., Harris, R., Panos, I., Miralles, B., Acosta, N., Galed, G., Heras, A. (2009). Functional characterization of chitin and chitosan. Current Chemical Biology, 3, 203-230.
• [6] Nwe, N., Stevens, W. (2002). Production of fungal chitosan by solid substrate fermentation followed by enzymatic extraction. Biotechnology Letters, 24, 131-134.
• [7] Teng, W.L., Khor, E., Tan, T.K., Lim, L.Y., Tan, S.C. (2001). Concurrent production of chitin from shrimp shells and fungi. Carbohydrate Research, 332, 305-316.
• [8] Wu, T., Zivanovic, S., Draughon, F.A., Conway, W.S., Sams, C.E. (2005). Physicochemical properties and bioactivity of fungal chitin and chitosan, Journal of Agricultural and Food Chemistry, 53, 3888-3894.
• [9] Pochanavanich, P., Suntornsuk, W. (2002). Fungal chitosan production and its characterization, Department of Microbiology, King Mongkut’s University of Technology Thonburi, Bangkok, Thailand, 35, 17-21.
• [10] Kaya, M., Cakmak, Y. S., Baran, T., Asan-Ozusaglam, M., Mentes, A., Tozak, K.O. (2014). New chitin, chitosan, and o-carboxymethyl chitosan sources from resting eggs of Daphnia longispina (Crustacea); with physicochemical characterization, and antimicrobial and antioxidant activities. Biotechnology and Bioprocess Engineering, 19, 58-69.
• [11] Donald, H.D., Hayes, E.R. (1988). Determination of degree of acetylation of chitin and chitosan. Methods in Enzymology, 161, 442-446.
• [12] Menon, K.K.G., Mulky, M.J., Sharma, V.S. (1993). The tea industry in India: how to redesign a native tea culture, processing and marketing, Oxford and IBH, New Delhi, pp. 3-10.
• [13] Selvakumar, P., Ashakumary, L., Pandey, A. (1998). Biosynthesis of glucoamylase from Apergillus Niger by solid-state fermentation using tea waste as the basis of a solid substrate. Bioresource Technology, 65, 83-85.
• [14] Moore-Landecker, E. (1996). Fundamentals of the fungi. 4th ed. Englewood Cliffs, NJ: Prentice-Hall, 251-278.
• [15] Nwe, N., Chandrkrachang, S., Stevens, W.F., Maw, T., Tan, T.K., Khor, E., Wong, S.M., (2002a). Production of fungal chitosan by solid state and submerged fermentation. Carbohydrate Polymers, 49, 235-237.
• [16] Jaworska, M.M., Konieczna, E. (2001). The influence of supplemental components in nutrient medium on chitosan formation by the fungus Absidia orchidis, Applied Microbiology and Biotechnology, 56, 220-224.
• [17] Rane, KD, Hoover, DG. (1993). An evaluation of alkali and acid treatments for chitosan extraction from fungi. Process Biochemistry, 28, 115-118.
• [18] Shimahara, K., Takiguchi, Y., Kobayashi, T., Uda, K., Sannan, T. (1989). Screening of Mucoraceae strains suitable for chitosan production. In Chitin and Chitosan ed. Skjak-Braek, G., Anthonsen, T. and Sanford, P. pp. 171-178. London: Elsevier Applied Science.
• [19] Crestini, C., Kovac, B., Giovannozzi-Sermanni, G. (1996). Production and isolation of chitosan by submerged and solid-state fermentation from Lentinus edodes. Biotechnology Bioengineering, 50, 207-210.
• [20] Hang, Y.D. (1990). Chitosan production from Rhizopus oryzae mycelia. Biotechnology Letters, 12, 911-912.
• [21] Xu, Y., Du, Y. (2003). Effect of molecular structure of chitosan on protein delivery properties of chitosan nanoparticles. International Journal of Pharmaceutics, 250, 215-226.
• [22] Wang, X., Du, Y., Liu, H. (2004). Preparation, characterization and antibacterial activity of chitosan-Zn complex. Carbohydrate Polymers, 56, 21-26.
• [23] Arof, A.K., Osman, Z. (2003). FTIR studies of chitosan acetate based polymer electrolytes. Electrochimica Acta, 48, 993-999.
• [24] Salokhe, V.M., Rakshit, S.K., Pranoto, Y. (2005.) Enhancing antimicrobial activity of chitosan films by incorporating garlic oil, potassium sorbate and nişin. Lebensmittel-Wissenschaft & Technologie, 38, 859-865.
• [25] Mincheva, R., Manolova, N., Sabov, R., Kjurkchiev, G., Rashkov, L. (2004). Hydrogels from chitosan crosslinked with poly(ethyleneglycol) diacid as bone regeneration materials. e-Polymers 058, 1-11.
• [26] Han, J., Zhou, Z., Yin, R., Yang, D., Nie, J. (2010). Alginate-chitosan/ hydroxyapatitepolyelectrolyte complex porous scaffolds: preparation and characterization. International Journal of Biological Macromolecules, 46, 199-205.
• [27] Brugnerotto, J., Goycoolea, F.M., Arguelles-Monal, W., Desbrieres, J., Rinaudo, M. (2001). An infrared investigation in relation with chitin and chitosan characterization. Polymer, 42, 3569-3580.
• [28] Velde, V., Kiekens, K.P. (2004). Structure analysis and degree of substitution of chitin, chitosan and dibutyrylchitin by FT-IR spectroscopy and solid state 13C NMR, Carbohydrate Polymers, 58, 409-416.
• [29] Koçer, İ., (2015). Farklı Yöntemlerle Kitosan Eldesi ve Karakterizasyonu. Yüksek Lisans Tezi. Hacettepe Üniversitesi, Kimya Mühendisliği Anabilim Dalı, Ankara.
• [30] Goy, R.C., De Britto, D., Assis, O.B.G. (2009). A Review of the antimicrobial activity of chitosan, polímeros. Ciência e Tecnologia, 19, 241-247.
• [31] Kong, M., Chen, X.G., Xing, K., Park, H.J. (2010). Antimicrobial properties of chitosan and mode of action: a state of the art review. International Journal of Food Microbiology, 144, 51-63.
• [32] Guo, Z., Xing, R., Lıu, S., Zhong, Z., Ji, X., Wanga, L., LI, P. (2007). Antifungal properties of Schiff bases of chitosan, n-substituted chitosan and quaternized chitosan. Carbohydrate Research, 342, 1329-1332.
• [33] Guo, Z., Xing, R., Lıu, S., Zhong, Z., Ji, X., Wang, L., LI, P. (2008). The influence of molecular weight of quaternized chitosan on antifungal activity. Carbohydrate Polymers, 71, 694-697.
• [34] Khalaf, S.A. (2004). Production and characterization of fungal chitosan under solid-state fermentation conditions. International Journal of Agriculture and Biology, 6, 1033-1036.
• [35] No, H.K., Lee, S.H., Park, N.Y., Meyers, S.P. (2003). Comparison of physicochemical, binding, and antibacterial properties of chitosans prepared without and with deproteinization process. Journal of Agricultural and Food Chemistry, 51, 7659‐7663.
• [36] Kucukgulmez, A., Celik, M., Yanar, Y., Sen, D., Polat H., Kadak, A.E. (2011). Physicochemical characterization of chitosan extracted from Metapenaeus stebbingi shells. Food Chemistry, 126, 1144-1148.
• [37] Kaya, M., Tozak, K.Ö., Baran, T., Sezen, G., Sargin, I. (2013). Natural porous and nano fiber chitin structure from Gammarus argaeus (Gammaridae crustacea). Excli Journal, 12, 503-510.
• [38] Wang, Y., Chang, Y., Yu, L., Zhang, C., Xu, X., Xue, Y., Li, Z., Xue, C. (2013). Crystalline structure and thermal property characterization of chitin from Antarctic krill (Euphausia superba). Carbohydrate Polymers, 92, 90-97.
• [39] Yen, M.T., Mau, J.L. (2007). Physico-chemical characterization of fungal chitosan from Shiitake stipes. LWT-Food Science and Technology, 40, 472-479.
• [40] Cahn, H.Y., Chen, M.H., Yuan, G.F. (2001). Fungal chitosan. Fungal Science, 16(1-2), 39-52.
• [41] Yen, M.T., Mau, J.L. (2004). Physicochemical properties of chitin from shiitake stipes and crab shells. Annual Tainan Woman’s Coll. Arts Technol. 23, 229-240.
• [42] Ifuku, S., Nomura, R., Morimoto, M., Saimoto, H. (2011). Preparation of chitin nanofibers from mushrooms. Materials, 4, 1417-1425.
• [43] Czechowska-Biskup, C., Jarosińska, D., Rokita, B., Ulański, P., Rosiak, J.M. (2012). Determination of Degree of Deacetylation of Chitosan-Comparison of Methods. Progress on Chemistry and Application of Chitin and its Derivatives, 17, 5-20.
• [44] Kasaai, M.R. (2009). Determination of the degree of N-acetylation for chitin and chitosan by various NMR spectroscopy techniques: A review. Carbohydrate Polymers, 79, 801-810.
• [45] Yang, B.Y., Montgomery, R. (2000). Degree of acetylation of heteropolysaccharides. Carbohydrate Research, 323, 156-162.
• [46] Rinaudo, M. (2006). Chitin and chitosan: Properties and applications. Progress in Polymer Science, 31, 603-632.
• [47] Park, P.J., Je, J.Y., Byun, H.G., Moon, S.H., Kim, S.K. (2004). Antimicrobial activity of hetero-chitosans and their oligosaccharides with different molecular weights. Journal of Microbiology and Biotechnology, 14, 317-323.