İnülinaz Üretimi İçin Fermantasyon Parametrelerinin İstatistiksel Yaklaşımla Optimizasyonu

Çalışmanın amacı katı faz fermantasyonunda yeni bir fungus izolatı olan Rhizopus oryzae HBF351’dan aktivitesi yüksek ve düşük maliyetli inülinaz üretiminin gerçekleştirilmesidir. Bunun için ilk olarak yüksek enzim üretimini destekleyen en iyi substratın belirlenmesi ve fermantasyon ortamını etkileyen bazı parametrelerin istatistiksel bir yaklaşımla optimize edilmesi hedeflenmiştir. Maksimum enzim üretimini destekleyen en iyi ortamı bulmak için 5 farklı substrat (buğday kepeği, yulaf kepeği, soğan kabuğu, elma posası ve marul kökü tozu) araştırılmıştır. İnülinaz üretimi için bazı fermantasyon koşullarını (başlangıç pH (4-7), inkübasyon sıcaklığı (25°C-45°C), başlangıç nem seviyesi ( %50-100) ve inkübasyon süresi (3-7 gün)) optimize etmek amacıyla Box-Behnken tasarımı (BBD) kullanılmıştır. Çalışmada buğday kepeğinin enzim aktivitesini destekleyen en iyi substrat olduğu bulunmuştur. pH, inkübasyon sıcaklığı, nem seviyesi ve inkübasyon süresinin enzim üretimi üzerinde oldukça etkili parametreler olduğu gösterilmiştir (p<0.0001). Çalışmada modelin uygunluğu R2 (regresyon katsayısı) (0.99) kullanılarak değerlendirilmiştir ve deneysel ve tahmin edilen sonuçlar arasındaki iyi korelasyon elde edilmiştir. Maksimum inulinaz aktivitesini (385.1 IU gds-1) sağlayan optimum koşullar pH 5.18, 33.4 °C, % 72.1 nem seviyesi ve 4.7 gün olarak bulunmuştur. Sonuç olarak, bu çalışma Rhizopus oryzae'den inülinaz üretmek için uygun maliyetli bir yöntem sunmaktadır. Ayrıca, yüksek enzim aktivitesi elde edilen inülinazı, fruktoz şurubu ve diğer endüstriyel alanlarda kullanım için güçlü bir potansiyel aday yapmaktadır.

Anahtar Kelimeler:

İnülinaz, Box-Behnken Tasarımı, Katı Faz Fermantasyonu, Optimizasyon

Optimization of Fermentation Parameters for Inulinase Production by Statistical Approach

The aim of the study was to carry out high-activity and low-cost inulinase production from Rhizopus oryzae HBF351, a new fungal strain, in solid phase fermentation. For this, it was firstly aimed to determine the best substrate that supports high enzyme production and to optimize some parameters that affect the fermentation condition by a statistical approach. Five different substrates (wheat bran, oat bran, onion peel, apple pulp and lettuce root powder) were investigated to find the best substrat supporting maximum enzyme productions. Box–Behnken design (BBD) was employed to optimize fermentation conditions (initial pH (4-7), incubation temperature (25°C-45°C), initial moisture level (50-100%) and incubation time(3-7 days)) for the production of inulinase. In the study, wheat bran was the best substrate that supports enzyme activity. pH, incubation temperature, moisture level and incubation time have been shown to be highly effective parameters on enzyme production (p <0.0001). In the study, the fitness of the model was evaluated using R2 (regression coefficient) (0.99) and a good correlation were obtained between experimental and predicted results. Optimum conditions providing maximum inulinase activity (385.1 IU gds-1) were found as pH 5.18, 33.4 °C, 72.1 % moisture level and 4.7 days. As a result, this study provides a cost-effective method to produce inulinase from Rhizopus oryzae. Also, it can be suggested that the produced inulinase has strong potential for usage in the production of fructose syrup and other industrial areas due to its high activity.

Keywords:

Box-Behnken Design, Inulinase, Optimization, Solid Phase Fermentation,

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Abou-Taleb, KA, Galal, GF, 2018. A comparative study between one-factor-at-a-time and minimum runs resolution-IV methods for enhancing the production of polysaccharide by Stenotrophomonas daejeonensis and Pseudomonas geniculate. Annals of Agricultural Sciences, 63(2):173-180.
Ayyachamy M, Khelawan K, Pillay D, Permaul K, Singh S, 2007. Production of inulinase by Xanthomonas campestris pv phaseoli using onion (Allium cepa) and garlic (Allium sativum) peels in solid state cultivation. Letters in applied microbiology, 45(4):439-444.
Dinarvand M, Rezaee M, Foroughi M, 2017. Optimizing culture conditions for production of intra and extracellular inulinase and invertase from Aspergillus niger ATCC 20611 by response surface methodology (RSM). Braz J Microbiol 48(3):427-441.
El Aty AAA, Wehaidy HR, Mostafa FA, 2014. Optimization of inulinase production from low cost substrates using Plackett–Burman and Taguchi methods. Carbohydr Polym 102:261-268.
Flores-Gallegos AC, Veana F, González-Herrera SM, Muñiz-Márquez DB, Sáenz-Galindo A, Rodríguez-Herrera R, 2018. Fungal Inulinases: An Interesting Option for Food Sweetener Production. In Enzymes in Food Technology (pp. 79-101). Springer, Singapore.
Garai D, Kumar VA, 2013. Box–Behnken design approach for the production of xylanase by Aspergillus candidus under solid state fermentation and its application in saccharifcation of agro residues and Parthenium hysterophorus. L Ind Crops Prod, 44:352-363.
Germec M, Gürler HN, Ozcan A, Erkan SB, Karahalil E, Turhan I, 2020. Medium optimization and kinetic modeling for the production of Aspergillus niger inulinase. Bioproc Biosyst Eng, 43(2):217-232.
Germec M, Ozcan A, Turhan I, 2019. Bioconversion of wheat bran into high value-added products and modelling of fermentations. Ind Crops Prod, 139:e111565. https://doi:10.1016/j.indcrop.2019.111565.
Germec M, Turhan I, 2019. Evaluation of carbon sources for the production of inulinase by Aspergillus niger A42 and its characterization. Bioprocess and Biosystems Engineering, 42(12):1993-2005.
Govarthanan M, Park SH, Kim JW, Lee KJ, Cho M, Kamala-Kannan S, Oh BT, 2014, Statistical optimization of alkaline protease production from brackish environment Bacillus sp. SKK11 by SSF using horse gram husk. Prep Biochem Biotechnol, 44(2):119–131.
Ilgın M, Germec M, Turhan I, 2019. Inulinase production and mathematical modeling from carob extract by using Aspergillus niger. Biotechnol Prog e2919. https://doi.org/10.1002/btpr.2919.
Kalaiyarasi M, Vijayaraghavan P, Raj SRF, Vincent SGP 2017. Statistical approach for the production of protease and cellulase from Bacillus cereus KA3. Bioprocess Eng 1(4):93–103.
Karatop R, Sanal F, 2013. A Potential Resource in Fructose Production from Inulin: Aspergillus wentii Inulinase. Journal of Cell and Molecular Biology, 11:1/2: 21.
Karam EA, Kansoh AL, Moharam ME, Hassan ME, Kansoh AL, 2018. Immobilization of Inulinase Produced by Rhizopus oligosporus NRRL 2549 for continuous fructose production. J Mater Environ Sci, 9 (8): 2315-2321.
Krishna C, 2005. Solid-state fermentation systems—an overview. Crit Rev Biotechnol 25(1–2):1–30.
Mazutti M, Bender JP, Treichel H, Di Luccio M 2006. Optimization of inulinase production by solid-state fermentation using sugarcane bagasse as substrate. Enzyme and Microbial Technology, 39(1), 56-59.
Mohamed SA, Salah HA, Moharam ME, Foda MS, Fahmy AS 2015. Characterization of two thermostable inulinases from Rhizopus oligosporus NRRL 2710. Genet Eng Biotechnol 13(1):65-69.
Narayanan M, Srinivasan B, Gayathiri A, Ayyadurai A, Mani A, 2013. Studies on the optimization and characterization for the biosynthesis of inulinase under solid state fermentation. Int J ChemTech Res, 5(1): 376–384.
Niyonzima FN, More S, 2015. Detergent-compatible proteases: microbial production, properties, and stain removal analysis. Prep Biochem Biotechnol, 45(3):233–258.
Onilude AA, Fadaunsi IF, Garuba EO, 2012. Inulinase production by Saccharomyces sp. in solid state fermentation using wheat bran as substrate. Annal Microbiol, 62(2):843-848.
Pandey A, Selvakumar P, Soccol CR, Nigam P, 1999. Solid state fermentation for the production of industrial enzymes. Curr Sci, 77(1):149–162.
Rawat HK, Soni H, Kango N, Kumar CG, 2017. Continuous generation of fructose from Taraxacum officinale tap root extract and inulin by immobilized inulinase in a packed-bed reactor. Biocatal Agric Biotechnol, 9:134-140.
Rodrigues CJ, Pereira RF, Fernandes P, Cabral JM, & de Carvalho CC, 2017. Cultivation‐based strategies to find efficient marine biocatalysts. Biotechnol J 12(7): 1700036. https://doi.org/10.1002/biot.201700036
Sheng J, Chi Z, Gong F, Li J, 2009. Purification and characterization of extracellular inulinase from a marine yeast Cryptococcus aureus G7a and inulin hydrolysis by the purified inulinase. Appl Biochem Biotechnol, 144(2):111-121.
Singh S, Bajaj BK, 2016. Bioprocess optimization for production of thermoalkali-stable protease from Bacillus subtilis K-1 under solid-state fermentation. Preparative Biochemistry and Biotechnology, 46(7): 717-724.
Singh RS, Chauhan K, 2017. Inulinase production from a new inulinase producer, Penicillium oxalicum BGPUP-4. Biocatal Agric Biotechnol, 9:1-10.
Singh R, Singh R, 2017. Inulinases. Current Developments in Biotechnology and Bioengineering. Elsevier, 423-446. Current Developments in Biotechnology and Bioengineering: Production, Isolation and Purification of Industrial Products http://dx.doi.org/10.1016/B978-0-444-63662-1.00018-X.
Singh RS, Chauhan K, 2018. Production, purification, characterization and applications of fungal inulinases. Curr Biotechnol, 7(3):242-260.
Singh RS, Chauhan K, Jindal A, 2018. Response surface optimization of solid state fermentation for inulinase production from Penicillium oxalicum using corn bran. Journal of Food Science Ad Technology, 55(7):2533-2540.
Singh RS, Chauhan K, Kaur K, Pandey A, 2020. Statistical optimization of solid-state fermentation for the production of fungal inulinase from apple pomace. Bioresource Technology Reports, 9: 100364. https://doi.org/10.1016/j.biteb.2019.100364.
Soccol CR, da Costa ESF, Letti LAJ, Karp SG, Woiciechowski AL, de Souza Vandenberghe LP, 2017. Recent developments and innovations in solid state fermentation. Biotechnol Res Inn, 1(1):52–71.
Trivedi S, Divecha J, Shah A, 2012. Optimization of inulinase production by a newly isolated Aspergillus tubingensis CR16 using low cost substrates. Carbohydr Poly, 90(1): 483-490.
Xiong C, Jinhua, W, Dongsheng L, 2007. Optimization of solid-state medium for the production of inulinase by Kluyveromyces S120 using response surface methodology. Biochemical Engineering Journal, 34(2), 179-184.