Sequential optimization of aspergillus flavus kla-80 for the production of naringinase using stastical experimental designs

Paper Details

Research Paper 18/05/2023
Views (408) Download (29)

Sequential optimization of aspergillus flavus kla-80 for the production of naringinase using stastical experimental designs

Ananda Sindhe, K. Lingappa
Int. J. Biosci.22( 5), 149-159, May 2023.
Certificate: IJB 2023 [Generate Certificate]


Naringin is a flavanone bitter glycoside which is found in flowers and fruit of the grapefruit, which on further hyrdolysis yields naringenin and glucose. As naringin is responsible for the bitterness of the fruit, to neutralize the bitterness by eco-freindly method, microbial method is performed by using naringinase enzyme. In the presesnt study naringinase producing strain Apergills flavus KLA-80 which was previously isolated from soil sample  of Kalyan-Karnataka region, India, and deposited in NFCCI with an accesion number of OQ152018 which was maintained in the lab conditions was used for optimization studies.Further to increase the activity of enzyme one factor at a time approach was carried out where the activity was found to be increased from 559 to 720U/mL in a temp of 350C, pH 4.5, carbon source 1.25% and nitrogen source of 0.15% at an agitation speed of 120rpm repectively. Eventually the production of naringinase from Aspergillus niger is optimized by Response Surface Methodology i.e., by CCD (Central Composite Design) method considering four factors at a time (pH,Agitation speed, Galactose, Peptone) as the variables. Based on the statstical method of optimization it is found that the enzyme activity was increased upto 20.41%.


Adami R, Russo P, Amante C, De Soricellis C, Della Porta G, Reverchon E, Del Gaudio P. 2022. Supercritical Antisolvent Technique for the Production of Breathable Naringin Powder. Pharmaceutics 14(8), 1623.

Alam MA, Subhan N, Rahman MM, Uddin SJ, Reza HM, Sarker SD. 2014. Effect of Citrus Flavonoids, Naringin and Naringenin, on Metabolic Syndrome and Their Mechanisms of Action. Advances in Nutrition 5(4), 404-417. 10.3945/an.113.005603

Amiri MJ, Bahrami M, Dehkhodaie F. 2019. Optimization of Hg(II) adsorption on bio-apatite based materials using CCD-RSM design: characterization and mechanism studies. Journal of Water and Health 17(4), 556-567. /10.2166/wh.2019.039

Borkar V, Chakraborty S, Gokhale JS. 2020. Fermentative Production of Naringinase from Aspergillus niger van Tieghem MTCC 2425 Using Citrus Wastes: Process Optimization, Partial Purification, and Characterization. Applied Biochemistry and Biotechnology 193(5), 1321-1337.

Chen R, Qi QL, Wang MT, Li QY. 2016. Therapeutic potential of naringin: an overview. Pharmaceutical Biology 54(12), 3203-3210.

Cook N. 1996. Flavonoids–Chemistry, metabolism, cardioprotective effects, and dietary sources. The Journal of Nutritional Biochemistry 7(2), 66-76.

Dutt K, Gupta P, Saran S, Misra S, Saxena RK. 2009. Production of Milk-Clotting Protease from Bacillus subtilis. Applied Biochemistry and Biotechnology 158(3), 761-772. /10.1007 /s12010-008-8504-9

Gupta AK, Yumnam M, Medhi M, Koch P, Chakraborty S, Mishra P. 2021. Isolation and characterization of naringinase enzyme and its application in debittering of Pomelo juice (Citrus grandis): A comparative study with macroporous resin. Journal of Food Processing and Preservation 45(5). Portico.

Jitolis JL, Awang Ali AN, Saad I, Taha NA, Idris J, Bolong N. 2021. Utilization of Response Surface Methodology and Regression Model in Optimizing Bioretention Performance. 2021 IEEE International Conference on Artificial Intelligence in Engineering and Technology (IICAIET). 10.1109/iicaiet51634.2021.9573644

Kalil SJ, Maugeri F, Rodrigues MI. 2000. Response surface analysis and simulation as a tool for bioprocess design and optimization. Process Biochemistry 35(6), 539-550.

Karuppaija S, Kapilan R, Seevaratnam V. 2016. Optimization of naringinase production by Rhizophus stolonifer in solid state fermentation media using paddy husk as support. Scholars Academic Journal of Biosciences 4(6). sajb.

Kranz P, Adler P, Kunz B. 2010. Sorption of citrus flavour compounds on XAD-7HP resin during the debittering of grapefruit juice. International Journal of Food Science & Technology 46(1), 30-36.

Lee HS, Kim JG. 2003. Effects of debittering on red grapefruit juice concentrate. Food Chemistry 82(2), 177-180.

Lee WC, Yusof S, Hamid NSA, Baharin BS. 2006. Optimizing conditions for enzymatic clarification of banana juice using response surface methodology (RSM). Journal of Food Engineering 73(1), 55-63. jfoodeng.

Manish Kumar, Veenya Pareek. 2022. Minimization of Limonin and Naringin Content in Kinnow Juice Using Response Surface Methodology. Annals of Biology 38(1), 88-95.

Mishra P, Kar R. 2003. Treatment of Grapefruit Juice for Bitterness Removal by Amberlite IR 120 and Amberlite IR 400 and Alginate Entrapped Naringinase Enzyme. Journal of Food Science 68(4), 1229-1233.

Mukesh Kumar DJ, Saranya GM, Suresh K, Andal Priyadharshini D, Rajakumar R, Kalaichelvan PT. 2012. Production and Optimization of Pectinase from Bacillus sp. MFW7 using Cassava Waste. Asian Journal of Plant Science and Research, 2012, 2(3), 369-375.https:// www. /publication/264851241_Production_and_ Optimization_of_Pectinase_from_Bacillus_sp_MFW7_using_Cassava_Waste/links/540c60140cf2f2b29a377e64/Production-and-Optimization-of-Pectinase-from-Bacillus-sp-MFW7-using-Cassava-Waste.pdf

Mukund P, Belur PD, Saidutta MB. 2013. Production of naringinase from a new soil isolate, Bacillus methylotrophicus: Isolation, optimization and scale-up studies. Preparative Biochemistry and Biotechnology 44(2), 146-163. 10.1080 /10826068.2013.797910

Muñoz M, Holtheuer J, Wilson L, Urrutia P. 2022. Grapefruit Debittering by Simultaneous Naringin Hydrolysis and Limonin Adsorption Using Naringinase Immobilized in Agarose Supports. Molecules 27(9), 2867. /molecules27092867

Oskouie SFG, Tabandeh F, Yakhchali B, Eftekhar F. 2008. Response surface optimization of medium composition for alkaline protease production by Bacillus clausii. Biochemical Engineering Journal 39(1), 37-42. /j.bej.2007

Puri M. 2011. Updates on naringinase: structural and biotechnological aspects. Applied Microbiology and Biotechnology 93(1), 49-60. /10.1007 /s00253-011-3679-3

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.

Puri M, Banerjee A, Banerjee UC. 2005. Optimization of process parameters for the production of naringinase by Aspergillus niger MTCC 1344. Process Biochemistry 40(1), 195-201.

Puri M, Kaur A, Barrow CJ, Singh RS. 2010. Citrus peel influences the production of an extracellular naringinase by Staphylococcus xylosus MAK2 in a stirred tank reactor. Applied Microbiology and Biotechnology 89(3), 715-722. /10.1007/s00253-010-2897-4

Puri M, Kaur A, Singh RS, Singh A. 2009. Response Surface Optimization of Medium Components for Naringinase Production from Staphylococcus xylosus MAK2. Applied Biochemistry and Biotechnology 162(1), 181-191. /10.1007/s12010-009-8765-y

Ribeiro M, Silveira D, Ferreira-Dias S. 2002. Selective adsorption of limonin and naringin from orange juice to natural and synthetic adsorbents. European Food Research and Technology 215(6), 462-471.

Sandon F. 1958. Experimental Designs. By W. G. Cochran and G. M. Cox. 2nd ed. Pp. xiv, 611. 82s. 1957. (John Wiley and Sons, New York; Chapman and Hall, London). The Mathematical Gazette 42(342), 334-334.

Sanjay Kumar, Vijay Kumar, Pankaj Gautam, Dinesh Puri. 2020. Isolation, screening and characterization of fungi from rotten pomelo (citrus maxima) for the production of debittering enzyme naringinase 11(5), pp.1152-1160, Article ID: IJARET_11_05_125.10.34218/IJARET.11.5.2020.

Shivalee A, Lingappa K, Mahesh D. 2018. Influence of bioprocess variables on the production of extracellular chitinase under submerged fermentation by Streptomyces pratensis strain KLSL55. Journal of Genetic Engineering and Biotechnology 16(2), 421-426.

Srikantha K, Kapilan R, Seevaratnam V. 2017. Kinetic Properties and Metal Ion Stability of the Extracellular Naringinase Produced By Aspergillus Flavus Isolated From Decaying Citrus Maxima Fruits. International Journal of Scientific Research in Environmental Sciences 5(3), 71-81. 10.12983/ijsres-2017-p0071-0081

Sukhvir Kaur, Harjot Pal Kaur, Bhairav Prasad, Tejaswani Bharti. 2016. Production and optimization of pectinase by bacillus sp. isolated from vegetable waste soil. Indo American Journal of Pharmaceutical Research, 6(01), 2016.https://www.

Supriya Chatla, Devalarao Garikapati, Abdul Rahaman, Iswarya Obilineni. 2022. Production of Naringinase by using Amla on Solid State Fermentation. Research J. Pharm and Tech 15(3), 1225-1229. DOI: 10.52711/0974-360X.2022.00204.

Tran AM, Nguyen TB, Nguyen VD, Bujna E, Dam MS, Nguyen QD. 2020. Changes in bitterness, antioxidant activity and total phenolic content of grapefruit juice fermented by Lactobacillus and Bifidobacterium strains. Acta Alimentaria 49(1), 103-110.

Vinoth Kumar V, Kayambu P, Revathi Babu 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. 10.1007/s10068-010-0116-9

Xia XK, Zhang YE, Lei SJ, Hu B, Fu CX. 2021. Optimization of process parameters for naringinase production by Aspergillus tubingensis UA13 and pilot scale-up study. Preparative Biochemistry & Biotechnology 52(2), 226-233. 10.1080 /10826068.2021.1925914

Zhang J, Zhang J, Shan Y, Guo C, He L, Zhang L, Ling W, Liang Y, Zhong B. 2022. Effect of harvest time on the chemical composition and antioxidant capacity of Gannan navel orange (Citrus sinensis L. Osbeck ‘Newhall’) juice. Journal of Integrative Agriculture 21(1), 261-272. /10.1016/s2095-3119(20)63395-0

Zhu Y, Jia H, Xi M, Li J, Yang L, Li X. 2017. Characterization of a naringinase from Aspergillus oryzae 11250 and its application in the debitterization of orange juice. Process Biochemistry 62, 114-121.