Studies On The Fermentation Of Naringinase From Aspergillus Niger DB056

Naringinase is a kind of glycosidase, which can hydrolyze naringin to naringenin. It is anenzyme containing α-L-rhamnosidase and β-D-glucosidase. Although, naringinase has manyimportant applications, such as debittering of citrus fruit juice, increasing aroma of grape wine,industrial preparation of rhamnosidase and flavonoids, its application has been limited duing tolow productivity and high producing cost. Therefore, researches on establishing efficientfermentation procedure for producing naringinase have important significance.The aim of the present work was to investigate the effects of medium composition andculture condition on naringinase produced by Aspergillus niger DB056so as to enhance thenaringinase productivity. Main content were focused on studing fermentation process in250mLflask and7L bioreactor, as well as procedure scale-up. In this study, α-rhamnosidase activity andnaringinase activity were adopted to evaluate the effecience of procedures.As showed by results, naringinase excreted by Aspergillus niger DB056was an inducibleenzyme. Among various carbon and nitrogen sources used, naringin and corn steep liquor werethe most effective. Besides, emulsifier Triton X-100could promote production of naringinase.The optimum medium of flask-fermentation naringinase was: naringin3.5g/L, corn steep liquor4.0g/L, MgSO₄·7H₂O1.0g/L, KH₂PO₄1.0g/L, KCl0.5g/L, CaCl₂0.1g/L, emulsifier TritonX-1001%（V/V）. Using the optimum medium, the activities of α-rhamnosidase and naringinaseincreased by42.4%and147.8%, and the enzyme activities of α-rhamnosidase and naringinasereached to994.58U/mL and276.47U/mL, respectively.Culture temperature, mycelial morphology, initial pH of the medium, inoculation amount,medium volume in flasks had an important impact on naringinase production. The optimumfermention condition of flask-fermentation naringinase was: initial pH of the medium8.0, adding5beadings,45mL medium in250mL flask, inoculation amount7%, culture temperature34℃and rotate speed of table concentrator190r/min. Under these conditions, the activities ofα-rhamnosidase and naringinase increased by72.3%and78.0%, and the enzyme activities ofα-rhamnosidase and naringinase reached to1076.32U/mL and420.68U/mL, respectively.The results of the fermentation naringinase in7L bioreactor showed that20g/L naringinwas better than other two carbon sources （20g/L glucose and20g/L shaddock peel powder） forAspergillus niger DB056producing α-rhamnosidase and naringinase. Furthermore, highernaringin concentrations in the medium could improve enzyme yield. One-time addition of naringin led to higer enzyme activity. Fixed pH6.0, agitation speed700r/min andfermentation temperature32℃were found to be better choice in terms of naringinaseproduction.The maximum α-rhamnosidase activity harvested in7L,20L and200L bioreactor were1038.20U/mL,932.21U/mL and1069.30U/mL, respectively. The maximum naringinaseactivity harvested in7L,20L and200L bioreactor were535.02U/mL,617.57U/mL and727.44U/mL, respectively. The result that the enzyme yield of scale-up test reached that of labexperiment indicated the procedure was easy to scale up.The results elucidated the effects of medium composition and culture condition onAspergillus niger DB056producing naringinase. After present work, procedures of high-yieldfermentation for naringinase was found to increase enzyme yield. It laid a well foundation fornaringinase industrial production.