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Screening, Producing-naringinase Characterization And Breeding Of High-yield Strain From Aspergillus Niger

Posted on:2010-07-01Degree:MasterType:Thesis
Country:ChinaCandidate:H G ChenFull Text:PDF
GTID:2121360275497078Subject:Food Science
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Naringin(naringenin 7-rhamnoglucoside) is present in the citrus plant of Rutaceae family, specially grapefruit, Guanximiyou, bitter orange. This flavonoid is by far the most bitter. The concentration in the Pomelo fruit is from 100 ppm~370 ppm, which has been a major limitation in the commercial acceptance of juice. The naringin level can be reduced by technologies such as adsorptive debittering, chemical methods, treatment withβ-cyclodextrin and enzymatic debittering. The enzymatic debittering of citrus fruit juice has some advantages, such as mild work conditions, noncontaminant operation and absence of secondary products. The debittering process could be most cost effective and economically viable if naringinase production is achieved industrilly using microorganisms. Currently, it lacks of commerical naringinase in industrial application, due to be short of high-yield- naringinase strain.In earlier research, our study group haved isolated 40 production-naringinase strains, used grapefruit bitter component as sole carbon source. In this paper, we screened a high-yield, safe producing-nanringinase strain from 40 wild strains. Subsequently, we researched the characterization of strain p??cting-naringinase, further, srceening high-yield mutants.We obtained three high-yield strains, named DB054, P-127 and 4-4-1, from the 40 wild strains. DB054 with the naringinase activity of 108.6 U/mL, identified as Aspergillus Mich.ex Fr. P-127 with the naringinase activity of 103.6 U/mL, identified as Penicillium Lk.ex Fr. 4-4-1 with the naringinase activity of 114.3 U/mL, identified as Fusarium Lk.ex Fr. separately.We studied characterization of A. niger DB054 producting-naringinase. The results show that the naringinase is exoemzyme and inducible enzyme. We obtained discipline of the A. niger DB054 producing-naringinase via shake flask experiments and 5 L bioreactor Fermentatio. A.niger DB054 has the phenomenon of Carbon Catabolite Repression(CCR). Glucose suppress the wild strain to biosynthesis naringinase. The mycelium modality has a significant impact on strain A.niger DB054 produceing naringinase. Filamentous mycelium is better to prdcuce naringinase than mycelial bead. To adjust the shaker and add emulsifier to medium can alter the mycelium modality or epicyte structure, enhance the membrane permeability, improve the naringinase to be liberated to extracellular.Studies on the mutation breeding of A. niger DB054, with a view to obtain the naringinase high-yield mutant. The spore-sprouted Aspergillus niger 8-hour were mutagenized 40 min by 30μL/mL ethyl methane sulphonate. 2-deoxy-glucose was used as inhibitor to eliminate low-yield mutants, added 5 mg/mL in the media. Plate transparent circle was introduced screening naringinase high-yield strain by Davis method. 11%mutants yield 40% higher of naringinase than the original strain. A high-yield strain, T-226 with the highest naringinase activity of 373.4 U/mL was finally selected from these potential high-yield mutants after rescreened by shake flask fermentation twice. When the T-226 strain was fermented in 5 L bioreactor, the enzyme activity could reach to 631.9 U/mL after 84 h. Thus, the established screening method is highly efficient to isolate naringinase high-yield mutant of A. niger...
Keywords/Search Tags:Aspergillus niger, Naringinase, Identification, Property of enzyme production, Induced mutation breeding, Fermentation
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