Studies On The Extraction And Purification Of Ascorbic Acid Oxidase From Squash And Its Application In The Storage Of Oxygen-eliminated Orange Juices

The extraction and purification of ascorbic acid oxidase (AAO) from squash (Cucurbita pepo medullosa) and the possibility of application of this enzyme in the storage of deaerated orange juices were investigated. Studies were emphasized on the extraction, purification, identification and some characteristics of A.AO. The effect of addition of AAO in orange juice on the removal of oxygen, and on the juice quality stored at 4 C and 25 XI? were also investigated. The results indicated:1. AAO isolated from squash was purified by ammonium sulfate precipitation at40 to 80% saturation, acetone precipitation, DEAE-Cellulose chromatography and Sephadex G- 150 chromatography. The purified enzyme obtained has been found to be homogeneous by eltrophoresis on polyacrylamide gel, and identified as AAO by comparing with the standard enzyme from Cucurbita (Sigma Co.) The purification procedure achieves a 173.03-fold purification with a 42.83% recovery of the total activity present in the crude squash juice.2. The thermostability of squash AAO is poor. Incubation of the enzyme at 60C for 20mm resulted in complete loss of activity. All the microbes in the enzyme solution can be removed by 0.45 p m micro filtration membrane without any AAO activity loss. No significant effect of sodium benzoate at 0 to 0.1% concentration on AAO activity was observed.3. The procedure of AAO-addition and pasteurization was designed as following:30.82mg L-ascorbic acid, 0.04% sodium benzoate. 2 units of AAO and about280m1 orange juice were fed and sealed in glass bottle with crown-cap. Oxygen dissolved in orange juice was eliminated by AAO at 25 ?C in 2 hours before pasteurized at 70(1? for 30mm.4. The storage experiment of enzymatically deaerated orange juice indicated that:(1)Elimination of oxygen dissolved in orange juice by AAO before storage resulted in greatly increased retention of L-ascorbic acid. Contrast to the controlafter 360 days StOrage at 4'C and 25'C, AAO-added orange julce has a 22.2l%and 3l.l0% higher L-ascorbic acid Hon resPecthely (2) ContraS to thecontrol, oxygen-eliminated orange juices resulted in the longer lag period beforebrowning and the lower broWnin pigments formation. The lag period beforbroedng increasd 25 days stord at 4'C,and 30 days at 25oC. The incrase ofbrowning index (420IUn) after 360 days storage only 36.90% relative tO the controlat 4'C,and 56.92% at 25'C. (3) No significant senSOry differenCe betWen theorange juice added only 2 units AAO per bottle and the cOntrl withut mpe.But during stOrage, AAO-added orange julce could iInProve thq sensory quality oncolor and flavoL (4) No significant differences of tuthidity soluble solid, titratabeacidity solid acid ratio, and aerobic baCterial coun between AAO-added orangejuice and the control, which could be attributed tO the AAO addition or oxygenelimination.5. No sighfican differenes of sensory quality was observed betwen orangejuice added 2 units of squash AAO in differen purification level and the control.The rate of oxygen elimination of lowe Purification level AAO was s1ighti haerthan that of higher one. lt is advisable to aPPly crude AAO or lower purified AAOto oxygen-elimillated orange julce manufacthe, so as tO reduce the production coSt.