ナリンギン

Naringin (naringenin-7-rhamnoglucoside), a bitter substance in some kinds of citrus fruitssuch as℃. Natsudaidai and grapefruit, can be hydrolysed by the action of naringinase, resultingin loss of bitterness. It was already reported that some commercial pectinase preparations containnaringinase too. Recently, a few excellent strains of molts, which produce naringinase ofhigh activity, were selected and isolated by several Japanese authors and trial preparations ofenzyme were prepared by them.In order to obtain the fundamental basis for employing the enzyme preparation to agriculturalmanufacture, naringinase was examined using a typical Japanese commercial pectinasepreparation, " Sankyo Sucrase " from Seler-otinia libertiana as well as two kinds of trial naringinasepreparations, A from Asp. niger and B from Asp. usami mut. shirousamii.Naringinase activity of preparations was estimated by deter m ining decrease of naringinand expressed with the rate of its decomposition. Determination of naringin was carried outcolorimetrically by Davis's method using a Hitachi electric colorimeter, type EPO-A, with bluefilter or a Hitachi spectrophotometer, type EPU-2, at 420 mu. Since the reaction product, naringenin,also develops the similar yellow color by the Davis's reagents, coloration of the reactionmixture must be corrected for naringenin to calculate the degree of naringin decompostionby means of the equation of approximation.It was found that "Sucrase" c ontains naringinase activity even though it was less activethan that in the naringinase preparations. Then the conditions for estimating the activity ofthree enzyme preparations cited above were discussed. The final concentration of naringinasepreparations A and B in the reaction mixture was suitable from 0.05 % to 0.10 % and that ofsubstrate, naringin, from 0.0125 % to 0.05 %, when the reaction was performed at 40℃ and pH4.0 for 30 to 60 min. However, since the naringinase activity of the pectinase preparation ismuch less than that of the naringinase preparations, concentration of enzyme must be at leastfrom 2.5 % to 5.0 % and reaction time from 120 to 180 min. Under these conditions, decompositionof naringin by enzyme was directly proportional to reaction time as shown in Fig. 2.Naringinase of the preparation A was already reported to hydrolyse naringin most activ e lybetween pH 4.0 and 4,5. Similary, naringinase of the preparation B and " Sucrase " was reactivebetween pH 4.0 and 5.0. However, activity was gradually reduced over pH 5.0 and belowpH 4.0, as indicated in Fig. 3.It had been also reported that optimum temperature of naringinase of the preparation A wasfrom 40℃ to 45℃. However, Fig. 4 showed that naringinase of the preparation B decomposednaringin most actively at 50℃ to 60℃ but not at 70℃. On the contrary, "Sucrase " exhibitedthe maximum activity at 50℃ and rather pretty high activity at 40℃ too, but very low at 60℃.Activity of the preparation A was also established to be stable from pH 4.0 to 6.0, u p perlimit tested, but inactivated almost completely below pH 1.7. From Fig. 5, it was indicated thatactivity of the preparation B was stable between pH 2.9 and about 7.0, decreased a little frompH 8.0 to 9.0 and inactivated at pH 10.0 at room temperature (20℃ to 25℃). Stable pH rangeof naringinase of " Sucrase " was a little narrower, with lower limit at pH 3.5, than that ofthe preparation B and activity was inactivated at pH 2.2. Whereas activity of the preparationA was stable at pH 8.0 while that was fallen to about 50 % at pH 9.0 and to about 25 % atpH 10.0.Of course naringinase activity of all three preparations was similary diminished by heating.As obvious from Fig. 6, activity was retained at 40℃ and 50℃ but reduced to below 50% at 60℃ for 30 min., while that was inactivated at 70℃.Stability of naringinase at various pHs was varie d with treating temperature and time.For example, Fig. 7 showed that naringinase of preparation B was fairly stable until pH 9 andinactivated sharply over pH 9 at 20℃, whereas that was stable until pH 8 but reduced to about25 % of original activity at pH 8.8 at 30℃. Similar phenomena were also indicated in Fig. 8 inmore detail at 5℃, 20℃, 30℃ and 37℃ concerning the preparation A.Fig. 9 showed that inactivation of naringinase in alkaline med i a proceeded in short time.At pH 10.0, naringinase activity was diminished within 120 min. even at 10℃. However, at pH9.0, activity was retained during first 120 min., but falling of activity was carried out forsuccessive 120 min. Of course lowering of activity did not take place at pH 7.0 and 8.0 within4 hrs. even at 30℃ and 37℃ as pointed out in Fig. 10.Rhamnose and glucose were formed from narin g in by molt's naringinase and enzymaticreaction was strongly retarded by these sugars. Such sugar inhibition disturbs the employmentof the naringinase in agricultural manufacture, since sugar was preferentially used. Therefore,fructose, glucose, sucrose and sorbitol were also examined. As shown in Table 1, all sugarstested inhibited remarkably the reaction, but sorbitol had the most least inhibiting ability.