Distribution of Ascorbate Oxidase in Citrus Fruits
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Food Sci. Techno!., Int., 2 (3), 154-156, 1996 No te Distribution of Ascorbate Oxidase in Citrus Fruits Nazamid bin SAARI,1* Shuji FUJITA,1 Selji YAMAGUCH12 and Tetsuzo TON03 1 Laboratory of Food Science, Faculty of Agricu!ture, Saga University, Saga 840, Japan 2Saga University Farm, Kubo-izumi, Saga 849, Japan 3 Department of Food Science and Nutrition, Nishikyushu University, Kanzak~ Saga 842, Ja pa n Received November 24, 1995 Twenty cultivars of citrus fruit were harvested at two stages of development: young fruit (weight about 2.5 g per fruit) and fully ripe fruit of which the ascorbate oxidase (AAO) activities were determined. Among the different cultivars, the specific activity of J~O in the young fruits ranged from 47 to 2120 units per mg protein, while fully ripe fruits had AAO specific activity ranging from 18 to 97 units per mg protein. Significant differences were also observed between tissue types in the AAO specific activity with the highest activity in the albedo (64-70%), followed by the flavedo (26-34%) and the pulp (2-3%). Further examination of the seasonal changes in ~~O specific activity in the peel (flavedo and albedo) of Satsunra mandarin fruit indicated the highest AAO activity when a fruit weight was between 2-4 g. The AAO activity then decreased as the fruit became more mature. Similar changes in the pattern of J~O specific activity were also observed for the different harvest years 1994 and 1995. Keywords: ascorbate oxidase, ascorbic acid, citrus fruit, distrlbution Ascorbate oxldase (AAO, EC 1.l0.3.3) is a well known orange ( Citrus sinensis), sour orange ( Citrus aurantium), enzyme which catalyzes aerobic oxidation of L-ascorbic acid Sanbokan (Citrus sulcata), Beni-amanatsu (Citrus natsu- (AsA) to dehydroascorbic acid. The enzyme was Isolated daidai), Beni-hassaku ( Citrus hassaku), Asahikan (Citrus from cucurblts vegetables such as zucchini squash (Dunn et asahikan), grapefruit ( Citrus paradisi), Tosa-buntan ( Citrus a/., 1951; Tokuyama et al, 1965; Lee & Dawson, 1973; grandis), Meyer lemon (Citrus meyerii), Iemon (Citrus Marchesini & Kroneck, 1979; Avigliano et a/, 1983) and limon), and Tahiti llme ( Citrus aurantlfolia). cucumber (Nakamura et a/., 1968; Aikazyan & Nalbandyan, A cetone powder preparation and enzyme extraction 1979; Sekiya et al, 1990). Recently, the cucumber AAO has Fruits were prepared as acetone powders by blending 25 g of been used as an analytical tool in the enzymatic method for frozen tissues with about 100 ml of ice cold acetone and ASA determination. In a previous paper (Saarl et a/, 1995), filtered under suction. The residues were washed repeatedly we reported the determination of ASA by Satsuma mandarin with the cold acetone_ The acetone powders were dried at AAO, In addition to the vegetables, there is a possibility that ambient temperature to remove residual acetone and stored other citrus fruits might also serve as a good source of AAO. at -20'C prior to use. AAOS were extracted by agitating 0.05 Because very few studies have been conducted on the g of acetone powders with 25 ml of Mcllvaine buffer (pH 7.0) occurrence and characterization of AAO from other citrus at 4'C for 30 min. The slurries were filtered through Toyo No. fruits except for Satsuma mandarin (Tono et a/, 1985), 2 filter paper (Advantec Toyo Co., Tokyo) and centrifuged at Sharnouti (Kanner et al, 1981) and Pineapple (Vines & 15,500Xg for 20 min at 0-4'C. The resulting supernatants Oberbacher, 1963) oranges, this work attempted to examine were used as the enzyme solution. the distribution of AAO activity in various fruit cultivars. AAO activity and protein assay AAO activity was measured by the spectrophotometric method of Tono & Materials and Methods Fujita (1982) summarized as follows: The assay mixture Citrus fruit Twenty cultivars of citrus fruits were contained 0.5 ml of 0.8 mM ascorbic acid in 2% metaphos- collected from the experimental orchard of Saga University at phoric acid (HP03) and I .O ml of AAO solution. After two stages: young fruit (mid-June, weight about 2.5 g per incubation at 35'C for 5 min, the reaction was stopped by fruit) and fully ripe fruit (October-November). The fruits adding 3.5 ml of 2% HP03' One unit of AAO activity was harvested were named according to Tanaka's system ( 1948): defined as that causing an increase of 0.01 in absorbancy per Clementine (Citrus reticulata), Ponkan ( Citrus reticulata), min at 243 nm. Protein was assayed by Hartree's method Shiikwasha (Citrus depressa), Satsuma mandarin (Citrus (1972) with bovine serum albumin as the standard. unshiu), Hanayu (Citrus hanaju), Hyuganatsu (Citrus Tamurana), Miyauchi-iyo (Citrus iyo), Funadoko ( Citrus Results and Discussion funadoko), Yoshida navel orange ( Citrus sinensis), Valencia Comparative studies of A A O activity in various citrus fruit The twenty cultivars of citrus fruit analyzed in this * Present address: Faculty of Food Science and Biotechnology, University of Agriculture Malaysla, 43400 Serdand. Selangor Darul Ehsan, Malaysia. study are either commercial citrus or used as genetic resources Citrus F~ruit Ascorbate Oxidase 155 3000 1 40 1 Zo = _ ZSOO o c ~ Q, ~ 1 Oo ~ I o o'a * E o)~ 2000 E \e] 80 Z \u, = ~E I SOO ~)~ 60 = p :~ > ~_ ,:; > u 40 I Y v'Is I OOO O~ O Zo < l SOO o .~ t') o a5 i_ .~ C-u)_ ~!~~S1:1"c~:]~ c~_ ,u) (:~ ~f~ u) a5 u) ':~c: E gg c~ :':~ ,: .u) :!~ 95 4J f~~: ~ '~~O~")q::, f~ '~ ' 'dl~O .~,~ ~ll'~; c~RS 'e '~:j 4J.~~,v) ~'c'Cg~~~\;,:~~S5~~g,q) ::l O:~J_.~ ,,)- c:,gL;~ ~ '9 u).~;~.~ ~; ~ C5 J: l~' .h .u)_ .~ - .-~ v) ~s :: ~ 95 l~a9 CL;9e C: C:E ~;:~::' ,,= ~S9'~~L; Oift~95,:-t'):, :~_V :,v) ag~:q! $J '~C: ':~u)Q):~v)u) f-' .~t" :~ '~ . ~;O g, _'!! aDc~~~'.~=tS E-~1(J J::~~i L;9a ebE gg ,9:' - C~ .~:'.-.u):'L; u'u) :' ~Odrl ~.~ ~ •~11~'~ 'J ~ C: ':~'_u) ~ a)~\q' L;815~~:,u) '- o L a8 v)~1~L::'La5._ u) v) ,~ y):j -:O ~!'~E'-~lS'Ji~ '- ~'L;v'- ~ ~u)~.~~ ~,~'F9 . ~~L;S~1 ~ _ ,: :, ~~ ~ (J :,O :D q't'~j~/~:'~'t!: ~ O\1:tl{::'~'~ ~.~u ~'):$ a! ._ ._ 95~]l~~ ae L u) ': ,a '-' L~i ~u;~1:'vc::L!a'::L ~L IJ~,-•(S":~~~C5$J-'LE.~v;~~ L! ~' . :~ ._ V'J ~5\-'a' 9'u)~LLL~; '- ~~'- ~;L ::v)~ ,JL ~":$ a9 95 f^ r _ y):~~(J\J~o~~c .~I~~a, '~ ._ c~C: ~'):'~~~~4Ji~~~~~~U\J~:~,~L~u)~C54J\_'~u)~~~ ~:~: . ._ ._ 95L: L e)E •:::t:':~'-":5L~~~_:"~L:- CJ '~'\/ v) ~, 4J h*' ~ .JL;'~"U Q'~tg~L)'-'- c:c~V 4J~ ~,y: >1'J~'9 ~ O) OIJ~~ ~$J '•~~9, :~u) c,U ._ _ ._ .~ •_ 'th_ ._~~~Ec9~4J ._ IJL='T O '~ c ~ c = = '!\J4J ~EO o ~u)~L~~=:5\/ \-'v) c5=~ '~'v'Uc5L!~ E c9ct'J::'0~a"JE 1~'~ :'u' O(J~,5Q'~t~;~CJ'-'~rL\_'>,\Jc:f L u'_4'(J__:'(J c:c'gE= ~ u= ~ ~ 'O o o cu ._ , 1: :: c a',~cDE(J4J oO)cc ~'O~' E~~u'cgQ"!1;:'-'* CEI CDc: LJD'~ '~ s:.~ !x c5 . 'DOIJ ~,\J c5'J oc 'Uu'C::~:'a'Ccl'-'4JcE~~ ~=0~ ~;~~>bc_l_^u'0'~0c~fQ'a'u'Locg~0==cc'v,~u'>,E!Esa 'E CJ su o~'~~ c5~~.Ec:Ic~L u'O '~ '- cJe~ I~oaJ)c9 ~ O~ U) c/)U)COo0<(!'H:~ ==cDE~~~~ .-I'C .- ,: .- ~~~~~c ~ = c~f c5 >,'- = >~~ '9~~~e UOAl' ~ c:1 ~~•~a' a'<u' d5 O a'c o a'c5 Fig. 2. Comparison of' AAO specific'- activity a' in fully ripec!' fruit of citrus := CD ,!f U' >1 E; cultivaTS. The fruits were preparcd as acetone powders and the activities were Fig. l. Comparison of AAO specific activity in young fruit ofcitrus cultivars. measured as described in Materials and Methods. AAO activity was expressed The fruits were prepared as acetone powders and the activities were measured as specific activity. as described in Materials and Methods. AAO activity was exprcssed as specific acuvity. 2;)'o in Japan. A comparison of AAO specific activity in the young fruit of varlous citrus cultivars is given in Fig. l. The results showed that the specific activity of AAO of the different 200 cultivars varied widely from 47 to 2120 units per mg protein. The relatively higher specific activity ( 1 570-2 120 units per mg ~o* 5C CL protein) was observed in the cultivars of Yoshlda navel c;) E orange, Valencia orange, Beni-amanatsu, Beni-hassaku, u) \d~ Asahikan and Tosa-buntan, while the cultivars of I)c OO Clementine, Ponkan, Shiikwasha, sour orange and Tahlti )~ ~> lime had a relatively low AAO activity with values of about d~ 1/3 or less of the highest range. Satsuma mandarin as well as o 5C < other citrus fruits had a fairly strong AAO activity. < Figure 2 shows a comparison of AAO specific activity in o the fully ripe stage of various citrus fruits. All the ripe cltrus Beni- Tosa- Grapefruit fruits examined showed variations in AAO activity ranging amanatsu buntan between 18-97 units per mg protein. The cultlvars of sour Fig. 3. Comparison of AAO activity in various parts of fully ripe citrus fruits. orange, Sanbokan, Beni-amanatsu, Iemon and Tahiti lime Flavedo ([:•1), albedo (It~:.~) and pulp (1) were removed from each ofthe fruits. The tissues were prepared as acetone powders as described in Materials and showed a relatively high AAO activity (80- 100 units per mg Methods. AAO activity was expressed as s pecific activity. protein). The lowest range of AAO activity was observed in the cultivars of Satsuma mandarin, Hanayu.