Food Structure

Volume 8 Number 1 Article 9

1989

The Effect of Irradiation on Starch Content in Golden Delicious Apples

E. Kovacs

A. Keresztes

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Recommended Citation Kovacs, E. and Keresztes, A. (1989) "The Effect of Irradiation on Starch Content in Golden Delicious Apples," Food Structure: Vol. 8 : No. 1 , Article 9. Available at: https://digitalcommons.usu.edu/foodmicrostructure/vol8/iss1/9

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THE EFFECT OF IRRADIATION ON STARCH CONTENT IN GOLDEN DELICIOUS APPLES 1 2 E. Kovacs and A. Keresztes

1oepartment of Microbiology, Central Food Research Institute, 1022 Budapest, Herman Ott6 u. 15 . Hungary

2oepartment of Plant Anatomy, EOtvOs Lorand University, 1088 Budapest, MUzeum krt . 4/A Hungary

Introduction

Starch content in apple (Golden Starch begins . to accumulate in a Delicious) skin and flesh was studied as developing apple fruit 3 to 4 weeks after a function of radiation dose (0, 0.5, 1, bloom . Over the next 2 months it accumu­ 2 and 5 kGy) after 8 weeks shelf life lates to a maximum value and subsequently (l60C, 80-90% RH). declines as the fruit matures. Starch Starch was generally not found in synthesis and decomposition are modified the flesh with the exception of the 5 kGy by different circumstances. Starch is a sample. Starch grains were observed, how­ reserve ~arbohydrate that is eventually ever, in plastids of the epidermis and quantitatively converted to soluble especially in the hypodermis in correla­ sugars, that are stored in the vacuole or tion with the radiation dose. 1 kGy dose metabolized by the cell through respira­ caused a significant effect (P~ 99,98%) tion. on retardation of starch breakdown meas­ Simons and Chu (1982; 1983) investi­ ured by electron microscopic morphometry gated corking, a physiological disorder and chemical analysis. of apples. Cytoplasmic membrane break­ Analysis showed that increasing radi­ down occurred in cortical cells adjacent ation dose increased glucose concentra­ to the vascular bundles at least 45 and' tion in the flesh. In the skin the con­ 60 days prior to fruit maturation . Cell centration of all three sugars, glucose, walls were thick in fruit exhibiting fructose and sucrose, increased with ir­ corking. Starch accumulation occurred radiation up to 2 kGy, but they decreased within l ocalized areas and was attached with higher doses. to the cell walls. Starch grain accumula­ tion on cortical cell walls and prolifera­ tion of starch grains in cells having thick walls was noticed 30 days before maturation (Simons and Chu, 1982; 1983) . Profiles of tissue development and sub­ sequent breakdown indicate that this Initial paper received June 21, 1988 physiological disorder has developed in Manuscript received December 15, 1988 formative and developmental stages of Direct inquiries to E. Kovacs growth, with extensive tissue breakdown. Telephone number: 36-1-152028/157341 Electron microprobe studies provided evidence that both potassium and calcium concentrations were low in all affected tissues, although the potassium level was higher than calcium in most of the sampled areas (Simons and Chu, 1982) . Mahanty and Fineran (1975) investi­ KEY WORDS: apple, shelf-life, irradiation, gated the cellulbr ultrastructure of Identification of irradiation treatment, control and calcium sprayed apples which transmission electron microscopy, ultra­ were cool-stored for 3 months. Cells of structure, chloroplast, amyloplast , starch, the control sample were less well pre­ prolamellar body-like structures, served compared to those of calcium sugar-starch interconversion. sprayed apples. Chromoplasts of the epidermis had prominent plastoglobuliand thylakoids which were scattered or some­ times alveolar rather than compacted. Cells from hypodermis showed increased lysosomal activity. Mitochondria and

67 E. Kovacs and A . Keresztes pl~stids containing starch appeared normal . In the chromoplasts of the calcium sprayed apples, compacted 1a fO(.tJEJJ b£LlCIOUJ thylakoids and plastoglobuli were present . The inner cortical cells had a large central vacuole , and the cytoplasm sur­ rounding this was usually granular and contained numerous plastids with starch . Gamma-irradiation has been reported to increase the sucrose content of potato tubers and s weet pota to roots (Hayashi and Ka wash Jma , 1982a , b ) . Sucrose accumula ­ tion was dep0ndent upon irr adiation dose , and the preferable dos e range was between 1-2 kGy for roots of sweet potato . The sucrose content of irradiated vegetables and fruits increased for a long period after irradiation . It v.•as suggested that an enhanced activity of sucrose synthase and sucrose-phosphate synthase played an important role in the sucrose accumulation in irradiated potatoes (llayashi and Kawashima , 1983 ; Hayas hi and Aoki , 1985 ) . Kovacs and co - workers (1988) inves­ tigated the microstructure of calcium treated and irradiated apples, a short time after treatment and after 3 months ~ubsequent cool storage . All tr~atments (calcium , irradiation , calcium trcatm~nt combined with irradia­ tion ) maintained th~ basic comparlmcn ­ tation of the cells in to cytoplasm, plastids and vacuoles . By con trast , and mainly .in the epidermis , the integ ­ rity of vacuoles had been lost in rhP control by lhe end of storage . Calcium treatment either alone or in combination with irradiation resulted in a som~what better preservation than irradiation alone. Irradiation and combined treatment preserved starch . The difference was ~vident already 4 days after the treat:l'rent , and the effect lasted for at least 3 months . It is known that metabol~c disorders Fig . 1 . Demonstration of starch by ~n apples always cause a decreased starch starch iodine test in apple (Golden breakdown. Early theories on cause of Delicious , stored at 16oc , 80-90\ Rll) bitter pit all were based on starch that a : 5 days after irradiation ; b : 8 weeks remained in the tissue reviewed by Faust after irradiation and Shear ( 1968 ) . The aim of the pr esen t work was to and 5 kGl doses , the dose ra Lc was study the effect of irradiation on t he l kGy . ff ) u sing facilities at the ultrastructure of plastids and the starch­ Instit u t e of Isotopes of Hu ngarian sugar intcrconversion in the apple . An Academy of Sciences . After irradiation , identification method of irradiation samples were stored for 8 weeks ( shelf treatment in apple has also been sought . life at 160C , 80-90% relative humidity ME_erials and Nethods [RH] ) . This tempera lure was chosen to hasten the physiological and biochemical Raw materials and treatments changes . The ripening stages of apple ----;;:ppfes (cv . Golden Delicious) were and the short time effect ( 5 days ) of harvested in an orchard of D~nszentmikl6s irradiation was monitored by the starch Horticultural Station near Budapest , iodine pattern index (Reid ct al . , 1982) Hungary . Harvest time was 100 - 1 10 days {Fig . 1 a . ) . All ultrastructural and following full bloom apples arc harvested chemical investigations were carried out in Hungary in this stage for winter ­ 8 weeks after treatments when the appl e storage . After picking at the green skin was yellow and the starch disap ­ skinned stage fruits were irradiated by peared from the fruit flesh (except a Co - 60 radiation source (0 , 0 . 5 , 1 , 2 5 kGy ) ( Fig . lb ) .

68 The effect of radiation on starch

Fig. 2. Ultrastructure of plastids in the epidermis of samples stored for 8 weeks (Golden Delicious appl e ) (a: control; b: lkGy; c : 2kGy ; d: 5 kGy ), s ~ starch grain. Bars = 1 ;urn . Electron microscopy and morhpometry enlarged prints , in 24 - 27 plastids from the outer hypodermis (h - h ) . The \!Olurre Fixation was carried out in 6 % 1 3 of starch grains was expressed as a ~~;~~h~~~t~~~i~~~Y~~ ~:~ ) 0 f~;s2MhK;~a4°C. percentage of plastid volume. After thorough washing in the buffer , petermination of sugar samples were postfixed in 1% (w/v) Os04 for 1.5 h, dehydrated in an acetone 10 g of the skin or flesh sample series and embedded in Spurr ' s resin. was homogenized with 50 ml of 80 % Using flat molds , samples from the skin ethanol for 5 minutes. Each filtrate was were oriented for subsequent transverse evaporated to dryness to remove ethanol , sectioning. then redissolved in 100 ml of water. Sections were cut with a Porter-Blum Each solution was analyzed for soluble ultramicrotome equipped with a diamond sugars by high pressure liquid chroma ­ knife, post - stained with uranyl acetate tography (HPLC) using a LABOR MIM and lead citrate and examined in a (Hungary) apparatus combined with a Tesla BS 500 electron microscope ope.rated Beckman pump (USA) and RI detector _ 5 at 60 kV. (Beckman) , having sensitivity of 2xl0 Starch grains were measured with a square lattice grid superimposed on ~~~x~~~u~) ~a~p;~~~~~i~~NH 250 ~~~~~h

69 E . Kovacs and A. Keresztes acetonitrile-water ( 75 : 25 ) as mobile phase at 1 ml.min-1 flow rate.

~etermination of starch 5 g of skin or flesh of apple was horrogenized with 100 ml of 1 N HCL for 5 minu tes . The homogenate was refluxed on a boiling water bath for 3 hours, made up to 1000 ml , glucose determined by HPLC and expressed as starch .

Results a nd Discussio n The skin of stored apples examined after different radiation doses showed several ultrastructural changes. Data concerning the epidermis and hypodermis will be evaluated separately. Plastids in the epidermis of the control sample were roundish with many electron ­ translucent vesicles . They generally contained dense inclusions but not starch grains (Fig. 2a) . Unevenly dis ­ tributed starch grains occured in the epidermis of samples irradiated with 1 kGy or higher doses (Fig . 2b-c ) . The normal organization o f the cell was broken down by an irradiation dose of 5 kGy (Fig . 2d) . Pl astids in hypodermal cells of the control sample contained small and l arge grana. In the outer hypodermis 1 ~~J~sl~~!~~:l~~~ ;ta;~~egr=~~~t~~e rare and small. In deeper hypodermal layer (h 2-5• according to Bain and Mercer, 1963) plastoglobuli are more electron dense , and plastids are free of starch (Fig . )a). In the 0 . 5 kGy sample the lumina of thylakoids in hypodermal plas­ tids were often electrondense (Fig . 3b) . Structures similar to prolamellar lx:xlies could be seen in inner layers of hypo ­ dermis {Fig . 3c). Earlier this structure was conside ­ red as a characteristic feature of the etioplasts ; later it was established that prolamellar bodies also appear to differentiate following exposure to continuons red light {Boardrran et al . , 1971 ) or white light (Rascio et al. , 1980). They are formed when the equilibrium of the membrane components (or their synthesis ) is disturbed. We suppose , that in our case the prolamellar body- like structure is the result of disturbance of the membrane equilibrium caused by ionizing radiation .

Fig . 3 . Ultrastructure of plastids in the hypodermis of samples stored for 8 weeks (Golden Delicious apple) (a : con ­ trol ; b , c : 0 . 5 kGy), t = thylakoid , g = granum , pb = prolamellar body-like structures. Bars = 1 JUm .

70 The effect of radiation on starch

Table 1 Starch content determined by morphometry and chemically i n hypodermis of Golden Delicious apples stored for 8 weeks after irradiation

St a r ch content Dose (kGy) as % of plastid as % of glucose after hydrolysis

X x 0 . 38 ~ 0.21 2 . 83 t 0 . 92 0.5 1. 52 ~ 0 . 69 2 .85 : 0 . 91

15.82 : 2 . 39xxx 6.32 ~ 0.90 XXX

28 . 53 ~ 3 . 16 XXX 6. 56 : 0 . 85 XXX

38 . 21 : 3 . 72 XX 1~ . 06 : 2.40 XXX

average; s ~ standard deviation; xx significa ntl y differed from the control , at level 95 % xxx significantly differed from the control, at l e vel 99 %

Plastids of t he hypodermis of 1 kGy apple cortex contained small and large starch grains {Fig. 4a ) in addition to occasional p r o l amellar body-like s ruc­ tures. Apples irradiated with 2 kGy contained conspicuous starch grains, mainly in the outer hypodermal cells (Fig . 4b) , but cells with damaged mem­ branes also occurred . Large starch grains remained in the outer layer of the hypo- ~~r~~~ l~~!r 0 ia~=~ ~h~~~o~f~:~!a~~~h~~:s- amounts of starch were observed (Fig. 4c). According to the results of morphometry, the starch content of the hypodermal cells had already differed significantly at an irradiation dose of 1 kGy from the control (Table 1 ) . According to the chemical analy s i s more starch remained in the skin at a dose 1 - 5 kGy. The data of morphometry and chemical analysis correlate well . Fruit flesh was also examined for starch grains . Both in the control and irradiated (0 . 5 - 2 kGy) flesh , the small plastids were free of starch (Fig. Sa~) . Starch grains were present in cells of 5 kGyirradiated apple ( Fig . 5e ) . This was also evident from the iodine test (Fig. lb) .

Fig. 4 . Ultrastructure of plastids in the hypodermis of samples stored for 8 weeks (Golden Delicious apple ) (a : 1 kGy , B : 2 kGy , c : 5 kGy) , s =starch . Bars : 1 ,AJID .

71 E . Kovacs and A. Keresztes

5a

The analysis of sugars indicated that glucose concentration increased in the flesh with the increasing radiation 5e doses , which means that most of the decomposed starch was con verted into g lucose during storage after irradiation (Fig . 6 ). At the same time in the skin of apple the glucose , fructose and sucrose concentration increased only up to 1 and 2 kGy , then decreased (F ig. 7 ) . Both phenome na have also been observed in s weet potato roots as a function of radiation dose (Hayashi and Kawashima , 1 982a , b. ). Several factors may have influence on preserving starch in the irradiated fruits. These include Ca mobilization from the f l esh to the skin (Kovacs et al ., 1985 ; Kovacs and Zackel , 1987) and effects involving the integrity of membranes fRoman1 et al., 1 968; Isherwood, 1 976} . The question is further complicated by the fact that Ca also has an effect on the state of the membranes . Fig . 5. Ultrastructure of p l astids in Further investigations are needed for the flesh of samples stored for 8 weeks underst a nding of the interaction of Ia ' control; b ' 0 . 5 kGy ; c' 1 kGy , these factors . D: 2 kGy, e: 5 kGy ), s = starch . Bars : 1 p m.

72 The effect of radiation on starch

FLESH

LSD 5-. =2 ,33 SKIN

15 LSD,., =2.23

fructose

2 dose lkGyl dose { kGy)

Fig . 6. The effect of irradi ation on the Fig . 7. The effect of irradiation on the sugar content in the flesh of apple stored sugar content in the skin of apple stored for 8 weeks for 8 weeks

Acknowledgements Hayashi T , Kawashima K. (1982a) . The effect of gamma - irradiation on the suc­ Mr . B . B6ka , Ms. J . Bencze B6cs , rose content in sweet potato roots and potato tubers . Agri c . Biol . Chern . 46, Ms. K. Lehofer and Ms . G. Gacsi are ~ed for their participation in the technical 1475 - 1479 . - work. Hayashi T , Kawas h i ma K . (1982b). Accu ­ mulation of suc r ose in gamma irradiated References ~~~: g~~~to root s . J . Food Sci . !l• Bain J M, Mercer FV . (1963). The submi cro ­ 2 scopic cytology of superficial scal d , a Hayashi T , Kawashi ma K . (1983). Activi ties ph ysiol ogical disease of apples , Aust . of e n zymes of sugar metabolism in g amma J. B iol . Sci . ~ , 4 4 2 - 4 4 9 . irradi ated potato tubers . J . Food . Sci . Boardman NK , Anderson JM , Kahn A , Thome SW , ~ . 12 4 2 -12 45 and 1 2 49 . Treffry TE . (1971 ) . Formati o n of photo­ Hayashi T , Aok i s. (1985 ) . Effec t of synthetic membranes dur i ng chloroplast irradiation on car bohydrate metabol ism development. In : Boardman NK , Linnane AW , responsible f or s ucrose accumulation in Smillte RM : (eds) . Anatomy and biogenesis potatoes . J . Ag r ic . Food Chern . ll• 14 - 17 . of mitochondria and chl oroplasts . North - Hol l and Publ . Co ., Am st erdam - ~ Isherwood F . (1976 ) . Mechanism of starch­ pp . 70 - 84 . sugar interconvers ion in Solanum tuberosum. Phytochemistry .!2_ , 33 - 41 . Faust , M. Shear CB . (1968 ). Corking dis ­ orders of apples : a physiological a nd Kov~cs E , Zacke1 E . (1987). The effect of biochemical review . Bot . Rev . .!i_ , 441 - 469 . irradiation and calcium treatmen t on the shelf - life extens ion of different appl e varieties ( in Hungarian ) . Kertgazdasag .!.2_ , 22 - 30 .

73 E . Kovacs and A . Keres ztes

Kovacs E , Keresztes A, Kovacs J . ( 1985). D. Lewis : How many samples did you exam­ Effect of low dose irradiation and Ca ine a nd how did you select the p l astids? treatment on the mi c r ostucture and the shelf -life extens i o n of fruits . Inter ­ Authors : Two fruits were selected from national Symposium o n Food I rradiation each group , 10 - 12 sampl es were cut ou t Processing , IAEA/FAO, Washington D. C . USA. , of t he s kin a nd flesh of each . Each 4 - 8 March 1 985 , Poster, I AEA (SH-271/198 ), plasti d in a mi crograph was measured for Extended synopses . star c h and t he micrographs were sel ec t ed on t he basis o f t heir quality . Ko vacs E , Ker e sztes A , Ko vacs J . ( 1988 ). The effect of gamma irradiatio n and cal ­ T. Hayashi : How d i d t he con ten ts of starch c i um t reatme nt on the ultras t r ucture of a nd sugars change d u ring s t ora ge aft e r i r radiation? f~i ! ~s a nd pears. Food Mic rostruc . 2, Authors : For the first r un we did not Mahanty Il K, Fineran BA. (1975). The examine time-dependence of these proces ­ effects of cal ciu m on the ultrastructure ses , bu t it wi ll be really impor tan t of Cox ' s orange apples with reference to when developing a method from these bitter pit disorder. Aust . J . Bot . 23 , resul ts for i dentifying irradiated ~les . 55 - 65 . - B . Fineran : What is the large dark area in your Figure 2a at the bottom right of Rascio N, ~~riani -c o l ombo P , Orsenigo M. ( 1980 ) . The u l tras t r uctural development the plast id? \-lhat are the other rounded of plastids in l eaves of maize plants struct ures in this plastid? The latter exposed to continuous illumination . look l i ke l ipid material to me . Protopl asma ..!..Ql_ , 1 31 - 1 39 . ~ Reid MS , Jadfield CAS , Watkins CB , Such l arge dark areas are called inclusi on Herman JE . (1 982 ). Star c h iodine pattern bodies by s ever a l authors , they may be as a matur ity i ndex f o r Granny Sm i t h of pr o teinaceous naturC' (A mes a nd Pi vorun , appl es . N.Z . J . Agric. R e s. ~ ' 229 - 237 . 19 74 : Amer . J . Bot . 61 , 79 4-7 97 ; Roma n i RJ , Yu IK, Ku U, Fischer LK, Varke y and Nada k a vukFren, 1 982 : Ne w Phytol. Dehgan N. (19 68 ) . Cellular sencesc nee , 92 , 273 -279 ). The other .rounded structures radiation damage t o mitocho ndr i a , a nd are plastoglo bul i . t h e compen sator y response i n ripen i ng Rev i~w_e_r__!_Y_: In Fig . 6 . glucose concentra­ pear fruits . Pl a nt Phys i o l. ~ ~ 1089-1096 . tion i n f l esh at 5 kGy is greatl y in ­ Simons RK , Chu MC . (1982 ). Cellular i.Jrage creased ove r the contr o l level. I n Fig .lb . profile analysis of appl es exhibiting the starch level at 5 kGy is a l so i n­ corking disorders as r e l ated to calcium creased . Norma lly , in starch - sugar inter­ and potassium . Sci. Hortic . (Amsterdam) conversion if one componen t increases a ~ 217-231. there should be decrease in the other . For both components to increase is Simons RK , Chu MC . (1983 ). Anomalous char­ anoma l ous . This point should be adressed . acteristics of cell u l ar structure related to corking in apples . Sci . Hortic. Authors : On Fig . 6 . a significant glucose accumulation is perceptible . Possibl y the (Amsterdam). ~ ~ 1 1 3 - 124 . glucose o riginating from the starch break­ down cannot be used in the me t abolic Discussion with Revi ewers pathway due to the consi derabl e radia t ion T . Huyash i : Wha t i s the purpose of irra ­ damage to t he c e ll s ; wh ile in sampl es diating appl e? If it is disi nfestation, irradiated with smaller doses g l ucose t he dose r equire d is in the range of 0 . 1 does not accumulate but it is u sed u p in to 1 kG y, which is lower t han that u sed t he increased r espiration. (Dh arkar and i n your study . Why did yo u c hoose the Srcenivasan , 1966 : Food I r r adiation dose range i n your study? Proc . of symp ., 6 -10 June , Karlsruhe , pp : 635 - 649 ; Van Kooy , 1966 : Preservati on Au thors : The 0 - 5 kGy dose range was chosen of fruit and vegetables by irradiation , torOIIOw up the e ffe c t of irradiation on Proc . o f Panel, 1-5 August , Vienna , starch . The aim of a ppl e i rradiation is pp . 29 - 43 ; Kovacs and Vas , 1974 : Acta either disinfestation ( 0 . 1 - 1 kGy ) or to Alimentaria , 3 , 19 - 25 ). extend s helf life us i ng irradiation (1 kGy } in combinati on with Ca - t r eatment . Reviewer IV : Coul d you give some informa ­ tion on the locati o n of the samples with respect to the c r o ss-section of the fruit? Authors : Sampl es f o r bot h the skin and flesh were col lect ed f r om the region of the l a r gest diameter , t he flesh samples were cut out ha l f way be tween the carpel s and the skin.

74