EFFECT OF TEMPERATURE ON PIGMENT DEVELOPMENT IN RED BLUSH GRAPEFRUIT AND RUBY BLOOD ORANGES

Transcription

1 EEDNGS FRST NTERNATONAL CTRUS SYMPOSUM, VOL. 1,1969 pf!o C 71 EFFECT OF TEMPERATURE ON PGMENT DEVELOPMENT N RED BLUSH GRAPEFRUT AND RUBY BLOOD ORANGES F.. Meredith 1 and R. H. Yong NTRODUCTON The red-fleshed grapefrit has been the predominant grapefrit type planted in Texas since the freees of 1949 and The increased prodction of red grapefrit in the Rio Grande Valley has presented a major color problem to the citrs processing indstry of the area. The major pigments of the red-fleshed grapefrit are lycopene and carotene (Matlack 1935, Kahn and Mackinney 1953, Crl and Bailey 1957). t as reported by Prcell (1959) that the red pigment, lycopene, in red-fleshed grapefrit reaches a maximm abot September 1. After this time the lycopene of the endocarp sloly disappears so that by the beginning of the processing season the endocarp appears a yelloorange. Prcell and Schlt (1964) fond that age of frit and environmental factors sch as temperatre inflence the seasonal decline of lycopene. t as observed by Prcell et a!. {1968) that ith Red Blsh grapefrit a 95 F day temperatre and an 85 F night temperatre ill inhibit the normal seasonal decline of lycopene hile a temperatre of 6 F day and 4 F night ill case a loss of lycopene. These reslts raised the qestion of hat old happen to the lycopene and carotene in the endocarp of the Red Blsh grapefrit hich ere transferred from the 6 F j 4 F temperatre to the 95 F/85 F temperatre. The blood orange is not in commercial prodction in the United States as in some Eropean contries. The blood orange is niqe among citrs in that the endocarp contains anthocyanin pigments (Matlack 1931 a, b, and Chandler 1958). The anthocyanins are pigments that are chemically different from the lycopene-carotene pigment fond in red grapefrit. The anthocyanin pigments are ater-solble hile lycopene and carotene are hydrocarbon solble, ater-insolble. Carrante (1941) has reported the formation of anthocyanins in blood oranges dring the cool eather periods of November to Janary. As far as is knon no stdies on anthocyanin formation in blood oranges have been condcted nder controlled temperatres. Tnjs posed the qestion of hat old happen to the anthocyanin and carotenoid pigments in the endocarp of the blood orange hen sbjected to the 95 F/85 F temperatre and to the 6 F/4 F temperatre. Experiments ere designed to determine the changes in chlorophyll and carotenoids in the flavedo, and in lycopene and carotene in the endocarp of the Red Blsh grapefrit, hen the frit ere exposed for varying intervals of time to controlled temperatre. Experiments ere also designed to determine changes in chlorophyll and carotenoid in the flavedo, and of carotenoids and anthocyanins in the endocarp of Rby Blood oranges. MATERALS AND METHODS Small sor orange seedlings ere removed from nrsery plots in Febrary and placed into 1-qart containers filled ith soil. n late May and early Jne Red Blsh grapefrit and Rby blood oranges ere grafted (Erickson 1957, 196, Yong and Erickson 1961, and Olsen 1965) onto the vigorosly groing seedlings. Prcell eta!. (1968) has shon that pigment changes in the endocarp of grafted Red Blsh grapefrit are comparable to frit gron in an orchard. t as therefore decided that grafted frit old be sed as it alloed large nmbers of frit to be placed into the small area of the environmental chamber. On Jne the diameter of the 7 red-fleshed grapefrit ere measred. 1 Sothern Utiliation Research and Development Division, Food Crops Utiliation Research Laboratory, Weslaco, Texas. Crops Research Division, Agricltral Research Service, United States Department of Agricltre, Weslaco, Texas. For red grapefrit ith the same diameter ere given identical nmbers and each one placed into a separate set. This as repeated ntil 1 frit ere placed in each set. The blood oranges ere treated in the same manner except that three sets ere sed ith 75 frit per set. The for red-fleshed grapefrit sets ere treated as follos: Set 1 as placed into an environmental chamber ith 95 F day temperatre and 85 F night temperatre. Set as placed into an environmental chamber of 6 F day temperatre and 4 F night temperatre. Set 3 as placed otside nder natral conditions and as labeled control. Set 4 as placed otside nder natral temperatre bt ith restricted light and as labeled shade. The light intensities of the otside control and shade frit ere recorded on a pyrheliometer ith the control frit receiving an average for five days of 1. g cal/sq em/min hile the shade frit received an average of. g cal/ sq em/min. On October 16

2 7 thirty grafted red grapefrit ere removed from the 6 F/4 F temperatre and placed into the 95 F/85 F temperatre chamber. The frit as labeled 95 F/85 F - 6 F/4 F. The three blood orange sets ere given the same treatment as sets 1,, and 3 in the above. On November 14 otside control blood oranges ere placed into the 6 F/4 F temperatre. The environmental temperatre chambers ere programmed so that day temperatres ere held from 8:a.m. ntil 4: p.m., ith night temperatres held from 4: p.m. to 8:a.m. The light in the chamber as provided by banks of G.E: 3 poer-grove florescent tbes and 1 att incandescent blbs. llmination as 8 foot candles at the top of the leaves. The lights ere on from 6:a.m. to 6: p.m. Nine frit ere harvested every three eeks except for the third sampling period. t as felt that sfficient frit might not be available later on in the experiment if frit sampling as condcted at the reglar interval time. t as decided to extend this sampling time to 41 days. The eight and diameter of each individal red grapefrit and blood orange ere recorded. The nine frit of each citrs vareity ere divided into three samples ith sample 1 containing the 3 largest frit by eight, sample the next 3 largest frit, and sample 3 the remaining frit. The flavedo as removed by grating on a vegetable grater, thoroghly mixed, and 1 g dplicate samples taken. The samples ere blended ith 5 mg CaC 3 and 8% aqeos acetone in a VirTis 45 homogenier. The extract as vacm filtered, blended again in 8% acetone, filtered and made to volme. The chlorophyll in this extract as determined spectrometrically on a Cary Model 15 UV visible spectrophotometer at 663 nm and 645 nm by the procedre of Holden (1965). The aqeos acetone extract as placed in a separatory fnnel along ith 5 ml of hexane, and the mixtre as ashed ith ater to remove the acetone. The chlorophyll as removed so that it old not interfere ith the spectrometric determination of the carotenoids (Bacon and Holden 1967). The chlorophyll and saponifiable lipids ere removed from the hexane phase by treatment ith rn1 of -. methanol satrated ith KOH. After mintes th 1 phase as removed, ashed ith diethyl either and tie ' \l.e: extract as added back to the hexane. The he,. 1 e ether h d. h 1. "-dne ll.a as e 1t ater severa tlffies to remove any. ' alkali and saponified material. The prified hexan/esidal of carotenoids as made to volme and dried With ex;ract ros Na S4. The absorbancy as determined at 4 nyct. and the carotenoid content as expressed as B-e :> nm sing the specific absorbancy index of 5. aroter.e The albedo on the frit as removed and the ind.d. lv U1J en d ocarp as e1ghed. The carotene-lycopene isolati. the red grapefrit and the carotenoid isolation in the boln m. Od orange ere earned ot by the procedre of Prcell o 95 () Prcell and Stephens (1959), Prcell and Schlt o 96 / and Prcell et al. (1968). The endocarp as blended ), Waring Blendor ith 3 parts ater to 1 part frit. While 1 h 3 blendor motor as stil rnning, dplicate 1 g sample ere taken for carotenmd and for anthocyanin analysis. The carotene and lycopene content of the red grapefrit endocarp extract ere determined spectrometrically by the procedre of Lime et al. (1957). The carotenoid content oi the blood orange endocarp extract as determined by the same method employed for the flavedo. The sample t'}ken for anthocyanin analysis had 5 ml of 1% methanolic HCl (Francis, et al. 1966) added. The sample as 'vacm filtered and the mat as resspended for a second time in a mixtre of methanol, ater, and 11\1 1:49': 1, the slrry again filtered and the filtrate added to the original filtrate. The combined.filtrates ere concentrated on a rotary evaporator and chromatographcd on polyamide sing the method of Chandler and Sain ( 959). The anthocyanins on the chromographic colmn ere ashed ith 3 to 4 volmes of l %HC1 in ater. The prified anthocyanins ere elted as a tight band from the colmn ith 1% rnethanolic HCl. the elate as adjsted to ph 3.5 (Harborne 1958), 'made to a volme of 5 ml, and the absorbancy determined at 55 nrn. De to frit droppage the last nine blood oranges \vere sed from the 95 F/85 F temperatre chamber on December 4 and as conseqently the last data point for this temperatre treatment. RESULTS Red Blsh Grapefrnit The Total chlorophyll (Fig 1) in the flavedo of the red-fleshed grapefrit increased and then decreased ith the 95 F/85 F temperatre, shade, and control treatments, hile the 6 F/4 F temperatre treatment cased a loss of chlorophyll all throgh the experiment. The external color of the peel of the red-fleshed grapefrit as a bright lemon-yello after only three eeks in the 6 F/4 F temperatre environmental chamber hile the color of the frit nder the other treatments as a dark green. At the end of the experiment the red grapefrit in the 3 Use of a company and/or prodct named by the Department does not imply approval or recommendation of the prodct to the exclsion of others hich may also be sitable. 95 F/85 F temperatre as still green-yello corresponding to E on Harding's (1945) color chart for grapefrit. Figre shos ho the carontenoid levels ere affeted in the red grapefrit flavedo by the varios treatmens. fhe 6 F/4 F temperatre treatment cased a loss ot caro tenoid content in the flavedo ntil September 5 after hich the level remained constant. The carontenoid con_t; in the flavedo of the frit in the other three sets mcrea> d nil Agst _14 at hich point the carotenoids dec_:ean (M1ller and Wmston 1939, Yokoyama and White )._ U November 6 the carotenoid content in the flavedo ot a three sets started to increase. The lycopene m. red-fleshed grapefruit. as a ff ec ted. verv.; drmatically by the varios temperatre treatments (Fig '. o o. reas ll The frit in the 95 F /85 F temperatre had an me

3 73 j.4 >.3. (.). ' "/ A_SHA!:JE 8.95/85 C. CONTROL D. 6/4 LSD 5 (,!) '(,!) 3. a. (.) >- 1...J A.95/85 8_ SHADE C. CONTROL.95/85-6/4 E _ 6/4 LSD 5 o-r_1 8/ /-51o; Fig 1. Total chlorophyll in the flavedo of red-fleshed grapefrit KY._/6--i/V----/ Fig 3. Lycopene in the endocarp of the red,grapefrit..15!.1 1/) 6 b.5 [%: <! A.95/85 B. CONTROL C.SHADE D. 6/4 (,!) Q '(,!) " 1-- :: q: (.) A_SHADE B. CONTROL c_ 6/4 D_ E _ 95/85-6/4 o _ f1 8/ Jl/6 11/7 V l:'ig.. Carotenoids in the flavedo of red-fleshed grapefrit. lycopene and did not ndergo the normal seasonal decline as did the lycopene in the endocarp of the shaded frit and the control frit. The 6 F4 F temperatre treatment cased a loss of lycopene to a level of abot.1 mgl1 g hich then remained constant from October 16 throgh the remainder of the experiment. The 95 F85 F temperatre prodced an increase of lycopene in the 95 F 185 F- 6oF4 F frit. The increase of lycopene in the 95 F85 F -6 F/4 F red grapefrit over the 6 F4 F as significant ith an f vale of 85. and an LSD.o 5 of.1 mgl 1 g. Both shade and control grapefrit increased in 1ycopene concentration and then passed throgh the normal seasonal d:cline of lycopene. The red grapefrit nder the shade as shghtly higher in lycopene content per frit yielded crves Which ere very similar to the lycopene crves (mgl 1 g) shon in Fig 3. The carotene (Fig 4) in the flesh of the red grapefrit from the 95 F85 F, shade, and control treat ents increased at a slo rate all throgh the experiment. he 6o F 4 F temperatre cased a very rapid increase of carotene hich passed throgh a maximm and then 1 8_/14 9_/5----/6--/7----/ Fig 4. Carotene in the endocarp of the red grapefrit. decline. The 95 F85 F - 6 F4 F frit lost carotene at a mch faster rate than 6 4 F frit. The loss of carotene in the 95 F85 F - 6 F4 F temperatre treatments as significant ith an f test of 3.4 and an LSD.os of.14 mg/1 g. Rby Blood Oranges Rby Blood Oranges n the blood orange, chlorophyll a and b (Fig 5 and 6) decreased nder all three treatments. The 6 F4 F temperatre treatment cased the most rapid loss of chlorophyll a and b, the sloest rate of destrction of chlorophyll a occrred ith the 95 F85 F temperatre treatment. The control frit had the least loss of chlorophyll b of any of the treatments. De to the small amonts of chlorophyll a and b, the analysis as discontined in the 6 F4 F temperatre treatment after September 9, hile the analysis as contined ntil December 4 for the other to treatments. The otside peel color of the blood oranges on Agst 16 in the 6 F4 F temperatre as a dark yello. The peel color, in comparison ith the color chart on oranges of

4 74 ' 5.4 A_ 95/85 B_CONTROL c_ 6/4 LSD 5 ' :;; 3 <( _j _j >-.. :: _j 3 (f) f- :: <1 :... 6Ci 4-- 8_ co.-;, C.9SS o _ /16 9/9 1/3 11/14 1/4 Fig 5. Chlorophyll a in the flavedo of the blood orange. 7/1 8/ i/ /4 1/6 Fig 7. Carotenoids in the flavedo of blood oranges. ' Cil _J _J >-.. :: _J A_95/85 B_CONTROL _6/4 LSDO' --._ /1 8/16 9/9 1/3 J/14 1/4 Fig 6. Chlorophyll b in the flavedo of the blood orange. ' lc f- :: <1 1 r, 8-5U,' 1 ( fl CONTROl, c 9/ A'. 6 ; ; C. SO 4,., H...,.,. c Fig 8. Carotenoids in the endocarp of the blood orange. Harding (194), had an eqivalent vale of H. At the completion of the experiment the peel of the blood oranges in thr 6 F/4 F temperatre environment ere a deep orange color and had a vale of L on Harding's color chart. The flavedo carotenoids of the blood oranges held at 95 F/85 F temperatre treatment (Fig 7) decreased ntil the November 14 sampling at hich point they started to increase. The carotenoids in the flavedo of blood oranges in the 6 F/4 F temperatre treatment decreased and then increased. The carotenoids in the flavedo of the blood oranges sed as control decreased all throgh the experiment. Miller and Winston (1939) reported that the carotenoids in Valencia oranges increased all dring the interval from November to Febrary hile the carotenoids in grapefrit decreased. The carotenoids in the endocarp of the blood orange (Fig 8) from the 6 F/4 F temperatre increased rapidly in concentration hile the carotenoids of the control increased very sloly. The drop in concentration of the carotenoids on December 4 in the 6 F/4 F blood or:l!1ge as de to siing of the frit and diltion. The mg/frit carotenoid crve of this data is a smooth crve ithot an inflection. The carotenoids in the 95 F /85 F environment increased slightly and then decreased.. Anthocyanin pigments (Table 1) ere not detected Hl the blood oranges from the three treatments th:ogh November 14. On December 4 anthocyanins ere fond both in the blood oranges kept all throgh the experime:t at 6 F/4 F and in the blood oranges that ere placed mto the 6 F/4 F temperatre on November 14. Anthocyanlll pigments ere not fond in the 95 F/85 F temper::tre or in the otside control blood oranges dring the experiment.

5 75 Table 1. Effect of climate on absorbancya of anthocyanin in blood oranges. 7/1 8/16 9/1 1/3 11/14 1/4 1/ Treatment a 55 nm in 1 em cell and 5 ml volme. DSCUSSON.5. The accmlation of lycopene in the red-fleshed grapefrit appears to be temperatre dependent. The net increase of lycopene in the endocarp of the red grapefrit from the 95 f/85 F - 6 F/4 F temperatre clearly shos that the 95 F/85 F temperatre enchances lycopene synthesis, hereas the 6 F/4 F temperatre inhibits or retards lycopcne synthesis. The seasonal loss of lycopene in the endocarp of the control and shade red grapefrit indicates that a destrctive mechanism is present for lycopene. The 6 F/4 F temperatre or other cool temperatres may act as an activator to trn the destrctive mechanism on for lycopene in the endocarp of the red grapefrit. The 6 F/4 F temperatre apparently enchanced the synthesis of carotene at the beginning of the experiment. The loss of carotene later in the experiment in the 6 F/4 F temperatre indicates that the synthesis of carotene as retarded, inhibited, or an active destrctive mechanism for the destrction of the cyclic carotenes as initiated. The 95 F/85 F temperatre did not appear to inhibit the synthesis of carotene. Apparently the destrctive mechanism for carotene active in the 6 F/4 F temperatre as not active in the 9S F/85 F temperatre at the end of the experiment. The 6 F/4 F temperatre apparently stimilated the prodction of carotenoids in the blood orange, hereas the 95 F/85 F temperatre apparently inhibited carotenoid prodction. The prodction of anthocyanin pigments in the to different sets of blood oranges indicate that anthocyanin synthesis is dependent on matrity as ell as cool temperatre. The carotenoids in the flavedo of the red grapefrit in the 6 F/4 F temperatre treatment decreased to a constant level hile the carotenoids in the flavedo of the blood range ith the same conditions shoed a decrease then a lllcrease in levels. The rapid increase of the carotenoids in he blo?d.orange flavedo and not in the red grapefrit avedo md1cates the possibility of to different controlling mechanisms for carotenoid prodction. CONCLUSON The net increase of lycopene in red grapefrit transterred from the 6 F/4 F temperatre to the 95 F/85 F emperatre shos that lycopene prodction is dependent on arm temperatre. n the endocarp of red grapefrit earlier observations ere confirmed that 6 F/4 F temperatre reslts in a net increase of carotene and decrease of lycopene hile the 95 F/85 F temperatre reslts in a net increase of lycopene and a decrease of carotene. The 6 F/4 F temperatre prodces an increase of carotenoids in the endocarp of the blood orange, hile the 95 F/85 F temperatre decreases prodction of endocarp carotenoids. The formation of anthocyanins as fond to be dependent on matrity and cool temperatres. LTERATURE CTED BACON, M. F., and M. HOLDEN Changes in chlorophylls reslting from varios chemical and physical trestments of leaves and leaf extracts. Phytochem. 6: CARRANTE, V Slle pigmentaioni delle arrance italiane. Acireale (Sicily) Regia Stax. Sper. Frtticolt e Agrmicolt Ann. 16:193., CHANDLER, B. V., and T. SWAN Separation of anthocyanins from plant extracts. Natre 183 (4666):989. CURL, A. L., and G. F. BALEY The carotendoids of Rby Red grapefrit. Food Research : ERCKSON, L. C Citrs frit grafting. Science 15(35) :99. ERCKSON, L. C Color development in Valencia oranges. Proc. Amer. Soc. Hart. Sci. 75: FRANCS, F. J., J. B. HARBORNE and W. G. BARKER Anthocyanins in the lo bsh bleberry, Vaccinim angstifolim. Jor. Food Sci. 31(4): HARBORNE, J. B Spectral methods of characteriing anthocyanins. Biochem. Jor. 7(1):-9. HARDNG, P. L., and D. F. FSHER Seasonal changes in Florida grapefrit. U. S. Dept. Agr. Tech. Bll. 886:1. HARDNG, P. L., J. R. WNSTON, and D. F. FSHER 194. Seasonal changes in Florida oranges. U.S. Dept. Agr. Tech. Bll. 753:11. HOLDEN,M Chemistry and biochemistry of plant pigments (edited by T. W. Goodin), (p. 465). Academic Press, Ne York. KAHN, M. U. D., and G. MACKNNEY Carotenoids in grapefrit, Citrs paradisi. Jor. Plant Physiol. 8:55-55.

6 76 LME, B. J., F. P. GRFFTHS, R. T. O'CONNOR, D. C. HENZELMAN, and E. R. McCALL Spectrophotometric methods for determining pigmentation-beta-carotene and lycopene in Rby Red grapefrit. Jor. Agr. Food Chern. 5: MATLACK, M. B a. The jice sac of the orange ith some observations on the plastids of citrs. Washington Acad. Sci. Jor. 1:438. MATLACK, M. B. 1931b. Observation of the red color of the blood orange. Plant Physiol. 6:79. MATLACK, M. B Pigments of pink grapefrit, Citrs grand is (L., Osbeck). Jor. Biol. Chern. 11: MLLER, E. V., and J. R. WNSTON nvestigation on the development of color in citrs frits. Florida State Hort. Soc. Proc. 5:87-9. OLSEN, E Grafting citrs frit to small potted seedlings. Jor. Rio Grande Valley Hort. Soc. 19:-4. PURCELL, A. E Seasonal development of carotene and Jv in grapefrit. Jor. Rio Grande Ho;tc Pt;;e 13 : ; PURCELL, A. E., and E. F. SCHULTZ, JR nflence of frit age on lycopene concent. in colored grapefrit. Proc. Amer. Soc \"11, Sci. 85: fo;: PURCELL, A. E., and T. S. STEPHENS Determining the effect of reciprocal grafts Of and hite varieties of grapefrit on the ace re lation of carotenoids. Proc. Amer. Soc. llj. Sci. 74: rt. PURCELL, A. E., R. H. YOUNG, E. F. SCHULT, JR and F.. MEREDTH (1968). The effect of artificial climate on the intn frit color of Redblsh grapefrit. Accepted -rna! f pblication in Proc. Amer. Soc. Hort. Sci. or YOKOYAMA, H., and M. J. WHTE Carotenoids in the flavedo of Marsh Seedless grapefrit. Agr. Food Chern. 15: YOUNG, L., and L. C. ERCKSON nflence of temperatre on color change in Valencia oranges. Proc. Amer. Soc. Hort. Sci. 78:197-.