Changes in the content of free amino acids In cherimoya fruits at various ripening temperatures

Changes in he con en o ee amino acids In che imoya ui s a a ious ipening empe a u es Cambios en el con enido de aminoácidos lib es en u os de chi imoyo a dis in as empe a u as de madu ación TORRES, M. N.; LÁZARO, J. J.; PERIAGO, J. L.; GIL, A. and FAUS, M. J. Depa amen o de Bioquímica y Biología Molecula . Facul ad de Fa macia, Uni e sidad de G anada. 18071 G anada, España. RESUMEN Las a iaciones en el con enido de aminoácidos lib es en u os du an e la madu a ción es un ema de in e és debido a la elación de es os compues os con su olo , sabo y es uc u a. Es e es udio se ha diseñado pa a de e mina los cambios en aminoácidos lib es du an e la madu ación de los u os del chi imoyo (Annona che imolia a o Fino de Je e), e aluando la in luencia de la empe a u a de almacenamien o después de la eco lección. Ex ac os me anólicos de epica pio y mesoca pio del u o se usa on pa a la cuan i icación de los aminoácidos lib es, los cuales ue on analizados po c oma og a ia de in e cambio iónico. Ci ulina y p olina se de ec a on en can idades ela i amen e al as. Glu ama o, glu amine, aspa a o, aspa agina y los no p o einogené icos au ina, ácido y aminobu í icoy ácido a-aminoadípico ue on encon ados en concen aciones que a ia on en e 2-6% del o al del con enido de aminoácidos lib es. La madu ación inc emen ó el con enido de aminoácidos, sob e odo de p olina, ci ulina y glu ama o, cuando los chi imoyos se almacena on a 20 oC o 8 oc. Es e inc emen o no ocu ió cuando la empe a u a de almacenamien o se man u o a 4 oC, sugi iendo que el daño po ío a ec a ma cadamen e a la ía de sín esis de la p olina a pa i de glu ama o. Palab as cla e: Aminoácidos. Annonacea. Chi imo�a. Madu ación. ABSTRACT The a ia ions in he con en o ee amino acids in ui s du ing ipening is a ma e o in e es since hese compounds a e ela ed o hei la ou , as e and s uc u e. This s udy was designed o de e mine he changes o ee amino acids in ipening che imoya ui s (Annona che imolia a . Fino de Je e), e alua ing he in luence o he s o age empe a u e a ie ha es ing. Me hanolic ui ex ac s o epica p and mesoca p we e used o quan i ica ion o ee amino acids which we e analyzed by ion-exchange ch oma og aphy. Ci ulline and p oline we e de ec ed in ela i ely high amoun s. Glu ama e, glu amine, aspa a e, aspa agine and he non p o einogenic au ine, y-aminobu y ic and y aminoadipic acids we e ound in concen a ions ha a ied be ween 2-6% o he o al ee amino acids con en . Ripening inc eased he con en s o amino acids, mainly p oline, A s Pha maceu ica, 36:1; 51-58,1995 52 TORRES, M. N., LÁZARO, J. J., PERJAGO, J. L., GIL, A. and FAUS, M. J. ci ulline and glu ama e when che imoya s o age a 20 oC o 80 oC. These inc eases did no occu when he s o age empe a u e was main ained a 4 oC sugges ing ha chilling inju y ma kedly a ec s he syn hesis pa hway o p oline om glu ama e. Key wo ds: Amino acids. Annonaceae. Che imoya. Ripening. Recibido: 20-10-94. Acep ado: 16-11-94. BIBLID [0004-2927(1995) 36:1; 51-58] INTRODUCTION Che imoya ui is one o he iches sou ces o ee amino acids, accoun ing o 10.4% o ui d y weigh . (1, 2). In he las ew yea s, a g ea deal o in e es has be en cen e ed on he ee amino acid con en o so ne ui s (3-6). Ven u a e al. (7) ha e examined he suga apple Annona squamosa, which p esen s he unusual and in e es ing me abolical cha ac e is ic ha y-aminobu y ic acid and ci ulline p edomina e among he o al amino acids, inc1uding a ginine and o ni hine. In he sou sop (Annona mu ica a) p oline and y-aminobu y ic acid a e he majo amino acids, oge he wi h nine o he s which appea in smalle amoun s (7). Recen ly Touche e al. ha e epo ed ha Annona c . c assi lo a and Annona paludosa a e cha ac e ized by a high con en o basic amino acids, pa icula ly his idine, lysine, a ginine and omi hine. (3). The a ia ions in he con en o ee amino acids in ela ion o me abolic changes occu ing du ing g ow h and ipening, p ocesses ha a e o undamen al impo ance in con olling he pos -ha es beha iou o ui s, ha e ecei ed li le a en ion. A p esen he e a e no da a conceming he quali a i e and quan i a i e pa ems o ee amino acids in ipenig che imoya ui s. The aim o his s udy was o de e mine he changes in he con en o ee amino acids o ipening che imoya ui s a e ha es a he g een- ipe s age. To e alua e he in luence o chilling inju y, che imoya ui s we e s o ed a 8 oC and 4 oC and he changes in he ee amino acid pa ems we e compa ed wi h hose ob ained in ui s s o ed a 20 oC. MATERlALS AND METHODS Plan ma e ial Uni o m weigh (500 + 50 g) che imoya ui s (Annona che imolia Mill) o he "Pino de Je e" a ie y we e ha es ed a he g een- ipe s age and subsequen ly ipened unde one o he ollowing empe a u es: 20 oC, 8 oC o 4 oC, and a 85% ela i e humidi y (RH). A s Pha maceu ica. 36:1; 51-58. 1995 CHANGES IN THE CONTENT OF FREE AMINO ACIDS IN CHERIMOY A FRUITS ... 53 The ui s we e s o ed o 12 o 40 days depending on he ipening empe a u e. Sampling o che imoyas o amino acid analysis was done a 4 day in e als du ing ipening. Amino acid analysis The ui s we e washed wi h ap wa e and d ied wi h il e pape . Mesoca p was sepa a ed o he epica p by a s ainless s eel spa ula and seeds we e emo ed. Each issue was cu in o small pieces and weighed. Samples homogeniza ion we e ca ied ou sepa a ely wi h app oxima ely 100 g o mesoca p and 50 g o epica p. Two olumes o 80% me hanol in wa e con aining 0.2% sodium disulphi e by epica p o mesoca p weigh we e added. Suspensions we e ob ained by disin eg a ion using a So all Omnimixe homogenize o 5 mino The suspensions we e il e ed wice h ough ou laye s o su gical gauze. The me hanolic ex ac was cen i uged a 1,200 g and acuum concen a ed a 40- 50 oC. Final olumes o epica p and mesoca p ex ac s we e espec i ely adjus ed o 100 mI. and 50 mI Samples o amino acid analysis we e dilu ed i e imes wi h a bu e pH 2.1, con aining 250 nmoles o no leucine as in e nal s anda d. Amino acids we e de e mined by ion-exchange ch oma og aphy using a Ch omaspeck-J-180 au oma ic amino acid analyze as desc ibed p e iously (8). RESULTS Table 1, 2 and 3 espec i ely show he con en s o mesoca p and epica p ee amino acids o che imoya ui s ipened a 20 oC, 8 oC, and 4 oC a e ha es a he g een- ipe s age. The majo ee amino acids de ec ed in bo h issues we e ci ulline and p oline. The concen a ion o ci ulline a.ccoun ed o 11-65% and 12-44% o o al ee amino acids in epica p and mesoca p espec i ely. The ela i e con en s o p oline a ied be ween 11-58% and 16-60%. Acidic amino acids and hei amides as well as se ine we e also de ec ed in ela i ely high amoun s (2-6%). On he con a y, a oma ic and basic amino acids, excep o ni hine, we e p esen in low amoun s. O ni hine ep esen ed 2- 20% o o al ee amino acids bo h in epica p and mesoca p o che imoya ui s. B anched chain and neu al alipha ic amino acids we e always p esen in he ui s bu hei le els we e e y low. Th eonine and hyd oxyp oline we e also de ec ed in low amoun s. The non-p o einogenic amino acids, au ine, y-aminobu y ic and a-aminoadipic we e egula ly ound in quan i ies ha a ied be ween 2-5% o he o al ee A s Pha maceu ica, 36:1; 51-58, 1995 54 TORRES, M. N., LÁZARO, J. J., PERJAGO, J. L., GIL, A. and FAUS, M. J. T ABLE 1.-Mesoca p and epica p ee amino acid con en s o che imoya ui s ipened a 20 °C-22 oC and 85% ela i e humidi y a e ha es ing a he g een- ipe s age . Epica p Mesoca p Amino acid Day a e ha es ing Days a e ha es ing 1 4 8 12 1 4 8 12 TAU 0. 42 1. 38 0. 39 1. 33 0. 51 0.11 0.45 0. 52 a-AA 0. 42 0.52 0.70 1. 03 0.17 0. 21 0. 60 0. 45 y-AB 0.33 0. 19 0.68 0. 52 ASP 0. 59 0. 31 0. 14 0. 30 0. 05 0. 15 0. 18 0. 12 THR 0.21 0. 19 0. 31 0.48 0. 12 0. 08 0. 17 0. 14 SER 0. 47 0. 26 0. 22 0. 46 0. 19 0.10 0. 16 0. 16 ASN 0. 82 0. 41 0. 65 . 0. 88 GLU 0. 11 0. 28 0. 32 0. 50 0. 16 0. 04 0.07 0. 06 GLN 0.38 0.71 0. 95 1. 43 0.06 0. 07 0. 07 0. 04 PRO 4.90 12.60 2.41 13.42 1.91 4.90 17. 12 7. 20 OH-PRO 0. 17 0. 21 0. 36 0. 56 0. 09 0. 15 0. 16 0.18 GLY 0. 11 0. 08 0. 16 0. 10 0. 06 0. 08 0. 18 0. 15 ALA 0. 10 0. 19 0. 21 0. 17 0. 21 0. 17 0. 14 0. 11 CIT 11. 82 14. 33 15. 83 14. 21 3. 81 1. 72 7. 30 6. 00 VAL 0. 25 0. 22 0. 58 0. 58 0. 11 0. 06 0. 23 0. 20 ILEU 0. 45 0. 23 LEU 0. 25 0. 20 0. 04 0. 04 0. 06 0. 11 TYR 0. 06 0. 15 0.11 0. 25 0. 07 0.08 0. 11 0. 15 PHE 0.27 0. 10 0. 32 0. 62 0.17 0.17 0. 14 0. 10 TRP 0.08 0. 72 0. 14 0. 23 0. 03 0. 08 0. 13 0. 05 HIS 0. 06 0. 08 0. 10 0.06 ORN 1. 21 1. 84 1. 60 2. 21 0. 90 0. 45 1. 05 1.30 LYS 0. 21 0. 28 0. 32 0. 36 0. 15 0. 07 0. 15 0. 18 ARG 0. 00 0.01 0.02 0. 03 -= No de ec ed. * Resul s a e exp essed as mean o ou de e mina ions in �moles o ee amino acids pe g o esh issue. amino acids. Ci ulline was egula ly de ec ed in highe concen a ions in epica p han in mesoca p wi h independence o he ipening empe a u e. P oline was also ound ele a ed in epica p wi h he inc eases mo e e iden a 8 oC and 4 oc. A 20 oC he concen a ion o glu ame e was inc eased in he epica p compa ed o ha o he mesoca p bu i did no p esen signi ican di e ences be ween bo h issues a 8 oC and 4 oc. The concen a ions o ci ulline and p oline we e inc eased a 8 oC and dec eased a 4 oC in ela ion o hose o 20 oC in bo h epica p and mesoca p, and glu amine and se ine we e ound a highe le els a 8 oC and 4 oC han a 20 oC. A s Pha maceu ica, 36:1; 51-58, 1995 CHANGES IN THE CONTENT OF FREE AMINO ACIDS IN CHERIMOYA FRUITS ... 55 TABLE 2.-Mesoca p and epica p ee amino acid con en s o che imoya ui s ipened a 8 oC and 85% e1a i e humidi y a e ha es ing a he g een- ipe s age . Epica p Mesoca p Amino acid Day a e ha es ing Days a e ha es ing 1 8 16 20 1 8 16 20 TAU 0.43 0.66 0.53 0.79 0.57 0.41 0.40 0.42 a-AA 1.21 1.37 0.82 3.08 1.21 0.85 0.92 2.05 y-AB 0.65 0.33 0.74 0.58 ASP 0.46 1.05 0.56 0.81 0.47 0.32 0.44 0.37 THR 0.22 0.58 0.31 0.75 0.37 0.33 0.54 0.60 SER 0.59 1.05 0.64 1.24 0.58 0.45 0.53 0.67 ASN 1.16 1.46 0.45 0.62 2.05 1.67 0.61 1.05 GLU 3.20 2.90 1.39 2.03 1.87 1.02 0.80 0.82 GLN 2.10 1.91 1.10 2.10 6.42 2.97 1.95 2.25 PRO 23.95 22.71 25.42 24.82 38.05 15.21 19.25 9.75 OH-PRO 0.31 0.24 0.32 0.38 0.23 0.46 0.69 0.32 GLY 0.17 0.22 0.15 0.10 0.02 0.01 0.05 0.07 ALA 0.21 0.34 0.15 0.17 0.32 0.41 0.20 0.18 CIT 20.92 19.21 15.13 24.08 8.72 11.33 7.40 7.52 VAL 0.32 0.47 ILEU 0.26 0.25 0.19 0.25 0.30 0.42 0.71 LEU 0.05 0.04 0.04 0.08 0.07 0.17 0.06 0.07 TYR 0.02 0.05 0.03 0.04 PHE 0.10 0.18 0.15 0.14 0.03 0.17 0.08 0.11 TRP 0.02 0.12 0.03 0.06 HIS 0.19 0.15 0.24 0.14 0.05 0.10 0.12 0.21 ORN 1.35 2.95 1.25 0.82 1.21 2.05 1.08 4.10 LYS 0.60 0.71 0.23 0.35 ARG 0.51 0.50 0.40 0.45 0.16 0.23 0.24 0.31 - = No de ec ed. * Resu1 s a e exp essed as mean o ou de e mina ions in Jlmo1e s o ee amino acids pe g o esh issue. DISCUSSION The amino acid p o ile o che imoya du ing ipening a e ha es a he g een- ipe s age was cha ac e ized by a high con en o ci ulline and p oline and mode a e concen a ions o omi hine, glu ama e, aspa a e, glu amine and aspa agine and low le els o b anched chain a oma ic and basic amino acids. So ne opical Annonaceae con ain high le els o basic amino acids in he lea es as well as in he ui s, ci ulline and omi hine being he mos equen ly isola ed (3). Fu he mo e, suga apple also con ain high le els o ci ulline (6) and many o he ui s no ela ed o che imoyas a e also cha ac e ized by he p esence o high concen a ions o basic amino acids (5, 6, 9, 10). P oline is A s Pha maceu ica, 36:1; 51-58,1995 56 TORRES, M. N., LÁZARO, J. J., PERlAGO, J. L., GIL, A. and FAUS, M. J. TABLE 3.-Mesoca p and epica p ee amino acid con en s o che imoya ui s ipened a 4 oC and 85% ela i e humidi y a e ha es ing a he g een- ipe s age Epica p Mesoca p Amino acid Day a e ha es ing Days a e ha es ing 1 12 24 32 1 12 24 32 TAU 0.95 0.48 0.98 0.83 0.26 0.39 0.63 0.03 a-AA 0.75 0.23 1.62 1.21 0.44 0.20 0.65 0.24 y-AB 0.45 0.40 0.82 2.71 0.16 0.44 0.55 0.42 ASP 0.98 0.70 2.15 1.06 0.97 0.81 0.75 0.31 THR 0.17 0.10 0.38 0.44 0.26 0.13 0.29 0.10 SER 0.68 0.35 1.41 l.l0 0.68 0.44 0.78 0.39 ASN 0.27 l.l2 0.61 0.50 0.23 0.13 0.23 0.22 GLU 1.40 0.91 3.05 l.l0 1.87 1.39 1.71 0.78 GLN 2.01 3.20 1.42 1.56 1.88 0.80 1.30 1.07 PRO 1.22 0.32 3.10 2.58 0.88 0.37 0.65 1.17 OH-PRO 0.98 0.65 1.85 2.02 0.26 0.37 1.07 0.10 GLY 0.12 0.17 0.08 0.15 0.26 0.05 0.10 0.11 ALA 0.57 0.25 0.93 1.04 0.24 0.29 0.58 0.28 CIT 11.20 6.62 3.95 19.35 2.48 3.67 2.36 5.72 VAL 0.12 0.17 0.22 0.34 0.10 0.03 0.05 0.05 ILEU 0.25 0.26 0.29 0.03 0.05 0.15 0.13 0.06 LEU 0.05 0.06 0.06 0.06 0.02 0.02 0.03 0.09 TYR 0.13 0.28 0.21 0.20 0.10 0.05 0.15 0.04 PHE 0.61 0.07 0.52 0.38 0.12 0.05 0.06 0.22 TRP 0.27 0.12 0.26 0.14 0.02 0.04 0.02 0.02 BIS 0.01 0.15 0.05 0.06 0.01 0.04 0.07 0.09 ORN 0.17 2.65 0.84 0.94 LYS 0.04 0.05 0.04 0.04 ARG 0.05 0.07 0.25 0.06 0.04 0.00 0.04 0.32 - = No de ec ed. * Resul s a e exp essed as mean o ou de e mina ion in ¡ moles o ee amino acids pe g o esh issue. usually p esen in ui s du ing he ipening p ocess (11) and he SOUTSOP, a kind o che imoya, con ains his amino acid a he highes le els among he s udied Annonaceae (7). y-aminobu y ic acid was p esen in che imoya bu i s le el was only mo e han 2% o he o al amino acids in con as o he esul s epo ed o o he Annonaceae (6). Tbe ipening p ocess has been obse ed o be in ima ely associa ed wi h he me abolism o a ginine, p oline and glu amic acid. The p oline le el in pa icu la being inc eased a he heigh o he ipening p ocess (11). In OUT s udy p oline and ci ulline as well as glu ama e inc eased quickly a e ha es in epica p and mesoca p o che imoya ui s. Tbe inc ease in A .� Pha mn .p.ll i .n. �6:1: 'il-'iR lQQ'i CHANGES IN THE CONTENT OF FREE AMINO ACIDS IN CHERIMOY A FRUITS ... 57 p oline was no co ela ed wi h a de c ease in a ginine and glu ama e as i has been desc ibed in g apes (6). Howe e , Pool el al. (12) ha e eco ded a ise in he le els o bo h a ginine and p oline du ing long e m s o age. The epica p o che imoya was iche han mesoca p in he concen a ion o ci ulline, p oline and glu ama e. This ac may be ela ed o a mo e ac i e syn hesis pa hway in he i s issue since p oline is one o he p incipal compounds in he main enance o he plan cell wall s uc u e. When he ipening p ocess o he che imoyas was ca ied ou a 8 oC, we ob ained he highes concen a ions o ci ulline, glu ama e and p oline, bo h o epica p and mesoca p. I seems ha dec easing in empe a u e o ipening is ela ed o an in c ease in he ac i i y o he enzy nic sys ems o p oduce mo e p oline. Howe e , chilling inju y occu ing a 4 oC ma ked1y dec eased he le els o ci ulline, glu ama e and p oline bo h in epica p and mesoca p. F om he da a ob ained in his s udy we may conclude ha he ipening p ocess and he beginning o he ui senescence in che imoya a e co ela ed wi h inc eases in he le els o p oline, ci uline and glu ama e, which appea s o be me abolically associa ed, and ha chilling inju y, conside ably a ec s he accumula ion o amino acids ha no mally occu du ing he ipening pe iodo ACKNOWLEDGEMENTS This wo k has been suppo ed by a g an om Caja Gene al de Aho os de G anada, G anada (Spain). REFERENCES (1) MANNINO, S. and AMELOTTI, G. (1974). 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