Guava (Psidium Guajava L.) Sugar Components and Related Enzymes at Stages of Fruit Development and Ripening

472 Nippon Shokuhin Kogyo Gakkaishi Vol. 29, No. 8, 472～476 (1982) (30)

Article

Guava (Psidium guajava L.) Sugar Components and Related Enzymes at Stages of Fruit Development and Ripening

Golam MOWLAH* and SaburoITOO LaboratoryofPostharvest Physiology andPreservation Fruits andof Vegetables, FacultyofAgriculture, Kagoshima University, 1-21-24Korimoto, Kagoshima, 890

Guava contained fructose, glucose, surcrose, and inositolin the decreasing order. Reducing sugars mainly composed of fructose and glucose increased slowly during the period of immature and mature stages, and increased sharply at ripening upto fullripe stage. Although the concentrations of both fructose and glucose were higher in white, C-1-16 seedling variety than in pink, C-1-40 seedling variety in either of the immature and mature stage, only with the exception of fructoseat maturation where it was higher in pink. At full ripe the pink variety contained more fructose and glucose than the white and at ripening the concentrations of froctose and glucose were almost nearing to equal to each other but at full ripe fructose comprised 55.93% of totalsugars in the white variety and 58.28% in the pink. Sucrose and inositolconstituted the minor sugar components and also increased gradually with insignificantrise of concentration during ripening. Activityof invertase began to develop during ripening and attained the maximum at full ripe. This enzyme showed a maximum activityat pH 3.5-4.0. Amylase activitystarted to increase as the fruitsreached the maturation and increased upto full ripe and showed the optimum pH 7.0. Although, at ripening the white variety had higher invertase activitythan the pink, amylase activity of the pink variety was 2.1 times higher than the activityof the white.

The chemical components depending on stagesof maturation and ripening depending varietiesand cultivarsat differentstages of on varietiesand cultivars.In this study two maturationand ripening play a vitalrole in varietiesof guava were investigatedon the judging the suitabilityof the fruitsas a raw sugar metabolism and invertaseand amylase materialin producing differentprocessedpro- activitiesthroughout the period of fruit ducts and to determine the qualityof the proc- development and ripening. essed products. Some fruitsshowed sudden changes of sugar content during development Materialsand Methods and ripening1)2).Informations regarding some The white, C-1-16 and pink, C-1-40 seedling fruitsare availableabout theirsugar metabolism varietiesof guava (Psidium guajava L.) were and concerning enzyme activityduring matu- classifiedas immature, mature, ripe and full ration and ripening3)4).Only insufficient ripe as statedpreviously9). evidencehas been elucidatedabout the invertase Extractionand identificationof sugars activityand sugar composition in fruits5)-8). This was done according to the method of Stillnow a few data are availableregarding SWEELY et al10)with gas chromatographyas the sugar metabolism and related enzymes described by YAMAKI et al3) and KAWATA11) activityin tropicalfruits at their different using a Shimadzu Gas Chromatograph, Model

* Present address where the correspondence is to be made: Laboratory of Food Science & Technology, Faculty of Agriculture, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156, Japan. (31) MOWLAH・ITOO: Guava Sugar Components and Related Enzymes 473

and trimethysiliyl (TMS) preparation of sugars are given in Fig. 1 and the retention time curve for the TMS-derivatives of the standard sugars under the above conditions of the experiments are shown in Fig. 2. The internal standard used was n-docosan. The TMS solution (13ml) consisted of pyridine (10ml), hexamethyldisi- lazane (HMDS), trimethylchlorosilane (TMCS) and n-doconsane (26mg).

Enzyme preparation Preparation of acetone powder from fresh

guava was carried out according to the earlier work9). Extraction of invertase was made from the acetone powder with 4.0% NaCl according to HASEGAWA and SMOLENSKY6) and CHAN et al8) and that of amylase according to YAMAKI et al3). Insoluble polyvinylpyrrolidone (0.5%) was used to prevent the formation of protein- tannin complex. Calcium acetate (0.2%) and cysteine hydrocloride monohydrate (0.3%) were added to the extracting media as calcium Fig. 1 Outline for extraction and trimethyl- and cysteine were helpful in maintaining the silyl (TMS) preparation for sugars activities of α-amylase and β-amylase, respec- from whole guava samples. tively12)13).

Enzyme assay

Enzyme assay were made according to YAMAKI et al3).

Invertase (β-D-Fructofuranoside fructohydro- lase, EC. 3.2.1.26): 3ml of the final enzyme reaction mixture consisted of 0.1M acetate buffer (pH 4.0), 1ml of 0.01M sucrose and 1ml of enzyme solution (31.45-56μg protein

per ml) incubated at 30℃ for 60min. The released reducing sugars were determined as

glucose by NELSON-SOMOGYI copper reduction Fig. 2 Retention time of gas chromatography method14). One unit of invertase activity was of trimethylsilyl of standard sugars defined as the amount of gm equivalent of reducing groups expressed as glucose under the above conditions.

Amlyase (1.4-glucan 4-glucan hydrolase, GC-4B with hydrogen flame ionization detector EC. 3.2.1.1.): The reaction mixture was by a stainless steel column (3 meters×3mm) , composed of 1ml of 0.75% soluble starch, 1ml packed with 5% silicon gum SE-30-Chromosorb of 0.1M phosphate buffer, pH 6.0, and 1ml rbw, with temperature ranges from 190 to of the enzyme solution (31.00-57.00μg protein 270℃ (5℃/min) and detector teperature was per ml), and the incubation temperature was 290℃ and recording charge speed was 5mm/ 30℃. At zero time and at the end of incuba- min. The carrier gas (nitrogen) flow rate tion time 0.2ml of reaction mixture was was 30ml/min and the hydrogen gas flow rate withdrwn and poured into the tubes containing was 35ml/min. The flow chart for extraction 0.4ml of 50% acetic acid to stop further enzyme 474 日本食品工業学会誌第29巻第8号 1982年8月 (32) action. Then 2ml of 50 fold solutionof 1% iodineand 10% potassiumiodide (KI) solution were added just 5min before the spectro- photometricreading at 575nm. The percent of reduction in absorbance was regarded as the activityof the amylase. Proteinanalysis Proteincontents in the extractswere determined by the method of Lowry et al15). Resultsand Discussion From the accumulation trends of sugars in white, C-1-16 seedlingand pink, C-1-40 seedling varietiesof guava, as shown in Table. 1, fructose,inositol and sucrose were the main sugar components in guava and fructose,the predominat sugar component, increased re- markably in all thestages of maturation and ripening in both varieties,whereas, glucose, Fig. 3 Invertase activity in two varieties of guava during maturation and ripening another predominant sugar of guava, increased graduallythroughout the periodof development and ripening. Among minor sugars,sucrose and inositolincreased slowly during all the stages untilfull ripe. In immature and mature stages the concentrationsof fructose and the hydrolysis of sucrose and subsequently slow down the increasing trend of sucrose during glucose in each varietywere nearing to each fruit ripening. On the other hand, for the other but in ripening fructosecomprised the major sugar fraction. This pattern of sugar white the ratio of sucrose to reducing sugars accumulationwas alsorecognized in datesand at immature and mature remained at 0.04 but at full ripe it decreased to 0.02. Similarly, grape varietyof fruits8)16)17). Invertaseactivity was detectableduring the the pink started with higher ratio values of 0.05 and 0.04 at immature and mature stages, period of ripening and increasedsuddenly at fullripe in both varities.This increased in- respectively. And these values decreased to vestase,as in Fig.3, might be responsiblefor 0.02 during full ripening. These findings corresponded to the fact that the ratio of Table 1 Sugar contentsof guava varietiesat sucrose to reducing sugars are inversely differentstages of maturationand proportional to the activity of invertase in ripening guava varieties. These phenomena are similar to those in tomatoes, tuberous roots and sugar canes18)-20). This like other fruit21), the accumulation of sugars in guavas changes thoughout the period of fruit development and ripening. This study also indicated the increasing invertase activity in guavas during ripening along with little accumulation of sucrose and at full ripe the accumulating trend was slow with a sharp increase of invertase activity which resulted the conversion of sucrose to glucose giving increased amount of glucose and fructose at full ripe. On the other hand, (33) MOWLAH・ITOO: Guava Sugar Components and Related Enzymes 475 the slightlyincreased accumulation of sucrose at fullripe was accountable to the increase of amylase activity(Fig. 4) at this stage. The increasedactivity of amylase caused a break- down of the storage carbohydrate to sugar substanceswhich seved as precusor pool for sucrosesynthesis at full ripe. YAMAKI et al3) also reported that in case of Japanese pear fruitsucrose accumulation was in parallel to the degradationof strach. Sugar accumulation in fruitsdepends mainly on the translocation in leafand bark3); and their contents varies depending on cultivars22).It is known that the carbohydrate accumulation in fruitsgreatly Fig. 5 pH specificity curves for invertase and influencedby the interrelatedphenomenon of amylase of guava the starch-sugarinterconversion and synthesiz- ing enzymes4)23)24).In translocation pathway synthesizingpathway4), or by the enzymes, such sucrose is converted into starch the main storage carbohydrate which in turn was con- as, sucrose synthetaseand sucrose-phosphate synthetase that were found in the case of verted into sucrose4)23)25). The sucrose accumulation was very small and grape2)4),pear27) and citrus28).It is also no sorbitolwas detected in guava varities. known that sorbitolis converted to fructose These findings also may be correlated to the during the metabolicpathway by the sorbitol reasons that the sucrose accumulation is in- dehydrogenase and sorbitol-6-phosphatede- fluenced by the cell permeability of sucrose hydrogenase and reportsare availableon the into the flesh without being converted to facts that sorbitolis partiallyconverted to sucroseduring fruitmaturation29)30). glucose and fructose by the membrane bound invertase26),and by the activation of sucrose From Fig. 4, it was observed that amylase activitywas detected and started to increase as the fruitsreached the maturationupto full ripe stage. At ripening the pink varietyin- dicatedamylase activityof 2.1 times higher than the white variety. The investaseshowed the highestactivity in the range of pH 3.5-4.0 and amylase had the pH optimum of 7.0 (Fig.5). Acknowledgement The authorsare gratefulto the management of the IbusukiBotanical Station for renderingscope to utilizethe guava samplesgrown in thatstation duringdifferent stages of the fruitgrowth. The authorsgratefully acknowledge Prof. T. Obara of Tokyo Universityof Agriculturefor kindlygoing through the manuscript and offeringvaluable Fig. 4 Amylase activity in two varieties of suggestions. guava at stages of maturation and ripening Reference 1) WENKAM, N.S. and MILLER, C.D.: Hawai, Agric.Exp. Stn.,Bull., 135, 1 (1965). 2) NAWKER, J.S. and DOWNTON, W.J.S.: Royal 476 日本食品工業学会誌第29巻第8号 1982年8月 (34)

Society of New Zeland, Bull. 12, 819 1974). 26) RICARDO, C.P.P.: Planta (Berlin), 118, 333 3) YAMAKI, S., KAJIURA, I. and KAKIUCHI, N.: (1974). Bull. Fruit Tree Res. Stn., 6, 15 (1979). 27) LATCHE, A, PECH, J.C. and FALLOT, J.: 4) HAWKER, J.S.: Phytochemstry, 8, 9 (1969). Physiol Veg, 13, 637 (1975). 5) ARNOLD, W.N.: Biochem. Biophys. Acta, 110. 28) BEAN, R.C.: Plant Physiol.,35, 429 (1960). 134 (1965). 29) BIELESKI, R.L: Aust. J. Biol. Sci, 22, 611 6) HASEGAWA, S. and SMOLENSKY, D.C.: J. Agric. (1969). Food Chem., 18, 902 (1970). 30) REID, M.S.: Royal Society of New Zealand, 7) NAKAGAWA, S. and KAWASAKI, Y., OGURA, N. Wellington, Bull. 12, 823 (1974), and TAKAHARA, H.: Agric. Biol. Chem., 36, 18 (Received Jan. 13, 1982) (1971). 8) CHAN, H.T.J., KWOK, S.C.M. and LEE, C.W. Q.: J. Food Sci.,40, 772 (1975). グァバ果実の成熟並びに追熟時における糖 9) MOWLAH, G. and ITOO, S.: Nippon Shokuhin 及び糖関連酵素について Kogyo Gakkaishi, 29, 413 (1982). 10) SWEELY, C.C., BENTLEY, R., MAKITA, M. and ゴーラム・モーラ*,伊藤三郎**

WELLS, W.W.: J. Am. Chem. Soc., 85, 2497 (*東京農業大学,**鹿児島大学) (1962). 11) KAWAMATA, S: Tokyo Agric. Exp. Stn., Bull., (1) グァバ果実の構成糖は,フラクトース,グルコー 10, 53 (1977). ス,イノシトールおよびシュークロースであり,その量

12) BAIJAL, M., SINGH, S., SHUKLA, R.N. and 的関係は,フラクトース>グルコース>シュークロース SANWAL, G.G.: Phytochmistry, 11, 929 (1972)., 13) THOMA, J.A., SPRADLIN, J.E. and DYGERT, S.: >イノシトールの順であった。 In "The Enzymes" Ed. Boyer, P.D., 3rd ed. (2) フラクトースおよびグルコース量は,成熟中に大 Vol. 5, p. 115, Academic Press, New York. きな変化は示さなかったが,追熟時に急激な増加が見ら

14) SPIRO, G.R.: Methods Enzymol., 8, 3 (1960). れた。一方,微量糖成分であるシュークロースおよびイ 15) LOWRY, O.H., ROSEBEROUGH, N.J. FARR, A.L. ノシトールは,追熟中にわずかに増加した。 and RANDALL, R.J.: J. Biol. Chem., 193, 265 (1951). (3) 未熟果並びに成熟果中のフラクトースおよびグル 16) KLIEWER, W.M.: Am. J. Enol. Vitic.,16, 101 コース量は,成熟果のフラクトースを除き,桃色種より

(1965 b). も白色種の方が高い値を示した。一方,完追熟果におい 17) KLIEWER, W.M.: Am. J. Enol. Vitic.,18, 33 ては,フラクトース,グルコース共に桃色種が高い値を (1967). 18) MANNING, K. and MAW, G.A.: Phytochemistry, 示した。 14, 1965 (1975). (4) 追熟中のフラクトースとググルコースとの量比 19) RICARDO, C.P.P. and SOVIA, D.: Planta (Ber- は,白及び桃色種各々ほぼ一定の値を示し,完追熟果で lin).,118, 43 (1974). は,フラクトースの割合は白色種55.93%,色種58.28% 20) SACHER, J.A., HATCH, M.D. and GLASZIU, K. T.: Plant Physiol.,38, 348 (1963). 桃であった。 21) KLIEWER, W.M.: Plant Physiol.,41, 923 1966). (5) インベルタゼ活性は,追熟中に現われ,完追熟果で 22) KAJIURA, I, YAMAKI, S., M., AKIHAMA, T. and 最大となった。また,その最適pHは3.5-4.0であった。 MACHIDA, Y.: Jpn. J. Breed, 29, 1 (1979). アミラーゼ活性は,成熟・追熟に従って増加し,その最 23) ISHERWOOD, F.A.: Phytochemistry, 15, 33 1976). 24) HATCH, M.D. and GLASZION, K.T.: Plant 適pHは7.0であった。追熟果においては,インベルタ Physiol.,38, 344 (1963). ーゼ活性は白色種が桃色種に比べ高く,一方,アミラー 25) MURATO, T.: Nippon Nogei kagaku Kaishi, 44, ゼ活性は桃色種が白色種の2.1倍の値を示した。 412 (1970).