A STUDY OF THE CARBOHYDRATE COMPOSITION OF PAPAYA FRUIT

I. IDENTIFICATiON OF PRINCIPAL SUGARS.II. REFRACTIVE INDEX AS A MEASURE OF SUGAR LEVEL.III. SEASONAL AND MATURITY RELATIONSHIPS.

A THESS SUBMITTED TO THE GRADUATE SCHOOL OF THE

UNIVERSITY OF HAWAII W PARTIAL FULFILLMENT

OP THB REQUIREMENTS FOR THE DECREE OF

MASTER OF SCIENCE

IN

TROPICAL CROP SCIENCE

June, 1962

by

You* if Abu-2* id

Thesis Committee:Toehio mrashige, Chairman Richard A. Hamilton John W. Hyiia r.ha» ic>m h . Uuaountut

ACKNOWLEDGEMENTS

The author expresses hie deepest appreciation to Dr. Toahio

uraahige, Advisor, lor suggesting the problem and lor guidance

and encouragement throughout the course of this study and m pre­

paring this manuscript.

Sincere appreciation im also extended to die Agency lor inter'

national Development, Department of State, U .S.A., for having

sponsored the author's graduate program.

Gratitude le also expressed to the Hewed Agricultural Experiment

Stat on, College of Tropical Agriculture, University of Hawaii, lor

use of its stair and facilities; to Dr. Richard A . Hamilton Cor supply­

ing some of die papaya fruit samples; and to Dr. John W. Hylin for

chromatographic materials end advice.

a .any thanks in extended to ail others who contributed to making

my stay in the United States most profitable and enjoyable.

TABLE OF CONTENTS

PAGE

LIST OF TABLES.......................................... ............................. iLIST OF ILLUSTRATIONS ..................... iiINTRODUCTION ........................................................... IREVIEW OF LITERATURE..................................................... 2EXPERIMENTAL SECTION

X, identification of Principal SugarsA . Materials and Methods

1. Sources and Kinds of Samples . . . . . . . . . 42. reparation of Extracts ............ 33. Chromatography. ............. .. . 64. Relative Content of Sucrose........................... 83. Starch Detection.......................... 8

B. Data and Discussiont. identity of Sugars in Diverse Geaatic Linea . . 92. Component Carbohydrate* in Solo Papaya aa

Related to Stags of Fruit Maturity . . . . . . II3. Smbo r/«n infliriftn y Kiiiitwd to *les mn of

Harvest .................. u4. Range of Sucrose Content is Soto Papaya Fruit • 13

II. Refractive index as a Measure of Sugar LevelA. Materials and Methods

1. Source and Kinds of Fruit Samplas . . . . . . . 162. Methods of Analyses .................. 17

B. Data and Discussion ................ 24

ill. Seasonal and Fruit Maturity Relationships1. Seasonal Relationships . . . . . . . . . . . . . 282. Fruit Maturity Relationship........................... 29

B. Data and Discussion1. Seasonal Relationships ............... 302. Fnit Maturity Relationships......................... 33

SUMMARY AND CONCLUSIONS.................. . ................... 36LITERATURE CITED. . ................................................................. 39

i.

LIST OF TABLES

TABLE PAGE

I Tbs Component Sugars in Ripe Fruits of NiasGenetic Lines of 'apaya . , ........................ 16

U Component Carbohydrates in Solo .apaya Fruitsss Related to Stags of aturity . . . . . 12

IH Component Sugars la Ripe Soto 'apaya Fruit asRelated to Season of H am s . . . . . . 14

IV Bangs of Sucrose Content to Ripe Soto PapayaFruit . . . . . . . . . . 15

V Relationship Between Refractive Index of FruitSep sod Sugar Content of Fruit ss Determined by Antbroos sad Reducing Sugar .'vtottnda . 26

Via Composition of Soto Papaya Fruit aa Related toSeason of Harvest . . . . . . . . . . . . . 21

VSb Composition of Soto Fspeya Fruit as Related toSeason of Harvest. . . . . . . . . . . . . 32

VII Sugar Concentration erf Solo a eya Fruit as Re*latsd to Maturity at Harvest . ...................... 24

u.

LIST OP ILLUSTRATIONS

FIGURE AGE

1 Standard Thioaulfate Equivalent aa Related to theAmount of Reducing Sugar la Reaction Mixture . . 22

3 Reletioassup Between Sugar Content and AnthroaeReaction..................................... . 25

3 Relationship Pateeeu Refractive index of Sap andSugar Content of ’apaya Fruit aa Determined by Anthroae end Reducing Sugar Methods................. 27

INTRODUCTION

Horticultural investigation* with papaya (Caries papaya L .) have

been la progress for a nu-.iber of years, particularly la the tropics

sad sub-tropics, but thus far 1 file information on tbs biochemistry or

physiology of this crop has been made available. It la hoped that this

investigation w 11 provide a renewed foundaton end serve ss an impetus

to further research in these aspects. Working with the fruit, specific

objectives pursued la due study are (I ) identification of constituent sugars,

(2) establishing reliability of refractive index ea a measure of sugar

level, end (3) determ niag die relationship of sugar content to season of

harvest and stage of maturity. It is evident from die review below diet

die sugars in ps.aya fruit were not accurately identified in previous investi­

gations . Moreover, no systematic attempt was made to demonstrate the

applicability to papaya of the common practice of estimating auger con­

tents by simply determining eep refractive index. Finally, it is known

that la Hawaii fruit flavor varies with season of harvest and other factors

but chemical evidence has not been presented to show any probable cause.

REVIEW OF LITERATURE

All previous reports revealed that la die papaya fruit reducing sugars

comprised die bulk erf the total sugar with sucrose being a minor consti­

tuent. ratt sad del Rosario (I I ) repotted a 1913 that total sugar averaged

6.0, reducing sugars 5.9, and sucrose 0.1%. These figures and all those

shown below ate on a fresh weight basis, hi 1915 Thom son (13) surveyed

several genetic lines and found that total sugar content ranged from 6.4 to

15.3; reducing sugar, 6.1 to 13.2; and sucrose, 0 to 2.0%. A few other

genetic lines subsequently examined by Rape (10) allowed a range la total

sugar concentration of 6.6 to 13.6; reducing sugar, 6.4 to 15.4; and sucrose

0.4 to 0.6%. In each of these inwest.gitiooa, tie compound# comprising tie

reducing sugar fraction were not specifically identified end all data were

expressed ae 'glucose '. Even the identification of sucrose wee besed only

upon the difference in reducing sugar contents before end after hydrolysis

erf sugar extracts.

Thompson (15) also reported that os Iodine reactive starch could be

observed in soy papaya line studied. Furthermore, sugar content increased

steadily during fruit maturation and even up to a day before full ripeness.

This relationship between fruit maturity and sugar content was subsequently

substantiated by Jones el a!. (4) in 1941. Thompson's observation revealed

that the increased auger content wee due solely to increase in reducing augurs;

sucrose showed so trend with maturation and ripening. On the other hand,

Jones et at. found the increased sugar content to be associated with decrease

in sucrose as well ae an increase la reducing sugar.

Awada and Uteda (I ) observed that the eager level la papaya fruit wan

inversely related to soil moisture content. Fruits obtained during Septem­

ber and October from orchards under lew rates of irrigation contained 9.6

to 11.5| sugar, whereas those obtained during October through February

from fields subjected to high irrigation contained 6.4 to 9.7%.

Refractive index determined with a hand ref tactometer has been used

widely among sugar and horticultural crops to estimate sugar concentra­

tions (31. in these instances, the sen content la dissolved

1m adequately correlated to auger concentration and/or flavor.

EXPERIMENTAL SECTION

IDENTIFICATION OF PRINCIPAL SUGARS

A. MATERIALS AND METHODS

1. Sources sad Kinds of Sam plea:

The fruit aampftea used for sugar identification were obtained

from die Waimaoalo. Poamobo and Mid-Pacific branch farms of

dm Hawaii Agricultural Experiment Statioa. klentificstioas were

performed on fruits representing nine genetic lines, flue stages

of fruit maturity, and two seasons of harvest. The genetic lines

considered were Solo, etereon 170, Hybrid *5, Guinea Gold,

IAC-VA 32, LAC-VIA-16, IAC-23. IAC-39, and Bettina 100-A.

In tfffffilnthwi of both gonotic lines seasons of herveot,

fruits holf yellow at picking and subsequently allowed to develop

M l color In dm laboratory were used. In the study with genetic

lines, only one fruit of each line wan used, wberaes in dm eee-

eooat study, three fruite et each harveet were examined. Of the

latter, only average observations era reported.

To consider identity of eugsrs associated with fruit maturity,

the stages full yellow, half yellow, a trace of yellow, mature

green, end immature green were considered* These stages

refer to maturity at time of picking; the colors describe ex­

ternal appearance of fruit. Mature green fruits were charac -

terised by brown, full-six* seeds, whereas the immature

green fruits contained email, white eeads. A complete act

of fruits depicting each etage of maturity was obtained from

each experimental tree. Two sets of fruits were picked et

each of two separate dates -* November 29, 1961, and

January 3, 1962. At each harvest the sugars of one set were

immediately extracted. The other set wee eltowed to attain

full yellowness ie the laboratory and then extracted. Only

Solo fruits were used in this and the seasonal study. in the

see tonal study two to three harvests were made during each

of summer and winter.

reparation of extracts:

The procedure developed end employed la this study to

prepare auger extracts from papaya fruit constated of die

following steps:

a. Fifty grama of pulp were removed from one half of

each fruit.

b. One gram CaCOg was added to maintain near neutral

pH and retard inversion of sucrose.

c. One-hundred milliliters of distilled water was tiea

added and the mixture wee homogenised for 5 minutes

in « Waring Mender.

d. The homogenete wee hoiled for 5 admass to inactivate

enzymes.

e. Five grams of washed F liter-Cei was added and the

hot mixture wee filtered.

f. The residue was riaeed with two SO-ml. portions of boil-

lag distilled water.

g. The combined filtrates were cooled and diluted to SO mi.

Storage erf die extracts over extended periods was achieved

by freezing m oiyethylene plastic bottles. The degree of thorough­

ness of auger extraction by this procedure wee evident in the virtual

absence of •isM wn - fisc tlvo substances extrectable from

the residue. The residue remaining after auger extraction wee re­

extracted by tie seme procedure, end la a typical teat tie remain­

ing contents in antirone-reactive substances raged from 0.033 to

0.144$ of the sugars already removed.

Chromatography:

la preliminary explorations diverse methods described by Block

d d . ( l ) s a d Lederer sad Lsderer (3) were tested. The following

a. apex:

Whatman * i, sheets 7 1/2 x it laches.

b. Solvent System:

Upper phase of a butanol; 95% ethanol; H O,

4:1:5, V:V:V.

c. Spotting:

Twenty-five .mcroltsra of tie frut extract

were a potted 2 inches from tie base of tie

ehect. Reference glucose, fructose, sucrose

end other augers were spotted la amounts of

500 micrograma each.

d. Development:

The descending technique wee used, and develop­

ment wee allowed to proceed over e 3 day period.

e. Detection:

A 3'f solution ef p-oaiatdtne >tK!l la n-butenol wee

sprayed upon the ehiometagrame sad yellow or

brown epota formed toy sugars were detected after

beating 10 minutes et lOtiPc.

Since it is often possible that sugar extracts prepared toy tie

above ontiUaed steps may contain artifacts, tbs data baaed upon

a.

these extract* were eventually verified by direct examinations

of unheated and otherwise unmodified fruit sap*. Tbe chromato­

graphic data axe quantitative only to the extent dud area and color

intensity of spot* were considered la estimating relative amounts

of sugars*

4. Relative Content of geeross

rogres* in the chromatographic identification work disclosed

the sucrose level in extracts to be ^significant la comparison to

that of either glucose or fructose. Thus the relative amounts of

sucrose in extracts from Solo fruits showing undetectable,

moderate, and high chromatographic levels were further deter­

mined by dm method of reducing sugars described elsewhere in dm

section entitled, Refractive Index es s Measure of Seanr Level.

5. Stanch Detection;

Alee apparent daring the course of investigation wee die con-

• ncuoua absence of reserve carbohydrate In papaya fruit. Thus

an attempt was made to detect starch hi fruit tissue of varying

stages of maturity and ripening. A drop of aqueous iodine reagent

(0.3 gimme i3 and 1.1 grama El to 100 ml distilled water) was

:«laced upon the tissues; immediate development of blue color indi­

cated presence of amyloae. More critical observations were

fffcrtvinut by microscopic of the treated tfTirt

sections.

DATA AND DISCUSSION

1. identity of Sugars la Diverse Genetic Line*:

Tbs anisidiae-reactive substances separated from sugar

extracts of ripe papaya fruits of several genetic lines axe shown,

with relative amounts, la Table 1. H e meet striking character!# -

tic of all lines examined was the prominence of both fructose sad

glucose sad virtual absence of sucrose. Sucrose was detected in

only the Solo lias and even then in only a small amount. The

data obtained bare, together with previously reported observations

of other genetic lines (10, 13), do not preclude widespread

occurrence of sucrose la Caries papaya, but emphasis# the

relative insignificance of sucrose as an accumulated sugar in the

species.

An unidentifiable substance, showing positive reaction with

p-snlaidioe • HCl and a low Ilf liwttcnl to lactose is butanol:

ethanol:water, was also obesrved in Solo but not other lines.

Neither maltose nor raffinose could be identified with thin sub­

stance. This unknown did not reduce silver nitrate, end thereby

eammm wmm at vsm wmm or wm mmrm m s or t& su

TABU: I

Genetic 14* rgactoea .-7 RMMH Saen* tfcSAnt&fSad**

fafemm 17D 3 3 0 0

Hyteid 19 3 3 0 0

3 3 0 6

XXMfe 33 3 3 0 0

iMSwlf&FdS '■ ^ 3 3 0 0

wmm 3 3 0 0

3 3 0 0

AvfeUaa 100-* 3 3 0 0

$•&» 3 3 1 9

•0 * ima-deteetftbta, 9 » tw e i, 3 « andante m a tt» 3 * Ix& w esacnst*

* “>iil fiTrT1aaiirmt>TTr aofeatMkm trittfe Rf In " I t r t tn iliry i soXv&sA go 2aet#em, iMfc e*1*1 ■**>■# ay

suggested S non-reducing compound.

Component Carbohydrates la Solo .apsya ss Related to Siege

of Fruit Maturity:

Regardless the stage of fruit maturity, fructose and

glucose were again the meet prominent soluble sugars. This

is evStoat to toe date of Table II. The detectability of sucrose,

st least to toe present tests with Solo materials, astmad to

depend upon fruit maturity. Sucrose eea present to low amounts

only to fruits which were harvested to e stage when some degree

of yellowing, s characteristic associated with ripening, wan

evident. The green fruits contained no detectable sucrose.

This contradicts toe finding by Jones et si. (4) who reported

an inverse relationship bstwssn fruit maturity and sucrose con­

tent. The basis for the discrepancy remains to be investigated.

emitting immature fruits to ripen to the laboratory caused

oo change to toe kinds of extxactabie sugars. Detectability of

toe unidentified naisidine -reactive substance paralleled that of

sucrose, i.e ., it was only detectable when sucrose wee present

and undetectable to the letter's absence.

Reserve carbohydrate to conspicuously absent in papaya

n13.

c o o e e o o s o o l i i t i i i | i «

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«*#•©© «*♦►**©© <******©© **•*«•©©

j) 21i

hiI0 •« r

i{|Ie s

r ?S

fruits. This is shown m part by tbs lack of starch in fruits of

varying stages of maturity (Table IQ. Absence of starch la

papaya fruit had been observed earlier by Thompson (13). Fur­

ther evidence showing lack of a sugar reserve in papaya is

described in a section to follow la this report.

3. Sugar Composition ss ftelatsd to S—son of Harvest:

Table Ul related die soluble sugar constituents to the season

of harvest. Analysis of Solo fruits obtained from ths three branch

farms •- Watmanalo, ttarooho and Mid actfic ** showed no con­

sistent seasonal difference in kinds of sugars. Fructose and

glucose were again very prominent and sucrose relatively iasigai-

fleant. According to these data, the nniHaatififrf substance was

absent is winter fruits; bid analysis of further asm plea did not

bear this out.

4. Range of Sucrose Content in Solo Papaya Fruit;

As is svidsnt In Table IV, tbs fruit eampls with s chromato-

graahicaily undstsctabls level of sucrose contained 0.125 ; that

with a moderate level, 1.79%; and that with a large amount, 2.65.

by flesh weight of thie sugar. These percentages represent a

range of only one to 20 percent of the total sugar in die extracts,

and agree with reports of previous investigators.

These (tots, which attach special prominence to the reducing

sugars fructose and glucose, may cam some doubt on die reported

13.

TABU in

CCMPGKST SUGARS IS K Pf SOU PAPAYA PROTT AS KEUftW TO SSASC* OF MMSSf

S u i t e r W i n t e r

Dateharvested

DateHarvested

r t i & t o - I m l j i L ^ w s s r A z - j A w t i S ,___Fructose Glaocee Sucre— Unidentified**

6/17/a7/u/a6/25/ a

7/ivaP/10/61

7/19/a6/i6/a

333

33

33

333

33

33

111

11

11

* A , * J U J A k £

o 12/Va1/17/62 2/14/62

g ,o ju m u B1/17/622/W &

mumpacimc

333

33

i/17/a 32/U/62 3

333

33

33

111

11

02

000

00

00

*0 * no»-detectable, 1 * truce, 2 * — derate a— ant, 3 * large a— out.

**ial«ld1ae react Ira eobetanee with Rf ia betaaol tethanol tuater e-parable to tte t of laetoee.

atmr m memm ctmrm m m$. p in ii m m

TAMM, St

FruitSttSple (SS S3— 1/

i: rmn frr^rJE—mISEZEZ S SI (— osmmmiM

jg m fiis s s S A .o f total

96

i t

153

3

2

0

9«8S

*6*73

12.125

M i

123*

12^5

9 f#

1*73

C.125

M i

1AJB0

1«C2

vitamin C content (60 mg/100 g) of papaya (6). This vitamin

la usually estimated by raeaettr ng (be reducing capacity oI

extracts, and inadequate removal of the reducing sugars con­

tained or Improper choice or reagent can possibly result in

erroneous data.

The exact proportion of glucose end fructose la papaya

fruit baa not been eetabiiebed la this study, bid chromato­

graphy suggeatad that theee sugsra occur la equal amounts.

Tha identification of the sniakh ne-reactive substance With

Rf la butanol :e€haooi; water identical to lactose may prove to

be significant. Whether or act this substance ta a auger re ­

mains to be established, ft is interesting that tb'.s com >ound

la evident ta frutt extracts only whan sucrose la present, and

the exact relationship between the two may provide challenging

study.

I!. REFRACTIVE INDEX AS A MEASURE OF SUGAR LEVEL

A. MATERIALS AND METHODS

1, Source aad Kinds of Fruit Samples:

One fruit each of 6 genetic lines of papaya were used to

16.

were

mad

e on

msi

lute

d,

uofil

tere

d >.

th #

be ng

the

usua

l

8

1!a*s II

II

m

%

I

I1

i

MI|II

ii

i

as well aa relatively non-specific and bias sensitive

than the enthrone method described below. The

method adopted in this study, except for clarifica­

tion and hydrolysis, was Method I of Ward end

Johnston (14). The method consisted of the follow

ing steps;

a. CiTificat on and Hydrolyse of Extracts:

Tbs method of Loomis (7) with alight modi -

fication was used. To 25 milliliter* of extracts, e

solution of saturated neutral lesd acetate

( was added drepwiee with con*

scant stirring untu a yellow precipitate wee obtained

in aa external indicator of S& otusium chromate

(KjCrO^). lbs suspensicm was either filtered

through acid-washed asbestos or centrifuged five

minutes at circa 1000 x G and the clear solution was

subsequently deleaded with crystalline potassium

oxalate (KjCjO^.HLjQ). A weight of potassium oxalate

equivalent to that of the lesd Metals required for

clarification was used. The deleaded suspension

was further filtered or centrifuged. Tbs clarified

solution was then hydrolysed by adding 2.5 mi.

concentrated HC1 and standing overnight (at

least 12 bouse) at zoom temperature. it me

subsequently neutralized to pH 7.0 with 2 N

NaOH# and diluted to 390 sal. One milliliter of

due solution diluted with 4 ml. distilled water

wee carried trirough the analytical procedure

described below. Mote that the fins! dilution of

extract used in sugar determination by the reduc­

ing sugar method was 5Q -fold,

b. jaesaeats:

1. otaas um ferocyaaide (K|Fe (C N ^ ), S .25 g . .

and sodium carbonate (NSgCQg*), 10.40 g.

Both salts ware dissolved ia one liter dsst.iled

water.

2. otasauun iodide (Kl), 12.S g .i sloe sulfate

(Za&O . 7HgO), 25*0 g.i sodium chloride

(NaCi), 125.0 g. The three eslts were dis­

solved ia 500 ml. distilled water.

2. Acetic acid (CgHgOg). t l (V/V).

20.

4. Starch indicator. Oat gram starch « * i

added to 20 ml. water, mimd tad poured

into 60 ad. boiling water, and boiled lor

2 minutes; 20 g. sodium chloride was than

added, and tfee solution was cooled and

made to a volume of 100 ml.

5. ota**mm Iodate (K IO j), O.OIOi eollltlee.

6. Qtassium iodide (Ki), 2$ solution.

7. Sodium Thioeulfste ( N a j S .Sr^Q), 0.333

solution.

S. Glucose

c. Standsrdizat.o ; of Na^S|0a soiut on.

A mixture of S ml. reagent S, 8 ml.

reagent 6 and 3 ml. reagent 3 was titrated with

reagent 7 using starch indicator. Normality

o f N e ^ -

g. K10^/liter X ml. KiO solution used 35.67 X ml. thioauUate solution required

d. re la tion of Standard Curve:

Pile milliliter solutions containing 0-4 mg.

glucose were carried through tbs procedure in

step <•) below, and die 0.O1N thioeuifete

equivalent determined:Ml

0.01N tiuasuliate jp ffiiiB t “ (WB-R) X

where WB * weter hlenk titer, R * sugar

solution titer, and N * normality of tiuo-

euifate auiutioa.

A typically obtained eteadard curve relating

glucose content end 0.01N thioaulfete equiva*

lent le ahown m Figure 1,

Analytical proeedure:

Five i iUUiter* of auger solution or

extract were mined witliS ml. reagent 1 la

n boiling tuba, covered loosely with e glass

bulb, end placed ia boiling water lor 15

miautea. The reaction mixture waa toen

cooled lor 3 minutes laa cold water beth,

3 ml. reagent 3 and 3 ml. reagent 3 were

added, end titration with standard sodium

thiosulfate (reagent 7) using starch ea indi­

cator (reagent 4), was performed. A bleak

determination wee made simultaneously in

place of sugar solution.

23.

Aathroae Method:\ .;

The enthrone method l« reportedly court epecific

for carbohydrates tnan reducag sugar methods. Fur­

thermore, It it much more sensitive, aim ole, tod

rapid, tad it measures all forma of carbooydratea.

The method itttd la thit study net adapted from thoea

of Morris (9) tad Loewu* (6), tad cootitttd of tht

flowing:

a. Reafltnt:

Too grama (2.0 g .) enthrone {9-oxyantnraceae)

wore dissolved la I liter of 90% of HgSOjg, pet*

pared by cautious addition of 1 liter concentrated

HjSO to 30 ml. distilled H^O.

b. hrocedurs:

Three miUiiitera of the solution to be deter­

mined were measured am as It x ISO mm. teat

tube, and 0 ml. of the reagent wee added. The

solutions were mixed thoroughly at ooca by swirling.V/v. ' xi' 1 ' -v : ; ; ; : s ;\ * • v'l' V '• v v \xhj - ’

The tube wee pieced is a boiling water bath for

3 mmutea and then cooled. The color developed

wee meeeared ia a Biutch myi i omh Spectrosic

20 colorU&etc r at a wsveleagtb of 320 millimi­

cron# compared against a blank containing

only water tad reagent. Linearity of Use re*

action In the range 0 - 136 micrcgram# glucose

in evident la Figure 2. A 30-mlcrogtam glucose

standard wee carried throughout *

All domical analyse# were performed is duplicate#. The co­

efficient# of correlation between refractive index and total sugar#■f <?Bvr

ee determined by reducing auger tty* enthrone method# were cal­

culated , The ye* were examined by constructing

regression lines. All etatietlcai analyeee performed here and else-

where in die work were baaed upon procedure# described by Saedecor (12).

S. DATA AND DISCUSSION

The date showing range#

auger content ef papaya fruit# ere presented in Table V. The correla­

tion between refractive index of fruit eap end auger content in the fruit

was indeed significant, the coefficients between refractive index and

total sugar were 0.997 by the enthrone and 0.99S by the reducing sugar

method. These relationships are further illustrated fay the rugreneion

lines in Figure 3. Note that sugar determination# by the enthrone method

were consistently lower than those by the reducing sugar method, and me

**}fg

. nr- ■____ __ _ ____ __

ABSORBANCE AT 620 m j i

w i t

: S 24XMNUP 1S8WES 9NNK (W FW Tf Hi? ASl SZX&fcM CWfSSJH? 43V fffllf'Wr

TOTA

L SU

GAR

IN

FRU

IT

(AS

GLU

CO

SE

, %

FRES

H

WE

IGH

T) 15 r

12

6 _

3 _

0

O = Anthrone Method: Refractive Index,Y = 0.898 X - 2.16

• = Reducing sugar Method: Refractive Index,

1.04 X - 1.94

✓

//

// /

/ ///

// /

//

12 16

REFRACTIVE INDEX OF SAP (% DISSOLVED SUBSTANCES). Figure i. Relationship between refractive index of sap and augar coat*# offrapstya fruit

determined by aathrotse and reducing augat methods.

mom accurate of the two needs to he further determined. By extra*

pointing the regression lines to bisect the refractive Index axis, it

is that the sap contained approximately 2pt of dissolved

substances other than sugars. While refractive index does act far-

aish an absolute measure of sugar content of papaya, its judicious

yafe n rn v t fk i £ SUllSidti iU S llilS *

tlons. foe example, ia genetic selection studies.

SEASONAL AND FRUIT MATURITY RELATlONShtFS

A* MATERIALS AND METHODS

I . Seasonal Relationships;

The aas** 6olo fruits employed ia the sugar jdenflflcsfl'M)

study (Section I) were used to explore the tfuestitstive relation-

«htn oetwean «• » » « « of harvest ■»*< aiztr content. After fruits

i> d f frein^f m ripeness hi the laboratory, refractive index

of >*»s **p ««d moieture contents were determined, extracts

were prepared for s^aysis#! ^ ngsr determination by the reducing

sugar sad enthrone methods described uadtr Section It,

Retractive Iir-.ex a* a ■" easure of Sugar Level.

Te determine moisture content, a cylinder ot pulp was oh*

taitted with a number 5 cork-borer from the uoextrected half

of each fruit, weighed, and rewefghad after 120 hours to an

80°C oven. According to preliminary trials constant weight

was obtained by this method. The loss to tissue weight after

desalcatloa was used ss index of tissue moisture content.

T-teet of group comparison! between summer sod winter

fruits was performed on data of each of toe three locations

from which fruit samples were obtained. Standard error of

trttrt was also calculated.

Fruit Maturity ftelatlooehto:

The asms fruits sad extracts employed to part of the identi­

fication study (Section 9 were also used to determine toe

quantitative relationship between maturity and sugar content.

Thus two sots of fruits xeprossntiag fivs state* of maturity,

each picked on each of two separata dates — November, 1961,

and January, 1962 - - were examined. One set was analysed

immediately after harvest and the other when the iruits had

ripened fully to the laboratory. The data obtained wars re*

tractive index sad total sugar by the enthrone method. Steps

to obtain each of toss# data ware identical to those previously

described.

DATA AND DISCUSSION

i. Seasonal Relationship:

Kafr active indices, moisture contents sad total sugars by

anti) roue sad reducing sugar methods of ripe Solo fruit obtained

during summer and winter of 1961 -62 from tbs three branch

farms are shown in Tables Via and VIb. T-teats showed highly

significant differences between summer and winter refractive

indices,moisture contents, and total sugars by the reducing;

auger method. Summer fruits consistently showed higher re­

fractive indices sad total augers and lower moisture contents.

It thus may be expected that fruits obtained during summer will

be sweater than those of winter • This relationship may be ex­

pected la Hawaii since higher intensity sad longer duration of

light is characteristic of tbs summer, whereas lass HgN and

greater rainfall ia mors typical of winter. These factors directly

in fiii*^ |be rates of synthesis eraf echifTvelstlofi of carbohydrate

ia Ml plants. Awada and Ifceda (1) observed earlier that sugar

content of papaya was inversely related to the supply of soil

moisture.

No difference was evident between seasons ia total sugar

as determined by tbs enthrone method. This ia perhaps

the method la so sensitive that a drastic dilution (1000 fold) of

TAB* m

c m s m t i * RCfto patau m u? as to m m m m ?j#m st

« r^adfoMitfw

t m s e , '■•; M oletor*, # Mi w mq! tftm m t

l A J L E A l A O

7/W fcl15**A * C«6 1 5 ^ 1 C,21 M 1 M * 4 ^ * * 4

f S t f f il

3 f a f i&

13w6 * Q «£ 12.3 * 0 .3I M j t W

8 6 .3 * 0 .4« l i Sa § . 3 * e a

a / tc fa i1 4 *4 * 0.31 4 .6 * 0.1

*5 .1 * 0 .1« W l W

\ f a t e2 / W &

1 2 .5 * 0 .21 2 .9 * 0 .3

8 9 .1 * 0.5 8 9 .5 * 0 .2

? / t ^ iV I6 / H

14.5 1 0 .1 15 *11 0*3

554 .9 *0 .3 «4 ^ S * 0.1

i / p M2 f W &

10.7 * OwR 1 2 ^ * 0.1

9 U 0 * < V » 8 % 3 * 0.1

«mm mcavamtm o r art/) ? *p a x a F u t a r a s s m to a u a o i m mmsr

V l i l t r

Burmet mm

fiwhii dapwby ABtoMM(«S

f M b

total »9apur IqrPAMowla Sugar Caw O M 0M f f ~*edb ttg * * }

WBwfwSate

total Sugar by a t ; I*’1?*'!©' Caa Glueroae,

$ FVaaftk Aalgbt)

total Sugar tgr OtPaMat Super Caa daeoaa» f Ttmtki *tel«£t)

X j U E d l J J l l J ?

y rr fa7/14/418/11/St

9 .2 * 0.1 10.5* CU3 9 .* * 0.3

13^ * C.3 1 3 ^ 1 CLO 12.3 * 0 .1

nyty&il/l7 * 22/I4/&2

9«£*0*>1 0 .5 * 0 .9io a * c .5

H .7 * 0.5 11.0* CL4 10.7 * 0.3

U L U U U U

jytoA'i9.5 * 0*4

KU2 * cmi3 d S * e u 13.5 * C.3

t/w M 10*1* 0.1 10.1 *<L3

12.2 * OU 12.5 * 0 .6

Klfc > PJCI71B

7/195^1V t ^ 1

F.5 ♦ 0.2 9d6* OgO

13.7* ^ 3 1 4 ^ * 0.3

1/17/532/M ^2

0.7 *10.11 0.3

10.7 * 1.3 11.9 * 0*5

extract was required and any error ia preparing the dilution

was magnified proportionately. In effect# these date suggest

that the enthrone method cannot ha applied to dames small

differences la sugar content of papaya fruit. However# when

large differences axe expected# such as among certain genetic

lines or as evidenced below among fruits differing widely ia

maturity# the enthrone » t * M snay be advantageous •

Fruit Maturity Relatioaahipa:

Sugar content with respect to stage of maturity of papaya

fruit ia shown in Table VH. Admittedly the data may not ha

based upon adequate replication; nevertheless# definite trends

axe evident. Total sugars wars substantially lower la fruits

picked before any externally evident yellow color. Allowing

grata fruits to ripen la the laboratory resulted In little if any

change in sugar level. These observations, together with that

In a preceding section where tissue amylose was shown to be

absent, establish that papaya fruits lack reserve carbohydrate.

Tima, the report by Thompson (11) la further substantiated, and

the implication ia made that hulk of the sugars ia a papaya fruit

must result from the continual mobilization of soluble sugars

from other parts of the plant and accumulation ia the fruit during

maturation and possibly during the xipeuing process. In

table m

mm &mcmmam or sm fm u mm m musm to hkrhuw at mmm

MfclS la p of Fruit v A. -- .. 1 * -- Stags of futurity_Jk ...- ■*..*.---<fc.fc% 1 A*' f j*; J yiE"*?, *

Haffeaafctes XniMtf 9

foiftl OHgMPB I f ABtfercgw (as daasaa. %

nWjpR wSGfct.

9 f 4M» 10.34 4 «M» 104I 3 «•*» 1142 2 MB < 41 1 — 3*9

9 5 «N* f0m 34 9 mm 12.13 5 mm 1142 5 mm 3.91 9 mm 2 4

i/j/te 5 9 13JB n .?i 4 15.0 12.1I 3 12.12 2 M 3 41 1 4 # 2 4

5 5 144 12.54 9 144 t»*33 9 334 10*72 9 9 4 4 #1 5 M 1.9

*1 * I— trim greee, 2 • —b w rsreen, $ m trsaa of ya&cm, 4 * half yellow, 9 * M l yellow.

commercial practice It would be critical that fruits be harvested

none too seen during maturation If maximum auger contest ia

deaired. Too early removal from trees la perhaps the reason

that fruits sold la markets substantially diatom from orchards

axe not as sweat as those sold ia nearby markets. Any evidence

of external yellow color should indicate adequate maturity.

36.

SUMMARY AND CONCLUSIONS

This study with p*?«ya fruits attempted to <|) identify the principal

sugar*. (2) establish the relationship betw ea sap refractive index and

actual sugar content, and (3) determine sugar content as related to sea­

son of harvest and Mags of maturity.

Regardless of genetic line, stage of fruit maturity, or season, die

reducing sugars fructoee and glucose were the moat prominent, the

exact proportions of each were not determined, although chromatography

suggested nearly equal levels. Sucrose, observed only la Solo, was de­

tectable in relatively small amount, ranging from one to 20 percent of the

total auger. Even in Solo, green fruits contained no sucrose. Os the

beais of these findings, together with similar observations made fay earlier

investigators, it is concluded dial sucrose is not a significantly accumulated

sugar in the papaya fruit.

Starch as amyiose was completely absent. An unidentified substance

giving positive reaction with p-snlsidtas - HCi but not eilver nitrate* sad

possessing an Rf in n-butanol tetbaaol: water Identical to lactose but sot

maltose or raffinoae, was detected in small amounts in Solo. Detectability

of diis substance coincided with the presence of sucrose, but its significance

end relationship to sucrose remain unestablished.

The prominence at reducing sugars in pa pays fruit extracts may cam

some doubt on the reported vitamin C content. Tide vitamin ia usually

estimated by the reducing ability at fruit extracts, and inadequate removal

of nugara or improper choice of reagent could result ia erroneous data.

A near perfect correlation between refractive index, ot fruit aap and

augar content of fruit ea measured by either the enthrone or reducing

auger method wee revealed. Nevertheless, refractive index coosietently

disclosed more than dissolved sugars, htwt -thee Id not be Inferred ee

absolute measure el augar. la comparative situations, for example, ia

genetic selection studies, its Judicious use furnishes w eqHtfe relative

information. Total augar as deterwdaed by the enthrone "wtVa* was ia*

variably lower than by the reducing augar method; furthermore, small

differences could not be detected. The enthrone method, while simpler

mat more rapid, has questionable reliability in soma work with papaya fruits.

Its extreme sensitivity requires evtraefe to be drastically diluted prior to

analysis and errors in preparing dilutions are magnified proportionately.

Papayas obtained in summer may be expected to be tweeter than those

of winter since In the former sugar ttfflwefty is and moisture level la

lower.

Fruits removed from trees

contained inriicetily lass than thnar which shtwidt some degree of

yellowing. Permitting green fruits to ripan after harvest did not result in

my change la sugar level. TMs fladiag, together with the observed abseace

of amylose starch ia fruit tissue *t any stage of maturity, estaoUsheu that

papaya fruits coataia so reserve carbohydrate. St implies that the bulk

of the augers la the fruit must result from costioued mobilUatiou from other

parts of the plaat mad accumulation ia fht fruit during matui atioa mad possibly

ripening process. Hsace, It la critical aot to pick fruits prematurely if

maximum sugar comem is desired.

LITERATURE CITED

AM dii M««ioii6. ia d i. 1957. Effect of water aad aitrogea apptice- tiocj on composition, growth, sugars ia fruits, yield sad sax expres­sion of tits papaya plants (Qiric* papaya L.), Haw. Agr. Exp. St*. Tech. Bull. 33.

Block, J., L . Durrum, aodiC. Zweig. 19ft.aad fJ*o*r Llectrophoroeis. Academic T «u , Now York.

Currence, T. hi., and R. Larson. 1941. Refractive Index *a so esti­mate of quality between *od withia muakmeloa fruits. last by*. 16:611-620.

Jones, W. W ., W. B. Storey, O. K. Partis, and F. C. HoWaway. 1941. 'spays production io the Hawaiian islands. Haw. Agr. Exp. Sts.Bull. 97.

Lsderer, E. aad MU Lederer. 1937, Chromatography, A Review of Principles sad Application. D. van Noetssad, Hew York.

Loews*, F . A . 1952. Improvemsot is unthrone method for determina­tion of carbohydrates. Anal. Cham. 24:219.

Loomis, W. E. 1925. A Study of the clearing of alcoholic plant extracts. Teat hys. 1:179-158.

Miller, C. D.» B. Braathoover, N. Sekiguchi, H. Denning, aad A . Bauer. 1956. Vitamin valttss of foods used la Hawaii. Haw. Agr. Expt. Sts. Tech. Bull. 30.

Morris, D. L . 1945. Quantitative determination of carbohydrates with Uiwywood's enthrone reagent. Sc tenet 107:254.

Pops, W. T . 1930. 'spays culture ia Hawaii. Haw. Agr. Exp. Sea. BeU.i l .

*ratt, D. S. and J. t. del Rosario. 1913. Tsilippias fruits: composition aad charactariattea. hil. Jour. Sc. 8:59-80.

40.

12. Saedftcor* G. W. 1900. Statistical Method*. Iowa Stats College rose, Ames.

12. Thompson* A. A. 1910. 1he composition of Hawaiian fruits sad nuts. Hew. Agr. Exp*. Sts. Asut. Rapt. 1914:02-73.

14. Ward, G. M. aad P. 8. johastoa. 1900. Chemical ractixids of plant analysis. d a * Dept. Agr. t'sfei. 1004.