'Kind' KA bdclittonetn rBasfiir

- - B.Sc..(Agile.) Honours (Khartoum)

A Thesis

Submitted in partial fulfilment of the requirement Tor (lie

degree of Master of Science (Agric.)

Supervisor: Dr. Abu-Bakr Ali Abu-Goukh

MSPARTMICNT OF HOUTICUI/I'UKIi

FACULTY OF AGRICULTURE

UNIVERSITY OF KHARTOUM

MAY, 1999

3 1 / 2 8

Please be aware that all of the Missing Pages in this document wereoriginally blank pages

(In the name of Allah, the Beneficent, the Merciful)

My Lord: "Increase me in 1(iiozi/(ed(je"

(20:114, Ta Ha, Holly Quran)

Acknowledgment

My special praise and unlimited thanks avtto ALLAH; who

supplied helped and gave me health and patience to complete this

study.

I wish to express my deep sense ol' gratitude and sincere thanks

to my supervisor Dr. Abu-Bakr AH Abu-Golikh for his

encouragement, excellent suggestions, continued interest and

guidance during my study.

My greal thanks to Dr. Farouk Hassan El Tahir, Dr. Mustafa

ElbalJa hn~ continuous interest, help and encouragement.

Thanks and also due to the members of Horticulture and

Bioclieuisir_y_De4iartmeut,

I am grateful to my family members for their help and

encoruagemenl.

(ii)

ABSTRACT

Compositional changes in fruit pulp and peel.during ripening of

white - and pink-fleshed guava fruit types were investigated.

Fruit tissue firmness decreased progressively, in a simMar

manner, in both guava types. The white and pink guavas exhibited a

typical climacteric pattern of respiration, with.climacteric peak al 7.6

kilogram-force (kg. f) flesh firmness in both types.

Total soluble solids (TSS) and total sugars increased in pulp and

peel of both guava types with decrease in flesh firmness.. More

increase in total sugars was observed after the climacteric peak.

Reducing sugars and titratable acidity increased up to the climacteric

peak and decreased afterwards. Total protein increased up to the

-fttthripe stage- antHftmr-decreiised. A s c o ^

compounds decreased continuously during ripening of the two types.

The peel showed higher values of ascorbic acid and phenolic

compounds compared to the pulp. The white-fleshed guavas had

higher levels of TSS, total sugars, reducing sugars, titralable acidity

and ascorbic acid content compared to the pink-fleshed fruits.

Changes in the activities of the cell wall degrading enzymes,

pectinesterase (PE), polygalacluronase (PG) and cellulasc were also

studied to find out the reason for tissue softening in g.uava fruit during

handling, transportation and storage.

(ii)

Pectinesterase activity, in both guava types, increased gradually

till the climacteric peak and then decreased. Polygalacturonase and

cellulase activities progressively increased during ripening and high

correlation was observed between the increase in enzyme activity and

the loss in fruit flesh firmness. It was concluded'.that PE, PG and

cellulase enzymes play an important role in controlling tissue

softening mechanism during fruit ripening in guavas.

(iii)

CONTENTS

Page

Abstract - .- ii

Arabic Abstract -. iv

List of Figures ix

Chapter 1 : Introduction I

Chapter 2 : Literature Review - 3

J2..I. Origin, Production and Distribution -• 3

2 .2. Fruit Characteristic 3

2.3. Fruit Ripening and Senescence ----- 4

2 .4. Respiration Rale 5

% .5. Compositional Changes During Fruit Ripening 7

J2.5.1. Total Soluble Solids 7

-.2.5.2. Total and Reducing Sugars 7

2 . 5.3. Acidity and pH 8

2 • 5.4. Amino Acids and Proteins 9

2. «5.5. Phenolic Compounds 10

2. • 5.6. Vitamins and Minerals 11

2 .6. Fruit Softening - 13

£•6.1. Role of Pcctinestcrase 15

£.6.2. Role of Polygalacturonase 16

£ . 6 . 3 . RoleofCellulase 18

(vi)

Page

Chapter 3 : Materials and Methods 20

3.1. Fruit Source and Ripening Procedure 20

3.2. Respiration and Firmness Determination .- 20

3.3. Total Soluble Solids Determination 20

3.4. Compositional Changes 21

3.4.1. Tissue Extraction 21

3.4.2. Titralable Acidity Determination 21

3.4.3. Total Sugars Determination 21

3.4.4. Reducing5Tugars Determination 21

3.4.5. Total proteins Determination - 22

3.4.6. Phenolic Compounds Determination 'l'l

3.4.7. Ascorbic/I cid Determination •-•- 22

3.5. Enzymatic Activity Changes - 22

3.5.1. Enzyme Extraction • 22

3.5.2. Pectineslerase Activity Determination 23

3.5.3. Polygalacturonase Activity Determination 24

3.5.4. Cellulase Activity Determination 24

Chapter 4 : Results 25

.4.1. Fruit Firmness and Respiration Rate. - 25

4.2. Compositional Changes During Ripening in Guava Fruit 25

4.2.1. Total Soluble Solids 25

(vii)

Page

4.2.2. Total Sugars - 26

4.2.3. Reducing Sugars - 26

4.2.4. Titratable Acidity 27

4.2.5. Total Proteins 27

4.2.6. Phenolic Compounds 27

4.2.7. Ascorbic Acid 28

4.3. Enzymatic Activily(^hanges During Ripening of

Guava fruit 28

4.3.1. Pectinesterase * 29

4.3.2. Polygalacluronasc 29

4.3.3. Cellulase -' 29

Chapter 5 : Discussion 43

Conclusion - 55

References 56

Appendixes - - 74

(viii)

List u£ FiguresPage

Figure :

1. Changes of fruit flesh firmness during ripening of while-and

pink-fleshed guava fruits. 31

2. Relationship between CO2 production and fruit flesh firmness

during ripening of white-and pink-fleshed guava fruits 32

3. Changes of total soluble solids (TSS) during ripening of

white-and pink-fleshed guava fruits 33

4. Total sugars changes during ripening of vvhitc-and pink-fleshed

guava fruits. - 34

5. Reducing sugars changes during ripening of white-and

pink-fleshed guava fruits ---• .35

6. Changes in titratablc acidity during ripening of while-and

pink-fleshed guava fruits 36

"?. TaTT0^nnntirhfjfinTn"n"cioTiteTit daring ripening of "white-and

pink-fleshed guava fruits 37 -

8. Changes in phenolic compounds during ripening oi' whilc-and

pink-fleshed guava fruit 38

9. Changes in ascorbic acid content during ripening of white-and

pink-fleshed guava fruits - 39

10. Changes in PE activity d^crign ripening of white-and

pink-fleshed guava fruits 40

11. Changes in PG activity during ripening of white-and

pink-fleshed guava fruits 41

12. Changes in cellulase activity during ripening of'white-and '

pink-fleshed guava fruits 42

(ix)

CHAPTER 1

CHAPTER 1

INTRODUCTION

Guava {psidium guajava L.) is native of tropical countries. It can

grow satisfactory even in adverse environmental conditions.

The fruit has a characteristic odour and is eaten fresh, processed

or cooked (Saiunkhe and Desai, 1984).

Ripe guava fruit is an excellent source of ascorbic acid (vitamin

C) and dietary fiber. It has about five times more vitamin C than

orange (guava, 242 mg. per lOOg. j orange, 50mg. per lOOg.) (Watt

and Merrill, 1975). Guava is also a good source of vitamin A,

phosphorus, calcium and iron as well as tln'amin and niacin (Wilson,

-1-986)—

Guava in Sudan is still a minor fruit crop, but it is commercially'

grown in every region in the Country. Sudan has great potentiality to

produce good Quality guava fruits. It has good marketability in the

middle east and European countries.

The fruit is delicate and many culttvars cannot withstand

long-distance transportation and may reach the market in a mushy,

over-ripe state. It can store for only few days at ambient conditions.

Ripening of the fruit is characterized-by different compositional

changes and by softening of the flesh. Development of proper

handling techniques and control of the ripening proc'ess are of crucial

importance for development of a sound guava industry in the Sudan.

This study was carried out with the following objectives:

1. To provide base-line information regarding the biochemical

nature of ripening and softening mechanisms in the guava fruit, to

assist in development of sound programmes for controlling fruit

ripening and loss of firmness during handling, transportation and

storage of guava fruits.

2 - To study compositional changes in pulp and peel of white -

and pink-fleshed guavas during fruit ripening.

3. To investigate changes in the activity of the cell wall

degrading enzymesjpcclincstcrasc (PE), poiygalacturonasc (PG), and

cellulase during guava fruit ripening.

CHAPTER 2

LITERATURE REVIEW

2..1- Origin, Production and Distribution:

Guava (Psidium guajava L.) is an important cultivated species of

the Myrtle family (Salunk he and Desai, 1984). It is native to the

tropical regions of America. It was spread first into the Philippines

and India, and then quickly extended to most of the tropical and

subtropical regions of the world, _where it has become naturalized

(Yadav, 1998). The crop tolerates a wide range of climates and is

fairly salt and drought resistant. There could be 2 to 3 harvest seasons

in one.year. It has been reported that fivc-ycar-old guava trees can

produce from 130 to 225 kilograms of fruit per tree (28 to 48 tons per

Guava is a minor fruit crop. The major producing countries arc :•

India. (200,000), Mexico (127,000), Egypt (34,000) and Venezuela

(17,000) metric tons (Anon, 1978). ,

In Sudan, guava is a popular fruit crop. It is grown in every slate.

Marketability is limited to local markets due to the delicate nature of

the fruit and poor transportation and storage facilities.

2 »2. Fruit characteristics:

Guava fruit has a rough yellow skin and is round, ovoid, or

pyriform shape. The flesh colour varies from white, deep pink to

salmon red, and the flavour has been described as sweet, mushy, and

highly aromatic. The fruit is eaten fresh, processed or cooked. It is

used for the preparation of juices, jams, jellies, pastes and other

similar products (Salunkhe and Desai, 1984). Guava fruit is very rich

in ascorbic acid (Wilson, 1980). It is a fair source of beta-carotene

(280 IU), calcium (23mg), phosphorous (42mg), riboflavin (0.05'mg),

thiamine (0.05mg) and niacin (1.2mg per lOOg) (Watt and Merrill,

1975). Reportedly, consumption • of'guava fruit reduces serum

cholesterol levels, triglycerides and hypertension, and increases the

level of high density lipoprotein. Both ripe and green fruits, leaves,

roots and bark are used in local medicine for treatment of

gatroenteritis, diarrhea and dysentery (Yadav, 1998).

1*3. Fruit Ripening and Senescence-

Ripening is the sum total of the physio—chemical changes which

makes fruit edible. Ripening is a dramatic event in the life of a fruit. It

transforms a physiological mature but inedible plant organ into a

—vJsuallyjitliacJL^^ the completion

of development of a fruit and the commencement of senescence, and it

is normally an irreversible event. Ripening is the resuU of a complex

of changes, many of them probably occurring independently of one

another. The number, complexity and commercial importance of these

changes make fruit ripening special case of plant organ senescence

(Wills etaL, 1981).

Ripening and senescence processes involve the action of a group

of chemical substances produced by the plant itself- plant hormones.

Chemists have synthesised compounds which function as the natural

plant hormones do, and have achieved some success in controlling the

vital processes of ripening and senescence of fruit. These chemicals

may hasten or delay ripening and senescence (Salunkhe et aL, 1975).

Mature green guavas can be stored at 8 to 10°C and 85 to 95%

relative humidity for 2 to 5 weeks (Panlaslico el aL, 1975).

Ripening of guava was markedly hastened by treatment with

growth regulators such as 2,4 - D and 2, 4, 5 - T. The rate of ripening

was doubled in guava treated with 200 ppm 2, 4, 5 - T (Saha, 1971).

Singh et ah, (1981) found that dipping of guava fruits in calcium nitrate

solution (0.5 to 2.0%) reduced weight loss, respiration rate, disease

occurrence and maintained the edible quality of guavas for more than 6

days compared with 3 days for untreated fruits. Prc-and post-harvest

application of morphactin (a chloroflurenol methyl ester) retained

chlorophyll content, increased soluble solids, and reduced weight loss

with both pre-harvest (2.5 ppm) and post-harvest (2.5 and 5.0 ppm)

application, and extended the shelf life of guava fruits (Goldstein and

Swain, 1963).

Waxing of fruits retarded ripening and senescence of guava

(Bhullar and Farmahan, 1980). The use of potassium permangenate

(KMnO4) or potassium ineta-bi-sulfite in muslin bags (at 10 g/kg fruit)

placed inside the storage polyethylene bags was reported to retard the

rate of guava fruit ripening and extended storage life (Bhullar and

Farmahan, 1980). Guava irradiated at 30 Krad and stored at ambient

temperature exhibited a marked delay in ripening up to 3 to 5 days

(Pablo etal , 1975).

% . 4. Respiration Rate:

Respiration is the process in which complex compounds are

broken down into simple end products, during which exchange of gases

take place. Respiration rate is an excellent indicator of metabolic

activity of the tissue. Thus a useful guide of potential storage life of

fresh fruit and vegetables. Baile (1960) reported that the chemical

changes that taken place in detached fruits are directly or indirectly

related to the oxidative and fermentative activities collectively refer to

as biological oxidation. He found that the-pattern of respiration was

classified into four distinct phases basetl on the observed changes :

(1) The pre-climactric phase, when the fruit is green and firm and

CO2 production is at minimum. (2) The climacteric rise phase, when a

sudden increase in CO2 production is observed and the fruit remains

green and firm. (3) The climacteric peak, which is marked by a peak

in CO2 production. During this stage the fruit tends'to break in colour,

becomes softer and develops an aroma characteristic to the variety.

Thb phase corresponds with the eating ripeness of the fruit. (4) The

post-Climacteric phase, when the CO2 release shows a sudden decline,

the fruit develops attractive colour and aroma and becomes soft and

"e irJle ripe. Mteintrfs^smge^eiTesceTi-cesets-in, andthe fruit becomes-

susceptible to infection by microogranisms, resulting in decay anil

death of fruit tissues.

The respiration process was classified into three distinct stages by

Phan et al (1975) : (a) Breaking down of polysaccharidcs into simple

sugars, (b) Oxidation of simple sugars to pyruvic acid, and (c)fterobic

transformation of pyruvale and other organic acids into CX>2, water and

energy. Proteins and fats can also serve as substrate in the breakdown

process.

Akamine and Goo (1979) studied the respiration and cthylcne

production in fruits of four guava cultivars. The fruits of all cullivars

had a climacteric pattern of respiration with clhylenc triggering tiie

respiration rise.

5,5. Compositional Changes During Fruit Ripening:

During ripening, a i'nut passes through a scries of overt changes

in colour, texture and flavour, indicating that compositional changes

are taking place. Attain of maximum eating quality of fruit

necessitates the completion of such chemical changes. Unripe fruits

are usually starchy and acidic in taste, hard in texture and sometimes

astringent. After ripening, they become sweet, soft, and highly

flavoured, so greatly acceptable as human food (Maltoo ct al, 1975).

These changes, are described in the text below.

2.5.1. Total Soluble Solids-

Total soluble solids (TSS) were reported to increase during fruit

ripening (Abu-Goukh and Abu-Sana, 1993, Ibrahim eta_L, 1994,

Rodriguez etaL, 1971). The total solids in the fruit were found to

mradn-consl ant dnringJOiiiLripejoLUig.,The,.solubLe_sLolids,__oii.the_other

hand were found to increase at the expense of insoluble solids

(Krishnamurthy etaJL, 1960). Popenoe ct aL (1958) attributed the

increase in TSS during fruit ripening to the hydrolysis.of the starch to

sugars. This was then confirmed by Baile (1960).

Rodriguez et ah, (1971) observed a gradual increase in TSS

during guava.fruit ripening. Similar findings were reported for

bananas (Ibrahim et aL, 1994), mangoes (Abu-Goukh and Abu-Sara,

1993, Minessy ct ah, 1984, Saccd ct ah, 1975b) and dates (Dowson and

Aten, 1962).

.2,5.2. Total and Reducing Sugars:

Climacteric fruit in particular, may show considerable changes in

sugar content during fruit ripening (Hulme, 1971). Starch and.sucrose

7

were found to convert into glucose during fruit ripening (Wills et

aL, 1981). Mowlah and I too (1982) showed that glucose, fructose

and sucrose were the main sugars in white-and pink - fleshed guava

varieties. Chan and Kwok (1975) reported that the total, reducing

and non-reducing sugars were present in guava fruit in amounts of

5.8, 5.5 and 0.3%, respectively, and the gas-chromatographically

determined contents of fructose, glucose and sucrose were 3.4, 2.8

and 0.3%, .respectively.

Total and reducing sugars were found .tc increase during

ripening of guava fruit and then decreased (Subrahmanyam and

Acharya, 1957). The level of fructose increases during ripening and

then decreases in the over-ripe fruits (Le-Riche, 1951). Seshadri

and Vasistha (1964) reported that glucose, arabinose and maltose

increased during guava fruit ripening. Ogata et ah, (1972) found

that unripe pulp and peel of guava fruits contained trace amounts ol

sedoheptulose, which increased to 0.04% in the ripe pulp.

J2-5.3. Acidity and pH:

The non-volatile organic acids arc among the major cellular

constituents undergoing changes during ripening of fruits (Mattoo

etaL, I975). Organic acids play important role in development,

maturity, ripening and post-harvest life of fruits (Agnihotri et ah,

1964). Citric acid is the predominant acid in guava, while malic

acid present in appreciable amounts (Hulme 1970). Other organic

acids reported toc<x<AYw.guava include; tartaric, oxalic, succinic and

ascorbic acids.The reported acidity and pH of guava fjesh range

from 0.33 to 0.99% and 4.7 to 5.4, respectively (Salunkhe and

Desai, 1984).

8

Usually organic acids decline during ripening of fruits as they are

respired or converted to sugars (Wills et ilL 1981). Great variations

were reported in changes in organic acids during ripening of different

types of fruits. Krishnamurthy et aL, (196Q) reported 6-fold decrease

in total acids in "Raspuri" mango. A reduction of 35 - folds was

reported in "Alphonso" mango (Subramanyairret a_L, '1976). Medlicott

and Thompson (1985) found a profound decrease in citric acid and

small reduction in malic acid during ripening of "Kcitt" mango.

Reduction in total acidity was also reported during ripening of pears

and peaches (Vlrich, 1958). Total acidity was shown to increase

during fruit ripening in banana (Ibrahim et al , 1994), and pineapple

(Gortner et ah, 1967). Rodriguez etaL, (1971) observed a decline in

pH values of guava fruit extracts during fruit ripening.

Changes in acidity during fruit ripening were reported to follow

the re s p iratory climacter ic~ur bairanatAimTednEmd "Tmgw a, -19 95 -;•

Desai and Deshpande, 1975; Palmer, 1971) and mango (Abu-Goukh.

and Abu-Sarra, 1993; Krishnamurthy etaL 1971).

51-5.4. Amino Acids and Proteins:

Proteins and free amino acids "are minor constituents of fruits and

have no role in determining eating quality. Changes in amiRO acids

and proteins however, indicate variations in metabolic activity during

the different developmental stages. During the climacteric phase of

many fruits, there is a decrease in free'amino acids which often

reflects an increase in protein synthesis. During senescence, the level

of free amino acids increases reflecting a breakdown of enzymes and

decreased metabolic activity (Wills et ah, 1981).

The observation that the amino acids mcthionine and/or [3-alaninc

may possibly act as the immediate precursors orelhylene in the tissues of

fruits and vegetables (Burg and Clagett, 1967; Liebcrman et al., 1966;

Yang, 1985) signifies the importance ofamino acids metabolism in fruit

ripening.

A small net increase in protien content was observed during

ripening of mango (Mattoo and Modi 1969), pear (Li and 1 lansen, 1964),

avocado and tomato (Spencer, 1965). Quantitative changes insoluble

, proteins during mango ripening have been repeatedly demonstrated (Ghai

and Modi, 1972). Abu-Goukh and Abu-Sarra (1993) reported that the

total protein in pulp and peel of three mango cuitivars increased up to the

full-ripe stage and then decreased at the over-ripe stage. That decline was

explained as breakdown of proteins during senescence, which supported

the view that proteins in ripening fruits are enzymes required for the

ripening process (Frcnkcl ct ah, 1968).

5.5 Phenolic Compounds1:

Phenolic compounds are important factors of fruit quality. The

term "tannins" was reserved for those phenolic compounds of sufficiently

higher molecular weight to form strong complex with protein and other

polymers (Nezain lil-Din et al., 1983). Tannins represent the primary

substrate for browning reaction in fruit tissues (Ilulme, 1971). The

tannins arc also responsible for the astringent taste in unripe fruit which

disappears due to polymerization of tannins during fruit ripening (Al-

Ogaidi and Mutlak, 1986). Tannins have been repeatedly demonstrated lo

play a vital role in plant disease resistance and protect fruits and

vegetables against insect pests (Nezam lil-Din eJ.ajL, 1983) and diseases

(Walker and Stahmann, 1955, Raa, 1968, Mclcan ctaK, 1961)

Abers and Worlstad (1979) suggested that the more reactive

phenols, such as leuco anthocyanins and flavanols, play a major role in

the colour determination of strawberries preserves.

Factors such as size of the fruit, location on the tree and locality

of the tree were reported to influence the fruit phenolic content (Mann

et aJL, 1974). It was reported that phenolic compounds in the peel of

mango were higher than those in the pulp (Abu-Goukh and Abu-Sarra,

1993, Lakshmiriarayana, 1980). Tannins in dates are present just

below the skin (Dowson and Atcn, 1962).

It was reported that phenolic compounds decreased during

ripening of bananas (Ibrahim etal., 1994, Abu-Goukh e taL 1995),

mango (Abu-Goukh and Abu-Sarra, 1993) and dates (Al-Ogaidi and

Mutalk, 1986). Spayal and Morris (1981) reported that phenolic

cjonipounds were present in relatively high quantities m the immature

green fruit as compared to other maturities. Mowlah, and Itoo (1982)

reported that in both types of guavas^white and pink, the total"

polyphenol decreased all along from the immature stage to the to full

ripe stage.

The decreasing • in astringency with guav'a ripening was

associated with the increased polymerization of leucoanthocyanidins

and hydrolysis of the astringent arabinose ester of hexahydrodiphenic

acid (Goldstein and Swain, 1963, Misra and Seshadri 1968)

5,6. Vitamins and Minerals:

Vitamin C (ascorbic acid) is only a minor constituent of fruits

and vegetables, but is of major importance in human nutrition.

Virtually all man's dietary vitamin C (approximately 90 percent) is

obtained from fruits and vegetables. The daily requirement of man

from vitamin C is about 50 milligrams, and many fruits and vegetables

contain this amount of vitamin C in. less than ]()() grams of tissue

(Wills etaL 1981).

Guava fruit is extremely rich in vitamin C. It has about five times*v*

than orange (242 nig per 100 g for guava v$ . 50 mg per 100 g for

orange) (Watt and Merrill, 1975). According to Wilson (1980), the

levels of ascorbic acid in guava are subject to wide variation due to

geographical location, horticultural practices, season and cultivay

Reported values vary from 10 to 1160 mg of ascorbic acid per" 100 gin

fruit (Dedolph, 1966; Wilson, 1980). Variable reports are available

about the amounts of ascorbic acid in white - and pink - fleshed

guavas. El-Fake and Saeed(l975) reported higher values of ascorbic

acid in white - fleshed than pink-fleshed fruit, while other investigators

reported the reverse (Agnihortri et &L 1962; El-Zorkani, 1968).

The peel of guava fruit was reported to contain most of the

ascorbic acid in the fruit (Wilson, 1980). Similar results were repotted

for mangoes (Lakshminarayana etaL, 1970, Siddappa and Bhatia,

1954). The higher values of ascorbic acid in the peel were attributed to

interference of phenolic compounds, with the dye, 2, 6-dichlorophenol

indophenol used in the assay (Lakshminarayana et a_L, 1970). In

contrast, Abu-Goukh and Abu-Sarra (1993) reported lower values for

ascorbic acid in peel compared to the pulp in three mango fruit

cultivars.

The ascorbic acid content in guava fruit reaches a maximum in

the green, fully mature fruit and starts to decline rapidly as the fruit

ripens (Agnihorti etaL, 1962, El-Zorkani, 1968). The decline in

ascorbic acid content during ripening was also reported for. mango

12

fruits (Abu-Goukh and Abu-Sana, 1993, Minessy etaJL, 1984,

Chikkasubbaniia and Hudclar, 1982).

Guava fruits are fair source of provitamin A (P-Carotene) (280

International Units), Calcium (23 ing), phosphorous (42 mg),

riboflavin (0.05 mg), thianiin (0.05 mg) and niacin (1.2 mg per lOOg

fresh weight) (Watt and Merrill, 1975). French and Abbott (1948)

reported that the edible portion of pink-fleshed fruit had carotene

content of 3.1 nig per lOOg, while the white-fleshed fruit contained no

measurable carotene (at 450 nm). These investigators also showed that

about one-half of the colour of the pink-fleshed guavas was due lo

lycopene. Cordoba (1961) found that the thiamin content of

pink-fleshed guava was higher (0.05 ing) than that of the

white-flcshcd fruits (0.03 nig per lOOg).

X' 6. Fruit Softening: ,

Fruit softening is characterized by changes in flesh firmness and

has long been associated with fruit ripening (Dostal, 1970). These

changes in frilit firmness determine shelf-life and quality of the

commodity. Control of fruit texture is a major objective in modern

food technology (Van Buren, 1979).

Fruits undergo progress decline in flesh firmness with fruit

ripening. Abu-Goukh and Abu-Sarra (1993) observed a rapid decrease

in flesh firmness during ripening of three mango cullivars. They found

that more than 80% of firmness decline occurred over two days and

coincided with climacteric peak of respiration. Similar pattern of

change was reported for bananas (Abu-Goukh eiah., 1995), pears

(Luton and Holland, 1986), Apples, peaches, and apricots (Salunkhc

andWu, 1973) and dates (Barrcvellcd, 1993).

13

Histological studies and chemical analysis revealed a

considerable loss of cell wall materials during ripening - associated

fruit softening (Tandon and Kalara, 1984; Hall, 1964; Hultin and

Lcvine, 1965; Nour, 1978). As the Iruit ripens, the contents of

soluble pectins increase while the total pcclic substances decrease.

This trend was found in guava (Luh, 1971), melon (Rosa, 1928),

bananas (Van Loesecke, 1950), citruf (Sinclair and Joliffc, 196!),

strawberry (Neal, 1965) and mango (Tandon and Kalara, 1984).

There are evidences indicating that two processes are at work on

pectic substances during fruit ripening: (1) depolymerization or

shortening of chain length, and (2) de-esterification or removal of

methyl groups from the polymer (Mattoo et aL, 1975). The general*

observation is that softening is accompanied by solublization of

pectin substances involving the sequential action of pectinesterase

(PE) and polygalacturonase (PG) enzymes. This notion was

supported by reports on changes in cell wall pectic materials in

ripening mango (Tandon and Kalara, 1984; Roe and Buremmer,

1981) tomato (Besford and Hobson, 1972; Arad etaL, 1983), pear

(Ahmed and LabavifeU, 1980a), Peach, apple (McCready and Me

Comb, 1954) and strawberry (Kertcsz, 1951).

Very limited degradation of Cellulase was reported to occur

during softening of many fruits, including banana (Barnell, 1943),

peach (Sterling, 1961), pear (Ahmed and Labavitch, 1980a) and

apple (Kertesz el ah, 1959; Knee, 1973), Tahir and Malic (1977)

reported that the Cellulose and hcmicellulQse contents of mango fruit

did not show appreciable changes during ripening, therefore, appear

to have insignificant importance in lextural changes during ripening. ,

However, Ceilulase and hemicelluose were reported to change

14

substantially during softening of dates (Coggins ejLaJL, 1967;

Hasegawa and Smolcnslfy, 1971) and bananas (Barnell, 1943).

Besford and Hobson (1972) observed some degree of softening in

tomato fruit before commencement of the PH and PG action. A

non-enzymatic reaction was suggested to occur in plants which might

be responsible for some of the changes that occur in pectic substances

as well as in other polysaccharides (Kertesz, 1943). A wall-modifying

eiizyme was reported by Karr and Albcrshcim (1969) to be an

important prerequisite for the action of the polysaccharide degrading

enzyme. Mattoo ejtaL, (1975) stated that softening in some fruits

occurs through hydrolysis of starch (as in squash) or fats (as in

avocado).

. 6.1. Role of Pec tines terase:

^

hydrolysis of methyl and other ester groups of the polygalacluronide.

chains of pectin (Lee and MacMillan, 1970; Lineweaver and Janscn,

1951). A direct role of PB in fruit softening has been demonstrated;

Buescher and Tigchelaar (1975) reported that PE activity increased

during ripening of normal tomato fruit, while that of rm fruit (non

ripening mutant) remained constant.•The failure of the nou ripening

tomato mutant (rin) was evaluated in terms of PB activity (Buescher

and Tigchelaar, 1975). PH activity has been reported to increase

during ripening of banana (Ilultin and Levine, 1965; IX\Swardl and

Maxi, 1967), date (Al-Jasim and Al-Delaimy, 1972) and tomato

(Hobson, 1963;'Pelhawala el aL, 1948). Mattoo and Modi (1969)

related the softening of chilling injured mango fruits to the increase

activity of PIS. Barmare and Rouse (1976) suggested that PB activity

15

Could be used to monitor changes in softening time in avocado fruits

during storage. In pears, PE activity was reported to decrease during fruit

maturation (Nagei and Patterson, 1967) and level up during ripening

(Ahmed and Labavitch, 1980b) Abu-Sarra and Abu-Goukh (1992)

reported that, PE activity in "Kitehner" and "Dr. Knight" mango fruits

decreased steadily during ripening at similar rates, whereas that in "Abu

Samaka" fruits it increased up to shortly before the climacteric peak of

respiration, then decreased. Such decrease in PB activity was also found

in ripening mangoes by other workers (Roe and Buremmer, 198!; Tahir

and Mafic, 1977; Van Lelyveld and Smith, 1979). However, Ashrafet aL,

(1981) found inconsistent pattern of change in PI.7, activity of some

Pakislane mango cullivars.

•The—peehV^i-ibstaim^s-xxll^L^ showed

different degrees of esterifiealion. Saeed el aL, (1975a) reported 58,70

and 75% degree of estcrfication for "Alphonso", "Kitchncr" and "Abu-

Samaka" mango cultivars, respectively,. A very high correlation was

observed between the degree of eslrification and fruit firmness (121-

Nabawi et ah, 1970). Alni-Goukh and Abu-Sarra (1993) observed slower

rale of fruit softening in "Abu-Samaka" mangoes in spite of its higher PG

activity coparcd to "Kitchncr" and "Dr. Knight", cullivars. They

suggested a key role for Pit in controlling the rate of fruit softening

during mango fruit ripening.

6.2 Role of Polygiihicfuronnsc:

polygalacluronascs are enzymes responsible for the cleavage

of the a- 1,4 glucosidic bond between adjacent galacturonic acid units

in (he polymeric backbone of iJic pectic substances. There was a clear

evidence that P(i act on I he de-csterificd portion of (he

polygalacluronide chain. It was not clear, but it seemed more, likely,

that both carboxyl groups adjacent to the 1,4 glucosidic bond5ko<\l<lhc

free from linkages in order to be labile to PG enzyme (Benkova and

Markovic, 1976; Line weaver and Jans-en, 1951).

The role of PG in ripqning - associated fruit softening has been

discussed in many reviews (Pilnik and Voragen, 1970; Van Burcn,

1979). PG activity was reported to increase at the onset of ripening in

pears (Ahmed and Labavifch, 1980b)^peaches (Pressey et at., 1971)

and tomatoes (Hobson, 1962). PG activity progressively increased

during mango fruit ripening and a very high correlation was observed

between the increase in PG activity and loss of flesh firmness

(Abu-Sarra and Abu-Goukh, 1992; Roe and Burmmer, 1981).

Tigchelaar and McGlasson (1977) stated that the inhibition of ripening

in rin and nor, singlegene non-ripening mutants of tomato is proposed

"to occur Titn5clty^tTrtJirgtrthe d'1ircts~of thtiTre -geTTes-oir fruit P<x

biosynthesis or activation. Similar observations were reported by,

Buescher and Tigchclaar (1975) and Poovaish and Nukaya (1979).

The physiological disorder "blotchy ripening11 in tomato was also

evaluated in terms of PG activity. The red areas of blotchy fruit

contain less PG than in even red fruits and the activity is even lower in

non-red areas (Hobson, 1964). Ahmed and Labavfo^(1980a) reported

that treatment of cell wall from unripe fruits with highly purified PG,

changed the sugar composition to resemble that of cell wall from ripe

fruits. Pressey ct at., (1971) stated (hat in peaches, fruit firmness began

to decrease before PG was detected, however, the fruit softened

rapidly after the appearance of the. enzyme. Miller (1987) related the

softening of mechanically stressed cucumbers to the increase in PG,

PE and xylanase activities.

17

%.6.3. Role of Cellulase:

Cellulascs are enzymes responsible for the hydrolysis of {3 -1 ,

4-glucosidic bonds of cellulose as well as the P -1, 4 glucanic linkages

of xyloglucan, which is a hemicellulosic polysaccharide prominent in

cell walls of dicotyledonous plants (Keegstra ct at., 1973).

The role of cellulase in fruit softening was not fully understood.

Babbitt et at., (1973) found that tomato fruits treated with gibberellic

acid showed very low PG activity, while cellulase activity increased

steadily. From the loss of firmness in the treated fruits, they suggested

that softening was initiated by the action of cellulolytic enzymes and

that the"pectolytic enzymes were involved in subsequent changes of

texture. Pesis et at., (1978) reported that ccllttlase activity in detached

avocadoes was directly correlated with fruit softening. In contrast,

Hobson (1968), found that cellulase activity was not correlated with

loss of firmness during ripening of tomato. The enzyme activity was

reported to increase during ripening of normal tomato fruit and similar

change was observed in rin and nor non-ripening mutants (Poovaish

and Nukaya, 1979, Buescher and Tigchelaar, 1975). No eellulase

activity was delected in Sapodilla fruit (Selvaraj et at., 1982) and.pears

(Ahmed and Labavi'+di, 1980b). However, during maturation and

ripening the cellulase activity increased markedly in "Deglet Noor"

date fruits (Masegawa and Smolensky, 1971). The amount of fiber

content, which consist of Cellulose, hemicellulose and lignin,

decreased during maturation and ripening of dales (Mustafa et at.,

1986). The cellulose in the ripening dates is converted into glucose

(Barrevclled, 1993). The increase in collulase activity during IVuil

ripening was demonstrated in avocado (Pesis et at., 1978), dates

(Hasegawa and Smolensky , 1971), Strawberry (Maurice and

Palchctt, 1976), and tomato (I Jail, 1964, 1965; Hobson, 1968).

In mangoes, it was reported that cellulase activity steadily

increased with the decrease in.tissue firmness with high correlation

(Abu-Sarra and Abu-Goukh, 1992; Roe andBuremmer, 1981). They

suggested that PG, PE and cellulase are the degrading enzymes

responsible for fruit softening during mango fruit ripening.

CHAPTER 3

CHAPTER 3

Materials and Methods

3.1. Fruit Source and Ripening Procedure:

Mature green fruits of white-and pink-fleshcd guava fruits were

obtained from the University of Khartoum orchard at Shambal (Lai.

15° 40 'N, Long. 32° 22'E). Fruits were selected for uniformity in size,

colour and freedom from blemishes. About 400 fruis, from each guava

fruit type were washed, dried, placed in carton boxes, and stored in the

ripening room at 22 + 1°C and 90-95% RH.

3.2. Respiration and Firmness Determination:

Random samples of sixteen fruits from each type were removed

-daily-forr^spimtioi:^ rate

was determined for each fruit of the sample separately using the total

absorption method of Charlmcrs (1956) (Appendix 1). Hesh firmness

was measured by the Magness and Taylor firmness tester (D. iiallauf

Meg. Co.) equipped with an 8mm diameter plunger tip and expressed

in kilogram - force (kg. I"). Two readings were laken fromopp ff sides

of each fruit after the peel was removed. The fruits were labelled and

stored at - I2"C and later soiled into seven groups (13.4, 1 1.5, 9.5, 7.6,

5.7, 3.8 and 1.9 kg. f.) according lo iheir flesh firmness. Hach group

consisted of 10 fruits.

3.3. Total Soluble Solids Determination:

The frozen fruits of (he different groups were thawed separately

for 90 min. Total soluble solids (TSS) were measured directly from

20

the fruit juice using Kruss hand refractometer (Model HRN-32). Two

leadings of individual fruits of each group were determined by placing

a drop of the fruit juice from opposite sides in the refractometer prism

and the TSS was determined directly as percentage.

3.4. Compositional Changes:

3.4.1. Tissue Extraction:

Thirty grants of pulp or peel from each group were homogenized

in lOOmls. of distilled water for one minute in a Sanyo Solid State

blender (Model SM 228P) and centrifuged at 6,000 rpm for 10 min in

a Gallenkamp portable centrifuge (CP 400). The volume of the

supernatant, which consistutcd (he pulp or peel extracts, were

determined.

3.4.2. Titratablc Acidity:

Titratable acidity in pulp and peel extracts were determined

according to the .method described by Ranganna (1979). Five mis. of

pulp or peel extract were taken. Twenty mis. of distilled water were

added and titrated against 0.IN NaOH using phenophthalcin as

indicator. Tilratablc acidity was expressed as percent cilrie acid.

3.4.3. Total Sugars:

Total sugars were determined in pulp and peel extracts, using the

Anthrone method of Ycnun and Willis (1954) (Appendix 2). Total

sugars were expressed in grams per lOOg fresh weight.

3.4.4. Rcducing$ugars: /

The reducing sugars were determined in pulp and peel extracts

according to the technique described by Nelson (1944) as modified by

21

Somogyi (1952) (Appendix 3). Reducing sugars were expressed in

grams per lOOg fresh weight.

3.4.5. Total Proteins :

The total proteins, in the pulp and peel of guava fruit extracts

were determined by the protein - dye binding procedure described by

Bradford (1976) (Appendix 4). The protein content was expressed in

grams per lOOg fresh weight.

3.4.6. Phenolic Compounds:

The Folin - Ciocalue method of Singleton and Rossi (1965) was

used for determination of the phenolic compounds in the pulp and

peel extracts (Appendix 5). Phenolic compounds were expressed in

grams per lOOg fresh weight.

3A7. Ascorbic Acid:

Ascorbic acid in the pulp and peel of guava fruit extracts was

determined by the 2, 6 - dichlorophenol - indophcnol liiration method

described by Ruck (1963) (Appendix 6). Ascorbic acid content was

expressed in milligramsper lOOg fresh weight.

3.5. Enzymes Extraction and Assays:

3.5.1. Enzyme Extraction:

Enzymes were extracted according lo (he procedure described by

Ahmed and Labavilch (1980b). Guava fruits al designated stages of

firmness were peeled, and 5()g of flesh were homogenized in two

volumes of 100 mM sodium acetate buffer pH 6.0 containing 0.2%

sodium dithionile (Na2 S2 0.,) and .1% poly-vinyl pyrrolidone (IJVI\

MW. 44,000) for one minute using Sanyo Solid State blender. The

homogcnatc was ccnlrifugccl at 10,000 rpm for 20 min. The

supernatant was stored at - 12°C (buffer extract).

The residue was suspended in two volumes of I M sodium

acetate buffer.pH 6.0 containing 6% NaCI. The pH of the suspension

was adjusted to 8.2 with 2N NaOH. The sample was kept overnight at

4 C with continuous stirring and then centrifuged at 10,000 rpm for 20

min. The residue was discarded and the supernatant was filtered twice

using Whatman No. I, 9-cm filler paper. The fillcntfc (salt extract) was

dialyzed against distilled water for 48 hours with four changes. All

operations were carried out in an ice bath. This dialyzed sample

constituted the enzyme extract.

3.5.2. Pcctincstcrnsc Activity Assay:

Peclinesterasc (PE) activity was determined according to the

H:eelinkjire-{-les€ril5ed^y-^^

used was a 1% (w/v) solution of pectin (Citrus; 150 grade; H.P.

Bulmer Ltd., Hereford, England). The pM of the pectin solution was

adjusted to 7.0 with 0.02 N NaOH. The reaction mixture contained

25mls. of the crude enzyme, 5mls. of 0.2M-sodium oxalale and 25mls.

of the substrate. The reaction mixture was incubated al 3()"C and

continuously stirred by bubbling C()2- free air through it. Dining (he

course of the reaction, the pll of the reaction mixture was maintained

at 7.0 with 0.02N NaOH. The amount of 0.02 N NaOH added was

recorded every \5 nun. Enzyme activity was determined in terms of

milliquivalenl of ester hydrolyzed per kilogram of original fruit fresh

weight. The activity was then expressed in units. One unit of PE

activity was defined as one millicquivalenl of ester linkages cleaved

per niin per kg fresh weight.

3.5.3. Polygalacturonasc Activity Assay:

Polygatacturonase (PG) activity was determined by measuring

the reducing groups released from polygalacluronic acid (Orange;

Sigma Chem - Co.)- Reducing groups released were measured

according to the technique described by Nelson (1944) as modified

by Somogyi (1952) (Appendix 3). The reaction mixture containing

0.25 ml of crude enzyme, 0.25 ml of 100 mM sodium acetate buffer

pH 4.5, and 0.5 ml of 0.1% polygalacturonic acjd solution, was

•incubated at 37°C for 30, 60, 90, 120, and 150 min. At the end of

each incubation period the amount of reducing groups released was

determined. A calibration curve was obtained using d-galacturonic

acid (MW 212.2, Sigma Chem. Co.) as a standard. PG activity was

determined as micromoles of galacturonsyle reducing groups

liberated-per minute per kilogram original fruit fresh weight. The

activity was then expressed in units. One unit of PG activity was

defined as one/* mole ol galacturonasyl reducing groups liberated

per min. per kg fresh weight.

' 3.5.4. Cellulase Activity Assay:

Cellulase activity was determined by measuring the reducing

groups released from carboxymcthyj Cellulose. The concentration of

the reducing groups was determined, as in Ihe PG activity assay, with

p-glucose as a standard. The reaction mixture contained 0.25 ml of

crude enzyme, 0.5 ml of 0.1% carboxymethyl cellulase, and 0.25 ml

of 100 mM sodium acetate buffer pII 5.0. Incubation was carried out

at 37°C for 2, 4, 6 and 12 hours. Cellulase activity was determined as

micromoles of glueosyl reducing groups catalyzed per hour per

kilogram fresh weight. The activity was then expressed in units. One

unit of Cellulase activity was defined as one M. mole glucosyl

reducing groups liberated per hour per kg fresh weight.

24

CHAPTER-4

CHAPTER 4

RESULTS

4.1. Fruit Flesh Firmness and Respiration Rate :

Changes in fruit flesh firmness during ripening of white - and

pink - fleshed guava fruits a.t? shown in Fig. 1. Flesh firmness varied

within the individual fruits of the same type and at the same ripening

stage. Therefore, the values for flesh firmness in Fig. 1. were

calculated as means of 10 fruits in each group sample determined in

both sidesof the fruit.

During fruit ripening, the two guava fruit types had shown typical

pattern of changes in flesh firmness. The white - fleshed guava fruit

Jiajd^pix^rej^wejy declined in firmness from 28.2 kg.f. in the firm

mature green stage to 3.1 kg.f. in the soft ripe stage, while the pink

flesh guava decline from 27.5 to 3 kg.f. in the same stages. It look the

fruit 24 days to reach that final ripening stage.

Fig. 2 shows respiration rate of the while - and pink - fleshed

guavas. The respiration curves of the Iwo guava types exhibit a typical

climacteric pattern, wilh climacteric peak al 7.6 kg. f. flesh firmness

for both types. The respiration rate was significantly higher in the pink

- fleshed guava during all stages of ripening. (Appendix 8, (able I).

4.2. Compositional Charges During Fruit Ripening:

4.2.1. Total Soluble Solids:

Changes in total soluble solids (TSS) during guava fruil ripening

are shown .in Fig. 3. In both guava types, TSS increased wilh the

25

decrease in flesh firmness. The white fleshed guava had shown

significantly higher values compared to the pink - fleshed ones.

During fruit ripening, TSS had increased about 1.2 - guava folds in

both types. (Appendix 8, table 2).

4.2.2. Total Sugars:

Fig. 4. Shows changes in total sugars in pulp and peel during

ripening of white - and pink - fleshed guava fruits. Total sugars in the

white guavas were about 3 times higher than that in the pink ones. The

peel in both types contained significantly more total sugar than the

pulp. (Appendix 8, table 3,4, 5, 6).

The total sugars increased in the two guava types with decrease

in flesh firmness. More increase was noticed after fruit firmness of 7.6

kg. f. which coincided with the climacteric peak of respiration (Fig. 2).

Tfre~~pnS^tlTrar^ was-9^2% and

30.4% in the white - and pink - fleshed types compared to

post-climacteric increase of 31.7% and 56.7% in the white and pink

types, respectively. (Fig. 4).

4.2.3. Reducing Sugars:

Fig. 5. Shows the changes in reducing sugars in pulp and peel

during fruit ripening of white and pink guava types. The reducing

sugars in pulp and peel had increased up lo the climacteric peak

(firmness 7.6 kg. f.) in the white-fleshed or shortly before thai

(firmness 9.5 kg. f.) i/i the pink - fleshed guavas and subsequently

decreased. However, the amounts of reducing sugars at the beginning

and end of the ripening period were almost similar. Reducing sugars

in the peel of the while guavas were significantly higher than in the

26

pulp, while in the pink guava type the reducing sugars were higher in the

pulp compared to the peel (fig. 5). (Appendix 8, table 7,8).

4.2.4. Titnihifolc Acidity:

Tilralable acidity was expressed as percent citric acid. Fig. 64}

shows the changes in tilralable acidity in pulp and peel during fruit

ripening of whilc-and pink-ficshed guava fruits. Tilralablc acidity in pulp

and peel of both guava types had increased up to the climacteric peak and

then decreased afterwards. There were a significant differences between

the peel and pulp in white- Meshed guava fruit, but no significant

differences were observed in the pink guava fruits (Appendix 8, table

9,10).

Changes in total protein content in pulp and peel of while and pink guava

fruit types during fruit ripening are shown in Fig. 7. Total protein

increased systematically up to the full-ripe stage (fruit firmness 3.8. kg.f.)

and suddenly decreased. The pink-lleshed guava fruits had significantly

higher total protein content in pulp and peel compared to the white-

ileshed. ones. (Appcndix8, table 11,12), The peel in both guava types had

shown higher values of total protein content compared to the pulp (Fig.7).

4.2.6. Phenolic compounds:

Fig. 8. Shows the changes in phenolic compounds in pulp and peel

of-the two guava fruit types. 'I'he phenolic compounds decreased with

decreased flesh firmness. The decrease in the white guava types was

much drastic than that in the pink type. The decrease in total

phenolic in the white guavas was 7- and 5 folds

in the peel and pulp, respectively, compared to the pink guavas with 3-

- and 4 - folds decrease in peel and pulp (Fig. 8). The white guavas

had significantly higher values of phenolic compounds in pulp and

peel compared to the pink guava type. The peel in both types had

shown higher values of total phenolic* than the pulps. (Appendix 8,

table 13, 14).

4.2.7. Ascorbic Acid:

Fig. 9. Shows the changes in ascorbic acid content in pulp and

peel during fruit ripening of white and pink guava types. Ascorbic

acid decreased steadily during ripening. At the final - ripe stage (flesh

firmness 1.9 kg. f.) the amount of ascorbic acid retained was 86.3% in

the pulp and 85.6% in the peel of the while-fleshed guava fruils,

compared to 76.6% and 78.1% in pulp and peel of the pink - fleshed

guavas, respectively (Fig. 9). The white guava fruils had 19.2%; and

22.3% much more ascorbic acid in pulp and peel/respectively, than

the pink guava type. The peel in both lypcs showed significantly

higher values of ascorbic acid content compared to the pulp.

(Appendix 8, table 15, 16, 17, 18).

4.3. Enzymatic Activity changes During Fruit Ripening :

The activities of cell wall degrading enzymes, pcclinesterase

(PE), polygalacturonasc (PG) and Cellulase were measured in extracts

prepared from pulp of the white - and pink - fleshed guava fruits

during fruit ripening.

Enzyme activities in the buffer extracts were extremely low

compared to the activities in the corresponding salt extracts.

Moreover, the buffer extracts contained lar<;e amounts oi' reducinu

28

sugars which resulted in high back-ground readings in the reducing

group assay. Although that problem can be lessened by dialysis of the

extract, the time required was excessive. Only the data of enzyme

activities measured in the NaCl washes of the cell wall pattets will be

presented hens.

4.3.1. l'cclinelorasc Activity:

Changes in pectinesterase activity during fruit ripening of white

and pink guava types are presented in Fig. 10. The pectinesterase

activity, in both guava fruit types, had increased rapidly during the

onset of ripening and reached the maximum level with the climacteric

peak of respiration (flesh firmness 7.6 kg. f.) and then decreased to a

minimum value. The white guava fruit type had shown significantly

higher HE activity compared to the pink fruit type. (Appendix 8, table

19).

4.3.2. I'olygalacturonase Activity:

Pig. 11. Shows changes in polygalactuionasc activity during

"ripening of white - and pink -fleshed guava fruits. PG activity

progressively increased during fruit ripening in a similar manner, in

both fruit types. High correlation was observed between the increase

in PG activity and loss of flesh firmness. The PG activity had

increased 4.2 - and 3.8 - folds in the white and pink guava types,

respectively.There was a significant difference obscrvecjbelween

the two types. (Appendix 8, table 20).

4.3.3. Cellulasc Activity:

Changes in Cellulase activity during ripening of white and pink

guava fruits are shown in l-'ig. 12. Cellulase activity had progressively

increased during fruit ripening. The correlation was very high between

29

the increase in ccllulasc activity and the decrease in fruit flesh firmness.

The ccllulasc activity had increased 3.5-and 4.8 folds in the white and

pink guavas, respectively (Fig. 12). High significant differences in

ccllulasc activity were observed between the two guava types. (Appendix

8, table 21).

30 n

25-

U)

WWCDcEi lszV)

u.

20

15

U-

0 +1 2 3 4 6 6 7 8 9 10 1112 13 1415 16 17 18 19 20 2122 23 24

Days After Picking

Fig. I : Changes of fruit flesh firmness duringripening oi: white~(-«-) and pink - fleshed

guava al 22"C and 90-95% RI-l.

31

45

40

35

JC•

JC

OOCO

.8"TO

oc

ion

2"a

</>0)

30*

25*

20-

15-

in.

13.3 11.4 9.5 7.6 5.7 3.8

Fruit Flesh Firmness (kg.f)

1.9 0

Fig. 2 : Relationship between CO2 Production andfruit flesh firmness during ripening ofwhite - (-•-) and pink - fleshed (•*-) guavafruits.

32

CO(0

(00)3

15co

3o

14

12

10

8

0

13.3 11.4 9.5 7.6 5.7 3.8

Fruit Flesh Firmness (kg.f)

1.9 0

Fig. 3 : Changes in total souble solids (TSS)during ripening of white -(••-) and pink -fleshed guava fruits.

12

10

fsO)oo

CO

en3e3 4*orco"O

a:2 '

" A - - - A

13.3 11.4 9.5 7.6 5.7 3.8 1.9 0

Fruit Flesh Firmness (kg.f)

Fig. 5 : Reducing sugars changes in pulp (- )ami peel ( ) during ripening of white- (-«-) and pink - fleshed ( •*- ) guavafruils.

35

0.24

0.16

£ 0.08opO

0

: 13.3 11.4 9.5 7.6 5.7 3.8 1.9 0

Fruit Flesh Firmness (kg.f)

Fig. 6 : Changes in titraUible acidity in pulp (—)and peel ( ) during ripening of white( -«- ) and pink - fleshed ( +• ) guavafruits.

36

fJC

wt+Mi

O)oo

s

ote

inT

ota

l Pij

0.48*

0.44*

0.4«

0.36 •

0,32-

0.28*

0.24-

0.2-

0.16 •

0.12-

0.08*

0.04-

0-

m m

13.3 11.4 9.6 7.6 5.7 3.8 1.9 0

Fruit Flesh Firmness (kg.f)

Fig. 7 : Changes in total protein content in pulp( ) and peel ( ) during ripening ofwhite - (-«- ) and pink - fieshcd ( -A.- )uuava fruits.

37

CO

Ooo

3«

C3OQ.

O

oo

"ocro>

JZQ.

0.380.360.340.32

0.3

0.28

0.26

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0.12

0.1

0.08

0.06

0.04

0.02

0

2 13.3 11.4 9.5 7.6 5.7 3.8

Fruit Flesh Firmness (kg.f)

1.9 0

Fig. 8 : Changes in phenolic compounds in pulp(—) and peel ( ) during ripening ofwhite - (-*-) and pink - fleshed (•*-) guavafruits.

fres

h.

o>oo

"oCO

'bic

Asc

oi

95*

90'

85 •

80 •

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70*

65*

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13.3 11.4 • 9.5 7.6 5.7 3.8

Fruit Flesh Firmness (kg.f)

1.9

Fig. 9 : Changes in ascorbic acid content in pulp• (—) and peel ( ) during ripening of

white - (* ) and pink - fleshed (•*•) guavafruits.

39

j

c

73OTOOV)

23

OO

CL

13.3 11.4 9.5 7.6 5.7 3.8

Fruit Flesh Firmness (kg.f)

1.9 0

Fig. l() : Changes in Peetinesterase (PC) activityduring ripening of while - ( ^ ) and pink -Jleshed (-«-) guava fruit.(One unit of PB uctivily will cleave I milliequivalenlof ester linkages per minute).

40

35

30

J9?

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8=

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owS 10TOcooJ5 5to

13.3 11.4 9.5 7.6 5.7

Fruit Flesh Firmness (kg.f)

9

3.8 1.9 0

Fig. I I : Changes in PG activity during ripeningof white - ( - • - ) and pink - fleshed (•*• )guava fruits.(One unit of PCI nclivily will l iberate une (.t molegahicluronsyl recluciiui, giou|>s per minute).

32

30

28

.26

£ 24-

I 22x:w on

18

a 16

I 14 •

!* 12

1 10(0 a

"55 6o

13.3. 11.4 9.5 7.6 5.7 3.8

Fruit Flesh Firmness (kg.f)

1.9 0

Fig. 12 : Changes in Ccllulase activity duringripening of white - (-«-) and-pink - fleshed(•*-) guava fruits.(One unit of cellulase activity will liberate one jjmole reducing groups per minute).

CHAPTER 5

CHAPTER 5

DISCUSSION

Guava is a.popular fruit crop in Sudan. It is grown in every slate.

Although Sudan has great potentiality of producing high quality guava

fruits and good potentiality for exporting guavas to other countries, its

marketability is limited to local markets due to the delicate nature of

the fruits, poor handling practices, and inadequate transportation and

storage facilities. In this study, the biochemical and enzymatic

changes that occur during ripening of the white - and pink - fleshed

guava fruits were followed. The aim of the study was to provide a

base-line information regarding the biochemical nut lire of ripening

and softening mechanisms in guava fruits to assist in development of

sound prjo^rairumibiix^ of

firmness during handling, transportation and storage.

Fruit softening is characterized by changes in flesh firmness ami

has long-been associated with fruit ripening (Dostal, 1970). These

changes in fruit firmness determine shelf-life and quality of the

commodity. Control'of the fruit texture is a major objective in modern

food technology (Van Buren, 1979). During fruit ripening, the white -

and pink-fleshed guava fruit types had shown typical pattern of

changes in flesh firmness. l;ruit flesh firmness had progressively

declino/during fruit ripening (l;ig. i). The decline observed was about

8-folds from the hard mature green stage to the final soft ripe stage.

Similar drop in guava fruit firmness was reported in earlier studies

(Rodriguez ct al., 1971). Abu-Goukh and Abu-Sana (1993) observed

a rapid decrease in flesh firmness during ripening of three mango

43

cultivars. They found that more than 80% of the firmness decline hatf

occurred over two days and coincided with the climacteric peak of

respiration. Similar pattern of change'was-reported for bananas

(Abu-Goukh et al., 1995), pears (Luton and Holland, 1986), apples,

peaches and apricots (Salunkhe and Wu, 1973) and dates (Barrcvcllcd,

1993).

Respiration rate of the white - and pink-fleshed guavashad shown

a typical climacteric pattern of respiration, with climacteric peak at 7.6

kg. f flesh firmness for both types (Fig. 2). The...respiration rate was

higher in the' pink-fleshed guava fruits during all stages of ripening.

The climacteric pattern of respiration was also reported earlier by

Akamine and Goo (1979), who observed that clhylene had triggered

the respiratory rise.

changes

in colour, texture and flavour, indicating that compositional changes

are taking place. Attainment of maximum eating quality of a fruit"

necessitates completion of such chemical changes. Unripe fruits, are

usually hard in texture, starchy and acidic in taste and sometimes

astringent. After ripening, they become soft, sweet, non acidic>lost

astringeney and highly flavoured, so greatly acceptable as human

food. These changes are generally coincided wilh I lie peak of

respiration.

Total soluble solids (TSS) and total sugars had increased in ho(h

guava types wilh decrease in flesh firmness, Rodriguez et al. (1971)

observed-gradual increase in TSS and total sugars during guava fruit

ripening. Similar findings were reported for bananas (Ibrahim el al.,

1994), mangoes* (Abu-Goukh and Abu-Sana, 1993; Mincxsy el al.,

1984, Saeed et a]., 1975b) and dates (Dowson and Aten, 1962). Biale

(1960) attributed the increase in TSS and total sugars during ripening

to the hydrolysis of starch to sugars. The peel in both guava types

contained more total sugars than the pulp. This may be due to the less

amounts of moisture content in the peel compared to the pulp, since

sugars were expressed in g per lOOg fresh weight (Fig. 4).

More increase in total sugars in pulp and peel was observed after

fruit firmness of 7.6 kg.f, which coincided with the climacteric peak of

respiration (Fig. 2). This remarkable increase in total sugars observed

after the climacteric peak (Fig. 4) may be attributed to the increase in

activity of enzymes responsible for starch hydrolysis and/or decline in

the rate of sugar breakdown by the respiration process.

Climacteric fruits in particular may show considerable changes in

sugar content during fruit ripcjiingXHiU^£^19JQ^

were round to convert into glucose during fruit ripening (Wills et al.,

1981). The reducing sugars in the pulp and the peel had increased up

to the climacteric peak (firmness 7.6 kg. f) in the white-fleshed or

shortly before that (firmness9.5 kg. f) in the pink-fleshed guavas and

subsequently decreased. Maximum valves reached were 6 and 8

(g/IOOg f.wl) in the pulp and 5 and 10 (g/lOOg. f. wt) in (he peel of the

pink and while guavas, respectively (Fig. 5). Mowlah and lloo (1982)

showed that glucose, fructose and sucrose were the main sugars in (he

white-and pink- fleshed guavas. The level o! fructose sugars increased

during guava fruit ripening and their decreased in the over-ripe fruits

(Le-Riche, 1951). Subrahmanyam and Acharya (1957) also (bund that

reducing sugars increased during guava fruit ripening and then

decreased; Similar results were also reported in mangoesj,where

reducing sugars increased up to the climacteric peak and decreased

45

afterwards (Abu-Coukh and Abu-Sarra, 1993, Subranianyam ct al.,

1976).

The non-volatile organic acids are among the major cellular

constituents undergoing changes during ripening of fruits. Taste in

fruits is a function of sugar-acid blend (Mattoo et al.,' 1975). The

white fruit type had shown higher acidity in the pulp and peel than the

pink fruit type. (Fig. 6). The white-fleshed type had also, the highest

TSS. (Fig. 3); total sugars (Fig. 4) and reducing sugars (Fig. 5-),nuiking

it more tasty and preferred by most consumers compared to the

pink-fleshed fruit typeJltratable acidity in pulp and peel of both guava

types studied increased up to the climacteric peak (firmness 7.6 kg.f)

and then declined afterwards (Fig. 6). Similar results were reported

during ripening of bananas (Ahmed and Tingwa, 1975; Desai and

Deshpande, 1975; Palmer, 1971) and mangoes (Abii-Jao_ukh -and

Abu-Sarnrrl"995rKrishnamurthy ct al., 1971). The pulp in both guava

types showed lower titratable acidity compared to the peel. Similar

findings were reported in mangoes (Abu-Coukh and Abu-Sarra, 1993).

Proteins and free qmino acids arc minor constituents of fruits and

have no role in determining eating quality. Changes in ami no acids and

proteins however, indicate variations in metabolic activity during the

different developmental stages. During the climacteric phase of many

fruits, there is a decrease in free amino acids which ofW\ reflects an

increase in protein synthesis. During senescence, (he level of free

amino acids increases reflecting breakdown of enzymes and decreased

metabolic activity (Wills et al., 1981). The total protein in pulp and

peel of the white and pink guava types increased systematically up to

the full-ripe stage (fruit firmness 3.8 k.g) and suddenly.decreased

(fig.7). Abu-Goukh and Abu-Sana (1993) reported thai

46

total protein in pulp and peel of»Mn<)ocul1:ivars increased up to the

full-ripe stage and then decreased at the over-ripe stage. That decline

was explained as breakdown of proteins during senescence; which

supported the view that proteins in ripening fruits are enzymes

required for the ripening process (Frenkel et al., 1968).

The pink-fleshed guava fruits had higher total protein content in

pulp and peel compared to the white-fleshed ones. The peel in both

guava types, had shown higher values of total protein content

compared to the pulp (Fig. 7). The peel in mangoes was also reported

to have higher protein content than the pulp (Abu-Goukh and

Abu-Sarra, 1993). Although the peel of both guava type studied had

higher protein content, no higher metabolic activity canbe ascribed

for it at this stage, but can be explained for the lower moisture conlcnl

of the peel compared to the pulp, since the piolein content was

Phenolic compounds are important factors of fruit quality.-

Tannins arc high molecular weight phenolic compounds that form

strong complex with proteins and other polymers. The tannins

represent the primary substrate for browning reaction in fruit tissues

(Hulme, 1971). They are also responsible for the astringent taste in

unripe fruits which disappears due to polymerization of tannins during

fruit ripening (Al-Ogaidi and Mullak, 1986). Tannins play a vital role

in plant disease resistance (Kaa, 1968; McLean et al., 1961) and

protect plants against insect pests (Ne/.am FI-I)in et al., 1983).

The phenolic compounds in pulp and peel of while and pink

guava types progressively decreased with decreased fruit flesh

firmness (Fig.8). Molah and lloo (1982) reported that in both while

and pink guavas the total polyphenol decreased all along from the

immature stage to the full-ripe stage. The decrease in phenolic

compounds with fruit ripening was also reported in bananas (Ibrahim

et al., 1994; Abu-Goukh et al., 1995), mangoes (Abu-Goukh and

Abu-Sarra, 1993) and dales (Al-Ogaidi and Mutlak, 1986).

The decrease in phenolic compound in the white guava type was

much drastic than that in the pink type. The decrease in total phenolics

was 7 - and 3 - folds in the pulp of white and pink types (Fig. 8). This

may add for the better taste of the white-fleshed gLiava type compared

to the pink-fleshed type. The decrease in astringency in guava during

ripening was associated with the increased polymerization of

leucoanthocyanidins and hydrolysis of the astringent arabinose ester

of hexahydrodiphenic acid (Goldstein and Swain, 1963; Misra and

Seshadri, 1968). The peel in both types lui 'sIiowiiJiiglici-vaJ-ueK-i-^1-

tettrl^"lTeTiotics"than the pulp. This may have more significance in

plant disease resistance and protection of the fruit against insect pests-.

Ascorbic acid (vitamin C) is only qminor constituent of fruits and

vegetables, butifc&fmajor'importance in human nutrition. Virtually all

man's dietary vitamin C is obtained from fruits and vegetables. The

daily requirement of man from vitamin C is about 50 milligrams and

many fruits contain this amount of vitamin C in less than 100 grams of

tissue (Wills et al., 1981). Guava fruit is extremely rich in vitamin C

(242 mg per lOOg) (Watt and Merrill, 1975).

Ascorbic acid in pulp and peel of white and pink guava (ypes

decreased steadily during fruit ripening. Al the final ripe stage (flesh

firmness 1.9 kg.f) the amount of ascorbic acid retained was 86.3% in

the pulp and 85.6% in the peel of the white-fleshed guava fruits

48

compared to 76.6% and 78.1% of pulp and peel of the pink-fleshed

guavas, respectively (Fig. 9). The ascorbic acid content in guava fruit

reaches a. maximum level in the mature green stage and starts to

decline rapidly as the fruit ripens (Agnihotri et al., 1962; El-Zorkani,

1968). The decline in ascorbic acid content during ripening was also

reported in mango fruits (Abu-Goukh and Abu-Sana, 1993; Mincssy

etal., 1984; Chikkasubbanna and Huddar, 1982).

The white guava fruits had 19.2% and 22.3% much more

ascorbic acid than the pink guavas, in pulp and peel, respectively

(Fig.9). Variable reports are available about the amounts of ascorbic

acid in the white - and the pink - fleshed guavas. El-Fake ct al. (1975)

reported higher values for ascorbic acid in white-fleshed than

pink-fleshed fruits, while other investigators reported the reverse

(Agnihotri et al., 1962; El-Zorkani, 1968).

Th"e~peerUf FotfT types showed much higher values of ascorbic-

acid content compared to the pulp (Fig. 9).The peel of the guava fruit •

was reported to contain most of ascorbic acid in the fruit (Wilson,

1980). Similar result were reported for mangoes (Lakshminarayana et

al., 1970; Siddapa and Bahatia, 1954). The higher values of ascorbic

acid in the peel were attributed to interference of phenolic compounds

with the dye, 2,6-dichlorophenol indophcnol, used in the assay

(Lakshminarayana et al., 1970). In contrast, Abu-Goukh and

Abu-Sarra (1993) reported lower values for ascorbic acid in Ihe peel

compared to the pulp in three mango eultivars.

Fruit softening is characterized by changes in flesh firmness and

has long been associated with fruit ripening. Fruits undergo progress

decline in flesh firmness. This decline was demonstrated in mangoes

(Abu-Goukh and Abu-Sarra, 1993), bananas (Abu-Goukh and et al.,

49

1995) pears (Luton and Holland, 1986), apples, peaches and apricots

(Salunkhe and Wu, 1973) and dates (Barrevclled, 1993). The general

observation is that softening is accompanied by soiublization of pectic

substances : involving the sequential action of pectincstcrase (PE) and

polygalacturonase (PG) enzymes. This notion was supported by

'reports on changes in cell wall pectic material in ripening mango

(Tandon and Kalara, 1984, Roe and Buremmer, 1981), tomato

. (Besford and Hobson, 1972; Arad et al., 1983), pear (Ahmed and

Labavitch, 1980a), peach and apple (McCready and McComb, 1954),

and strawberry (Kertesz, J951).

A direct role of PE in fruit softening has been demonstrated.

Buescher and Tigchelaar (1975) reported that PE activity increased

during ripening of normal tomato fruit, while ihal'of rin fruit (a non

ripening mutant) remained constant. The failure of the uon ripening

loiirato~rrrataiTr("j^)""wTiis"evaliiate"d'~iii"ten'iis of PE activity (Buescher

and Tigchelaar, 1975). Mattoo and Modi (1969) related the softening

of chilling injured mango fruits to the increase activity of PE. Barmorc

and Rouse (1976) suggested that PE activity could be used to monitor

changes in softening time in avocado fruits during CA storage.•

The exlractable PE activity, in the white - and pink - fleshed

guava had increased rapidly during the onset of ripening, reached the

maximum level with the climacteric peak of respiration (flesh firmners

7.6 kg.f.) and then decreased to a minimum value (Fig. 10). Abu-Sana

and Abu-Goukh (1992) reported that PE activity in "Abu-Samaka"

mango increased up to shortly before the climacteric peak of

respiration and then decreased. That finding agrees with previous

reports on mangoes (Van Leiyveld and Smith, 1979; Tahir and Malik,

1977; Roc and Buremincr, 1981). The increase in PE activity during

50

fruit ripening has been reported in bananas (Hultin and Levine, 1965;

De Sward and Maxie, 1967), dates (Al-Jasim and Al-Delaimy, 1972)

and tomatoes (Hobson, 1963; Pctliawala et al., 1948). The

white-fleshed guava fruit had show higher values for PE activity

compared to the pink-fleshed ones. This could be explained for

difference in constituents of the white and pink guava types. White

guava types had shown higher values for TSS (Fig. 3), total sugars

(Fig. 4), reducing sugars (Fig. 5) titralablc acidity (Fig. 8) and most

probable poetic substance compared to the pink guavas.

Polygalacturonas.es (PGs) are enzymes responsible for (he

cleavage of the a -1,4 glucbsidic bond between adjacent galacturonic

acid units in the polymeric backbone of the pectic substances. There

was a clear evidence that PG act on the dc-cslerified portion of the

polygalacturonide chain. It was not clear, but it seems more likely, that

Iree

from linkages in order to be labile to PG enzyme (Benkova and

Markovic, 1-976; Linewcaver and Janscn, 1951).

The role of PG in ripening-associated fruit softening is well

documented (Pilnik and Voragen, 1970; Van Buren, 1979). Tigchelaar

and McGlasson (1977) stated that the inhibition of ripening in rm and

nor, single gene, non-ripening mutants of tomalo is proposed to occur

directly through the effects of these genes on fruit PG biosynthesis or

activation. Similar observations were reported by Bucschcr and

Tigchelaar (1975) and Poovaish and Nukaya (1979). The physiological

disorder "blotchy ripening" in tomalo was also evaluated in terms of

PG activity. The red areas of blotchy fruits contain less PG than in

even red fruits imd the activity is even lower in non-red areas (Hobson,

1964).

51

PG activity progressively increased during fruit ripening in a

similar manner in both guava fruit types. High correlation was

observed between the increase in PG activity and loss of fruit flesh

firmness (Fig. 11). The PG activity had increased 4.2 - and 3.8 - folds

in the white and pink guaya types, respectively. Similar results were

reported for mangoes (Abu-Sarra and Abu-Goukh, 1992; Roe and

Burmmer, 1981), pears (Ahmed and Labavitch, 1980b), peaches

(Pressy et al., 1971), dates (Hasegawa et al., 1969) and tomatoes

(Hobson, 1962).

Pressey et al., (1971) stated that in peaches, fruit firmness began

to decrease before PG was detected, however, the fruit softened

rapidly after the appearance of the enzyme. Miller (1987) related the

softening of mechanically stressed cucumbers to the increase in PG,

PE and xylanase activities. Ahmed and Labavitch ^98()a)repoji.cd_

that treatment of cell wall from unripe fruits with highly purified PG,

changed the sugar composition to resemble that of cell wall from ripe

fruit.

Besford and Hobson (1972) observed some degree of softening in

tomato fruit before commencement of the PK and PG action. A

non-en/.ynialic reaction was suggested to occur in plants which might

be responsible for some of the changes that occur in peclic substances

as well as in oilier polysaccharides (Kcrlesz, 1943). A wall-modifying

enzyme was reported by Karr and Aibersheim (1969) to be an

important prerequisite for the action of the polysaecharide-degrading

enzyme. Matlo et al. (1975) slated that softening in some fruits occurs

through hydrolysis of starch (as in squash) or of fat (as in avocado).

Cellulases are enzymes responsible for the hydrolysis of

(3-1,4-glucosidic bonds of cellulose as well, as (3-1,4-glucanic linkages

of xyloglucan, which is a hcmiccllulosic polysaccharide prominent in

cell wall of dicotyledonuous plants (Keegstra el al., 1973).

The role of celluiase in fruit softening was not fully understood.

Babbit et al., (1973) found that tomato fruits treated with gibberellie

acid showed very low PG activity, while cellulase activity increased

steadily. From the loss in tissue firmness in the treated fruits, they

suggested that softening was initiated by the action of celluloiytie

enzymes and that the pectolytic enzymes were involved in subsequent

changes of texture. Pesis et al., (1978) reported that celluiase activity

in detached avocado fruits was directly correlated with fruit softening.

Celluiase activity increased markedly during maturation and ripening

in "Deglet Noor" date fruits (Hasegawa anc^Smplenste^ 497-1-). The

-aniotmrui'ltbcFcontent, which consist of cellulose, hcniiccllulo.se and

lignin, decreased during maturation and ripening of dates (Mustafa et

al., 1986). The cellulase in the ripening dates was converted into

glucose (Barrevelled, 1993).

In contrast, Hobson (1968), found that cellulase activity was not

correlated with loss of firmness during ripening of tomatoes. The

enzyme activity was reported to-increase .during ripening of normal

tomato fruit and similar change was observed in rj_n and nor

non-ripening mutants (Poovish and Nukayu, 1979; Bucscher and

Tigchelaar, 1975). No cellulase activity was detected in sapodilia fruit

(Selvaraj el al., 1982) and pears (Ahmed and Labavitcli, 1980b). Very

I i in {degradation of cellulose was reported to occur during fruil

softening in bananas (Barhell, 1943), peaches (Sterling, 1961), apples

(Kcrtcsz et al., 1959; Knee, 1973) and pears (Ahmed and Labavitch,

53

1980a). Tahir and Malik (1977) reported that the cellulose and

hcmiccllulose content in mango fruit did not show appreciable

changes during ripening, therefore, appear to have insignificant

importance in textural changes during fruit ripening. However,

cellulose and hemicellulose were reported to change substantially

during fruit softening of bananas (Barncll, 1943) and 'dales (Cogginc

et al., 1967; Hasegawa and Smolensky, 1971).

Cellulase activity had increased progressively during fruit

ripening of white-and pink-fleshed guavas (Fig. 12). The correlation

was very high between the increase in celulase activity and the

decrease in fruit flesh firmness. The cellulase activity had increased

3.5 - and 4.8 - folds in the white and the pink guava types,

respectively. Abu-Sarra and Abu-Goukh (1992) also found that.

cellulase activity steadily increased with the decrease in tissue

It was

suggested that PG and cellulase are the degrading enzymes

responsible for fruit softening during mango fruit ripening. Such

increase in cellulase activity during fruit ripening is well demonstrated

in avocado (Pesis et al., 1978), date (Hasegawa and Smolenskcy,

1971), Strawberry (Maurice and PalehcU, 1976) and tomato (Hall,

1964, 1965;Hobson, 1968).

54

CONCLUSION

Ripening in guava fruit is characterized by and increase in tola

sugars, total soluble solids, total proteins, cellulase and polygalacturonase

activities and decrease in tissue firmness, ascorbic acid and pectinesterase

activity. l'Yuit tissue showed a typical climacteric rise in respiration.

Reducing sugars content followed the respiration pattern and lilratable

acidity decreased only after the climacteric peak.

The two types differ among themselves and between pulp and peel

in quantity of chemical constituents present at particular stage of ripening

and/or the iiuniberof days Taken to reach that stage.

Pcctincstcrase enzyme plays a major role in controlling softening

rate during ripening of white- and pink-fleshed guava fruit by controlling

polygalacluronasc action. Due to the high correlation observed between

the increase in activities of polygalacturonase and cellulase enzymes and

loss of fruit firmness, thatindicatc the 3 enzymes-pectineslerasc (PI2),

polygalacturonase (PG) and ccllulase are the main enzyme responsible

for fruit softening during guava fruit ripening.

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Appendix

Appendix 1

Respiration Rate Assay

(Total Absorbilion Method)

Procedure :

1 - Place a known weight ol" fruit or vegetable in a respiration jar

cquiped t with an inlet and outlet.

2 - Pass a constant air flow through :

(a) 40%.NaOH (to absorb atmospheric CO2).

(b) Distilled water to absorb any NaOl I fumes and to humidify

the air.

3 - Pass the humidified, CO2 free, air through the respiration jar.

4 - Trap CO2 of respiration in 50ml of O.lN.NaOJl for a

predetermined period of time.

5 - Titrate 15ml of the NaOH Sol'n. (containing CO2 of respiration)

with O.1N IIC1 using mixed indicator to whitish yellow end

point. •

6 - Add methyl orange indicator and proceed in (itration to pink

colour end point.

Calculations :

The acid used for the second part of the (illation (V2) is equivalent

to half the total carbonate.

74

.-. mey. of carbonalc = 2 V r N- HC i

Equivalent Wt. of carbonate = formula Wt./charge

, = 106/2 = 53

Wt. of Na2CO3 = (meg. of Na2CO3) (Bqui. Wt. of Nu2CO3).

44Wt. of CO2 absorbed (ing) = (Wt. of Na2CO3) (Dil. factor)

Dil. factor = 50315

Wt. of CO2 absorbedRespiration Rate =

(nig CO /K"-hr) S a m P l e W L (K^-) X Ra t ion (hr)

Appendix 2

Total Sugars Assay(Anlhcrone Method)

Preparation ol* Standard Curve :

1 - Dissolve lOOmg glucose in 100ml of sol'n to gel slock (A) of

concentration 1000[ig/ml.

2 - Prepare stock sol'n. (B) by diluting 10ml of slock (A) wifh

40ml distilled water. Concentration of slock (13) will be

200|Ag/ml.

3 - Prepare standard curve concentrations as follows :

0.0ml of stock sol'n + 1.0ml H20 -» O.Ofig sugars (Blank)

25 ).il of slock (13)+ 0.975ml H20 -> 5 » »

50 - - " " + 0.950 " " • -> 10 " "

75 » » " " + 0.925 " " -> 15 »

100 » » » " + 0.950 " " -» 10 » »

25 (.L1 of stock (A) + 0.975 " » -> 25 » "

75 »» » " " + 0.950 " » ->• 50 » "

100 » - " - + 0.900 " " -» 100" "

Autherone Solution : - 2()()ing anlhrone/IOOml cone. HSCh

Procedure :

1 - Place lml of the tested solution in test tube (containing

I ()-l()0|.lg sugars)

2 - Add 2ml of anthrone solulioj).

3 - Mix well on lube mixture and leave lo cool at roomtemperature.

4 - Read O.D. al 62()mu.

76

Appendix 3

Nelson-Somogyi(reducing groups and reducing sugars assay)

Preparation of Standard Curve :

1 - Dissolve lOOmg glucose in 100ml of sol'n to get slock (A) of

concentration 1000f.ig/ml.

2 - Prepare stock sol'n. (B) by diluting 10ml of slock (A) with

40ml distilled water. Concentration of slock (B) will he

200|ig/ml.

3 - Prepare standard curve concentrations as follows :

0.0ml from slock sol'n + 1.0ml U20 —> O.Oug sugars (Blank)

25j.il of slock (B)-i- 0.975m! H20 -> 5 »

50 » " " " + 0.950 " » -> 10 » "

75 " " " " + 0.925 " •• -> 15 » »'

|()() „ „ „ „ .,- 0.900 " » -> 20 " »

25 (11 of slock (A) + 0.975 " »• -> 25 » »

50 " " " " + 0.950 " » ~> 50 " »

100 " » " " -i- 0.900 " " -> 100" -

Reagents :

Solution 1 : Dissolve (I2g KNa-larlral.c -I- 24g anhydrous

Na2CO, + I6g NalICO-, + 144g Na,SO4) in distilled water

and make to final volume of 800.

Solution II : Dissolve (4g CuSO,,.5l 1,0 -I- 36g Na,SO,) in

distilled water and make (o final volume of 200ml.

Nelson reagent :

1 - 25g Ammonium Molyhdale in 450ml water.

2 - Add 21 ml concentrated 11OSO,.

77

3 - Add 3g Na2HASO4.7H2O dissolved in 25ml H2O.

4 - Wrap bottle in foil and let sit for 48 hours before use.

Procedure :

1 - Place one ml of sample in test tube

(containing 10-100 g sugars)

2 - Mix one part of sol's 11 with four parts of sol's 1 and add to

sample tube one ml.

3 - Boil in water bath for 25-30 minutes.

4 - Cool under running lap water and add one ml of Nelson

reagent.

5 - Read O.D. at 520mu.

Appendix 4

Titratable Acidity

Equivalent Weight of Aculs :

Citric

Malic

Tartarie

Acetic

I'roeedure :

64

67

75

60

1 - Take 10ml of tesled sol'n. in a beaker.

2 - Add 20ml distilled water.

3 - Add 3 drops of phenophthalein indicator.

4 - Titrate with 0.1 NaOl I to faint pink colour

(pH =0=8.2)

Calculations1:

%Acid =

(ml of NaOll) (N.NaQll) (dil. factor) (Kqu. Wl. of acid) x 100100 (Wt. of sample g)

dilution factor =

Sample total volumeSample volume used in tilration (10)

Appendix 5

Protein Assay By Dye-Binding

(Bradford, 1976)

Preparation of Standard Curve :

1 - Dissolve lOOmg of protein (Bovine serum albumin) in lOOiul

of sol'n. to get slock (A) of concentration 1000 jig/ml.

2 - Prepare stock solution (13) by diluting 10ml of stock (A) with

40ml distilled water (Cone. 200 |ig/ml).

3 - Prepare standard curve concentrations as follows :

0.0ml from stock sol'n + 0.3ml H20 -> 0.0|.lg protein' (Blank)

25 ji I of slock (B)+ 0.275ml Ii,0 -> 5 » »

50 "

75 "

100 »

25f.il

50 »

Procedure He

" " " +„ „ „ +

„ „ „ +

of slock (A) +

„ -|.

0.250

0.225

0.200

0.975

0.250

it

n

If

t l

H

- * 10

-> 15

- * 20

~> 25

-> 50

1 - Dissolve lOOmg of Coomassie Brilliant Blue dye in 50ml

ethanol (95%).

2 - Add 100ml 85% (W/V) phosphoric acid.

3 - Complete the volume to one liter and filter.

Procedure :

1 - Place 0.3ml of tested sol'n. in test lube.

2 - Add 3nil of protein reagent and shake well.

3 - Read O.D. at 595 imi.

Appendix 6

Total Pheuolics Assay(Folin - Ciocatcu)

Preparation of Standard Curve :

1 - Dissolve lOOmg (annic acid in 100ml of sol'n. to get stock (A)

of concentration 1.000 (.Lg/inl.

2 - Prepare stock sol's (B) by diluting 10ml of stock (A) with

40ml distilled water (Conccenration of stock (B) will be

200 |ig/ml).

3 - Prepare standard curve concentrations as follows :

0.0ml from stock sol'n + 0.3ml 112O -> O.Ojig tannic (Blank)

25 Hi of slock (13)+ 0.275ml H2() -> 5 » »

50 »

75 »

100 »

25j.il

50 "

Procedure :

» » " + 0.250 " »

" » " + 0.225 " "

" " » + 0.200 " »

of stock (A) + 0.275 " »

» " » + 0.250 " »

- > 10

- > 15

-> 2 0

- > 2 5

-> 50

1 - Place 0.3ml of the tested solution in a lest tube

(containing 5-2 g phenolics).

2 - Add 1.5ml of diluted l*olin - Cioealcu reagent (I + 9 distilled

water) and mix well.

3 - Add 1.2ml sodium carbonate solution (7.5g per 100ml of

sol'n.) between 30 seconds and before K minules elapsed.

4 - Place Lest tubes for one hour at 30°C (86T') and for another

hour at O"C (32°l:0. Usually 30 minutes is sufficient.

5 - Read O.D. at 760 mil.

Appendix 7

Ascorbic Acid(Tilration Method)

Preparation :

1 - Take 30 g of fruil tissue and blend with reasonable amount of

0.4% oxalic acid.

2 - Filler by Whatman No. 1 filler paper.

3 - Make up lo 250ml with 0.4% oxalic acid.

4 - Pipette 20ml of fi Iterate into a beaker.

5 - Titrate with dye 2,6-diehlorophenol-indophenol to a faint pink

colour.

Calculations :

Ascorbic acid = liter (ml) x dye strength x 100

(mg/l()0g) factor

Factor = Sample Wl. (30u) x Sample volume I'orT Ural ion (20ml)

Total volume of smaple (250ml)

Reagents :

1 - 0.4% oxalic acid :- 4g oxalic acid/liler of sol'n.

2 - 10% oxalic acid :- 50g oxalic acid/500rnl of sol'n.

3 - Standard ascorbic acid :- 0.05g ascorbic acid/25()ml of 10%

oxalic acid.

4 - Dye sol'n. : 0.2g of 2,6-dichIorphenol-mdoplienol dye per

500ml ofsol'n. and filter.

Determination of dye strength :

Add 5ml of standard ascorbic acid sol'u. lo 5ml of 10% oxalic

acid sol'n. in a beaker and tilralc with the dye sol'n. to faint

pink colour.

Dye strength = —tiler

S2

. Anova Tables lor Statistical Analysis

General Linear Models ProcedureClass Level Information

ClassGroupRep.Color

Levels7

102

Values1 2 3 41 2 3 4Pink White

55

66

77 8 9 10

Number of observations in data set = J40General inear Models Procedure

•pendent Variable : Respiration Rateuree DP Sum of Sqaurcs Mean Square P. Value Pr < I7

odcl 7 9708.494786 1386.927827 66.62 0.0001ror 132 2748.03749 20.818465>rrected Total 139 12456.532214

R-Square C.V. Root MSB RliSRA Mean0.779390 18.95888 4.562726 24.0664286

Table (1)

General Linear Models Procedure

^pendent Variable : TSS

jurcc • DP Sum of Squures Mean Square P. Value- Pr < Fodel 7 1622.592357 231.798908 2.54 0.0174ror 132 12038.951857 91.204181jrrecled Total 139 13661.544214

R-Square C.V. Root MSE ' TSS Mean0.118771 91.33222 9.550088 10/1564286

Table (2)

General Linear Models ProcedureClass Level Information

ClassGroupRep.SourceColor

Levels7312

Values1 2 3 4 5 6 71 2 3PeelPink White

Number of Observations in by group = 42

General inear Models Procedure

Dependent Variable : Total Sugars

Source DV Sum of SqauresModel 7Error 34

Corrected Total 41

R-Squarc0.986779

O.3O53OOOO

0.00409048

0.30939048

C.V.5.073538

Table (3)

Mean Square0.043614290.0001^031

Root MSB0.010969

1\ Value P r < F362.52 0.0001

lolal Sugaur Mean0.21619048

General Linear Models ProcedureClass Level Information

ClassGroupRep.SourceColor

Levels7312

Values1 2 3 A1 2 3PulpPink While

5 6 7

Number of Observations in by group = 42General inear Models Procedure

Dependent Variable : Tola! Sugars

Source DV Sum ol'Sqaures0.477835710.004861900.48269762

C.V.5.441409192

Table (4)

Model 7Error 34Corrected Total 41

. R-Squarc0.989928

Mean Square0.068262240.00014300

Root MSB0.0! 1958

Value477.37

P r < l ;

0.0001

Potal Sugaur Mean(U1976190

GroupRep.SourceColor

7321

1 21 2PeelPink

3 4 53

Pulp

6 7

Number of Observations in by group = 42General inear Models Procedure

Dependent Variable : Tola! Sugars

SourceModellirrorCorrected

\W1

34Total 41

R-Squarc0.953366

Sum of Sqaures0.027550000.001347620.02889762

C.V.4,960965

Mean Square0.003935710.00003964

Root MSB0.006296

1\ Value99.30

Total Sug"0.

Pr< 1-0.0001

aur Mean12690476

Table (5)General Linear Models Procedure

Class Level Information

ClassGroupRep.SoureeColor

Levels7321

Values1 2 31 2 3Peel PulpWhite

4 5 6 7

ModelBrrorCorrected' Polal

R-Squ0.918

73441

are1(7

Number of Observations in by group = 42General inear Models Procedure

Dependent Variable : Total Sugars

Source Dl; Sum of Sqaurcs Mean Square I1'. Value Pr<l-"0.06129524 0.00875646 54.46 0.00010.00546667 0.00016078O!O6676I9O

C.V. Root MSB Total Sugaur Mean4.102952 0.012680 0^30904762

Table ((>)General Linear Models Procedure

Dependent Variable : Reducing Suguars

Source 1)1' Sum of Sqauics Mean Square I1'. Value IV<I ;

Model . 7 0.25625000 0.03660714 470.10 0.0001terror 34 0.00264762 0.00007787Corrected Total 41 0.25889762

R-Sqiiare C.V. Root MSB UI-DU Mean0.989773 1.311027 0.008824 0.67309524

Table (7)

General Linear Models ProcedureClass Level Information

ClassGroupRep.SourceColor

Levels7321

Values1 2 31 2 3Peel PulpPink

4 5 6 7

Number of Observations in by group = 42General incar Models Procedure

Dependent Variable : Reducing Sugars

Source DP Sum of Sqaures Mean Square P'. Value Pr<P'Model 7 0.18163333 0.02594762 40.69 0.0001Error ' 34 0.02168095 0.00063768Corrected Total 41 0.20331429

R-Square C.V, Root MSIi RliDU Mean0.893362 5.757837 0.025252 0.43857143

I able (8)

General Linear Models ProcedureClass Level Information

4 5 6 - 7ClassGroupRep.SourceColor

Levels7321

Values1 2 31 2 3Peel PulpWhite.

Number of Observations in by group = 42General incar Models Procedure

Dependent Variable : Tilratable acidily

Source DV Sum of Sqaures Mean Square F. Value Pr < FModel 7 1.60928571 0.22989796 34,13 0.0001L-iTor 34 0.22904762 0.00673669Corrected Total 41 1.83833333

R-Square C.V. Rool MSB TIT Mean0.875405 4.875883 0.082077 1.68333333

Table (9)

General Linear Models ProcedureClass Level Information

ClassGroupRep.SourceColor

Levels7321

Values1 2 3 4 51 2 3Peel PulpPink

6 7

Number of Observations in by group = 42General incar Models Procedure

Dependent Variable : Tkratable acidity

Source DV Sum of Sqaurcs29.14696429

142.64404762171.79101190

C.V.138.6420

Table (10)

Model 7Error 34Corrected Total 41

R-Squarc0.169665

Mean Square4.163852044.19541317

Root MSE2.048271

F. Value Pr < F0.99 0.4533

TIT Meani.47738095

General Linear Models ProcedureClass Level information

ClassGroupRep.SourceColor

Levels73•2

1

Values1 2 3 4 5 6 71 2 3Peel PulpWhile

Number of Observations in by group = 42General incar Models Procedure

Dependent Variable : Protein

SourceModellirrorCorrected

DV1

34Total 41

R-Squarc0.356996

Sum of S qau res0.018152380.032695240.05084762

C.V.6.225732

Mean Square0.002593200.00096162

Root MSI:0.031010

F. Value Pr < F2.70 0.0246

Protein Mean0.49809524

Table (11)

87

General Linear Models ProcedureClass Level Information

ClassGroupRep.SourceColor

Levels7321

Values1 2 3 41 2 3Peel PulpPink

5 6 7

Number of Observations in by group = 42General incar Models Procedure

Dependent Variable : Protein

SourceModelErrorCollected

DV7

34Total 41

R-Square0.664026

Sum ol Sqaures0.009580950.004847620.0(442857

C.V.2.148687

Mean Square0.001368710.00014258

Root MSB0.011941

R Value P r < i ;

9.60 0.0001

Protein Mean0.55571429

Table (12)

General Linear Models ProcedureClass Level Information

ClassGroupRep.SoLUVO

Color

Levels7321

Values1 2 3 4 51 2 3Peel I'ulpWhite

6 7

Number of Observations in by group. = 42General inear Models Procedure

Dependent Variable : Phenolic

Source • Dl; Sinn of Sqaurcs Mean SquareModel 7Error 34Corrected Total ' 41

R-Squarc0.875938

0.420266670.059523810.47979048 .

C.V.35.00672

Table (13)

0.060038 JO0.00175070

Rool MSIi0.041841

Value P r < F34.29 0.0001

Phenolic Mean0.11952381

General Linear Models ProcedureClass Level Information

ClassGroupRep.SourceColor

Levels7321

Values1 21 2Peel 1Pink

3 4 53

\\\p

6 7

Number of Observations in by group = 42General inear Models Procedure

Dependent Variable : Phenolic

Source ' DP Sum of Sqaures Mean SquareModel 7Error 34Corrected Total 41

R-Square0.929217

0.090330950.006880950.0972! 190

C.V.19.21205

Table (14)

0.012904420.00020238

Rool MSL0.014226

Value P r < F63.67 0.0001

Phenolic Mean0.07404762

General Linear Models ProcedureClass Level Information

ClassGroupRep.SourceColor

Levels7321

Values1 2 31 2 3Peel PulpWhite

4 5 6 7

Number of Observations in by group = 42General inear Models Procedure

Dependent Variable : Ascorbic AcidSource DPModel 7Error 34Corrected Total 41

R-Squarc0.903074

Sum of Sqaures6.380000000.684761907.06476190

C.V.

8.120515

Table (15)

Mean Square0.911428570.02014006

Rool MSE0.141916

Value Pr < F45.25 0.0001

ASCO Mean1.74761905

89

General Linear Models ProcedureClass Level Information

ClassGroupRep.SourceColor

Levels7321

Values •1 2 3 41 2 3Peel PulpPink

5 6 7

Number of Observations in by gruup = 42

General inear Models Procedure

Dependent Variable : Ascorbic Acid

Source DF Sum oCSqaurcs Mean SquareModel 7Error 34Corrected Total 41

R-Square0.563777

0.243095240.188095240.43119048

C.V.27.16445

Table (16)

0.034727890.00553221

Rool MSE0.074379

Value P r < l ;

6.28 0.0001

ASCO Mean0.27380952

General Linear Models ProcedureClass Level Information

ClassGroupRep.SourceColor

Levels731

Values1 2 3 4I 2 3PulpPink White

5 6 7

Number of Observations in by group = 42General inear Models Procedure

Dependent Variable : Ascorbic Acid

Source DV Sum ol'Scjaurcs(6.073988100.74202381 .

16.81601190C.V.

16.52376198

Table (17)

ModelErrorCorrected

734

Total 41

R-Squarc0.955874

Mean Square2.296284010.02182423

Rool MSE' 0.147730

Value P J - < J :

105.22 0.0001

ASCO Mean0.89404762

90

General Linear Models ProcedureClass Level Information

ClassGroupRep.SourceColor

Levels7312

Values1 2 31 2 3PeelWhite I

4

'ink

5 6 7

Number of Observations in by group = 42General incar Models Procedure

Dependent Variable : Ascorbic AcidSource DFModel 7Error 34Corrected Total 41

R-Square0.966295

Sum of Sqaures33.96625000

1.1847619035.15101190

C.V.16.55791161

Table (18)

Mean Square4.852321430.03484594

Root MSfi0.186671

Value Pr < F139.25 0.0001

ASCO Mean1.12738095

General Linear Models ProcedureClass Level Information

ClassColorGroupRep.

Levels272

ValuesPink White1 2 3 41 2

5 6 7

Number of Observations in by group = 28Analysis of Variance Procedure

Dependent Variable : Pectincsterasc

Source DF Sum of Sqaures Mean SquareModel • 7Error 34Corrected Total 41

R-Squarc0.893733

0.005142290.000611430.00575371

C.V.19.95052

Table (19)

0.000734610.00003057

Rool MSI7.0.005529

Value24.03

Pr < F0.0001

Pectincsleiase Mean0.02771429

General Linear Models ProcedureClass Level Information

ClassColorGroupRep.

Levels273

ValuesPink. While1 2 3 41 2 3

5 6 7

Number of Observations in by group = 28Analysis of Variance Procedure

Dependent Variable : Polygalacturonasc

Source DF Sum of Sqaures Mean SquareModel 7Error 34Corrected Total 41

K-Sc|imre0.825753

0.235166670.049623810.28479048

C.V.8.608133

Table (20)

0.033595240.00145952

Root MSE0.038204

F. Value23.02

Pr < F0.0001

POLY Mean0.44380952

General Linear Models ProcedureClass Level Information

ClassColorGroupRep.

Levels273

ValuesPink White1 2 3 41.2 3

5 6 7

Number of Observations in by group = 28Analysis of Variance Procedure

Dependent Variable : Ccllulasc

Source Dl ; Sum ol' Sqaures Mean SquareModel 7Error 34Collected Total 41

R-Square0.806387

0.383650000.092114290.47576429

C.V.10.54567

Table (21)

0.054807140.00270924

Root MSE0.052050

Value P r < F20.23 0.0001

Cellulasc Mean0.49357143

92