Fruit Pulp and Peel.During Ripening of White - and Pink-Fleshed Guava Fruit Types Were Investigated

'Kind'K A 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 1/28 Please be aware that all of the Missing Pages in this document were originally 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. Asco^ 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£ Figures Page 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

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 etaL 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

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

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% J sodium dithionile (Na2 S2 0.,) and .1% poly-vinyl pyrrolidone (I VI\ 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. 6 4 } 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-

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Fig. I : Changes of fruit flesh firmness during ripening oi: white~(-«-) and pink - fleshed guava al 22"C and 90-95% RI-l.

31 45

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13.3 11.4 9.5 7.6 5.7 3.8 1.9 0 Fruit Flesh Firmness (kg.f)

Fig. 2 : Relationship between CO2 Production and fruit flesh firmness during ripening of white - (-•-) and pink - fleshed (•*-) guava fruits.

32 14

CO (0 8

(0 0) 3 15 co 3 o

13.3 11.4 9.5 7.6 5.7 3.8 1.9 0 Fruit Flesh Firmness (kg.f)

Fig. 3 : Changes in total souble solids (TSS) during ripening of white -(••-) and pink - fleshed guava fruits. 12

10 f s O) o o

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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 ( •*- ) guava fruils. 35 0.24

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

36 0.48*

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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 of white - (-«- ) and pink - fieshcd ( -A.- ) uuava fruits.

37 0.38 0.36 0.34 0.32 0.3 0.28 CO 0.26

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2 13.3 11.4 9.5 7.6 5.7 3.8 1.9 0 Fruit Flesh Firmness (kg.f)

Fig. 8 : Changes in phenolic compounds in pulp (—) and peel ( ) during ripening of white - (-*-) and pink - fleshed (•*-) guava fruits. "o o o Ascoi'bic CO o> fresh. 45- 50- 70* 75' 85 • 60« 65* 55- 80 • 95* 90' Fig. 9:Changesinascorbicaci d contentinpulp 13.3 11.4•9.57.65.73.8 • (—)andpeel(during ripeningof fruits. white -(*)andpinkfleshe(•*• ) guava " jfr-A Fruit FleshFirmness(kg.f) 39 1.9 j

73 O TO O V) 2 3

O O CL

13.3 11.4 9.5 7.6 5.7 3.8 1.9 0 Fruit Flesh Firmness (kg.f)

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

40 35

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o w S 10 TO c o o J5 5 to

9 13.3 11.4 9.5 7.6 5.7 3.8 1.9 0 Fruit Flesh Firmness (kg.f)

Fig. I I : Changes in PG activity during ripening of white -(-•-) and pink - fleshed (•*• ) guava fruits. (One unit of PCI nclivily will liberate une (.t mole gahicluronsyl recluciiui, giou|>s per minute). 32

.26 £ 24- I 22 x: w on

18 a 16 I 14 • !* 12 1 10 (0 a o"55 6

13.3. 11.4 9.5 7.6 5.7 3.8 1.9 0 Fruit Flesh Firmness (kg.f)

Fig. 12 : Changes in Ccllulase activity during ripening of white - (-«-) and-pink - fleshed (•*-) guava fruits. (One unit of cellulase activity will liberate one jj mole 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. References References

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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 = 2Vr N- HC i

Equivalent Wt. of carbonate = formula Wt./charge

, = 106/2 = 53

Wt. of Na2CO3 = (meg. of Na2CO3) (Bqui. Wt. of Nu2CO3). 44 Wt. of CO2 absorbed (ing) = (Wt. of Na2CO3) (Dil. factor)

Dil. factor = 50315

Wt. of CO2 absorbed Respiration Rate = (nig CO /K"-hr) SamPle WL (K^-) X Ration (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 room temperature. 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 64

Malic 67

Tartarie 75

Acetic 60

I'roeedure :

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 100 100 (Wt. of sample g)

dilution factor =

Sample total volume Sample 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 " " " " + 0.250 it -* 10 75 " „ „ „ + 0.225 n -> 15

100 » „ „ „ + 0.200 If -* 20

25f.il of slock (A) + 0.975 tl ~> 25 50 » „ -|. 0.250 H -> 50 Procedure He 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 » » » " + 0.250 " » -> 10 75 » " » " + 0.225 " " -> 15 100 » " " » + 0.200 " » -> 20 25j.il of stock (A) + 0.275 " » -> 25 50 " » " » + 0.250 " » -> 50 Procedure : 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 Procedure Class Level Information Class Levels Values Group 7 12 3 4 5 6 7 Rep. 10 12 3 4 5 6 7 8 9 10 Color 2 Pink White Number of observations in data set = J40 General inear Models Procedure •pendent Variable : Respiration Rate uree DP Sum of Sqaurcs Mean Square P. Value Pr < I7 odcl 7 9708.494786 1386.927827 66.62 0.0001 ror 132 2748.03749 20.818465 >rrected Total 139 12456.532214 R-Square C.V. Root MSB RliSRA Mean 0.779390 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 < F odel 7 1622.592357 231.798908 2.54 0.0174 ror 132 12038.951857 91.204181 jrrecled Total 139 13661.544214 R-Square C.V. Root MSE ' TSS Mean 0.118771 91.33222 9.550088 10/1564286 Table (2) General Linear Models Procedure Class Level Information Class Levels Values Group 7 12 3 4 5 6 7 Rep. 3 1 2 3 Source 1 Peel Color 2 Pink White

Number of Observations in by group = 42 General inear Models Procedure Dependent Variable : Total Sugars Source DV Sum of Sqaures Mean Square 1\ Value Pr

General Linear Models Procedure Class Level Information Class Levels Values Group 7 1 2 3 A 5 6 7 Rep. 3 1 2 3 Source 1 Pulp Color 2 Pink While

Number of Observations in by group = 42 General inear Models Procedure Dependent Variable : Tola! Sugars Source DV Sum ol'Sqaures Mean Square Value Pr

Number of Observations in by group = 42 General inear Models Procedure Dependent Variable : Tola! Sugars Source \W Sum of Sqaures Mean Square 1\ Value Pr< 1- Model 1 0.02755000 0.00393571 99.30 0.0001 lirror 34 0.00134762 0.00003964 Corrected Total 41 0.02889762 R-Squarc C.V. Root MSB Total Sug"aur Mean 0.953366 4,960965 0.006296 0. 12690476 Table (5) General Linear Models Procedure Class Level Information Class Levels Values Group 7 12 3 4 5 6 7 Rep. 3 1 2 3 Souree 2 Peel Pulp Color 1 White Number of Observations in by group = 42 General inear Models Procedure Dependent Variable : Total Sugars Source Dl; Sum of Sqaurcs Mean Square I1'. Value Pr) General Linear Models Procedure Dependent Variable : Reducing Suguars Source 1)1' Sum of Sqauics Mean Square I1'. Value IV

Number of Observations in by group = 42 General incar Models Procedure Dependent Variable : Reducing Sugars Source DP Sum of Sqaures Mean Square P'. Value Pr

General Linear Models Procedure Class Level Information Class Levels Values Group 7 1 2 3 4 5 6-7 Rep. 3 1 2 3 Source 2 Peel Pulp Color 1 White.

Number of Observations in by group = 42 General incar Models Procedure Dependent Variable : Tilratable acidily Source DV Sum of Sqaures Mean Square F. Value Pr < F Model 7 1.60928571 0.22989796 34,13 0.0001 L-iTor 34 0.22904762 0.00673669 Corrected Total 41 1.83833333 R-Square C.V. Rool MSB TIT Mean 0.875405 4.875883 0.082077 1.68333333 Table (9) General Linear Models Procedure Class Level Information Class Levels Values Group 7 12 3 4 5 6 7 Rep. 3 1 2 3 Source 2 Peel Pulp Color 1 Pink

Number of Observations in by group = 42 General incar Models Procedure Dependent Variable : Tkratable acidity Source DV Sum of Sqaurcs Mean Square F. Value Pr < F Model 7 29.14696429 4.16385204 0.99 0.4533 Error 34 142.64404762 4.19541317 Corrected Total 41 171.79101190 R-Squarc C.V. Root MSE TIT Mean 0.169665 138.6420 2.048271 i.47738095 Table (10)

General Linear Models Procedure Class Level information Class Levels Values Group 7 12 3 4 5 6 7 Rep. 3 1 2 3 Source •2 Peel Pulp Color 1 While

Number of Observations in by group = 42 General incar Models Procedure Dependent Variable : Protein Source DV Sum of S qau res Mean Square F. Value Pr < F Model 1 0.01815238 0.00259320 2.70 0.0246 lirror 34 0.03269524 0.00096162 Corrected Total 41 0.05084762 R-Squarc C.V. Root MSI: Protein Mean 0.356996 6.225732 0.031010 0.49809524 Table (11)

87 General Linear Models Procedure Class Level Information Class Levels Values Group 7 12 3 4 5 6 7 Rep. 3 1 2 3 Source 2 Peel Pulp Color 1 Pink

Number of Observations in by group = 42 General incar Models Procedure Dependent Variable : Protein Source DV Sum ol Sqaures Mean Square R Value Pr

General Linear Models Procedure Class Level Information Class Levels Values Group 7 12 3 4 5 6 7 Rep. 3 12 3 SoLUVO 2 Peel I'ulp Color 1 White

Number of Observations in by group. = 42 General inear Models Procedure Dependent Variable : Phenolic Source • Dl; Sinn of Sqaurcs Mean Square Value Pr

Number of Observations in by group = 42 General inear Models Procedure Dependent Variable : Phenolic Source ' DP Sum of Sqaures Mean Square Value Pr

General Linear Models Procedure Class Level Information Class Levels Values Group 7 1 2 3 4 5 6 7 Rep. 3 1 2 3 Source 2 Peel Pulp Color 1 White

Number of Observations in by group = 42 General inear Models Procedure Dependent Variable : Ascorbic Acid Source DP Sum of Sqaures Mean Square Value Pr < F Model 7 6.38000000 0.91142857 45.25 0.0001 Error 34 0.68476190 0.02014006 Corrected Total 41 7.06476190 R-Squarc C.V. Rool MSE ASCO Mean 0.903074 8.120515 0.141916 1.74761905 Table (15)

89 General Linear Models Procedure Class Level Information Class Levels Values • Group 7 12 3 4 5 6 7 Rep. 3 1 2 3 Source 2 Peel Pulp Color 1 Pink

Number of Observations in by gruup = 42 General inear Models Procedure Dependent Variable : Ascorbic Acid Source DF Sum oCSqaurcs Mean Square Value Pr

General Linear Models Procedure Class Level Information Class Levels Values Group 7 12 3 4 5 6 7 Rep. 3 I 2 3 Source 1 Pulp Color Pink White

Number of Observations in by group = 42 General inear Models Procedure Dependent Variable : Ascorbic Acid Source DV Sum ol'Scjaurcs Mean Square Value PJ-

90 General Linear Models Procedure Class Level Information Class Levels Values Group 7 1 2 3 4 5 6 7 Rep. 3 1 2 3 Source 1 Peel Color 2 White I'ink

Number of Observations in by group = 42 General incar Models Procedure Dependent Variable : Ascorbic Acid Source DF Sum of Sqaures Mean Square Value Pr < F Model 7 33.96625000 4.85232143 139.25 0.0001 Error 34 1.18476190 0.03484594 Corrected Total 41 35.15101190 R-Square C.V. Root MSfi ASCO Mean 0.966295 16.55791161 0.186671 1.12738095 Table (18)

General Linear Models Procedure Class Level Information Class Levels Values Color 2 Pink White Group 7 12 3 4 5 6 7 Rep. 2 1 2 Number of Observations in by group = 28 Analysis of Variance Procedure Dependent Variable : Pectincsterasc Source DF Sum of Sqaures Mean Square Value Pr < F Model • 7 0.00514229 0.00073461 24.03 0.0001 Error 34 0.00061143 0.00003057 Corrected Total 41 0.00575371 R-Squarc C.V. Rool MSI7. Pectincsleiase Mean 0.893733 19.95052 0.005529 0.02771429 Table (19) General Linear Models Procedure Class Level Information Class Levels Values Color 2 Pink. While Group 7 12 3 4 5 6 7 Rep. 3 12 3 Number of Observations in by group = 28 Analysis of Variance Procedure Dependent Variable : Polygalacturonasc Source DF Sum of Sqaures Mean Square F. Value Pr < F Model 7 0.23516667 0.03359524 23.02 0.0001 Error 34 0.04962381 0.00145952 Corrected Total 41 0.28479048 K-Sc|imre C.V. Root MSE POLY Mean 0.825753 8.608133 0.038204 0.44380952 Table (20)

General Linear Models Procedure Class Level Information Class Levels Values Color 2 Pink White Group 7 12 3 4 5 6 7 Rep. 3 1.2 3 Number of Observations in by group = 28 Analysis of Variance Procedure Dependent Variable : Ccllulasc Source Dl; Sum ol' Sqaures Mean Square Value Pr