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Separation of cytokinin nucleosidase from adenosine nucleosidase by affinity chromatography and the study of postharvest calcium treatments on sliced tomato
Tamg, Jessica Jiashi, Ph.D.
The Ohio State University, 1992
UMI 300 N. ZeebRd. Ann Arbor, MI 48106 SEPARATION OF CYTOKININ NUCLEOSIDASE FROM ADENOSINE
NUCLEOSIDASE BY AFFINITY CHROMATOGRAPHY AND THE STUDY OF
POSTHARVEST CALCIUM TREATMENTS ON SLICED TOMATO
DISSERTATION
Presented in Partial Fulfillment of the Requirements for
the Degree Doctor of Philosophy in the Graduate School
of The Ohio State University
By
Jessica J. Tarng, B.S., M.S.
*****
The Ohio State University
1992
Dissertation Committee Approved by
J.B. Allred
M.E. Mangino Adviser A. Proctor Department of Food Science and Technology Copyright by
Jessica Jiashi Tarng 1992 To My Parents
and Dear Husband Jimmy
ii ACKNOWLEDGMENTS
I wish to express my sincere gratitude and appreciation to Dr.
Grady W. Chism III, as my academic adviser, for inspiring and caring
for me throughout my research and life in The Ohio State University.
Thanks to the members of my reading committee, Drs. J.B. Allred,
M.E. Mangino, and A. Proctor, and to the Department of Food Science
and Technology for the financial assistance.
To my colleague Madge Player, Daniele Korleskind, Isabelle
Laye, and Serena Laroia, thank you for sharing, caring, and
encouraging me.
Special acknowledgment goes to all the faculty, staff, and fellow students who all have helped in their own way. VITA
August 20,1962 Born - Taipei, Taiwan, Rep. of China
1980-1984 ...... B.S., The National Chung-Hsing University, Taichung, Taiwan, Rep. of China.
1984-1986 ...... M.S., The National Taiwan University, Taipei, Taiwan, Rep. of China.
1986-1987 ...... Research Assistant, The National Taiwan University, Taipei, Taiwan, Rep. of China.
1987-Present .... Graduate Research Associate, The Ohio State University, Columbus, Ohio.
Major Field: Food Science and Technology TABLE OF CONTENTS
DEDICATION...... i i ACKNOWLEDGEMENTS...... iii
VITA...... iv LISTOFTABLES...... vi
LIST OF FIGURES...... vii ABSTRACT...... x i
CHAPTER I. ISOLATION OF CYTOKININ NUCLEOSIDASE FROM ADENOSINE NUCLEOSIDASE IN RIPE TOMATO FRUIT BY AFFINITY CHROMATOGRAPHY AND THEIR KINETIC CHARACTERISTICS...... 1 Introduction...... 1 Literature Review...... 3 Materials and Methods ...... 20 Results and Discussion...... 30
II. EFFECTS OF VACUUM INFILTRATION AND DIPPING OF CALCIUM SOLUTIONS ON THE QUALITY OF MINIMALLY PROCESSED TOMATO SLICES...... 67 Introduction...... 67 Literature Review...... 69 Materials and Methods ...... 78 Results and Discussion...... 81
LIST OF REFERENCES...... 103
v LIST OF TABLES
TABLE PAGE
1. The protocol for silver staining...... 29
2. Purification of adenosine nucleosidase in each peak fraction of affinity chromatography from ripe tomato fruit...... 43
3. Kinetic constant for cytokinin nucleosidase and adenosine nucleosidase activity in the four peak fractions in Fig. 10 and 11...... 49
4. Acceptability scores for calcium vacuum-infiltrated tomato slices...... 102 LIST OF FIGURES
FIGURE PAGE
1. Chemical structures, trivial names and abbreviations for some naturally occurring and synthetic cytokinins...... 4
2. Cytokinin biosynthesis interconversion and degradation in plant tissues: key role of N6-[isopent-2-enyl]adenosine...... 6
3. Enzymes of N6-[isopent-2-enyl]adenosine metabolism...... 8
4. The steps of affinity chromatography...... 16
5. Chemical structure of bicinchoninic acid ...... 20
6. The reaction scheme shows how the BCA combines the biuret reaction to form the purple color complex...... 22
7. The comparison of protein recovery percentages attained by the widely used Bradford method and the recently developed BCA protein assay method 32
8. Affinity chromatography of cytokinin nucleosidase on an i6Ado-Agarose column (Elution gradient 1)...... 35
9. Affinity chromatography of adenosine nucleosidase on an i6Ado-Agarose column (Elution gradient 1)...... 37
10. Affinity chromatography of cytokinin nucleosidase on an i6Ado-Agarose column (Elution gradient 2)...... 39
vii 11. Affinity chromatography of adenosine nucleosidase on an i6Ado-Agarose column (Elution gradient 2)...... 41
12. Affinity chromatography of cytokinin nucleosidase on an Ado-Agarose column (Elution gradient 2...... ) 46
13. Affinity chromatography of adenosine nucleosidase on an Ado-Agarose column (Elution gradient 2)...... 48
14. Lineweaver-Burk plot of adenosine nucleosidase activity in peak fraction of NS I against adenosine concentration...... 51
15. Lineweaver-Burk plot of cytokinin nucleosidase activity in peak fraction of NS I against cytokinin concentration...... 53
16. Lineweaver-Burk plot of adenosine nucleosidase activity in peak fraction of NS II against adenosine concentration...... 55
17. Lineweaver-Burk plot of cytokinin nucleosidase activity in peak fraction of NS II against cytokinin concentration...... 57
18. Lineweaver-Burk plot of cytokinin nucleosidase activity in peak fraction of i6Ado NS III against cytokinin concentration...... 59
19. Lineweaver-Burk plot of adenosine nucleosidase activity in peak fraction of Ado NS III against adenosine concentration...... 61
20. SDS-polyacrylamide electrophoregram of partially purified nucleosidase attained from i6Ado-Agarose affinity chromatography column in Fig. 10 and ...... 11 65
viii 21. The calibration curve of the molecular weight versus relative mobility f R value on a semi-logarithmic scale...... 66
22. Effect of calcium lactate and calcium gluconate vacuum infiltration treatments on the firmness of tomato slices at day zero...... 83
23. Effect of calcium lactate and calcium gluconate vacuum infiltration treatments on the firmness of tomato slices at day ten...... 85
24. Effect of calcium lactate dip treatments on the firmness of tomato slices...... 87
25. The comparison of calcium lactate and calcium gluconate dip treatments on the firmness of tomato slices at the same concentration of 250 mM...... 88
26. Effect of calcium lactate vacuum infiltration treatments on the color of tomato slices...... 90
27. Effect of calcium lactate and calcium gluconate dip treatments on the color of tomato slices...... 91
28. Effect of calcium gluconate vacuum infiltration treatments on the color of tomato slices...... 93
29. The appearance of tomato slices vacuum infiltrated with 50 mM and 100 mM calcium lactate, and 100 mM calcium gluconate after 11 days storage...... 94
30. Effect of calcium vacuum infiltration treatments on total plate counts of tomato slices...... 97
31. Effect of calcium vacuum infiltration treatments on yeast counts of tomato slices...... 99
ix 32. Effect of calcium vacuum infiltration treatments on mold counts of tomato slices...... 101
x SEPARATION OF CYTOKININ NUCLEOSIDASE FROM ADENOSINE NUCLEOSIDASE BY AFFINITY CHROMATOGRAPHY AND THE STUDY OF POSTHARVEST CALCIUM TREATMENTS ON SLICED TOMATO
By
Jessica J. Tarng, Ph.D.
The Ohio State University, 1992 Professor Grady W. Chism, III, Adviser
Adenosine (Ado) nucleosidases were extracted from ripe tomato fruits and separated by affinity chromatography on an N6-
[isopent-2-enyl]adenosine (i6Ado)-Agarose column. Three distinct
forms of adenosine nucleosidase were present in the extracts. Two
fractions of the nucleosidases were capable of deribosylating Ado and i6Ado, and one of these contained the majority of the total
nucleosidase activity. The third fraction, cytokinin nucleosidase, deribosylated i6Ado but not Ado and had a Km of 55.0 uM for i6Ado.
Cytokinin nucleosidase showed a single band having a molecular weight of 40 Kd after SDS electrophoresis and silver staining. Postharvest calcium treatments by dipping and vacuum infiltration increased the firmness of tomato slices. The action of calcium seemed to be dependent upon concentration with the higher concentration having a greater influence on texture of tomato slices.
All calcium infiltration treatments improved both the visual appearance and the reflected color of tomato slices at time zero.
The effect of calcium lactate on reflected color decreased with storage time. However, the overall visual appearance of tomato slices treated with calcium lactate was superior to that of tomato slices treated with calcium gluconate and untreated slices at the end of 11 days storage. 100 mM and 250 mM calcium lactate appeared to have the greatest antimicrobial effects. The sensory evaluation at day zero showed no significant difference (a=0.01) for overall acceptance between the samples infiltrated with 50 mM, 100 mM calcium lactate, 100 mM calcium gluconate, and untreated.
xii CHAPTER I
SEPARATION OF CYTOKININ NUCLEOSIDASE FROM ADENOSINE
NUCLEOSIDASE BY AFFINITY CHROMATOGRAPHY
INTRODUCTION
Cytokinins are a group of N6 substituted adenine derivatives which have been shown to be involved in various aspects of plant regulation, including senescence, ripening and growth (Miller, 1956; Letham, 1971; Davey and Van Staden, 1978). The most fundamental control mechanisms are presumably operating at the levels of enzymatic regulation of metabolism (biosynthesis, interconversion, and degradation). The metabolism of cytokinins has been studied in a variety of plant tissues and some enzymes which regulate the metabolic pathways of cytokinins have also been isolated (Chen et al., 1976; Laloue et al., 1977; Chen and Petschow, 1978; Chen et al.,
1982; Letham et al., 1983; McGaw and Horgan 1983a and b; Chen and
Leisner, 1984).
1 2
Most of the enzymes explored are cytokinin-specific, whereas enzymes which mediate cytokinin base-nucleoside-nucleotide
interconversions also act on other purines and their derivatives. Due to the similar features between cytokinins and unsubstituted
purines, it is suggested that the steps common to the metabolism of
these two groups of compounds may be important control points at
which cytokinin metabolism is regulated (Laloue and Pethe, 1982;
Letham et al., 1982; Burch and Stuchbury, 1987).
It has been well documented that unripe tomato fruits
(Lycopersicon esculentum L) exhibit high endogenous cytokinin
activity (Desai and Chism, 1978; Davey and Van Staden, 1978;
Mapelli, 1981). However, very few studies have emphasized the
enzymatic activities regulating cytokinin levels in these fruits. An
understanding of what enzymes regulate cytokinin metabolism and how they function is vital to further control the physiological
processes, extend storage period, and improve the overall quality of tomato fruits. The present study is designed to confirm the presence of cytokinin-specific regulatory enzymes which mediate the interconversion of cytokinin base-nucleoside in ripe tomato fruit. LITERATURE REVIEW
The first naturally occurring cytokinin isolated from plant tissue was 6-(4-hydroxy-3-methyl-trans-2-butenyl)purine (Zeatin,
Z) (Letham, 1963). It was found to induce cell division at concentrations as low as 5x10"11 M. Figure 1 represents the general structure of cytokinins along with their various substitutents.
The major structural feature of cytokinins that appears to be most siginificant is that the adenine base should have an N6 alkyl substitution of 5-6 carbons in length containing an alpha double bond. Nishikawa et al. (1986) suggested that the alpha double bond was required for cytokinin activity.
The presence of cytokinins in different tomato cultivars is well established. Zeatin (Z) and zeatin riboside (ZR) were found to be the predominant cytokinins in normal tomato fruits (Desai and
Chism, 1978; Davey and Van Staden, 1978).
Mapelli (1981) observed high levels of N6-[isopent-2- enyljadenosine (i6Ado) and its free base N6-[isopent-2-enyl]adenine
(i6Ade) activities in normal tomato varieties and lower levels of these in parthenocarpic varieties. Seeds seem to be functionally important with respect to cytokinin metabolism since
3 4
H'N 'ni
SUBSTITUENTS TRIVIAL NAME ABQR
R 1 r 2 r 3
H H NG(02-isopen1cnyl) l5 A de H 3 a d e n in e
H nOoluranosyl N^tn^-isopenlenyl) 16 Ado CH2 CH3 a d e n o s in e CH3S nboluranosyl 2 melhyllliio N^lA^.isopemenyij mszl('Ado a d e n o s in e
H2OM H H Zeatin Z
CH2 CH 3 H ribofuranosyt Zeatin riboside ZR
y C H 2 0 \i / \ It II Oiiiydro2cann Dill? CH 3 C H 3
H H Gbcn/ylnmmo- Cll2 ^ / p u n n c
CM2" H l< o
I* 0 CM, 0 O nbofuranosyl Ad0 a d e n o s in e
Figure 1. Chemical structures, trivial names and abbreviations for some naturally occurring and synthetic cytokinins. 5
parthenocarpic varieties exhibit lower levels of cytokinin activity than do normal seeded fruits (Mapelli, 1981; Palmer et al., 1982).
Studies on normal ripening and non-ripening mutant tomato
varieties have indicated that cytokinins may be involoved in ripening processes (Abdel-Rahman et al., 1975; Desai and Chism, 1978; Davey
and Van Staden, 1978; Mapelli, 1981) These data show endogenous
cytokinin content in tomatoes decreases with the onset of ripening.
The change in cytokinin activity are presumably enzyme-mediated.
Burch and Stuchbury (1987) showed that within the tomato plant, the distribution of purine and cytokinin degrading enzymes varies and the activities of these enzymes change substantially during development.
The metabolism of i6Ado and its free base i6Ade has been studied in several plants. A scheme showing the key role of i6Ado in cytokinin metabolism is presented in Figure 2. In general, i6Ado and i6Ade undergo one or more of four basic reactions. The enzymes associated with i6Ado or i6Ade degradation cleave at various sites of their structures (Fig. 3). The first reaction is cleavage of the isopentenyl side chain via cytokinin oxidase (Paces et al., 1971; Whitty and Hall, 1974; Brownlee et al.,
1975; Laloue e t al., 1977; Terrine and Laloue, 1980; McGaw and Horgan, 1983a; McGaw and Horgan, 1983b; Chatfield and Armstrong,
1986; Chatfield and Armstrong, 1987) which causes irreversible inactivation of the cytokinin molecules. The second pathway is trans-hydroxylation of the terminal methyl group in the side chain which leads to an increase in the cytokinin activity (Laloue et al., 6
Ade MVA
Ado
5 '-AMP A -iPP
Cytokinin- containing vi 6AMP- ATP tRNA
v glycosyl derivatives
0 no i r ZR ^ Z v A de
3-m ethyl- 2-butenal 11
A la n in e 7-G-Z & 9-G-Z O-G-Z & O-G-ZR Conjugates
DiH-Z & DiH-ZR
O-G-DiH-Z & O-G-DiH-ZR
Figure 2. Cytokinin biosynthesis interconversion and degradation in plant tissues: key role of N6-[isopent-2-enyl]adenosine (thick arrows designate direction of an increased biological activity) 7
Figure 2. (continued) Cytokinin biosynthesis interconversion and degradation in plant tissues: key role of N6-[isopent-2- enyl]adenosine.
Enzymes involved in cytokinin metabolism 1. Adenosine phosporylase
2. Adenosine kinase 3. Adenosine phosphoribosyltransferase
4. A2-isopentenylpyrophosphate:
AMP-A2-isopentenyltransferase
5. 5'-Nucleotidases
6. cytokinin oxidase
7. cytokinin nucleosidase
8. Adenosine deaminase
9. Microsomal mixed function oxidases
10. Microsomal mixed function oxidases
11. Lupinic acid synthase (or fc-(9-cytokinin)alanine synthase) 12. Cytokinin glucosyltransferase
13. Adenylate kinase (?)
14. Nucleases
15. Nucleoside diphosphokinase (?) 16. Cytokinin glycosyltransferases 8
CYTOKININ OXIDASE 'CH 3 HN CH — CH ADENOSINE CH3 DEAMINASE
CYTOKININ NUCLEOSIDASE HOCH
OH OH
Figure 3. Enzymes of N6-[isopent-2-enyl]adenosine metabolism (Paces et al., 1977) 9
1977; Palni and Horgan, 1983; Chen and Leisner, 1984; Einset, 1984;
Einset, 1986; Einset, 1987). The third reaction is ring substitution which results in a slight lowering of cytokinin activity (Miura and
Miller, 1969; Miura and Hall, 1973). The forth pathway is the
interconversion of i6Ado and i6Ade. The cleavage of the ribose
moiety of i6Ado via nucleosidase activity is considered as an
activation step in cytokinin metabolism, because i6Ade was found to
be more active than i6Ado in promoting the growth of plant tissue. (Dyson et al., 1972; Schmitz et al., 1972; Whitaker and Kende, 1974;
Hecht et al., 1975; Laloue et al., 1977; Erion and Fox, 1981; Letham
et al., 1983). On the other hand, the ribosylation of i6Ade mediated
by cytokinin phosphorylase results in a slight decrease in activity.
Enzymatic activity catalyzing the removal of ribose moiety was investigated in immature corn kernels (Whitty and Hall, 1974),
barley leaves (Paces, 1976), rape seedlings (Paces et al., 1977),
tobacco cells (Terrine and Laloue, 1980), wheat germ (Chen and
Christopeit, 1981), tomato roots and leaves (Burch and Stuchbury,
1986b), and ripe tomato fruit (Rolle and Chism, 1986). Chen and Kristopeit (1981) purified adenosine (Ado) nucleosidase activity
from wheat germ and proved that Ado was a better substrate than
i6Ado for this enzymatic activity. The presence of Ado and other
naturally occurring cytokinins inhibited the deribosylation of i6Ado.
The Km values for i6Ado and Ado were 2.4 uM and 1.4 uM,
respectively. Rolle and Chism (1986) isolated and partially purified a cytokinin nucleosidase activity from ripe tomato fruit using acetone 10
powder preparation and DEAE column chromatography. These
preparations also caused rapid degradation of Ado. The reaction
velocity with Ado as substrate was 16 times as rapid as i6Ado in Tris/HCI buffer, pH 7.5. Burch and Stuchbury (1986b) reported that two distinct forms
of adenosine nucleosidase (R1 and R2) were present in tomato roots
and one form (Lf) in leaves. They all had a better affinity for Ado
than ZR. It was observed that cytokinin ribosides acted as competitive inhibitors of all three enzymes although the enzymes
showed different patterns of inhibition.
It is still uncertain whether plants contain cytokinin-specific enzymes which catalyze cytokinin base-nucleoside-nucleotide
interconversions. These may be caused by enzymes which catalyze analogous reactions for Adenine (Ade) and its derivatives. Chen and co-workers (Chen and Eckert, 1977; Chen and Petschow, 1978; Chen,
1981; Chen et al., 1982) purified three other enzymes from wheat
germ (Km values in parentheses): adenosine phosphorylase, which favors nucleosides formation (i6Ade 57.1 uM, Ade 32.2 uM); adenosine kinase (i6Ado 31.0 uM, Ado 8.7 uM), and adenosine phosphoribosyl transferase (i6Ade 130.0 uM, Ade 74.0 uM). In each case, the Km's for Ado or Ade was less than that for i6Ado and i6Ade. Burch and Stuchbury (1987) suggested that if the interconversion of cytokinin base-riboside-ribotide is catalyzed by enzyme which also act on other purines, the steps common to the metabolism of these two groups of compounds may be important control points at which cytokinin metabolism is regulated. Because 11
of the similar features between cytokinins and unsubstituted purines, the enzymes mediating purine metabolism may play a significant role in the formation of biologically active cytokinins (Laloue and Pethe, 1982), in the uptake and release of cytokinins
(Letham et al., 1982), and in the maintenance of an adequate level of
active cytokinin in plant cells (Chen and Kristopeit, 1981).
The enzymatic activity which catalyzes the cleavage of the isopentenyl side chain has been detected in various plant tissues (Paces et al., 1971; Whitty and Hall, 1974; Brownlee et al., 1975;
Laloue et al., 1977; Summons et al., 1980; McGaw and Horgan, 1983a; McGaw and Horgan, 1983b; Chatfield and Armstrong, 19 87).
Cytokinin oxidase was partially purified by Whitty and Hall (1974) and was shown to have a molecular weight of 88,000 and required molecular oxygen. Only the naturally occurring cytokinins i6Ado and
ZR were found to serve as substrates of this enzymatic activity and the nucleoside or free base worked equally as well. It has been postulated (Whitty and Hall, 1974) that this oxidation reaction appeared to be accomplished through the formation of an unstable imine intermediate, and this intermediate was isolated later by
Laloue and Fox (1985) from reaction mixtures containing 2- mercaptoethanol. This enzyme activity was enhancedin vitro in the presence of copper-imidazole complexes, and the reaction was not inhibited by anaerobic conditions when the reagents were present
(Chatfield and Armstrong, 1987).
Hydroxylation of i6Ade or i6Ado has been reported in at least four plant systems: immature corn kernels (Miura and Hall, 1973), 12
microsomal fraction from tobacco tissue (Chen, 1982), microsomes
from cauliflowers (Chen and Leisner, 1984), and several different
tissues from Actinidia and other woody plants (Einset, 1984; Einset,
1986). Hydroxydation steps are specific for thetrans form which is considerably more active than the c/s form (Hall and Srivastava,
1968). Z and ZR, the products of the trans-hydroxydation of i6Ade
and i6Ado, are believed to exhibit greater cytokinin activity than
their precursors (Miura and Hall, 1973; Chen and Leisner, 1984;
Einset, 1986). Einset (1986) reported that the reactions converting i6Ade to Z was 02-requiring and specific for i6Ade over the i6Ado.
He suggested that i6Ado normally may be metabolized via i6Ade and
i6AMP to Z. The microsomal cytochrome P-450 enzymes were suggested to be involved in the hydroxylation of i6Ado and i6Ade in cauliflowers in the presence of NADPH (Chen and Leisner, 1984). Ring substitution of i6Ade normally causes slight decrease of
cytokinin activity. Cytokinins have been reported conjugated in four different positions and with several different groups which led to the formation of ribosides, ribotides, glucosides, and amino acid conjugates (Chen and Eckert, 1977; Laloue et al., 1977; Summons et al., 1977; Chen and Petschow, 1978; Entsch et al., 1979a and 1979b;
Scott e t al., 1980; Summons et al., 1980; Summons et al., 1981; Scott et al., 1982). These conjugates are generally of enhanced metabolic stability, yet less active than the parent cytokinins (Letham and Palni, 1983).
Chen and Eckert (1977) and Chen et al. (1982) in their studies on preparations from wheat germ cells have discovered an adenosine 13
kinase which converted i6Ado to i6AMP and adenosine
phosphoribosyltransferase which converted i6Ade to i6AMP directly.
These enzymes exhibited lower affinities for the cytokinin structures than the corresponding purine structures. The production
of i6ADP and i6ATP along with i6AMP has been reported in tobacco
cells by Laloue et al. (1974). The physiological significance of
ribotides is still unknown, but the existence in vivo of nucleoside
5'-triphosphate is of theoretical importance as they could be
incorporated in tRNA molecules (Laloue et al., 1974). Hence, the ribotides probably play a key role in cytokinin metabolism in many tissues (Laloue et al., 1977; Burch and Stuchbury, 1986a; Burch and
Stuchbury, 1987).
The glucosides are also widespread, but, unlike the ribosides, the glucose residue is not confined to the 9 position of the purine ring. Laloue et al. (1977) observed the occurrence of 7- glucosylation with i6Ade but not i6Ado. From radish cotyledons, an enzyme has been partly purified (Entsch et al., 1979b) which synthesizes the 7-glucopyranoside of zeatin using UDPglucose as a glucose donor and an unsubstituted position 9 of the purine ring was essential for the activity. The 7-glucosides of cytokinin are known to be very weakly active in cytokinin bioassays (Letham et al.,
1983), and extremely metabolically stable (Entsch et al., 1979a;
Summons et al., 1980; Letham et al., 1982). Laloue et al. (1977) suggested that the cytokinin-7-glucoside may be a protected or storage form of the cytokinin which could account for the higher biological activity of cytokinin bases than their corresponding 14 ribosides. Conjugation of alanine to the 9-nitrogen atom of i6Ade and other cytokinin bases leads to lupinic acid formation. The enzyme mediating this metabolic interconversion was isolated from developing lupin seeds and designated 8-(9-cytokinin)alanine synthase or lupinic acid synthase (Entsch et al., 1983). Like the 7- glucosides, the 9-alanyl cytokinin conjugates are extremely stable in tissues where they have been examined as metabolites of other cytokinins and where they have been exogenously applied (Parker et al., 1978). McGaw and Horgan (1985) suggested that these compounds and the 7- and 9-glucosides may very well be detoxification products incapable of further contribution to the active cytokinin pool in tissues where they accumulate.
As mentioned previously in this review, it is still uncertain whether plants contain cytokinin-specific enzymes which catalyze the interconversion of i6Ado and i6Ade. Although it has been recognized that the steps common to metabolism of unsubstituted purines and cytokinins may be important points at which cytokinin metabolism is regulated, research has not been reported to date to provide strong evidences for the distinction of cytokinin nucleosidase from adenosine nucleosidase in any plant sources.
Previous research done in our laboratory by Rolle and Chism (1986) suggested that the partially purified enzyme extracts from ripe tomato were either contaminated with adenosine nucleosidase activity, or that they contained an adenosine nucleosidase capable of utilizing cytokinins as substrate. Our attempts at further 15
purification to eliminate the contaminating activity,
characterization of this enzyme systems are reported in later
section. The technique of affinity chromatography has been successfully applied on the separation of a variety of biological substances including proteins, enzymes, antibodies, nucleic acids,
and nucleotides in the past decade. The basis for this technique, as illustrated in Figure 4, is relatively simple. The enzyme or
biomolecule of interest has a bioselective attraction for the bioligand attached to an insoluble, porous support. When a crude enzyme extract is passed through the column, the enzyme of interest will be retained on the column as an enzyme-bioligand complex.
Unwanted proteins without any affinity for the bioligands will be eluted with buffer, and subsequently the desired protein can be eluted by a suitable concentration of soluble bioligand or by changing the conditions (pH, ionic strength, temperature etc.) which result in dissociation of the complex. Affinity chromatography has been utilized to isolate cytokinin-binding protein (CBP) from wheat germ, barley embryos and leaves, and tobacco leaves (Takegami and Yoshida, 1975;
Yoshida and Takegami, 1977; Erion et al., 1978; Moore, 1979; Chen et al., 1980; Reddy et al., 1983; Kharchenko et al., 1984). Moore (1979) reported a rapid affinity chromatographic purification for partially purified CBP from wheat germ by coupling kinetin riboside to AH
Sephadex 4B. 16
Suitable inert matrix > Oioliqand
\ Suitable chemical coupling from matrix to bioligand, ollcn termed a "spacer arm"
fa)
Crude cell extract, plasma, etc.
— Exchange buffer
(b)
Desired protein is retained
Chromatographic packing of Extraneous proteins do not bind immobilized bioligand from step 1
(0
Figure 4. The steps of affinity chromatography, (a) Step 1: Immobilize a bioligand, (b) Step 2: Prepare crude extract and free it from any endogenous substrate, (c) Step 3: Apply the substrate free extract to a column of the bioselective adsorbent, (d) Step 4: Wash away unwanted proteins, (e) Step 5: The desired protein in eluted, possibly with a soluble bioligand. Figure4. (continued)
Unwanted contaminating protein
(<0
Desired protein being eluted f with a suitable concentration of free bioligand
(e)
Figure 4. The steps of affinity chromatography. 18
Affinity adsorbents of 6-benzylaminopurine (BAP) attached to
Epoxy-Sepharose or CNBr-Sepharose were prepared by Kharchenko et al. (1984) for purifying CBP. It was found that the columns possess
nonspecific sorption. Elution of the enzyme required 1 mM BAP to
avoid the contamination of substances retained as a result of
nonspecific sorption.
Affinity chromatography has been employed to isolate and
purify several enzymes in tomato. Tomato D-galacturonanase was separated from pectinesterase by affinity chromatography on pectic
acid cross-linked by epichlorohydrine (Rexova-Benkova et al., 1977).
Both enzymes were adsorbed to the column and then stepwise eluted
by buffers of increasing pH. Alcohol dehydrogenase from tomato fruits has been purified 99-fold by Nicolas and Crouzet (1980) using affinity chromatography on Blue Sepharose CL-6B with 37% yield.
Signoret and Crouzet (1982) reported a 263-fold purification of tomato peroxidase by using an immobilized concanavalin A Sepharose column. The enzyme was eluted using an a-methyl-D- mannoside gradient. We take an interest in the preparation of an adenosine-epoxy- agarose column by Schrader et al. (1976) due to the same affinity adsorbent was made for our experiment, even though it was not used in the field relative to tomato or cytokinin. This immobilized ado- epoxy-agarose column was employed as the final step in the purification of adenosine deaminase from human erythrocyte and a dramatic 468,000-fold purification was achieved. The crude enzyme extract was partially purified by gel filtration and DEAE-sephadex 19 column before being subjected onto the affinity column and an increase of 312-fold purification (from a purification factor 1,500 to 468,000) was obtained in this single step of affinity chromatography. A similar affinity column prepared using inosine
(Ino), the product from the reaction of adenosine deaminase on Ado, as the ligand did not retard adenosine deaminase. MATERIALS AND METHODS
I. Plant Materials
Tomato fruits (Lycopersicon esculentum Mill. cv. Floradade)
were obtained from local wholesaler. The tomato fruits were
washed with distilled water and stored under room temperature.
II. Determination of Protein Content
Protein content was determined according to the method of Smith et al. (1985) using bicinchoninic acid (BCA) with bovine serum
albumin (BSA) as standard.
The molecular structure of BCA is shown in Figure 5. BCA, in
BCA MW 388.27
Figure 5. Chemical structure of bicinchoninic acid.
2 0 21
the form of its water soluble sodium salt, is a sensitive, stable, and
highly specific reagent for the cuprous ion (Cu+). Macromolecular structure and four peptides (cysteine, cystine, tryptophan, and tyrosine) have been reported to be responsible for color formation in
protein samples when assayed with BCA (Wiechelman et al., 1988).
The reaction scheme showing how the BCA protein assay reagent reacts with protein and Cu+2 in an alkaline medium to form the purple product, BCA-Cu+ complex, is presented in Figure 6. The purple product, formed by the interaction of two molecules of BCA with one cuprous ion (Cu+), is w ater soluble and exhibits a strong absorbance at 562 nm. This allows the spectrophotometric quantitation of protein in aqueous solutions.
Two reagents (A and B) were mixed at a ratio of A:B = 50:1 to make the working BCA assay reagent. Reagent A contains sodium carbonate, sodium bicarbonate, BCA detection reagent and sodium tartrate in 0.2 N NaOH. Reagent B is 4% copper sulfate solution. The working reagent is stable at least a day at room temperature. Each standard or unknown protein sample (0.1 ml) was pipetted into the test tube. For blanks ,0.1 ml of buffer was used. Then, 2 ml working BCA reagent were added to each tube and mixed well. All tubes were incubated at 60°C for 30 min in a water bath. All tubes were allowed to cool to room temperature after incubation. The absorbance of the purple product at 562 nm was measured utilizing a
Perkin-Elmer Colemam 124D double beam spectrophotometer. The standard curve was plotted by the net absorbance of BSA at 562 nm against protein concentration. Using this standard curve, the protein BCA Protein Assay Reagent ~ooc—( On i O v- coo \ / protein + Cu+2 OH 7 H20 Cu
0 0 C —\ ( ) N
BCA - Cu+1 complex
Figure 6. The reaction scheme shows how the BCA combines the biuret reaction to form the purple color complex.
t'j 23
concentration for each unknown protein sample was calculated.
III. High Performance Liquid Chromatography
The analyses of loss of substrates and formation of products
were carried out using a Waters HPLC unit equipped with a Waters system interface module. Fifty ul samples were injected via the WISP 700 satellite autosampler and chromatographed on an ODS- silica column (silica with n-octadecyl groups bound to the surface, Selectosil Cl 8, 0.46 ID x 25 cm column from Phenomenex) fitted with a reverse-phase guard column (0.2 ID x 1 cm). Solvent delivery was controlled by two Waters model 501 pumps. Separation of substrates and products was achieved by a several segment linear gradient starting from water to 20% methanol over 5 min, held at
20% methanol for 2 min, from 20% to 40% over 2 min, held at 40% methanol for 3.5 min, from 40% to 78% methanol over 3.5 min, held at 78% methanol for 10 min, from 78% back to water over 3 min, and equilibated with water for 10 min, at a flow rate of 1 ml/min. After passage through the column, effluent was monitored by a
Waters model 484 tunable absorbance detector at a wavelength of 254 nm and sensitivity of 0.1 absorbance units full scale (AUFS).
Data were managed by a Water Maxima 820 chromatography data work station equipped with a NEC PowerMate SX Plus computer and a
NEC Pinwriter P5200 printer.
The amounts of Ado and i6Ado lost and Ade and i6Ade formed from them were determined by comparing the area of the peak for 24
each compound to those of standard curves generated using standard
solutions in 10 mM Mes/NaOH buffer, pH 6.0. The enzyme activity
was then calculated from the mole fraction of products formed.
IV. Preparation of Enzyme Extract
Tomato fruits were diced and frozen with liquid nitrogen immediately. The tomato pieces were subsequently homogenized in
a Waring Blender in cold acetone (-20±2°C). The homogenate was filtered on a Buchner Funnel using vacuum and the residue which
comprised the acetone power was washed with 1-2 liters cold acetone (-20±2°C). Vacuum was applied for an additional 30 min to remove excess acetone completely. The dry acetone powder was wrapped by aluminum foil and stored at -20°C.
Acetone powder (10 g) was extracted with 80 ml of 10 mM 2-
[N-morpholine]ethane sulfonic acid (Mes/NaOH) buffer, pH 6.0 by using a Potter-Elvehjem homogenizer. The homogenization was carried out within the carefully controlled 0-4°C range in an ice bath. The homogenate was filtered through double layers of cheesecloth while the filtrate was maintained on ice. The filtrate was centrifuged for 25 min at 12,577xg in an Eppendorf Centrifuge kept in a walk-in refrigerator at 4°C. The supernatant is referred to as crude extract which was purified in the further affinity chromatography step. 25
V. Preparation of Bioselective Adsorbent Gel
Because a suitable group selective adsorbent is not
commercially available, a coupling procedure was carried out in our
laboratory. Epoxy-activated-agarose freeze-dried powder (1 g of
epoxy-activated-agarose powder equivalent to 3 ml swollen gel) was reswelled and washed with water (100 ml/g powder) on a
sintered glass filter. The washed moist gel was added to a solution
of Ado or i6Ado in 0.1 M sodium bicarbonate buffer pH 11.0 and allowed to react for 24 hours in a water bath shaker at 37°C. The substituted gel was thoroughly washed with 250 ml of distilled water to ensure complete removal of the excess ligand. The remaining excess active groups then were blocked with 1.0 M ethanolamine for 4 hours at 37°C. Ethanolamine was washed away with 250 ml of distilled water and then alternately three times with sodium phosphate buffer (0.2 M, pH 8.0) and sodium acetate buffer (0.2 M, pH 4.0) containing 0.5 M NaCI. The gel was subsequently packed in a 0.7 x 15 cm column (BIO-RAD Econo-
Column) and equilibrated with starting buffer (10 mM Mes/NaOH, pH
6.0). Based on the amount of Ado or i6Ado which was removed from solution during the 24 h coupling period, approximately 1 umole of
Ado or 0.6 umole of i6Ado was bound per g of the epoxy-activated agarose powder. 26
VI. Adsorption and Elution from Affinity Chromatography
The crude extract was applied onto the Ado-Agarose (or i6Ado-
Agarose) column (containing 3 ml gel) which had previously been
equilibrated with 10 mM Mes/NaOH buffer, pH 6.0. The column was
eluted at a rate of 20 ml.cm-2h-1, and eluted first with 10 bed
volumes of 10 mM Mes/NaOH buffer solution, followed by various
concentrations of Ado and i6Ado in the same buffer. Fractions of 4
ml were collected. The fractions were dialysed against 2 liters of
10 mM Mes/NaOH to free the solutes of Ado and i6Ado used in elution solutions before being determined for adenosine nucleosidase and cytokinin nucleosidase activities and protein content.
VII. Assays for Nucleosidase Activities
The assays of adenosine nucleosidase and cytokinin nucleosidase were based on the conversion of nucleosides to the corresponding free bases. The incubation mixture (0.7 ml) contained 0.35 ml of enzyme preparation and 0.35 ml of 1.5 mM substrate solution (Ado or i6Ado in 10 mM Mes/NaOH buffer, pH 6.0). After 2 h incubation at 32°C in a water bath, the reaction was terminated by heating in boiling water for 10 min. An aliquot (0.3 ml) of the reaction mixture was withdrawn at 0 and 2 h and 50 ul of the mixture was subjected to HPLC to determine the loss of substrates and formation of products from the enzymatic reaction. The enzyme activity is defined as the formation of 1 nmole of Ade or i6Ade per 27 min per mg protein at 32°C with Ado or i6Ado as substrate, respectively.
VIII. Kinetic Analysis
Fractions with adenosine nucleosidase and cytokinin nucleosidase activities were collected from two independent runs of affinity chromatography elution. The concentrations of Ado or i6Ado solutions used as substrate were 25, 35, 50, 100, 200, and 300 uM in
Mes/NaOH buffer, pH 6.0. The enzyme activity assay was carried out as described in VII. The values of apparent Km and Vmax were determined by plotting 1/V against 1/Conc. The intercept on X axis is defined as -1/Km and the one on Y axis is equal to 1/Vmax.
IX. SDS-polyacryamide Electrophoresis
Electrophoresis was performed by using a Bio-Rad Mini-
PROTEAN II Dual Slab Cell apparatus. T w enty to th irty ul concentrated enzyme solution was electrophoresed on 12% polyacrylamide Mini-PROTEAN II ready gels (Bio-Rad). A constant electric current of 200 volts was applied during the electrophoresis with Tris-glycine buffer, pH 8.3, and bromophenol blue as indicator.
Proteins were first stained in a 0.25% solution of Coomassie
Brilliant Blue R-250 in m ethanol-acetic acid-water (4:1:5, v/v) for
30 min and destained in 40% methanol/10% acetic acid followed by silver staining. The procedures for silver staining are described in
Table 1. The molecular weight marker proteins used were: Carbonic anhydrase (29,000), Ovalbumin (45,000), Bovine plasma albumin
(66,000), Phosphorylase B (97,400), G-Galactosidase (116,000), and Myosin (205,000). 29
Table 1. The protocol for silver staining.
Solution Volume (ml) Duration (min)
1. Fixative 40% methanol/10% acetic acid 400 30 2. Fixative 10% ethanol/5% acetic acid 400 15 3. Fixative 10% ethanol/5% acetic acid 400 15 4. Oxidizer 200 5 5. Deionized water 400 6 6. Deionized water 400 5 7. Deionized water 400 — Repeat washes 5, 6, 7, until all the yellow color is removed from the gel. 8. Silver reagent 200 20 9. Deionized w ater 400 1 10. Developer 200 0.5 Develop until solution turns yellow or brown 'smokey1 precipitate appears. Then pour off the developer, and add fresh developer. 11. Developer 200 5 12. Developer 200 5 13. Stop 5% acetic acid 400 5 RESULTS AND DISCUSSION
The use of BCA protein assay method to quantify the protein
content in our enzyme extract was found to be more suitable than the widely used the Bradford method (1976). The biggest hurdle encountered using the Bradford method was the rapid formation of
insoluble precipitates which interfered considerably the absorbance
measurement of the color complex formed. Hence, the protein data obtained utilizing the Bradford method was very unstable and inconsistent with time and much lower than that detected by BCA assay method. This made the accurate determination of protein content in our fractions very difficult. The BCA method was found to be very sensitive, and the purple color complex formed was stable for up to 3 hours. The comparison of quantitation data obtained from both methods is shown in Figure 7. The total protein recovery measured by BCA method ranges from 85 to 97%. However, results from Bradford method showed an exaggerated recovery percentage ranging from 200 to 600% and a large variation existed among several different trials of the same batch of fractions.
Two types of coupling ligands, Ado and i6Ado, immobilized affinity columns and various elution programs were investigated in
30 Figure 7. The comparison of protein recovery percentages attained by the widely used Bradford method and the recently developed BCA protein assay method.
31 O Q_ Q. CO o O o PROTEIN RECOVERY BY BCA METHOD (S) BCA BY METHOD RECOVERY PROTEIN ro tri o K) cn O o co o PROTEIN RECOVERY BY BRADFORD METHOD (X)
FRACTION NUMBER Z£ 33
an attempt to separate adenosine nucleosidase (Ado NS) activity
from cytokinin nucleosidase (i6Ado NS) activity in the crude enzyme extract from ripe tomato fruit. The fractionation of the crude enzyme extract on the column of epoxy-activated agarose with i6Ado as ligand is shown in Figure 8-11. When 2 mM Ado was added to the eluting buffer, Ado NS and i6Ado NS activities were co-eluted as a triplet peak (Fig. 8 and 9), fraction Nos. 7 to 16, which might indicate the existence of multiple molecular forms of the enzymes.
All the enzymatic activities were eluted from the column with the wash of 2 mM Ado solution and no activities were detected later even after 2 mM i6Ado was applied. The results indicated that 2 mM
Ado may be a too strong eluent which decreased the selectivity and specificity, thus, washed out all enzyme activities at the same time. The nucleosidase activities extracted from tomato fruits were eluted in three peaks on the i6Ado-Agarose column when a weaker eluent, 1 mM Ado was utilized instead of 2 mM Ado. The profile of cytokinin nucleosidase activity eluted from i6Ado-Agarose column is shown in Figure 10, while Figure 11 represents the elution profile of adenosine nucleosidase activity from the same column. Addition of adenosine at a concentration of 1 mM to the eluting buffer, caused the minor first peak to emerge. The second peak was eluted by the addition of i6Ado in buffer to a concentration of 1 mM.
The third peak appeared when the concentration of i6Ado in the eluting buffer was increased to 1.5 mM.
Fraction Nos. 12 and 17, which represented peak I (designated as nucleosidase I, NS I) and peak II (NS II), respectively, Figure 8. Affinity chromatography of cytokinin nucleosidase on an i6Ado-Agarose column. Column was equilibrated with 0.01 M Mes/NaOH buffer, pH 6.0, at a flow rate of 20 ml/cm2h and fractions (4 ml) were collected. After applying the enzyme extract from ripe tomato fruit, column was eluted with the above buffer. At the point indicated the eluent in 0.01 M Mes/NaOH buffer, pH 6.0, was changed to: A, 2.0 mM Ado; B, 2.0 mM i6Ado; C, 0.01 M Mes/NaOH buffer, pH 6.0, nothing added. ♦, cytokinin nucleosidase activity; □, Protein content.
34 o j o CD o o o n —* —* —* n o r o ro o FORMATION OF OF I6ADE FORMATION (nmol/min/mg protein) PROTEIN CONTENT (ug/m CONTENT PROTEIN l) <-n <-n O tn O cn O O O O O O O O o O O “ O o
FRACTION NUMBER ££ Figure 9. Affinity chromatography of adenosine nucleosidase on an i6Ado-Agarose column. Column was equilibrated with 0.01 M Mes/NaOH buffer, pH 6.0, at a flow rate of 20 ml/cm2h and fractions (4 ml) were collected. After applying the enzyme extract from ripe tomato fruit, column was eluted with the above buffer. At the point indicated the eluent in 0.01 M Mes/NaOH buffer, pH 6.0, was changed to: A, 2.0 mM Ado; B, 2.0 mM i6Ado; C, 0.01 M Mes/NaOH buffer, pH 6.0, nothing added. ♦, adenosine nucleosidase activity; □, Protein content.
36 PROTEIN CONTENT (ug/m l)
0 - r 200 r —1 —1 f\) ro -i300 cn o cn O cn O O O O O O
o cn O O cn
FORMATION OF ADE (nmol/min/mg protein) Figure 10. Affinity chromatography of cytokinin nucleosidase on an i6Ado-Agarose column. Column was equilibrated with 0.01 M Mes/NaOH buffer, pH 6.0, at a flow rate of 20 ml/cm2h and fractions (4 ml) were collected. After applying the enzyme extract from ripe tomato fruit, column was eluted with the above buffer. At the point indicated the eluent in 0.01 M Mes/NaOH buffer, pH 6.0, was changed to: A, 1.0 mM Ado; B, 1.0 mM i6Ado; C, 1.5 mM i^Ado. ♦, cytokinin nucleosidase activity; □, Protein content.
38 400 CO cn O CO >— Q_ O O c n O IX)O rx) CO CO cn i — O FORMATION OF OF I6ADE FORMATION c nO O cn o r o (nmol/min/mg protein) PROTEIN CONTENT (ug/m CONTENT PROTEIN l) CO cn o O o O O O cn O cn c n CO c n
FRACTION NUMBER 6£ Figure 11. Affinity chromatography of adenosine nucleosidase on an i6Ado-Agarose column. Column was equilibrated with 0.01 M Mes/NaOH buffer, pH 6.0, at a flow rate of 20 ml/cm2h and fractions (4 ml) were collected. After applying the enzyme extract from ripe tomato fruit, column was eluted with the above buffer. At the point indicated the eluent in 0.01 M Mes/NaOH buffer, pH 6.0, was changed to: A, 1.0 mM Ado; B, 1.0 mM i6Ado; C, 1.5 mM i6Ado. ♦, adenosine nucleosidase activity; □, Protein content.
40 PROTEIN CONTENT (u g /m l) 200 250 0 0 3 100 150 50 0 5 10 RCIN NUMBER FRACTION NS 15 Ado -600 -800 - -400 -200 1200 1000
FORMATION OF ADE (nmol/min/mg protein) 42
exhibited both adenosine nucleosidase and cytokinin nucleosidase
activities. However, the third peak which had cytokinin
nucleosidase activity was eluted two fractions earlier (fraction Nos.
24, i6Ado NS III) than the one (fraction Nos. 26, Ado NS III) which
exhibited adenosine nucleosidase activity.
The Ado NS and i6Ado NS activities detected in peak I and peak
II might suggest the existence either of a single enzyme with two separate catalytic sites, or of two separate enzymes of similar structure and properties, one specific to adenosine, the other to cytokinin. The presence of cytokinin nucleosidase activity in tomatoes suggests that this enzyme system indeed has a role in the metabolism of cytokinin in tomatoes. These findings seem to support the hypothesis of Chen (1981), that this enzyme system may function to regulate the pool of active cytokinins.
Table 2 is a summary of the purification factors for each peak fraction in Figure 10 and 11 on the i6Ado-Agarose column. Ado NS activity was purified about 55-fold in the NS II peak fraction.
Almost 41-fold purification of cytokinin NS was gained in the i6Ado
NS III peak fraction. The total recovery of Ado NS activity was
94.01% with 0.16% from peak I, 90.44% from peak II, and 3.41% from peak III. The total recovery of cytokinin nucleosidase activity was
56.61% and was 0.14%, 47.73%, and 8.74% from peak I, peak II, and peak III, respectively. The low recovery of cytokinin nucleosidase activity could be due to the extremely low concentration of cytokinin nucleosidase in enzyme extract, in addition to the instability of purified nucleosidase in dilute solution. 43
Table 2. Purification of adenosine nucleosidase in each peak fraction of affinity chromatography from ripe tomato fruit.
Peak Fraction Specific Activity Purification Factor /S u b s tra te (umol/min/mg protein)
Crude enzyme extract Ado 18.79 i6Ado 4.15
NS 1 Ado 140.54 7.48 i6Ado 26.08 6.28
NS II Ado 1036.76 55.18 i6Ado 82.02 19.76
i6Ado NS III 169.82 40.92
Ado NS III 213.46 11.36 44
The multiple forms of Ado NS were also observed by Imagawa
et al. (1979) in extracts from tea leaves. According to the elution volume of the three adenosine nucleosidases from gel filtration, it
was found that they all have the approximately same molecular
weight of 68,000. The partially purified Ado NS from tomato roots
was also found to have two forms, which were designated as form
R1 and R2, by Burch and Stuchbury (1986b). The Km values for R1
and R2 were 25 uM and 9 uM, respectively. A similar affinity column prepared using adenosine as the
immobilized ligand did not separate the different forms of adenosine
nucleosidase and cytokinin nucleosidase although it did retard the
enzymes (Fig. 12 and 13). The two enzyme activities were co-eluted
as a single peak with the application of 1 mM i6Ado in the eluting solution. The fractions which exhibited nucleosidase activities from
i6Ado-Agarose (in Fig. 10 and 11) column were collected for kinetic
analyses and results are shown in Table 3 and Figure 14-19. The data plotted are representative of the means and standard deviation of duplicates. The best fitting lines were determined by least squares analysis. Overall, fairly good correlation (r=0.97-0.99) was obtained for all double reciprocal plots. The lower apparent Km values for Ado (38.66 uM and 62.65 uM for NS I and NS II, respectively) than that for i6Ado (98.18 uM and 145.00 uM for NS I and NS II, respectively) suggested that NS I and II have higher affinity to Ado than to i6Ado. The third component of NS, i6Ado NS
III, exhibited a much lower apparent Km value to i6Ado, that is higher Figure 12. Affinity chromatography of cytokinin nucleosidase on an Ado-Agarose column. Column was equilibrated with 0.01 M Mes/NaOH buffer, pH 6.0, at a flow rate of 20 ml/cm2h and fractions (4 ml) were collected. After applying the enzyme extract from ripe tomato fruit, column was eluted with the above buffer. At the point indicated the eluent in 0.01 M Mes/NaOH buffer, pH 6.0, was changed to: A, 1.0 mM Ado; B, 1.0 mM i6Ado; C, 1.5 mM iSAdo. ♦, cytokinin nucleosidase activity; □, Protein content.
45 PROTEIN CONTENT (ug/m l) 5 T450 — J no no CO CO cn o cn O cn O cn O o O o O o o o O O 1 250 r
O c n O cn O O
FORMATION OF I6ADE (nmol/min/mg protein) Figure 13. Affinity chromatography of adenosine nucleosidase on an Ado-Agarose column. Column was equilibrated with 0.01 M Mes/NaOH buffer, pH 6.0, at a flow rate of 20 ml/cm2h and fractions (4 ml) were collected. After applying the enzyme extract from ripe tomato fruit, column was eluted with the above buffer. At the point indicated the eluent in 0.01 M Mes/NaOH buffer, pH 6.0, was changed to: A, 1.0 mM Ado; B, 1.0 mM i6Ado; C, 1.5 mM i6Ado. ♦, adenosine nucleosidase activity; □, Protein content.
47 450 -i 1 r 800 l
o c n CT) i i i i i i o ___ i— > c n ___ *. 4 o i— ■—i— __ FORMATION OF ADE OF FORMATION o o o o o o —* —* —* i\) no co u> -t* (nmol/min/mg protein) PROTEIN CONTENT (ug/m CONTENT PROTEIN l) cn O cn ______OOOOOOOOO O INJ o -4— ■—i— '— i— ■ cn "1 1 I 1 I 1 r~ to o CO
FRACTION NUMBER 49
Table 3. Kinetic constant for cytokinin nucleosidase and adenosine nucleosidase activity in the four peak fractions in Fig. 10 and 11.
Peak Fraction Apparent Km V max V max/Km /Substrate (uM) (nmol/min/mg protein)
NS I Ado 38.66 12.66 0.33 i6Ado 98.18 8.37 0.09
NS II Ado 62.65 89.58 1.43 iSAdo 145.00 66.80 0.46
i6Ado NS III 55.00 14.76 0.27
Ado NS III 94.30 17.71 0.19 Figure 14. Lineweaver-Burk plot of adenosine nucleosidase activity (V) in peak fraction of NS I against adenosine concentration. The rate of enzymatic reaction is expressed as nmol adenine produced per minute per mg protein.
50 0.26 r\j no 00 1/V (nmol/min/mg protein) O O cn o p o - co o“ p o o cn
1/[Ado] (uM) IS Figure 15. Lineweaver-Burk plot of cytokinin nucleosidase activity (V) in peak fraction of NS I against i6Ado concentration. The rate of enzymatic reaction is expressed as nmol i6Ade produced per minute per mg protein.
52 cn CO ro 1/V (nmol/min/mg protein) O o ro o co o cn
1/[i6Ado] (uM) Figure 16. Lineweaver-Burk plot of adenosine nucleosidase activity (V) in peak fraction of NS II against adenosine concentration. The rate of enzymatic reaction is expressed as nmol adenine produced per minute per mg protein.
54 0.05 co ro l/V (nmol/min/mg protein) ro co o cn
1 1 /[Ado] (uM) Figure 17. Lineweaver-Burk plot of cytokinin nucleosidase activity (V) in peak fraction of NS II against i6Ado concentration. The rate of enzymatic reaction is expressed as nmol i6Ade produced per minute per mg protein.
56 0.12 o oo o CT) 1/V (nmol/min/mg protein) » p o ° o -o - o o b ro o o o OJ - © o o p
1/[i6Ado] (uM) Figure 18. Lineweaver-Burk plot of cytokinin nucleosidase activity (V) in peak fraction of i6Ado NS III against i6Ado concentration. The rate of enzymatic reaction is expressed as nmol i6Ade produced per minute per mg protein.
58 0.28 ro CD ro © 00 1/V (nmol/min/mg protein) o o o
l/[i6Ado] (uM) 6 £ Figure 19. Lineweaver-Burk plot of adenosine nucleosidase activity (V) in peak fraction of Ado NS III against adenosine concentration. The rate of enzymatic reaction is expressed as nmol adenine produced per minute per mg protein.
60 0.35 O j C ro Cn ro 1/V (nmol/min/mg protein) O r\j co o cn
1 /[Ado] (uM) 19 62
affinity, than NS I and NS II. These results indicate that i6Ado NS III may be the form which is specific for the deribosylation of
cytokinins (e.g., i6Ado). NS I and NS II might be cytokinin nucleosidases which are either contaminated with Ado NS activity,
or they are an Ado NS capable of utilizing cytokinins as substrates.
The second hypothesis seems to be more likely due to the facts that
both NS I and NS II have lower Km values and higher Vmax/Km ratios
for Ado than for i6Ado. Hence, the enzyme activity appear to favor Ado as substrate over i6Ado. It was found that NS II exhibits the highest activity (1036.76 umol/min/mg protein) to adenosine and presents the majority (90.44% recovery) of the enzymatic activity capable of hydrolyzing adenosine. It was also found that NS II has the highest Vmax/Km ratio to adenosine which indicates that NS II can utilize Ado much more efficiently than the other two forms (NS I and Ado NS III). Therefore, we conclude that the fraction of NS II appeared to be the major component of adenosine nucleosidase which might play the most significant role in adenosine metabolism.
The higher Vmax to Km ratio (Table 3) of NS II for i6Ado than that of i6Ado NS III indicated that NS II can utilize i6Ado sightly more efficiently than i6Ado NS III, which is a specific form to utilize only i6Ado. Hence, these results demostrate that adenosine nucleosidase plays an important role as well as cytokinin nucleosidase in cytokinin metabolism in plant tissues.
Previous reports have shown that Ado NS from various plants also acted on cytokinins and always expressed a lower affinity to cytokinins than to adenosine (Chen and Kristopeit, 1981; Rolle and 63
Chism, 1986; Burch and Stuchbury, 1986b). Because of the similar
features between cytokinins and unsubstituted purines, the steps
common to the metabolism of these two groups of compounds play a significant role in the uptake and release of cytokinins and in the
maintenance of an adequate level of active cytokinin in plant
tissu es.
The peak fractions designated as NS II, i6Ado NS III, and Ado NS
III were concentrated in dialysis bags surrounded b y
polyvinylpyrrolidone (PVP) powder. Five ml enzyme solution was concentrated to about 0.2 ml in an hour by placing the dialysis bag in
PVP powder, occasionally stripping off the hydrated gel forming on the outside. The purity of the peak fractions containing NS II, i6Ado
NS III, and Ado NS III were examined by SDS-polyacrylamide gel electrophoresis as shown in Fig. 20. Due to the extremely low concentration of enzyme present in our samples, silver staining was carried out following Coomassie Brilliant Blue R-250 in the hope of increasing sensitivity. Every peak fraction shows a nearly homogeneous state, that is, only one major band was observed for each fraction. By comparing the f R values with that of molecular weight markers shown in Figure 21, the major band for NS II and Ado
NS III are almost identical at molecular weight of 43,500. The major band for i6Ado NS III was fairly close to the one for NS II and
Ado NS III at molecular weight of 40,000.
If the observed polypeptide is present as a single subunit, the molecular weight found for Ado NS is slightly smaller than that reported previously. Burch and Stuchbury (1986b) proposed the 64 major form of Ado NS from tomato roots (R2) had a molecular weight of approximately 68,000. The molecular weight of partially purified Ado NS from barley leaf was about 66,000 (Guranowski and
Schneider, 1977). Clark et al. (1972) isolated an Ado NS from potato leaf with a molecular weight of 62,400, whereas Chen and
Kristopeit (1981) partially purified the same enzyme with a molecular weight of 59,000 from wheat germ. Figure 20. SDS-polyacrylamide electrophoregram of partially purified nucleosidase attained from i6Ado-Agarose affinity chromatography column in Fig. 10 and 11. The slots from left to right are crude extract, NS II, i6Ado NS III, and Ado NS III. Figure 21. The calibration curve of the molecular weight markers markers weight molecular the of curve calibration The 21. Figure
MOLECULAR WEIGHT x 1 ,0 0 0 scale. versus relative mobility Rf value on a semi-logarithmic semi-logarithmic a on value Rf mobility relative versus 0 0 0 1 0 0 1 0.0 - hshrls B Phosphorylase 0.1 Myosin EAIE OIIY (Rf) MOBILITY RELATIVE 0.2 B-Galactosidase 0.3 0.4 o ie lsa albumin plasma Bovine 0.5 Carbonic anhydrase Ovalbumin 0.6 0.7 0.8 6 6 CHAPTER II
EFFECTS OF VACUUM INFILTRATION AND DIPPING OF CALCIUM
SOLUTIONS ON THE QUALITY OF MINIMALLY PROCESSED
TOMATO SLICES
INTRODUCTION
Minimally processed fruits and vegetables are produced by the
unit operations of washing, sorting, and slicing but not blanching, therefore, they are still living tissues. Mechanical injury or wounding due to cutting causes some physiological consequences which may result in the loss of edible quality (i.e. color, texture, and flavor). Hence, control of wounding and the consequences of it becomes a major challenge to the postharvest life of minimally processed fruits and vegetables. In addition to inducing the wounding response, cutting exposes additional surfaces to microorganisms and therefore enchances the growth of microbials.
67 68
Calcium has long been associated with regulation of the
ripening process of fruit and postharvest storage life. Maintenance
of relatively high calcium concentration in fruit and vegetable
tissues results in a lower respiration rate (Bangerth et al., 1972; Watkins et al., 1982; Sams and Conway, 1984), slower rate of
ripening (Poovaiah and Leopold, 1973; Poovaiah, 1979a; Lieberman and Wang, 1982), slower softening of fruit flesh (Bangerth et al.,
1972; Mason, 1976; Scott and Wills, 1977; Conway and Sams, 1983; Sams and Conway, 1984; Lasekan, 1990), and suppressed ethylene
production (Lougheed et al., 1979; Watkins et al., 1982; Conway and
Sams, 1987).
Many studies of postharvest calcium application have been
performed on fresh whole fruits but not on minimally processed vegetables or fruits. The aim of this research is to evaluate calcium treatment on maintaining the storage quality of tomato slices and in
increasing their shelf life before they are consumed or further
processed. LITERATURE REVIEW
Postharvest calcium treatm ents have been utilized to control
or alleviate degradative changes and prolong the storage life of fruits and vegetables. Calcium (Ca) has been applied before or after
harvest to prevent physiological disorders (Shear, 1975) and to delay ripening of tomatoes (Wills et al., 1977), mangoes (Tirmazi and Wills, 1981; Wills et al., 1989), avocados (Wills and Tirmazi, 1982; Eaks, 1985; Wills and Sirivatanapa, 1988), and pears (Wills et al., 1982) . The importance of Ca in the regulation of fruit ripening and vegetable maturation is well established (Ferguson, 1984;
Poovaiah, 1986). It has been reported that high Ca concentration is associated with low respiration rate (Bangerth et al., 1972; Faust and Shear, 1972; Tingwa and Young, 1974; Watkins et al., 1982; Sams and Conway, 1984), retardation of senescence (Lieberman and Wang,
1982; Poovaiah and Leopold, 1973; Poovaiah, 1979a), hardening of tissues (Tagawa and Bonner, 1957; Bangerth et al., 1972; Mason,
1976; Scott and Wills, 1977; Simon, 1978; Conway and Sams, 1983;
Sams and Conway, 1984; French et al., 1989; Lasekan, 1990), and suppression of ethylene production (Lougheed et al., 1979; Watkins et al. 1982; Sams and Conway, 1984; Conway and Sams, 1987).
69 70
Postharvest introduction of Ca has been achieved by dipping, vacuum infiltration, or pressure infiltration. The amount of calcium
uptake is dependent on the concentration of calcium salts solution,
the degree of vacuum or pressure applied, and the soaking time for the fruits or vegetables in the solution. Conway and Sams (1 9 8 3 )
compared the three methods and found that the concentration of Ca
in the flesh of apples dipped in calcium chloride (CaCl 2) increased as
the concentration of calcium solution increased but not enough to
reduce decay. Vacuum infiltration of a 12% CaCl2 solution doubled the Ca content of apple fruit compared with a dip treatment in the same solution and resulted in 30% less decay. Pressure infiltration of the 12% CaCl2 solution increased the Ca content of fruit to more than twice that resulting from vacuum infiltration, and treated fruits had 50% less decay area than untreated fruits.
Introduction of Ca into apples after harvest has been shown to increase firmness immediately and to maintain firmness during storage (Bangerth et al., 1972; Mason, 1976; Scott and Wills, 1977;
Poovaiah et al., 1978; Poovaiah, 1979b; Poovaiah, 1980; Drake and
Spayd, 1983; Conway and Sams, 1983; Sams and Conway, 1984).
Dipping 'Jonathan1 apples prior to storage in either 4 or 6% CaCl2 solution greatly reduced the development of internal breakdown from over 60% in the water-dipped control fruits during 19 weeks at
2.2°C plus 1 week at 23°C (Bangerth et al., 1972). Calcium chloride solutions also improved the retention of fruit flesh firmness by 1.0 or 1.5 lb over that observed for the control fruits. 71
Mason (1976) reported that dipping apples in a solution of 4%
CaCl2 and 0.3% Keltrol, a commercial food thickener based on xanthane gum, increased the Ca concentration of the flesh and slowed the loss of firmness in cold storage. Batts and Bramlage
(1977) determined that a CaCl 2 dip treatment reduced softening and delayed breakdown of 'McIntosh' apples but did not influence the quality of 'Baldwin' or 'Cortland' apples.
Apples pressure-infiltrated with 1-4% of CaCl 2 were found to be 7-15% firmer and have 20-35% less decay than the control fruit not treated with CaCl2 (Sams and Conway, 1987). Pressure- infiltration of CaCl2 solutions also resulted in decreased ethylene production in apples (Sams and Conway, 1984). Scott and Wills
(1977) proposed that vacuum infiltrating CaCl2 after harvest reduced bitter pit, a calcium-related physiological disorder, and senescence of apples. Uptake of Ca solution by fruit varied widely between the open-calyx and closed-calyx cultivars. Calcium was found significantly more effective than Mg or Sr at reducing decay and ethylene production while maintaining firmness. Calcium was also the most effective cation for preventing decay caused by Penicillium expansum (Conway, 1982;
Hopfinger et al., 1984; Conway and Sams, 1987). In other studies, Ca was more important than Mg, K, Na, and P in affecting apple (Faust and Shear, 1968; Poovaiah and Leopold, 1973; Bramlage et al., 1985) and bean (Bateman, 1964) quality. However, the correlation between
Ca concentration and firmness of tomato fruit between firm and soft cultivars were not observed by Brady et al. (1985). They suggested 72
that direct analyses of cell wall Ca by a method which avoids
interaction with the intracellular acid are required if the
relationship is to be seen.
Studies on other fruits showed that the firmness of
strawberries was increased by soaking or vacuum infiltrating in 1 or 2% Ca lactate solutions (Morris et al., 1985; Main et al., 1986).
Calcium lactate was more effective in firming sliced strawberries
than in firming whole strawberries.
Wills et al. (1977) reported that small uptake of Ca achieved
by dipping tomatoes at ambient pressure in 4 and 8% CaCl2 solutions tended to stimulate ripening, but the greater uptakes of Ca obtained
by dipping tomatoes in higher concentrations at reduced pressure retarded ripening. Moline (1980) investigated the effects of vacuum
infiltration of CaCl2 on ripening rate of tomato fruits. They pointed out that the fruit color change was dependent upon the amount of vacuum applied, concentration of CaCl2 used, maturity of fruit selected, and temperature. Calcium applied at 380 mm vac. had no significant effect on internal color change but reduced external color change of fruit.
Wills and Tirmazi (1979) demonstrated that ripening of green tomatoes, as expressed by change of color and increased ethylene evolution and respiration, was inhibited when the Ca content of the fruit was raised to greater than 40 m g /100 g fresh weight. The fruits showed no sign of ripening even after 6 weeks of storage at
20°C, and application of 1000 ul/l ethylene for 3 weeks had no effect. Other divalent cations than Ca+2 , Mn+2, Co+2, and Mg+2 were 73
as effective as Ca+2 in retarding tomato ripening. The monovalent
metal ions, Na+ and K+, were less effective than Ca+, but did give some retardation of ripening.
Vacuum infiltration of pears (Wills et al., 1982) with CaCl2 delayed fruit ripening. The time to ripen was extended from about
11 days in control fruit to about 15 days for fruit infiltrated with
12% CaCl2 - However, infiltrating unripe bananas in CaCl2 solution accelerated ripening. The breakdown of pectic substances in the cell wall middle lamella is a critical factor in fruit ripening and results in fruit softening. Polygalacturonase (PG) is believed to be the major enzyme responsible for the breakdown of pectic substrances in the middle lamella and cell wall. Polyuronides, probably arising from the middle lamella, are liberated by the action of PG during ripening (Knee, 1974). Other enzymes such as cellulase, pectinmethylesterase, and 8-galatosidase which are active in tomatoes have been implicated in cell wall degradation, but their importance in overall softening is thought to be minimal (Hobson,
1968; Wallner and Walker, 1975; Gross and Wallner, 1979; Brady et al., 1982; Pressey and Avants, 1982). Edgington et al. (1961) observed that pectic substances were more easily released by PG from calcium deficient tissue than from normal tissue. Later, high concentration of Ca was found to inhibit the activity of mitochondria and pectic enzymes in tomato (Wills and Rigney, 1979). The effects of calcium ions and the chelating agents EDTA and citrate on the ability of partially purified PG from 74
ripe tomato fruit to degrade polygalacturonate were examined by Buescher and Hobson (1982). They reported that the action of PG on
polygalacturonate was effectively inhibited by calcium ions. The
inhibition of degradation by calcium ions was counteracted by the
presence of EDTA or citrate. They suggested that calcium
associated with the cell wall middle lamellae and its removal
regulate the rate and extent of degradation by PG during normal tomato fruit ripening.
Brady et al. (1985) investigated the interactions between the
amount of polygalacturonase, calcium and firmness in tomato fruit.
They found that firm cultivars were firmer than the soft cultivars throughout ripening , and generally they contained less PG activity at each stage examined. Uronic acid polymers in isolated cell wall were degraded rapidly by endogenous PG when citrate was present to complex Ca. In the presence of sufficient citrate, cell wall uronic acids of a firm and soft cultivar were equally susceptible to hydrolysis, suggesting that differences in the digestion of the walls by PG were dependent upon differences in Ca content or distribution.
Sams and Conway (1984) studied the effect of postharvest Ca infiltration on soluble polyuronide content in 'Golden Delicious1 apple fruit. They found that soluble polyuronide content was negatively correlated to Ca concentration and fruit firmness was positively correlated to Ca concentration. They suggested that if loss of Ca ions is the change which leads to the breakdown of the aggregation between the cell wall and middle lamella, then postharvest Ca application may prevent the dissociation and result in firmer fruit 75
and lower soluble polyuronide content. Recently, Glenn and Poovaiah (1990) investigated changes in
texture, cell wall structure and composition during storage of calcium treated and untreated 'Golden Delicious1 apple fruit. It was
observed that the cell wall region of Ca treated fruit showed no
swelling during storage and cell-to-cel contact was m aintained,
whereas regions of the middle lamella in untreated tissue stained
lightly, appeared distended, and eventually separated. In control
fruit, microfibril orientation was lost in distended regions of the cell wall, especially in the outer wall region sdjacent to the middle
lamella. These changes during storage of control fruit were
accompanied by a decrease in arabinose and galactose moieties of the cell wall and an increase in soluble pectin. Calcium treatment of fruit inhibited solubilization of polyuronide and arabinose moieties and reduced the loss in galactose content during storage.
Because Ca+2 affects fruit ripening so dramatically, the movement of Ca+2 in the cell wall middle lamella has been implicated as a factor that may control ripening. Rigney and Wills
(1981) measured cell wall middle lamella Ca+2 content in ripening tomato pericarp with an electron microprobe and proposed that solubilization of cell wall bound calcium is a prerequisite for the initiation of ripening and tissue degradation by PG. A shift in Ca+2 from bound to soluble forms during ripening was observed, which did not occur in the non-ripening mutant rin (Suwaan and Poovaiah,
1978; Poovaiah, 1979a). 76
Most of the Ca+2 introduced into fruit tissues was found
apparently accumulated in the cell wall middle lamella region, and it
is here that Ca+2 is thought to have its antisenescent effects (Demarty et al., 1984; Ferguson, 1984). Research on apples showed
that Ca from postharvest treatments enters the fruit through
epidermal openings, primarily lenticles (Batts and Bramlage, 1977).
Ferguson and Watkins (1981) reported that most of the Ca taken into apple fruits during postharvest vacuum infiltration was found in the
intercellular spaces. By autoradiographically localizing 45Ca and 36CI, Wienke (1980) suggested that the Ca applied on apple fruits by
postharvest dipping was incorporated into the cell wall in the same
manner as native calcium. The possible mechanism explaining the role of calcium in reducing decay and maintaining firmness in stored fruit probably is that Ca binds to the pectins in the middle lamella and improves the cohesiveness of the fruit (Norton and Witter, 1963; Woodward, 1972; Grant et al., 1973; Clarkson and Flanson, 1980; Buescher and Hobson,
1982; Rolle and Chism, 1987). Pectic substrances, cross-linked inter- and intra-molecularly by calcium, are though to be largely responsible for tissue rigidity, and the enchanced stability of the complex could limit its vulnerability to attact by the enzymes occurring naturally in the fruit or from fungal pathogens . The formation of the Ca pectate crosslinkage stabilize or strengthen cell walls of fruits, perhaps by interference with the active site on the molecule and decreased accessibility to the polymer for enzymes.
(Grant et al., 1973). 77
As fruits mature, there is an exchange of monovalent for polyvalent cations, particularly Ca, and as a result the pectic substances in the cell wall were less extensively cross-linked and became more accessible to degradative enzymes (Wallace et al.,
1962). When ion exchange takes place in the cell walls, the cell walls have always exhibited a large preference for Ca (Demarty et al., 1978; Demarty et al., 1984). Although postharvest Ca treatments have shown many benefits on controlling storage quality of apples, it has also been reported that excessive application or uptake of Ca did not further suppress decay of apple fruits but did cause skin injury (Bangerth, 1972; Conway and Sams, 1985). Studies done by Conway and Sams (1985) on apples showed that the stage of fruit maturity at the time of harvest and treatment with Ca can significantly affect the benefits achieved by Ca treatments. If the fruit was harvested and treated too early, too little CaCl2 solution was taken into the fruit and little decay inhibition was realized. If the fruit was harvested too late, however, more Ca was taken up than was needed for optimum decay control and severe fruit injury resulted. It has also been suggested that large amounts of supplemental Ca induced patterns of textural change during storage different from those which occurred under the influence of the endogenous Ca alone (Abbott et al., 1989). MATERIALS AND METHODS
I. Plant Materials
Tomato fruits (Lycopersicon esculentum Mill. cv. Floradade)
were purchased from a local wholesaler. Fruits were kept at room
temperature for a few days to let them ripen. Fruits were sorted for
size and uniformity of color manually. Fruits were washed with
0.05% sodium hypochlorite and the slicer was sanitized with 0.5% sodium hypochlorite. Washed fruits were cut into 0.7 cm slices
using a Hobart meat slicer. Slices which weighed between 25 and 50
g were sorted out for calcium treatments.
II. Preparation of vacuum infiltrated tomato slices
Slices were randomly placed in the following solutions, then were vacuum-infiltrated for 2 minutes. a. Control (no vacuum infiltration)
b. 50 mM of Calcium lactate
c. 100 mM of Calcium lactate
d. 250 mM of Calcium lactate
e. 50 mM of Calcium gluconate
78 79
f. 100 mM of Calcium gluconate
g. 250 mM of Calcium gluconate
III. Preparation of dipped tomato slices
Slices were randomly placed in the following solutions for 5
minutes. a. Control (no dip)
b. 250 mM of Calcium lactate
c. 500 mM of Calcium lactate
d. 250 mM of Calcium gluconate
Slices were removed from the solutions, drained and placed into polyethylene bags. Five slices were placed in each bag and the bags were stored at 8°C in the dark in an incubator. A bag for each treatment was removed at each sampling day.
IV. Microbial counts
Microbial examination for standard plate counts (SPC) and yeast and mold counts (PDA) was carried out using standard methods. Duplicate platings were made at each dilution.
V. Color measurement
Reflected color was determined on homogenates (1:1 w/w with water) after vacuum degassing. L, a, b values were determined utilizing a Hunter LabScan colorimeter. Hue angle value (0) was 80
calculated from the relationship of 0=tan_1b/a. A lower 0 value
indicates a higher degree of redness.
VI. Texture measurement
Texture analyses were conducted using an Instron Model 1000
equipped with 0.6 cm probe having a drive speed of 50 mm/min. The
load cell force range used was 0-50 kg. The force required for
puncture was determined at four sites on each of four slices.
VII. Preference test
The preference test in terms of the overall acceptance
(including color, texture, and flavor) was conducted right after the tomato slices were vacuum treated. Thirty judges were selected at
random from the faculty and students of the Food Science and
Technology Department. Each judge was asked to circle one category from 'dislike extremely1, which indicates 1 point, to 'like extremely', which indicates 9 points, for each tomato slice sample provided.
The F-test and multiple comparison were carried out from the hedonic rating scores given by each judge. Tomato slices which were vacuum infiltrated with 50 mM calcium lactate, 100 mM calcium lactate, 100 mM calcium gluconate, and no vacuum controls were tested. RESULTS AND DISCUSSION
Introduction of calcium by either vacuum infiltration or dipping was found to be very effective in increasing the firmness of tomato slices and preventing their texture from softening during the subsequent storage at 8°C. The initial firmness of untreated tomato slices was 32.8 N and dropped gradually to 15.8 N after 10 days.
Figure 22 and figure 23 represent the effects of vacuum infiltration of calcium lactate and calcium gluconate on the texture of tomato slices at day zero and day 10, respectively. Calcium treated tomato slices were firmer than controls immediately after vacuum treatment. Slices treated with calcium lactate and calcium gluconate increased their firmness with storage time. Calcium lactate appeared to have a greater effect on the firmness of tomato slices than did calcium gluconate at the same concentrations. The action of calcium seemed to be dependent upon concentration with the higher concentration having a greater influence in texture of tomato slices. The treatment of 50 mM Ca gluconate resulted in a least increase of 3.5 times in firmness after 10 days. Slices treated with 250 mM calcium lactate had the greatest increase in firmness throughout 10 days storage and were almost 10 times
81 Figure 22. Effect of calcium lactate and calcium gluconate vacuum infiltration treatm ents on the firmness of tomato slices at day zero.
82 200
180 I untreated
160 H Ca lactate
140 I Ca gluconate
120
100 80 I_T 60
40 ✓ f
20 0 1 untreated 50 mM 100 mM 250 mM
CONCENTRATION OF CALCIUM Figure 23. Effect of calcium lactate and calcium gluconate vacuum infiltration treatm ents on the firmness of tomato slices at day ten.
84 FIRMNESS (N) 200 200 140 140 160 180 100 120 0 - 80 0 H 40 60 20 20 0 - - untreated Ca lactate lactate Ca Ca gluconate Ca untreated OCNRTO O CALCIUM OF CONCENTRATION 0 mM 50 T T 0 mM 100 5 mM 250 cn CO 86
firmer than untreated control. Calcium lactate dip treatment led to a pronounced increase in
the firmness of tomato slices relative to controls at time zero (Fig.
24). Throughout 10 days storage period, calcium dip treatm ent
appeared to effectively prevent the sliced tomatoes from losing their firmness. The results are consistent with the observation of
Mason (1976). A dip solution of calcium chloride (4%, w/w) was observed to slow the loss of firmness of the flesh of apple in cold
storage. It was observed that the calcium effect was dependent upon concentration significantly. Calcium lactate appeared to
be more effective than calcium gluconate at the concentration of
250 mM (Fig. 25). The dependence of concentration on the effectiveness of
calcium in firming has been reported in many other studies. Sams
and Conway (1987) observed that apples treated with 1% CaCl2
solution was 7% firmer than the control, while 4% CaCl2 solution
resulted in a 15% increase in firmness of apples. Lasekan (1990)
estimated the effect of Ca on the storage life of oro(Antiaris
africana) and found that an increase of CaCl2 from 2 to 4% increased the firmness of the fruits. Tomato slices vacuum infiltrated with either calcium lactate or calcium gluconate were visibly more red than no vacuum controls until the end of 10 days storage. However, the reflected color of the homogenates measured only showed consistent effects at time zero.
The controls had higher hue angle values than that of the tomato slices treated with calcium lactate or calcium gluconate at time iue 4 Efc o clim att dp ramet o te firmness the on ents treatm dip lactate calcium of Effect 24. Figure FIRMNESS (N) 200 5 - 250 - 300 i 350 100 - 150 50 I 50 - f oao slices. tomato of 0 2 TRG TM (DAYS) TIME STORAGE Control 0m C lactate Ca 500mM lactate Ca 250mM 4 6 8 10 12 87 iue 5 Te oprsn f acu lcae n clim gluconate calcium and lactate calcium of comparison The 25. Figure
FIRMNESS (N) ae ocnrto o 20 mM. 250 of concentration same i tetet o te imes f oao lcs t the at slices tomato of firmness the on treatments dip 100 200 i 0 5 2 5 - 150 0 - 50 - 0 2 TRG TM (DAYS) TIME STORAGE 5m a gluconate Ca 250mM Control 5m C lactate Ca 250mM 4 8 6 10 12 88 89
zero. The development of red color of tomato slices infiltrated with all three concentrations of Ca lactate was retarded during the first
week of storage and began after one week (Fig. 26). However,
calcium lactate dip treatm ents accelerated the appearance of red color at the beginning of storage as compared with controls (Fig.
27). According to the reflected color measured, the accelerating
effect of calcium on pigment synthesis disappeared after 4 days. It has been found that a small uptake of Ca by dipping tomatoes in 4 or
8% CaCl2 stimulated ripening, but vacuum infiltrating in 8% CaCl2 retarded ripening in whole fruits (Wills et al., 1977).
Red color development has been recognized as one of the major
components of ripening occurring in most fruit in conjunction with other climacteric changes. Therefore, the delay of color development indicated delay in ripening. Moline (1980) found that vacuum infiltrating tomato with 4% and 6% CaCl2 at 380 mm vac. reduced external color change of fruit. They suggested that the color change was dependent upon the amount of vacuum applied, maturity of fruit selected, concentration of Ca solution, and temperature. It has been reported that more internal and external color development was observed while chilled tomato fruit was vacuum infiltrated with 6% CaCl2 at 380 mm vac. degree (Moline, 1980). Calcium applied at 190 mm vac. had variable effects on color of chilled tomato as affected by maturity. The pigment synthesis was accelerated by 100 and 250 mM calcium gluconate infiltration treatment in the first two days, thereafter, pigment degradation occurred slowly. However, color development somehow was not seen iue 6 Efc o clim att vcu iflrto tet ents treatm infiltration vacuum lactate calcium of Effect 26. Figure
HUE ANGLE (°) n h clr ftmt slices. tomato of color the on 8 - 28 0 - 30 32 4 - 34 - 6 3 - 38 -i 40 0 2 TRG TM (DAYS) TIME STORAGE 5 m C lactate mM Ca 250 0 M a lactate mM Ca 50 untreated 0 M a lactate mM Ca 100 4 6 8 10 12 90 iue 7 Efc o clim att ad acu guoae dip gluconate calcium and lactate calcium of Effect 27. Figure
HUE ANGLE (°) ramns n h clr f oao slices. tomato of color the on treatments 36 - 36 - 40 38 - 38 - 44 8 - 48 50 - 50 -i 2 5 0 2 TRG TM (DAYS) TIME STORAGE 250mM Ca gluconate Ca 250mM 500mM 500mM Control 5m C lactate Ca 250mM 4 Ca lactate 6 8 10 12 92
on slices infiltrated with 50 mM Ca gluconate throughout the 1 0
days storage (Fig. 28). This indicated that pigment degradation as
well as pigment synthesis was enhanced by Ca gluconate treatments. During the 10 days storage period, the tomato slices treated with
100 mM and 250 mM calcium gluconate were always more red than
controls as the reflected color measured. According to the hue angle data, the patterns of color change
differed between samples treated with Ca lactate and Ca gluconate. In order to explore the explanation, the Ca content uptaken by slices should be measured. It has been suggested that an optimum amount of Ca should be applied and uptaken in order to get the desire effects
(Wills et al., 1977; Scott and Wills, 1977; Moline, 1980). The maturity of fruit selected also affect the extent of Ca uptaken and the effectiveness of Ca treatments greatly (Moline, 1980; Conway and Sams, 1985). The large variation of initial color among the raw materials may have obscured effects of Ca treatments.
After 11 days, the visual appearance of tomato slices treated with Ca lactate became paler. However, their overall appearance was good in comparison with the slices treated with calcium gluconate or with untreated control slices which had an apparent brown edge (Fig. 29). In order to know the actual relationship between the red color and pigment synthesis or degradation, changes in the quantitation of various pigments during storage should be determined.
By the end of 10 days storage, 100 mM and 250 mM Calcium iue 8 Efc o clim lcnt vcu infiltration vacuum gluconate calcium of Effect 28. Figure
HUE ANGLE (°) ramns n h clr f oao slices. tomato of color the on treatments 8 - 28 32 4 - 34 i 36 0 2 TRG TM (DAYS) TIME STORAGE 250 mMgluconate Ca 250 50 mMgluconate Ca 50 untreated 100 mMgluconate Ca 100 4 6 8 10 12 94
^OmlVI CALCIUM GLUCONATE
>3 50mM CALCIUM LACTATE
VSM h 100mM CALCIUM LACTATE
NO VACUUM CONTROL
11 DAYS
Figure 29. The appearance of tomato slices vacuum infiltrated with 50 mM and TOO mM calcium lactate, and 100 mM calcium gluconate after 11 days storage. 95
lactate appeared to have the greatest antibacterial effect (Fig. 30).
The growth of yeast was not observed on the slices infiltrated with
100 mM calcium lactate until after one week storage. Figure 31
shows that 100 mM and 250 mM calcium lactate exhibited the
greatest effect on inhibiting the growth of yeast after 10 days
storage. The mold counts were maintained at a fairly low level in
all treatments except the untreated control up to 4 days. However,
by the end of 10 days storage, no treatments showed pronounced effects on inhibiting mold growth (Fig. 32). Very few investigation
has been reported to date about the effect of postharvest Ca
treatment on the inhibition of microbial growth on vegetables or
fruits. Our results here show that calcium lactate treatments have
slight antibacterial and antiyeast effects and are more effective than calcium gluconate in terms of their antimicrobial capability.
The overall acceptability of tomato slices infiltrated with 5 0
mM calcium lactate, 100 mM calcium lactate, and 100 mM calcium
gluconate were tested by 30 judges. The hedonic rating scores data
at day zero showed no significant difference between these three
treatments and untreated control at alpha=0.01. After 3 days, the slices treated with 100 mM calcium lactate was significantly
inferior (alpha=0.05) to the other three samples due to the
development of an off flavor (Table 4). Figure 30. Effect of calcium vacuum infiltration treatments on total plate counts of tomato slices.
96 MEAN LOG 10 (CFU/G) - 0 1 - 8 - 6 - 0 MC lactate mMCa 50 B untreated H 0 m a lactate mMCa 100 B □ 50 mMgluconate Ca 50 □ lactate mMCa 250 ^ B B HI 250 mMgluconate Ca 250 mMgluconate Ca 100 0 TRG TM (DAYS) TIMESTORAGE 10 4 o i s " Figure 31. Effect of calcium vacuum infiltration treatments on yeast counts of tomato slices.
98 ■ untreated
50 mM Ca lactate
6 ~ i 100 mM Ca lactate
250 mM Ca lactate 5 - □ 50 mM Ca gluconate
CD 100 mM Ca gluconate -V. 3 U. 250 mM Ca gluconate u
o 3 - CD o
Z - 6001^3161660 < 2
Ll I viss H
STORAGE TIME (DAYS) o v£> Figure 32. Effect of calcium vacuum infiltration treatments on mold counts of tomato slices.
1 0 0 MEAN LOG 10 (CFU/G) - 4 5 2 - 3 - untreated H □ 50 mMgluconate Ca 50 □ 5 m C lactate mM Ca 250 lactate mMCa 50 0 m C lactate mM Ca 100 250 mMgluconate Ca 250 100 mMgluconate Ca 100 6867 TRG TM (DAYS) TIMESTORAGE 6 o Table 4. Acceptability scores for calcium vacuum-infiltrated tomato slices.
Overall Time Treatment Acceptability1
0 day No vacuum control 6.19 a 2 50mM Ca lactate 5.78 a 100mM Ca lactate 5.69 a 10OmM Ca gluconate 6.12 a
3 day No vacuum control 5.36 a 50mM Ca lactate 5.88 a lOOmM Ca lactate 4.16 b 100mM Ca gluconate 5.80 ad
5 day No vacuum control 4.72 c 50mM Ca lactate 3.61 be lOOmM Ca lactate 3.11 b 100mM Ca gluconate 5.00 cd
1. 9-point scale: 1 = extremely dislike, and 9 = extremely like. 2. Any two values in the column not followed by the same letter significantly different at the 5 percent level. LIST OF REFERENCES
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