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THESIS

ATE U LIBRARIES

mlll’ll’illmuInlllﬁlﬁlln l um

3 1293 01399 2262

 

 

 

 

 

 

This is to certify that the

dissertation entitled

THE ROLE OF ALLIINASE AND V-GLUTAMYL TRANSPEPTIDASE
0N FLAVOR ENHANCEMENT IN ONION (Allium cepa L.)

 

presented by

Tirza Hanum

has been accepted towards fulﬁllment
of the requirements for

Ph.D.

degree in

 

 

Food Science

4,—7ﬂcw/

 

/ /Aajor professor

Date December 15, 1994

 

MS U i: an Afﬁrmative Action/Equal Opportunity Institution 0-12771

THE ROLE OF ALLIINASE AND y-GLUTAMYL TRANSPEPTIDASE
ON FLAVOR ENHANCEMENT IN ONION (Allium cepa L.)

By

Tirza Hanum

A DISSERTATION
Submitted to
Michigan State University

in partial fulﬁllment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Food Science and Human Nutrition

1995

ABSTRACT

THE ROLE OF ALLIINASE AND y-GLUTAMYL TRANSPEPTIDASE
ON FLAVOR ENHANCEMENT IN ONION (Allium cepa L.)

B y
Tirza Hanum

The onion ﬂavor is developed by the action of alliinase on nonvolatile
precursors S-alk(en)yl substituted L-cysteine sulfoxides when onion cells are
macerated to produce pyruvate, ammonia and many volatile sulfur compounds
associated with ﬂavor and odor. In onion. cells, about ﬁfty percent of the ﬂavor
precursors are believed to be present as y-glutamyl S-alk(en)yl L-cysteine
sulfoxides. Gamma glutamyl transpeptidase can act on these and liberates S-

alk(en)yl-L-cysteine sulfoxides for action by alliinase to produce onion ﬂavor.

The objectives of this study were to characterize alliinase and y-glutamyl
transpeptidase from onion and to determine the efﬁcacy of y-glutamyl
transpeptidase in combination with alliinase on the enhancement of onion
ﬂavor.

Alliinase and transpeptidase were extracted and characterized. The onion
ﬂavor of enzyme treated onion macerates extracted using supercritical carbon
dioxide was analyzed by gas chromatography- mass spectrophotometry.

Purified alliinase and y-glutamyl transpeptidase showed a single protein
band on SDS-PAGE corresponding to protein bands ranging from 46 to 66 kDa
and 116 to 205 kDa. The estimated molecular weight of these dissociated

proteins suggested that alliinase consisted of four equal subunits of 50 kDa and

Tirza Hanum

transpeptidase had an isoelectric point of 4.8 and 5.2, pH optima of 7.5 and 9.0,
a similar temperature optima of 40°C and activation energy (Ea) of 16.6 and
15.8 kJ/mole, respectively.

Transpeptidase and alliinase enhanced pyruvate production in onion
macerates by approximately 2.5 fold of the control. The effect of y-glutamyl
transpeptidase in conjunction with alliinase on onion ﬂavor proﬁle was shown
by a shift of major ﬂavor compounds from methyl propyl disulﬁde, methyl 1-
propenyl trisulfide, dimethyl tetrasulﬁde and propyl l-propenyl trisulﬁde, into
new major ﬂavor components methyl l-propenyl disulﬁde, dipropyl di sulfide,
propyl l-propenyl disulﬁde, methyl l-propenyl trisulﬁde and propyl l-propenyl
trisulﬁde. This increase in l-propenyl containing ﬂavor compounds may affect

overall ﬂavor of transpeptidase treated onion extracts.

 

 

ACKNOWLEDGMENT

I am highly indebted to Allah S.W.T Who enabled me to complete this
manuscript.

I wish to express my heart felt gratitude and appreciation to my academic
advisor Dr. J. N. Cash, for his kind help, consistent encouragement and excellent
guidance during my doctoral program and in the preparation of the whole
manuscript.

My appreciation is extended to Dr. MJ. Zabik, Dr. M.A. Uebersax and Dr.
P. K.W. Ng for serving on my guidance committee and for critically reviewing the
manuscript and making many constructive suggestions.

Appreciation is also extended to Dr. M.A. Uebersax for allowing me to use
cooling room and centrifuge in his Laboratory, to Dr. D.E. Guyer for technical
assistance and allowing me to use the supercritical carbon dioxide extraction unit,
and also to Dr. D. M. Smith, Dr. J. J. Pestka for allowing me to use equipment in
their Laboratories.

Special acknowledgment goes to Dr. Nirmal K. Sinha and Dr. Muhammad
Siddiq for valuable suggestions, technical assistance, critical evaluation and timely
help in preparation of this disser‘ation. Thanks are also due to all my lab patners
and friends at Michigan State University. Aknowledgment also goes to the HEDS
Project of the Government of the Republic of Indonesia and USAID for ﬁnancial
support.

Finally and for many reasons, I owe the greatest debt to my husband
Zoelﬁkar Zoebir, my son Vicky, my daughters Tizzy and Viera, for their support,
understanding, patience and help during this doctoral program.

iv

TABLE OF CONTENTS

LIST OF TABLES .................................................................................................... vii
LIST OF FIGURES ................................................................................................. viii
INTRODUCTION ........................................ . ............................................................ 1
REVIEW OF LITERATURE
A. Flavor Precursors of Onion ...................................................................... 4
B. Alliinase of Onion. ..................................................................................... 8
C. Gamma-Glutamyl Transpeptidase of Onion. ........................................ 15
D. Volatile Flavor Components of Onion ................................................... 19
E. Method of Measurement of Flavor ........................................................ 27
F, Flavor Enhancement by y-Glutamyl Transpeptidase ........................... 30
G. Flavor Extraction and Identiﬁcation ...................................................... 31
MATERIALS AND METHODS
MATERIALS ..................................................................................................... 36
Onion bulbs ............................................................................................... 36
Chemicals and mgents ........................................................................... 36
METHODS ......................................................................................................... 37
Relationship Between Alliinase Activity
and Pyruvate Content. ............................................................................ 37
Extraction of enzymes ............................................................................. 39
Assay of alliinase. ..................................................................................... 42
Assay of y-glutamyl transpeptidase ....................................................... 43
Protein determination ............................................................................... 44
Characterization of enzymes .................................................................. 45
The Effect of transpeptidase and
alliinase on pyruvate production ........................................................... 48
SC-COz Flavor extraction and analysis ............................................... 51

 

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RESULTS AND DISCUSSION
Relationship between Alliinase Activity

and Pyruvate Content. ............................................................................ 53
Concentration of pyruvate, Alliinase, and
Transpetidase during storage .................................................................. 56
Characteristics of Alliinase ...................................................................... 56
Characteristics of y-Glutamyl transpeptidase Transpeptidase ........... 72
Effect of Transpeptidase and Alliinase of
Onion on Pyruvate Production. ............................................................. 85
Effect of kidney transpeptidase and I
onion alliinase on Pyruvate production ............................................... 89
Effect of incubation time on pyruvate production. ............................. 89 -
Effect of enzyme concentration on pyruvate production ................. 91
Eﬂ'ect of Transpreptidase and Alliinase on Flavor Proﬁle .................. 93
Flavor compounds of onion extracts. ................................................... 94
Flavor diﬁ'erential of onion .exuacts ...................................................... 99
Effect of transpeptidase on ﬂavor proﬁle ............................................ 101
Characteristic ﬂavor of onion. .............................................................. 106
SUMMARY AND CONCLUSIONS .................................................................. 108
BIBLIOGRAPHY ................................................................................................. l l l
APPENDICES ....................................................................................................... 124

vi

LIST OF TABLES

Table Page
1. S-Alk(en)yl cysteine sulfoxides in onion ............................................... 5
2. Sulfur containing y-glutamyl peptides in onion .................................... 9
3. Compounds identiﬁed in steam distilled onion oil .............................. 23
4 Pyruvate content and alliinase activity in onions ............................... 54
5. Puriﬁcation of alliinase ............................................................................ 58
6. Substrate speciﬁcity of alliinase ............................................................. 64
7. Effect of inhibitors on alliinase activity ................................................ 69
8. Puriﬁcation of transpeptidase ................................................................ 73
9. Effect of inhibitors/promoters on transpeptidase activity .................. 83
10. Effect of transpeptidase and alliinase on pyruvate

production in onion macerates. ............................................................. 88
11. The yield of onion ﬂavor extracted from onions by

supercritical C02 extraction .................................................................. 95
12. Compounds identiﬁed in the supercritical

C02 extract of onions ............................................................................ 96

vii

LIST OF FIGURES

Figure Page
1. Cleavage of y-glutarnyl peptides by peptidase

and transpeptidase. ................................................................................. l6
2. Formation of volatile sulfur compounds in onion ............................... 20
3. Typical pressure-temperature phase diagram ....................................... 33 -
4. Schematic of the supercritical ﬂuid

extraction unit. ......................................................................................... 45
5. Correlation between alliinase activity and

pyruvate content of onions .................................................................... 55
6. Effect of storage on pyruvate content, alliinase

and transpeptidase activity

and sprouting index of onion. ............................................................... 57
7. Elution proﬁle of alliinase on CMC column ........................................ 59
8. Elution proﬁle of alliinase on Sephadex G-200 .................................. 60
9. Elution proﬁle of alliinase on Polybuﬁ‘er 94 ........................................ 61
10. Lineweaver Burk of alliinase for kinetic

constant determination ........................................................................... 63
11. Effect of pH on alliinase activity ........................................................... 66
12. Effect of temperature on alliinase activity ............................................ 67
13. Heat inactivation of allinase at different temperatures ....................... 68
14. Arrhenius plot for activation energy (Ea) determination ................... 70
15. SDS-PAGE of reduced alliinase : A: standard protein markers;

B and C: puriﬁed alliinase (10 mg protein) ........................................... 71
16. Elution proﬁle of transpeptidase on CMC column ............................. 74
17. Elution proﬁle of transpeptidase on Sephadex G-150 ....................... 75

viii

 

29.

30.

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31

32.

Elution proﬁle of transpeptidase on Sephadex G-150 ....................... 75

Elution proﬁle of transpeptidase on PBE 94 ....................................... 76
Lineweaver Burk plot of transpeptidase for

kinetic constant determination .............................................................. 78
Effect of pH on transpeptidase activity ............................................... 79
Eﬁect of temperature on transpeptidase activity ................................ 80

Heat inactivation of transpeptidase at
different temperatures .............................................................................. 81

Arhenius plot of transpeptidase for activation energy
(Ea) determination. .................................................................................. 82

SDS-PAGE of reduced transpeptidase; A: protein markers
B and C: puriﬁed transpeptidase (20pg protein) ................................ 84

Effect of transpeptidase and alliinase on conversion of
GMCS to pyruvate ................................................................................. 86

Effect of incubation of transpeptidase- alliinase
treated onion macerates on production of pyruvate .......................... 90

Effect of enzyme concentrations of transpeptidase- alliinase
treated onion macerates on production of pyruvate .......................... 93

Differential ﬂavor proﬁle of onion cv Spartan Banner
and cv Yellow Sweet. ............................................................................ 100

Effect of transpeptidase and alliinase on ﬂavor proﬁle of onion
cv Spartan Banner ................................................................................. 102

Effect of transpeptidase and alliinase on peak area changes
compared to control .............................................................................. 103

Effect of transpeptidase and alliinase on
ﬂavor proﬁle of onion cv Spartan Banner ........................................ 104

Effect of transpeptidase and alliinase on peak area changes
compared to control .............................................................................. 105

 

Appendix

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LIST OF APPENDICES

Appendix Page
1. Synthesis of standard compound ......................................................... 124
2. Standard curve for pyruvate for alliinase assay ................................. 128
3. Standard curve of aniline for transpeptidase assay ........................... 129
4. Standard curve for Bovine serum albumin (BSA)

for protein determination ....................................................................... 130
5. Direct plot of substrate concentration (S)

vs rate of reaction (V)for kinetic constant determination ................. 131
6. Relationship between elution volume and

MW of protein markers for alliinase ..................................................... 132
7 Relationship between elution volume and

molecular weight of transpeptidase ..................................................... 133
8. Peak area of ﬂavor compounds of onion extracts ............................. 134

 

 

Onion (.4
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1987).

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Gamma?

INTRODUCTION

Onion (Allium cepa L.) which belongs to Allium genus is prized for its ﬂavor
and pungency' and is widely used as ﬂavoring agent in food processing. The
ﬂavor of Alliums, and in particular of the onion has been extensively studied, in
terms of the chemical structure, properties, their origin, and formation within the
plant (Abraham et al., 1976; Whitaker, 1976; Fenwick and Hanley, 1985; Carson,
1987).

The characteristic onion ﬂavor is developed by the action of ﬂavor
producing enzyme alliinase (S-alk(en)yl L-cysteine sulfoxide lyase, EC. 4.4.1.4)
on non volatile, odorless precursors (alk(en)yl substituted L-cysteine sulfoxide)
when onion cells are cut or macerated to produce pyruvate, ammonia and many
volatile sulfur compounds associated with ﬂavor and odor (Whitaker, 1976).
Several attempts have been made to purify and characterize alliinase from onion
(Schwimmer and Mazelis, 1963; Tobkin and Mazelis, 1979; Nock and Mazelis,
1987).

Gamma-glutamyl transpeptidase (EC. 2.3.2.2) in sprouting onion also plays a
key role in onion ﬂavor chemistry since a signiﬁcant proportion of the ﬂavor
precursors are bound to glutamic acid as y-glutamyl peptides and are not
susceptible to the action of the endogenous alliinase the ﬂavor producing
enzyme. Gamma-glutamyl transpeptidase which has transferase activity should
liberate the free cysteine sulfoxide derivates, the ﬂavor precursors. The free
cysteine sulfoxide derivates should thus be susceptible to conversion to the
volatile sulfur ﬂavor components of onion by either the endogenous alliinase or

by exogenous alliinase from a suitable source of enzymes. This enzyme has been

1

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isolated from germinating seeds of chiVes (Matikkala and Virtanen, 1965a),
sprouting onion bulbs (Matikkala and Virtanen, 1965b; Schwimmer and Austin,
1971a), Ackee plant (Kean and Hare,l980), yeast (Penninckx and Jasper, 1985),
and from garlic (Ceci et al, 1992).

The enzymatic reactions to produce sulfur volatiles are central to the onion
ﬂavor biochemistry (Lancaster and Boland, 1990). While it is not always desirable
to retain the full enzymatic ﬂavor potential of the fresh onion in some uses
(pickled or when eaten raw), in dehydrated or onion oil, it is desirable to do so.
Schwimmer and Austin (1971a; 1971b) and Schwimmer (1973) reported that
pyruvate production of onion was shown to be extended by addition of y-
glutamyl transpeptidase. Although the increase in pyruvate was shown to
correlate with onion ﬂavor, few reports have proﬁled the qualitative aspect of the
volatile compounds generated during enzymatic reaction catalyzed by alliinase
alone or in combination with y-glutamyl transpeptidase in onion eultivars.
Previously, alliinase and y-glutamyl transpeptidase have neither been puriﬁed to
homogeneity nor characterized for their detailed physicochemical properties.

The objectives of this research were: a) to characterize alliinase, the enzyme
responsible for the development of ﬂavor in onion, b) to characterize y—glutamyl
transpeptidase, the enzyme responsible for the liberation of free alk(en)yl-L-
cysteine sulfoxides from a potential ﬂavor precursor y-glutamyl peptides which
then becomes available for alliinase, and c) to study the efﬁcacy of y-glutamyl
transpeptidase in combination with alliinase on the enhancement of ﬂavor as
shown by the enhancement of pyruvate production and the change of ﬂavor

proﬁle of onion ﬂavor extracted by super critical carbon dioxide.

REVIEW OF LITERATURE

Onions have been grown for many thousands of years for their therapeutic
and prophylactic properties, their religious signiﬁcance, and their ﬂavor and taste
(Fenwick and Hanley, 1985). Today onions are used for their ﬂavor, aroma, and
taste, being prepared domestically or forming raw materials for a variety food
manufacturing processes (dehydration, freezing, canning, and pickling).

Intact onion bulbs have little distinctive ﬂavor or odor characteristic and no
lachrimatory properties. However, on cutting or crushing onion tissue, alliinase
(alkyl cysteine sulfoxide lyase, C-S lyase, EC. 4.4.1.4) which is present in onion
tissue hydrolyzes the odorless ﬂavor precursor S-alk(en)yl-L—cysteine sulfoxides
to produce pyruvate, ammonia and the many volatile sulfur compounds associated
with ﬂavor, odor, and lachrimatory factor.

Another enzyme, y-glutamyl transpeptidase (transpeptidase, EC. 2.3.2.2)
was also reported to play a role in onion ﬂavor chemistry since this enzyme acts
upon the peptides and liberates the amino acid derivatives which serve as ﬂavor
precursors. Schwimmer (1971) showed that both L-cysteine sulfoxide lyase and
y—glutamyl transpeptidase acted sequentially to produce pyruvate from synthetic
y-glutamyl-S-methyl L-cysteine. Schwimmer and Austin (1971a) reported that
rehydrated onion ﬂavor can be enhanced by the application of y-glutamyl
transpeptidase. These enzymatic reactions to produce sulfur volatiles are central
to onion ﬂavor biochemistry (Lancaster and Boland, 1990). This chapter reviews,

the chemistry of the ﬂavor precursors, the characteristics of alliinase and y-

glutamyl transpeptidase and the role of both enzymes in onion ﬂavor generation.

 

 
  
  
   
  
 
  

1. Flavor P
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A. Flavor Precursors of Onion
S-Alk(en)yl-L-cysteine sulfoxides

The non-volatile sulfur compounds in the onion cell which give rise to
ﬂavor and pungency when tissue is disrupted are organosulfur compounds,
referred to as S-alk(en)yl-L—cysteine sulfoxide. The relative levels of sulfur
components in onion depend greatly on species, cultivar, maturity, cultural and
environmental conditions, and sample preparation methods (Saghir et al., 1965).
Table 1 summarizes the alkyl cysteine sulfoxide compounds present in onion and
the following are their characteristics which make a signiﬁcant contribution to the
pungency, lachrimatory, and ﬂavor of onion.

(+)—§-Methyl-L-cystcine sulfoxide: This compound was the ﬁrst sulfoxide
compound isolated from onion and appears to have the widest distribution in
Alliium species (V irtanen and Matikkala, 1959; Carson and Wong, 1961a; 1961b).
Hine and Roger (1956) showed that this compound possesses the S-conﬁguration
at the asymmetric center. According to Whitaker (1976), this compound may be
the major producer of fresh onion ﬂavor and presented in about 0.9 :t: 0.25 mg/ g
fresh weight (Lancaster and Kelly, 1983).

(+)-§-Propyl-L—cysteine sulfoxide : First isolated from onion in 1959 by
Virtanen and Matikkala. In contrast to (+)-S- methyl-L-cysteine sulfoxide, this
compound is considered to be restricted to a number of Allium species. The level
of this compound in white onion was 2.9 :r: 0.4 mg/ g fresh weight (Lancaster and
Kelly, 1983).

(+)-§-(z-Propgnyl)-L-cysteine sulfoxide: Commonly called alliin or S-allyl
L-cysteine sulfoxide, was the ﬁrst sulfoxide compound isolated from garlic (Allium
sativum) by Stoll and Seebeck in 1951. While it presents in especially large
amount in garlic, Freeman and Whenham (1975) reported that this compound was

absent in onion.

 

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Table l. S-Alk(en)yl-L-cysteine sulfoxides in onion*

 

 

Compound Formula
(+)—S-Methyl- L-cysteine sulfoxide CH3—S-CH2-CH-COOH
O NH2
(+)-S-Propyl-L-cysteine sulfoxide CH3-CH2-CH2-S-CH2-CH-COOH
O NI-I2

(+)-S-(2—Propenyl)-L-cysteine sulfoxide CH3=CH-CH-S'-CH2-C'H-COOH

O NH2
trans (+)-S-(l-Propenyl)-L-cysteine CH3-CH=CH-S-CHZ-C'H-COOH
sulfoxide ' ‘ . o NH2

 

* From Whitaker ( I_ 976)

 

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trans (+)-§-( l-Erggnyn-L-cysteine sulfoxide : This compound shown to
differ from alliin by the position of the double bond C1-C2 versus C2-C3. Carson et

a1. (1966) studied the structural conﬁguration of S-(l-propenyl)-L-cysteine
sulfoxide by nuclear magnetic resonance spectroscopy and concluded that the
vinylic double bond of the neutral compound has the trans configuration.
Schwimmer (1968) reported the enzymatic degradation of this compound and
found that it is the major precursor involved not only in eye tearing but also in
development of the sensory factors of odor, bitter taste, and tongue biting
sensation in fresh onion, and odor of cooked onion (Schwimmer, 1969).
According to Matikkala and Virtanen (1967), S-(l-propenyl)—L-cysteine sulfoxide
compound is the major S-substituted cysteine sulfoxide found in onion where it
occurs at levels up to 200 mg/ 100 g fresh weight but the concentration of this
compound is very low or absent in garlic.

From the kinetic constant of the enzymatic reaction and the fact that this
compound is the predominant L—cysteine sulfoxide derivate in onion, Schwimmer
(1969) concluded that this compound is the principal endogenous substrate for
alliinase in onion which is largely responsible for the development of the sensory
attributes perceived upon communition of onion tissue. In vitro study showed
the cyclitation of trans-S-(1-propenyl)-L-cysteine sulfoxide to form cycloalliin in
alkaline conditions (Carson and Boggs, 1966). However, Muller and Virtanen
(1965) were unable to demonstrate this process occurring in viva. Granroth
(1970) found that cycloalliin is neither hydrolyzed by alliinase nor contributes to

onion ﬂavor.

Gamma glutamyl peptides
Although y-glutamyl peptides are well represented in the Leguminoceae

and Alliaceae, their function is not fully understood. They may function as

 

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storage compound of N and S and also may have a role in the transport of amino
acids across cell membranes (Kasai and Larson, 1980). In Allium species, about 24
y-glutamyl peptides have been isolated with nine of these peptides occurring as
intermediates in the biosynthesis of S-alk(en)yl L-cysteine sulfoxides (onion ﬂavor
precursors) including y-glutamyl trans-(+)-S-(l-propenyl)-L-cysteine sulfoxide and
8-2 carboxypropyl glutathione as the major peptides in onions (Shaw et al., 1989).
Table 2 summarizes y—glutamyl peptides found in onion.

The concentration of peptides in onion vary widely from the abundant y-
glutarnyl S-propenyl L-cysteine sulfoxide (between 1.24 and 2.18 mg/g fresh
weight, followed by y-L-glutamyl-S-2-carboxy-propyl glutathione (0.45-0.60
mg/g fresh weight), v-glutamyl phenylalanine (009—0. 16 mg/g fresh weight) to the
sparse y-glutamyl S-2 carboxy propyl cysteine (2.1 mg/ 13.5 g fresh weight)
(Matikkala and Virtanen, 1967). Gamma glutamyl peptides of S-propyI-L-cysteine
sulfoxide and S-allyl-L—cysteine sulfoxide have not been isolated, and one may
speculate that this is because of their low concentration rather than their absence
from the tissue (Lancaster and Boland, 1990). Whitaker (1976), reported the
presence of y-glutamyl-L-methionine (5-12.7 mg/ 100 g fresh weight) and y-
glutamyl-S-methyl-L-cysteine (5-19 mg/100 g fresh weight) in onion.

In a study on metabolism of y-glutamyl peptide during storage, Lancaster
and Shaw (1991) reported that y-glutamyl trans (+)-S-(l-propenyl)-L-cysteine
sulfoxide and 8-2 carboxy propyl glutathione were absent prior to bulb formation
and then at bulbing accumulated to levels of 2.1 and 0.4 mg/g fresh weight,
respectively. During sprouting, level of both compounds decreased by 50% . The
signiﬁcance of y-glutamyl peptides in the metabolism of onion plant is unclear.
They occur in highest concentration in bulbs, and are rapidly hydrolized by y-
glutamyl peptidase during sprouting. Kasai and Larson (1980) raised the
possibility that y-glutamyl peptides may play a role in the transport of amino acids

 

across cell
sum’lt‘d tl
mpounds

A 53

00

found in oni
onion (Writ;
not at ailablc
presence of 1
Was found i
lmfstigation
with the allii
dehydrated

33d sulfur vc

across cell membranes. The decrease in peptide compounds during storage

supported the hypothesis that the major y-glutamyl peptides function as storage
compounds in stored onion bulbs (Lancaster and Shaw, 1991).

A signiﬁcant portion of y-glutamyl (+)-S-l-propenyl-L-cysteine sulfoxide
found in onion represents about ﬁfty percent of the potential ﬂavor precursors of
onion (Whitaker, 1976) but it is not susceptible to the action of alliinase, therefore,
not available for ﬂavor production (Matikkala and Virtanen, 1967). However, the
presence of y-glutamyl transpeptidase (EC. 2.3.2.1), which can act on this peptide,
was found in sprouted onions by Austin and Schwimmer (1971). In a related
investigation, it was shown that the sequential action of y-glutamyl transpeptidase
with the alliinase on the onion extract (Schwimmer, 1971) and the suspension of
dehydrated onion powder resulted in the enhancement production of pyruvate

and sulfur volatiles.

B. Alliinase. of Onion
Enzyme nomenclature

The isolation of alliinase in onion, together with the isolation of substrate for
this enzyme, established the major enzymatic pathway of the characteristic ﬂavor
developed when the cells are ruptured (Schwimmer et al., 1960; Kupiechi and
Virtanen, 1960). This enzyme has been given several names including alk(en)yl
-L-cysteine sulfoxide lyase, cysteine sulfoxide lyase, C-S lyase and alliin lyase.
However, the Enzymes Commission has assigned the systematic name: alliin alkyl-

sulfenate lyase (EC. 4.4.1.4) with a trivial name alliin lyase or alliinase.

Occurrence of alliinase in nature
Alliinase occurs in most, if not in all members of the genus Allium (T suno,

1958). It has been isolated from garlic (Stoll and Seebeck, 1951; Kazaryan and

- an"

 

Table 2 S

—-——.‘-

No

 

a) Glut;

9

Table 2. Sulfur containing y-glutamyl peptides in onion“

 

No. Compound

 

1. y—L-Glutamyl-S-methyl- L—cystein sulfoxide

HOOC-CH-(NI-IQ-(CHQZ-CO—I\'lH-CH-CH2--S'-CH2
COOH O
2. y—L-Glutamyl-S-methyl- L-cystein
HOOC-CH(Nl-lz)-(Cllz)z-CO-NH-('IH-CH2 -S-CH2
COOH
3. y—L-Glutamyl-S-(1-propenyl)-L—cystein-sulfoxide
HOOC-CH(NH1)-( CH2)2-CO-NH-(‘ZH-CH2-S‘-CH=CH-CH2
. COOH O
4. y—L—Glutamyl-S(2-carboxy- 1 -propyl)-L- cysteinyl glycine
Hooc-CH(NH2)-(CH)2-coIsIH-qH-coNH-CH2 coon
‘ CHz-S-CH2 -C‘H-COOH
CH.
5. y-L-glutamyl phenylalanine
HOOC-CH(NH2)- (C141)2 -C0NH-CH-CH2- CGI-l5
COOH
6. Glutathione y-glutamyl-L-cysteine disulﬁde
CHz-S-S-CH2-CH-NH-CO-(CH2)2-
HOOC-CH(NI12)-(CH2)2-CO-NH-CH COOH CH(NH,)-OOH
I

CO-NH-CHz-COOH

7. Glutathione-cysteine disulﬁde
CHz-S-S-CHz-CHmHz)-COOH
HOOC-CH(NHZ)—(CHZ)2-CO-NH-CH
CO-NH-CHz-COOH

 

* Whitaker (1976); Lancaster and Boland ( I 990)

10

Goryachenkova, 1979; Nock and Mazelis, 1987; Jansen et al., 1989), white Globe
onion (Schwimmer and Mazelis, 1963), Spanish white onion (Tobkin and Mazelis,
1979), and yellow onion (Hanum et al., 1993). It has also been reported in related
genera of the Liliceae such as Ipheion (Tsuno, 1958) and Thulbaghia (Jacobsen et
al., 1968). Alliinase-like activity has also been found in Brassica species (De Lima,
1974), Albizia lophanta (Schwimmer and Kjaer, 1960), Acacia farnesiana (Sweet
and Mazelis, 1987), and in a variety of bacterial species (Nomura et al., 1963). In

onion cells, this enzyme is present in the vacuole (Lancaster and Collin, 1981).

Purification of alliinase

Alliinase was ﬁrst extracted in a' relatively crude state from garlic by Stoll and
Seebeck in 1951. Sufﬁcient puriﬁcation of this enzyme was not achieved until the
late 1970’s. Extensive work by Tobkin and Mazelis on onion bulbs in 1979
obtained 64 fold puriﬁcation and-a speciﬁc activity of 289 mkatal/kg. Although
the enzyme was reported as homogenous, the electrophoretic pattern showed
contaminants on sodium dodecyl sulfate polyacrylarnide gel electrophoresis (SDS-
PAGE). In 1987, Nock and Mazelis using similar puriﬁcation method obtained a
speciﬁc activity of 23.3 Unit/mg protein, 16.6 fold puriﬁcation and 8% recovery of
alliinase from onion bulbs. However, isoelectric focusing in a broad pH gradient
(pH 3-10) of puriﬁed enzyme showed some charge heterogeneity.

Kazaryan and Goryachenkova (1979) also developed a method for the
isolation of alliinase from garlic bulbs and obtained a highly puriﬁed preparation
of alliinase which showed one band in SDS-PAGE. Similar puriﬁcation to
apparent homogeneity of this enzyme with respect to native PAGE, SDS-PAGE,
Isoelectric focusing (IEF) and Electrophoretic titration curve analysis obtained

from garlic, was reported by Jansen et al. (1989).

11

Lancaster and Boland (1990) concluded that solubility and stability of
alliinase were the major causes of considerable frustration in puriﬁcation studies.
The requirement for co-solvent to maintain enzyme activity during puriﬁcation is

an indication of the difﬁculties in maintaining this enzyme in aqueous solution.

Physicochemical properties of alliinase

Spectral studies on alliinase from onion and garlic revealed an absorption
peak at 420 nm which is the characteristic of pyridoxal phosphate (T obkin and
Mazelis, 1979). The stimulation of alliinase activity by pyridoxal phosphate has
been shown in onion (Schwimmer and Mazelis, 1963; Schwimmer, 1964;
Schwimmer, 1969; Schwimmer and Guadagni, 1968). Quantitative analysis
indicated that the onion enzyme contains three pyridoxal phosphate moieties per
150kDa molecular weight or one per equivalent subunit (T obkin and Mazelis,
1979). The stimulation effect of pyridoxal phosphate on alliinase activity of garlic
was also shown by Kazaryan and Goryachenkova (1979) and Nock and Mazelis
(1987). Kazaryan and Goryachenkova (1978) found that about six equivalents of
pyridoxal phosphate co-enzyme were found in the enzyme molecule of 130 kDa
molecular weight. However, Nock and Mazelis (1987) showed the presence of
one coenzyme molecule per subunit of alliinase extracted from garlic while Jansen
et al. (1989) failed to show any stimulation of reaction with added pyridoxal
phosphate (0.25-2.50 mM ﬁnal concentration).

Careful chemical and spectroscopic studies suggested that pyridoxal
phosphate molecules were strongly bound and could not be removed during the
purification. Even though the presence and concentration of pyridoxal
phosphate in garlic alliinase has not been fully resolved, alliinase from onion or
garlic is considered a pyridoxal phosphate dependent enzyme. The positive

periodic acid Shiff-base staining of the single band native gel and a subunit band

 

 

I")

(T

ﬁr)

’Y’

(5..

SP,

12

in SDS-PAGE suggested the presence of a carbohydrate constituent and indicated
that the alliinase of onion is a glycoprotein (Tobkin and Mazelis, 1979). However,
Jansen et al. (1989) could not conﬁrm the presence of carbohydrate in garlic
enzyme as reported by Nock and Mazelis (1987).

In large part, due to lack of sufﬁcient puriﬁcation of the enzyme to permit
deﬁnitive studies, the data reported demonstrated conﬂicting results with respect
to physicochemical and kinetic properties. Schwimmer and Mazelis (1963) found
that alliinase was very unstable at acid pH and pH optima depended on the buffer
used. Tobkin and Mazelis (1979) found optimum pH of 7.4-8.5 depending on the
buffer used. Nock and Mazelis (1987) also reported that alliinase of onion and
garlic although from closely related species, have many differences such as their
pH optima of 6.5 and 8.5, respectively- The isoelectric pH of onion alliinase was
4.0 (Nock and Mazelis, 1987), compared to 4.9 (Jansen et al., 1989) and 6.2
(Kazaryan and Goryachenkova, 1979) in garlic. Alliinase from garlic was reported
to be stable at temperature range 30-35°C with activation energy of 14.7 kJ/mol
(Jansen et al., 1989). However, studies on the thermal stability of alliinase from
onion or garlic have not been done as extensively as for temperature optima.

Bello (1972) reported that crude onion alliinase from Spartan Banner cultivar
contained sixteen protein bands and identiﬁed three activity bands by disc gel
electrophoresis. Tobkin and Mazelis (1979) in more detailed examination found
that onion alliinase had a molecular weight of 150 kDa determined by
sedimentation equilibrium centrifugation. Gel electrophoresis determination
showed 3 subunits of molecular weight 50 kDa. Later, Nock and Mazelis (1987)
reported that onion alliinase had molecular weight of 200 kDa and was composed
of 4 subunits of 50 kDa. Kazaryan and Goryachenkova (1979) fond that the
molecular weight of garlic alliinase was 130 kDa and consisted of two subunits

while Nock and Mazelis (1987) found that alliinase from garlic had molecular

13

weight of 85 kDa with two subunits of 42 kDa. Jansen et al. (1989) reported that
the molecular weight of alliinase from garlic was 111 and 53 kDa under
dissociating conditions.

There appears to be little doubt that the subunit molecular weight of alliinase
is close to 50 kDa, however, the molecular weight of the native enzyme molecules
is unresolved. The most likely explanation for this may be that rather than
existing as a native molecule with a set number of subunits, the subunits
aggregate into dimers and tetrarners, and what is seen as the “native" enzyme is a
time-averaged rapid equilibrium between those forms (Lancaster and Boland,

1990).

Catalytic properties of alliinase
The reaction catalyzed by alliinase is beta-elimination of the S-alk(en)yl-L—

cysteine sulfoxide group from the substrate as shown by the following reaction

(Abraham et al., 1976 :Whitaker, 1976).

alliinase
R-S(O)-CHz-CH(NH2)-COOH + Hzo _. R-SOH + CH2=C(Nl-Iz)-COOH (1)
CH2 =C(N1{2)-COOH -> CH3C(O)-COOH + NH3 (2)
2 R-SOH —- R-S-R+ R-S-S-R (3)

On treating with alliinase, alkyl cysteine sulfoxide decomposes into sulfenic
acid and a-arninoacrylic acid (equation 1). Both reaction products are chemically
unstable. The a-arninoacrylic acid spontaneously hydrolyzes to pyruvate and
ammonia (equation 2) while the unstable sulfenic acid (RSOH) will spontaneously
undergo non enzymatic rearrangement leading to the formation of mono, di, and

trisulﬁdcs of alkyl groups R (methyl, propyl and propenyl) to give a range of

 

i
- .
‘15)
s «all

l4

ﬂavor compounds (equation 3). Thus the reaction is commonly described as

follow:

alliinase
2R-SO-CHz-CH(NHz)-COOH + H20 -’ R-S-S-R + 2 CI-l3-CO-COOH + 2 Nl-l3

The general catalytic properties of alliinase were well described long before
the enzyme was puriﬁed to homogeneity. In an extensive study of the speciﬁcity
of garlic alliinase, Stoll and Seebeck (1951), Mazelis and Crew (1968) and Jansen
et al. (1989) found that its activity depended upon speciﬁc properties which
included: 1) the compound had to be derived from L-cysteine amino acid; 2) the
sulfur atom of cysteine had to be linked to an aliphatic group such as methyl,
ethyl, propyl, isopropyl, allyl or butyl; 3) the amino group of the cysteine portion
of the compound could not be substituted; 4) the sulfur atom of the cysteine
derivate had to be present in the sulfoxide forms and the sulfur atom had to be
either the (+) or the (-) conﬁguration. Subsequent work of several groups on
partially puriﬁed alliinase has established that alliinase from onion has a speciﬁcity

similar to garlic as mentioned above (Schwimmer et al., 1964; Brosin, 1969).

Inhibitors of alliinase

Inhibitor studies of alliinase fall into two classes: those involving pyridoxal
phosphate antagonist and those using substrate analogs as competitive inhibitors.
In the former group, inhibition by hydroxyl amine, aminooxyacetate and sodium
cyanate has been demonstrated on onion (T obkin and Mazelis, 1979) and garlic
(Jansen et al., 1989). In the second group of inhibitors, studies indicate the nature
of the binding site for the substrate analogs mostly to the derivatives of
unoxidized cysteine such as methyl, ethyl, propyl, butyl and ally] cysteine
(Schwimmer et al, 1964; Jansen et al., 1989).

15

C. Gamma Glutamyl Transpeptidase of Onion
Enzyme nomenclature
Cleavage of ﬂavor precursor y—glutamyl peptides is brought about by both

the transferase (y-glutamyl transpeptidase) and the hydrolase (y-glutamyl
peptidase). The reactions catalyzed by both enzymes are indicated in Figure 1.
The presence of y—glutamyl peptidase in onion and its role in ﬂavor enhancement
have not been resolved and also this enzyme has not gained ofﬁcial Enzyme
Commission (EC) recognition. In this study the discussion will be focused on the
characteristic and role of y-glutamyl transpeptidase. According to Enzyme
Commission the systematic name of y-glutamyl transpeptidase is y-

glutarnylpeptide: amino acid y-glutamyl transferase (EC. 2.3.2.2).

Occurrence in nature

Gamma glutamyl transpeptidase is ubiquitous in some growing plants
(Thompson et al., 1964) and in mammalian kidney (Orlowski and Meister, 1965)
but is absent or present only in trace amounts in dormant onion (Matikkala and
Virtanen, 1965a). Initial studies on the active preparation isolated from sprouting
onion reported the possibility of a y—glutamyl peptidase being present (Matikkala
and Virtanen, 1965a). Austin and Schwimmer (1971) prepared y—glutamyl
transpeptidase from sprouting onion bulbs and showed that the enzyme acts as a
transferase. Transpeptidase type enzyme capable of releasing amino acids from
the y-glutamyl peptide bond had also been reported in yeast (Penninckx and
Jasper, 1985), ackee plant (Kean and Hare, 1980), tobacco suspension cultures
(Steinkamp and Rennerberg, 1984), legumes, asparagus, and radish (Kasai et al.,
1982; Thompson et al., 1964).

l6

L-glutamyl-S-alk(en)yl-L-cysteine sulfoxide 4» H20 (1)

l
y- glutamyl
peptidase

 

V

L-glutamic acid + S-alk(en)yl-L-cysteine sulfoxide

L-glutamyl-S-alk(en)yl-L-cysteinc sulfoxide + R-CH-OR’ (2)
l (amino acid or
peptide)

y- glutamyl
transpeptidase

 

l
y—glutamyl-NHCHCOR + S-a1k(en)yl-L-cysteine sulfoxide

Figure l. Cleavage of y-glutamyl peptides by peptidase and transpeptidase
(Fenwick and Hanley, 1985).

 

l7

Puriﬁcation of y-glutamyl transpeptidase

Gamma glutamyl transpeptidase from plants and mammalians have been
studied by a number of investigators and several attempts have been made to
purify this enzyme. Matikkala and Virtanen (1965b) extracted crude
transpeptidase with M/15 phosphate buffer pH 7.4. Austin and Schwimmer (1971)
extracted transpeptidase from insoluble form in homogenate with high
concentration of NaCl. Puriﬁcation of 800 fold by ammonium sulfate
fractionation was reported by Schwimmer and Austin (1971a), however, an
attempt to apply chromatography on diethylaminoethyl-cellulose (DEAE-

cellulose) was unsuccessful due to unrecoverable enzyme activity.

Physicochemical properties of y-glutamyl transpeptidase

Transpeptidase isolated from kidney bean fruit (Phaseolus vulgaris) has a
pH optimum of about 9.5 (Thompson et al., 1964). The activation by sodium
citrate and the large number of amino acid compounds which would act as
glutamyl acceptors is in agreement with transferase activity. Orlowski and Meister
(1965) isolated y-glutamyl transpeptidase from hog kidney, followed by Szewezuk
and Baranowski (1965), isolating transpeptidase from beef kidney. These
transpeptidases exhibited optimal pH of 9.0 and the activity was affected by
magnesium ion. Partially puriﬁed transpeptidase from germinating chive seeds
(Allium schoenoprasum) had a pH optimum of 8.0 (Matikkala and Virtanen,
1965b).

Austin and Schwimmer (1971) isolated a relatively crude transpeptidase
from sprouting onion bulbs and reported a pH optimum of 9.0, kinetic constanth
(using synthetic y-glutamyl-p-nitroanilide) of 14.3 mM. Even though pH optima
of transpeptidase of onion is 9.0, at pH 5.0 - 6.0 in “crushed onion” y-glutamyl

18

transpeptidase was still active enough to account for the slow disappearance of

peptides in sprouting onion (Matikkala and Virtanen, 1967).

Catalytic properties of y-glutamyl transpeptidase

Since a signiﬁcant portion of ﬂavor precursors in onion tissue exist as y—
glutamyl peptides which are not susceptible to the action of alliinase, the presence
of y-glutamyl transpeptidase which catalyzed the transferase and releases of the
free amino acid derivate has been suggested to enhance onion ﬂavor formation.
The reaction catalyzed by y—glutamyl transpeptidase (a transferase) are shown in
Figure 1, equation 2. The transpeptidase enzyme catalyzes the transfer of the
glutamyl moiety of a y-glutamyl peptide to either amino acids or other peptides to
form new peptide bond and releases S-alk(en)yl-L-cysteine sulfoxides which then
become available for alliinase to produce ammonia, pyruvate and thiosulﬁnates.

Gamma glutamyl transpeptidase from sprouting onion bulbs shows a
transferase activity by displacing the amino acid of the peptides with another
amino acid to form a new peptides and releasing free amino acid derivates. In this
case another amino acid or derivate must be available for the reaction to proceed.

While the data suggested that the puriﬁed enzyme is y-glutamyl transpeptidase
(transferase); y-glutamyl peptidase (hydrolase) may also present in crude extract
from sprouting onions. However, the evidence for the presence of a hydrolase in
the crude enzyme is not conclusive (Austin and Schwimmer, 1971). The mode of
action of the enzyme in viva remain to be clariﬁed. It may then be established

whether hydrolase or transferase is present in sprouting onions or one enzyme

with a dual role.

l9

Inhibitor of transpeptidase

In contrast to the kidney, Mg“ did not appear to affect the activity of
onion transpeptidase. As for the Lima bean enzyme (Goore and Thompson, 1967),
borate also inhibited the onion transpeptidase. However, neither citrate nor
carbonate reported to inhibit. Free amino acid was thought to stimulate activity,
presumably due to their function as glutamyl acceptors (Schwimmer and Austin,
1971a). Glutamic acid and its derivates were expected to inhibit since they are
probably better substrate for transpeptidase.

D. Volatile Flavor Components of Onion

The genesis of ﬂavor compounds in Alliium could be divided into those
compounds formed directly on crushing (mainly sulfur containing) and those
which arise via secondary reactions (primarily carbonyl compounds). Figure 2
illustrates the formation of the major sulfur containing volatile in onion (Abraham
et al, 1976; Mazza et al., 1980; Lancaster and Boland, 1990) while the
characteristics of each compound are brieﬂy discussed here. The fresh ﬂavor of
onion and other members of the Allium genus is produced by enzymatic
decomposition of ﬂavor precursors (methyl, propyl and propenyl) derivates of L-
cysteine sulfoxide (1) from which the primary product are sulfenic acid (2) and
pyruvate (10). The unstable sulfenic acids with a half life of about 90 seconds
(Moisio et al., 1962) will undergo rearrangement to form thioalkanal S-oxide (3)
and thiosulﬁnates containing allyl and alkyl substituents (4). On standing (or
more rapidly with heating), the thiosulﬁnates decompose to yield a mixture of
monosulﬁde (5), disulﬁde (6), trisulﬁde containing methyl, propyl and l-propenyl
groups, smaller quantities of thiophene derivates (8), and other cyclic sulfur

containing compounds.

 

 

A;

R...

Q
\~

20

O

11"nase ,
Ra‘s-CH 2-CH(NH2)COOH E—IL—r R-SOH + CH.3 CO COOH + NH:3
(t) (to)
:\( R «CH3 CH- CH 1
c. 5.0
CH3 CH:

0 . (3)
l O\ CHaCHZCHO + s

R-s-s-R
(4) \ (11)
I

RSR + RSS)R + $02

I
t
I
.' o
+ t
RSSR+ 8- 88-80)
(6)
0 (R- CH3 CH- CH)

(Me UMe + 83638 RSR + 83339 etc.
' (5)

Figure 2. Formation of volatile sulfur compounds in onion (Abraham et al.,
1976; Mazza etal., 1980; Lancaster and Boland, 1990).
1. S-alk(en)-yl cysteine sulfoxide 5. monosulﬁde 9. trisulﬁde;

2. S-alk(en)-yl sulfenic acid 6. disulﬁde 10. pyruvate
3. thiopropanal S-oxide; 7. thiosulfonate 11. prOpanal
4. thiosulﬁnate;; 8. dimethyl thiophene

R: methyl, propyl, or propenyl.

21

Thiosulﬁnates and thiosulfonates are thought to give the characteristic
ﬂavor to freshly cut onion. The propyl and propenyl-containing di and trisulﬁde
are responsible for the ﬂavor of cooked onion and are also present in a steam
distilled onion oil (Boelens et al, 1971). Dimethyl thiophenes found in fresh onion
and also in steam distilled onion oil, have been described as having a fried onion
ﬂavor. The characteristic sweet taste of boiled onions was thought to be due to
increasing amounts of n-propanthiol (Boelens et al., 1971).

Over 80 volatiles have been reported in fresh, cut, and steam distilled
extract of Allium, particularly onion and garlic (Boelens et al, 1971; Abraham et al,
1976; Lancaster and Boland, 1990). The sulfur volatiles and the carbonyl type
volatiles formed directly and indirectly from enzymatic cleavage of ﬂavor
precursors are shown in Table 3. The amount of these compounds formed
depends on variety, maturity, cultural and environmental conditions, and sample

preparation method.

Enzymatically formed compounds
Sulfenic acid

Under the inﬂuence of alliinase, Allium ﬂavor precursors are cleaved to
yield alk(en)yl thiosulﬁnate, pyruvate and ammonia. It was suggested by Stoll
and Seebeck (1951) that alk(en)yl sulfenic acid (RSOH) and a-amino acrylic acid
are intermediate in this reaction. However, examination by mass spectrometry did
not reveal the intermediary of 2-propenylsulfenic acid when (+)-S(2- propenyl)-L-
cysteine sulfoxide was decomposed by alliinase (Dabritz and Virtanen, 1965).
Based on their ﬁnding, they proposed a different mechanism which involves a
bimolecular reaction between two molecules of (+)-S-Qlyj-L—cysteine sulfoxide to
form d_i_al_Lvl thiosulﬁnate directly. Therefore, the failure to detect alk(en)yl sulfenic

22

intermediary does not necessarily rule out their being ﬁrst product formed by
alliinase (Whitaker, 1976).

Thiosulﬁnates

Symmetrical (diallyl-alkyl-alkan)-thiosulfinates and unsymmetrical
thiosulﬁnate are considered to result from the condensation of sulfenic acids
possessing the same alk(en)yl radical or two different sulfenic acids respectively
(Cavallito et al., 1952; Fujiwara et al., 1958; Lukes, 1971; Yagami et al., 1980).
Thiosulﬁnates are pungent, smelling, rather thermally unstable and may dissociate
to form disulﬁdes and thiosulfonates (Boelens et al., 1971). As an example of the
thermal instability of thiosulﬁnate, Brodnitz et a1 (1971) reported that when
thiosulﬁnate was kept at 20°C for 24 hours it decomposed almost completely to
yield diallyl disulﬁde (66%), diallyl sulﬁde (14%) and diallyl trisulﬁde (9%) in
addition to S0,.

Signiﬁcant differences are found in the way trans(+)-S-(l-propenyl)—L-
cysteine sulfoxide is degraded by alliinase. While mixed (l-propenyl)-S-alk(en)yl
thiosulﬁnate apparently occurs, the occurrence of di-(l-propenyl)-thiosulﬁnate
and its product is open to question (Fenwick and Hanley, 1985). However,
Lawson and Hughes (1991) meanwhile characterizing the formation of allicin and
other thiosulﬁnates from garlic, using high performance liquid chromatography
(HPLC) technique identiﬁed all thiosulﬁnates including dipropenyl thiosulﬁnates,
methyl propenyl thiosulﬁnates, and dimethyl thiosulﬁnates. For the ﬁrst time
Sinha et al. (1992) reported the presence of diallyl thiosulﬁnate or its isomer in
onion ﬂavor. They also identiﬁed di-(l-propenyl)-thiosulﬁnate and propyl
methane thiosulfonate from supercritical carbon dioxide extract of onion

measured by gas chromatography-mass spectrometry (GS-MS).

Table 3. Compounds identiﬁed in steam distilled onion oil*

Oxygen compounds

Propanal

Dimethyl furan
2-Methyl pentanal
2—Methyl-pent-2-enal
Tridecan-Z-one
S-Methyl-Z-n—hexyl-ZS-
dihydrofuran-3one

Dkulﬁdes

Dimethyl disulﬁde

Methyl propyl di sulﬁde

Allyl methyl disulﬁde

Methyl cis-propenyl disulﬁde
Methyl trans-propenyl disulﬁde
Isopropyl propyl di sulﬁde
Dipropyl disulﬁde

Allyl propyl disulﬁde
gig-Propenyl propyl disulﬁde
trans-Propenyl propyl disulﬁde
Diallyl disulﬁde

Allyl propenyl disulﬁde (2)

Thiophene derivates

2,5-Dimethylthiophene
2,4-Di methylthiophene
3,4-Dimethylthiophene
3,4—Dimethy12,5-dihydro-
thiophene-Z-one

Tetrasulﬁdes

Diallyl tetrasulﬁde
Dimethyl tetrasulﬁde

Thiosulfonates

Methyl methanethiosulfonate
Propyl methanethiosulfonate
Propyl propanethiosulfonate

Monosulfides

Dimethyl sulﬁdes

Allyl methyl sulﬁde

Methyl propenyl sulﬁde (2 )
Allyl propyl sulﬁde
Propenyl prOpyl sulﬁde (2)
Dipropenyl sulﬁde (3)

Trisulfidas

Dimethyl u'isulﬁde

Methyl propyl trisulﬁde

Allyl methyl trisulﬁde

Methyl cis-propenyl trisulﬁde
Methyl trans-propenyl trisulﬁde
Diisopropyl trisulﬁde

Isopropyl propyl trisulﬁde
Dipropyl trisulﬁde

Allyl propyl trisulﬁde

Diallyl trisulﬁde

cis-propenyl propyl trisulﬁde
trans-Propenyl propyl trisulﬁde

Thiols

Ethanethiol
Methanethiol
Propanethiol
Allylthiol

Carbonyl

Propanal
2-Methylpentanal

Thiosulﬁnates

Diallyl thiosulﬁnate
Di-l-propenyl thiosulﬁnate

a“(Boelens et al., 1971; Abraham et al., 1976; Lancaster and Boland, 1990; Sinha etal.,

1992: Block et al., 1992b).

CL
1‘
p?

 

 

SI".

0!

a 8
m.

.3

 

Block et al. (1992a) also identiﬁed eight different thiosulﬁnates include diallyl
thiosulﬁnate in the room temperature vacuum distilled onion extract analyzed by
HPLC. However, they reported that even under the gentlest gas chromatography
condition diallyl and other allylic thiosulﬁnate failed to survive (Block et al,
1992b).

Lachrirnatory factors

The lachrimatory or tear-producing characteristic is the hallmark of onion.
Virtanen and Spare (1961) showed that the lachrimator was formed enzymatically
during the hydrolysis of S-l-propenyl-L-cysteinc and tentatively identiﬁed as 1-
propenyl sulfenic acid. Wilkins (1961) proposed thiopropanal S-oxide as a
possible structure for the lachrimator. Brodnitz and Pascale (1971) using
synthesized thiopropanal S-oxide established the structure as thiopropanal S-
oxide CH3CH2-CH= :0 rather than sulfenic structure R-SOH. Block et al. (1979)
reﬁned the structure of the lachrimatory factor as a mixture of approximately 95%
of the Z and 5% of the E-isomer of thiopropanal S-oxide.

Nonenzymatically formed volatiles
Thiosulfonates

Several authors have suggested the formation of thiosulfonates as a
secondary reaction in the enzymatic cleavage of S-alkenyl-L-cysteine derivates.
In vitro hydrolysis of S-methyl-L-eysteine sulfoxide showed that almost all of the
material was converted to pyruvate, ammonia, dimethyl disulﬁde and methyl
methane thiosulfonate (Osterrnayer and Tarbell, 1960). The same reaction using
propyl derivative found dipropyl disulﬁde and propyl propane thiosulfonate.

However, the propenyl derivative gave neither disulfide nor thiosulfonate. These

compounds are apparently not present in onion oil, but have been identiﬁed in
freshly cut onion (Boelens et al., 1971).

Propyl methane thiosulfonate was identiﬁed in super critical CO2 extract by
Sinha et al (1992). It has been suggested that the absence of these compounds in
steam distilled onion was because they are soluble in water and could not move to
vapor phase during steam distillation (Boelens et al., 1971). The odor of fresh
onion is ascribed to thiosulﬁnates and thiosulfonates. Even though thiosulfonates
are considered to be less signiﬁcant as intermediate compounds than the
corresponding thiosulﬁnates, thiosulfonate with four or more carbon atoms

displays a powerful and distinctive odor of freshly cut onions.

Sulﬁdes

The ﬁrst major investigation of onion ﬂavor using modern technique of
analysis may be considered to be that of Carson and Wong (1961). These workers
used gas chromatography and other techniques for the identiﬁcation of a series of
disulﬁde and trisulﬁde containing methyl and propyl groups, in an isopentane-
soluble fraction of chopped onions. Propyl disulﬁde was the major component
followed by propyl trisulﬁde but no compound with an allyl group was detected.
Brodnitz et al. (1969) dentiﬁed several cis and trans-propenyl alkyl disulﬁdes in
onion oil, then Brodnitz and Pollock (1970) analyzed eight important sulﬁde
components namely, methyl propyl disulﬁde, cis-l-propenyl methyl disulﬁde,
trans-l-propenyl methyl disulﬁde, propyl di sulﬁde, cis-l-propenyl propyl di sulﬁde,
trans-propenyl propyl disuﬁde, methyl propyl trisulﬁde, and propyl trisulﬁde. The
largest fraction was propyl disulﬁde followed by propyl trisulﬁde. In spite of the
fact that there is much less propenyl than propyl in these compounds, the
propenyl derivates make a very strong contribution to onion ﬂavor. Sulﬁde

compounds are reported to posses the ﬂavor of cooked onion.

26

Thiophenes

Several sulfur-containing heterocyclic compounds were isolated from onion
volatiles, the interesting thiophene derivates 2,4; 2,5 and 3,4 dimethyl -2,5
dihydrothiophene were identiﬁed by Brodnitz et al. (1969) and Albrand (1980).
Boelens et al. (1971) reported that by the action of heat and or ultra violet
irradiation, methyl propenyl disulﬁde and propyl propenyl disulfide were
converted into dimethyl thiophene and saturated disulﬁde. Of the thiophenes
formed, the major product is 3,4—dimethyl thiophene. Thiophenes have been

evaluated as having a fried onion odor.

Carbonyls

Several carbonyl compounds were identiﬁed in steam distilled onion oil and
include propanal, 2-methyl pentanal, and 2-methyl-2-pentenal. This appears to be
a result of the formation and spontaneous decomposition of thiopropanal S-oxide
as shown in the equations below (Spare and Virtanen, 1963). Vinyl alcohol
decomposed from thiopropanal S-oxide (equation 1) are known to form aldehyde
(equation 2). The small amount of 2 methyl-2-pentenal may result from aldol

condensation of propanal (equation 3).

o o

l I
CHa-CH=CH-SH -» CH3-CH=CHOH + H—S-H (1)
CH3-CH=CH0H - CHa-CHz-CHO (2)
2 Cig-CHz-CHO —. ens-enfcnzc-Cno (3)

CH.

Propanal is stated to be one of the more important ﬂavor compound in raw onion.
Boelens et al. (1971) have proposed a scheme for formation of most aldehydes

27

following alliinase action on trans-(+)S-(1-propenyl)-L-cysteine sulfoxide which
involves the conversion of pyruvate to aldehydes and appears to be formed much

more rapidly in cut onion than are the sulﬁde compounds.

Methods of Measurement of Flavor

The assessment and comparison of ﬂavor in onions requires a suitable
method for measuring ﬂavor. Numerous methods have been published and these
range from a very simple to the use of complex physicochemical methods.
According to Lancaster and Boland (1990) the diversity of methods can be
attributed to the three facets of ﬂavor biochemistry. First the net ﬂavor depends
upon the presence and action of both enzyme and substrate, second the net ﬂavor
is produced by numerous chemically diverse volatiles and third the sulfur
compound within the plant are chemically complex, with some of them being very
unstable in vitro. The following methods are widely used for estimating ﬂavor in
onions however, there are advantages and disadvantages to each of them.
Selecting a suitable method of ﬂavor measurement depends on the use of analysis

and kind of plant materials available.

E. Measurement of reaction products
The cleavage of the ﬂavor precursors by alliinase produces thiopropanal S-
oxide, sulfenic acid, sulfur volatiles, pyruvate and ammonia. There is no better
judge of ﬂavor quality than the human being. However, sensory analysis may
suffer from fatigue, indifference, time constraints and lor variability on the part of
the evaluator. Numerous other external variables may also confuse the judgment
of the sensory evaluation. Quite often it is desirable to supplement sensory

analysis wit the use of an instrumental technique.

Three different methods to measure thiopropanal S-oxide and thiosulﬁnates
(thin layer chromatography (TLC), spectrophotometer and HPLC) have been
developed by Lukes (1971); Freeman and Whenham ( 1975), Lawson et al. (1990a;
1990b) and Lawson and Hughes (1991) respectively. However, the instability of
these compounds causes difﬁculties with the reproducibility of measurement.

Measurement of pyruvate has been used in various experiments for ﬂavor
assay. This acid is not itself a ﬂavor component but provides a measure of ﬂavor
because when the primary ﬂavor precursors are converted by alliinase into ﬂavor
components, pyruvate is released in chemically equivalent amount. Schwimmer
and Weston (1961) found that over 95% of the maximum amount of enzymatically
developed pyruvate is produced within 6 minutes after the start of communition.
A highly signiﬁcant correlation (=0.97) was shown between the amount of
enzymatically developed pyruvate presented in the juice of comminuted onion
and the olfactory threshold concentration of the juice ( Schwimmer and Guadagni,
1968). A similar relationship between pyruvate analysis and ﬂavor perception
was reported by Wall and Corgan (1992). The test which is based on the color
formation of carbonyl compound with 2,4 dinitrophenyl hydrazine has been used
for screening large numbers of samples as in breeding or selection program
(Randle and Bussard, 1993). This test provides a measurement of total ﬂavor, but
does not provide any information about relative amounts of individual ﬂavor
precursors or ﬁnal ﬂavor volatiles. This method cannot be used with dehydrated
onion, because carbonyls other than pyruvate was formed during storage (Saguy
et al., 1970).

The volatile sulfur and carbonyl compounds formed in crushing onion may
be measured by gas chromatography. In this method, the volatile mixture is
separated on a liquid phase, and the components are identiﬁed by retention time

or by mass spectrometry. The peak area is used to determine the amount of a

29

volatile. Several reports on this method have been published (Mazza et al., 1980;
Boelens et al., 1971; Brodnitz et al., 1969; Block et al., 1992b; Calvey et al., 1994).
Recently separation of heat sensitive volatile thiosulﬁnates compounds by HPLC
was reported (Lawson et al., 1990a and 1990b; Lawson and Hughes, 1991; Block
et al., 1992a; Calvey et al., 1994). Since the volatile compound was affected by
method of sample preparation, incubation time, temperature and type of onion
product, in order to obtain reproducibility, it is important to standardize the
procedure used.

Measurement of ﬂavor precursors

Measurements of the amounts of ﬂavor precursors have been used as
indicators of ﬂavor strength in onion. In order to measure these compounds,
enzymes acted upon this ﬂavor precursor must ﬁrst be inactivated. Freeman and
Whenham (1975), Lancaster and Kelly (1983), and Shaw et al. (1989) developed
quantitative analysis of the major y-ylutarnyl peptides in onion bulbs and each of

the S-alk(en)yl-L-cysteine sulfoxides.

Measurement of alliinase and transpeptidase activities

This measurement has usually been made by extracting the enzymes into a
suitable buffer solution than removing the endogenous substrate and pyruvate by
dialysis or gel chromatography. The activity of alliinase is determined by
measuring the initial rate of pyruvate formation. The latter is estimated by the
intensity of the color of its 2,4-dinitrophenylhydrazone in alkali or alternatively
may be estimated by coupling the lyase to lactic dehydrogenase and measuring
the rate of reduction of NADH (Schwimmer and Mazelis, 1963).

The activity of transpeptidase is determined by the measurement of aniline
released by the action of y-glutamyl transpeptidase on the synthetic substrate 7-

30

glutamyl-p-nitroanilide in the presence of a suitable acceptor amino acid. The
enzyme assay is based on the measurement of colored product obtained by the

coupling of diazotized aniline with naphthylenediamine (Thompson, 1970).

F. Flavor Enhancement by y-Glutamyl Transpeptidase

Lukes (1971) emphasized that the enzymatic developed compounds are not
the only one important in onion ﬂavor. In fresh onion, the enzyme reaction does
not go to completion. The author demonstrated by adding puriﬁed alliinase to
macerated onion after it had stood several hours and noting the production of
more lachrimator. The reason that some onions lack of pungency may due to the
lack of alliinase; however, they may posses onion ﬂavor (Schwimmer and
Guadagni, 1968).

Schwimmer (1971) reported that Albizzia alliinase and kidney
transpeptidase acted sequentially in a coupled enzymatic reaction to convert v-
glutamyl cysteine peptides to pyruvate. Using synthetic y-glutamyl S-methyl L-
cysteine (GMC) and cation eluate of dried onion suspension as the substrate, the
author found that the conditions for optimal lyase activity did not depress the
activity of transpeptidase preparation acting on synthetic substrate glutamyl-p-
nitroanilide. He also found that under optimal conditions of alliinase, no pyruvate
was produced by transpeptidase in the absence of alliinase. However, in the
presence of alliinase, transpeptidase enhanced pyruvate production beyond that
formed in the presence of alliinase alone.

Subsequent investigation by Schwimmer and Austin (1971) on dehydrated
onion conﬁrmed that transpeptidase enhanced pyruvate release. They concluded
that added transpeptidase may be useful in enhancing the value and quality of
dehydrated onion, garlic and other Allium species, improving the ﬂavor of onion-

containing foods as well as providing a use (as the enzyme extraction medium) for

 

 

31

culled, sprouted onions which would ordinarily be a waste product. They also
suggested that other source of transpeptidase such as mammalian kidney and
microbial may also be suitable for enhancing onion ﬂavor. Although Schwimmer
(1973) was granted a patent for ﬂavor enhancement of Allium products by
applying exogenous transpeptidase, an investigation of ﬂavor proﬁle of enzyme

added onion extract was sparsely reported.

G. Flavor Extraction and Identiﬁcation

Flavor studies of onion have been done on different media search, such as,
head space vapor ( Mazza et al., 1980; Kallio and Salorinne, 1990; Oshumi, et al.,
1993), steam distillation (Brodnitz et al, 1969; Boelens et al., 1971; Martin-Lagos et
al., 1991; Kuo and Ho, 1992), vacuum distillation (Block et al., 1992a), organic
solvent extraction (Boelens et al, 1971; Lukes, 1971; Martin-Lagos et al., 1991;
Block et al., 1992a ; 1992b). Each technique has been used for ﬂavor
identiﬁcation combined with GC, GC-MS, HPLC, TLC as well as Nuclear
Magnetic Resonance Spectroscopy (NMR) and Infrared Spectrophotometry (IR).

Supercritical ﬂuid extraction

Besides the techniques above, extraction using supercritical ﬂuid CO2 as

the solvent (SC-C02) was used in onion ﬂavor study (Sinha et al., 1992). This

supercritical ﬂuid extraction (SFE) technique is a relatively new unit operation
that exploits the dissolving power of ﬂuids at temperature and pressure above
their critical values to extract soluble components from a mixture. Currently, this
technique offers the potential advantages of higher yield and better quality
products (Rizvi et al., 1986a).

A supercritical ﬂuid is any ﬂuid at a temperature above its critical value.

Figure 3 shows a generalized pressure-temperature phase diagram for COzin

32

which the region indicates solid, liquid and gas respectively. Also are shown on
. the diagram lines indicating co-existence of two phases. The liquid-gas line
extends from the triple points to the critical point, where the properties of liquid
and vapor become identical. The region represented by the supercritical ﬂuid
state is shown as the shaded area A supercritical ﬂuid exhibits physicochemical
properties intermediate between those of liquid and gas, which enhance its role as
a solvent. Its relatively high density gives good solvent power while its relatively
low viscosity and diffusivity values provide appreciable penetrating power into
the solute matrix. These properties give rise to higher rates of mass transfer of
solutes into a supercritical ﬂuid than into a normal liquid. Separation using
supercritical ﬂuid often be carried out at relatively moderate temperature (40-
60°C) making the process advantageous for separation heat-labile materials in
food processing. In addition, the solvent can easily be separated from the
products. Besides carbon dioxide, some of the supercritical solvents used in
supercritical extraction are methane, ethylene, propylene and diethyl ether. These
solvents cover a wide range of critical temperature, molecular size, and polarity.
Among them carbon dioxide which has received a lot of attention, is the ideal
supercritical solvent for food application. It is nontoxic, nonﬂammable,
noncorrosive, inexpensive and readily available, and has low critical temperature
(31.6°C) and pressure (73.9 Bar) (Rizvi et al., 1986a; 1986b).

A supercritical ﬂuid extraction system consists of four basic components: a
solvent compressor or pump, an extractor, a temperature/pressure-control system
and a separator or adsorber. Additionally, other equipment, including auxiliary
pumps, valves, back pressure regulators, ﬂow meters, and heater/coolers for proper
operation of the process are also required (Figure 4)

There are three common processing methods used for SC-CO2 extraction;

the ﬁrst method is temperature manipulated to remove the desired extract from the

PRESSURE

Pc

33

SUBCRITICAL REGION

 

 

 

 

 

 

 

TEMPERATURE

Figure 3. Typical pressure-temperature phase diagram.

 

 

 

 

 

1 '—&M —l 00 Vent
Ll Pressure 1 ‘ Flow
control meter

valve 1

Pump . L J

CO .
“W95“ Extractor trap

 

 

 

 

 

 

 

 

 

 

 

 

Préheater

 

Figure 4. Schematic of the supercritical ﬂuid extraction unit.

 

35

solvent. The second method uses pressure regulator to perform the separation,
under this scheme the solvent contained solute is passed through a valve where
the pressure is decreased, and the solute separates out. The solvent may be
recompressed and recyled, or simply vented from the system. The third method
involves isotherm and isobaric conditions (Rizvi et al, 1986a; Cohen, 1984;
Swientek, 1987). The second method was used by Sinha et al. (1992) to extract
onion ﬂavor. The authors reported that extraction with SC-CO2 produced fresh
onion-like ﬂavor components from onion and identiﬁed thermally unstable
compounds that were not reported before. However, very limited data are

available for the solubility of organic compounds in SC-COZ.

Flavor identiﬁcation

Identiﬁcation of ﬂavor compounds in onion extracts was carried by
comparing mass spectral data obtained from Mass Spectrometry with the data of
published paper or mass spectral data base.

MATERIALS AND METHODS

Materials
Onion

Onions from ten cultivars/selections (Norstar, Northstar, Sweet Sandwich,
Spartan Banner-80, Spartan Banner, Magna Sweet, K. Downing, Granite, MSU
3506, MSU 8450) grown and harvested at the Michigan State University Muck
Farm, and ﬁve market cultivars (Michigan Sweet, Texas Sweet, Yellow Sweet,
Imperial Sweet, Vidalia) were used in this study. Two sets of onions harvested at
different times were used; the ﬁrst set was obtained from 1992 harvest and the
second set from the 1993 harvest. All samples were stored at 410°C, prior to use.

Chemicals and reagents

Substrates for enzme assays: Synthetic substrates for alliinase assay including, S-
methyl, S-ethyl, S-propyl and S-l-propenyl L-cysteine sulfoxide were synthesized
from commercially available S-methyl, S-ethyl and S-propyl cysteine (Sigma
Chemical Co., St Louis, M0.) by oxidation with hydrogen peroxide (Stoll and
Seeback, 1951) and crystallized according to Bello (1972) (Appendix 1).
Synthetic substrate y-glutamyl-p-nitroanilide for transpeptidase assay was
obtained from Aldrich Co.

Column chromatography: Sephadex G-150 (40-120 p), and G-200 (40-120 )4),
carboxy methyl-cellulose (CMC), polybuffer exchanger 94 (PBE 94) and standard
mixture protein marker (MW range 29-660 kDa) for gel ﬁltration were obtained
from Sigma Chemical Co.

37

SDS-PAQE: Electrophoretic grade acrylamide, NN'-methylene(bis)acrylamide
N,N,N,N’-tetramethylethylenediamine (T EMED), sodium dodecyl sulfate (SDS),
ammonium persulfate, 2-mercaptoethanol (2-ME), standard mixture protein marker
(MW range 29-200 kDa) and Commasie brilliant blue R-250 were purchased from
Sigma Chemical Co.

Buffer and sm’ﬁc chemicals: All buffer solutions were prepared according to
Stoll and Blanchard (1990). All solvents were ACS reagent grade and all other
chemicals used were of analytical reagent grade.

METHODS
Relatioship Between Alliinase Activity and Pyruvate Content
Onion preparation

Onion bulbs harvested in early October 1993 were divided into three sets.
The ﬁrst set which was used for alliinase assay and pyruvate determination of
onion cultivars, was packed in paper bags (about 25 bulbs each) and stored at 4—
10°C. The second set for transpeptidase study was planted in damp sand to
induce sprouting. Rooting occurred after 10-12 days and green shoots were
visible after one month. The third set used for storage studies (Spartan Banner
and Yellow Sweet onion) was packed in a wooden box and stored at ambient
temperature During storage period of 32 weeks, the mean temperature was 20°C.
At two week intervals, randomly selected bulbs with no signs of spoilage and
without external sprouts were drawn for analysis.

Determination of pyruvate in onion bulbs

Pyruvate concentration in onion samples was determined in triplicate based
on the method described by Thomas et al.(l992) and Wall and Corgan (1992).
Dry outer scales were removed, and bulbs were sliced. A 20 g portion of onion

38

tissue was blended with an equal volume of distilled H20 for 3 min, allowed to
stand for 15 min, ﬁltered, and diluted in distilled H20 (1:20). Background

measurement of pyruvate level (control) was determined by quickly slicing 20 g
onion tissue into 60 ml of 5% trichloro acetic acid (T CA) to inactivate the alliinase.

After 1 hour, control was blended for 3 min and ﬁltered. Filtrate was diluted (1:10)
with distilled H20. Sample and control were analyzed for pyruvate. Each reaction

tube of pyruvate measurement contained 1 ml of diluted ﬁltrate, 1 ml distilled H20,

and 1 ml of 0.0125% 2,4—dinitro phenyl hydrazine (DPNH) in 2 N HCl. A blank
was prepared with 2 ml H20 and 1 ml DPNH. All reaction tubes were vortexed

and placed in a waterbath at 37°C for 10 min. After incubation, 5 ml of 0.6 N
NaOH was added and test tubes were vortexed and let stand for 5 min. Pyruvate
was measured at 420 nm using a spectrophotometer (Ultraspec II, Pharrnacia LKB
Biotechnology Inc. Maryland, USA) at 420 nm. Standards were prepared from
sodium pyruvate. Final pyruvate concentration was calculated from the
difference between pyruvate level in the sample and control. Results were

expressed as mmole pyruvate per g fresh weight.

Sprouting index (SI) determination

i Sprouted bulbs were deﬁned as having visible green shoots emerging from
the neck. Sprout growth measured as sprouting index of onion was determined
according to the method of Ceci and Curzio (1992). Three bulbs were used in
each triplicate. Sprout were separated by a longitudinal cut. Bulb and sprout
were separately weighed. Sprouting index corresponds to sprout weight in g per
100 g of onion bulbs.

39

Extraction of Enzymes
Extraction of alliinase

The procedure for alliinase preparation was based on the methods
described by Jansen et al.(l989) and Fujita et al. (1990). All Operations were
carried out at 24°C.

Extraction :

Peeled and chopped onions (600 g) were blended with 1:1 (w/v) 0.05 M
potassium phosphate buffer, pH 7.5 containing 0.1 M KCl, 0.5% ascorbic acid,
0.5% (w/v) polyvinylpirrolidon (PVP), 10%(w/v) glycerol in chilled Waring
blender for 3 min at high speed. The homogenate was ﬁltered through two layers
of cheese cloth, followed by ﬁltration through nylon cloth and the ﬁltrate was
centrifuged at 8,000 x g for 30 min at 4°C. The supernatant was brought to 35-
65% saturation with ammonium sulfate. The precipitate was centrifuged at 16,000
x g for 30 min at 4°C. The pellet obtained was re-suspended in 30 ml 0.05 M
phosphate buffer pH 7.0 and was dialyzed against the same buffer for 16 hr at
4°C. The dialyzate was centrifuged at 14,000 x g to remove insoluble material.
Puriﬁcation:

Puriﬁcation of alliinase was canied in several steps. In the ﬁrst step, sample
was passed through CMC column (2.5 x 35 cm) which was prepared as follows.
Five gram CMC resin was suspended in 5 volume of 0.05M NaOH containing 0.5
M NaCl for 20 min. The slurry was neutralized with 5 volumes of distilled water in
a Buchner funnel. The resin was removed from the funnel, resuspended with 5
volume of 0.75 M HCl, poured back into the funnel and neutralized with distilled
water until the efﬂuent showed a pH of approximately 5.0. Activated resin was
then packed into the column and equilibrated by passing through 10 volumes of
elution buffer until the efﬂuent pH reached the pH of the equilibrated buffer
(Cooper, 1985). Alliinase protein was eluted with 100 ml 0.05 M phosphate

buffer pH 7.0 followed by 600 ml gradient of 00.4 M potassium chloride in 0.05
M phosphate buffer pH 7.0 (ﬂow rate 60 ml/hr). Fractions containing enzyme
were combined and dialyzed against 0.025 M Tris-HCl pH 7.5 and concentrated
by ultraﬁltration (molecular weight cut off 10 kDa, Amicon Co.).

The second column puriﬁcation step was gel exclusion chromatography on
a Sephadex G-200 column prepared as follows. Ten g of dried gel was slowly
sprinkled onto the surface of 500 ml 0.05 M phosphate buffer while gently
stirring the buffer with a glass rod intermittently for one hour, then allowing the
suspension to stand for 72 hr. If ﬁne particles were generated they should be
removed. The slurry then was transferred to a 300 ml vacuum ﬁlter ﬂask and
evacuated for 10 to 15 minutes. A silanized column (1.5 x 50 cm) was attached to
a sturdy support and was veriﬁed that it was perfectly vertical. The slurry was
packed into the column using attached extended column reservoir followed by
equilibration with elution buffer. The performance of column packing was
checked by using blue dextran dissolved in brom phenol blue solution. Protein
fractionation was started by applying about 1 ml of concentrated CMC eluates to
the surface of the gel. The protein was eluted by a linear gradient NaCl from 0.1
M to 0.35 M in 0.025 M Tris-HCI pH 7.5. The active peak fractions were
combined and dialyzed against 0.025 M histidine-HCI pH 6.5 for 16 hr.

The last step of puriﬁcation was chromatofocusing on polybuffer
exchanger. In chromatofocusing a pH gradient is formed by equilibrating the
column with starting buffer and eluting with polybuffer at a lower pH. The
polybuffer is operated over a maximum interval of 3 pH units. One pack
polybuffer exchanger of two hundred ml suspension in 24% ethanol was
dispersed in 20 ml of 0.025 M histidine-HCI pH 5.5 to make a slurry and was
degassed prior to packing. The slurry was packed into a column (1x16 cm) which

was mounted vertically, and any air bubbles present were ﬂushed from the bed

41

support. The column was equilibrated with 150 ml of initial buffer of 0.025 M
histidine-HCI pH 5.5. The performance of column packing was checked using
bovine cytochrome c as a marker. Strongly basic cytochrome c of 2-3 mg/ml
starting buffer were repelled from the gel easily. The protein (8 mg) was eluted
with 90 ml polybuffer 74 HCl, pH 4.0 diluted 1: 8 with distilled water. The active
enzyme fractions were combined and dialyzed against 0.03 M phosphate buffer,
pH 7.0 containing 10 mM EDTA and 20 mM pyridoxal phosphate. Finally, the
enzyme was precipitated from polybuffer with ammonium sulfate (0-80%
saturated) followed by dialysis for 16 hr against 0.03 M phosphate buffer to
obtain 1.2 mg of puriﬁed alliinase preparation.

Extraction of y—Glutamyl transpeptidase
Extraction

All operations were carried out at 2-4° C. Extraction and puriﬁcation of y-
glutamyl transpeptidase from sprouting onion was carried out using a modiﬁcation
of the method of Schwimmer and Austin (1971) and Kean and Hare (1979).
Sprouted onion bulbs (800 g) were blended with equal w/v of 0.05 mM Tris-HCl,
pH 7.5 containing 5% NaCl (w/v), 0.05% (v/v) 2-ME, 10 mM EDTA and 10%
(w/v) polyethylene glycol. in a chilled Waring blender for 3 min. The
homogenate was ﬁltered through two layers of cheese cloth and the ﬁltrate was
centrifuged at 6,000 x g for 30 min at 4°C. The supernatant was brought to 35-
85% saturation with ammonium sulfate. The precipitate was centrifuged at 16,000
x g for 30 min at 4°C. The pellet obtained was re—suspended in 0.05 M Tris-HCI
pH 7.5 and dialyzed against the same buffer for 16 hr at 4°C.

42

' rcatio

The enzyme was puriﬁed in three steps. In the ﬁrst puriﬁcation step, sample
was passed through CMC prepared as previously described. To use the resin, the
slurry was packed into the column (1.5 x 40) and equilibrated with 0.05 M Tris-
HCI pH 7.5. Inactive proteins were washed with 50 ml of the same buffer, after
which gradient elution was started using 0.05-0.50 M Tris-HCl buffer pH 7.5. The
active fractions were collected , dialyzed against 0.05 M Tris-HCl pH 7.5 for 16 hr
and concentrated by ultraﬁltration (Amicon, MW cut off 10,000).

The next puriﬁcation step was gel permeation in Sephadex G-150 column
(1.5 x 50 cm) prepared as described above and was equilibrated with 0.05 M Tris-
HCl buffer pH 7.5. The protein was eluted with gradient concentration of 0.25-
0.50 M NaCl in 0.05 M Tris-HCI pH 7.5. The active fractions were collected and
dialyzed against 0.05 M Tris-HCI pH 7.5 for 16 hr and concentrated by
ultraﬁltration.

The last step of puriﬁcation was chromatofocusing on polybuff er
exchanger prepared as described above. The slurry was packed into a column
(1x20cm) which was mounted vertically. The column was equilibrated with 200
ml of initial buffer of 0.025 M histidine-HCI pH 5.5. The protein (18 mg) was
eluted with 100 ml polybuffer 74 HCl, pH 4.0 diluted 1: 8 with distilled water. The
active enzyme fractions were combined and dialyzed against 0.05 M Tris HCl, pH
7.5. Finally, the enzyme was precipitated from polybuffer with ammonium sulfate
(0-80% saturated) followed by dialysis for 16 hr against 0.05 M Tris HCl, pH 7.5
to obtain 1.4 mg of puriﬁed transpeptidase preparation.

Assay of alliinase
Alliinase activity was assayed in triplicate according to the modiﬁcation of

the methods of Schwimmer and Mazelis (1963) and Fujita et al.(l990). The

43

standard assay mixture of 1.0 ml ﬁnal volume, consisted of 0.1 ml of 0.5 mM
pyridoxal phosphate, 0.4 ml of 200 mM ethyl cysteine sulfoxide, 0.4 ml of 30 mM
phosphate buffer pH 7.0. An aliquot of 0.1 ml puriﬁed alliinase was added to
initiate the reaction. The reaction mixture was incubated at 37°C for 10 min and
was terminated by addition of 1 ml of 2 N HCl containing 0.0125 % (w/v) 2,4
DPNH and was incubated at 37°C for 10 min, 5 ml of 0.6 N NaOH was added to
the solution and the absorbance at 420 nm was measured against a blank
containing above reaction mixture without the substrate. Pyruvate production
under assay condition (10 min incubation and 80 mM substrate concentration)
was linear. Enzyme activity was calculated using sodium pyruvate as standard
(Appendix 2). One unit of alliinase activity is deﬁned as the amount of enzyme
that will produce 1 mmole pyruvate per min under standard enzyme assay

conditions.

Assay of y-glutamyl transpeptidase

Gamma glutamyl transpeptidase was assayed in triplicate according to the
method of Thompson (1970) and Lancaster and Shaw (1991). The method is
based on the measurement of colored product obtained by the coupling of
diazotized aniline with naphthyl ethylenediamine. The aniline is released by the
action of y-glutamyl transpeptidase on the synthetic substrate L—y-glutamyl p-
nitroanilide.

Enzyme solution (0.4 ml) was added to 0.5 ml L-y-glutamyl p-nitroanilide
solution (4.0 mM in 0.1 M Tris-acetate buffer pH 9 containing 3 M sodium citrate)
and 0.1 ml of 0.1 M methionine. Reaction mixture was incubated at 37°C for 3 hr.
The assay was terminated by adding 2 ml of 40% (w/v) TCA. The liberated p-
nitroaniline was diazotized to form a pink dye by sequential addition of 1 ml 0.1%
(w/v) NaNO2 , 1 ml 1% (w/v) ammonium sulfamate, 1 ml naphthyl ethylene di-amine

di-HCl solution (3 mg/ml in 95% ethanol) at 3 min interval. Absorbance was
measured at 560 nm spectrophotometrically. Enzyme activity was determined
using aniline standard curve (Appendix 3). One unit of transpeptidase activity is
deﬁned as the amount of enzyme that will liberate one mmole nitroaniline from L-

y-glutamyl p-nitroanilide per min under standard enzyme assay condition.

Protein determination

Protein determination was assayed qualitatively from its absorbance at 280
nm and quantitatively by the method of Lowry et al. (1951).

The ﬁrst step of the Lowry method involves the formation of a copper-
protein complex in alkaline solution. This complex then reduces a
phosphomolibdic-phosphotungstate reagent to yield an intense blue color.
Standard bovine serum albumin (BSA) solution (3 mg/ml) for standard curve was
pipetted to the tube in a volume of 0-1 ml. The volume was brought to 1 ml by
adding an appropriate amount of distilled water. In the meantime, 15 m1 reagent A
(100 g NaZCO3 in 1 liter 0.5 N NaOH), 0.75 ml reagent B (l g CuSO45H20 in 100
ml distilled water), and 0.75 ml reagent C (2 g potassium tartrate in 100 ml distilled
water), were throughly mixed. One ml of the above solution made as above was
added to each tube, vortexed and incubated for 15 min at room temperature. At
the conclusion of the incubation period, 3 ml of Folin reagent (5 ml 2 N Folin-
phenol in 50 distilled water) was forcibly pipetted to each tube and the resulting
solution was vortexed immediately. After 45 min. at room temperature, the
absorbance of the colored samples was recorded at 540 nm using a
spectrophotometer. Protein concentration of the sample was determined using the
standard curve of BSA (Appendix 4).

45

Characterization of Enzymes
Effect of pH

The effect of pH on alliinase and transpeptidase activities was determined
between pH 3.5 and 10 using 0.05 M acetate buffer, 0.05 M phosphate buffer,
0.05 M Tris-HCl buffer for pH range 3.5-5.5; 6.0-7.5 and 8.0-10, respectively.
Aliquots of alliinase (0.1 ml) was incubated in 0.4 ml buffer solutions. Similarly,
0.4 ml transpeptidase was incubated in 0.5 ml buffer solutions pH 3.5-10 for 10
min at 37° C. After incubation period, the substrate was added and the residual

activities were measured.

Effect of temperature

The effect of temperature on alliinase activity was determined by
incubating alliinase mixtures containing 0.4 ml of 0.05 M phosphate buffer pH 7.0
and 0.1 ml alliinase for 15 min at temperature interval ranging from 0 to 75°C.
Similar incubation was carried out on transpeptidase mixtures containing 0.5 ml of
0.05 M Tris-HCl buffer pH 7.5 and 0.4 ml transpeptidase. After incubation, each
substrate was added and the residual activities were determined as described in
the enzyme assays.

Thermal stability of both enzymes was determined by incubating tubes
containing aliquots of enzyme at 30, 40, 50, 65 and 75°C at pH 7.0 using 0.03 M
phosphate buffer for allinase and at 40, 55, 60, 65 and 75°C at pH 7.5 using 0.1 M
Tris-acetate buffer pH 7.5 for transpeptidase, for a period of 0-25 min. At every 5
minutes, a tube was drawn and residual activity was determined as described in
the enzyme assay.

Assuming pseudo ﬁrst order reaction kinetics, rate constants for

inactivation were calculated and the calculated rate constants were used to derive

the activation energy Ea for inactivation alliinase and transpeptidase. The

calculation was based on the Arrhenius plot for dependence of reaction velocity

on temperature, and carried out according to the method of Price and Stevens

(1982).

Effect of inhibitors/promoters

The effect of various substances on the activity of alliinase on homolog
series of substrates was tested using several substrate analogs (S-methyl, ethyl, S-
propyl and S-l-propenyl-L-cystein), a series of free amino acids (L-methionine, L-
serine, L-alanine and L-cysteine) and antagonist pyridoxal phosphate compounds
(aminoxy acetate, hydroxylamine-HCI and sodium cyanate).

The effect of various substances on the activity of transpeptidase on
synthetic substrate y-glutamyl-p-nitro anilide was tested using several free amino
acids (L-methionine, L-glutamate, L—glutamine), S-substituted free amino acids (S-
methyl, S-propyl, S-l-propenyl L-cysteine), peptides (gluthathione), and inorganic
salts (MgClz, CaClz, NazCO3, Na-citrate, Na-borate).

Inhibitor constant for substrate analogs was determined by the graphic
method of Dixon et al. (1979) from the plot of the reciprocal of substrate
concentrations vs the reciprocal enzymatic activities in the presence and absence
of inhibitor. Inhibition effect of other compounds was calculated as relative
activity in the presence and absence of inhibitors. Alliinase activity was

determined according to the standard alliinase assay described above.

Enzyme Kinetics
Kinetic constants Km and me for alliinase and transpeptidase were

determined using synthesized substrate methyl, ethyl, propyl, l-propenyl-L-
cysteine sulfoxide and y-glutamyl-p-nitro anilide respectively. The effect of

substrate concentration on the rate of pyruvate production by alliinase acting on

foreleg
the star
with:
{here

{1911.

SDSl

330“
3035

down

Subs
305;
45 a
has

tho

llo

47

homolog series of substrate from methyl to propenyl was carried out according to
the standard assay for alliinase using different final concentration of each
substrate ranging from 2.0 to 10.0 mM. Kinetic constant Km was calculated from
Lineweaver—Burk reciprocal plot (1934) and direct plot of Eisenthal and Bowden
(1974).

SDS-PAGE

Electrophoretic patterns of alliinase and transpeptidase were determined
according to the method of Laemmli (1970) as described by Garﬁn (1990). The
SDS-PAGE system was made up of four components. From the top of the cell
downward there were the electrode buffer pH 8.3, sample protein, 4% stacking gel
prepared in pH 6.8 Tris buffer, and 12% resolving gel prepared in pH 8.8 Tris
buffer. The gels were cast in a Mini gel apparatus (Bio Rad Lab, Richmond, CA).
Subsequently, enzyme sample (10ml) and a mixture of protein markers (myosin,
205; b-galactosidase, 116; phosphorylase, 97.4; albumin bovine, 66; albumin egg,
45 and carbonic anhydrase, 29 kDa) was loaded to each well. Electrophoresis
was carried out at 120 V in the stacking gel and 200 volt in the running gel for
about 60 min. The presence of subunits of reduced protein was determined in
12% SDS-PAGE containing 2-ME (Weber and Osborn, 1960).
Commasie blue staining, the gel was stained with 0.1% Commasie blue R-250
(w/v) and destained with 40% methanol-10% acetic acid solution until

background was satisfactorily removed.

Molecular weight determination
Molecular weight of the onion holoenzyme and subunits were determined

by gel exclusion column chromatography (1.5 x 40 cm) using Sephadex G-200
equilibrated with 0.05 M phosphate buffer pH 7.0 containing 0.05% 2-ME and

 

to.
cot

83E

Th.
Pn

en

Lt

48

20 umole pyridoxal phosphate (Weber and Osborn, 1960). All experiments were
carried out at 24°C and ﬂow rate was maintained at 30 ml/hr. Column was
standardized with a gel ﬁltration standard protein mixture consisting of carbonic
anhydrase( 29 kDa) , albumin bovine serum (66 kDa), alcohol dehydrogenase (150
kDa) and thyroglobulin bovine (660 kDa). The molecular weight of alliinase was

determined from a standard curve (log mol mass vs v/vo where v is elution volume
and Va is a void volume) of marker proteins (Weber and Osborn, 1960; Whitaker,
1963; Andrews, 1964).

The molecular weight of subunits were obtained after prior dissociation of
the enzyme in 6 M urea and 0.1% SDS at 65°C for 45 min. Column was
equilibrated with the same buffer. The activity of the enzyme and protein
concentration in each elution fraction was tested using standard enzyme assay

and absorbance at 280 nm Spectrophotometer respectively.

The Effect of Transpeptidase and Alliinase on Pyruvate Production

Preparations of enzyme solution

The effect of transpeptidase alone or in combination with alliinase on the
enhancement of pyruvate production in synthetic substrate y—glutamyl S-mcthyl
L-cysteine sulfoxide (GMCS) and in macerated onion was carried out according
to the method of Schwimmer (1971). Alliinase (8 Unit/mg protein alliinase; 0.0
Unit/mg protein transpeptidase) and onion transpeptidase (2.6 Unit/mg
transpeptidase; 520 tnUnit/mg alliinase) were prepared according to the enzyme
extraction methods previously described from stored and sprouting onion bulbs,
respectively. Kidney transpeptidase (4.9 Unit/mg protein) was obtained from
Sigma Chemical Co., St Louis, MO). Stock enzyme solutions used in this study
were 16 Unit onion alliinase/ml 0.05 M phosphate buffer, pH 7.0, 6.0 Unit onion
tranSpeptidase/ ml 0.05 M Tris buffer, pH 7.5 and 4.8 Unit kidney

49

transpeptidase/ml 0.05 M Tris buffer, pH 7.5. The concentration of pyruvate

formed was determined according to the method of pyruvate deterrni nation.

Preparation of onions

Onion bulbs, weighing approximately 750 g were peeled, cut, and
immediately processed in Acme Juicerator (Model 6001, Acme Juicer
Manufacturing Co., Sierra Madre, CA). Except for the incubation study,
macerated onion tissue was covered and held at room temperature for 2 hr to
complete endogenous enzymatic alliinase action. Standard mixtures for pyruvate
formation studies and ﬂavor analysis were prepared based on Schwimmer (1971)

where each ml of macerated onion contained 100 mM L-methionine, 20 uM
pyridoxal phosphate and 1 mM MgC12.

Effect of transpeptidase and alliinase on GMCS

The effect of transpeptidase and alliinase on synthetic substrate using
glutamyl S-methyl L-cysteine sulfoxide (GMCS) was carried out by adding 6 Unit
transpeptidase , 8 Unit alliinase or both to 4 ml of 2mM GMCS /ml 0.05 M Tris-
HCl buffer, pH 7.5 containing 100 mM L-methionine and 20 uM pyridoxal
phosphate. Pyruvate production was determined according to the method
described earlier and the conversion of GMCS to pyruvate was expressed in

percentage.

Effect of transpeptidase and alliinase on onion macerates

The effect of onion transpeptidase alone or in combination with exogenous
alliinase on pyruvate production in macerated onion was canied out in triplicate.
To each standard mixture of 4 ml macerated onion was added 6 Unit

transpeptidase, 8 Unit alliinase, or both. Effect of pH was determined by adjusting

all

rll

tea

[lie

0
r":
- .

its

na'
s.

Siill
tier

lbs!

lhl

Tm

Slat.-

ﬁller

pH with 1.0 M sodium chloride solution. Enzymatic pyruvate formed was

measured in the reaction mixtures after 20 hr of incubation at 37 °C.

Effect of incubation and enzyme concentration on pyruvate production

The effect of kidney transpeptidase alone or in combination with
exogenous onion alliinase on pyruvate production in macerated onion was carried
out in triplicate according to a modiﬁcation of the method described by
Schwimmer (1971). Stock enzyme solutions used in this study were 16 Unit onion
exogenous alliinase/ml of 0.05 M phosphate buffer pH 7.0 and 4.8 Unit kidney
transpeptidase/ml of 0.05 mM Tris buffer pH 7.5. The effect of incubation time on
pyruvate production in macerated onion treated with exogenous alliinase alone,
transpeptidase alone, and transpeptidase in conjunction with exogenous alliinase
aws determined by adding 8 Unit Exogenous alliinase, 2.4 Unit transpeptidase
and 2,4 Unit transpeptidase in conjunction with 8 Unit exogenous alliinase to a
standard mixture of macerated onion respectively. Pyruvate was determined
every 0, 2, 4, 10, 20, and 36 hr. Control containing macerated onion alone was run
to show pyruvate production due to endogenous alliinase.

To study the effect of enzyme concentrations on pyruvate production; 0,
0.48, 0.96, 1.92, 2.40 and 2.88 Unit transpeptidase and 0, 1.6, 3.2, 6.4, 8.0, and 9.6
Unit alliinase were added to a standard mixture of macerated onions respectively.
Transpeptidase in conjunction with exogenous alliinase were prepared by adding
9.6 Unit alliinase to 0.48, 0.96, 1.92, 2.4 and 2.88 Unit transpeptidase in the

standard mixture respectively. Pyruvate was measured in the reaction mixtures
after 20 hr of incubation at 37°C.

51

Supercritical Carbondioxide (SC-C0,) Flavor Extraction and Analysis
SC-CO2 ﬂavor extraction

Due to availability of enzyme, kidney transpeptidase was used in ﬂavor
study. Enzyme treated macerated onions were prepared by adding 1000 Unit
kidney y-glutamyl transpeptidase and 600 Unit exogenous onion alliinase into
500 ml macerated onion containing 100 mM L-mcthionine, 20 11M pyridoxal
phosphate and 1 mM MgC12. Control and enzyme treated macerated onion were
held at 37°C in 1 liter glass stoppered Erlenmeyer ﬂask for 20 hr before ﬂavor
extraction. Duplicate extractions were made. Macerated onion was used in this
study as it allowed for good mixing with the solvent in the extraction vessels.

The SC-CO2 extraction apparatus consisted of industrial grade CO2 gas
99.5% purity from a gas cylinder compressed with a gas booster. A pressure
regulator positioned between the reservoir and the extraction vessel controlled
the extraction pressure. The total volume of the stainless steel extraction vessel
was 750 ml. The temperature of the vessel was maintained at 37°C. Macerated
onion in the volume of 500 ml was poured into the vessel. The extraction process
was started by slowly raising the pressure in the extraction vessel while the
system outlet was closed. Carbon dioxide passed through the vessel contents at a
ﬂow rate of about 1 liter/min (STP) and was monitored with a ﬂow meter and a wet
test meter. After the extraction vessel pressure reached 204 atm a heated
micrometering outlet was opened. Onion ﬂavor was collected in an armed trap

tube, as the CO2 solvent and the onion solute separated, the solvent returned to

atmospheric pressure. The extraction was performed for about 24 hr or after 800-
1000 liter CO2 had passed through.

52

GC-MS Analysis and Identiﬁcation

For the purpose of ﬂavor analysis, 100 mg onion extract was diluted with
100 pl methylene chloride and injected into the GC-MS. The GC-MS system
consisted of JEOL AX 505H double focusing mass spectrometer coupled with a
Hewlett-Packard HP 5890J gas chromatography. The column was a fused silica
DB-l capillary column (30 m x 0.25 mm x 0.25 mm). The operating condition was
injector and detector 150°C, oven 35—150°C at 5C°lmin linear and GC-MS transfer
line 150°C. Helium carrier gas ﬂow rate was 1 ml/min. The mass spectra were
obtained by electron ionization at 70 eV. Kovat retention indices (1k) were
calculated against the retention time of normal hydrocarbon of (L,-C16 co-injected
with the sample (Ettre, 1964).

Identiﬁcation of each compound was based on comparisons of the mass
spectral fragmentation pattern and GC retention with published works or mass
spectral data base.

RESULTS AND DISCUSSION

Relationship Between Alliinase Activity and Pyruvate Content
Pyruvate content of onions analyzed ranged from 4.66 to 10.73 umoles/g

fresh wt (Table 4). Ten onion cultivars/selections, Norstar, Northstar, Sweet
Sandwich, Spartan Banner-80, Spartan Banner, Magna Sweet, K.Downing,
Granite, MSU 3506, and MSU 8450, had pyruvate content between 8-10
umoles/g fresh wt while in ﬁve onion cultivars, Michigan Sweet, Texas Sweet,
Yellow Sweet, Imperial Sweet and Vidallia it was between 4.66-6.50 umoles/g
fresh wt. The ﬁrst 10 onions can be catagorized as pungent-cooked type and the
later ﬁve as sweet-salad onions (Schwimmer and Weston, 1961). Pyruvate is the
initial product formed in a chemically equivalent amount along with ammonia and
sulfenic acid by the action of alliinase on alk(en)yl-L—cysteine sulfoxides. It is a
measure of pungency and ﬂavor strengths of onion (Schwimmer and Weston,
1961; Schwimmer and Guadagni, 1968; Schwimmer et al., 1964; Lancaster and
Boland, 1990; Wall and Corgan, 1992; Randle and Bussard, 1993).

The speciﬁc activity of alliinase assayed from crude enzyme extracts ranged
from 1.6 to 5.7 Unit/mg protein (Table 4). The onion cultivars/selections which
were categorized as pungent exhibited higher alliinase activity (3.00 —5.70
Unit/mg protein) as compared to onions which were sweet (1.60-2.23 Unit/mg
protein). There was a positive correlation (=0.95) between pyruvate formed and
the activity of enzyme alliinase (Figure 5) which suggesting that in onions the
greater the alliinase concentration, the higher the pyruvate, as well as ﬂavor

volatiles formed.

Table 4. Pyruvate content and alliinase activity in onions

 

 

vate Speciﬁc ActivityM

Onion cultivar (pmole/ g fresh wt) (Unit/mg protein)
Norstar 10.13 :1: 0.94 5.70 :1: 0.10
Northstar 9.37 :1: 1.12 4.60 :1: 031
Sweet Sandwich 10.47 :1: 1.12 5.23 :i: 0.45
Spartan Banner-80 9.73 :t: 0.75 4.90 :t: 0.40
Spartan Banner 8.60 :1: 1.10 4.06 :1: 1.10
Magna Sweet 9.13 :1: 0.30 3.66 :t 1.25
K.Downing 9.13 :t 1.05 4.20 :1: 0.60
Granite 10.73 a: 1.00 5.63 d: 0-22
MSU 3506 10.37 :1: 1.25 5.52 :1: 0.10
MSU 8450 8.53 :t 1.95 3.00 :1: 0.50
Michigan Sweet 5.60 :1: 0.20 2.23 :1: 0.04
Texas Sweet 5.60 :1: 1.15 2.17 :1: 0.11
Yellow Sweet 5.70 :1: 0.78 2.22 :1: 0.36
Imperial Sweet 6.50 :1: 0.36 2.00 :1: 0.10
Vidalia 4.66 i 0.61 1.60 :i: 0.10

 

** Alliinase activity from 65% ammonium sulfate precipitates

3906. activity [Unﬁlmo prof ain]

55

 

 

m2 = 0.905 ,

 

 

Pyruvate (pmololg trash wt)

“gums Correlationbetweenallnme“ m"
arulpynrvatecommonions.

12

Concentration of Pyruvate, Alliinase and Transpeptidase During Storage

Spartan Banner, a pungent onion and Yellow Sweet a sweet onion with
pyruvate concentration of 8.60 :1: 1.10 and 5.70 :r: 0.78 umoles/g fresh wt,
respectively were stored for 32 weeks at 20° C. Pyruvate concentration in Spartan
Banner onion increased up to 24 weeks from 8.6 to 11.8 pmoles/g fresh wt. In
Yellow Sweet the increase was from 5.7 to 9.6 umoles/g fresh wt (Figure 6).
Thereafter in both type of onions pyruvate level decreased.

Transpeptidase and alliinase extracted from stored onions were puriﬁed
using CMC column chromatography and assayed for their activity changes
during storage There was a slight increase in alliinase activity in both types of
onion analyzed which was similar to a pattern of an increasing in pyruvate level.
After 12 weeks, the activity of y-glutamyl transpeptidase showed a rising trend
(Figure 6). Similar results were reported by Lancaster and Shaw (1991) and Ceci
and Pomilio (1992). There was no measurable sprouting characterized by
formation root and green shoots in Spartan Banner and Yellow Sweet cultivars up
to 8 and 12 weeks, respectively. Thereafter sprouting commenced in Spartan
Banner onions up to 16.5% of the bulbs while Yellow Sweet onion which

showed a similar pattern had 11.5% sprouting at 32 weeks (Figure 6).

Characterizatics of Alliinase

Puriﬁcation

According to the scheme summarized in Table 5 thirty eight fold
puriﬁcation and 2% recovery of puriﬁed alliinase was obtained. The elution
pattern on CMC, Sephadex G-200 and Polybuffer 94 are shown in Figures 7, 8,
and 9, respectively. Much of the difﬁculty previously found in preparing pure
preparation of alliinase due to its instability under the usual condition of

extraction and puriﬁcation (Lancaster and Boland, 1990).

g sprouted/100 g bulbs

 

l8

  
   
 
  

—O— Spartan Banner
—0— Yellow Sweet

 

 

 

Unlt transpeptidase/mg protein

 

—0— Spartan Banner
2* —0— Yellow Sweet

 

 

 

Unit alliinase/mg protein

 

 

 

3 I I I I I .I _f I I
Pyruvate (parole/g rr wt)
1 3

 

11-

9-1

 

 

 

S I I I I I I I I

0 4 8 12 16 20 24 28 32

Week

Figure 6. Effect of storage time on pyruvate content, alliinase
and transpeptidase activities, and sprouting index of onions.

J

3’;

:‘.§

Table 5. Puriﬁcation of alliinase

 

 

Total Total Speciﬁc Puriﬁcation Recovery
Puriﬁcation Step protein activity activity Fold (%)
(mg) (Unit) (Unit/mg met)
1. Homogenate 2078 2620 1.26 1 100
2. 456596 (NH4)2804 454 1920 4.22 3 73
fractionation
3. CMC 75 630 8.40 7 24
4. Sephadex G-200 8 315 39.38 31 12
5. Polybuffer94 1.2 58 48.33 38 2

 

CMC: carboxy methyl cellulose

Absorbance at 42. nm

59

 

0.8-

0.6-

0.4-

0.2-

 

 

 

IIIIIIIIII 280 “m

-0.8

-0.6

-O.4

-0.2

 

 

 

0.0

Flglue7. EhrlionproﬁleofaliimseonCMCcohunn.

 

Elutlon volume (ml)

0.0

170

Absorbance at 2811 till

EIN [IN I 0‘ N t'OIIIII -ll‘hll‘

Absorbance at 420 n.

 

 

0.8-

0.6‘

0.4-

 

 

 

 

 

 

 

 

 

1.0
loo-cacao- 280 nm
420 nm -o,3
-0.6
-0.4
-0.2
t t . "I 0.0
100 125 175 200 225

 

Elutlon volume (ml)

Figlu'eS. ElulionproﬁleofalliinaseonSephadexG-ZOO.

Absorbance at 280 an

61

0.4

E... omN we 855:2;

7m 1.
0 0
- D -

-0.3

0.0

 

 

 

420 nm

280 nm

 

 

 

0.4

.
2
0

- -
3 I
o .
0 0

E: ON? no aocaaceoac

 

60

Fraction number

Egure9. EluﬁonproﬁleofalliinaseonPolybuﬂ'erM

62

The rapid loss of activity could be alleviated by working at low temperature (0-

4°C), addition of 5% (v/v) polyethylene glycol and low level of 2-ME (0.05%).
Homogeneity of the ﬁnal preparation was indicated by one single protein band
after staining with Commasie blue R-250 ( Figure 18). The speciﬁc activity of
puriﬁed alliinase on S-ethyl-L-cysteine sulfoxide was 48.33 Unit/mg protein. Nock
and Mazelis (1987) reported alliinase activity of 23.3 Unit/mg protein in white
onion. In a related species, Welsh onion leaves and garlic, the alliinase activity of
97.5 Unit/mg protein and 103.6 Unit/mg protein, respectively has been reported
(Fujita et al., 1990; Jansen et al., 1989).

Substrate speciﬁcity

The effect of concentrations of the homolog series of substrate, S-alkyl
derivatives from methyl to propenyl, on the rate of pyruvate production catalyzed
by alliinase and kinetic constants calculated using Lineweaver-Burk (1934)
reciprocal plot and direct plot of Eisenthal and Bowden (1973) are shown in
Figure 10 and Appendix 5, respectively. The maximal velocity (VIM) under the
condition of the reaction showed relatively little variation with respect to the

chain length of the S substituent (1.39-1.46 pmoles of pyruvate/min), whereas
values for Km varied widely (Table 6). The result indicated that in the homolog

series of S-alkyl derivatives, K!n increased with increase in C atom. Relatively
lower Km value of S-(l-propenyl)-L-cysteine sulfoxide (2.14 mM) compared to
other compounds indicated the higher speciﬁcity of alliinase on this compound.
This result was in agreement with the result of Schwimmer et al. (1964), who used
relatively crude alliinase, that trans (+)-S-(1-propenyl)-L-cysteine sulfoxide is the
principal substrate for alliinase. In general, KIn values of onion alliinase obtained in
this study were slightly lower than Km values of alliinase reported for white globe

onion (SchWimmer et al., 1964).

Iapgruvate-I)

IIIII. llllll

lh'f

 

 

 

 

 

 

 

8
y-0.72143+12.214x W-OSSB
y-0.68571 +4.1S71x W-QSSZ
y-M+2.0m RAZ-m a methyl
6‘ y-Q73571+1.4871x W-OSBB o ethyl
I propyl
« 0 1-propenyl
4 -1
. o
2 -1
d I
o I ' I I F I ' I T I C
0.0 0.1 0.2 0.3 0.4 0.5 0.6
1/5 (H)

Figtu'e 10. UneweavenBurk plots ofalliinase

 

Table 6. Substrate spesiﬁcity of alliinase

 

Lineweaver Burk* Direct Plot"
Substrate Km Vmax Km Vmax

(mM) pmole/min) (mM)( umol/min)

 

 

S-methyl-L-cysteine sulfoxide 16.90 1.39 19.0 1.5
S-ethyl -L-cysteine sulfoxide 6.03 1.45 6.0 1.5
S-propyl-L—cysteine sulfoxide 3.02 1.45 2.8 1.5
S-l-propenyl-L-cysteine sulfoxide 2.14 1.40 1.8 2.0
* calculated from Frgure 10

** calculated from Appendix 7

Effect of pH

Alliinase showed a pH optima of 8.0 and was relatively stable at a wide pH
range of 5.5 - 8.0 (Figure 11). About 70% inactivation of enzyme activity
occurred at pH 4.0 in contrast to only 25% at pH 10. Tobkin and Mazelis (1979)
reported that the pH optima of relatively pure onion allinase was 7.4 - 8.5. At pH
range of macerated onion (5.0-6.0), alliinase activity was 65-85% of its maximal

activity.

Effect of temperature

Aliinase retained 80% of its maximum activity over 25- 50°C with optimal
activity at 39°C (Figure 12). Above 50°C the activity declined rapidly but the
enzyme was not completely inactivated even at 70°C. Heating of alliinase at
35°C for 25 minutes, showed relatively no observable inactivation. After heating
at 55, 60, 65, and 75°C for 10 min, alliinase lost about 18, 42, 88 and 100 % of its
activities respectively (Figure 13). Arrhenius plot (Figure 14) for reaction velocity

showed linearity in the temperature range from 30 to 65°C. The activation energy

65

(Ea) of reaction for S-ethyl-L-cysteine sulfoxide as the substrate was 16.6 k]

lmole.

Effect of Inhibitors/promoters

Inhibitor studies of alliinase fall into two classes: (i) involving pyridoxal
phosphate antagonists and (ii) involving substrate analogs as competitive
inhibitors. In the former group, reversible inhibition of alliinase by hydroxylamine
has been demonstrated by several authors (Tobkin and Mazelis, 1979 and
Kazaryan et al., 1979). In this study inhibition effect of these compounds as well
as other inhibitors (sodium cyanate, aminooxyacetate, iodoacetate and iodine) was
investigated. Some homolog alkyl substituents and free amino acids which have
inhibiting effect on the substrate of this enzyme were also studied (Table 7).
Percent inhibition of homolog alkyl substituent increased as the number of
carbon atom C increased, while free amino acids, except L—cysteine did not show
any inhibitory effect. L-cysteine, a competitive inhibitor of alliinase, has been
reported to decrease the absorption maximum of alliinase (Mazelis and Crew,

1968; Jansen et al., 1989).

Molecular weight and SDS-PAGE of alliinase

Molecular weight of native alliinase determined by Sephadex G-200 column
chromatography using mixed protein standards was estimated to be 200 kDa
(Appendix 6). However, electrophoresis of reduced alliinase protein on SDS-
PAGE indicated a single protein band of dissociated enzyme corresponding to
protein marker bands ranging from 45 to 66 kDa. The estimated molecular weight
of this dissociated protein was 50 kDa(F1gure 15). These results

 

 

12

10

18

ms

 

 

100-

u . a
0 0
8 6

A! $8.50. 05:83"-

40-

20

pH

Figurell. Eﬁ'ectopronallﬁmseactivity.

Relative activity (an

67

 

 

 

 

 

20 r r I
0 20 40 60

Temperature (°C)

Figrlelz. Hectoftemperatmeonaliimseacliv'ty.

80

Relative activity

 

1.0

0.8

0.6-r '

0.4-

0.2‘

0.0-

 

 

Figure13.

 

 

30' c
40'c
50'c
65' c
7s'c

 

10 20

Time (min)

Heathnclivationofalliinaseatdifferent

unperatures.

30

 

69

Table 7. Effect of inhibitors on alliinase activity

 

 

Substance Effect Relativea Kib

activity (mM)
(%)

L-cysteinec competitive inhibition 97 1.20

S-mcthyl-L-cysteine competitive inhibition 91 2.70

S-ethyl-L—cysteine competitive inhibition 86 2.75

S-propyl L-cysteine competitive inhibition 78 2.40

L-cystine no inhibition 100

L-methionine no inhibition 100

L-serine no inhibition 100

Magnesium chloride no inhibition 100

Hydroxylarnine sulfated pyridoxal S-P-antagoni st 20

sodium cyanate pyridoxal S—P-antagoni st 85

aminooxyacetate pyridoxal S-P-antagonist 15

 

a relative activity compared to control (%)
b concentration of inhibitor 1 mM
c concentration of inhibitor 2.0 mM, substrate 200 mM

d concentration of inhibitor 0.05 mM

In ( rate of inactivation)

70

 

2.2-

 

21

y=9.1437-1993.Qt W-OSBI

 

 

 

0.0032

1 T U

j I ' I
0.0033 0.0034 0.0035

VT ('0

Figtu'e14. Arrheniusplotofallﬁnasefor
activationenergy(Fa)delerminalion.

0.0036

71

 

Figure 15. SDS-PAGE of reduced alliinase
A: protein markers, B and C: purified alliinase (10 pg),

72

suggested that onion alliinase consists of four subunits of same size. There
appears to be little doubt that the subunit molecular weight is close to 50 kDa,
however, the molecular weight of the native enzyme molecule is unresolved. The
most likely explanation or the observed behavior is that rather than existing as a
native molecule of a set number of subunits, the subunits aggregate into dimers
and tetrarners, and what is seen as the "native" enzyme is a time-averaged rapid

equilibrium between those forms (Lancaster and Boland, 1990).

Characteristics of v- Glutamyl Transpeptidase

Puriﬁcation

By the puriﬁcation procedure described and summarized in Table 8,
homogenous transpeptidase was obtained from sprouting onion. The elution
patterns on CMC, Sephadex G-150 and Polybuffer 94 are presented in Figures 16,
17, and 18, respectively. Transpeptidase was puriﬁed twenty seven fold, while the
recovery of the homogenous preparation was 2%. Homogeneity of the ﬁnal
preparation was shown by a single protein band after staining with Commasie
blue R-250 ( Figure 27). The speciﬁc activity of puriﬁed transpeptidase on y-
glutamyl p-nitro anilide was 15 Unit/mg protein. This is higher than the speciﬁc
activity of ammonium sulfate precipitation of 77 mUnit/mg protein reported by
Schwimmer.
(1971). The difference in enzyme activity may be due to the difference in enzyme
assay employed.

Substrate speciﬁcity
Kinetic constant Km and Vmax with L-y-glutamyl p-nitro aniline as a

substrate was 12.17 mM and 1.37 umole/2 hr respectively (Figure 19 ).

Table 8. Puriﬁcation of transpeptidase

 

 

Total Total Speciﬁc Puriﬁcation Recovery
Puriﬁcation Step protein activity activity Fold (%)
(mg) (Unit) (Unit/mg met)
1. Homogenate 1584 871 0.55 1 100
2. 358572» (NH4)2804 187 453 2.42 4 52
fractionation
3. CMC 112 294 2.63 5 34
4. Sephadex G-150 18 83 4.60 8 10
5. Polybuffer94 1.4 21 15.00 27 2

 

CMC: carboxy methyl cellulose

Absorbance at 560 nm

 

74

 

 

 

 

 

 

Fraction number

1.5
---------~~ 280 nm

560 nm -1.2
-0.9
”0.6
-0.3
r ; ”r 0.0

40 50

I‘Igm'elti. ElmionproﬁleoflrarspeptidaseonCMCcolmnn.

Absorbance at 2" nm

Absorbance at 56. nm

75

 

 

 

 

 

0.5
i ~~~~~~~~ 280 nm '
0 4d 560 nm
0.3'1
0.2- “
1 3:3. "
’:
0.1'l _r z:
i: ':
0.0 . 4’. . , . -.
0 10 20 30 40 60

Fraction number

Figure17. EulionproﬁleoflrarqreplitmonSqrhadexG-lso.

Absorbance at 280 um

Absorbance at 560 nm

76

 

 

 

0.8 _ a 0.8
d — 560 nm 1
..................... 280 nm
0.6- ‘ 7 -0.6
0.4:
0.2-
0.0

 

 

 

 

 

Fraction number

Figure18. ElulionproﬁleoftrampeptidaseonPBEM.

 

Absorbance at 280 um

Effect of pH

This enzyme showed an optimal activity at pH 9.0 and was relatively
stable at a narrow range pH (8-9.5). Within pH range 5-6, the pH of macerated
onion, the activity of y-glutamyl transpeptidase was only 30-40% of its maximal
activity (Figure 20).

Effect of temperature

This enzyme retained 80% activity over 25-55°C temperature range with
optimal activity at 40°C (Figure 21). After heating at 55, 60 and 65°C for 10 min,
glutamyl transpeptidase lost 10, 60 and 90% of its activity, respectively (Figure
22). Arrhenius plot of activity versus 1/T revealed a linear relationship in the
temperature range of 40-65°C. Activation energy Fa was 15.8 kJ/mole, using 7-
glutamyl p-nitro aniline as the substrate (Figure 23). Unlike pH stability, thermal
stability of y-glutarnyl transpeptidase was similar to alliinase.

Effect of inhibitors/promoters

Table 9 shows the effect of various inhibitors/promoters on the activity of
puriﬁed transpeptidase. In contrast to mammalian kidney transpeptidase, MgM
and carbonate did not appear to affect onion transpeptidase activity. Borate and
citrate inﬂuenced the enzyme activity. Inhibition of transpeptidase by glutamyl
peptides may be due to competitive inhibition of peptides upon glutamyl p-nitro
anilide. Added free amino acids stimulated the activity, presumably due to their

function as glutamyl acceptors.

Molecular weight and SDS-PAGE of transpeptidase
Molecular weight of native and reduced transpeptidase determined in

Sephadex G-200 column chromatography using mixed protein standard was

Ilv( uncle PllAIZ hr)

 

 

 

10

y- -07zass+ssrzsu ale-0.983
8‘
6-

O
4-
O

O

Z-r
O
0 I ' r ' a ' r
0.2 0.4 0.6 0.8 1.0 1.
1/(S) (mM)

 

79

 

 

12

10

v8

 

 

1001

q . . . q
0 o 0
8 6 4.

33 33.38 2.233.

204

pH

[lumen Eﬁ’ectoprontranspeptidmeaetivity.

Relative Activity m

 

 

 

 

 

0 20 40 60 80

Temperature (’C)

Figrn'e21. Eﬁ'ectoflemperatureonlrampeptidmeacﬁvity.

Relative activity (I)

81

 

 

 

 

1.0 -

0.8-1

0.6d

0.4- —O— SS'C
——o—- 60'c
—I— GS'C

0.2- - ,
—-o— 75 C

0.0 w - 3F

0 5 10 15

Time (min)

Pigmezz Heatimcﬁvaﬁonofunnspepﬁdase

atditramtmmpaamm

 

In (rate of inactivation)

 

2.5

2.4-

2.3-

2.2-

2.1-

 

y 2 mm - 1907.4): m2 = 0.994

 

 

 

2.0
0.0032

V Y

I I ' I '
0.0033 0.0034 0.0035 0.0036

1/T (°K)

mun-223. Arrtnniusplotofu'ampeptidasefor

acuvaﬁonmay(Ea)demminaﬁon.

Table 9. Effect of inhibitors and promoters on transpeptidase activity

 

 

Substance Effect Relative‘ -b

activity (mM)
(%)

S-methyl L-cysteinec g1 utam y] acceptor l 15

S-propyl L-cysteine glutamyl acceptor 130

L-methionine glutamyl acceptor 125

Magnesium chloride no inhibition 100

Sodium carbonate no inhibition 108

Glutathione competitive inhibition 10 0.24

y—L—glutamyl S-methyl competitive inhibition 20 2.50

L-cysteine ‘

Hydroxylamine sulfated 98

Sodium citrate 97

Sodium borate 86

L- glutamate 70

L-glutamine 9O

 

' relative activity compared to control (%)

" concentration of inhibitor 1 mM

° concentration of inhibitor 0.01 mM, substrate 4.0 mM
" concentration of inhibitor 0.05 mM

 

Figure 24. SDS-PAGE of reduced y-glutamyl transpeptidase
A: protein markers, B and C: purified transpeptidase (20 pg),

85

estimated to be 120 kDa (Appendix 7). Similarly, electrophoresis of reduced
transpeptidase protein on SDS-PAGE indicated single protein band of enzyme
which correspond to protein marker band ranging from 116 to 205 kDa (Figure
24). These results may suggest that transpeptidase consisted of only one

monomer.

Effect of Transpeptidase and Alliinase of Onion on Pyruvate Production
Glutamyl S-methyl L-cystein sulfoxide as substrate

Figure 25 shows the effect of onion transpeptidase in conjunction with
onion alliinase on the conversion of y-glutamyl S-methyl L-cysteine sulfoxide
(GMCS) to pyruvate. In the absence of alliinase, transpeptidase did not catalyze
the production of pyruvate from GMCS. Conversely, no pyruvate was formed in
the presence of alliinase alone. With alliinase and transpeptidase working
together simultaneously, pyruvate was produced at a constant rate until 1.6
umole/ml or 90% of GMCS was converted to pyruvate.

The good evidence for couple reaction of transpeptidase and alliinase was
shown by the effect of the order of enzyme addition to GMCS solution. When
alliinase was added ﬁrst, presumably nothing happened because the GMCS is not
a substrate for alliinase action. Upon the addition of transpeptidase, the
production of pyruvate increased parallel to that produced when the two
enzymes were added simultaneously. When transpeptidase is added ﬁrst, it
presumably liberates S-methyl L-cysteine sulfoxide (equation 1). Accumulation of
S-methyl L-cysteine sulfoxide available to the alliinase added after 30 minute
would result in an increased rate of pyruvate production (equation 2).

Small increase in pyruvate after the addition of transpeptidase most

probably due to the alliinase contained as impurity of transpeptidase extraction.

Conversion to pyruvate (I)

 

 

 

 

‘l 00
80 -
60 -
40 4
,
Z 0 _ —0— AI|+TP
—o— Alliinase (All)
—0— Transpeptidase (TP)
0 A: ' ' ' I ' I ' l f
0 30 60 90 1 20 1 50

Incubation time (min)

Figune25. Eﬂ‘ectofharspepﬁdaseandallﬁnase
onconvem'onofGMCSmpymvate.

transpeptidase
(1) y -glutamyl—S-methyl-L-cysteine sulfoxide + H20» L—glutamic acid +

S-methyl-L-cysteine sulfoxide
alliinase
(2) S-methyl-L-cysteine sulfoxide+ H20 -’ NH3 + Pyruvate + S(volatile)

This result may suggest that the addition of transpeptidase in conjunction with

alliinase resulted in an increasing of conversion of GMCS to pyruvate.

Macerated onion as substrate

Stored onions have been shown to contain ﬂavor precursor alk(en)yl L-
cysteine sulfoxide (Lancaster and Kelly, 1983) and y-glutamyl peptides (Shaw et
al., 1989) as well as alliinase, the enzyme responsible for conversion of ﬂavor
precursors to sulfur contained ﬂavor compounds. However, transpeptidase which
is required to liberate free alk(en)yl L-cysteine (sulfoxide) from v-glutamyl
peptides to become available to the allinase is absent in dormant onion.
Exogenous transpeptidase in conjunction with endogenous and/or exogenous
alliinase was expected to enhance pyruvate production in macerated onion as
well as affect ﬂavor profile of the onion ﬂavor extract.

The effect of transpeptidase and alliinase of onion on pyruvate production
in onion macerates were presented in Table 10. Exogenous onion alliinase alone
enhanced the production of pyruvate, however, transpeptidase by alone showed
a very small increase in pyruvate as compared to control. When added
simultaneously, the combined action of alliinase and transpeptidase resulted in
increase in pyruvate formation over and above that of catalyzed by either enzyme
alone (Table 10). Pyruvate formation at pH 9.0 was higher than at pH 7.5 which
was higher than pH 5.6. Yu et al (1989a) showed that the concentration of ﬂavor

compounds increased with increasing pH of macerated onion.

Table 10. Effect of transpeptidase and alliinase on pyruvate production
in onion macerates

 

 

 

 

pyruvate (umoles/ g fresh wt)
transpeptidase

pH additives ' without with
5.6 none 8.5 10.5
alliinase 12.4 18.6

alliinase +L—methioninea 12.6 18.8
alliinase+ Mg++b 12.4 18.6

7.5 none 8.0 l 1.0
alliinase 12.0 19.0

alliinase + L-methionine 12.8 19.8

alliinase + Mg'H' 12.2 19.2

9.0 none 7.8 10.8
alliinase 12.0 20.8

alliinase + L-methionine 13.2 21.0

alliinase + Mg++ 12.0 20.4

 

a L-methionine, 0.2 M
b Mgc12, 1 mM

89

Difference in pyruvate formation with and without L—methionine suggested that
this amino acid was required by transpeptidase to act upon the substrate. Unlike
alliinase from Albizzia (Schwimmer and Austin, 1971b), there was no inhibition of
transpeptidase by exogenous onion alliinase. In agreement with inhibitor study
mentioned above, Mg++ did not enhance pyruvate formation.

The enhancement of pyruvate production by exogenous lyase may
indicate that in fresh macerated onion, the enzymatic reaction does not go to
completion. The increase in pyruvate due to transpeptidase alone or in
conjunction with exogenous alliinase may be the result of the action of
transpeptidase on y-glutamyl peptides to release L-cystein sulfoxide which
becomes available for alliinase to produce pyruvate, ammonia and sulfur
compounds. More pyruvate was produced by transpeptidase in conjunction with
exogenous alliinase, suggesting that addition of exogenous alliinase in addition to

transpeptidase was necessary to enhance pyruvate production in macerated

onions.

Effect of kidney transpeptidase and onion alliinase on pyruvate production

Effect of incubation time on pyruvate production

Schwimmer and Austin (1971b) reported that in the presence of kidney
transpeptidase, exogenous aliinase from Albizzia lophanta inhibited the formation
of pyruvate in fresh dehydrated onion suspension. The interaction between
endogenous onion alliinase which was assumed to be present in onion suspension
with exogenous alliinase from a different source was concluded as to be the
reason. The effect of kidney transpeptidase and onion alliinase on the
enhancement of pyruvate production in fresh macerated onion has not been

reported. The effect of both enzymes on pyruvate enhancement in fresh

Pg ruvate (panel In fresh wt)

 

 

 

 

 

 

 

 

20
—a
—4.>
——a
a
-—t-— Blank
54 —o— Alliinase (All)
—-t— Transpeptidase (GTP)

—O— AII+GTP

 

 

o I t T ' I ‘ I

0 10 20 30 . 40

Time (hour)

FigureZ6. Eﬂ‘ectofincubaﬁonﬁmeofuampeptidase-alliinase
heatedonionmacamonpyruvatepmduﬁon.

91

macerated onion as well as on ﬂavor profile of onion ﬂavor extract are described
asfollows.

Spartan Banner cultivar was selected for the pyruvate production study.
Pyruvate production of 8.5 umole/ g fresh wt, catalyzed by endogenous alliinase
(control) in macerated onion was achieved 2 hr after maceration (Figure 26).
Adding the exogenous onion alliinase (4 Unit) was delayed the maximum
production of pyruvate to 4 hr. While transpeptidase alone (2.4 Unit), and in
conjunction with exogenous alliinase (4 Unit) continuously increased the
production of pyruvate until 20 hr of incubation. Exogenous onion alliinase was
added to ensure that total endogenous and exogenous alliinase was sufficient
enough to convert the available ﬂavor precursors. Prolonged incubation up to 36

hr showed little noticeable increase in pyruvate production.

Effect of enzyme concentration on pyruvate production

The effect of enzyme concentrations on pyruvate production was analyzed
(Figure 27). Pyruvate production by the endogenous alliinase (control),
exogenous alliinase, transpeptidase alone, and transpeptidase in conjunction with
exogenous alliinase in macerated onion was 8.5, 12.7, 10.0 and 20.8 umoles/ g
fresh wt respectively. The increase in pyruvate due to transpeptidase in
conjunction with alliinase from control was 12.3 umoles/g fresh wt or 1.5 fold of
the blank. Hence, maximal pyruvate formed was 2.5 fold greater than control.
This increase in pyruvate production by exogenous alliinase (4.2 umoles/g fresh
wt) may indicate that in macerated onion the enzyme does not go to completion.
Schwimmer and Guadagni (1968) found that lack of pungency in some onion
preparations is due to lack of alliinase. The increase in pyruvate due to
transpeptidase alone (1.5 umoles/g fresh wt) or in conjunction with exogenous

alliinase (12.3 umoles/g fresh wt) may be the result of the action

 

 

 

 

 

 

 

 

 

g 15
a: —c— Alliinase (4)
an
E
g 10 -
5
3
9
E
a. 5 I ' I ‘ I ' I ' I ' I
0.0 1.8 3.2 4.8 8.4 8.0 9.6
Alliinase (Unit)

30
E , (b)
t —0— WM
g 20 - —I— 11» Alliinase
o
E
3
8 1O - M
9
E
R‘

0 I r ﬂ I I I I ﬁ I

0 00 0.48 0 96 1.44 1 92 2 40 2 88

mmpepttdaae (Unit)

Figure27. Eﬂ'ectofenzymeconcenhationoftranspeptidme-alliirme
treatedonionmaoeratesmpynwatepmducﬁon.

of transpeptidase on y-glutamyl peptides to release S-alk(en)yl-L-cysteine
sulfoxide which becomes available for alliinase to produce pyruvate, ammonia and
sulfur compounds. More pyruvate was produced by transpeptidase in
conjunction with exogenous alliinase, suggesting that adding exogenous alliinase
in addition to transpeptidase was necessary to enhance pyruvate production in
macerated onion.

Pyruvate which has been shown to have a high correlation with ﬂavor
perception is used as a measure of ﬂavor strength (Wall and Corgan, 1992). It

provides a measurement of total ﬂavor, but does not provide any information
about relative amount of individual ﬂavor volatile. Analysis of SC-CO2 extract of

macerated onion may provide the effect of y-glutamyl transpeptidase on individual

ﬂavor volatile.

Effect of Transpeptidase and Alliinase on Flavor Proﬁle
Table 11 shows the yields of onion ﬂavors extracted by supercritical carbon
dioxide from Spartan Banner and Yellow Sweet onions in the absence or in the

presence of transpeptidase.

Table 11. The yield of onion ﬂavors extracted by supercritical carbondioxide

 

 

No. Variety Treatment Yield
(%)*
1 Spartan Banner control 0.05
2 Spartan Banner transpeptidase 0.06
3 Yellow Sweet control 0.04
4 Yellow Sweet transpeptidase 0.05

 

* average of two extractions (g /100 g fresh onion)

Flavor compounds of onion extracts

Identiﬁcation of the ﬂavor compounds of the control and transpeptidase
treated, Spartan Banner and Yellow Sweet onions is based on comparisons of
mass spectral data with published works. Table 12 shows the ﬂavor components
in these onion cultivars identiﬁed by GC-MS analysis along with their mass
spectral data (including the isotope distribution pattern of the molecular ion
region). A relatively constant retention time of each sample analyzed, made the
quantitation of the compounds possible.

The SC-CO2 onion extract contained many ﬂavoring compounds which
can be classified into the following categories: oxygen compounds, thiosulfonate,
alkyl dithienes, thiophenes, monosulﬁdes, disulﬁdes, trisulﬁdes, and tetrasulﬁdes.
Qxygeg compounds: Two carbonyl compounds thiopropanal S-oxide and 2-
methyl pentanal were found in SC-CO2 extract. The presence of thiopropanal S-
oxide was identified by comparing its retention time and mass spectral pattern
with
those of published by Brodnitz and Pascale (1971), Nishimura (1973) and Block et
al. (1992), and 2-methyl pentanal to the data of Boelens et al. (1971). According
to Brodnitz and Pascale (1971), these aliphatic aldehydes are present in onion
ﬂavor by the action of alliinase on the major precursor S-propenyl L—cysteine-
sulfoxide lead to the formation of thiopropanal-S-oxide, an unstable compound
known as lachrimatory factor. This compound then rearranged spontaneously to
form propanal and sulfur. 2-Methyl pentanal then formed by aldol-condensation
of two molecules of propanal.

Thiosulfonate: Methyl methane thiosulfonate was identiﬁed in onion extract by
comparing with mass spectra published by Boelens (1971). Several authors have
suggested the formation of thiosulfonate as the secondary reaction in the

enzymatic cleavage of S-alkyl-L-cysteine derivatives. In vitro study using S-

95

methyl L-cysteine sulfoxide showed that most of these compounds were
converted to pyruvate, ammonia, dimethyl disulﬁde, and methyl methane
thiosulfonate (Ostermayer and Tarbell, 1960; Yu, et al., 1994). Thiosulfonates have
never been found in onion oil nor in Allium species, this may be caused by their
low vapor pressure and fairly good water solubility. Consequently they were
absent in steam distilled onion. However, these compounds were found in
chopped raw onion (Boelens et al., 1971) and in present work. The presence in
SC-CO2 extract. may be due to the good solubilizing power of supercritical C02-
Alﬂl dithiene: Compound 3-ethyl-1,2-dithi-4~ene and its isomer 3-ethyl-1 ,2-dithi-
5-ene were identiﬁed by comparing with mass spectral published by Brodnitz et
al. (1971) and Kallio and Salorinne (1990). The presence of these compounds in
headspace of crushed onion was ﬁrst reported by Kallio and Salorinne (1990) and
Nishimura et al. (1988). Brodnitz et al. (1971) assumed that the compounds were
dehydration products formed nonenzymatically from diallyl thiosulfinate or 1-
propenyl propyl thiosulﬁnate during gas chromatography and that they were not
original components of garlic. However, if this dehydration occurs spontaneously
in crushed onions, the compounds formed are no more “artiﬁcial” than any of the
major ﬂavor compounds (Kallio and Salorinne , 1990).

Thiophene derivatives: 3,4-dimethyl thiophene and 3,4 dimethyl-2,5
dihidrothiophene were identiﬁed by comparing their spectral patterns with those
of published by Weast and Graselli (1989) and Boelens et al.(1971). According to
Boelens (1971), heat and ultraviolet irradiation decomposed alkyl l-propenyl
disulfide (methyl propenyl disulﬁde, propyl propenyl disulfide) into
dimethylthiophenes, saturated disulfide, minor quantities of anti-unsaturated

monosulﬁde, and saturated trisulﬁde as illustrated in equations 1 and 2.

Table 12. Compounds identiﬁed in the supercritical CO2 extract of onions

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

No a Mass b
1k compound weight mass spectral data Ref
1 760 thiopropanal S-oxide 90 92(2), 91(1), 90(55), 41(100), 3,
42(30), 45(20), 48(18), 73(16), 13,
43(10), 44(6), 72(5), 47(4), 14
_ 75(2), 74(2). _
2 768 dimethyldisulﬁde 94 96(8). 95(1), 94(90), 45(100), 5,7,
79(60), 46(52), 47(38), 65(32) 12,
13
3 777 2- methyl pentanal 100 100(40), 43(100), 5§(75), 2,4
85(45), 57(35), 41(22), 55(15),
71(1 1).
4 867 3,4- dimethylthiophene 112 111(100), 114(10), 113(8), 9,4
112(76), 97(50), 5,(2) 2,
69(22), 59(22), 39(22), 77(12), 12
71(10), 76(9).
5 872 diallylsulﬁde 114 116(3), 115(2), 111““), 8,
(di-l-propenyl sulﬁde) 99(100), 113(41), 41(40), 12
45(30), 39(22), 71(18), 59(18),
.5 84(10.2), 65(5).
6 895 methyl propyl 122 124(7), 123(2), 122(7T17, 9,
disulf id e 80(100), 43(70), 41(50), 39(40), 12,
45(38), 47(22), 64(22), 61(17), 13
105(8). _
7a 902 methyl l-propenyl 120 122(20), 121(12), 120(87), 1,
disulfide" 45(100), 39(40), 75(40), 47(25), 10
12.5). 12
7b 910 methyl l-propenyl 120 123(9),122(2),120(100), 1,7
disulfide“ 45(90),105(76),39(70),75(38),10
47_2 25.)
8 931 dimethyluisulﬁde 126 128(18),127(10),126(100), 9,12
45(61), 79(59)8, 47(30), 46(27),
111(22) 64(18 .840)
9 1010 methylmethane 126 128(25), 127(10), 126(75), 2,12
thiosulfonate 47(100), 45(95), 81(75), 63(65),
79(55), 64(30). _
10 1023 dipropyldisulfidc 150 152(5), 151(2), 150(45), 4,6
43(100), 45(80), 75(52), 73(47), 12,
47(41), 59(41), 108(22), 39(18), 13
66(15).
11a 1050 l-propenylpropyl 148 150(9), 149(3), 148(100), 10,1
d1ulf41(100), 42(25), 44(60 ), 2,
__ 106(57), 72(18), 64(20), 38(13). 13
11b 1099 l-propenylpropyl 148 150(10), 149(7),148(100), 10,1
disulfide“ 43(100), 42(80), 44(62), 2,
), 73(35), 47(35), 72(40), 13

 

 

 

 

106(54
64(38), 37(13).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

12a 1113 3-vinyl-l,2—dﬁhi- 146 148711), 147(7), 146(62.5), 10,1
4-ene* 45(100), 39(55), 113(42.5), 2,
73(32), 82(30), 101(17.5), 13
59(12), 147(8).
12b 1117 3-viny1-l,2-dithi- 146 148(11), 147(7), 146(72), 10,
5—ene* 45(100), 39(47), 113(37.5), 8
73(32), 41(32), 82(26), 101(21), 12
59(21), 131(13). _
13 1143 methylpropyl 154 156(22), 155(10), 154(100), 4, 6,
trisulﬁde 41(85), 43(77), 47(54), 112(45), 12
138(21), 45(18), 79(18), £07).
14a 1153 methyll-propenyl 152 154(18), 153(10), 152(100), 6,12
trisulﬁde" 45(95), 88(48), 73(42), 79(33),
47(27), 39(27), 41(18), 64(16),
_ 103(10). _
14b 1158 methyl l-propenyl 152 154(12), 153(6), 152(65), 8,6,
m'sulﬁde" 45(100), 88(43), 73(42), 39(30), 12
79(23), 47(18), 41(15), 64(13),
_ 105(10 .
15 1205 dimethyltetrasulfide 158 160(20), 159(9),15§(100), 2, 6,
45(100), 79(80), 64(38), 12
47(22.5), 46(21), 94(18), 111
#_ (12.5), 61(6), 48(6).
16 1213 3,4 dimethyl-2,5- 128 130(7), 129(5), 128(53), 2,12
dihidrothiophene 45(100), 43(82), 41(62), 39(58),
85(30). 73(28). 99(25). 55(23).
112(10), 81(10), 67(17).
17a 1325 l-propenylpropyl 180 182(22), 181(15), 180(100), 4,12
trisulﬁde" 45(100 ), 145(60), 106 (46)
74(38), 73(37. 5), 39(37.,5)
83(33_), 116(30), 151(22), 59(7)
18 1342 dipropyltrisulﬁde 182 184(8),183(5),182(97), 2,
43(100), 75(68), 45(23), 39(22), 6,
98(10), 131(8), 117(5). 12
17b 1356 l-propenylpropyl 180 182(20), 181(18), 180(100), 6,
trisulfide“ 45(100), 73(78), 43(55), 41(48), 12
115(42), 116(39), 75(30),
_ 74(30.5), 47(26), 87(12), 138(7).
19 1364 diallyltn'sulﬁde 178 180(10), 179(8), 178(72), 7,8,
113(100), 41(100), 45(90), 12
73(58). 58(48). 79(34). 99(28).
61(28), 52(22), 133(20).
20 1505 methyl3,4—dimethyl- 190 192(14), 191(14), 190(78), 11,
2-thienyldisulﬁde 143(100), 59(8.1), 41(47), 12
45(47), 99(33), 111(20), 67(20),
65(18).

 

b

a e

Kovat index; m/z with intensity in parentheses. *may be isomer Brodnitz et al, 1969; 2.Boelens et al,
1971', 3. Brodnitz and Pascale. 1971; 4.Schreyen et al, 19765. Heller and Milne.1980; 6. Wu ct al. 1990; 7.
Vernin et al. 1986: 8. Yu et al, 1989; 9. Weast and Graselli. 1989; 10. Kallio and Salorinne, 1990; 11. Kuo
and 1101992; 12. Sinha ct 111.1992; 13. Oshumi, 1993. ‘

 

CH3 -CH=CH-S-S-R - BBQ-C113 + R—S-S-R + 1123 (1)
s

CHI-CHzCH-S-S-R -* CHa-CH=CH-S-R + R-S-S-S-R + (2)
CI-I,-CH=CH-S-CH=CH-CI-I3
(R: methyl or n-propyl)

However, further investigation by headspace analysis of crushed onion showed
that thiophenes already present in freshly cut onion as well as after processing -
such as in a steam distilled onion. The authors also found that of the thiophenes
formed, the major product is 3,4 dimethylthiophene, less than 10% 2,4
dimethylthiophene, and only a trace (<1%) of the 2.5-dimethyl is formed. In this
experiment only trace amount 3,4 dimethylthiophene was found in SC-CO2
extract. This could be due to a low level of thiophene in freshly cut onion,
relatively mild temperature of extraction (37°C), or relatively low Gas
Chromatography analysis (30 -150°C).

Monosulﬁde: Only diallyl sulﬁde of monosulﬁde compounds was found in SC-
CO2 onion extract. Its structure was conﬁrmed by comparing mass spectral
pattern to mass spectral published by Yu et a1 (1989) and Stenhagen (1969).
According to Boelens et a1 (1971), the disproportionation of unsaturated disulﬁde
in vitro gives rise to the formation of unsaturated monosulﬁde.

Disulﬁdes: Seven disulﬁdes were found in onion extract by comparing their mass
spectral to the published data. The compounds were dimethyl disulﬁde (Heller
and Milne .1980), methyl propyl disulﬁde (Weast and Graselli, 1989), methyl 1-
propenyl disulﬁde and its isomer (Brodnitz et al., 1969), dipropyl disulﬁde
(Schreyen et al., 1976), l-propenyl propyl disulﬁde (Brodnitz et al., 1969), 3-vinyl-
1,2-dithi-4ene and its isomer 3-vinyl-l,2-dithi-5-ene (Brodnitz et al., 1971; Kallio
and Salorinne,.1990), and methyl 3,4—dimethyl-2-thienyl disulﬁde (Kuo and Ho,

1992). Brodnitz et al. (1971) using IR and NMR analysis, reported diallyl
thiosulﬁnate to be a major constituent of fresh garlic extract. These authors
indicated that during gas chromatography diallyl thiosulﬁnate undergoes
dehydration, forming two isomeric disulﬁdes; 3-vinyl-1,2-dithi-5-ene and 3-vinyl-
1,2-dithi-4-ene. These compounds also tentatively identiﬁed by Kallio and
Salorinne (1990) n the head space of crushed onions. In the present study GC-
MS analysis of SC-CO2 extract failed to detect the presence of diallyl
thiosulﬁnate. However, the dehydration products, dithienes, were found in a
relatively low concentration.

Trisulﬁdes: Seven trisulﬁdes were identiﬁed in this experiment by comparing their
mass spectral patterns to published data. They were dimethyl trisulﬁde (Weast
and Grazelli, 1989), methyl propyl trisulﬁde (Schreyen, 1976), methyl l—propenyl
trisulﬁde and its isomer (Wu et al., 1990), dimethyl trisulﬁde, (Weast and Grazelli,
1989), dipropyl trisulﬁde (Boelens et al, 1971), l-propenyl propyl trisulﬁde and its
isomer (Schreyen, 1976), and diallyl trisulﬁde (Y 11 et al, 1989).

Tetrasulﬁdes: The ﬁrst dimethyltetrasulﬁdes identiﬁed in onion, was found in SC-
CO2 extract. The identiﬁcation of this compound was based on mass spectral of
synthetic tetrasulﬁde published by Boelens (1971).

Flavor differential of onion extracts

Peak area and relative abundance of ﬂavor compounds detected by Gas
chromatography was presented in Appendix 8. From 20 compounds (including
the isomers) identiﬁed in the SC-CO2 extracts (Table 12), only 15 compounds
(including the isomers) could be measured accurately enough. Five compounds
(dimethyl disulﬁde, 3,4-dimethylthiophene, diallyl sulﬁde, methyl methane
thiosulfonate, methyl 3,4-dimethyl-2-thienyl disulﬁde) were present in trace

amount so they were excluded from the quantitative calculation.

Peak area (8)

100

 

 

 

 

 

 

 

 

 

 

 

 

 

 

15
4 U Yellow Sweet
0 Spartan Banner #-
IZ —-
9 ﬂ _.
5
5
6 _— I )-
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a 4 a
3 ~ /~ / /- ~
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1 2 3 4 5 6 7 8 9101112131415

Flavor compound

ﬁgure 28. Diﬂ‘erential ﬂavor proﬁle ofonion
cvSpaerBannerarﬂchellowaeet.

1. thiopropanal S-oxldo 2. 2-methyl pentanal 3. 6methyl pro yl disulfide

4.11101 yl1-propenyldlsulﬂdo 5. dlmethyl trlaultlde 6.pdlpro yld sulfide

7. pro yI1-propenyldlsultlde83,-ethyl-12-dlthl-5-eno 9. math ylypr trlsultlde

10.111 yl1-propenyltrlsulﬂde 11. dimethyl tetrasulﬂde 12.3 ,Hlmethyrés-dihydrothlophene
13.propyl1-propenyltrlsulllde 14. dlpropyl trlsultlte 15. diallyl trlsultlde

101

The relative abundance of each ﬂavor compound in Spartan Banner and
Yellow Sweet onion were shown in Figure 29. In both cultivars, eight major
compounds presented in a proportion clearly above the others (>5%) including
thiopropanal S-oxide (1), methyl propyl disulﬁde (3), dimethyl trisulﬁde (5),
propyl l-propenyl trisulﬁde (13) and dipropyl trisulﬁde (14). Except for dimethyl
tetrasulﬁde, the same result was reported by Kallio and Salorinne (1990).

Effect of transpeptidase on ﬂavor proﬁle

Total peak area of 15 ﬂavor compounds of transpeptidase treated Spartan
Banner onion macerates increased by about 60% from that of control. The ﬂavor
proﬁle showed an increase in relative abundance of some compounds such as
thiopropanal S-oxide(1), dipropyl disulﬁde (6) and methyl l-propenyl disulﬁde (4)
propyl l-propenyl disulﬁde (7), methyl l-propenyl trisulﬁde (10), and propyl 1-
propenyl trisulﬁde (13). The increase in relative abundance of compounds listed
above were 2.7 to 4.3%, 6.7 to 9.4%, 5.8 to 11.1%; 4.3 to 9.0%; 9.5 to 16.3% and
13.1 to 22.5%, respectively (Figures 30 and 31). The increase in trans-l-propenyl
containing ﬂavor compounds in transpeptidase treated onion extract compared to
control were higher than that of the remaining ﬂavor compounds.

Total peak area of 15 ﬂavor compounds in transpeptidase treated samples
of Yellow Sweet onion was only 20% higher than the blank. Like Spartan
Banner cultivar, the increase in relative abundance of dipropyl disulﬁde (6),
methyl propyl trisulﬁdes (9), methyl l-propenyl disulﬁde (4), propyl l-propenyl
disulﬁde (7), methyl l-propenyl trisulﬁde (10), and propyl l-propenyl trisulﬁde (13)
were observed in transpeptidase treated Yellow Sweet onion (Figures 32 and 33).
The increase in relative abundance of ﬂavor compounds in transpeptidase treated

macerated onion from that of control may be due to the increasing of availability

Peak area (S)

102

 

25

 

Control

a Transpeptidase

 

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Flavor compound

ﬁgure29. Eﬁ'ectofhanspeptidaseonfhvorproﬁle
donioncvSpartanBarln;

«1. till ropanal S-oxlde 2. 2-mothyl pentanal 3. methyl propyl dlsulﬂde

*4. met yI1-proponyl disulfide 5. dlmothyl trlsultldo “6. derop ylrdlsultlde

*7. p yl 1-propenyl disulfide 8. 3440*513-dth-5me >9. methyl prorltrlsulﬂde

.10, "101g yI1-propenyl trIsulﬂde 11. dlm Itetrasumda 12. 3 ,4-dlrxethy- ,S-dlhydrothlophene
313.propyl1-proponyltr|sulllde 14. dipropyyl trlsultlte 15. diallyl trlsulﬂde

a Peal: area [S]

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

200
Z Spartan Banner
150 % l
g x Z
4 2 4
.. —¢—-¢——2 4—
2 4 2 2
o /7}I // g%% / 744.22er
7 ‘44 Q
2
-1004..a-.........
123456789101112131415
Compounds
Figure30. Eﬁ'ectoftrarqrept’ﬂaseandalliinme
onpeakareachangecomparedtnconunl.
1.1hIopropanal S-oxlde 2. 2-methyl pentanal 3. methyl pro yI dleulflde

4. methyl 1-propenyl disulfide 5.dlmethyltrlsul1|de 6. dlpropyl deulﬂde
oyl1-propenyldlsulﬂde 8. 3-et «651-, -1 ,2-dIthI-5-ene 9. methylp ItrIsuIIIde

10.111r yI1-propenyltrleulﬂde 11. dlm yl tetrasulﬂde 12.3 ,4-dlmethytzs-dlhydrothlophene

13.propyl1-propenyltrleulllde 14. dlpropyl trleulﬂte 15.dlallyltrlsul1|de

Peak area (S)

104

 

25
Control
I] Transpeptidase

 

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12 3 4 5 6 7 8 9101112131415

 

 

 

Flavor compound

Figure31. Effectoftranspeptidmeononbntlavorproﬁle
ofonioncheIowaeet.

1. 1111 ropanal S-oxlde 2. 2-methyl pentanal 3. math ro dlsultlde

4. m yI1-propenyldlsumde 5. dlmethyl trleumde 6. dlprogy'ld sglﬂde

7. pro yl 1-propenyldlsul1lde 8. 3-eth l-1,2-dlthl-5-ene 9. methyl pro ltrlsulﬂde

10. m yl1-propenyltrlsulﬂde 11. dlmet yl tetrasulllde 12.3,4-dlmethyfzs-dihydrothlophene
13. propyl 1-propenyl trlsulllde 14. dlpropyl trIeultlte 15. diallyl trleulﬂde

1 Peak ueaelrange [x]

105

 

200

 

Z Yellow Sweet

 

 

150

 

 

 

100

 

 

 

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Compounds

P1gure32. Eﬁectoftrarqrept'llaseandalliinase
onpeakareachangecompmedmcontml.

1. thl ropanal S-oxlde 2. 2-methyl pentanal 3. methyl pro yl dleulﬂde

4. m yI1-propenyldleulllde 6. dlmethyl trlsulllde 6. dlpropyl d sulfide

7. pro yl1-propenyldleultlde 8. 3-eth l-1,2-dlthl-5-ene 9. methyl pro ltrlsulﬂde

10. m yl1-propenyltrlsulﬂde 11. dlmet yl tetrasulﬂde 12.3,4-dlmeth - s-dlhydrothlophene
13. propyl 1-propenyl trlsulllde 14. dlpropyl trleulflte 15. diallyl trlsultlde

106

of ﬂavor precursors. Most prominent increasing of l-propenyl containing sulfur
compounds in transpeptidase treated samples compared to other compounds may
be due to a relatively higher level of y-glutamyl-S-l-propenyl-L-cysteine sulfoxide
in onion sample. Shaw et al. (1989) showed that of total y-glutamyl peptides
present in onion, the major proportion is y-glutamyl-S-l-propenyl-L-cysteine
sulfoxide at levels between 1.24 and 2.18 mg/ g fresh wt, equivalent to 5-6 umole/g
fresh wt or about 50-60% of total peptide compounds in fresh onion (Matikkala
and Virtanen, 1967).

The effect of transpeptidase on onion ﬂavor proﬁle was shown by a shift of
major components from methyl propyl disulﬁdes (3), dipropyl disulﬁdes (6), methyl
l-propenyl trisulﬁde (10), dimethyl tetrasulﬁde (l 1) and propyl l-propenyl
trisulﬁde (13), into new major components of methyl-l-propenyl disulﬁde (4),
dipropyl disulﬁde (6), propyl-l-propenyl di sulﬁde (7), methyl-l-propenyl trisulﬁde
(10) and propyl l-propenyl trisulﬁde (13). This increase in l-propenyl
containing ﬂavor compounds may contribute to the alteration of ﬂavor

composition of enzyme treated samples and their overall aroma.

Characteristic ﬂavor of onion

Various ﬂavor compounds have been suggested as imparting characteristic
ﬂavor to raw and processed onion. The odor threshold of ﬂavor compounds were
all low and similar (Boelens et al., 1971). Yamanishi and Orioka (1955) considered
the sweetness of cooked onions to be due to propanthiol. Galetto and
Bednarczyk (1975) found that methyl propyl disulﬁde, methyl propyl trisulﬁde,
and dipropyl trisulﬁde to make the greatest contribution to the ﬂavor of onion oil.
The ﬂavor of raw and fresh onion has been attributed to alk(en)yl thiosulﬁnates
(Brodnitz et al, 1973) and thiosulfonates (Boelens et al, 1971). Characteristic

ﬂavor of steam distilled onion oil was considered to be mainly due to propyl and

107

l-propenyl di and trisulﬁde, while fried onion odor was considered to result from
dimethylthiophene formation.

Almost all of the compounds described above were found in ﬂavor extract
of control or transpeptidase treated onion macerates. The shift in relative
abundance of ﬂavor compounds (mainly of l-propenyl containing polysulﬁde
compounds) due to transpeptidase action may affect the overall ﬂavor. Since the
taste sensation perceived is a complex mixture of sweetness, pungency, bitterness,
and volatiles, correlation between the relative abundance of the compound being

measured and ﬂavor perception can provide a valuable information.

108

SUMNIARY AND CONCLUSION

In this study 15 onion cultivars/selections were evaluated for relationship
between alliinase activity and pyruvate concentration. Pyruvate which has been
used as the measure of pungency showed a positive correlation (1:0.95) with
alliinase activity. Depending on pyruvate content, ten cultivars/ selections were
catagorized as pungent-cooked onion and 5 cultivars as sweet-salad type onions.

Two onion enzymes, alliinase and y-glutamyl transpeptidase, were
extracted, puriﬁed and characterized. They were also studied for storage stability,
and their effect on onion ﬂavor development. During 32 weeks storage at 20°C,
the activity of alliinase showed a rising trend while the activity of transpeptidase
in sprouted onions after 12 weeks showed a gradual rising reaching peak activity
of 2.4 Unit/mg protein. Pyruvate concentration also showed a rising trend during
32 weeks storage.

A thirty eight and twentyseven-fold puriﬁcation of alliinase and y-glutamyl
transpeptidase were achieved with a recovery of 2% and 2% respectively.
Homogeneity of ﬁnal enzyme preparations was shown by one single protein band
following staining with commasie blue R. The isoelectric point of alliinase and
transpeptidase determined by isoelectricfocusing were 4.8 and 5.2 respectively.
The Km of alliinase toward S-methyl, ethyl, propyl, l-propenyl substituted L-
cysteine sulfoxide were 16.9, 6.03, 3.02, and 2.14 mM repectively, whereas Vm,
showed little variation with respect to the chain length of the S-substituent. The

Km and Vum of transpeptidase with y-glutamyl p-nitro aniline as a substrate were

12.17 mM and 1.37 moles/2 hr respectively.

109

Alliinase had a pH optimum of 7.5 and was stable within a wide range pH
of 5.5-8.0. Transpeptidase had a pH optimum of 9.0 and was relatively stable at a
narrow range pH (8.0-9.5). Both enzymes had optimum activity at 40°C. Their
activation energies (Fa ) were 16.6 and 15.8 kJ/mole respectively. Free amino
acids and S-alk(en)yl susbtituted L-cysteine showed a competitive inhibition of
alliinase, however due to their function as glutamyl acceptor, free amino acids and
their derivatives stimulated the activity of transpeptidase.

Molecular weights of native alliinase and transpeptidase were estimated to
be 200 and 120 kDa, respectively. SDS-PAGE of reduced alliinase and
transpeptidase indicated a single band corresponding to protein marker band
ranging from 45 to 66 kDa and 116 to 205 kDa, respectively. SDS-PAGE and gel
permeation of reduced alliinase suggested that this enzyme consisted of four
subunits with estimated molecular weight of 50 kDa.

Gamma glutamyl transpeptidase in conjunction with exogenous alliinase
enhanced pyruvate production 2.5-fold greater than that of the control was
obtained in y-glutamyl transpeptidase treated macerated onions held for 20 hr at
37°C. Relative abundance of ﬂavor compounds in Spartan Banner, a pungent
onion, varied from a Sweet Yellow salad type onion.

The effect of y-glutamyl transpeptidase on onion ﬂavor proﬁle was shown
by a shift of major components from methyl propyl disulﬁde, methyl propenyl
trisulﬁde, dimethyl tetrasulﬁde and propyl l-propenyl trisulﬁde, into new major
components, methyl l-propenyl disulﬁde, dipropyl disulﬁde, propyl l-propenyl
disulﬁde, methyl l-propenyl trisulﬁde, and propyl l-propenyl trisulﬁde. This
increase in l-propenyl containing ﬂavor compounds may affect the overall ﬂavor

proﬁle of transpeptidase treated onion extracts.

 

110

In conclusion, transpeptidase in conjunction with onion alliinase enhanced
pyruvate formation in macerated onion and affected a change in overall ﬂavor

proﬁle of supercritical carbondioxide onion extracts.

 

BIBLIOGRAPHY

BIBLIOGRAPHY

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Albrand, M., Dubois, P., Etievant, P., Gelin, R. and Tokarska, B. 1980.
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lll

 

 

 

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Boelens, M., de Valois, P., Wobben, H. J. and van der Gen, A. 1971. A volatile
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Brodnitz, M. H., Pascale J. V. and Vock, M. H. 1973. Propyl propene
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Brodnitz, M. H., Pascale J. V. and Van Derslice, L 1971. Flavor components of
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APPENDICES

124

Appendix 1

Synthesis of Standard Compounds (Belle, 1972)
(a) S-methyl-L-cysteine sulfoxide

This compound was synthesized from commercially available S-methyl-L-
cysteine by oxidation (Stoll and Seebeck, 1951). A suspension containing 1.18
parts of (:1:) S-methyl-L-cysteine in 1.2 parts of H20 was prepared. One part by
volume of 25% 11202 was added with constant stirring to the suspension. The
mixture was heated to 50°C and stirred until it became clear. The mixture was
allowed to stand at room temperature for 24 hr and then ﬁltered to remove small
amount of insoluble materials. The ﬁltrate was heated to 50°C and added with
3.3 volume of acetone. After standing for 24 hr at room temperature, (:1:) S-
methyl-L-cysteine sulfoxide crystals were separated by ﬁltration. The crystals
were dissolved in H20 and then recrystallized from acetone. The resulting crystal

were dried over anhydrous CaSO4 and stored in the freezer (-20 °C) until further

use.

(:1:) S-ethyl-L-cysteine sulfoxide
Method used to synthesize (1) S-methyl-L-cysteine sulfoxide was used to

synthesize S-ethyl-L-cysteine sulfoxide from commercially available S-ethyl-L-
cysteine.

(t) S-n-propyl-L-cysteine sulfoxide

The synthesis of this compound involved a two step procedure:

Preparation and isolation of (:1:) S-propyl-L-cysteine and oxidation of the latter to

the sulfoxide.

 

125

(x) S-n-propyl-L-cysteine was prepared by the method described by
Schwimmer ( 1970). Sodium (0.92 g) was added to a suspension of 1.75 g (0.01
mole) of L-cysteine hydrochloride monohydrate in 30 ml of absolute ethanol
within 30 min at room temperature. When Sodium was about to disappear, 0.79 g
(0.01 mole) of n-propyl chloride was added with stirring. After 2 min the mixture
was diluted with water until it was clear and the solution was adjusted to pH 5.0-
5.5 with glacial acetic acid. The resulting suspension of (:1:) S-n-propyl-L-
cysteine was stored overnight at 0-4°C ﬁltered and washed with cold 1120 and
ethyl ether. The precipitate was dried over CaSO4

(1) S-n-propyl-L-cysteine sulfoxide was obtained by oxidation 1.18 parts
by weight (:1:) S-n-propyl-L-cysteine in 1.2 parts of H20 with one part of 25%
H202 with constant stirring . The suspension was heated to 50°C and stirred until
it became clear. The mixture was allowed to stand for 24 hr at room temperature
and then ﬁltered to remove some insoluble materials. The ﬁltrate was heated to
50°C, added with 3.3 volume of acetone and allowed to stand for 24 hr at room
temperature. The (a) S-n-propyl-L-cysteine sulfoxide formed are separated and

then recrystallized twice from acetone. The crystals were then dried over CaSO4
and stored in freezer (-20 °C) until further use.

(1) §-( l-prognyl )-L-cysteine sulfoxide

The synthesis of this compound involved a two step procedure:
preparation and isolation of (a) S-(l-propenyl)-L-cysteine and oxidation of the
latter to the sulfoxide. The synthesis of S-(l-propenyl)-L-cysteine was based on
the base isomerization procedure of Price and Synder (1962) for isomerization of
allylic sulﬁdes and of Carson and Wong (1963) for the isomerization of (:)S-allyl-
L-cysteine. Potassium butoxide (0.5 g) in 50 ml of dimethyl sulfoxide was added
to a suspension of 2.0 g of S-allyl cysteine in 50 ml deionized distilled water. The

mixture was stirred for 24 hr at room temperature and ﬁltered to remove some

 

126

insoluble materials. The ﬁltrate was concentrated by ﬂash evaporation and the
concentrate allowed to crystallize. The crystals were separated and recrystallized
from water twice. The resulting (:1:) S-(l-propenyl)-L-cysteine crystals were dried
over anhydrous CaSO4.

(:1:) S-(l-propenyl)-L-cysteine sulfoxide crystals was synthesized by
preparing a suspension containing 1.18 parts of (:1:) S-(l-propenyl)-L-cysteine in
1.2 parts of HQO . One part by volume of 25% H202 was added dropwise to the
suspension with constant stirring. The mixture was allowed to stand for 24 hr at
room temperature and then ﬁltered . The ﬁltrate was then heated to 50°C and
immediately 3.3 volume of acetone was added and then the mixture allowed to
stand for 24 hr at room temperature. The crystals formed were separated by

ﬁltration. After two recrystallization from acetone, the crystals were dried over
anhydrous CaSO, and stored in the freezer (-20 °C) until further use.
y-L-glutamyl S-methyl-L-cysteine sulfoxide (GMCS)

Synthesized of this compound was carried out according to the method
described by Orlowski and Meister (1965) using commercially available
substances. L-glutamic anhydride (13.2 g; 0.05 mole) was suspended in 35 ml of
glacial acetic acid, 0.05 mole of methyl-L-cysteine were added. The mixture was
heated at 60-65° C for 30 min and then evaporated under reduced pressure at
25°C to yield a crude product which was dried in a vacuum desiccator over
CaCl2 and NaOH. The product was ground to a ﬁne powder and dissolved in
250 ml methanol. Hydrazine hydrate (80%; 0.1 mole) was added, and the
solution was ﬁltered to remove a small amount of insoluble material. It was
allowed to stand at room temperature (26°C) for 48 hours, during which time a
crystalline product separated. The crystals were washed on a Buchner funnel
with cold distilled water and with ethanol, after which they were dried in a

vacuum over CaClz. The dried material was suspended with vigorous agitation in

 

127

10 parts of cold 0.5 M HCl, and the mixture was ﬁltered into a separatory funnel.
Free methyl cysteine was removed by extraction with 5 volumes ethyl acetate,
after which the aqueous layer was quickly separated out and brought to pH 7.0
by addition of 1 M NaZCO3. A crystalline product promptly separated, washed
with water and ethanol, and then dried in a vacuum desiccator over CaClz.

Sulfoxides form was obtained by oxidizing cysteine derivatives with peroxide

Jamaam; "2 '1

 

Absorbance at 420 nm

1.

 

128

Appendixz

Standard curve of pyruvate for alliinase allay,

 

 

y-15818e-2+0.92596x 1142:0393

 

 

Alla orbance [S4 llnm]

129

Appendix3

Standardcm'veofanilinefortranspeptidaseaamy

 

0.5

y: -a952293+4.9215.ex 34230.98

 

 

 

 

0.0.,.,.,.r
o 20 40 so so

Aniline (ymole)

I
100

120

Abs orbance at 540 run

130

M4

Standardctn'veofbovinesermnalbumin(BSA)

forpromindetermhraﬁonanwry

method)

 

0.8

0.6-

0.4 -

0.2-

 

0.0

y=7.3123e3+2.4000oex 5142:0907

 

' I
100 200

Protein (pg)

 

300

‘P. :ﬁitt'n-an-‘l- 3. , .

 

I31

AppendixS

Directplotofsubstrateooncenuaﬁon(S)vsrateof
reaction(V)forkineticcomtantdetermination.

 

5 ..

4.5 -

”.s‘ 4 "

g 3.5 --
O

8 as --
a
>

a 2 --
3

1.5 -‘-

ethyl-L-cys-sulfoxide

 

I I
1 I

d-
-

-0.02 -0.01 0 0.01 0.02 0.03 0.04 0.05 0.06

Substrate concentration (M)

 

loo Molecular weight [MW]

132

Appearix6

andMWofpmteinmarkersforalliirme.

 

6.0 '

y=7.1242-12979x 1142:1111)

 

 

 

 

4.6 ' I V I ' I '
1.0 1.2 1.4 1.6 1.8

Elution volume (V/Vo)

2.0

loo moleculu weight

5.8

133

W7

arllMWofprominmarkersfortlampeptilme.

 

5.6‘

5.4-

5.2-

5.0-

4.8-

4.6-

 

4.4

pence-0.199461 9‘230.“

 

‘

-
q
I
-
.

Elution volume (VNo)

 

APPENDIX 8

134

Peak area of ﬂavor compounds of onion

 

 

 

No. Compound MW Area‘ Relative Abundance (%)
vs vs", s3 88,, YS vs", SIT s3...
1. Thiopmpanal S-oxide 90 14630 11295 38.41 105.04 6.43 4.87 2.69 430
2. 2-rnethyl pentanal 100 32.88 51.11 4051 56.71 1.45 221 2.83 232
3. methyl propyl di-S 122 181.68 75.82 107.06 43.98 7.98 3.27 7.49 1.80
4. methyl l-propenyl di-S 120 67.63 185.43 83.03 27134 2.97 8.00 531 11.10
s. dimethyl 111-8 126 207.80 121.45 86.18 9450 9.13 5.24 6.03 3.87
6. dipropyl 111-8 150 107.10 139.70 189.79 426.43 4.71 6.03 6.73 9.45 -
7. propyl l-propenyl 01-8 148 36.04 7454 61.75 104.12 1.58 5.22 432 8.96
8. 3-ethyl-12-dithi-5ene 146 11232 8821 67.18 10225 4.94 3.81 4.70 4.19
9. methyl propyl di-S 154 121.48 185.61 48.61 97.81 534 8.01 3.40 4.00
10. methyl l-propenyl tri-S 152 216.87 350.17 135.15 25428 8.93 15.11 9.45 16.32
11. dimethyl tetra-S 158 288.20 31637 132.61 127.08 12.66 13.65 9.28 520
12. 3,4-dimethyl thiophene 128 99.40 61.03 9294 131.96 437 2.63 6.50 5.40
13.propyl-l-propenyltri-S 180 281.88 463.79 187.64 539.16 1239 20.01 13.12 2207
14. dipropyl his 182 146.61 54.46 83.89 9923 6.53 235 5.87 4.06
15. diallyl til-s 178 127.23 36.78 74.93 8937 559 159 5.24 3.66

 

" Average of 2 extractions.

 

.- --uma} -l

 

"71111111111111111711“