levels and thermostability of peroxidase extracted from some

Transcription

LEVELS AND THERMOSTABILITY OF PEROXIDASE
EXTRACTED FROM SOME VEGETABLES
By
Suha Osman Ahmed Osman
B.Sc. (Agric.) Honours 2003
University of Khartoum
A dissertation submitted in partial fulfillment for the requirement of the
degree of Master of Science in Food Science and Technology
Supervisor:
Dr. Babiker El Wasila Mohamed
Department of Food Science and Technology
Faculty of Agriculture
University of Khartoum
November – 2005
DEDICATION
To my dear family
With love and respect
Suha
i
Acknowledgement
My faithful thanks to Allah who gave me health and strength throughout
this study.
I profoundly acknowledge and appreciate my supervisor, the
prominent scientist Dr. Babiker El Wasila Mohamed for his tireless support
during the course of the study. However, whilest his assistance has been
invaluable, responsibility for any errors rests solely on the student.
I would love to acknowledge and appreciate Dr. Widad Elshafie for her
support and help during the practical.
I am also indebted to my sincere thanks to the staff of Department of
Food Science and Technology, Faculty of Agriculture, University of Khartoum
for their cooperation and help.
My deep thanks are due to all those friends and acquaintances who
have encouraged me to finish my work. Particular thanks to my intimate
friends for their unforgotten help.
I would like to extend my thanks to staff of Food Research Centre
(FRC), Shambat for their help and support.
Last but not least my thanks and grateful appreciation go to my family
for their support.
ii
ABSTRACT
This study aimed to extract peroxidase enzyme from some
vegetables and subsequency to study its heat stability under various
conditions. Unspecified varieties of potato, carrot, eggplant and tomato
were selected as enzyme sources. Peroxidase was found in all vegetables
investigated at different levels.
Peroxidase enzyme with reasonable enzymic activity was extracted
and detected at various pH values namely 5.0, 6.0, 7.0 and 8.0. Relatively
higher levels of peroxidase activities were extracted at pH 5.0 from
potato and tomato while those with higher levels from carrot and
eggplant were extracted at pH 6.0. Potato tuber was shown to contain the
highest level of peroxidase at all pH values investigated, whereas carrot
had the lowest peroxidase levels at the same pH values. Soluble
peroxidases extracted from the four vegetables were subjected to thermal
inactivation at 60, 70, 80 and 90ºC, and varying duration of heating times
2, 4, 6, 8 and 10min under different values of pH 5.0, 6.0, 7.0 and 8.0.
The results showed that the rate of loss of peroxidase activity from
the four vegetables investigated increased with both increase in
temperature and heating time. Biphasic inactivation curves were
observed for the enzymes extracted from all samples, where the initial
heat inactivation is rapid followed by a much slower inactivation periods.
The patterns of inactivation of peroxidases extracted from the four
vegetables were similar. The rate of loss of peroxidase activity was
shown to be pH dependant.
Potato peroxidase was observed to be more stable to heat as it was
iii
not completely inactivated at all tried conditions. A less severe heat
treatment is required to inactivate carrot, eggplant and tomato
peroxidases.
Complete
inactivation
of
carrot
peroxidase
was
accomplished in 4–10 min at 80ºC and in 2–10 min at 90ºC, while
peroxidase inactivation in eggplant required 8 – 10 min at 90ºC, both at
pH 8.0. Complete inactivation of tomato peroxidase required 6 – 10 min
at 90º C and pH 6.0.
iv
‫ﺧﻼﺻﺔ اﻟﺪراﺳﺔ‬
‫ﺃﺠﺭﻴﺕ ﺍﻟﺩﺭﺍﺴﺔ ﺒﻐﺭﺽ ﺇﺴﺘﺨﻼﺹ ﺇﻨﺯﻴﻡ ﺒﻴﺭﻭﻜﺴﺩﻴﺯ ﻤﻥ ﺒﻌﺽ ﺃﻨﻭﺍﻉ ﺍﻟﺨﻀﺭﻭﺍﺕ ﻭﻤﻥ ﺜﻡ ﺩﺭﺍﺴﺔ‬
‫ﺜﺒﺎﺘﻴﺘﻬﺎ ﺍﻟﺤﺭﺍﺭﻴﺔ ﺘﺤﺕ ﻋﺩﺓ ﻅﺭﻭﻑ‪ .‬ﺘﻡ ﺇﺴﺘﺨﺩﺍﻡ ﺃﺭﺒﻊ ﺃﺼﻨﺎﻑ ﻏﻴﺭ ﻤﺤﺩﺩﺓ ﻤﻥ ﺍﻟﺒﻁﺎﻁﺱ‪ ،‬ﺍﻟﺠﺯﺭ‪ ،‬ﺍﻟﺒﺎﺫﻨﺠﺎﻥ‬
‫ﻭﺍﻟﻁﻤﺎﻁﻡ ﻜﻤﺼﺩﺭ ﻟﻺﻨﺯﻴﻡ ‪.‬ﺃﻅﻬﺭﺕ ﺍﻟﻨﺘﺎﺌﺞ ﺃﻥ ﺍﻹﻨﺯﻴﻡ ﻤﻭﺠﻭﺩ ﻓﻲ ﻜل ﺍﻟﺨﻀﺭﻭﺍﺕ ﺒﻤﺴﺘﻭﻴﺎﺕ ﻤﺘﻔﺎﻭﺘﺔ ‪.‬ﺘﻡ ﺘﺤﺩﻴﺩ‬
‫ﻨﺸﺎﻁ ﻤﻘﺩﺭ ﻤﻥ ﺍﻹﻨﺯﻴﻡ ﻓﻲ ﻤﺴﺘﻭﻴﺎﺕ ﻤﺨﺘﻠﻔﺔ ﻤﻥ ﺍﻷﺱ ﺍﻟﻬﻴﺩﺭﻭﺠﻴﻨﻲ ‪ 7.0 ،6.0 ،5.0‬ﻭ‪ .8.0‬ﻜﺫﻟﻙ ﻓﻘﺩ ﺘﻡ‬
‫ﺍﻟﺤﺼﻭل ﻋﻠﻰ ﺃﻋﻠﻰ ﻤﺴﺘﻭﻯ ﻟﻨﺸﺎﻁ ﺍﻹﻨﺯﻴﻡ ﺍﻟﻤﺴﺘﺨﻠﺹ ﻋﻨﺩ ﺍﻷﺱ ﺍﻟﻬﻴﺩﺭﻭﺠﻴﻨﻲ ‪ 5.0‬ﻤﻥ ﺍﻟﺒﻁﺎﻁﺱ ﻭﺍﻟﻁﻤﺎﻁﻡ‪،‬‬
‫ﺒﻴﻨﻤﺎ ﻜﺎﻥ ﺃﻋﻠﻰ ﻤﺴﺘﻭﻯ ﻟﻪ ﻓﻲ ﺍﻟﺠﺯﺭ ﻭﺍﻟﺒﺎﺫﻨﺠﺎﻥ ﻋﻨﺩ ﺍﻷﺱ ﺍﻟﻬﻴﺩﺭﻭﺠﻴﻨﻲ ‪ .6.0‬ﺃﺜﺒﺘﺕ ﺍﻟﺩﺭﺍﺴﺔ ﺃﻥ ﺃﻋﻠﻰ ﻤﺴﺘﻭﻯ‬
‫ﻟﻨﺸﺎﻁ ﺍﻹﻨﺯﻴﻡ ﻓﻲ ﻜل ﻤﺴﺘﻭﻴﺎﺕ ﺍﻷﺱ ﺍﻟﻬﻴﺩﺭﻭﺠﻴﻨﻲ ﺍﻟﻤﺫﻜﻭﺭﺓ ﻭﺠﺩﺕ ﻓﻲ ﺍﻟﺒﻁﺎﻁﺱ ‪.‬ﺒﻴﻨﻤﺎ ﺴﺠل ﺍﻟﺠﺯﺭ ﺃﻗل‬
‫ﻤﺴﺘﻭﻯ ﻟﻨﺸﺎﻁ ﺍﻹﻨﺯﻴﻡ ﻋﻨﺩ ﻜل ﻤﺴﺘﻭﻴﺎﺕ ﺍﻷﺱ ﺍﻟﻬﻴﺩﺭﻭﺠﻴﻨﻲ ﺘﺤﺕ ﺍﻟﺩﺭﺍﺴﺔ‪ .‬ﺘﻡ ﺘﻌﺭﻴﺽ ﺇﻨﺯﻴﻤﺎﺕ ﺒﻴﺭﻭﻜﺴﻴﺩﻴﺯ‬
‫ﺍﻟﺫﺍﺌﺒﺔ ﻭﺍﻟﻤﺴﺘﺨﻠﺼﺔ ﻤﻥ ﻋﻴﻨﺎﺕ ﺍﻟﺨﻀﺭﻭﺍﺕ ﺘﺤﺕ ﺍﻟﺩﺭﺍﺴﺔ ﻟﻠﺘﺜﺒﻴﻁ ﻋﻠﻲ ﺩﺭﺠﺎﺕ ﺤﺭﺍﺭﺓ ‪ 80 ،70،60‬ﻭ‪º 90‬ﻡ‬
‫ﻭﺃﻭﻗﺎﺕ ﺘﺴﺨﻴﻥ ﻤﺨﺘﻠﻔﺔ ‪ 8 ،6 ،4 ،2‬ﻭ ‪10‬ﺩﻗﻴﻘﺔ ﻋﻠﻰ ﺩﺭﺠﺎﺕ ﻤﺨﺘﻠﻔﺔ ﻤﻥ ﺍﻷﺱ ﺍﻟﻬﻴﺩﺭﻭﺠﻴﻨﻲ ‪7.0 ،6.0 ،5.0‬‬
‫ﻭ‪ . 8.0‬ﺃﻅﻬﺭﺕ ﺍﻟﺩﺭﺍﺴﺔ ﺃﻥ ﻤﻌﺩﻻﺕ ﻓﻘﺩ ﺇﻨﺯﻴﻤﺎﺕ ﺒﻴﺭﻭﻜﺴﺩﻴﺯ ﻟﻨﺸﺎﻁﻬﻤﺎ ﻴﺯﻴﺩ ﺒﺯﻴﺎﺩﺓ ﺩﺭﺠﺔ ﺍﻟﺤﺭﺍﺭﺓ ﻭﺯﻤﻥ‬
‫ﺍﻟﺘﺴﺨﻴﻥ‪ .‬ﻜﺫﻟﻙ ﻓﻘﺩ ﻟﻭﺤﻅ ﺃﻥ ﻤﻨﺤﻨﻴﺎﺕ ﺍﻟﺘﺜﺒﻴﻁ ﺒﺎﻟﺤﺭﺍﺭﺓ ﻟﻜل ﺍﻟﻌﻴﻨﺎﺕ ﺍﻟﻤﺴﺘﺨﻠﺼﺔ ﺜﻨﺎﺌﻴﺔ ﺍﻟﻁﻭﺭ‪ ،‬ﺤﻴﺙ ﻴﺘﻡ‬
‫ﺍﻟﺘﺜﺒﻴﻁ ﺍﻹﺒﺘﺩﺍﺌﻲ ﺴﺭﻴﻌﹰﺎ ﻴﺘﺒﻌﻪ ﺘﺜﺒﻴﻁ ﺒﻁﺊ ﻟﻺﻨﺯﻴﻡ‪ .‬ﻭﺠﺩ ﺍﻥ ﻨﻤﻁ ﺘﺜﺒﻴﻁ ﺇﻨﺯﻴﻤﺎﺕ ﺒﻴﺭﻭﻜﺴﺩﻴﺯ ﻤﻥ ﺍﻟﺨﻀﺭ ﺍﻷﺭﺒﻊ‬
‫ﻤﺘﺸﺎﺒﻪ‪ .‬ﻜﺫﻟﻙ ﺘﺒﻴﻥ ﺃﻥ ﻤﻌﺩﻻﺕ ﻓﻘﺩ ﻨﺸﺎﻁ ﺇﻨﺯﻴﻡ ﺒﻴﺭﻭﻜﺴﺩﻴﺯ ﻟﻠﺤﺭﺍﺭﺓ ﺘﻌﺘﻤﺩ ﻋﻠﻰ ﺍﻷﺱ ﺍﻟﻬﻴﺩﺭﻭﺠﻴﻨﻲ ‪.‬ﺃﻅﻬﺭﺕ‬
‫ﻼ ﺘﺤﺕ ﺍﻟﻅﺭﻭﻑ ﺍﻟﺘﻲ‬
‫ﻼ ﻟﻠﺤﺭﺍﺭﺓ ﺤﻴﺙ ﺃﻨﻪ ﻟﻡ ﻴﺘﻡ ﺘﺜﺒﻴﻁﻪ ﺘﺜﺒﻴﻁﹰﺎ ﻜﺎﻤ ﹰ‬
‫ﺍﻟﻨﺘﺎﺌﺞ ﺃﻥ ﺒﻴﺭﻭﻜﺴﻴﺩﻴﺯ ﺍﻟﺒﻁﺎﻁﺱ ﺃﻜﺜﺭ ﺘﺤﻤ ﹰ‬
‫ﺘﻡ ﺘﺠﺭﻴﺒﻬﺎ‪ .‬ﻭﻋﻠﻴﻪ ﻓﺈﻥ ﺒﻴﺭﻭﻜﺴﻴﺩﻴﺯﺍﺕ ﺍﻟﺠﺯﺭ‪ ،‬ﺍﻟﺒﺎﺫﻨﺠﺎﻥ ﻭﺍﻟﻁﻤﺎﻁﻡ ﺘﺤﺘﺎﺝ ﻟﻤﻌﺎﻤﻼﺕ ﺤﺭﺍﺭﻴﺔ ﺒﺩﺭﺠﺔ ﺤﺭﺍﺭﺓ‬
‫ﺃﻗل ‪.‬ﻭﻟﻘﺩ ﻭﺠﺩ ﺃﻥ ﺍﻟﺘﺜﺒﻴﻁ ﺍﻟﻜﺎﻤل ﻟﺒﻴﺭﻭﻜﺴﻴﺩﻴﺯ ﺍﻟﺠﺯﺭ ﺒﺎﻟﺘﺴﺨﻴﻥ ﻴﺘﻡ ﻟﻤﺩﺓ ‪ 10-4‬ﺩﻗﺎﺌﻕ ﻋﻠﻰ ﺩﺭﺠﺔ ﺤﺭﺍﺭﺓ ‪º80‬‬
‫ﻡ ‪ ،‬ﻭﻟﻤﺩﺓ ‪10-2‬‬
‫ﺩﻗﻴﻘﺔ ﻋﻠﻰ ﺩﺭﺠﺔ ﺤﺭﺍﺭﺓ ‪ º90‬ﻡ ﺒﻴﻨﻤﺎ ﺘﺜﺒﻴﻁ ﺒﻴﺭﻭﻜﺴﻴﺩﻴﺯ ﺍﻟﺒﺎﺫﻨﺠﺎﻥ ﻴﺤﺘﺎﺝ ‪ 10-8‬ﺩﻗﻴﻘﺔ‬
‫ﻋﻠﻰ ﺩﺭﺠﺔ ﺤﺭﺍﺭﺓ ‪ º90‬ﻡ ﻭﻜﻼﻫﻤﺎ ﻋﻠﻰ ﺃﺱ ﻫﻴﺩﺭﻭﺠﻴﻨﻲ ‪.8.0‬ﺍﻟﺘﺜﺒﻴﻁ ﺍﻟﻜﺎﻤل ﻟﺒﻴﺭﻭﻜﺴﺩﻴﺯ ﺍﻟﻁﻤﺎﻁﻡ ﻴﺤﺘﺎﺝ ‪-6‬‬
‫‪ 10‬ﺩﻗﻴﻘﺔ ﻋﻠﻰ ‪ º90‬ﻡ ﻭﺃﺱ ﻫﻴﺩﺭﻭﺠﻴﻨﻲ ‪.6.0.‬‬
‫‪v‬‬
LIST OF CONTENTS
Page
Dedication…………………………………………………………………………………..
i
Acknowledgement ………………………….……………………………………………
ii
Abstract ………………………………………………………………………………………
iii
Arabic Abstract …………………………...………………………………………………
v
List of Contents ………………………..…………………………………………………
vi
List of Tables………………………………….……………………………………………
ix
List of Figures …………………………..…………………………………………………
x
CHAPTER ONE: INTRODUCTION………...………………………………
1
CHAPTER TWO: LITERATURE REVIEW…….………………………
3
2.1. Definition and general characteristics of enzymes……………………
3
2.2. Classification and numbering of enzymes………………………..………
4
2.3. Enzymatic browning in fruits and vegetables…………..………………
6
2.4. Biochemistry of higher plant peroxidases………………………………
7
2.5. Natural occurrence of peroxidases……………………………………..……
10
2.5.1. Sources and classification of peroxidases……………………………
10
2.5.2. Localization of peroxidases…………………………………………………
11
vi
2.5.3. Multiple forms (Isoenzymes) of peroxidases………………………
11
2.6. Mechanism of enzymic action of peroxidases …………………………
12
2.6.1. Peroxidatic reactions…………………………………………...………………
13
2.6.2. Oxidatic reactions………………………………………………………………
13
2.6.3. Catalytic reactions…………………………………………….…………………
13
2.6.4. Hydroxylation reactions……………….…………………………..…………
13
2.7. Physiological function of peroxidase………………………………………
16
2.8. Heat inactivation and regeneration of peroxidase……..………………
16
2.9. Effect of peroxidase action on food…………………………...……………
19
CHAPTER THREE: MATERIALS AND METHODS……..………
21
3.1. Materials………………………………………………………………….……………
21
3.1.1. The vegetables samples ……..…………………………….…………………
21
3.1.2. Chemicals…………………………………………………………………..………
21
3.1.3. Preparation of substrates………………………………………..……………
21
3.1.3.1. The hydrogen peroxide solution……………………….………………
21
3.1.3.2. The guaiacol solution…………………………………………….…………
21
3.1.4. Preparation of buffer solutions……………………………………………
21
3.2. Methods……………………………………………………………………..…………
22
vii
3.2.1. Extraction of peroxidase…………………………………...…………………
22
3.2.2. Enzyme assays……………………………………………………………………
22
3.2.2.1. Guaiacol method of assay…………………………………………………
23
3.2.3. Heat treatment…………………………………………………………….………
24
CHAPTER FOUR: RESULTS AND DISCUSSION …………..……
25
4.1 Levels of peroxidase activity………………………………..…………………
25
4.2 Heat inactivation of peroxidase……………………………..…………………
27
4.2.1 Thermal stability of potato peroxidase………………..…………………
28
4.2.2 Theraml stability of carrot peroxidase………...…………………………
34
4.2.3 Thermal stability of eggplant peroxidase …………...…………………
40
4.2.4 Thermal stability of tomato peroxidase…………………….……………
46
4.3 Conclusions and recommendations…………………………...……………
54
4.3.1. Conclusions……………………………………………………...…………………
54
4.4.2 Recommendations ………………………..………………...…………………
55
REFERENCES………………………...…………………………………………………
56
viii
LIST OF TABLES
Table Title
No.
4.1
Peroxidase activity of vegetables investigated ………..………
26
4.2
Heat inactivation of potato peroxidase……………………………
29
4.3
Heat inactivation of carrot peroxidase………….…………………
35
4.4
Heat inactivation of eggplant peroxidase…….…..………………
41
4.5
Heat inactivation of tomato peroxidase………...…………………
47
ix
LIST OF FIGURES
Fig.
Title
No.
2.1
Structure of Ferriprotophyrin III (Protohemin) ……………….
2.2
Proposed scheme for mechanism of peroxidase action in
9
four types of reactions in which the enzyme is involved…
15
4.1
Heat inactivation of potato peroxidase at pH 5.0………….…
30
4.2
Heat inactivation of potato peroxidase at pH 6.0…………..…
31
4.3
32
4.4
33
4.5
Heat inactivation of carrot peroxidase at pH 5.0…………..…
36
4.6
Heat inactivation of carrot peroxidase at pH 6.0…………...…
37
4.7
Heat inactivation of carrot peroxidase at pH 7.0…………...…
38
4.8
Heat inactivation of carrot peroxidase at pH 8.0…………..…
39
4.9
Heat inactivation of eggplant peroxidase at pH 5.0……….…
42
4.10
43
4.11
44
4.12
45
4.13
Heat inactivation of tomato peroxidase at pH 5.0..…………... 48
4.14
Heat inactivation of tomato peroxidase at pH 6.0………….…
49
4.15
Heat inactivation of tomato peroxidase at pH 7.0………….…
50
4.16
Heat inactivation of tomato peroxidase at pH 8.0……………
51
x
CHAPTER ONE
INTRODUCTION
In developing countries fruit and vegetable processing is among
the most important agricultural activities, without questions, this activity
plays an important role in the world of food economy by supplying
wholesome, safe, good quality and acceptable food to consumers
throughout the year. Dauthy (1995) claimed that the deterioration
reaction in fruits and vegetables could be due to enzymic, chemical and,
biological changes. It is well-known that the presence of residual
endogenous enzyme in either raw or processed fruit and vegetable
product may cause a loss of quality during storage. These changes can
affect the texture, colour, flavour and nutritional quality of product (Luh
and Doauf, 1971).
Peroxidase appears to be one of the most heat stable enzymes
present in many fruits and vegetable. Peroxidase is usually the indicator
enzyme of choice in fruit and vegetable freezing operations because of its
high concentration in most plant tissues and its high thermal stability, as
well as its ease of assay. The high thermal stability of peroxidase can be
seen as either an advantage or a problem in food industry. On the one
hand, it provides natural margin of safety in that if peroxidase is
inactivated, it is a reasonable assumption that other quality–related
enzymes have also been inactivated. On the other hand, the reliance on
peroxidase as an indictor may lead to an excessive heat treatment of the
product and cause other quality problems (Anthon and Barrett, 2002).
There is an increasing interest in the study of peroxidases, not only
1
in order to establish their physiological, but also for their possible
industrial and analytical application.
Objective of the study
The objective of this work has been to extend the knowledge of
vegetables peroxidase in particular.
The attainment of the study objective required the following:

The extraction of crude peroxidase enzyme from
some vegetables namely potato, carrot, eggplant and
tomato.

The estimation of peroxidases levels in the four
vegetables named above.

The investigation of thermostability of the extracted
peroxidases
under
various
conditions
temperature and length of heating time.
2
of
pH,
CHAPTER TWO
LITERATURE REVIEW
2.1. Definition and general characteristics of enzymes
Baedle (1948) defined enzymes as indispensable compounds that
play a key role in metabolism by bringing direction and control to the
physiological processes of living cells. Any change in enzyme
complement of living cells is immediately reflected in the physiological
and biochemical processes of the cell. According to Lehninger (1975),
enzymes are also defined as proteins specialised in catalysing biological
reactions. Another definition of enzymes was stated by Devlin (1986)
who claimed that enzyme are protein evolved by the cells of living
organisms for the specific function of catalyzing chemical reactions. All
enzymes are proteins, but not all proteins are enzymes.
Certain enzymes contain non-protein components such as
carbohydrates, lipids, phosphate, metal ions or small organic moieties.
The complete enzyme system usually includes both the protein and nonprotein parts, called holoenzyme. The protein part is termed the
apoenzyme and non-protein part, the prosthetic groups or cofactors. The
type of cofactor or coenzymes concerned in the enzymic process aids in
classification (White et al., 1973).
Enzymes cause chemical reactions to occur at their fastest rates
when the temperature is at an optimum level. For most enzymes, this is in
the range of 15.6ºC to 65.5ºC, but some reaction may occur at
temperatures above or below the optimum range. Thus, some enzymes
are able to react slowly at temperatures well below that of the freezing
3
point of water and other at temperatures above 71.1ºC, because proteins
are changed chemically and physically and coagulated by high
temperatures, especially when moisture is present, enzymes are usually
inactivated at temperature between 71.1 and 93.3ºC. Enzymes also have
an optimum pH at which they cause reaction to occur at the fastest rates,
the optimum pH for most enzymes was found to be in the range of pH
7.0 – 8.0 (Vieira, 1996)
2.2. Classification and numbering of enzymes
Verma (1995) reported that, since the enzymes are specific for a
particular reaction, they are named according to the substrate on which
they act or on the nature of the reaction they catalyse. The most common
method for naming them is to suffix-ase at the end of the name of the
substrate attacked. Thus, peptide is attacked by peptidase, lipid by lipase,
urea by urease and tyrosine by tirosynase. However, this nomenclature
has not always been practiced and many enzymes have been given
chemically uninformative trivial names. For example pepsin, trypsin and
catalase.
A commission of enzymes (International Union of Biochemistry,
I.U.B., 1972) has developed a complete, systematic, but rather a complex
system of nomenclature and classification.
The commission established a numerical system of classification
with the following recommendations:
a)
Reaction and the enzyme that catalyze them are
divided into six classes, each with 4-13 subclasses.
b)
Each enzyme has a systematic code number (E.C.) of
four digits. The first digit of the four figures
4
indicates the main class. The second digit indicates
the
sub-class.
The
third
digit
indicates
the
subdivision of the sub-class (sub-subclass). The
fourth digit designates the serial number of the
specific enzyme in the fourth sub-subclass.
For example, the code number E.C. 2.7.1.1 denotes main class 2
(transferase), subclass 7 (transfer of phosphate), sub-subclass 1 (an
alcohol functions as the phosphate acceptor), the fourth digit 1 indicates
hexokinase, or ATP: D-hexose 6-phosphotransferase. The following are
the six classes into which all enzymes may be divided.
1- Oxidoreductases:
Enzymes catalysing oxidoreductions between two substrates,
including dehydrogenases, oxidases, and deoxygenases. Peroxidase
belongs to this class.
2- Transferases:
These enzymes are involved in transferring functional groups
between donors and acceptors. The amino, acyl, phosphate, one-carbon,
and glycosyl groups are the major moieties that are transferred.
3- Hydrolases:
This group of enzymes can be considered as a special class of the
transferases in which the donor group is transferred to water. The
generalized reaction involves the hydrolytic cleavage of C-O, C-N, O-P,
and C-S bonds.
4- Layases:
Enzymes that catalyse removal of groups from substrates by
mechanisms other than hydrolysis leaving double bonds.
5
5- Isomarases:
Includes all enzymes catalysing interconversion of optical,
geometric, or positional isomers.
6- Ligases (synthetases):
Enzymes catalysing the linking together of two compounds
coupled to the breaking of pyrophosphate bond in ATP.
2.3. Enzymatic browning in fruits and vegetables
Enzymatic browning is one of the most important colour reactions
that affects fruits and vegetables. It is catalysed by polyphenoloxidases
and peroxidases (Marshall et al., 2000).
It was estimated that over 50 percent losses in fruits and vegetables
occur as result of enzymatic browning (Whitaker and Lee, 1995).
Phenolic compound and browning enzymes are in general, directly
responsible for enzymatic browning reaction in damaged fruits during
post-harvest, handling and processing. Once tissue is damaged by slicing,
cutting or pulping, however, the formation of brown pigments occurs.
Both the organoleptic and biochemical characteristic of fruits and
vegetables are altered by pigment formation. Marshall et al. (2000)
reported that the rate of enzymatic browning in fruits and vegetables is
governed by the active polyphenoloxidase and peroxidase content of the
tissues, the phenolic content of the tissues, pH, temperature and oxygen
availability with the tissue. Lee et al. (1990) reported that the relationship
of the rate of browning to phenolic content and polyphenoloxidase
activity could be positively related to discolouration of peaches.
According to Khan and Robinson (1993a) the peroxidase is directly
responsible for enzymatic browning in mangoes.
6
2.4. Biochemistry of higher plant peroxidases
Peroxidases (E.C. 1.11.1.7) are a group of heamcontaining
enzymes that present wide substrate specificity (Agostini et al., 2002).
Hematin peroxidases are consisting of colourless protein
(apoenzyme) combined with an iron–porphyrin. Whitaker (1972)
reported that the iron in prophyrin has six co-ordination positions, four of
which are taken up by prophyrin nitrogens and the fifth by protein
attachment. The six positions can be occupied by water or other radicals,
and the enzyme appears to operate by the exchange of groups at this
position.
The
enzyme
is
brown
in
color
and
contains
Ferriprotophyrin III (protohemin) group per molecule (Fig. 2.1).
7
one
Fig. 2.1. Structure of Ferriprotophyrin III (Protohemin)
(Whitaker, 1972)
8
Peroxidases are oxidoreductases that catalyze the oxidation
of a diverse group of organic compound using hydrogen peroxide an
ultimate electron acceptor (Dawson, 1988). Several of the enzymes in the
oxidored-uctase group are very important in food processing. Many
undesiarable change occurring in foods are due to the action of enzymes
in this group indigenous to the food. This includes enzymatic browning
(polyphenoloxid-ase), bleaching (lipoxygenase), destruction of ascorbic
acid (Ascorbic acid oxidase), and oxidative flavour deterioration
(Peroxidase). Also included in this group are some of the unique
enzymes added to foods for various specific purposes such as catalase for
the elimination of residual hydrogen peroxide after low temperature
pasteurization of milk (Reed, 1975).
Lepedus et al., (2004) claimed that peroxidases are found in plant
tissues and animal, as well as in microorganisms. Krell (1991) reported
that the horseradish (Armoracia sp.) roots represent the traditional source
for commercial production of peroxidases.
9
2.5. Natural occurrence of peroxidases
2.5.1. Sources and classification of peroxidases
Peroxidases are found in bacteria, fungi, plants and animals. On
the basic of sequence similarity, fungal, plant and bacterial peroxidases
can be viewed as numbers of a superfamily consisting of three major
classes (Welinder, 1992). These are:
Class I, the intracellular peroxidases which includes:
i.
Yeast cytochrome C peroxidase (CCP), a soluble
protein found in the mitochondrial electron transport
chain, where it probably protects against toxic
peroxidases.
ii.
Ascorbate peroxidase (AP), the main enzyme
responsible for hydrogen peroxide removal in
chloroplasts and cytosol of higher plants (Dalton,
1991).
iii. Bacterial
catalase
peroxidase,
exhibiting
both
peroxidase and catalase activities.
Class II, consists of the secretory fungal peroxidase, this includes:
i.
Ligninases, or lignin peroxidases (lips).
ii.
Manganese depend peroxidases (MnPs).
These are monomeric glycoproteins involved in the degradation of lignin.
Class III, consists of the secretary plant peroxidases, which have multiple
tissue specific functions, for example
i.
Removal of hydrogen peroxide from chloroplasts and
cytosol.
ii.
Oxidation of toxic compounds.
10
iii. Biosynthesis of the cell wall.
iv. Ethylene biosynthesis etc.
2.5.2. Localization of peroxidases
Peroxidase was shown to occur in most fruits and vegetables in
soluble and bound (ionically and covalently) forms (Silva et al., 1990) as
for example in banana (Haard, 1973), orange (Mclellan and Robinson,
1984) and spring cabbage (Mclellan and Robinson, 1987).
Reports have shown that peroxidase is localised in various sectors
of the cell including cytoplasm (Lee, 1973), Ribosomes (Darimont and
Baxter, 1973), nucleus and nucleolus (Raa, 1973), cell wall (Brownleader
et al., 1994) and also mitochondria (Prasad et al., 1995).
2.5.3. Multiple forms (Isoenzymes) of peroxidases
Contain enzymes which are formed by genetical change specially
by the processes which form alleles and iso-alleles, are known as
isoenzymes. The isoenzymes show very small differences in the
molecular structure with that of original enzyme. Physically and
chemically, the enzyme and isoenzymes are very similar and they
catalyse the same reactions. Isoenzymes can be separated by
electrophoretic techniques. Welinder (1992) reported that, plants have a
large number of peroxidase isoenzymes that may differ by more than
50% in amino acid sequence.
Peroxidase activity has been related to the existence of cationic
and/or anionic isoenzyme (Van Huystee, 1987). Shannon et al. (1966)
isolated seven peroxidase isoenzymes from horseradish roots and
purified
to
homogeneity
as
ascertained
by
chromatography,
ultracentrifugation and polyacrylamide disk electrophoresis. They
11
reported that, the seven isoenzymes may be segregated into two groups
on the basis of their chromatogramphic behaviour, electrophoretic
migration, spectrophotometric properties, amino acid and carbohydrate
composition.
Hoyle, (1977) isolated 24 peroxidase isoenzymes from horseradish
by isoelectric focusing. Aibara et al. (1981) obtained six basic
isoenzymes. EI to E6, of horseradish peroxidase which were isolated and
purified by CM. sephadex coloum chromatography, they also found same
differences in their amino acid composition.
Van Loon (1986) reported that, the Barley grains accumulate at
least three different cationic peroxidase isoenzymes during development.
Wheat and rye seeds may contain 10 or more peroxidase isoenzymes
distributed in the embryo, endosperm and scutellum. (Rebmann, et al.,
1991). Mazza et al. (1968) separated five peroxidase isoenzymes, (three
anionic and two cationic) from turnip roots. Mclellan and Robinson
(1987), isolated two peroxidase isoenzymes (anionic and cationic) from
spring cabbage. They reported also, the anionic isoenzyme was relatively
heat stable, while the cationic isoenzyme was more readily inactivated by
heat.
Lepedus et al. (2004) obtained two peroxidase isoenzymes from
carrot root using polyacrylamide gel electrophoresis (PAGE). Chatterjee
et al. (1999) isolated five peroxidase isoenzymes from hairy roots of
Cucumis melo using electrophorsis.
2.6. Mechanism of enzymic action of peroxidases
Peroxidase catalysis is associated with four types of activation
(Whitaker, 1972). These are:
12
2.6.1. Peroxidatic reactions
Peroxidatic reactions involves the oxidation of hydrogen donor by
H2O2 in the presence of peroxidase.
The general peroxidatic reaction can be written as follows:
2AH + H2O2
HAAH (polymerised product) + 2H2O
Under the usual, assay conditions in vitro where phenolic substrate
is used, only the peroxidatic reaction is of importance. Peroxidatic
reactions occur when p-cresol, guaiacol, o-dianisidine, resorcinol and
anilline are used as substrate.
2.6.2. Oxidatic reactions
Peroxidase catalyses oxidation of indole-3 acetic acid (IAA),
hydroquinone, dihydroxy fumarate and other compound by molecular
oxygen. This reaction is catalysed by trace amount of H2O2 and can be
inhibited by ascorbate. Oxidogenic molecule such as phenols promote the
reaction by increasing the rate of free radical formation of substrate
molecules.
2.6.3. Catalytic reactions
Peroxidase can catalyse the reaction:
2H2O2
2H2O + O2
In the absence of hydrogen donor, this reaction is, however, more than
1000 time slower than peroxidatic and oxidatic reactions (Whitaker, 1972).
2.6.4. Hydroxylation reactions
In the presence of certain hydrogen donor, particularly
dihydroxyfu-marate and molecular oxygen, peroxidase can hydroxylate a
variety of aromatic compounds, including tyrosine, phenylalanine-pcresol, p-coumeric acid, and benzoic and salicylic acid.
13
The mechanism of action of peroxidase incorporates all four types
of reaction. A general mechanism for the action of peroxidase was
proposed by Whitaker (1972) as shown in figure 2.2.
14
Fig. 2.2. Proposed scheme for mechanism of peroxidase action in four
types of reactions in which the enzyme is involved (Whitaker,
1972).
15
2.7. Physiological functions of peroxidase
The ubiquitous occurrence of peroxidase and its wide spread
distribution in higher plants has promoted many suggestions concerning
its physiological role. Peroxidase has been implicated in a variety of
physiological process such as ethylene biogenesis, cell development,
mebrane integrity, response to injury and disease resistance (Abeles and
Biles, 1991). Peroxidase has been also linked with respiratory control,
gene control and hormone metabolism (Haard, 1977).
Peroxidase is believed to participate in various oxidative processes
including lignification and degradation of auxin (Normanly et al., 1995).
2.8. Heat inactivation and regeneration of peroxidase
Adams (1978) defines the term inactivation of enzyme as the loss
of activity of an enzyme as a result of the application of a given heat
process. This inactivation may or may not be reversible and regeneration
is therefore, defined as the regain of activity after partial or completes
inactivation of the enzyme.
As in the case with all proteins, enzymes can be easily denatured in
several ways, among them, heat. Peroxidase from a variety of vegetable
sources has been shown to be very stable to heating (Chang et al., 1988;
Khan and Robinson, 1993b; Neves and Lourenço, 1998), and has been
claimed to be the most heat stable enzyme in plants (Burnette, 1977).
Thermal stability of peroxidases extracted from different sources
was observed to differ, for example, cabbage peroxidase activity was
shown to be more inactivated than brussels sprout peroxidase activity
(Mclellan and Robinson, 1981).
Tamura and Morita (1975) reported that inactivation of peroxidase
16
occurred upon exposure to temperatures higher than 60ºC. According to
them, three processes might be involved in the heat inactivation of
peroxidase. These processes are the dissociation of protohemin from
holoenzyme, a conformation change in the apo-peroxidase and the
modification or degradation of protohemin.
Several authors claimed that heat inactivation of peroxidase is
biphasic, i.e. the heat inactivation curves show two almost linear sections
of differing gradient. These sections corresponded to an initially rapid
inacti-vation phase followed by a second straight line segment of smaller
rate of descent. Among those authors are Adams (1978); Mohamed
(1983); Elshafie (1993) and Yemenicloglu et al. (1999).
Williams et al. (1986) reported that lipoxygenase rather than
peroxidase is the primary causative enzyme in development of the offflavour in English beans. Pea and green bean lipoxygenases were more
heat sensitive than peroxidases. Therefore, a less severe heat treatment
required to inactivate lipoxygenase was recommended for English green
peas and green beans. Barrett and Theerakulkait (1995) found that
lipoxygenase inactivation in super sweet corn at 93ºC was accomplished
in 6 to 9 minutes, while peroxidase inactivation under the same
conditions required 18 to 20 minutes. Inactivation of peroxidase and
liopxygenase at 93ºC in green beans required times of 2.0 and 0.5
minutes respectively.
Baardseth and Slinde (1980) reported that there were differences in
the heat stabilities of the peroxidases from carrot, swede and brussels
sprouts, but all peroxidases were more heat stable than the catalases.
Following heat inactivation of peroxidase, regeneration of activity
17
can occur. Many investigators have reported that peroxidase in
vegetables and other materials partly recover its enzymic activity when
the materials are cooled to room temperature (Lu and Whitaker, 1974;
Tamura and Morita, 1975).
Joffe and Ball (1962), in studying the kinetics and energetics of
thermal inactivation and regeneration of a peroxidase system, concluded
that the change in the tertiary structure of the protein moiety of the
enzyme molecule during inactivation involves more than hydrogen bond
and disulphide rupture.
Heat treatment applied to a particular food product for long time
duration than usual is needed to prevent the regeneration of peroxidase
activity in foods. Any regeneration which does occur is probably due to
the enzyme not being completely or irreversibly inactivated by heat.
Several factors affecting the regeneration of peroxidase activity are the
method used for detecting the activity, the severity of the heat treatment
combined with the time treated, and the condition of storage of the
inactivation enzyme prior to regeneration (Reed, 1975).
Schwimmer (1944) first separated peroxidase into two parts in
aqueous solution. One part was denatured protein which precipitates
during centrifugation. The second was the hemin group that was
originally attached to the protein and remained in solution. He suggested
that regeneration involves the precipitated compound redissolving,
combining with some soluble factor and the resulting complex then
resorts to its native, active state. Park and Fricker (1977) observed the
regeneration of peroxidases from horseradish and spinach during storage,
the extent of regeneration depended on the pH.
18
2.9. Effect of peroxidase action on food
Active enzyme system can spoil fruits and vegetables at sub-zero
temperatures, as low as – 18ºC and at low moisture levels, as low as
12.5% water as reported by Burnette (1977). Consequently, most
vegetables and even some fruits, to be preserved by canning, freezing or
even dehydration, are given a blanching treatment to inactivate these
enzymes and destroy bacteria in an attempt to prevent loss of quality of
stored fruits and vegetables. Peroxidase appear to be one of the most heat
resistant enzymes present in many fruits and vegetables. Therefore, it has
been used as index of blanching vegetables prior to canning and freezing.
It has been well established that peroxidase, one of the most stable
enzymes, can contribute to deteriorative changes in quality of the
processed products (Stanley et al., 1995). The relationship between
peroxidase activity and off-flavour production in green beans and turnip
has been well established (Zoueil and Esselen, 1959). Guyer and
Holmquist (1954), have also reported that the peroxidase activity in
processed
vegetables
was
closely
associated
with
off-flavour
development in products during storage.
High temperature short time (HTST) processing of vegetables is
now widely used in place of traditional blanching methods which involve
more prolonged treatment at less extreme temperatures. While HTST
processing is efficient and results in a better quality products (Llano et
al., 2003), regeneration of peroxidase activity is greater in HTST
processed vegetables, and in some cases, quality deteriorates more
rapidly (Adams, 1978) than in conventionally blanched vegetables.
Guyer and Holmquist (1954) showed that peroxidase activity and
19
off-flavour could be readily detected in canned peas which had been
(HTST) processed. Therefore, a small amount of residual peroxidase
activity, 1-5% for specific product, may or may not cause deterioration in
canned products, but not in frozen vegetables (Burnette, 1977).
Peroxidase can catalyse the oxidation of vitamins, growth
regulating substances and phenolic acids, but none of these reactions was
considered by Bruemmer et al. (1976) to be responsible for quality loss
in orange juice. This is because all these reactions were found to occur
extremely slow. Miesle et al. (1991) reviewed the roles of peroxidase
after harvesting which may lead to deterioration of certain fruit and
vegetable products. Lignification, for example, may be controlled by
peroxidase, and can lead to loss of quality of fruits and vegetables
following harvesting.
20
CHAPTER THREE
MATERIALS AND METHODS
3.1. Materials
3.1.1. The vegetables sample
Unspecified varieties of four vegetables (Potato, carrot, eggplant
and tomato) were obtained from the local market.
3.1.2. Chemicals
All chemicals used in this study were of analytical grade.
3.1.3. Preparation of substrates
3.1.3.1. The hydrogen peroxide solution
A solution of hydrogen peroxide was freshly prepared from 35%
(w/v) hydrogen peroxide (analar grade), which had previously been
stored under refrigerator, in an appropriate buffer.
3.1.3.2. The guaiacol solution
Guaiacol solution was prepared in 0.01 M phosphate buffers pH
(5.0, 6.0, 7.0 and 8.0).
3.1.4. Preparation of buffer solutions
Sodium phosphate buffers were prepared from 0.1M solution of
monobasic sodium phosphate (13.8g NaH2.PO4.2H2O in 1L) and 0.1M
solution of dibasic sodium phosphate (26.8g Na2HPO4 in 1L). The
solution was then adjusted to the required pH values.
21
3.2. Methods
3.2.1. Extraction of peroxidase
Extraction of soluble crude peroxidase from each vegetable
(potato, carrot, eggplant and tomato) was performed according to the
method described by Elshafie (1993).
Sixty grammes of samples were homogenized for three minutes in
100 ml of ice-cold 0.01M phosphate buffer at pH 5.0. The resultant suspension was filtered through a double layer of cheese cloth. The filtrate
was centrifuged at 15.000 r.p.m for 20 minutes at 4°C using Heraeus
Sepatech Suprafuge 22 Centrifuge.
The supernatant fluids were collected and retained for further
analysis. The same extraction procedure was repeated at variable pH
values (6.0, 7.0 and 8.0).
3.2.2. Enzyme assays
Various methods have been used to estimate peroxidase activity.
Acolourmetric method based on the rate and extent of pigment formed by
oxidation of phenolic and other aromatic substances was used in this
study.
This method for assaying peroxidase activity involves the use of
hydrogen peroxide and various hydrogen donors such as O-dianisidine
(Mohamed, 1983; Quesada et al., 1990), guaiacol (Chen and Whitaker,
1986; Elshafie, 1983; Marangoni et al., 1995), dihydroxyfumarate (Chen
and Schopfer, 1999), 3.3َ-diaminobenzidine tetrahydro-chloride (Herzog
and Fahimi, 1973).
The guaiacol method for the assaying of peroxidase activity is
simple and widely used method. Therefore, guaiacol method was chosen
22
in this study to estimate peroxidase activity.
3.2.2.1. Guaiacol method of assay
The guaiacol method for the assay of peroxidase activity is a
simple and widely used method requiring only guaiacol, H2O2 and buffer
in the assay mixture (Elshafie, 1993). The reaction is started by the
addition
of
peroxidase
and
the
absorbance
changes
followed
spectrophotometrically as a function of time. The major product of the
reaction is tetraguaiacol (Whitaker, 1972).
The substrate mixture for this peroxidase assay contained 99.8 ml
0.01M phosphate buffer, 0.1ml guaiacol and 0.1ml 35% hydrogen
peroxide solution.
OCH3
OCH3
O
OCH3
O
OH
+ 8 H2O
+ 4 H2O2
4
Guaiacol
O
O
OCH3
OCH3
Tetraguaiacol
23
The total of the reaction mixture in each of the cuvettes was 3.0 ml. Into
the reference and sample cuvettes, 2.9 ml of the reaction mixture were
pipetted. A (JENWAY 6305 UV/Vis.) spectrophotometer was set to zero
at 470 nm. Into the sample cuvettes the reaction was initiated by the
addition of 0.1ml enzyme solution and then absorption was measured.
Peroxidase activity was expressed as U.ml-1.
3.2.3. Heat treatment
Heat
inactivation
experiments
were
carried
out
at
four
temperatures in the manner described by Elshafie (1993). These
temperatures were 60, 70, 80 and 90°C. Enzyme extracts at pH 5.0 were
diluted 1 in 10(v/v) with 0.01M sodium phosphate buffer at each of the
pH values investigated. Aliquots (0.2ml) of the enzyme solution (at each
pH values) were placed in glass test tubes.
The test tubes containing the enzyme solution were transferred to
water bath set at the desired temperature. At various time intervals (2, 4,
6, 8 and 10 minutes), the tubes were removed and rapidly cooled in iced
water and held at-18°C until required for enzymic assay. Triplicate
samples were used for each time/temperature treatment. The same
procedure was repeated for the enzyme extract at pH 6.0, 7.0 and 8.0.
24
CHAPTER FOUR
RESULTS AND DISCUSSION
In this study, peroxidase enzymes were extracted from some
vegetables, namely potato, carrot, eggplant and tomato. Levels of
peroxidase activity were determined for each vegetable. That was
followed by determination of the heat stability of each enzyme under
various conditions including variable lengths of heating time, heating
temperature and pH.
The results of this investigation were presented and discussed in
this chapter. Peroxidases were extracted at pH 5.0, 6.0, 7.0, and 8.0 from
each of the four vegetables following the procedure described in section
3.2.1.
It is well known that peroxidase enzyme exists in soluble and bond
(ionically and covalently) forms (Mohamed, 1983; Silva et al., 1990;
Neves, 2002). The soluble fraction was reported to be the dominant form
in many plant tissues (Mohamed, 1983; Elshafie, 1993; Osman, 1993;
Neves, 2002). Consequently the soluble fraction is the from which will
be considered during the course of this study.
4.1 Levels of peroxidase activity
The soluble peroxidase fraction from each vegetable, extracted at
variable pH values, was determined and the results were summarized in
table 4.1.
The results indicated clearly that peroxidase was found in all
samples of vegetables investigated. The presence of peroxidase in
various fruits and vegetables was observed by many workers (Gorin and
25
Hemidema, 1976; Haard, 1977; Müftügil, 1985; Meclellon and
Robinson, 1987; Rhotan and Nicolas, 1989; Miesle et al. 1991; Neves,
2002 and Llano et al., 2003).
Table 4.1: Peroxidase activity of vegetables investigated
PH values
Peroxidase activity (U ml-1)
Potato
Carrot
Eggplant
Tomato
5.0
2.40
1.22
1.90
1.96
6.0
1.99
1.53
1.98
1.86
7.0
1.96
0.83
1.67
1.74
8.0
1.90
0.37
0.52
1.92
• Each value is a mean of three measurements.
• One unit of peroxidase activity (U) was defined as a change
of one absorbance unit (ml) per minute.
A reasonable enzyme activity was extracted and estimated at all
pH values. Relatively higher levels of peroxidase activities were
extracted at pH 5.0 from potato and tomato while those with higher levels
from carrot and eggplant were extracted at pH 6.0.
Potato tuber was shown to contain the highest peroxidase levels at
all pH values investigated whereas peroxidase from carrot had the lowest
peroxidase levels at all pH values investigated. Relatively low
peroxidases levels were observed in enzyme extracted at pH 8.0 from
carrot and egg-plant.
Müftügil (1985) who estimated the peroxidase enzyme activity of
26
some fresh vegetable found that peroxidase activities in all sample of
fresh vegetables investigated. According to him cabbage and green beans
had high enzyme activities whereas in onion and carrots the peroxidase
enzyme activity was low.
Osman (1993) found that the pH 5.0 and 6.0 are optimum pH for
detecting potato and onion peroxidase activities respectively. Other
investtigators found that the optimum pH values for detecting peroxidase
activities to be 5.0 for peach (Neves, 2002); 4.5 – 6.0 for lettuce
(Bestwick et al., 1998); 4.4 – 5.0 for potato (Mohamed, 1983); 5.0 for
potato and sweet potato (Elshafie, 1993) and 5.5 – 6.0 for peroxidase
extracted from papaya fruit (Silva et al. 1990).
4.2 Heat inactivation of peroxidase
Peroxidase is reported to be one of the most heat stable enzyme in
plant, hence can influence the flavour, texture and colour in row and
processed fruit and vegetables (Haard, 1973; Burnette, 1977; Osman,
1993 Clemente and Pastore, 1998).
Anthon and Barrett (2002) stated that as peroxidase is very
resistant to thermal inactivation, it is widely used as an index of
blanching and other heat treatments. Heat inactivation experiments of
peroxidase extracted from the four vegetables under consideration were
carried out at four tempera-tures (60, 70, 80 and 90º C) as described in
section 3.2.3.
Samples were heated at these temperatures for varying length of
time (2, 4, 6, 8, and 10 min.) under different pH values (5.0, 6.0, 7.0 and
8.0) in the present work. The effect of these factors on rates of
inactivation of peroxidases from the different vegetables under
27
consideration was examined.
4.2.1 Thermal stability of potato peroxidase
Potato peroxidase was subjected to thermal inactivation for
varying heating temperatures and length of heating times under different
conditions of pH. The results were presented in table 4.2 and figures 4.1
– 4.4.
The results obtained showed that the rate of loss of peroxidase
activity increases with both increased temperatures and heating times.
The initial heat inactivation of peroxidase enzyme is rapid followed by a
much slower inactivation period. For instance, heating at 60°C for 10 min
resulted in a loss of 54.5, 46.4, 43.1 and 42.5 enzyme activity at pH 5.0,
6.0, 7.0 and 8.0 respectively. This pattern was more or less true for other
temperatures. It was observed that the stability of the enzyme to heat
increased with increased pH values.
Potato peroxidase was not completely inactivated when it was
exposed to varying temperatures, length of heating times and at variable
pH values.
28
Table 4.2. Heat inactivation of potato peroxidase
a) pH 5.0
Temperature
% remaining peroxidase activity
0 min
2 min
4 min
6 min
8 min
10 min
60º C
100
61.3
52.0
49.7
48.8
45.5
70º C
100
43.3
39.3
35.0
32.3
30.1
80º C
100
20.4
16.8
15.2
13.0
12.2
90º C
100
12.6
9.0
8.19
7.9
6.3
b) pH 6.0
Temperature
0 min
2 min
4 min
6 min
8 min
10 min
60º C
100
73.0
59.7
57.7
54.7
53.6
70º C
100
39.0
34.3
29.1
25.2
24.9
80º C
100
24.4
19.7
14.7
14.2
13.9
90º C
100
14.2
11.5
9.5
5.8
4.3
c) pH 7.0
Temperature
0 min
2 min
4 min
6 min
8 min
10 min
60º C
100
83.8
73.1
61.2
60.0
56.9
70º C
100
45.0
32.9
28.2
23.4
18.7
80º C
100
32.6
25.5
21.7
16.8
9.6
90º C
100
23.2
17.8
7.4
5.4
3.2
d) pH 8.0
Temperature
0 min
2 min
4 min
6 min
8 min
10 min
60º C
100
84.9
76.1
67.8
62.2
57.5
70º C
100
42.9
31.4
22.6
16.6
14.8
80º C
100
30.5
20.5
15.2
14.5
12.9
90º C
100
7.8
7.5
7.1
5.4
5.2
Each value is a mean of three determinations
Enzyme assays with guaiacol.
29
Fig. 4.1. Heat inactivation of potato peroxidase at pH 5.0
100
90
Original activity (%)
80
70
60
50
40
30
20
10
0
0
2
4
6
8
Time (min)
60º C
70º C
30
80º C
90º C
10
100
90
80
70
60
50
40
30
20
10
0
0
2
4
6
8
Time (min)
60º C
70º C
80º C
31
90º C
10
100
90
80
70
60
50
40
30
20
10
0
0
2
4
6
8
Time (min)
60º C
70º C
80º C
32
90º C
10
100
90
80
70
60
50
40
30
20
10
0
0
2
4
6
8
10
Time (min)
60º C
70º C
33
80º C
90º C
4.2.2 Theraml stability of carrot peroxidase
Carrot peroxidase was subjected to thermal inactivation for
varying heating temperatures and length of heating time under different
conditions of pH. The results were shown in table 4.3 and figures 4.5 4.8.
activity increases with both increased temperature and heating time. The
initial heat inactivation of peroxidase enzyme was rapid followed by a
much slower inactivation period. Heat inactivation of carrot peroxidase
followed the usual pattern, which was also observed for potato
peroxidase, however some variations were observed when the effects of
the length of heating time were compared at the different pH values
studied. Complete inactiva-tion of carrot peroxidase was accomplished at
80°C for 4 to 10 minutes and at 90 º C in 2 to 10 minutes at pH 8.0.
34
Table 4.3. Heat inactivation of carrot peroxidase
a) pH 5.0
Temperature
0 min
2 min
4 min
6 min
8 min
10 min
60º C
100
50.8
36.8
24.8
23.2
21.5
70º C
100
32.7
26.2
21.8
19.1
18.0
80º C
100
22.4
14.2
11.2
10.9
9.2
90º C
100
12.8
7.3
6.5
5.7
4.3
b) pH 6.0
Temperature
0 min
2 min
4 min
6 min
8 min
10 min
60º C
100
31.3
20.9
16.9
15.0
14.3
70º C
100
20.4
14.8
13.0
11.7
11.3
80º C
100
13.7
9.1
7.4
6.7
6.5
90º C
100
8.9
5.4
5.0
4.1
3.0
c) pH 7.0
Temperature
0 min
2 min
4 min
6 min
8 min
10 min
60º C
100
50.6
35.3
29.2
25.2
22.7
70º C
100
32.1
20.7
16.6
13.4
13.0
80º C
100
22.7
13.0
12.1
10.5
9.7
90º C
100
11.3
5.2
4.8
3.6
2.4
d) pH 8.0
Temperature
0 min
2 min
4 min
6 min
8 min
10 min
60º C
100
37.8
21.6
16.2
13.5
12.6
70º C
100
17.1
3.6
2.7
1.8
0.9
80º C
100
2.7
0
0
0
0
90º C
100
0
0
0
0
0
35
Fig. 4.5. Heat inactivation of carrot peroxidase at pH 5.0
100
90
80
70
60
50
40
30
20
10
0
0
2
4
6
8
Time (min)
60º C
70º C
80º C
36
90º C
10
100
90
80
70
60
50
40
30
20
10
0
0
2
4
6
8
Time (min)
60º C
70º C
80º C
37
90º C
10
100
90
80
70
60
50
40
30
20
10
0
0
2
4
6
8
Time (min)
60º C
70º C
80º C
38
90º C
10
100
90
80
70
60
50
40
30
20
10
0
0
2
4
6
8
Time (min)
60º C
70º C
80º C
39
90º C
10
4.2.3 Thermal stability of eggplant peroxidase
Eggplant peroxidase was subjected to thermal inactivation for
varying heating temperatures and length of heating time under different
conditions of pH. The results were shown table 4.4 and figures 4.9–4.12.
The results obtained showed that the rate of loss of peroxidase activity of
peroxidase increases with both increased temperature and heating time.
The initial heat inactivation of peroxidase enzyme was rapid followed by
much slower inactivation period, which is similar to the results obtained
for potato and carrot.
Complete inactivation of eggplant peroxidase required 8 to 10 min.
at 90°C and pH 8.0, and therefore comes second to potato peroxideease
interm of heat stability.
40
Table 4. Heat inactivation of eggplant peroxidase
a) pH 5.0
Temperature
0 min
2 min
4 min
6 min
8 min
10 min
60º C
100
76.1
63.8
59.8
58.2
57.7
70º C
100
44.7
36.8
30.1
27.1
25.2
80º C
100
25.6
17.5
14.7
13.8
12.4
90º C
100
12.6
6.8
4.3
3.6
3.3
b) pH 6.0
Temperature
0 min
2 min
4 min
6 min
8 min
10 min
60º C
100
87.7
75.7
73.0
72.7
70.7
70º C
100
57.7
43.2
38.7
36.7
34.3
80º C
100
40.0
25.7
19.8
17.6
13.4
90º C
100
25.5
18.8
10.1
7.5
5.0
c) pH 7.0
Temperature
0 min
2 min
4 min
6 min
8 min
10 min
60º C
100
86.8
76.0
72.4
70.6
68.8
70º C
100
53.2
39.5
35.5
32.1
28.9
80º C
100
30.3
15.3
14.7
9.3
7.1
90º C
100
13.1
6.5
3.3
2.1
1.7
d) pH 8.0
Temperature
0 min
2 min
4 min
6 min
8 min
10 min
60º C
100
83.9
67.3
50.9
48.0
47.1
70º C
100
46.1
36.5
30.7
26.9
23.0
80º C
100
17.3
5.7
5.1
3.8
1.9
90º C
100
5.0
1.9
1.9
0
0
41
Fig. 4.9. Heat inactivation of eggplant peroxidase at pH 5.0
100
90
80
70
60
50
40
30
20
10
0
0
2
4
60º C
Time (min)
70º C
6
80º C
42
8
90º C
10
100
90
80
70
60
50
40
30
20
10
0
0
2
4
6
8
Time (min)
60º C
70º C
80º C
43
90º C
10
100
90
80
70
60
50
40
30
20
10
0
0
2
4
6
8
Time (min)
60º C
70º C
80º C
44
90º C
10
100
90
80
70
60
50
40
30
20
10
0
0
2
4
6
8
Time (min)
60º C
70º C
45
80º C
90º C
10
4.2.4 Thermal stability of tomato peroxidase
Tomato peroxidase was subjected to thermal inactivation for
varying heating temperature and length of heating time under different
conditions of pH. The results were shown in table 4.5 and figures 4.13 –
4.16.
activity increases with both increased temperature and heating time, a
result which is similar to those of potato, carrot, and eggplant peroxidase.
The initial heat inactivation of peroxidase enzyme was rapid followed by
a much slower inactivation period.
Complete inactivation of tomato peroxidase require 6 to 10
minutes at 90º C and pH 6.0.
46
Table 4.5. Heat inactivation of tomato peroxidase
a) pH 5.0
Temperature
0 min
2 min
4 min
6 min
8 min
10 min
60º C
100
74.8
57.3
55.1
53.9
52.2
70º C
100
42.5
31.1
28.5
27.7
26.0
80º C
100
25.1
13.7
11.2
10.7
10.2
90º C
100
5.4
0.8
0.8
0.51
0.34
b) pH 6.0
Temperature
0 min
2 min
4 min
6 min
8 min
10 min
60º C
100
88.3
77.9
75.2
74.7
73.1
70º C
100
67.5
55.5
52.6
51.6
49.4
80º C
100
17.0
5.1
3.7
3.7
3.7
90º C
100
1.6
0.3
0
0
0
c) pH 7.0
Temperature
0 min
2 min
4 min
6 min
8 min
10 min
60º C
100
81.9
70.3
67.4
64.9
63.2
70º C
100
50.9
33.9
31.9
28.5
26.8
80º C
100
19.5
6.7
4.2
2.8
1.9
90º C
100
8.2
2.2
1.7
1.7
1.1
d) pH 8.0
Temperature
0 min
2 min
4 min
6 min
8 min
10 min
60º C
100
89.7
78.6
73.2
70.3
66.4
70º C
100
68.7
56.2
46.1
42.0
39.7
80º C
100
19.7
7.2
6.4
3.9
2.9
90º C
100
8.3
3.1
2.0
1.5
1.0
47
Fig. 4.13. Heat inactivation of tomato peroxidase at pH 5.0
100
90
80
70
60
50
40
30
20
10
0
0
2
60º C
4
Time (min)
70º C
6
80º C
48
8
90º C
10
100
90
80
70
60
50
40
30
20
10
0
0
2
4
6
8
Time (min)
60º C
70º C
80º C
49
90º C
10
100
90
80
70
60
50
40
30
20
10
0
0
2
4
6
8
10
Time (min)
60º C
70º C
50
80º C
90º C
100
90
80
70
60
50
40
30
20
10
0
0
2
4
6
8
Time (min)
60º C
70º C
80º C
51
90º C
10
From the results presented in tables 4.2-4.5 and figures 4.1– 4.16,
it was clearly observed that the patterns of heat inactivation of peroxidase
from the four vegetables were similar. Potato peroxidase was observed to
be more stable to heat and therefore a less severe heat treatment is
required to inactivate carrot, eggplant and tomato.
Several authors, among them are Adams (1978); Mohamed (1983);
v
Osman (1993); Elshafie (1993); Yemenicloglu
et al. (1999) and Neves
(2002), have reported that the initial heat inactivation of peroxidase
enzyme was rapid followed by a much slower inactivation period and
have concluded that the heat inactivation process is biphasic. Although
Shannon et al. (1966) claimed that the biphasic heat inactivation might be
due to the presence of peroxidase isoenzyme with different sensitivities
to heat, Vamos -Vigyazo (1981) suggested that the non- linear
inactivation curve are due to the formation of new higher thermostable
complexes formed from thermally denatured enzyme protein and groups
of peroxidase that remain active.
The results obtained when potato, carrot, eggplant and tomato.
Peroxidase were heated show good correlation with those described by
other workers for different vegetables, for example Mohamed (1983)
working on potato, Osman (1993) working on potato and onion and
Elshafie (1993) working on potato and sweet potato.
Tamura and Morita (1975) reported that, inactivation of peroxidase
Occurred upon exposure to temperatures higher than 60°C. They claimed
that three processes might be involved in heat inactivation of peroxidase:
the dissociation of protohemin from the holoenzyme; conformation
change in the apo-peroxidase; and the modification for degradation of
52
protohemin. The results obtained in this study, showed that, potato
peroxidase is thermally more stable than carrot, eggplant and tomato
peroxidase. In addition, rates of heat inactivation were found to be pH
dependent. Mclellan and Robinson (1981) reported that thermal stability
of peroxidases extracted from different sources was found to be different,
also they observed that cabbage peroxidase activity was more inactivated
than brussels sprout peroxidase activity.
Deepa and Arumughan (2002) reported that the resistance to heat
treatment depends on the source of the enzyme as well as the assay
condition, especially pH and nature of substrate employed.
Neves and Lourenço (1998) reported that peach peroxidase,
soluble and bound, showed distinct heat lability, that fact was also
observed for isolated enzyme from apple (Moulding et al., 1989) and
papaya (Silva et al., 1990). Deepa and Arumughan (2002) observed that
the thermal stability of oil palm fruit peroxidase was greater than that
reported for cotton by Triplett and Mellon (1992), strawberry by Civello
et al., (1995) and coconut by Mujer et al., (1983). It has been shown that
the thermal stability of peroxidase was due to the presence of a large
number of cystein residues in the polypeptide chain (Deepa and
Arumughan, 2002).
53
4.3. Conclusions and recommendations:
4.3.1. Conclusions:
The following conclusions were drown from this study:
•
All vegetables investigated contained peroxidase enzyme.
•
The differences in levels of extracted peroxidases were
linked to its source.
•
The activity of peroxidase enzyme was found to be
dependant on the pH values used.
•
Potato tuber had the highest peroxidase level at all pH
values investigated, whereas carrot peroxidase had the
lowest peroxidase levels extracted under the same
conditions.
•
The rate of loss of peroxidase activity increased with both
increased temperatures and heating time in all vegetables
investigated.
•
Heat inactivation of peroxidase enzyme is biphasic, i.e.
the initial heat inactivation of peroxidase enzyme is rapid
followed by much slower inactivation period.
•
The patterns of inactivation of peroxidases from the four
vegetables are similar.
•
The stability of potato peroxidase to heat increase with
increased pH value.
•
Complete
inactivation
of
carrot
peroxidase
was
accomplished at 80ºC in 4 to 10 min and 90º C in 2 to 10
min at pH 8.0.
54
•
Complete inactivation of eggplant peroxidase required 6
to 10 min at 90ºC and pH 8.0
•
Complete inactivation of tomato peroxidase required 6 to
10 min at 90ºC and pH 6.0
•
Potato peroxidase was observed to be more stable to heat
and therefore a less severe heat treatment is required to
inactivate carrot, eggplant and tomato peroxidase.
4.3.2. Recommendations:
Further work is needed to give a better understanding of this heat
stable enzyme. Further suggested work includes
•
The study of the regeneration of the heat inactivated
enzyme.
•
Separation of the individual forms of the enzyme and
subsequently the determination of the heat stable and
heat labile ones.
55
REFERENCES
Abeles, F. B. and Biles, C. L. (1991). Characterization of peroxidase in
lignifying peach fruit endocrop. Plant physiology. 95:269–
273.
Adams, J. B. (1978). Inst. of food Sci. and tech. proceedings. 11 – 71.
Agostini, E.; Hernandez-Ruiz, J.; Amao, M. B.; Milrad, S. R.; Tigier, H.
A. and Acosta, M. (2002). A peroxidase isoenzyme secreted by
turnip (Brassica napus) hairy-root cultures: inactivation by
hydrogen peroxide and application. In diagnostic kits.
Biotechnol. App. Biochem. 35: 1 – 7.
Aibara, S.; Kobayashi, T. and Morita, Y. (1981). Isolation and properties
of basic isoenzymes of horseradish peroxidase. J. Bio. Chem.
90(2): 289 – 496.
Anthon, G. E. and Barrett, D. M. (2002). Kinetic parameters for the
thermal inactivation of quality – related enzymes in carrot, and
potatoes. J. Agric. Food. Chem. 50: 4119.
Baardseth, P. and Slinde, E. (1980). Heat indication and pH optima of
peroxidase and catalase in carrot, swede and brussels sprouts.
Food Chem. 5(2): 169 – 174.
Baedle, B. W. (1948). Genes and biological enigmas. Amer. Scientist 36:
69 – 74.
Barrett, D. M. and Theerakulhait, C. (1995). Quality indicators in
blanched, frozen, stored vegetables. Food Technol. 1(62): 64–
65.
56
Bestwick, C. S.; Brown, I. R. and Mansfield, J. W. (1998). Localized
changes in peroxidase Activity Accompany Hydrogen peroxide
Generation during the Development of a No host Hypersensitive
Reaction in lettuce. Plant physiol. 118: 1067 – 1078.
Brownleader, M. D.; Ahmed, N.; Trevan, M.; Cahplin, M. and Dey, P.
M. (1994). A study of extension and extensin peroxidase plant
perox. Newslett., 4: 3 – 15.
Bruemmer, J. H.; Roe, B. and Bower, E. R. (1976). Peroxidase reactions
and orange juice quality. J. Food Sci. 141: 186 – 189.
Burnette, F. S. (1977). Peroxidase and its relation to food flavour and
quality. A review. J. Food Sci. 4: 1 – 6.
Chang, B. S.; Park, K. H. and Lund, D. B. (1988). Thermal inactivation
kineties of horseradish peroxidase. J. Food Sci. 53: 920 – 923.
Chatterjee, P.; Golwalkar, S. and Mukundan, U. (1999). Peroxidase
production from Cucumis melo Hairy Root cultures. Plant
perox. Newslett. 13: 19 – 24.
Chen, A. O. and Whitaker, J. R. (1986). Purification and characterization
of lipoxygenase from immature English peas. J. Agric. Food
Chem. 34: 203 – 211.
Chen, S. and Schopfer, P. (1999). Hydroxyl-radical production in
physiological reactions Anovel function of peroxidase. Eur. J.
Biochem. 206: 726 – 735.
57
Civello, P. M.; Martinez, G. A.; Chaves, A. R. and Anon, M. C. (1995).
Peroxidase from strawberry fruit (Fragaria ananassa Duch.):
partial purification and determination of some properties. J.
Agric. Food chem. 43 : 2569 – 2601.
Clemente, E. and Pastore, G. M. (1998). Peroxides and polyphenols
oxidese. The importance for food technology, Bol. Soc. Brasil.
Cience. Tecnol. Aliment. 32(2): 167 – 171.
Dalton, D. A. (1991). Ascorbate Peroxidase. In Everse, J.; Everse, K. E.
and Grisham, M. B.; Eds. Peroxidase in chemistry and biology.
Boca Raton, Vol. II, pp. 139 – 153.
Darimont, E. and Baxter, R. (1973). Ribosomal and mitochondrial
peroxidase isoenzymes of the lentil (Lens culinaris) root.
Planta (Berl.). 110: 205 – 212.
Dauthy, M. E. (1995). Fruit and vegetable processing FAO Agricultural
services Bulltin No. 119.
Dawson, J. H. (1988). Probing structure. Function relations in hemecontaining oxygenases and peroxidases. Science 240: 433 –
439.
Deepa, S. S. and Arumughan, C. (2002). Oil plam fruit peroxidase:
purification and characterization. J. Food Sci. Technol. 39: 8 –
13.
Devlin, T. (1986). Textbook of Biochemistry with clinical correlations.
2nd ed. John Wiley and sons. Inc. New York, p. 118.
Elshafie, W. A. (1993). Isolation and Characterization of the Browning
58
Enzymes of Some Starchy Tuber. M. Sc. Thesis, Faculty of
Agriculture, University of Khartoum.
Gorin, N. and Heidema, F. T. (1976). Peroxidese activity in Golden
Delicious apples as a possible parameter of ripening and
senescence. J . Agric. Food chem.. 24 : 200 – 201.
Guyer, R. B. and Holmquist, J. W. (1954). Enzyme regeneration in high
temperature-short time sterilized canned foods. Food Technol.
8(12): 547 – 550.
Haard, N. F. (1973). Upsurge of particulate peroxidase in ripening
banana fruit, phytochemistry. 12: 555 – 560.
Haard, N. F. (1977). Physiological roles of peroxidase in post-harvest
fruit and vegetables. In: Enzymes in food and beverages
processing, eds. Ory. R. L. and St. Angelo, A. J. ACS, pp. 143.
Herzog, V. and Fahimi, H. D. (1973). A new sensitive colorimetric assay
for peroxidase using 3, 3َ – diaminobenzidine as hydrogen
donor. Anal. Biochem. 55: 554 – 562.
Hoyle, M. C. (1977). High resolution of peroxidase indolacetic Acid
oxidase isoenzymes from Horseradish by isoelectric focusing.
Plant physiol. 60(5): 787 – 793.
Joffe, F. M. and Ball, C. O. (1962). Kinatics and energetics of thermal
inactivation and the regeneration rates of a peroxidase system.
J. Food Sci. 27: 587 -592.
Kerll, H. W. (1991). Biochemical, Molecular and Physiological Aspects
of Plant Peroxidases (Lobarzewski, J.; Greppin, H.; Penel, C.
59
and Gaspar, Th, eds.), pp. 469 – 478, University of Geneva,
Geneva.
Khan, A. A. and Robinson, D. S. (1993a). Purification of anionic
peroxidase isoenzyme for mango (Mangifera indica L. var.
chaunsa). Food Chem. 46: 61 – 64.
Khan, A. A. and Robinson, D. S. (1993b). The thermostability of purified
mango isoperoxidase. Food Chem. 47: 53 – 59.
Lee, C. Y.; Kagan, V.; Jaworski, A. W. and Browns, S. K. (1990).
Enzymatic browning in relation to phenolic compounds and
chemical structure of tripeptide containing an unusual thioether
fungi. J. Biol. Chem. 257: 6414 – 6419.
Lee, T. T. (1973). On Extraction and Quantitation of Plant Peroxidase
Isoenzymes. Physiol. Plant 29: 198 – 203.
Lehninger, A. L. (1975). Biochemistry, 2nd ed. worth publishers, Inc.
New York, p. 183.
Lepedus, H.; Cesor, V. and Krsnik-Rosol, M. (2004). Guaiacol
peroxidases in carrot (Daucus corota L.) Root, Food Technol.
Biotechnol. 42(1): 33 – 36.
Llano, K. M.; Haedo, A. S.; Gerschenson, L. N. and Rojas, A. M. (2003).
Mechanical and biochemical response of kiwifruit tissue to
steam blanching. Food research international. 36(8): 767 – 775.
Lu, A. T.; Whitaker, J. R. (1974). Some factors effecting rates of heat
inactivation of horseradish peroxidase. J. Food Sci. 39: 1173 –
1178.
60
Luh, B. S. and Doauf, H. N. (1971). Effect of break temperature and
holding time on pectin and pectic enzymes in tomato pulp. J.
Food Sci. 36: 1030 – 1043.
Marangoni, A. G.; Jackman, R. L. and Stanley, D. W. (1995). Chillingassociated softening of tomato fruit is related to increased
pectinmehylesterase activity. J. Food Sci. 60: 1277 – 1281.
Marshall, M. R.; Kim, j. and Wei, C. (2000). Enzymatic browning in
fruits,
vegetables
and
sea
foods.
FAO,
Rome.
http://www.fao.org.
Mazza, G.; Charles, C.; Bouchet, M.; Ricord, J. and Raynaud, J. (1968).
Biochem. Biophys. Acta. 167: 89 – 98. (Cited by Aogstini et
al., 2002).
Mclellan, K. M. and Robinson, D. S. (1981). The effect of heat on
cabbage and brussels sprout peroxidase enzymes. Food Chem.
7(4): 257 – 266.
Mclellan, K. M. and Robinson, D. S. (1984). Heat stability of peroxidase
from orange. Food chem. 13(2): 139 – 147.
Mclellan, K. M. and Robinson, D. S. (1987). The heat stability of
purified spring cabbage peroxidase isoenzymes. Food chem.
26(2): 97–107.
Miesle, T. J.; Proctor, A. and Lagrimini, L. M. (1991). Peroxidase
activity isoenzymes, and tissues localization in developing high
bush bluberry fruit. J. Amer. Soc. Hortic. Sci. 116: 827 – 830.
Mohamed, B. E. (1983). A study of the Peroxidase of the Potato. Ph.D.
61
Thesis, Procter Department of Food Science, University of
Leeds, UK.
Mouldin, P. H.; Goodfellow, J.; Mclellan, K. M. and Robinson, D. S.
(1989). The occusence of isoperoxidases in conference pears.
Interm. J. Food Sci. Technol., 24: 269 – 275.
Müftügil, N. (1985). The pereoxidase Enzyme Activity of some vegetables
and its Resistance to Heat. J. Sci food Agric. 36 : 877 – 880.
Mujer, C.V.; Menzdoza, E.M. and Ramirez, D.A. (1998). Coconut
peroxidase Isoenzymes: Isolation partial purification and
physicochemical properties. Phytochem 22: 1335 – 1340.
Neves, V.A. (2002). Ionically Bound peroxidase from peach fruit.
Brazilian Archives of Biology and Tech. 45: 7 – 16.
Neves, V.A. and Lourenço, E. J. (1998). Peroxidase from reach fruit:
Thermal stability. Braz. Arch. Biol. Technol. 41(2): 179 – 186.
Normanly, J. Solvin, J. P.; Cohen, J. O. (1995). Rethinking auxin
biosynthesis and metabolism. Plant physiol. 107: 323 – 329.
Osman, A. M. (1993). The Effect of Added Peroxidase on the Quality of
Dried Potato and Onion. M. Sc. thesis, Faculty of Agriculture,
University of Khartoum.
Park, K. H. and Fricker, A. (1977). Thermal inactivation and storage
behaviour of technologically important enzymes. Horseradish
and Spinach peroxidase. Z Ernahrung Swiss. 16 (2): 81 – 91.
Prasad, T. K.; Anderson, M. D. and Stewart, C. R. (1995). Localization
62
and characterization of peroxidases in the mitochondria of
chilling-Acclimate Maize seedlings, Physiol. Plant. 108: 1597:
1605.
Quesada, M. A.; Tigier, H. A.; Bukovac, M. J. and Valpuesta, V. (1990).
Purification of an anionic isoperoxidase from peach seeds and
its
immunological
comparison
with
other
anionic
isoperoxidase. Physiole plant. 79: 623 – 628.
Raa, J. (1973). Cytochemical localization of peroxidase in plant cells.
Physiol. Plant. 28: 132 – 133.
Rebmann, G.; Hertig, C.; Bull, J.; Mauch, F. and Dudler, R. (1991).
Choning and sequencing of cDNAs encoding a pathogen
induced putative peroxidase of wheat (Triticum aestivum L.).
Plant Mol Biol. 16: 329 – 331.
Reed, G. (1975). Enzymes in Food Processing, Academic Press, New
York, p. 243.
Rhotan, C. and Nicholas, J. (1989). Changes in acidic and basic peroxidase
activity during tomato fruit ripening. Hortic. Sci. 24: 340 – 342.
Schwimmer, S. (1944). Regeneration of hear inactivated peroxidase. J.
Biol. Chem. 154: 487 – 495.
Shannon, L. M.; Kay, E. and Lew, J. (1966). Peroxidase isoenzymes
from horseradish roots. J. Biol. Chem. 241(9): 2166.
Silva, E.; Lourenço, E. J. and Neves, V. A. (1990). Soluble and bound
peroxidase from papaya fruit. Phytochemistry. 29: 1051 –
1056.
63
Stanley, D. W.; Bourne, M. C.; Stone, A. P. and Wismer, W. V. (1995).
Low temperature blanching effects on chemistry, firmness and
structure of canned green beans and carrots. J. Food Sci. 60:
327 – 333.
Tamura, Y. and Morita, Y. (1975). Thermal denaturation and
regeneration of Japanese – radish peroxidase. J. Biochem. 78:
561 – 571.
Triplett, P. A. and Mellon, J. E. (192). Purification and characterization of
anionic peroxidase from cotton (Gossypium hirsutum ) . plant
Sci. 81 : 147 – 154.
Vamos–Vigyazo, L. (1981). Polyphenol oxidase in ferit and vegetables.
CRC crit in food Sa. Nutri. 15 : 49 – 127 .
Van Huystee, R. B. (1987). Some molecular aspects of plant peroxidase
biosynthetic studies. Annu. Plant physiol. 8: 205 – 219.
Van Loon, L. C. (1986). The significance of changes in peroxidase in
diseased plants. In: Greppin, H.; Penel, C., Gaspar, T.; eds,
Molecular and physiological aspects of plant peroxidases,
Univ. Geneva, pp. 405 – 418.
Verma, S. K. (1995). A Textbook of Plant Physiology and Biochemistry.
First ed. Chan, S. and Company Ltd. New Dalhi, p. 94.
Vieira, E. R. (1996). Elementary food science. Fourth ed. Chapman and
Hall, Inc. New York, p. 123 – 124.
Welinder, K. G. (1992). Superfamily of plant, fungal and bacterial
peroxidases. Curr. Opin. Struct. Biol. 2: 288 – 393.
64
Whitaker, J. R. (1972). Principle of Enzymeology for the Food Science.
Marcel. Dekker Inc. New York, p. 592.
v
Whitaker, J.v R. and Lee, C. Y. (1995). Recent advances in chemistry of
enzymatic browning. In Lee, C. Y. and Whitaker, J. R. eds.
Enzymatics Browning and it’s prevention, p. 2 – 7. ACS
symposium series 600, Washington, DC, America Chemical
Society.
White, A.; Handler, P. and Smith, E. (1973). Principles of biochemistry.
Fourth ed. McGraw-Hill, Inc. New York, p. 215 – 216.
Williams, D. C.; Lim, M. H.; Chen, A. O.; Pangborn, R. M. and Witaker,
J. R. (1986). Blanching of vegetables for freezing – which
indicator Enzyme to choose. Food Technol. 40(6): 130 – 140.
Yemenicloglu, A.; Özkan, M. and Cemeroglu, B. (1999). Some
characteristics of polyphenoloxidase and peroxidase from taro
(Colocasia antiquorum). J. Agri. and Forestry. 23: 425 – 430.
Zoueil, M. E. and Esselen, W. B. (1959). Thermal destruction rates and
regeneration of peroxidase in green beans and turnips. Food
research. 24 p. 119.
65

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