riboflavin photosensitized oxidation of amino acids

154 Pages·2008·1.09 MB·English

R P O
IBOFLAVIN HOTOSENSITIZED XIDATION

A A
OF MINO CIDS

Dissertation

Presented in Partial Fulfillment of the Requirements
for the Degree Doctor of Philosophy in the Graduate
School of The Ohio State University

By

Yettella V Ramesh Reddy, M.S.
* * * * * *

The Ohio State University
2008

Dissertation Committee: Approved by

Dr. David B. Min, Advisor _____________________
Dr. M. Monica Giusti Advisor
Dr. V.M. Balasubramaniam Food Science & Nutrition
Dr. Luis E. Rodriguez-Saona Graduate Program
ABSTRACT

Milk is an important source of riboflavin and essential amino acids such as
phenylalanine, tryptophan, leucine, isoleucine and valine. Riboflavin, a well known
photosensitizer could rapidly destroy essential amino acids in milk through oxidation.
The effects of Trolox and ascorbic acid on the riboflavin photosensitized oxidation of
tryptophan, tyrosine, phenylalanine, methionine, arginine and valine were studied.
Riboflavin is a water soluble sensitizer and produces singlet oxygen under
light. Aqueous samples with 25 ppm added riboflavin were prepared separately to
contain 0, 100, 250, 500, 750 or 1000 ppm of Trolox or ascorbic acid. Samples were
stored under light (1000 lux) at 30 °C for 10 hours. As the concentration of Trolox
and ascorbic increased from 0 to 1000 ppm, the head space oxygen depletion
increased. This was due to the oxidation of Trolox and ascorbic acid along with
amino acids in the presence of riboflavin. High performance liquid chromatography
analysis of the samples indicated that both Trolox and ascorbic acid decreased the
degradation of phenylalanine, tryptophan and tyrosine significantly (p<0.05).

Trolox and ascorbic acid acted as singlet oxygen quenchers and can protect
tryptophan and tyrosine. However, the quenching mechanisms are different between
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Trolox and ascorbic acid. Trolox quenched both singlet oxygen and excited triplet
riboflavin under light, where as ascorbic acid quenched singlet oxygen only. The
singlet oxygen quenching rate of Trolox in the presence of tryptophan and tyrosine
were 1.55 x 107 M-1s-1 and 1.32 x 107 M-1s-1 respectively. The singlet oxygen
quenching rate of ascorbic acid in the presence of tryptophan and tyrosine were 1.16
x 107 M-1s-1 and 1.10 x 107 M-1s-1. Singlet oxygen quencher alone could not
completely protect tryptophan and tyrosine. Trolox, which is a quencher of both
singlet oxygen and excited triplet riboflavin, can better protect tryptophan and
tyrosine.

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Dedicated to my family

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ACKNOWLEDGMENTS

There is one person above all others who deserve my deepest respect and thanks; he
gave me the opportunity to join his research group and accepted to advise my
research work; he fostered a stress-free working relationship which was crucial to the
completion of this work, my advisor Dr. David Min. I am grateful to him for
supportive supervision. It has been a pleasure and honor to be his advisee.

I am thankful to my committee members Dr.Barringer, Dr.Bala and Dr.Rodriguez for
their support and encouragement.

I must thank Dr. Hyun Jung Kim for the thought provoking conversations we have
had and her support and advice throughout my research. I owe a great deal to her.

I wish to express my warmest thanks to all my faithful friends.

I acknowledge and return love of my mom; she believed in me all these years no
matter what I have chosen to do and my dad for giving me his vision of life and my
sense of self.

Finally, I am thankful to the Almighty for his grace.

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VITA

1999 ……………………….B.Sc., (Ag) A.N.G.R.Agricultural University
Hyderabad, India

2001……………………….. M.Sc.,(Ag) A.N.G.R.Agricultural University
Hyderabad, India

2004……………………….. M.S. in Food Science & Technology
Virginia Tech, Blacksburg, VA

2004 - 2008 ……………… Ph.D in Food Science and Technology
The Ohio State University, Columbus, OH

FIELD OF STUDY

Major Field: Food Science and Nutrition

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TABLE OF CONTENTS

Page
Abstract…………………………………………………………………. ii

Dedication………………………………………………………………… iv

Acknowledgments……………………………………………………… v

Vita……………………………………………………………………… vi

List of Tables…………………………………………………………… x

List of Figures……………………………………………………………… xii

Chapter 1: Literature Review……………………………………………… 1

1. Literature Review…………………………………………………………. 1
1.1. Lipid Oxidation………………………………………………………. 1
1.1.1. The chemistry of triplet and singlet oxygen oxidation………… 1
1.1.2. Singlet oxygen formation…………………………………….. 2
1.1.3. Photosensitization……………………………………………… 3
1.1.4. Type-I and Type-II pathways…………………………………. 4
1.1.5. Triplet oxygen oxidation………………………………………. 4
1.1.6. Singlet oxygen oxidation with fatty acids……………………... 6
1.1.7. Singlet oxygen oxidation with amino acids…………………… 6
1.2. Photosensitizers………………………………………………………. 8
1.2.1. Riboflavin……………………………………………………… 8
1.2.2. Stability of riboflavin…………………………………………. 9
1.2.3. Degradation of riboflavin……………………………………… 11
1.3. Singlet oxygen quenching mechanisms in foods…………………… 12
1.3.1. Singlet oxygen quenchers……………………………………… 13
1.3.2. Carotenoids……………………………………………………. 13
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1.3.3. Phenols………………………………………………………… 14
1.3.4. Trolox…………………………………………………………. 16
1.3.5. Ascorbic Acid…………………………………………………. 18
1.4. Singlet oxygen oxidation of milk products…………………………… 19
1.4.1. Sunlight flavor in milk…………………………………………. 20
1.5. Determination of quenching mechanisms……………………………. 24

2. Effects of Trolox and ascorbic acid on the photosensitized oxidation of
amino acids in the presence of riboflavin
2.1. Abstract………………………………………………………………. 57
2.2. Introduction………………………………………………………….. 58
2.3. Materials and Methods………………………………………………. 60
2.4. Results and Discussion……………………………………………….. 65
2.5. Conclusion……………………………………………………………. 72
2.6. Bibliography………………………………………………………….. 73

3. Quenching mechanisms and kinetics of Trolox and ascorbic acid on the
riboflavin photosensitized oxidation of tryptophan and tyrosine
3.1. Abstract……………………………………………………………… 92
3.2. Introduction…………………………………………………………. 93
3.3. Materials and Methods………………………………………………. 95
3.4. Results and Discussion………………………………………………. 97
3.5. Conclusion…………………………………………………………… 105
3.6. Bibliography…………………………………………………………. 106

Conclusion…………………………………………………………………… 120

Bibliography………………………………………………………………….. 121

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LIST OF TABLES

Table Page

1.1. Differences in the chemical properties of singlet oxygen and triplet
oxygen…………………………………………………………………. . 54

1.2. Relative oxidation rates of triplet oxygen and singlet oxygen with
oleate, linoleate and linolenate………………………………………….. 54

1.3. Hydroperoxides of fattyacids by singlet oxygen and triplet oxygen……. 55

1.4. Destruction of riboflavin in milk by sunlight…………………………… 56

1.5. Destruction of ascorbic acid in milk by sunlight……………………….. 56

2.1. Effects of 25 ppm riboflavin on the retention of phenylalanine,
tryptophan, and tyrosine under light…………………………………… 75

2.2. Effects of Trolox on the retention (%) of phenylalanine (1.25%)
under light……………………………………………………………… 75

2.3. Effects of Trolox on the retention (%) of tryptophan (1.25%)
under light……………………………………………………………… 76

2.4. Effects of Trolox on the retention (%) of tyrosine (1.25%)
under light……………………………………………………………… 76

2.5. Effects of ascorbic acid on the retention (%) of phenylalanine (1.25%)
under light……………………………………………………………… 77

2.6. Effects of ascorbic acid on the retention (%) of tryptophan (1.25%)
under light……………………………………………………………… 77

2.7. Effects of ascorbic acid on the retention (%) of tyrosine (1.25%)
under light……………………………………………………………… 78

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2.8. Effects of light or dark on riboflavin (25 ppm) photosensitized
oxidation of Trolox at 30 oC………………………………………… 78
2.9. Effects of light or dark on riboflavin (25 ppm) photosensitized
oxidation of ascorbic acid at 30 oC 79

3.1. The intercepts and slopes from regression lines in Figure 3.1 and Figure
3.3 for determining quenching rate of Trolox on singlet oxygen
oxidation of tryptophan and tyrosine. …………………………………. 1 1 7

3.2. The intercepts and slopes from regression lines in Figure 3.5 and Figure
3.7 for determining quenching rate of ascorbic acid on singlet oxygen
oxidation of tryptophan and tyrosine………………………………… 1 1 8

3.3. Reaction rate constants of singlet oxygen and tryptophan and tyrosine
(K) and quenching rates of tryptophan and tyrosine………………… 1 1 9
r

x