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EFFECT OF HIGH PRESSURE PROCESSING & DAIRY ON THE ANTIOXIDANT ACTIVITY OF STRAWBERRY BASED BEVERAGES

BY RAVI KIRAN TADAPANENI

DEPARTMENT OF CHEMICAL & BIOLOGICAL ENGINEERING

Submitted in partial fulfillment of the requirements for the degree of Master of Science in Food Process Engineering in the Graduate College of the Illinois Institute of Technology

Approved _________________________ Advisor

Chicago, Illinois December 2010

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ACKNOWLEDGEMENT

This thesis arose in part out of days of research that has been done since I joined Nutrition group. By that time, I have worked with a great number of people whose contribution in assorted ways to the research and the making of the thesis deserved special mention. In the first place I would like to record my gratitude to my mentors, Dr. Britt Burton Freeman and Dr. Indika Edrinsinghe for their supervision, advice, and guidance from the very early stage of this research as well as giving me extraordinary experiences through out the work. Above all and the most needed, they provided me unflinching encouragement and support in various ways. Their passion for science and research, being a constant oasis of ideas, exceptionally inspired and enriched my growth as a student, a researcher and a scientist want to be. I am indebted to them. I gratefully thank thesis committee members, Dr. Wei Zhang and Dr. Satish Parulekar for their constructive comments on this research work. I am thankful that in the midst of all their activity, they accepted to be members of the thesis defense committee. It is a pleasure to pay tribute also to the supporting collaborators. I would like to thank Eduardo Patazca for his high quality time in training me to handle High Pressure Processing equipment and to Dr. Artemio Tulio, Jr. for his valuable guidance and help in developing the antioxidant assays. I greatly acknowledge Katie Banaszewski’s timely help in analyzing the samples by HPLC techniques. My heartfelt gratitude goes to each one in the Nutrition group (Sandhya Krishnankutty, Eunyoung Park, Julie Rock Talbot, Claire Chang, Rima Juskelis, Dr. Joseph E. Jablonski, Jack Cappozzo, Fadwa Al-Taher,

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Dr. Yang Chen, Dr. Lauren Jackson and Varsha Muttagikar) for their steady support and motivation. I gratefully acknowledge my senior, Dhanesh Liyanage for his advice, supervision, and crucial contribution to this thesis. Many thanks go in particular to Dr. Rukma Reddy and Dr. Kathiravan Krishnamurthy. I am much indebted to their valuable advice in science discussion, supervision in processing methods and furthermore, using their precious times to answer some of my unintelligent questions about High Pressure Processing. Many thanks go to Dr. Ravinder Reddy, Dr. Stephen Grove and Dr. Jason Wan for their valuable suggestions in performing microbiological analysis. My special thanks go to Renee McBrien, for her indispensable encouragement and emotional comfort provided me, the home feel atmosphere. My time at Illinois Institute of Technology was made enjoyable in large part due to the many friends and groups that became a part of my life. The informal support and encouragement of friends has been crucial, and I would like particularly to acknowledge the

involvement

of

Neeraj

Maddi,

Srinivasa

Chandra,

Anuraga

Mandava,

Balasubramanyam Puli, Annamalai Suri, Satish Seerapu and Mythili Kotapalli. My Parents & Sister have been a constant source of support – emotional, moral and of course financial – during my graduate program, and this thesis would certainly not have existed without them. No words will suffice to thank my father who ignited my initial interest in science through his deep insight and research experience. Although our fields are now divergent – it is to him that this thesis is dedicated. Finally, I would like to thank everybody who was important to the successful realization of thesis, as well as expressing my apology that I could not mention personally one by one.

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TABLE OF CONTENTS

Page ACKNOWLEDGEMENT LIST OF TABLES LIST OF FIGURES

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...................................................................................................

vii

................................................................................................. viii

LIST OF ABBREVIATIONS ABSTRACT

..................................................................................

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.............................................................................................................

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CHAPTER 1. INTRODUCTION

..............................................................................

2. LITERATURE REVIEW 2.1 2.2 2.3 2.4 2.5 2.6

...................................................................

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Antioxidants ................................................................................ Fruits/vegetables and antioxidants – Health promoting effects .. Strawberries (Fragaria x ananassa) ........................................... Processing Technology ............................................................... Characterization and Measurement of Antioxidant Activity ...... Study Objectives .........................................................................

11 15 16 20 27 29

3. MATERIALS & METHODS 3.1 3.2 3.2 3.4 3.5 3.6 3.7 3.8

1

.............................................................

Chemical, ingredients & equipment ........................................... Beverage Preparation .................................................................. Beverage Processing Design ....................................................... High Pressure Processing............................................................ Measure of Antioxidant Activity in beverages ........................... Polyphenolic compound analysis in beverage using LC-MS/MS Microbiological Analysis ............................................................ Statistical Analysis ......................................................................

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31 31 31 33 33 36 41 43 46

4. RESULTS ................................................................................................

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4.1 Effect of dairy constituents on the antioxidant activity of the beverages..................................................................................... 4.2 Effect of processing on the antioxidant activity ......................... 4.3 Effect of storage on the antioxidant activity ............................... 4.4 Effect of processing on the polyphenolic compounds ................ 4.5 Food safety bbjective ..................................................................

47 49 52 56 58

5. DISCUSSION

.....................................................................................

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5.1 Effect of dairy constituents on the antioxidant activity .............. 5.2 Effect of processing on the antioxidant activity ......................... 5.3 Effect of storage on the antioxidant activity ...............................

63 65 69

6. CONCLUSION ........................................................................................

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APPENDIX A. POLYPHENOLIC PROFILE OF FREEZE DRIED STRAWBERRY POWDER BIBLIOGRAPHY

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....................................................................................................

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LIST OF TABLES

Table

Page

2.1. Major categories of phenolic compounds ........................................................

13

2.2. Anthocyanins in fruits ......................................................................................

15

2.3. Food sources rich in antioxidants ....................................................................

18

3.1. Beverage formulation recipe ............................................................................

32

3.2. Experimental design for beverage HPP ...........................................................

35

4.1. Shelf life stability of D-NStr as measured by ORAC (μM/mL) ......................

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4.2. Shelf life stability of ND-Str as measured by ORAC (μM/mL) ......................

53

4.3. Shelf life stability of D-Str as measured by ORAC (μM/mL) .........................

54

4.4. Shelf life stability of D-NStr as measured by FRAP (mM Fe2+/mL) ..............

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4.5. Shelf life stability of ND-Str as measured by FRAP (mM Fe2+/mL) ..............

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4.6. Shelf life stability of D-Str as measured by FRAP (mM Fe2+/mL) .................

55

4.7. Microbial counts (log10 CFU/mL) values of D-NStr ......................................

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4.8. Microbial counts (log10 CFU/mL) values of ND-Str .......................................

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4.9. Microbial counts (log10 CFU/mL) values of D-Str ..........................................

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LIST OF FIGURES

Figure

Page

2.1. Well known antioxidants .................................................................................

12

2.2. Phenolic antioxidant mechanism .....................................................................

14

2.3. Basic structure of anthocyanins .......................................................................

15

2.4. Structure of phenolic compounds ....................................................................

19

2.5. Schematic presentation of HPP range ..............................................................

26

2.6. Horizontal HPP unit .........................................................................................

26

3.1. Schematic of variation of pressure and temperature during a typical HPP run

35

3.2. 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) ...............

37

3.3. Behavior of antioxidant molecule ....................................................................

38

3.4. ORAC assay pathway ......................................................................................

39

3.5. Procedure of TEMPO® reading of samples .....................................................

44

3.6. Microbial quantitative analysis of sample using Petrifilm™ plating ..............

45

4.1. Effect of different matrices on antioxidant activity of unprocessed beverages

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4.2. Major polyphenolic compounds in beverage samples .....................................

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4.3. Antioxidant values of D-NStr beverage with different processing conditions

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4.4. Antioxidant values of ND-Str beverage with different processing conditions

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4.5. Antioxidant values of D-Str beverage with different processing conditions ...

52

4.6. Composition of D-Str beverage as determined by LC-MS/MS .......................

56

4.7. Effect of HPP on pelargonidin-3-O-glucoside .................................................

57

4.8. Effect of 5-weeks storage period on pelargonidin-3-O-glucoside ...................

58

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LIST OF ABBREVIATIONS

Abbreviation

Definition

ORAC

Oxygen Radical Absorbance Capacity

FRAP

Ferric Reducing Antioxidant Power

μM

Micromole

mM

Millimole

MPa

Megaascal

CVD

Cardiovascular Disease

GI

Glycemic Index

LDL

Low-density Lipoprotien

TEAC

Trolox Equivalent Antioxidant Capacity

PET

Polyethylene Terephthalate

LLDPE

Linear Low-density Polyethylene

v/v

Volume/Volume

LC-MS

Liquid Chromatography-Mass Spectrometry

TVC

Total Viable Count

CFU

Colony Forming Unit

ANOVA

Analysis of Variance

SD

Standard Deviation

TE

Trolox Equivalents

AUC

Area Under Curve

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ABSTRACT

Fruits contain a large family of polyphenolic compounds that have antioxidant properties. These compounds are known to associate with lower risk of major chronic diseases. Fruits are consumed as raw and in processed forms such as shakes and beverages. The perishable nature of fruits/fruit-based products demands processing strategies to enhance shelf-life and assure food safety, while maintaining nutritional quality. Dairy has been suggested to interfere with the antioxidant activity of polyphenolic compounds when included in the formulation of fruit-based beverages. Therefore, we propose to develop a model system for testing food matrix effects with standard and novel processing strategies on the antioxidant capacity (AC) of fruit-based beverages containing appreciable amounts of polyphenolic compounds. In the present study, we used strawberry (Str) beverages as the model to investigate the effect of dairy and high temperature-short time processing (HT–72°C-20 sec) and high pressure processing (HPP – 200 to 600 MPa; holding time 1 to 15 minutes) on beverage AC as measured by Oxygen Radical Scavenging Capacity (ORAC) and Ferric Reducing Antioxidant Power (FRAP). Three beverages were prepared and subjected to HT or HPP processing: 1) Str + Dairy beverage (D-Str), 2) Str + Distilled Water beverage (ND-Str), 3) Dairy (no Str) beverage (D-NStr). All beverages were matched for energy, macro- and micro- nutrients. HPP and HT application resulted in significantly reduced ORAC & FRAP values compared to their respective unprocessed beverages (P