an abstract of the thesis of · 2017-08-03 · members, especially dr. dan selivonchick, ... isomer...

AN ABSTRACT OF THE THESIS OF

G. Astrid Garz6n for the degree of Doctor of Philosophy in Food Science and

Technology presented on June 30, 1998. Title: The Stability of Pelargonidin-based

Anthocyanins in Natural and Model Systems.

Abstract approved:

= ^

Ronald E. Wrolstad

Pelargonidin 3-glucoside (pgd 3-glu), pelargonidin 3-sophoroside (pgd 3-soph),

and pelargonidin 3-sophoroside 5-glucoside acylated with cinnamic and malonic acids

(acyl-pgd 3-soph 5-glu) were extracted from strawberries (Fragaria anannassa cv,

Totem), nasturtium flowers (Tropaeolum majus), and radish peel (Raphanus sativus L.

cv, fuego), respectively. Their stability was studied in natural and model systems.

Natural systems consisted of strawberry juice at 8 "brix and strawberry concentrate at

65 0brix that were spiked with the anthocyanins (ACNS) to double the initial pigment

concentration. Model systems at low, intermediate, and high water activity levels

consisted of pH 3.4 citrate buffer, glycerol, and pigment. Changes in pigment,

degradation index, color, and relative peak area were monitored during storage in the

dark at 25 "C. Ascorbic acid degradation was also monitored in the natural systems.

Anthocyanin (ACN) degradation followed first order kinetics. No difference in stability

of the samples was found with fortified pgd derivatives; however, there was significant

difference in the degradation of ACNS between natural and model systems. The half life

(ti/2) of the ACNS ranged from 3.5 to 5 days in the concentrate, from 8 to 12 days in

juice, and from 58 to 934 days in model systems. In general, high Aw increased ACN

degradation. Ascorbic acid degradation followed first order kinetics and was

accompanied by ACN degradation.

The Stability of Pelargonidin-based Anthocyanins in Natural and Model Systems

by

G. Astrid Garz6n

A THESIS

Submitted to

Oregon State University

In partial fulfillment of the requirements for the

degree of

Doctor of Philosophy

Completed June 30,1998 Commencement June 1999

Doctor of Philosophy thesis of G. Astrid Garz6n presented on June 30, 1998

APPROVED:

■rrr igi^i^^r .^y ,

Major Professor, representing Food Science and Technology

y—>*" / *- =—'^^ j, \J ^ Head 01 Department of Food Science and Technology

'\__r J'*'*^— *"- -w*—mi

Dean of Graduate School

I understand that my thesis will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my thesis to any reader upon request.

G. Astrid Garz6n, Author

ACKNOWLEDGEMENTS

I would like to acknowledge Dr. Ron Wrolstad, my major professor, for having

welcomed me to his research group and for his support and patience during these years

of my stay at Oregon State University. I also want to thank the rest of my committee

members, especially Dr. Dan Selivonchick, Dr. Phil McFadden and Dr. Fred Ramsey

for both their guidance and friendship over the last few years.

I would like to express acknowledgement to the Small Fruit Research Center for

having financed part of this research project and to Conroy Packing Inc. (Salem, OR)

for providing the fruit necessary for this work.

I would like to express thanks to Bob Durst and Ken Stewart for their assistance

in juice processing and laboratory techniques and also to Tim McCann at Food

Laboratories (Portland, OR) for his information and assistance on water activity

analysis.

Finally, I would like to thank my family for believing in me through this

journey.

TABLE OF CONTENTS

Page

CHAPTER 1. INTRODUCTION 1

CHAPTER 2. LITERATURE RE VIEW 5

ANTHOCYANINS 5

Structure » 5 Anthocyanins in strawberry 6 Stability 7 Kinetics of anthocyanin degradation 19

REFERENCES 23

CHAPTERS. CHARACTERIZATION OF TWO PELARGONIDIN-BASED ANTHOCYANINS FROM STRAWBERRY (Fragana anannassa) AND NASTURTIUM FLOWERS (Tropaeolum majus) 33

ABSTRACT 34

INTRODUCTION 34

MATERIALS AND METHODS 35

Plant material and anthocyanin juices 35 Reagents 36 Pigment extraction and isolation 36 Pigment analysis 36 HPLC conditions for anthocyanin analysis 36 Separation and collection of pigments via

preparatory HPLC 37 Purification of pigments 37 MS analysis of anthocyanins 38 Alkaline hydrolysis of anthocyanins 38 Acid hydrolysis of anthocyanins 38 HPLC conditions for anthocyanindin analysis 39 Identification of anthocyanins and anthocyanidins 39 HPLC conditions for analysis of organic acids 39 Color specifications of pelargonidin derivatives

from strawberry 39 Preparation of trimethylsilyl (TMS) derivatives 40 Capillary Gas Chromatography 41

TABLE OF CONTENTS (continued)

Page

RESULTS AND DISCUSSION 41

Anthocyanin content 41 HPLC separation of anthocyanins and anthocyanidins 41 Organic acids analysis 47 Mass spectrometry 50 Identification of sophorose from the acid hydrolysis of

pgd3-soph 50 Color specifications of anthocyanins 55

CONCLUSIONS 55

REFERENCES 56

CHAPTER 4. COMPARATIVE STABILITY OF PELARGONIDIN-B ASED ANTHOCYANINS IN JUICE SYSTEMS 59

ABSTRACT 60

INTRODUCTION 60


Plant material 61 Processing of juice and concentrate 62 Compositional analysis 64 Pigment extraction and isolation 64 Anthocyanin purification 64 HPLC for separation of major anthocyanins 65 Analytical HPLC 65 Experimental design 65 Pigment analysis 66 Color and haze analysis 66 Changes in pigment profile during storage 66 Data analysis 67


Strawberry juice and concentrate before storage 67 Pigment analysis 69 Changes in haze during storage 74


Color analysis 75 Changes in HPLC proffle 75

CONCLUSIONS 82

REFERENCES 82

CHAPTER 5. COMPARISON OF THE STABILITY OF PELARGONIDIN- BASED ANTHOCYANINS IN STRAWBERRY JUICE AND CONCENTRATE 86

ABSTRACT 87

INTRODUCTION 87


Processing of juice and concentrate 89 Compositional analysis 89 Pigment extraction, isolation, and purification 91 Separation and collection of pelargonidin derivatives 91 Experimental design 92

Juice and concentrate fortification 92 Pigment analysis 93 Color and haze analysis 93 Analytical HPLC 93 Ascorbic acid analysis 93 Statistical analysis 94


Juice and concentrate prior to storage 95 Changes of total anthocyanin with time 98 Changes in color indices with storage 102 Changes in haze 105 Changes in anthocyanin pigment profiles during storage... 105 Changes in ascorbic acid during storage 110

CONCLUSIONS 116

REFERENCES 116


Page

CHAPTER 6. THE STABILITY OF PELARGONIDINANTHOCYANINS AT VARYING WATER ACTIVITY 120

ABSTRACT 121

INTRODUCTION 121


Plant Materials 123 Chemicals and reagents 123 Pigment extraction and isolation 124 Anthocyanin purification 124 Semipreparative HPLC 124 Analytical HPLC 125 Experimental design 126

Preparation of model systems 126 Pigment analysis 126 Statistical analysis 129


Changes in total anthocyanin pigment during storage at different Aw 129

Changes in HPLC profile during storage 141

CONCLUSIONS 149

REFERENCES 149

CHAPTER?. SUMMARY 153

BIBLIOGRAPHY 155

LIST OF FIGURES

Figure Page

2.1 Chemical structure of the most common anthocyanidins found in nature.... 6

2.2 Structural changes in anthocyanins with changes in pH 9

3.1 HPLC chomatogram of anthocyanins from (a) red nasturtium petals, (b) orange nasturtium petals, and (c) their characteristic UV-visible spectra.... 42

3.2 (a) Anthocyanidin profile of the pigments present in petals of nasturtium flowers and (b) their respective UV-visible spectra 44

3.3 (a) HPLC profile of strawberry anthocyanins and (b) UV-visible spectra of (2) pelargonidin 3-glucoside, (4) pelargomdin 3-glucoside-succinate - isomer 1, and (5) pelargonidin 3-glucoside-succinate -isomer 2 46

3.4 Changes in HPLC profile after alkaline hydrolysis of the two late-eluting peaks present in strawberry anthocyanins. (a) pelargonidin 3-glucoside- succinate-isomer 1, (b) pelargonidin 3-glucoside-succinate -isomer 2, (c) shift in time of elution for both compounds, (d) absorption UV-visible spectra for the three peaks 48

3.5 HPLC chromatogram of (a) succinic acid standard, (b) succinic acid from saponification, and (c) succinic acid from saponification after spiking with standard 49

3.6 Mass Spectrum of pelargonidin 3-sophoroside present in nasturtium flowers 51

3.7 Mass Spectrum of succinyl-pelargonidin 3-glucoside -isomer 1 present in strawberry 52

3.8 Mass Spectrum of succinyl- pelargonidin 3-glucoside -isomer 2 present in strawberry 53

3.9 GLC chromatogram of TMS derivatives of (a) glucose standard and (b) glycosidic substituents of the orange-colored nasturtium petals 54

4.1 Unit operations for the production of strawberry juice and concentrate 63

4.2 Changes in pigment during storage of juice systems 70

LIST OF FIGURES (continued)

Figure Page

4.3 Changes in pigment during storage of concentrate systems 71

4.4 Changes in color during storage of strawberry juice and concentrate 76

4.5 HPLC profile of the anthocyanins in unfortified juice and juice spiked with pelargonidin derivatives before storage. Peak identification: (1) cyanidin 3-glucoside; (2) pelargonidin 3-glucoside; (3) pelargonidin 3- rutinoside; (4) pelargonidin 3-glucoside acylated with succinic acid (isomer 1); (5) pelargonidin 3-glucoside acylated with succinic acid (isomer 2); (6) pelargonidin 3-sophoroside; (7) pelargonidin 3-sophoroside 5-glucoside; (8) pelargonidin 3-sophoroside 5-glucoside acylated with cinnamic acids; (9) pelargonidin 3-sophoroside 5-glucoside acylated with cinnamic and malonic acids 77

4.6 UV-visible spectra of (a) pelargonidin 3-glucoside (b) pelargonidin 3- sophoroside 5-glucoside from strawberry juice fortified with radish anthocyanin 78

4.7 Changes in anthocyanin profile during storage of (a) juice, (b) juice fortified with pelargonidin 3-glucoside, (c) juice fortified with pelargonidin 3-sophoroside, (d) concentrate, (e) concentrate fortified with pelargonidin 3-glucoside, (f) concentrate fortified with pelargonidin 3- sophoroside 80

4.8 Changes in anthocyanin profile during storage of (a) juice fortified with acyl- pelargonidin 3-sophoroside 5-glucoside, (b) concentrate fortified with acyl-pelargonidin 3-sophoroside 5-glucoside 81

5.1 Unit operations for the production of strawberry juice and concentrate 90

5.2 Changes in pigment during storage of juice systems 99

5.3 Changes in pigment during storage of concentrate systems 100

5.4 Changes in color during storage of juice systems 103

5.5 Changes in color during storage of concentrate systems 104

5.6 Changes in haze during storage of (a) juice systems and (b) concentrate systems 106


Figure Page

5.7 HPLC chromatograms of the anthocyanins in unfortified juice and juice spiked with pelargonidin derivatives before storage. Peak identification: (1) cyanidin 3-glucoside; (2) pelargonidin 3-glucoside; (3) pelargonidin 3- rutinoside; (4) pelargonidin 3-glucoside acylated with succinic acid (isomer 1); (5) pelargonidin 3-glucoside acylated with succinic acid (isomer 2); (6) pelargonidin 3-sophoroside; (7) pelargonidin 3-sophoroside 5-glucoside acylated with cinnamic acid; (8) pelargonidin 3-sophoroside 5-glucoside acylated with/?-coumaric acid and malonic acids; (9) pelargonidin 3-sophoroside 5-glucoside acylated with ferulic and malonic acids 107

5.8 Changes in peak area during storage of (a) juice, (b) juice + pelargonidin 3-glucoside, (c) juice + pelargonidin 3-sophoroside, (d) juice + acyl- pelargonidin 3-sophoroside 5-glucoside 108

5.9 Changes in peak area during storage of (a) concentrate, (b) concentrate + pelargonidin 3-glucoside, (c) concentrate + pelargonidin 3-sophoroside, (d) concentrate + acyl-pelargonidin 3-sophoroside 5-glucoside 109

5.10 Changes in ascorbic acid concentration during storage of (a) juice systems and (b) concentrate systems Ill

5.11 Changes in molar ratios of ascorbic acid/anthocyanin during storage of (a) juice systems and (b) concentrate systems 114

6.1 Flow chart of the preparation of model systems for evaluation of stability of pelargonidin derivatives at varying Aw 128

6.2 Changes in (a) monomeric anthocyanin and (b) polymeric color (%) during storage of model systems containing pelargonidin 3-glucoside. Means in the same column with unlike superscripts indicate significant difference (p-value < 0.05) 132

6.3 Changes in (a) monomeric anthocyanin and (b) polymeric (%) color during storage of model systems containing pelargonidin 3-sophoroside. Means in the same column with unlike superscripts indicate significant difference (p value < 0.05) 135

6.4 Changes in (a) monomeric anthocyanin and (b) polymeric (%) color during storage of model systems containing acyl-pelargonidin 3-


Figure Page

6.4 sophoroside 5- glucoside. Means in the same column with unlike superscripts indicate significant difference (p value < 0.05) 136

6.5 Chromatographic profile at time (a) zero and (b) 242 days of storage of model systems at Awl containing purified pelargonidin 3-glucoside 147

6.6 Chromatographic profile at time (a) zero and (b) 242 days of storage of model systems at Awl containing purified pelargonidin 3-sophoroside 143

6.7. Changes in peak area during storage of model systems containing acyl- pelargonidin 3-sophoroside 5-glucoside 145

6.8 Chromatographic profile at time (a) zero, (b) 242 days-replicate 1, (c) 242 days-replicate 2 of storage of model systems at Aw 1 containing acyl- pelargonidin 3-sophoroside 5-glucoside 146

6.9 Comparative UV spectra of pgd derivatives present in model systems at Aw 1 containing purified acyl-pelargonidin 3-sophoroside 5-glucoside. (a) pelargonidin 3-sophoroside 5-glucoside, (b) pelargonidin 3-sophoroside, (c) pelargonidin 3-sophoroside 5-glucoside acylated with cinnamic acids... 147

6.10 Chromatographic profile at time (a) zero, (b)142 days, (c) 242 days of storage of model systems at Aw 0.44 containing purified containing acyl- pelargonidin 3-sophoroside 5-glucoside 148

LIST OF TABLES

Table Page

2.1 Anthocyanin content of some major strawberry cultivars used in food processing 14

2.2 Water activity values corresponding to the most common processed strawberry products 19

2.3 Linear regression parameters of monomeric anthocyanin degradation in some strawberry systems 21

3.1 Characteristics of anthocyanins present in nasturtium flowers 43

3.2 Characteristics of anthocyanidins present in nasturtium flowers 45

3.3 Characteristics of anthocyanins present in Totem strawberries 47

3.4 Chromatographic properties of the sugar substituent present in the pgd- based anthocyanin of orange petals from nasturtium flowers 55

3.5 Color specifications of pelargonidin 3-glucoside and acylated pelargonidin 3-glucoside (oxonium forms) 56

4.1 Compositional and color indices of strawberry juice and concentrate before fortification 68

4.2 Anthocyanin and color characteristics of fortified strawberry juice and concentrate 69

4.3 Linear regression parameters of monomeric anthocyanin degradation in strawberry juice and concentrate 72

4.4 Changes in polymeric anthocyanin (%) in strawberry juice and concentrate during storage 73

4.5 Changes in degradation index (A 510/A 420) during storage of strawberry juice and concentrate systems 73

4.6 Changes in haze during storage of strawberry juice and concentrate systems 74

5.1 Compositional and color indices of strawberry juice and concentrate before fortification with pelargonidin derivatives 96

LIST OF TABLES (continued)

Table Page

5.2 Compositional and color indices of strawberry juice and concentrate after fortification with pgd derivatives 97

5.3 Linear regression parameters of monomeric anthocyanin degradation in juice and concentrate systems 101

5.4 Linear regression parameters of ascorbic acid degradation in juice and concentrate systems 113

5.5 Correlation coefficients for Ln anthocyanin concentration and Ln ascorbic acid concentration during storage 115

6.1 Composition of model systems used for evaluation of anthocyanin stability at varying Aw 127

6.2 Linear regression and first order reaction parameters for the degradation of model systems containing pelargonidin 3-glucoside 131

6.3 Linear regression and first order reaction parameters for the degradation of model systems containing pgd 3-soph 134

6.4 Linear regression and first order reaction parameters for the degradation of model systems at Aw 0.66 and 0.44 containing acyl-pelargonidin 3- sophoroside 5-glucoside 137

6.5a Time for 15% of the initial anthocyanin to degrade during storage of model systems at high Aw levels (1,0.90,0.89) 138

6.5b Rate constants for anthocyanin degradation during storage of model systems containing acyl-pgd 3-soph 5-glu at intermediate and low Aw

levels (0.66,0.44) : 139

6.6 Changes in degradation index (A510/A420) during storage of model systems spiked with pelargonidin-based anthocyanins 142

THE STABILITY OF PELARGONIDIN-BASED ANTHOCYANINS IN NATURAL AND MODEL SYSTEMS

CHAPTER 1

INTRODUCTION

Consumers first judge the quality of a food product by its color; experience

conditions us to associate color with quality and sensory properties (Newsome, 1986).

Studies have shown that the appearance of a food product affects our perception of its

odor, flavor, and texture and that food does not taste as it should when it is not colored

"right" (Hall, 1958). Sherbets that were white and made with different flavors were

unsuccessfully identified by individuals in a testing panel. Likewise, when the sherbets

were deceptively colored, most judges mistakenly identified the flavors. From these

studies, the researchers concluded that color far outweighs flavor in the impression it

makes on the consumer, even when the desirable flavors are present and the food is a

popular one.

The flavor of strawberry is highly prized and food products derived from this

fruit are popular with consumers. Unfortunately, the bright red color of the fresh fruit

deteriorates with processing and storage, and a dull, pale, brown color appears. This

change appears to the consumer as an indication of inferior quality and deterioration of

the fruit. Since an attractive and stable color is an important quality parameter for the

consumer, the instability of color and pigment degradation in strawberry products has

been of concern to researchers and the food processing industry.

For the last few years, strawberries have remained the largest single berry crop

in the State of Oregon. In 1996, 5,285 acres were harvested, representing a value of 22.3

million dollars. Ninety percent of this production was processed by the food industry

and only 10% was sold fresh (Oregon State University Extension Service, 1996). This

already high marketability of strawberry products could be expanded if the original

color of the fruit were maintained during the processing operations. Browning reactions

that occur upon processing of strawberry are very complex with several mechanisms

contributing to color degradation, e.g., Maillard browning, enzymatic browning.

ascorbic acid degradation, and ACN polymerization. These reactions along with ACN

degradation result in reduced red color and increased browning in strawberry juice

(Wrolstad et al, 1980).

Influences of temperature, pH, ascorbic acid, and phenolic substances on the

main ACN responsible for the color of fresh strawberries have been studied in model

systems (Sondheimer and Lee, 1949; Meschter, 1953; Sondheimer and Kertesz, 1953;

Huang, 1955; Lukton et al., 1956; Markakis et al., 1957; Goodman and Markakis, 1964;

Poei-Langston and Wrolstad, 1981; Wesche and Montgomery, 1990). However, most of

the studies on stability of strawberry ACN have been done in natural systems, e.g., fresh

fruit (Skrede, 1982; Gil et al., 1997), strawberry preserves (Kertesz and Sondheimer,

1948; Mackinney and Chichester, 1952; Abers and Wrolstad, 1979; Garcia-Viguera and

Zafrilla, 1998), frozen strawberries (Wrolstad et al, 1990), freeze-dried strawberries

(Erlandson and Wrolstad, 1972), strawberry juice and concentrate (Wrolstad et al.,

1980; Rwabahizi and Wrolstad, 1988; Bakker and Bridle, 1992; Bakker et al., 1992;

Skrede et al., 1992), and strawberry wine (Pilando et al., 1985). The complexity of

natural systems makes it difficult to control and monitor the influence of a single factor

that accounts for changes in ACNS. Thus, model systems are advantageous in

investigating the contribution of individual factors to pigment degradation.

The purpose of this project was to study and compare the stability of strawberry

ACNS in both natural (juice and concentrate) and model systems resembling strawberry

juice. The influence of three factors was evaluated: pigment concentration, chemical

structure of the ACN, and water activity (Aw).

REFERENCES

Abers, J. E. and Wrolstad, R. E. 1979. Causative factors of color deterioration in strawberry preserves during processing and storage. J. Food Sci. 44 (1): 75-81.

Bakker, J. and Bridle, P. 1992. Strawberry juice colour: The effect of sulfur dioxide and EDTA on the stability of Anthocyanins. J. Sci. Food Agric. 60: 477-481.

Bakker, J., Bridle, P. and Koopman A 1992. Strawberry juice colour: The effect of some processing variables on the Stability of Anthocyanins. J. Sci. Food Agric. 60: 471-476.

Erlandson, J. A. and Wrolstad R. E. 1972. Degradation of anthocyanins in limited water concentration. J. Food Sci. 37: 592-595.

Garcia-Viguera, C. and Zafrilla, P. 1998. Anthocyanins in processed fruit products, p. 134, In Papers Presented at the Third International Symposium on Natural Colorants for Food,Nutraceuticals,Beverages,Confectionery, and Cosmetics. Princeton, New Jersey.

Gil, M. I., Deirdre, M. H., and Kader, A. A. 1997. Changes in strawberry anthocyanins and other polyphenols in response to carbon dioxide treatments. J. Agric. Food Chem. 45: 1662-1667.

Goodman, L. P. and Markakis, P. 1964. The effect of peroxidase on anthocyanin pigments. J. Food Sci. 29: 53-57.

Hall, R. L. 1958. Flavor study approach at McCormick and Company, Inc. In Flavor Research and Food Acceptance, p. 224. Reinhnold Pub. Corp., New York.

Huang, H. T. 1955. Decolorization of anthocyanins by fungal enzymes. J. Agric. Food Chem. 3:141.

Kertesz, Z. I., and Sondheimer, E. 1948. To reduce color losses in strawberry preserves. Food Inds. 20:106.

Lukton, A., Chichester, CO., and Mackinney, G. 1956. The breakdown of strawberry anthocyanin pigment. Food Technol. 10 (9): 427-432.

Mackinney, G. and Chichester, CO. 1952. Color deterioration in strawberry preserves. Canner. 114(12): 13.

Markakis, P., Livingston, G. E., and Fellers, R C 1957. Quantitative aspects of strawberry pigment degradation. Food Res. 22: 117-130.

Meschter, E. L. 1953. Effects of carbohydrates and other factors on strawberry products. J. Agric. Food Chem. 1: 574-579.

Newsome, R. L. 1986. Food Colors. Food TechnoL 40 (7): 49-56.

Oregon State University Extension Service, 1996. Oregon County and State Agricultural Estimates. Special report 790. Economic Information Office. Corvallis, OR 97331-3601.

Pilando, L. S., Wrolstad, R. E. and Heatherbell, D. A. 1985. Influence of fruit composition, maturity and mold contamination on the color and appearance of strawberry wine. J. Food Sci. 50(4): 1121.

Poei-Langston, M. S. and Wrolstad R. E. 1981. Color degradation in ascorbic acid- anthocyanin-flavanol model system. J. Food Sci. 46: 1218-1222,1236.

Rwabahizi, S. and Wrolstad, R. E. 1988. Effects of mold contamination and ultrafiltration on the color stability of strawberry juice and concentrate. J. Food Sci. 53(3): 857-861.

Skrede, G. 1982. Quality characterization of strawberries for industrial jam production. J. Sci. Food Agric. 33: 48-54.

Skrede, G., Lea, P., and Wrolstad R. E. 1992. Color stability of strawberry and blackcurrant syrups. J. Food Sci. 57 (1): 172-177.

Sondheimer, E. and Kertesz, Z. I. 1953. Participation of ascorbic acid in the destruction of anthocyanin in strawberry juice and model systems. Food Res. 18:475.

Sondheimer, E. and Lee, F. A. 1949. Color changes of strawberry anthocyanin with D- glucose. Science. 109: 331.

Wesche-Ebeling, P. and Montgomery, N. W. 1990. Strawberry polyphenoloxidase: its role in anthocyanin degradation. J. Food Sci. 55: 731.

Wrolstad, R. E., Lee, D. D., and Poei, M. S. 1980. Effect of microwave blanching on the color and composition of strawberry concentrate. J. Food Sci. 45:1573.

Wrolstad, R. E., Skrede, G., Lea, P., and Enersen, G. 1990. Influence on sugar on anthocyanin pigment stability in frozen strawberries. J. Food Sci. 55(4): 1064- 1065, 1072.

CHAPTER 2.

LITERATURE REVIEW

ANTHOCYANEVS

ACNS are secondary metabolites of higher plants responsible for the attractive

colors ranging from salmon and pink, through scarlet, magenta, and violet to purple and

blue of most fruits, flower petals, and leaves. ACNS are found mainly in the vacuoles of

plant cells (Goodwin and Mercer, 1983), although they may sometimes be located in

spherical vesicles called "anthocyanoplasts" (Strack and Wray, 1994). These are

pigments particularly characteristic of angiosperms or flowering plants, which provide

our major source of food crops. Families containing pigmented fruits are the Vitacea

(grapes), the Rosacea (apple, pear, apricot, cherry, plum, peach, sloe, blackberry,

raspberry, strawberry, and quince), the Ericaceae (blueberry and cranberry), the

Saxifragaceae (black and red currants), the Caprifoliaceae (elderberry), and the

Solanaceae (tamarillo, huckleberry, aubergine, and potato). The cruciferae, in red

cabbage, colored pods of legumes, and the red roots of radish also have ACNS

(Timberlake and Bridle, 1975).

Structure

The ACN structure is made of a C15 heterocyclic nucleus bearing at least one,

and often several, sugar residues. The sugar hydroxyls may be esterified by aliphatic

and or/and aromatic organic acids (Tom£s-Barber6n and Robins, 1997). (Figure 2.1).

Differences between individual ACNS are the number of hydroxyl groups in the

molecule, the degree of methylation of these hydroxyl groups, the nature and number of

sugars attached to the molecule and the position of the attachment, and the nature and

number of aliphatic or aromatic acids attached to the sugars in the molecule (Mazza and

Miniati, 1993). The major anthocyanidins found in nature are pelargonidin (pgd),

cyanidin (cyd), peonidin (pnd), delphinidin (dpn), petunidin (ptd), and malvidin (mvd).

The most common ACNS are 3-glucosides and 3,5-diglucosides. Many ACNS

OH

R1=H R2 = H Pelargonidin (pgd) Rl=OH R2 = H Cyanidin (cyd) Rl=OH R2 = OH Delphinidin (dpn) Rl = 0CH3 R2 = H Peonidin (pnd) Rl = OCH3 R2 = OH Petunidin (ptd) Rl = OCH3 R2 = OCH3 Malvidin (mvd)

Figure 2.1. Chemical structures of the most common anthocyanidins found in nature.

containing acylated sugar moieties are also known. The acyl groups are mostly

derivatives of cinnamic acid such as p-coumaric, caffeic, and ferulic acid, but aliphatic

acids such as malonic, succinic, and acetic can also be present (Goto, 1987).

Anthocyanins in strawberry

Botanically, the strawberry (Fragaria x anannassa Duch.) is an aggregate fruit

formed by the ripening together of several ovaries, all belonging to a single flower and

adhering as a unit on a common, pulpy receptacle. The fruit is greenish white first and

ripens to a bright red. Chlorophyll and carotenoids are synthesized up to the 28th day

after petal fall; between the 25th and the 29th day, synthesis of ACNS begins

(Woodward, 1972).

The main ACN pigment in cultivated strawberries was identified as pgd 3-glu by

Robinson and Robinson (1932). This identification was confirmed by Sondheimer and

Kertesz (1948), Akuta and Koda (1954), and Robinson and Smith (1955). Lukton et al.

(1955) found a second pigment to be cyd 3-glu. Co and Markakis (1968) confirmed the

presence of pgd 3-glu and cyd 3-glu. Hong and Wrolstad (1990) revealed three

unidentified minor peaks eluting after pgd 3-glu. The first one was proposed as pgd 3-

rutinoside (pgd 3-rut), the second one was tentatively classified as a pgd derivative and

the third one was proposed as pgd 3-glu acylated with acetic acid. Bakker, et al. (1994)

confirmed the presence of pgd 3-rut and suggested that the second peak was pgd 3-glu

acylated with succinic acid.

StabiUty

The color of ACNS is deeply affected by the nature of their physicochemical

environment Tom£s-Barber£n and Robins (1997). Due to the complexity of the

environment present in living tissue and food systems several factors are responsible for

the degradation of these pigments. Temperature, pH, oxygen, concentration, enzymes,

metal ions, and ascorbic acid affect the stability of the ACNS (Mazza and Miniati,

1993).

The stability of ACNS is affected by heat (Markakis, 1974; Tom£s-Barber£n and

Robins, 1997). Early research on the reactions of strawberry pigment in juice and/or

partially purified pigment preparations by Kertesz and Sondheimer (1946); Nebesky et

al. (1949); Mackinney and Chichester (1952), and Meschter (1953) established that the

ACN in strawberry products was unstable in heating and storage and that many factors

affected the rate at which the pigment degraded. The exact mechanism of ACN

degradation is not fully known. Markakis et al. (1957) investigated the mechanism of

degradation of pgd 3-glu in pure pigment solutions and proposed opening of the

heterocyclic ring and formation of a chalcone as the first step of degradation. Further

degradation to a brown insoluble polyphenolic compound was proposed as the end

product contributing significantly to the darkening of strawberry products in storage.

Adams (1973) postulated hydrolysis of the glycosidic bond (position 3), followed by

conversion of the aglycon to a chalcone, which subsequently yields an a-diketone. It is

assumed that further degradation leads to brown products (Markakis, 1982). Piffaut et

al. (1994) proposed a hypothetical scheme for the thermal degradation of

8

malvidin 3,5-diglucoside in which initial hydrolysis of the glycosidic bonds destabilizes

the aglycon and leads to ring opening of the chalcone and subsequent rearrangement to

2,4,6 trihydroxybenzaldehyde and syringic acid.

Increase in temperature can contribute to monomeric ACN degradation and

polymerization of reactive phenolics (Markakis, 1982). Several studies on the

detrimental effect of temperature on processed strawberry products support this

hypothesis. As early as 1946, Kertesz and Sondheimer reported that the rate of color

degradation of strawberry preserves increased in proportion to the log of the

temperature. At room temperature (20 0C), the half life (tm) of the color was about 1300

hrs compared to 240 hrs at 38 0C and 6000-8000 hrs at 4 "C. This behavior was

confirmed by Meschter (1953) during processing of strawberry preserves, in which a

treatment of 100 0C for 1 hr resulted in 50% destruction of the fruit ACN; storage of the

preserve at 38 0C resulted in tmof the pigment of 10 days while storage at 25 0C

resulted in tmof 54 days. Abers and Wrolstad (1979) and Wrolstad et al. (1980)

reported increase in both color and ACN deterioration with increase in storage

temperature of strawberry preserves and after concentration of strawberry juice, •

respectively. During the study of the effect of sulphur dioxide and EDTA on the

stability of strawberry ACNS, Bakker and Bridle (1992) observed virtually no loss of

ACN in juice and puree samples kept at -20 "C while samples stored at 20 0C showed

high polymerization of the pigment.

The rate of ACN degradation is greatly affected by pH value (figure 2.2).

Increasing acidity has a protective effect on the stability of the pigment (Meschter,

1953). At pH below 2 the pigment exists primarily as the flavylium cation (F+) with the

positive charge delocalized through the pyrilium aromatic system, although carbons 2

and 4 were found to be the more positively charged positions of the chromophore (Amic

et al. 1990). As the pH is increased, proton loss occurs yielding the quinonoidal forms.

These quinoid forms are rapidly oxidized by air and destroyed; in neutral or slightly

acidic aqueous solution a colorless isomer ("pseudobase" or "carbinol base") is formed

(Robinson, 1991). In natural or processed food containing ACN, water is almost always

present and therefore, hydration of the flavylium cation to the colorless carbinol

pseudobase occurs. Water addition takes place at position 2, and chalcones are formed

xetfT OGlc

and/or

-H*

anhydrobase (violet-) ♦H* weakly acidic or neutral

OGlc

-H# ♦ H2O

• H*- H2O

OGlc

flavylium ion (red) acidic

pseudobase (colorless) weakly acidic

Figure 2.2. Structural changes in anthocyanins with changes in pH. Source: Francis, 1985.

from the carbinol pseudobase by a fast ring opening reaction and slow isomerization

process (Brouillard et al., 1978).

The influence of pH on the degradation rate of pure strawberry pigment and

strawberry juice was first researched by Lukton et al. in 1956. It was found that the rate

of pigment loss was pH-dependent under aerobic conditions, directly proportional to the

amount of the pigment in the pseudobase form, and inversely proportional to the

pigment in the cation form. When the pigment was present in nitrogen environment, the

rate of degradation was virtually independent of the pH. Similar results were obtained

by Markakis et al (1957), who tested the stability of pgd 3-glu in pH 2 and 3.4 buffered

solutions. Additional studies on the effect of change in pH on the rate of color loss in

strawberry concentrate reported 58 % color remaining at pH 1 after 1800 hrs of storage

compared to only 20 % at pH 5 at 700 hrs of storage Meschter (1953).

Chemical structure is another factor involved in the stability of ACNS. Sugars,

acylated sugars, hydroxyl groups, and methoxyl groups present in the aglycon all have

10

an effect on the stability of the pigments (Harbome, 1969; Brouillard, 1982; Mazza and

Brouillard, 1987). Increase in glycosylation and presence of acylation are believed to

retard degradation of the ACN molecule leading to higher stability of the pigment

(Markakis, 1982; Mazza and Miniati, 1993). Several studies have reported results which

agree with this hypothesis; Robinson et al (1966) observed lower decolorization of

anthocyanidin diglucosides as compared to the corresponding monoglucosides of stored

New York wines. In a corroborating study. Van Buren et al. (1968) found that the same

diglucosides were more stable to heat and light than the monoglucosides. Rommel et al.

(1990) and Rommel et al. (1992) hypothesized that substitution with the disaccharide

sophorose was a key factor contributing to the better color stability of red raspberry

wine as compared to strawberry wine.

Apparently, acylation of the ACN molecule usually lends stability (Bassa and

Francis, 1987). It is believed that the acyl groups can form a hydrophobic center with

the hydrophobic part of the aglycon which protects the molecule from attack of water

molecules at position 2 of the aglycon ring (Asen et al., 1977; Goto et al., 1979;

Hoshino et al., 1980; Brouillard, 1981). Guisti and Wrolstad (1996a) reported high color

and pigment stability when coloring brined cherries with ACNS extracted from radish

peel. The high stability of this model system was attributed to the nature of the major

ACN present in radish peel, which is an acylated triglycoside. In contrast, Baublis et al.

(1994) reported that no increase in stability was observed with increase in either

acylatiofi or glycosylation when comparing the ACNS of grape, red cabbage, and Ajuga

reptans in pH 3.5 citrate buffer solutions. ACNS from grapes include mono and

diglucosides of five different aglycons with the addition of monoacylation (Lea, 1988),

red cabbage contains diacylated triglucosides of cyanidin (Mazza and Miniati, 1993),

and Ajuga reptans contains glucosylated cyanidin, acylated with p-hydroxycinnamic

acid, ferulic acid, and malonic acid (Callebaut et al., 1990).

Chemical structure of the B ring also seems to influence ACN stability. From

the work of Hrazdina et al. (1970), it appears that the stability of the anthocyanidins 3,5-

diglucosides of grapes increases with the degree of methoxylation, but decreases with

an increasing hydroxylation of the aglycon. Substitution with a triglucoside in the B

ring of the major ACN present in Tradescantia pallida is believed to be the reason for

11

the higher stability of this pigment as compared to the main ACN inAjuga reptans

(Baublis et al. 1994).

Molecular oxygen has a deleterious effect on ACNS (Markakis, 1982). Nebesky

et al. (1949) called oxygen and temperature " the most specific accelerating agents" in

the degradation of the ACNS in blueberry, cherry, currant, grape, raspberry, and

strawberry juices. Daravingas and Cain (1965); Starr and Francis (1968); Clydesdale et

al. (1978) have also reported detrimental effect of oxygen in raspberry, cranberry, and

concord grape ACNS, respectively.

The detrimental effect of oxygen and ascorbic acid on ACN stability are related.

As early as 1953, Sondheimer and Kertesz reported that conditions favoring aerobic

oxidation of ascorbic acid in strawberry juice and model systems using pgd 3-glu from

strawberries caused maximal loss of ACNS, but when oxygen was excluded no color

deterioration was observed. Markakis et al. (1957) reported synergistic effect between

ascorbic acid and oxygen on pigment degradation in pure pgd 3-glu solutions. Bakker et

al. (1992) concluded that nitrogen protected the ACNS during preparation of strawberry

juice and puree; however, there was no difference in loss during storage between

aerobic and anaerobic systems as polymerization of pigments occurred under both

conditions.

The role of ascorbic acid on strawberry ACN deterioration was first pointed out

by Pederson et al. (1947) who found a high correlation between loss of the acid and

ACN in strawberry juice. Corroborative observations and conclusions were reported by

Nebesky et al. (1949). Sondheimer and Kertesz (1953) demonstrated that ascorbic acid

induced the destruction of ACNS in strawberry juice, both aerobically and

anaerobically. This acceleration of loss of pgd 3-glu when ascorbic acid is present was

confirmed in model systems stored under nitrogen and oxygen conditions by Poei-

Langston and Wrolstad (1981). During characterization of strawberries for jam

production, Skrede (1982) found that high contents of ascorbic acid in the fruit were

related to adverse effect on color stability and increase in degradation index (DI) value

upon storage of the jams.

The mechanism by which ascorbic acid accelerates ACN degradation is not fully

known. Sondheimer and Kertesz (1952); Sondheimer and Kertesz (1953); Harper et al.

12

(1969) postulated that hydrogen peroxide formed when ascorbic acid is oxidized to

dehydroascorbic could be responsible for the pigment degradation. It is known that

hydrogen peroxide is capable of oxidizing ACNS (Jurd, 1966; 1968). Another

mechanism in which ascorbic acid condenses directly with ACNS was proposed by Jurd

(1972). The reasoning for this hypothesis was the fact that the flavylium salt is

condensed with the 6-diketone dimedon at the 4 position of the anthocyanidin (Jurd,

1965) and that ascorbic acid is structurally similar to dimedone. Such condensation

products would be unstable and further degrade to colorless compounds. Poei-Langston

and Wrolstad (1981) also proposed condensation between the monomeric pigment and

ascorbic acid in model systems.

Non-oxidative degradation of ascorbic acid is also possible in food systems.

Huelin et al. (1971) found that the main products of the anaerobic decomposition of

ascorbic acid in the pH range of foods were furfural and 2,5 dihydro-2-furoic acid.

Furfural and a 6-unsaturated carbonyls are the precursors of browning (Braverman,

1963).

Sugars and their degradation products cause an effect on the stability of the

ACNS in food systems. Furyl aldehydes can also be formed by sugar degradation when

sugars are heated with acids (furfural mostly from aldo-pentoses and 5-hydroxymethyl

2-furaldehyde (HMF) from ketohexoses), as well as by the transformation of other

compounds such as ascorbic acid (Sloan et al., 1969). The deleterious effect of furfural

and HMF from sugar degradation on strawberry ACN has been reported by various

authors; fructose, arabinose, lactose, and sorbose were found to be the most deleterious

sugars (Meschter, 1953; Sondheimer, 1949). Jasna et al. (1983) studied the influence of

HMF on the degradation of cyd 3-glu in juice and model systems and found that

presence of such a compound accelerated pigment degradation. The pigment

degradation was more pronounced in juice than the model systems. They concluded that

pigment degradation occurred via several different reactions, which have not been

confirmed, and that the very same conditions favoring ACN degradation during

processing, e.g., high temperature favored the formation of HMF.

The concentration of ACN also influences pigment stability. Increased

concentration of ACNS in plant tissues intensifies their color and may enhance color

13

stability through the phenomena of intermolecular copigmentation and self-association

(Markakis, 1982). Copigmentation is defined as a molecular interaction between the

ACN colored forms and a colorless molecule, e.g. polyphenols, purines, alkaloids (Asen

et al, 1972; Osawa, 1982). Flavones, tannins, polysaccharides, and polypeptides also

produce copigmentation effects (Robinson, 1931; Robinson, 1939). This molecular

interaction reduces access of water molecules to the C2 and/or C4 electrophilic sites in

the flavylium and thus, the formation of carbinol pseudobases (Tom£s-Barber£n and

Robins, 1997). When the ACN concentration reaches high enough values, self

association between two flavylium cations, two quinonoidal bases or even between a

quinonoidal base and a flavylium cation may occur (Hoshino, 1992; Dangles et al,

1994). Skrede et al. (1992) reported that total ACN concentration was a major

parameter influencing stability of strawberry ACNS; strawberry syrup fortified with

pelargonidin 3-glucoside (pgd 3-glu) presented stability to same level as blackcurrant

syrup. Similarly, strawberry jams, to which elderberry colorant or pomegranate juice

was added to increase monomeric ACN content showed a slower loss of ACN and

smaller changes in color of the product as expressed by hue angle (Garcia-Viguera and

Zafrilla, 1998).

Concentration of ACN in strawberries varies depending on cultivar and stage of

maturity, which are key parameters when fruit is chosen for processing. Totem, the

predominant cultivar grown in the Pacific Northwest, is a US industry standard for

processing berries (Bakker et al., 1994). This cultivar is known for its fully red internal

and external color. The Hood variety, which also has intense red color, is considered to

have excellent quality for processing, but it is difficult to grow. Table 2.1 shows the

ACN content of some of the major strawberry cultivars used in the food industry.

Skrede (1980) investigated some compositional factors of twelve strawberry

varieties for potential use by the jam industry. Chemical, physical, and sensory analysis

were performed with fresh and thawed fruits and with the jams made from them and

compared to Senga Sengana, which is a variety widely used in Europe. Except for color,

no significant correlation existed between the chemical analysis and the sensory

parameters; there was a significant relationship between total ACN content and visual

color as judged by the panel. Based on these results, Totem was recommended for

14

Table 2.1. Anthocyanin content of some major strawberry cultivars used in food processing

Cultivar Anthocyanin content (mg/lOOg fruit)

Source

totem

Hood

66.6(1)

50.3 (2)

37.7 (3)

Benton

Tioga

32.7 (2)

24.0(4)

27.6 (3)

Senga Sengana 60.0(5)

Vancouver, BC, Canada Pacific Northwest, USA Pacific Northwest, USA

Pacific Northwest, USA Pacific Northwest, USA California, USA

Scotland

Reference:

(1) Bakker et aL, 1994

(2) Pilando et aL, 1985

(3) Abers and Wrolstad, 1979

(4) Rwabahizi and Wrolstad, 1988

(5) Bakker et al, 1992

industrial jam production at the same level as Senga. These findings confirmed those

reported by Nelson et aL (1976) where the only consistent correlation between chemical

parameters of fresh strawberries and sensory judgement in fruits after thawing was the

color. Similar results were found by Bakker et al. (1994) during the study of the

pigment composition of 39 strawberry genotypes. A good correlation between ACN

concentration and color of strawberry juice as expressed by hue was found. Among the

39 genotypes. Totem gave the highest color intensity (chroma).

Three different enzymes have been reported to contribute to ACN degradation.

Polyphenoloxidase (PPO) and peroxidase (POD) are oxygen-dependent and primarily

connected to enzymatic browning while glycosidases are connected to hydrolysis of the

15

aglycon. During handling, storage, and processing of fruits, cell damage occurs leading

to endogenous PPO and POD (located in the cytoplasm) and phenolics (located in the

vacuole) to interact (Tom£s-Barber£n and Robins, 1997). PPO has been reported to

cause pigment decolorization by Cash and Sistrunk (1971) and Pifferi and Cultrera

(1974). The most important natural substrates of PPO in fruits are catechins (Vdmos-

Vigy£z6, 1981), a phenolic compound, which has been identified as the major flavanol

in strawberries occurring in concentrations between 7 and 76 mg/Kg (Stohr and

Herrmann, 1975; Abers and Wrolstad, 1979; Wrolstad et al., 1980; Pilando et al., 1985).

Several authors have studied the detrimental effect of PPO on strawberry ACN; Cash

and Sistrunk (1971) reported degradation of color in heat-treated strawberry purees with

added strawberry PPO. Wesche and Montgomery (1990a) extracted two highly active

PPO isoenzymes from the pectin fraction of Tioga strawberries and characterized them

in further experiments (Wesche and Montgomery, 1990b) as PPO-Fi and PPO-F2. The

isoenzymes had no cresolase activity, but presented maximum activity with D-catechin

(CAT). To propose a degradation mechanism, Wesche and Montgomery (1990c)

prepared model systems containing each isoenzyme and CAT, pgd or cyd, or a

combination of either CAT-pgd or CAT-cyd. The results showed no activity of the

isoenzymes on pgd, very little activity on cyd, and high activity on both combinations

ACN-CAT; after 24 hrs at room temperature 50-60% of the ACN was lost and

formation of a brown precipitate was observed. From these findings it was proposed

that PPO does not have a direct action on ACNS; instead, PPO would promote

formation of quinones from CAT. Quinones, as monomers, can act directly on ACNS

causing their oxidation (Peng and Markakis, 1963; Pifferi and Cultrera, 1974).

Quinones are further polymerized to melanines, which can copolymerize with the

ACNS (Somers, 1971, Jurd, 1967). Sakamura et al. (1966) proposed that the brown

melanin formed during enzymatic browning would also mask the red ACNS.

Other studies support the effect of PPO on strawberry ACN. Timberlake and

Bridle (1976) observed color loss of ACNS in model systems of strawberry ACN and

CAT. Adams and Ongley (1973) found that sulphur dioxide (SO2) had a strong

stabilizing effect on pgd 3-glu in canned and bottled strawberries. In agreement with

these findings, when Bakker and Bridle (1992) added SO2 to strawberry juice and

16

puree, a reduction in ACN loss and formation of polymeric color was observed. SO2 has

longed been used as PPO inhibitor to prevent enzymatic degradation of tart cherry

ACNS (Goodman and Markakis, 1964) and in wine making as an inhibitor of oxidative

enzymes such as PPO (Allen, 1983). Abers and Wrolstad (1979) found higher quantity

of leucoanthocyanins and CAT in Tioga strawberries as compared to the Hood variety.

They suggested that such concentration may account for the greater susceptibility of

preserves made from Tioga to polymeric browning. Wrolstad et al. (1980) reported that

blanching of the strawberries with microwave energy before pressing for juice

production had a protective effect on ACNS and reactive phenolics, which resulted in

improved color stability. It was suggested that the protective effect was due to the

enzyme inactivation during blanching. Wrolstad et al, 1990 reported high reduction of

browning and formation of polymerized color in Senga Sengana strawberries to which

sucrose had been added. They proposed the inhibition of PPO by sucrose as a possible

mechanism.

Significant amounts of other phenolics that can serve as a substrate for PPO

have been detected in strawberries, e.g., ellagic acid (Rommel and Wrolstad, 1993),

quercetin 3-(9-glycoside, and kaempferol 3-Oglycoside (Mabry and Markham, 1970).

Abers and Wrolstad (1979) also found a higher concentration of flavanols in Tioga; the

flavanol reagent of Swain and Hillis (1959) reacts with compounds containing a

resorcinol nucleus. (Gil et al., 1997). Somers (1966) postulated the formation of a

leucoanthocyanin-ACN complex in wine.

POD are enzymes capable of oxidizing phenolics to quinones in presence of

hydrogen peroxide (H2O2). Due to the low level of H2O2 available in plant tissue, the

peroxidase activity has been considered limited in fruits (Nicolas et al, 1994). However,

recent research by Lopez-Serrano and Barcel6 (1996) and L6pez-Serrano and Barcelo

(1997) reports the presence of a peroxidase isoenzyme from strawberries. According to

the authors CAT is an excellent substrate for this isoenzyme, which is partially-heat

stable. Therefore, it would remain active after canning of strawberry products causing

browning reactions at low H2O2 concentrations. Upon oxidation of CAT by peroxidase,

CAT (9-quinones are formed and can interreact or further react with hydroquinones to

17

form condensation products which develop undesirable browning color (Cheyner et al.,

1989).

Pectolytic enzymes are widely used during fruit juice processing with the main

purpose of degrading the fruit pectin. Glycosidic activity or removal of the glycosidic

substituents of the ACNS by such enzymes has been reported in raspberry juice (Jiang

et al., 1990), raspberry wine (Withy et al., 1993), strawberry juice (Tanchev et al, 1969;

Blom, 1983), cherry nectar (Blom and Juul, 1982), blackberry wine, jellies, and jams

(Yang and Steele, 1958), raspberry ACN (Rommel et al., 1990), cranberry juice

(Wightman and Wrolstad, 1995), boysenberry and red raspberry juice (Wightman and

Wrolstad, 1996), and wine ACNS (Wightman et al., 1997).

Use of commercial pectinase preparations has been a common practice in the

production of strawberry juice as a way to facilitate pressing and ensure high yields of

juice and colored materials. Commercial enzyme preparations are fungal derivatives

from food-grades strains of Aspergillus niger and/or Trichoderma longibrachiatum.

These are crude preparations and therefore, undesirable side-activity can occur. In 1955,

Huang studied the decolorization of ACN by fungal enzymes by mixing pgd 3-glu and B

-glucosidase from Aspergillus niger in a buffer solution. He found that the overall

decolorization process involved an enzymatic hydrolysis of the ACN to the

anthocyanidin and sugar, and a spontaneous transformation of the aglycon into colorless

derivatives. Tanchev et al. (1969) treated strawberry juice with three different pectolytic

enzyme preparations. After four hours of incubation at 50 0C, all three preparations

caused destruction of the ACNS. With the aim of evaluating such detrimental effect,

Blom (1983) separated and purified B-glucosidase from a pectolytic enzyme preparation

(pectinol D). Chomatographic evaluation of the effect of the purified enzyme fraction

on strawberry ACNS gave clear indication of a lytic action on the B-glycosidic bond of

the pigment since the aglycon pgd was detected in the HPLC profile. The described

effect can also occur when mold contamination is present in the fruit. Pilando et al.

(1985) reported acceleration of color degradation in strawberry wine produced from

moldy fruit. Similarly, Rwabahizi and Wrolstad (1988) observed acceleration in the loss

of ACNS in juice and concentrate produced from moldy strawberries. Moldy fruit

18

contains mainly Botrytis cinerea, in which 6-galactosidase activity has been found (Pitt,

1968) although Dubemet et al (1977) also reported laccase activity from the same mold.

Sulfur dioxide causes bleaching of ACNS by reacting with the flavylium cation

(F") to yield a colorless compound, the 2 (or 4)-sulphonic acid (FSO3H), analogous to

the carbinol base (FOH) (Timberlake and Bridle, 1967; Parkinson and Brown, 1981).

This reaction, which can be reversible or irreversible, occurs in the food industry when

SO2 is added as preservative or during processes such the brining of red cherries that are

subsequently used for maraschino, candied, and glace cherries.

Concentrated aqueous solutions of metallic ions such as Na+ and Mg"^ strongly

stabilize ACN quinonoidal forms and flavylium ions avoiding the formation of colorless

pseudobases by hydration (Markakis, 1982). The color stabilization is believed to be

due to a reduction of the free water (water activity) by hydration of the metal ions

(Hoshino et al, 1982; Goto, 1987).

Among the factors affecting the stability of ACNS, water activity (Aw) is the

least studied. Since the Aw of processed strawberry products varies within a high range,

better understanding of the influence of this factor becomes relevant when trying to

solve the problem of pigment degradation. Table 2.2 lists the Aw values for the most

common strawberry products.

Despite the limited data on the effect of Aw on ACNS, the importance of the

physicochemical environment in which ACNS are present, even at the same Aw level

has been demonstrated. As early as 1957, Markakis suggested that water was involved

in decolorization reactions of strawberry ACNS; he observed that ACNS were more

stable when stored in dry crystalline form on dried paper chromatograms, but the effect

of water activity on strawberry ACN was not studied until 1972 by Erlandson and

Wrolstad. After storing samples of freeze-dried strawberry puree in desiccators at

different relative humidity (RH) levels, they found a pronotmced and direct effect of RH

on the rate of ACN degradation. They concluded that water availability was essential

for ACN degradation. In contrast to those findings, when Br0nnum and Flink (1985)

stored freeze-dried elderberry ACNS in desiccators at different RH levels, they

observed that loss of ACN was significant only at Aw levels above 0.51.

19

Table 2.2. Water activity values corresponding to the most common processed strawberry productsa

Product Aw

Strawberry juice 1.0 Strawberry concentrate 0.84-0.90 Strawberry preserves (jams, jellies) 0.75-0.80 Freeze-dried strawberries 0.60-0.65 Strawberry powder 0.20-0.50

a Modification from Beauchat, 1981

Kearsley and Rodriguez (1981) reported that ACN powder (unidentified source)

dissolved in different concentrations of glycerol-water solutions were relatively stable

to changes in Aw. However, studies conducted by Sian (1991) resulted in a slight

increase of retention of pineapple and papaya ACNS as the percentage of glycerol

increased from 10% to 20%, but a drop was observed when the percentage increased

from 20% to 30%.

Thakur and Arya (1989) tested the degradation of grape ACNS in isolated model

systems made of 80 % (v/v) aqueous methanol and micro-crystalline cellulose at 0,

0.33, and 0.73 Aw levels; results showed that after 100 days of storage the % of ACN

retention was higher in the samples with lower Aw.

Kinetics of anthocyanin degradation

First order kinetics for degradation of ACN has been reported by several

authors. In a model system, Markakis et al, 1957 determined that the thermal

degradation of strawberry ACN was a first order reaction with a linear phase between

45 and 110 0C. Similarly, Daravingas and Cain (1968) indicated the same trend for

black raspberry ACNS; Skrede et al (1992) for ACNS in strawberry and blackcurrant

juice; Cemeroglu et al. (1994) for sour cherry ACNS, Guisti and Wrolstad, (1996a) for

radish ACNS, and Baublis et al. (1994) for concord grape, red cabbage, and Ajuga

20

ACNS. Baublis et al (1994) also observed zero order kinetics for the degradation of

Tradescantia pallida ACNS at room temperature and exposed to light.

The change in the ACN following first order kinetics is described by the

equation:

Ln (C/Co) = -61 (time) + 60. Where C/ Co = monomeric ACN concentration at

any time/initial monomeric ACN concentration, 60 = intercept, and 61 = slope. This

order indicates that the rate of pigment degradation is exponential and that the time for

half of the reactant initially present to decompose t\n. is a constant which depends on the

initial concentration of the reactant (Voet and Voet, 1995). Despite this definition of

first order kinetics, it is important to remember that not only initial concentration

dictates the stability of the ACNS. An interaction of factors present in the system can

take place during storage and/or processing of strawberries. This is illustrated in table

2.3, which shows the linear regression parameters of monomeric anthocyanin

degradation in different systems.

To summarize, ACN degradation during processing of strawberry is a problem

that depends on the nature and physicochemical environment in which the pigments are

present. Most of the studies directed to understand and solve the problem of color

change due to the degradation of pgd 3-glu have been carried out in natural systems,

whose complexity may lead to interactions between several factors. Such interaction

makes it difficult to focus on parameters which may be more critical than others. The

effect of pH, ascorbic acid, temperature and some phenolic compounds have been

investigated, however the effect of other important variables such as POD, Aw and type

of chemical substitution of the pgd aglycon are still subjects of investigation. Only

recently it was demonstrated that POD may be responsible for color degradation in

processed strawberries (L6pez-Serrano and Barcel6, 1996; L6pez-Serrano and Barcelo,

1997), but it should be remembered that different varieties of the same fruit have

different levels of enzymes and substrates. In regards to Aw, the technique used to

lower the level of water availability seems to influence the behavior of the pigment to a

high degree. From all these facts, we conclude that evaluation of the mentioned factors

in model systems, as well as the evaluation of pgd substitution in strawberry systems

would generate useful information to food producers and processors. If either

Table 2.3. Linear regression parameters of monomeric anthocyanin degradation in some strawberry systems'

System Storage conditions Strawberry variety

Initial anthocyanin Rate of Half concentration degradation Life

(mg/L) (days'1) (days)

Pgd 3-glu

Pgd 3-glu

Pgd 3-glu

Pgd 3-glu

pH 3.4 buffer, 60 0C

pH 3.4 buffer + ascorbic acid, 60 0C (5 mM) (anaerobic conditions) pH 3.4 buffer, 60 0C (aerobic conditions)

pH 3.4 buffer + ascorbic acid, 60 0C (aerobic conditions)

Strawberry preserves 21 0C Strawberry preserves 21 0C

Strawberry juice 20 0C Strawberry concentrate 20 0C

Strawberry wine 25 0C Strawberry wine 25 0C Strawberry wine 25 0C Strawberry wine 25 0C Strawberry wine 25 0C Strawberry wine 25 0C

Strawberry syrup 25 0C

Hood Tioga

Hood + Benton Hood + Benton

Benton ripe Benton overripe Benton ripe/moldy Totem ripe Totem overripe Totem ripe/moldy

Unidentified

43

65

65

65

175 100

127 245

1.82 xlO"1

3.13 xlO"1

3.47 xlO"1

-1.062

7.16xl0'3

6.46 xlO"3

5.59 xlO"2

4.94 xlO"2

5.3 3.54 xlO"2

17.7 2.61 xlO2

7.95 2.55 xlO2

22.5 3.44 xlO2

31.2 3.06 xlO2

17.5 2.79 xlO2

4(1)

2 0)

2 0)

0.65(1)

97(2)

107 (2)

12(3)

14(3)

20(4)

27(4) 27(4)

20(4)

22(4> 25(4)

127 1.47 xlO-2 47 (5) to

Table 2.3. Continued

Strawberry syrup 20 "C (ascorbic acid added -774 mg/L) Unidentified 127 2.63 xlO"2 26(5)

Strawberry syrup 20 "C (strawberry anthocyanin added) Unidentified 1256 9.45 xlO"3 73(5)

Strawberry puree 20 0C Senga Sengana + unidentified Turkish

280 3.34xl0"2 21(6)

Strawberry juice 20 0C Senga Sengana + unidentified Turkish

220 4.36 xlO"2 16(6)

Strawberry juice 20 0C Senga Sengana 325 7.54 xlO"2 9(7)

Strawberry juice 20 oC(S02 added-140 mg/L) Senga Sengana 420 6.39 xlO'2 n0)

Data calculated from:

(1) Markakis et al., 1957(2) Abers and Wrolstad, 1979

(3) Wrolstad et al, 1980

(4) Pilando et al, 1985

(5) Skrede et al, 1992

(6)Bakkeretal.,1992

^Bakker and Bridle, 1992

to

23

diglycosydes or acylation of pgd increased the stability of the ACN, techniques such as

genetic engineering could be applied to modify pigment structure to give more a stable

ACN. As a result, consumers could have access to a more attractive and natural-looking

product.

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33

CHAPTERS

CHARACTERIZATION OF TWO PELARGONIDIN-BASED ANTHOCYANINS FROM STRAWBERRY (Fragaria anannassa) AND NASTURTIUM FLOWERS

(Tropaeolum majus)

G. Astrid Garz6n and Ronald E. Wrolstad

Dept. of Food Science and Technology Oregon State University, Corvallis, OR 97331

34

ABSTRACT

The anthocyanins in petals of red and orange nasturtium flowers (Tropaeolum

majus) and strawberries (Fragaria anannassa) were extracted and analyzed by High

Performance Liquid Chromatography (HPLC). Anthocyanin (ACN) content in petals

ranged from 25 to 30 mg/100 g petals. The pelargonidin-based pigment represented 35

% of the total peak area in red blossoms and 91 % in orange petals. ACN content in

strawberry was 28 ± 1 mg/100 g fruit; two minor peaks representing 9% of the total

peak area corresponded to pelargonidin 3-glucoside (pgd 3-glu) with succinyl acylation.

Pelargonidin 3-sophoroside (pgd 3-soph) in the petals of nasturtium flowers and two

isomers of succinyl-pgd 3-glu in strawberries were isolated and characterized by HPLC,

gas liquid chromatography (GC), spectral analysis, and mass spectroscopy (MS).

INTRODUCTION

The chemical structure of anthocyanins (ACNS) is one factor affecting pigment

stability. The nature of the sugar substitution, and whether this sugar is acylated or not,

are structural variations that influence anthocyanin (ACN) stability in food products.

Previous studies indicated that diglycosidic substitution provided more stability to the

ACN molecule than monoglycosidic substitution (Markakis, 1982). It has recently been

implied that substitution with the disaccharide sophorose contributes to the better color

stability of red raspberry products as compared to strawberry and blackberry products,

which have predominately monoglycoside substitution (Rwabahizi and Wrolstad, 1988;

Rommel et aL, 1990; Rommel et al, 1992; Boyles and Wrolstad, 1993). Acylation of

the sugar molecule with cinnamic acids is believed to improve stability of the ACN

molecule by preventing it from hydration (Asen et al., 1977; Brouillard, 1981; Goto et

al., 1979; Hoshino et al., 1980; Mazza and Miniati, 1993).

Because of the relationship between chemical structure of ACNS and their

stability, there is considerable effort on characterization of ACNS. HPLC, UV-visible

spectral analysis and Mass Spectrometry (MS) are techniques widely used presently for

this objective.

35

Among the non-characterized ACNS we find those present in the petals of

Nasturtium flowers (Tropaeolum majus), and some of the ones present in strawberry

(Fragaria anannassa). In 1963, Harbome reported the pigment present in the orange

petals of nasturtium flowers as pelargonidin 3-sophoroside (pgd 3-soph); however, no

additional literature on identification of this ACN has been found. Due to the low

stability of pelagonidin 3-glucoside (pgd 3-glu), the major ACN present in strawberry,

and our need to compare the stability of this ACN to pgd derivatives with diglycosidic

substitution and acylated substitution, nasturtium flowers became a potential source of

pgd 3-soph. Therefore, isolation and identification of the pgd pigment in nasturtium

flowers was necessary.

Earlier HPLC analysis of strawberry pigment by (Hong and Wrolstad, 1990)

revealed three unidentified minor peaks eluting after pgd 3-glu. The first one was

proposed as pelargonidin 3-rutinoside (pgd 3-rut), the second one was tentatively

classified as a pgd derivative and the third one was proposed as pgd 3-glu acylated with

acetic acid. Subsequently, Bakker, et al.(1994) confirmed the presence of pgd 3-rut and

suggested that the second peak was pgd 3-glu acylated with succinic acid. In addition,

they found UV-visible spectral evidence of the third peak being a pgd-based ACN.

The purpose of this study was to use chromatographic, spectral, and MS

techniques to confirm the presence of pgd 3-soph in the petals of nasturtium flowers and

to identify the two late-eluting ACNS of strawberry.

MATERIALS AND METHODS

Plant material and anthocyanin juices

Individually quick frozen (I.Q.F) strawberries (Fragaria anannassa cv, Totem)

were supplied by Conroy Packing Inc. (Salem, OR). Red and orange Nasturtium

flowers were collected from local gardens during the summer season in Corvallis, OR.

The petals were sorted according to color, frozen with liquid nitrogen and stored at -23 0C. Concord grape juice was bought at Safeway supermarket in Corvallis, OR, and

raspberry (Rubus idaeus cv, Willamette) juice was available from previous studies

(Boyles and Wrolstad, 1993).

36

Reagents

D-Glucose was obtained from Adams Chemical Co. (Round Lake, IL) and

trimethylsilyl reagent (TMS) was supplied by Pierce Chemical Co. (Rockford, IL).

Pigment extraction and isolation

Anthocyanins from IQF strawberries and frozen petals of nasturtium flowers

were extracted and isolated as described by Guisti and Wrolstad (1996a). Each plant

material was powdered in a stainless steel Waring blender after addition of liquid

nitrogen. The powder was mixed with 2 L acetone/Kg material and the extracted

material was separated from the cake by filtration on a Buchner funnel. Total

reextraction of the cake was done with aqueous acetone (30:10 v/v). Filtrates were

combined and partitioned with chloroform (1:2 acetone: chloroform v/v) and stored

overnight at 1 "C. The aqueous portion containing the ACNS was recovered and

separated from the residual acetone in a Buchi rotoevaporator at 40oC and resolubilized

in0.01%HCl.

Pigment analysis

Monomeric ACN pigment content was determined by the pH differential

method as described by Wrolstad (1976). ACN concentration was expressed as mg pgd

3-glu/L using a molar absorptivity of 22,400 and a molecular weight of 433.2.

Determinations were made on a Shimadzu UV visible spectrophotometer, model UV

160 U, using 1 cm disposable cells.

HPLC conditions for anthocyanin analysis

A Perkin-Elmer Series 400 chromatograph equipped with a Hewlett-Packard

1040A photodiode array detector and a Hewlett-Packard 9000 computer was used. The

mobile phase composition for ACN analysis was the following: (A) 100% acetonitrile,

(B) 5% acetonitrile, 10% acetic acid, 1% phosphoric acid. Conditions for separation of

ACNS from petals: linear gradient from 0% to 6% A for 15 min and from 6% to 10% A

37

for 10 min. Column: Supelco LC -18 (Supelco Inc., Bellefonte, PA), 25 cm x 4.6 mm

ID, particle size 5 n. Conditions for separation of ACNS from strawberry: linear

gradient from 2% to 13% A in 15 min, 13% to 15% A in 5 min, holding at 15% A for

15 min. Column ODS C -18 (PolyLC Inc., Columbia, MD), 25 cm x 4.6 mm ID,

particle size 5 \x. In both cases, the injection volume was 50 \il, flow rate 1 mL/min, and

simultaneous detection at 520 nm, 320 nm and 280 nm was applied. UV-vis spectra was

taken directly from chromatographic runs.

Separation and collection of pigments via preparatory HPLC

Pgd 3-soph was obtained from the petals of orange nasturtium flowers. Pgd 3-

glu and acylated pgd 3-glu (acyl-pgd 3-glu) were obtained from strawberries. The

ACNS were separated by preparatory HPLC with the same equipment and solvent

system used for the analytical section. The column used for separation was a Supelcosil

PLC-18 (Supelco Inc., Bellefonte, PA), 25 cm x 21.2 mm ID, particle size 5 \i. The

conditions for the chromatographic runs were: linear gradient from 0 % to 5 % A for 5

min, from 5% to 11 % A for 10 min, from 11 % to 28 % A for 6 min, and from 28 % to

5 % A for 8 min. Flow rate 10 mL / min.; injection volume 1 mL, primary detection at

520 nm.

Purification of pigments

Cleaning of the aqueous extract containing the ACN fractions was achieved with

a C-18 cartridge (high load C-18 tube), 20 mL capacity (Alltech Assoc, Inc., IL). The

minicolumn was activated with methanol and upon loading with pigment, washed with

0.01% HC1 (Wrolstad et al., 1990). The ACNS were eluted with methanol acidified

with 0.01% HC1 and concentrated by vacuum rotary evaporation at 35 0C. Purity of the

three ACN fractions was checked by analytical HPLC following the same methods

described for ACN analysis.

38

MS analysis of anthocyanins

Upon separation and purification, ACNS were analyzed by low resolution MS

(ionspray). The mass spectrometer was a Perkin Elmer SCIEX API DI + triple

quadrupole mass spectrometer equipped with an ion spray source (ISV = 4700, orifice

voltage of 80) and loop injection.

Alkaline hydrolysis of anthocyanins

For identification of the acylating group present in the pgd-based ACNS

obtained from strawberry, the method described by Hong and Wrolstad (1990) was

used. Approximately 10 mg of purified strawberry pigment was added to 20 mL of 10%

aqueous KOH in a screw-cap test tube. The mix was flushed with nitrogen to remove

oxygen. Hydrolysis of the acid was allowed during 8 min at room temperature in the

dark. For reconversion of the pigments to the oxonium ion form, 2 N HC1 was added.

Subsequent purification of the hydro lysate was carried out with a C-18 Sep-Pak

cartridge (Waters Assoc, Milford, MA) following the steps described before; both the

retained ACN and the eluate containing the acid were collected.

Add hydrolysis of anthocyanins

The method described by Hong and Wrolstad (1986) was applied. The ACNS

were recovered after purification of the hydrolysate from the alkaline hydrolysis.

Approximately 1 mg of the clean saponified pigment was mixed with fifteen mL of 2N

HC1 in a screw-cap test tube. The mix was flushed with nitrogen, capped and

hydrolyzed for 45 min at 100 "C, then cooled in an ice bath. Purification of the

hydrolysate was done using a C-18 Sep-Pak cartridge as described before. In the case of

flower sample, the eluate was collected for further identification of the sugar

substitution.

39

HPLC conditions for anthocyanindin analysis

For anthocyanidin analysis a Supelco LC -18 column (Supelco Inc., Bellefonte,

PA), 25 cm x 4.6 mm ID, particle size 5 |i was used. Conditions for the mobile phase:

(A) 15% acetic acid (aqueous), (B) 100% acetonitrile. Isocratic elution with 85% A and

15% B at 1 mL/min. Injection volume 50 |LIL. Simultaneous detection at 520 nm, 320

nm and 280 nm. UV-vis spectra taken directly from chromatographic runs.

Identification of anthocyanins and anthocyanidins

In order to compare retention times of anthocyanidins from the petals with

known standards, concord grape juice was used as source of five aglycons: delphinidin

(dpn), petunidin (ptd), pgd, peonidin (pnd), and malvidin (mvd) (Hong and Wrolstad,

1986). Strawberry juice was used as source of pelargonidin. Raspberry juice, which

contains minor amounts of pgd 3-soph (5.7 % by peak area), was used as a source of

this ACN to compare retention times of the pgd-based ACN present in the petals

(Boyles and Wrolstad, 1993).

HPLC conditions for analysis of organic adds

Organic acids obtained from saponification of the strawberry acylated ACNS

were analyzed using a Sperisorb ODS-2 column attached to a Sperisorb ODS 1 column,

both 250 mm x 4.6 mm, the solvent was 27.2 g/L H3PO4 pH 2.4, flow rate 0.7 ml/min,

isocratic conditions, injection volume 50 |iL. Primary detection at 214 nm.

Color specifications of pelargonidin derivatives from strawberry

Solutions of purified ACN (0.04 mg/ 3 mL) in pH 1 buffer were used to

determine transmittance of the ACNS in solution every 10 nm from 400 to 700 nm.

Determinations were made on a Shimadzu UV visible spectrophotometer, model UV

160 U, using 1 cm disposable cells. The weighted ordinate method was applied for

calculation of CEE tristimulus values X, Y and Z of the pigments (Hunter and Harold,

40

1987). Dominant wavelength and % purity were calculated from the CIE chromaticity

diagram, and the Munsell color specifications for both ACNS were obtained based on

the Munsell conversion chart.

CIE L*,a*,b* values, hue angle (H) and chroma (C) were interconverted from

the tristimulus values using the following equations (Hutchings, J. B., 1992):

L* = 116 (Y/Yn)173 -16, a* = 500 [(X/Xn)1/3 -(Y/Y.)m],

b* = 200 [(Y/YJ m - (Z/Z0 m, C = (a*2+b*2 )m, H = arctan (b*/a*).

Where: Xn = 97.30, Yn = 100, Z* = 116.14.

Differences in lightness, chroma and hue between the acylated pigment and pgd

3-glu were analyzed according to the following equations:

AL* (pgd 3-glu, acyl-pgd 3-glu) = L*( acyl-pgd 3-glu) - L* (pgd 3-glu)

AC = C(acyl-pgd 3-glu) - C (pgd 3-glu)

AH = [(AE*)2 - (AL*)2 - (AC)2]m

Where: AE* = [(AL*)2 + (Aa*)2 + (Ab*)2] *

Preparation of trimethylsilyl (TMS) derivatives

The sugar fraction previously obtained by Sep-pak cartridge isolation during the

acid hydrolysis step of nasturtium ACNS was freeze-dried and characterized by

capillary gas-liquid chromatography (GLC) analysis of TMS derivatives following the

methods described by Heatherbell (1974).

For derivatization, 10 mg of the freeze-dried fraction were dissolved in 100 ml

of methanol to yield a 0.1% (w/v) solution. An aliquot of the solution (500 |il) was

transferred to a 3 ml vial and rotoevaporated under vacuum to dryness. This resulted in

a crystallization of 0.05 mg of sugar in the vial that were converted to their TMS

derivatives by adding tri-sil reagent (300 \il), shacking vigorously in a Buchi shaker for

five min, heating at 70 0C for twenty min, and shaking for additional 15 min. Two ^il of

the final solution was injected into the gas chromatograph. The same treatment was

applied to the glucose standard.

41

Capillary Gas Chromatography

The gas chromatograph was a Hewlett Packard 5890 equipped with a Alltech SE

54 (30 meter 0.34 mm) capillary column and flame ionization detector. The GC

conditions for analysis were: injector temperature (190 0C), detector temperature (250 0C), nitrogen gas carrier flow rate 5 ml/min, injection volume 2 ^iL. The column was

operated at 200 "C under isothermic conditions for 30 min. A splitless injection sleeve

with purge off for the initial min following injection was utilized. Retention times were

determined with a Hewlett Packard 3393 recording integrator.

RESULTS AND DISCUSSION

Anthocyanin content

The total monomeric ACN content in the petals of nasturtium flowers and

Totem strawberries was comparable and is reported as the average of three

determinations. This average was 30 ± 2 mg/100 g for red flowers and 25 ± 3 mg/100 g

for orange flowers. The monomeric ACN yield from Totem strawberries was 28 ± 1

mg/100 g fruit. This result is within the range of monomeric ACN content found by

Wrolstad et al. (1970) in 18 strawberry selections (14.8-41.8 mg/100 g), but lower than

the content reported by Pilando et al. (1985) for the Totem variety (50.3 mg/100 g).

HPLC separation of anthocyanins and anthocyanidins

Figure 3.1 is a HPLC chromatogram of the ACNS from red and orange

nasturtium petals and their respective UV-visible spectra. The characteristics of the two

different colored flowers are summarized in table 3.1. Three major ACNS were

revealed and the retention times and maximum absorbance were the same for the two

samples. However, the percentage of each peak was different. For the red petals (figure

3.1 a), peak 3, which is the pgd-based pigment, represented 35% while for the orange

mm

DAD1, 4.040 (1366 mAU.Apx) of AREDFLA_.D DAD1. 6.1 • 0 (17S rnALJ.Apx') of AREDFLA_.0 DAD1. 3.3? mt. ■r:A:.:iA.'^; '.>\ ARfc'DF-'LA .0

Norm. "

1200-

1000-

800-

600-

400-

200-

T—r—i—i—I—i—i—r-

300 ' 1 ' ' ' 350

-I—j—\—i—r-

400 450 500 1 ' I '

550 nm

nm

Figure 3.1. HPLC chomatogram of anthocyanins from (a) red nasturtium petals, (b) orange nasturtium petals, and (c) their characteristic UV-visible spectra.

6

43

Table 3.1. Characteristics of anthocyanins present in nasturtium flowers

Peak Retention time % area Maximum E 440/E Amax

Plant material (min) Absorbance (nm) %

Red flower 1 5.93 59 528 29 2 7.66 7 513 34 3 10.4 35 502 45

Orange flower 1 5.99 8 528 31 2 7.71 1 513 34 3 10.3 91 502 45

petals (figure 3.1 b), it represented 86 % of the total peak area. During the analysis of

the absorption spectra we found that both colored-petals presented the same

characteristic spectra (figure 3.1 c); peak 3 showed a pronounced shoulder in the 410-

450 nm region and matched the spectra of pgd 3-glu from strawberry; in addition, this

peak presented the highest E 440/E X max (45 %) as compared to the other 2. The

description of peak 3 follows the characteristics found by Harbome (1958) for pgd-3-

glycosides; which is, having a X max at 505 nm, and a distinct shoulder to the main

absorption peak in the 410-450 nm region. Only weak UV absorption was detected in

the 320 nm region, which indicates no acylation with aromatic acids; as an additional

identification assay for the pgd-based ACN present in the petals of orange flowers,

spiking of red raspberry juice with the pgd-based ACN extract of the flowers resulted in

coelution of the previously identified pgd 3-soph fraction of the raspberry juice (Boyles

and Wrolstad, 1993).The anthocyanidin analysis for the pigment present in the petals

(figure 3.2 a) is reported in table 3.2. Three major peaks were found whose X max was

535 nm, 527 nm and 513 nm respectively as reported in figure 3.2 b. The spectral

characteristics of the anthocyanidins had the same basic pattern of the ACNS as found

by Hong and Wrolstad (1990). Again, peak 3 exhibited a pronounced shoulder in the

420 nm region, which suggested the presence of pgd. Evidently, there was a shift in the

^max of the anthocyanidins when compared to the X max of the ACNS, which agrees with

the results reported by Harbome (1958) who found a 6-12 nm longer visible X for the

m A U

mAU ' 1

1200-

- (a)

1000-

800-

600-

400- i 200-

\

0- ^JU Q V

5 i . i i i

10 mln

DAD1, 9.287 (502 mAU,Apx) of AREDFLA D DA01,6.110 {•■/& rtiAU.Apx} ot AREDRA.-D DA:';;. 4 i>6.) dOSft rnM; A-xO •;? ARJ:DR.A I;

Norm. - 1400- / W\

(b) / / AW 1200-

I' ••

1000-

i i t .

1 |i 800-

-v, / i i /; 1 '■ *

//V\ / '; 1 "i 600- ^l ///

\ t

400- ; 1 /^ 1 '^

\ i \ t 1 /"/ ",''' \ i \

200- \^/// \ \\

0-

V..-'-"'"%""*'' ^

300 350 400 450 ' 1 ' 500

1 ' ■ I ■ 550 nm

mm nm

Figure 3.2. (a) Anthocyanidin profile of the pigments present in petals of nasturtium flowers and (b) their respective UV-visible spectra.

fc

45

Table 3.2. Characteristics of anthocyanidins present in nasturtium flowers

Blossom Peak Retention % area Maximum Peak £440/ EXjan Color time (min) absorbance

(nm) assignment

Red 1 4.04 49 535 delphinidin 24 2 6.11 7.5 527 cyanidin 26 3 9.29 43 513 pelargonidin 31

Orange 1 4.07 6 534 delphinidin 24 2 6.14 2 527 cyanidin 27 3 9.32 92 513 pelargonidin 31

anthocyanidins in 0.01% methanolic HC1.

The HPLC profile of strawberry ACNS (figure 3.3 a) presents 5 major peaks

described in table 3.3. Peak 1 accounts for 2% of the total area and has been previously

identified as cyanidin 3-glucoside (cyn 3-glu) (Timberlake and Bridle, 1982), peak 2

accounts for 87% of the total area an has been identified as pgd 3-glu (Harbome, 1958),

peak 3 represents 2% of the total area and was tentatively identified as pgd 3-rutinoside

by Hong and Wrolstad (1990) and confirmed by Bakker et al. (1994). Peak 4 (2% of

total area)has been proposed to be pgd 3-glu acylated with succinic acid as determined

by mass spectrometry by Bakker et al. (1994) while peak 5 (7% of total area) is

unidentified and has been proposed as a pgd derivative (Hong and Wrolstad, 1990;

Bakker et al. 1994). Evidence of the nature of these two compounds was based partially

on their UV spectrum. Figure 3.3 b shows a A, ^a at 503 nm for peak 2 and a

X, max at 505 nm for peaks 4 and 5. The three spectra present a pronounced shoulder in

the 410-450 nm, which agrees with the previous description of pgd-3-glycosides spectra

Harbome (1958). There was no UV absorbance in the 310 nm, which suggests no

acylation with aromatic acids.

With the purpose of tentative identification of these anthocyanidins, Concord

grape and strawberry anthocyanidins were used as reference; Concord grape contains

five of the six common anthocyanidins, while strawberry contains the remaining one,

which is pgd. Wulf and Nagel, (1978), Wilkinson et al (1977), Casteele et al. (1983) and

m A U

mAU. 2 I

600- (a)

500-

400-

300-

200-

100-

1 3

0- . . A IL 4 A5

10 i ■ ■

20 mir

mm

*DAD1, "DAD1

26.773 (27.8 mAU, 21.151 (35.3rr;Ay,

-)of JUIC2014.D •5 of JUIC2014.D

■■DACV: i;ii^s<srjinvv.y:- JofJU^ri^P

Norm.

'T\ 500- ' '\

■ \ 1 \ 'J A

• V i if \

400- 1 i

. UX ■ i 1 / \| ■ \J .""': J / \1

/ 1

300- j j j \

t 1

• / j J j <

• :'

1 i i i /t^/ j •!

200- j i i i ■ i rJ

■ \ ^ //

v\ I'M

100- 1 i \_ / lA

- ^\J ■ ■-■■' -►2 K

0- ^^

1111,, i, i

300 350 111111111

400 450 1 | 1 1 1

500 1 1 1 1

550 nnn

nm

Figure 3.3. (a) HPLC profile of strawberry anthocyanins and (b) UV-visible spectra of (2) pelargonidin 3-glucoside, (4) pelargonidin 3-glucoside succinate-isomer 1, and (5) pelargonidin 3-glucoside succinate-isomer 2.

ft

47

Table 3.3. Characteristics of anthocyanins present in Totem strawberries

Peak Retention time % area i Maximum Peak ; assignment E MQ/E Xmu (min) absorbance (nm)

1 12.2 2 518 cyn 3-glu 33 2 14.5 87 503 pgd 3-glu 44 3 15.9 2 505 pgd 3-rut 49 4 20.6 2 505 Pgd 3-glu succinate

(isomer 1) 46

5 25.9 7 505 Pgd 3-glu succinate (isomer 2)

44

Hong and Wrolstad (1986) have demonstrated that the order of elution of grape

anthocyanidins is: dpn, cyd, ptd, pnd and mvd. When comparing the HPLC profile and

spectra of the aglycons from grape and petals, there was match in retention times and

^ max for dpn, cyd, and pgd. Determination of acylation of strawberry anthocyanin via

alkaline hydrolysis

Figure 3.4 shows the changes in the HPLC profile after alkaline hydrolysis of

the two late-eluting peaks present in strawberry ACN. Partial peak identification was

based on comparison of retention times with the starting material (figure 3.4 a). The

shift in time of elution was the same for both compounds and the new retention time

matched that one of pure pgd 3-glu; besides, the absorption spectra was identical for the

three peaks (figure 3.4 b). We found different results as compared to the ones reported

by Hong and Wrolstad (1990); they did not achieve hydrolysis during saponification of

peak 4. However, the presence of a shoulder in the 400-460 nm region and the lack of

elution of this compound from C-18 Sep-Pak with borate buffer supported their

assignment of its being a pgd derivative.

Organic adds analysis

HPLC analysis of the acids obtained from alkaline hydrolysis of the two

acylated pgd-based peaks showed the same retention time. Retention times of the

succinic acid standard and the two hydrolyzed peaks also matched, and peak area of the

mAUj (a)

125-j 4 10(H 75^ 50-3 251

o-i .Ji ■

10 20 mir

mAUj (b) 125^ 100-i 5 75-.

50-. 25-.

OH IM ' i

10 ■ ■■ i i i i i

20 mir

mAUj

125-i (c)

100 -i 75-.

50 -. 251

OH A. ^ i

1 10

i i i ! ... i

20 mir

*DAD1, 13.477 (173 mAU, -) of SUC1 AS2.D 'DAD1.. Zfi.l Hv {93.1 mAU, ■•) of Gi.USUCZ.i)

ARnorm

80-

1 l ' ' ' ' l ■ ' ' ' I ' ' ' ■ I ' 300 350 400 450

■ I ' ' ' ' I ' ' 500 550 nm

mm nm

Figure 3.4. Changes in HPLC profile after alkaline hydrolysis of the two late-eluting peaks present in strawberry anthocyanins: (a) pelargonidin 3-glucoside-suucinate-isomer 1, (b) pelargonidin 3-glucoside-succinate-isomer 2, (c) shift in time of elution for both compounds, (d) absorption UV-visible spectra for the three peaks.

00

49

acid from the saponified peak increased upon spiking with the standard as shown in figure 3.5

(a)

-V^

20 - (b)

' E

15 - 2 ■ * a :

cid

atio

10 -

s-

succ

in

sapo

n

0 -

•s -

/V. . /\ J\ rv ^

8 10 2 14 16 18 20 22 24 n*

m*U -) soo -

(c)

400 -

1 + * a

300 - -a .2 o «s

200 -

succ

inic

a

sapo

nific

st

anda

rd

100 -

0 - 1 ^ fK A --

S B 10 12 i* » 1 ' 4 ' rf ' ' rf ji mn

mm

Figure 3.5. HPLC chromatogram of (a) succinic acid standard, (b) succmic acid from saponification, and (c) succinic acid from saponification after spiking with standard.

50

Mass spectrometry

Figure 3.6 is the MS of the peak of interest present the orange petals of

nasturtium flowers. The analysis detected a parent peak at m/z 595, which is the MW of

pgd 3-soph and an ion at m/z 270.8 corresponding to the aglycon that was released

during the loss of sophorose.

The MS of the 2 purified peaks of interest (figures 3.7 and 3.8) present in

strawberry showed a parent peak at m/z 533, which is the molecular weight of pgd 3-glu

succinate and a ion at m/z 271 corresponding to the aglycon that was released during

the loss of glucose and succinate. These findings confirm the results obtained by Bakker

et al. (1994) in which fast atomic bombardment mass spectroscopy (FABMS) showed a

molecular mass of 533 for peak 4. We attribute the presence of two peaks with the same

characteristics to the possible presence of isomers with esterification in different

positions of the sugar molecule, one being more hydrophobic and hence, presenting

longer time of elution.

Identification of sophorose from the add hydrolysis of pgd 3-soph

Figure 3.9a shows the GLC chromatogram of the TMS derivatives of the

glucose standard as compared to those for glycosidic substituents of the orange-colored

nasturtium petals (figure 3.9 b). Two major peaks were detected for the standard and the

sample and the elution times for both are comparable as reported in table 3.4. As

mutarotation of sugars in solution is a common reaction, peaks 1 is believed to be the 6

anomer of glucose while peak 2 is believed to be the a anomer. During mutarotation,

higher amounts of the 6 form are expected due to the higher stability of this

conformational isomer (Southgate, 1976; Biermann and Mc Ginnis 1989).

100n

595.1

£

75-

270.8

C/3 a 50-

o .£ £ a &

25-

1 200 300 400 500 600

m/z 700 800 900 1000 1100

Figure 3.6. Mass Spectrum of pelargonidin 3-sophoroside present in nasturtium flowers.

#

00 C

100

75

50

25-

27).8

wJw

532.S

^Mf^H^JlSaHM^MMM^^^ U^t ̂Vffw^jyw*. 150 200 250 300 350 400

m/z 450 500 550 600

Figure 3.7. Mass Spectrum of succinyl-pelargonidin 3-glucoside -isomer 1 present in strawberry. to

100

75-

^

C/3 c

4)

04

50

25

ia.

270.8

AM ik 1 Ik.. . if , I, JU-

533.2

1 150 200 250 300 350 400

m/z 450 500 550 600 650

Figure 3.8. Mass Spectrum of succinyl-pelargonidin 3-glucoside -isomer 2 present in strawberry.

54

u->

(a)

UL

(b)

Figure 3.9. GLC chromatogram of TMS derivatives of (a) glucose standard and (b) glycosidic substituents of the orange-colored nasturtium petals.

55

Table 3.4. Chromatographic properties of the sugar substituent present in the pgd-based anthocyanin of orange petals from nasturtium flowers.

Compound Peak Retention time % area (min)

Glucose standard 1 7.91 54 2 10.05 46

Sample 1 7.87 49 2 9.99 51

Color specifications of anthocyanins

Table 3.5 reports the color specifications of the pgd 3-glu and acyl- pgd 3-glu.

The XYZ tristimulus values show that the relative amounts of each primary color is

about the same for both ACNS and therefore, the dominant wavelength (corresponding

to orange) and % purity are the same.

Inter conversion from the XYZ tristimulus values to Munsell specification gives

also the same notation for both pigments; the hue (yellow-red), value (degree of

grayness), and intensity of color (chroma) match for both anthocyanins. These

characteristics were very clear during the experiment as pgd 3-glu and acyl-pgd 3-glu

have the same visual response and color match when present in the oxonium form and

at the same concentration.

The chromaticity coordinates a* and b* and the degree of lightness were very

close when comparing the two ACNS and only slight differences were noticed in

chroma and hue angle. A AH* value of 2 indicates that the color of acyl-pgd 3-glu was

yellower, and a AC* of-1 indicates that acyl-pgd 3-glu was slightly paler than pgd 3-

glu.

CONCLUSIONS

Evidence of the presence of acyl-pgd 3-glu with succinic acid was found in

Totem strawberries; furthermore, 2 isomers of this compound were found. The

concentration of pgd 3-glu and succinyl-pgd 3-glu present in Totem strawberry

56

Table 3.5. Color specifications of pelargonidin 3-glucoside and acylated pelargonidin 3-glucoside (oxonium forms)

Value Pelargonidin Acylated pelargonidin 3-glucoside 3-glucoside

X 78.2 76.7 Y 67.8 65.6 Z 37.1 36.1 x 0.43 0.43 y 0.37 0.37 Dominant wavelength 590 590 % purity 43 43 Munsell notation 3.5 YR 8.42/8 3.5 YR 8.35/8 L* 85 85 a* 26 28 b* 39 38 Chroma 57 56 Hue angle 47 46

has an approximate ratio of 9:7. They present extremely close color specifications and

therefore match visually. It was possible to ascertain the presence of pgd 3-soph in

nasturtium flowers. This was the major pigment in orange-colored blossoms, whose

ACN extract was red. Conversely, the red petals yielded purple extracts since they

contain higher amounts of dpn and cyd as compared to the orange blossoms.

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Biermann, C and Mc Ginnis, G. D. 1989. Analysis of Carbohydrates by GLC and MS. Boca Raton, Fl, pp. 43-85.

57

Boyles, M. and Wrolstad, R. E. 1993. Anthocyanin composition of red raspberry juice: influences of cultivar, processing and environmental factors. J. Food Sci. 58:1135-1141.

Brouillard, R. 1981. Origin of the exceptional color stability of the Zebrina ACN. Phytochem. 20:143.

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•

58



Rommel, A, Heatherbell, D. A and Wrolstad, R. E. 1990. Red raspberry juice and wine: effect of processing and storage on anthocyanin pigment composition, color and appearance. J. Food Sci. 55 (4): 1011-1017.

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Rwabahizi, S. and Wrolstad, R.E. 1988. Effects of mold contamination and ultrafiltration on the color stability of strawberry juice and concentrate. J. Food Sci. 53(3): 857-861.

Southgate, D. A T. 1976. Determination of Food Carbohydrates, pp. 44,45, 121, 122.

Timberlake, C. F. and Bridle, P. 1982. Distribution of anthocyanins in food plants. In Anthocyanins as food colours. Markakis,. (Ed.), Academic Press, New York.

Wilkinson, M.; Swenny, J.G. and lacobucci, G. A 1977. High-pressure liquid chromatography of anthocyanins. J. Chromatogr. 132: 349-351.

Wrolstad, R. E., Putnam, T. P., and Varseveld, G. W. 1970. Color quality of frozen strawberries: effect of anthocyanin, pH, total acidity, and ascorbic acid availability. J. Food Sci. 35: 448.

Wrolstad, R.E. 1976. Color and pigment analysis in fruit products. Bull. 624, Oregon Agric. Exp. Stn., CorvaUis, Ore.

Wrolstad, R.E., Skrede, G., Lea, P. and Enersen, G. 1990. Influence of sugar on anthocyanin pigment stability in frozen strawberries. J Food Sci. 55 (4): 1064- 1065.

Wulf, L. W. and Nagel, C. W. 1978. High-pressure liquid chromatographic separation of anthocyanins in Vitis Vinifera. Am J. Enol. Vitic. 8:35.

59

CHAPTER 4

COMPARATIVE STABILITY OF PELARGONIDIN-BASED ANTHOCYANINS IN JUICE SYSTEMS



60

ABSTRACT

The stability of pelargonidin 3-glucoside (pgd 3-glu), pelargonidin 3-

sophoroside (pgd 3-soph), and pelargonidin 3-sophoroside 5-glucoside acylated with

cinnamic and malonic acids (acyl-pgd 3-soph 5-glu) was compared in juice systems.

Strawberry juice (8 0brix) and strawberry concentrate (57 "brix) were fortified with each

one of these anthocyanins (ACNS). Changes in pigment, degradation index, Hunter

color parameters, and % haze were monitored during sixteen week storage at 25 0C.

Changes in anthocyanin (ACN) profile were also monitored via High Performance

Liquid Chromatography (HPLC). Fortification increased stability of the ACNS in juice

samples whose half life (ti/2) ranged between 4 and 9 weeks. Fortification with acylated

pgd derivatives increased Unof the concentrate from 4 to 8 weeks. All samples

presented a decrease in degradation index and increase in haze. HPLC analysis

confirmed the protective effect of ACN fortification on juice samples.

INTRODUCTION

The attractive red color of fresh strawberries is a decisive factor influencing the

consumption of this fruit and the processed products derived from it. Unfortunately, the

original color of the fresh fruit is lost during processing and storage operations, and a

dull, brownish color develops. This change appears to the consumer as an indication of

inferior quality and deterioration of the fruit. Consequently, the market of strawberry

products is negatively affected, as its expansion becomes limited.

Factors responsible for color degradation in various strawberry products have

been widely investigated. Studies on strawberry preserves (Kertesz and Sondheimer,

1948; Mackinney and Chichester, 1952; Abers and Wrolstad, 1979), frozen strawberries

(Wrolstad et al., 1970), freeze-dried strawberries (Erlandson and Wrolstad, 1972),

strawberry juice and concentrate, (Wrolstad et al., 1980) and strawberry wine (Pilando

et al., 1985) have aimed to find explanation and solution to the color change of the fruit

during processing operations. From these studies several factors have been found to

responsible for such deterioration.

61

Chemical structure of the anthocyanins (ACNS) is one factor affecting the

stability of these pigments. It is hypothesized that increase in glycosylation and

presence of acylation delays degradation of the anthocyanin (ACN) molecule leading to

higher stability of the pigment (Markakis, 1982; Mazza and Miniati, 1993). Substitution

with the disaccharide sophorose is believed to be the key factor contributing to the

better color stability of red raspberry wine as compared to strawberry wine (Rommel et

aL, 1990; Rommel et al., 1992). Similarly, acylation of the ACN molecule is believed to

improve stability by preventing hydration of the molecule (Asen et al., 1977; Goto et

aL, 1979; Hoshino et aL, 1980; Brouillard, 1981). Guisti and Wrolstad (1996a) reported

high color and pigment stability when coloring secondary bleached cherries with a

ACNS extracted from radish peel. The high stability of this model system was attributed

to the nature of the major ACN present in radish peel, which is an acylated triglycoside.

Pigment concentration is another factor involved in the degradation of ACNS.

Skrede et al (1992) found that total ACN concentration was a major parameter

influencing stability of strawberry ACNS. Strawberry syrup fortified with pelargonidin

3-glucoside (pgd 3-glu) presented comparable stability to blackcurrant syrup whose

main ACN contains a disaccharide. Similarly, strawberry jams, to which elderberry

colorant or pomegranate juice was added to increase monomeric ACN content showed a

slower loss of ACN (Garcia-Viguera and Zafirilla., 1998). Both elderberry and

pomegranate fruits contain cyanidin 3-glu (Gil et al., 1995; Inami et al., 1996).

The purpose of this study was to determine the influence of chemical

composition and concentration of pgd-based ACNS on their stability. The stability was

compared when the pigments were present in single strength strawberry juice and

strawberry concentrate.


Plant material

Individually quick frozen (I.Q.F.) strawberries (Fragaria anannassa cv, Totem)

were supplied by Conroy Packing Inc. (Salem, OR). Orange nasturtium flowers

(Tropaeolum majus) were collected from local gardens during the summer season

62

(Corvallis, OR). Stems and leaves were separated from the petals, which were then

frozen with liquid nitrogen. Red radishes (Raphanus sativus L cv, fuego) were obtained

from the local market in Corvallis, OR. The radishes were peeled manually, the peel

was washed and frozen under liquid nitrogen. All frozen materials were stored at -23 0C

until further processing.

Processing of juice and concentrate

Figure 4.1 shows the unit operations for the processing of strawberry juice and

concentrate. 30 Kg of I.Q.F. Totem strawberries were thawed overnight at room

temperature and crushed using a hammermill (model D Comminuting machine, W. J

Fitzpatrick Co., Chicago, IL) equipped with a circular pore mesh, 1.27 cm in diameter

at a speed of 182 rpm. The pulp was depectinized at 45 0C for 2 hr by adding 0.135 mL

pectinase (ROHAPECT MB, Scott laboratories. Petaluma, CA) per Kg. Monitoring of

completion of depectination was carried out with a negative alcohol precipitation test

(1:1 juice: isopropanol +1% HC1). Pressing of the depectinized pulp was done at 4 bars

for 30 min in a Willmes press bag (Type 60, Moffet Co., San Jose, CA); one per cent

rice hulls was used as press aid. Filtration of the juice was performed with 2%

diatomaceous earth in a multipad filtration unit (Herman Strassburger K.G. presser from

Westhofen BEP worms); the unit was equipped with a pad filter SWK supra 2600 (Scott

Laboratories, Inc., San Rafael, CA). Subsequent pasteurization of the filtered juice was

done by high temperature short time treatment (HTST) at 85-90 0C for one min in a

tubular heater with screws (Wingear type, model 200WU, Winsmith Co, Springville,

NY). Half of the batch of the pasteurized single strength juice was concentrated to 57

"brix in a Centritherm (Alfa Laval type CT-1B, Sweden). Concentrate samples were

diluted to 8 "brix with distilled water prior to analysis.

63

Thaw 30 Kg I.Q.F. strawberries

Crush with hammermill

Depectinize with Rohapect MB (0.135 ml/Kg mash

Incubate (45 0C-2hr)

Alcohol test for pectin (incubate further if positive)

Add 1% rice hulls as press aid

Press at 4 bars for 30 min

Add 2% diatomaceous earth, pad-filter (SWK 'Supra 2600')

HTST pasteurization (95 0C for 15-30 sec)

Divide in two portions

Portion 2-concentrate STbrix

Figure 4.1. Unit operations for the production of strawberry juice and concentrate.

64

Compositional analysis

Total titratable acidity of the juice and concentrate was determined as g citric

acid per 100 ml by titration with 0.1 N NaOH to pH 8.2. Measurement of the pH was

done with a Fisher Accument pH meter, model 210. Soluble solids were determined on

an Auto Abbe refractometer 10500 (Reichert-Jung, Leica Inc., NY, USA); the

instrument was used under the % soluble solids with temperature compensate mode.


ACNS from strawberries, petals of orange nasturtium flowers, and radish peel

were extracted and isolated as described by Wrolstad et al. (1990). Frozen samples were

ground to a powdery state with liquid nitrogen using a stainless steel Waring blender.

The powder was blended with acetone (2 L/Kg powder) in order to extract the ACNS,

which were then separated from the cake by filtration on a Buchner funnel. Total

extraction of the ACNS from the cake residue was done with aqueous acetone (30:70

v/v). Filtrates were combined and shook in a separatory funnel with chloroform (1:2

acetone: chloroform v/v) and stored at 10 "C overnight for better separation between the

aqueous and the organic phases.

Anthocyanin purification

The aqueous extract was transferred to a Bucchi rotoevaporator to remove

residual acetone at 40 "C. The ACNS were purified subsequently using a C-18 cartridge

(high load C-18 tube), 20 mL capacity (Alltech Assoc, Inc., IL). The cartridge was

washed with 0.01% HC1 upon loading with pigment, and the ACNS were eluted with

methanol acidified with 0.01% HC1; elimination of methanol and concentration of

pigments were achieved by additional rotoevaporation at 40oC. In all cases, the aqueous

extract was diluted to a known volume with distilled water. The concentration of the

monomeric ACN was determined by the pH differential method as described by

Wrolstad (1976) and expressed as mg pgd 3-glu/L with molecular weight of 433.2 and a

molar absorptivity of 22,400.

65

HPLC for separation of major anthocyanins

A Perkin-Elmer Series 400 chromatograph equipped with a Hewlett-Packard

1040A photodiode array detector and a Hewlett-Packard 9000 computer was employed

for separation of the major anthocyanins. A reverse phase Supelcosil PLC-18 column

(25 cm x 21.2 mm) (Supelco Inc., Bellefonte, PA) was used for preparative isolation.

Chromatography was performed by elution with the following mobile phase: (A) 100%

acetonitrile, (B) 5% acetonitrile, 10% acetic acid, 1% phosphoric acid and a linear

gradient from 0% to 6% A in 5 min, from 6% to 11% A in 10 min, and from 11% to

28% in 6 min. The flow rate was 10 mL / min and injection volume 1 mL. Primary

detection at 520 nm. The purity of the pigments was determined by analytical HPLC.

Analytical HPLC

A Supelco LC -18 column (25 cm x 4.6 mm ID) (Supelco Inc., Bellefonte, PA),

particle size 5 ^ was used for analytical separation. The column was equilibrated with a

mobile phase containing: (A) 100% acetonitrile, (B) 5% acetonitrile, 10% acetic acid,

1 % phosphoric acid and chromatography was performed a linear gradient of 0% to 6%

A in 15 min and from 6% to 20% A in 25 min. The flow rate was 1 mL/min, injection

volume 50 |i L and primary detection at 520 nm.

Experimental design

Fortification with pgd derivatives was done at a level to approximate twice the

initial concentration of the pigment present in the strawberry juice and concentrate

respectively. Each portion of strawberry juice and concentrate was divided into 4

samples. Sample 1 was used as a control, sample 2 was spiked with pgd 3-glu, sample 3

was spiked with pgd 3-soph and sample 4 was spiked with acyl- pgd 3-soph 5-glu. 0.5%

potassium sorbate (w/w) and 0.5% sodium benzoate (w/w) were added to each sample

for preservation. Aliquots from each sample were transferred to 20-mL vials; vials were

sparged with nitrogen, screwcapped, and stored in the dark at 250C for 16 weeks.

66

Pigment analysis

Prior to analysis concentrate samples were diluted to 8 "brix. All samples were

centrifiiged to eliminate sediment from preservative. Monomeric ACN content was

determined by the pH differential method as described before. Color density, polymeric,

and degradation index were determined using spectral methods (Somers, 1977). Color

density and polymeric color were calculated as the sum of the absorbance at 420 mn

and that at the absorbance maximum of an untreated or of a bisulfite treated sample,

respectively. Polymeric color (%) was calculated as the ratio between polymeric color

and color density X 100. Degradation index was calculated as the ratio of the

absorbance maximum of the ACN/absorbance at 420 mn. Determinations were made on

a Shimadzu UV visible spectrophotometer, model UV 160 U, using 1 cm disposable

cells.

Color and haze analysis

Samples that have been previously diluted at 8 "brix and centrifuged were

rediluted to 2 "brix with distilled water and placed in a 0.5 cm-path length optical glass

cell (Helma, Germany). Hunter L*, a*, b* values, hue angle (arctan b*/a*), chroma (a*2

+ b*2)1/2> aad % haze were measured in a ColorQUEST Hunter colorimeter (Hunterlab,

Hunter Associates Laboratories Inc., Reston, VA) in the transmittance mode with a CIE

observer angle of 10 degrees, illuminant C. Hunter L* value expresses degree of

lightness from 0 = black to 100 = white, hue angle expresses the color nuance in

numerical terms (0 0= red-purple, 90°= yellow, 180°= bluish-green, 270°= blue), and

chroma expresses a measure of color intensity (McGuire, 1992).

Changes in pigment profile during storage

Monitoring of relative ACN degradation by peak area was performed applying

the same techniques described for analytical HPLC.

67

Data analysis

The ACN concentration was plotted against storage time. Linear regression

analysis was used to determine adequacy of the model describing kinetics of

destruction. Rate constants were calculated from slopes of the lines plotted, and half

lives (ti/2) were calculated from the equation: ti/2= Ln 0.5/k, where k = rate constant.


Strawberry juice and concentrate before storage

The compositional data of the juice and concentrate immediately after

production is shown in table 4.1. With the process of concentration of the juice, loss of

monomeric ACN, decrease in degradation index, and increase in % polymeric were

observed. No pronounced changes in the Hunter L* value and hue angle were detected

and only a slight increase in chroma was observed. Concentration did not have a

pronounced effect on pH of the products, but there was a small decrease in titratable

acidity in the concentrate.

The observed changes are in good agreement with previous observations during

concentration of strawberry juice made from Benton strawberries. Rwabahizi (1988)

reported degradation of the monomeric pigment, increase in polymeric color, and drop

in degradation index. Very small changes in color indices were reported. pH remained

constant while the total acidity dropped, which seemed to be due to loss of volatile acids

during vacuum concentration. Likewise, Wrolstad et al. (1980) reported degradation of

monomeric pigment, increase in browning and Hunter L* value after concentration of

juice made from Hood and Benton strawberries. Loss of monomeric ACN and

polymerization of pigments have been attributed to increase in temperature, which can

contribute to monomeric ACN degradation and polymerization of reactive phenolics

(Markakis, 1982)

68

Table 4.1. Compositional and color indices of strawberry juice and concentrate before fortification

Parameter Juice Concentrate

Monomeric Anthocyanin (mg/L) 276 242

PH 3.27 3.35

Titratable acidity (% citric acid) 0.85 0.67

COLOR INDICES

% Polymeric color Degradation index (A 510/ A 420) Hunter L* value Hue angle Chroma

1 0.79 41 46 74

12 0.98 40 48 89

Haze 13 8.1

Measurements on 80Brix basis

The values reported are average of three measurements

Table 4.2 lists the ACN pigment content and color characteristics of the initial

fortified samples. ACN fortification produced juice and concentrate samples with

similar respective ACN concentrations. Addition of the pigments caused small changes

in the initial content of polymeric color depending on the amount of polymers present in

the purified ACN.

Spiking with non-acylated pgd derivatives produced a decrease in the degree of

lightness of the juice, but very small changes in the L* value of the concentrate.

Chroma was notably increased upon fortification while hue angle was almost

unaffected. Samples fortified with pgd 3-glu and pgd 3-soph had similar color

characteristics while samples fortified with acylated ACN exhibited higher L* value

and hue. The color difference was probably due to light absorbance properties of the

ACNS of different sources (Francis, 1982).

69

Table 4.2. Anthocyanin and color characteristics of fortified strawberry juice and concentrate

Color indices

Sample Monomeric % polymeric L* Chroma Hue angle ACN (mg/L) pigment

Juice + pgd 3-glu Juice + pgd 3-soph Juice + acyl-pgd 3-soph 5-glu

525 489 476

Concentrate + pgd 3-glu 358 Concentrate + pgd 3-soph 325 Concentrate + acyl-pgd 3-soph 5-glu 371

2 5 2

9 16 2

37 34 43

39 40 42

89 84 98

91 92 97

44 43 47

45 48 47

All values measured on 8 0brix basis.

The values reported are average of two repeated measurements

Pigment analysis

Changes of pigment in strawberry juice and concentrate are presented in figures

4.2 and 4.3, respectively. Doubling the ACN concentration with non-acylated pgd

derivatives increased the stability of the pigment in the juice, but not in the concentrate.

Semilogaritmic plots indicated first order kinetics for ACN deterioration. Rate constants

and tic determined from logarithmic plots reported in table 4.3 show that the major

increase in tm of the juice ACN (4 to 9 weeks) was present when the sample was

fortified with pgd 3-glu. Fortification with acyl- pgd 3-soph 5-glu resulted in

improvement of stability of juice and concentrate. In both cases, the tm of the pigment

was increased from 4 weeks to 8 weeks.

Previous reports have shown findings similar to ours. Skrede and Wrolstad

(1992) found in their study that the differences in stability that had been observed for

different types of ACNS by other researchers was less important than the total ACN

concentration. When strawberry syrup was fortified with pgd 3-glu, the stability of pgd

3-glu became comparable to the delphinidin and cyanidin 3-diglycosides present in

blackcurrant syrup. The normal ACN concentration of strawberry is about 10 % of the

600

6 8 10 Weeks

6 8 10 Weeks

Figure 4.2. Changes in pigment during storage of juice systems.

o

400

a 300

control

concentrate +pgd 3-glu

concentrate + pgd 3-soph

concentrate + acyl-pgd 3-soph 5-glu

14 16

Figure 4.3. Changes in pigment during storage of concentrate systems.

72

Table 4.3. Linear regression parameters of monomeric anthocyanin degradation in strawberry juice and concentrate.

Sample Parameter

Bo Bi r2

(intercept) (slope) Half life Rateofdegradati on ti/2

(weeks ) (weeks)

Juice -0.483 -0.169 0.90 4 Juice + pgd 3-glu -0.123 -0.0797 0.90 9 Juice + pgd 3-soph -0.251 -0.141 0.90 5 Juice + acyl-pgd 3-soph 5- glu -0.0236 -0.0891 0.99 8

Concentrate -1.12 -0.168 0.65 4 Concentrate + pgd 3 -glu -1.015 -0.190 0.74 4 Concentrate + pgd 3 -soph -1.029 -0.159 0.67 4 Concentrate + acyl-pgd 3-; soph 5-glu -0.149 -0.087 0.94 8

one in blackcurrant (Skrede, 1987, Rwabahizi and Wrolstad, 1988). This finding was

similar to previously reported results (Skrede, 1987 and Taylor, 1989), in which the

variation of total pigment concentration between different blackcurrant cultivars was

more important with respect to loss of ACNS than the difference in stability of the

individual ACNS. More recently, when strawberry jam was fortified with elderberry

extract or pomegranate juice, the stability of the ACNS was extended from 160 to 194

days regardless of the pigment added (Garcia-Viguera and Zafrilla, 1998).

Contribution of polymeric color was reduced with ACN fortification; this

reduction was higher in the juice samples and the smallest contribution of polymeric

color was observed in the samples fortified with acylated ACN (table 4.4).

The change in degradation index with storage (table 4.5) was more noticeable in

the concentrates as compared to the single strength samples. Among all samples, the

juice fortified with acyl-pgd 3-soph 5-glu presented the smallest decrease in the

absorbance ratio. Higher values of A420 nm are indicative of presence of brown

pigments.

73

Table 4.4. Changes in polymeric anthocyanin during storage

%) in strawberry juice and concentrate

Sample Time of storage (weeks)

Juice Juice + pgd 3-glu Juice + pgd 3-soph Juice + acyl-pgd 3-soph 5-glu Concentrate Concentrate + pgd 3-glu Concentrate + pgd 3-soph Concentrate + acyl-pgd 3-soph 5-glu

0 12 16

28 67 79 86 100 100 2 8 14 19 41 28 5 20 47 56 62 65 2 5 10 13 19 23 12 78 87 85 83 87 9 51 67 76 74 100 16 67 78 84 86 75 2 26 29 36 26 46

The values reported are averages of two repeated measurements

Measurement on 8 "brix basis

Table 4.5. Changes in degradation index (A 510/A 420) during storage of strawberry juice and concentrate systems

Sample Time of storage (weeks)

0 2 5 8 12 16

Juice 0.8 0.5 0.5 0.3 0.5 0.6 Juice + pgd 3-glu 1.8 1.6 1.5 0.49 1.3 1.3 Juice + pgd 3-soph 1.6 1.2 0.9 0.33 0.8 0.8 Juice + acyl-pgd 3-soph 5- -glu 2.3 2.1 0.31 1.9 1.7 1.5

Concentrate 1.0 0.5 0.5 0.5 0.6 0.6 Concentrate + pgd 3- ■glu 1.4 0.7 0.6 0.7 0.6 0.6 Concentrate + pgd 3- -soph 1.2 0.6 0.6 0.6 0.6 0.6 Concentrate + acyl-pgd 3- soph 5-glu 10.7 1.3 1.3 1.1 1.3 1.0


Measurement on 8 "brix basis

74

Changes in haze during storage

Changes in haze through storage are reported in Table 4.6. Although the haze

content increased in all the samples, the highest increase was observed in the

concentrate fortified with acylated ACN while the lowest increase was obtained in the

single strength juice fortified with the same ACN. Guisti and Wrolstad (1996 a)

observed appearance of haze during the first week of storage of syrups containing

acylated ACN extracted from radish peel. This increase in haze during storage of these

model systems was directly proportional to the concentration of ACN added.

Table 4.6. Changes in haze during storage of strawberry juice and concentrate systems

Sample r rime of storage (weeks)

0 2 5 8 12 16

Juice 13 9 16 35 42 41 Juice + pgd 3-glu 3 4 6 17 8 18 Juice + pgd 3-soph 2 2 13 27 13 52 Juice + acyl-pgd 3-soph 5-glu 2 15 16 24 14 28 Concentrate 8 26 41 31 46 32 Concentrate + pgd 3- ■glu 6 9 11 51 47 47 Concentrate + pgd 3 -soph 3 19 46 44 37 40 Concentrate + acyl-pgd 3-soph 5-glu 3 82 87 87 90 91


Measurement on 8 0brix basis

Cemeroglu et al (1994) investigated the degradation of ACNS in sour cherry

juice and concentrate and haze formation depending on temperature and soluble solids

content. They reported that haze formation increased with increasing temperature and

soluble solids. Increase in both temperature and soluble solids content is inherent to our

concentration process. The increase in haze during storage of the concentrate sample to

75

which acyl-pgd 3-soph5-glu had been added made color measurements very difficult

light could not be transmitted through the sample.

Color analysis

Figure 4.4 shows the change in color parameters of the samples during storage.

Changes in hue angle were influenced by fortification of the juice samples, but little

effect of fortification on hue angle was noticed in the concentrate. Although there was

an overall shift of red towards orange, changes in color experienced by the spiked

samples were considerable smaller than the change experienced by the control. Increase

in hue angle is characteristic of strawberry products during storage (Wrolstad et al.,

1980); however, Skrede et al (1992) reported only a slight change in fortified strawberry

juice as compared to unfortified juice.

Unfortified concentrate along with juice and concentrate samples in which the

ACN concentration had been adjusted experienced drop in lightness indicating

development of brown color; however, the juice fortified with acyl-pgd 3-soph 5-glu

presented the lowest decrease. On the other hand, the unfortified juice presented

decolorization during storage (increase in L* value). Decolorization of ACNS indicates

decrease in pigment content and has been observed during storage of strawberry wine

(Pilando et al., 1985), and strawberry juice and concentrate (Wrolstad et al., 1980),

Lightening is believed to be due to accelerated oxidation, polymerization, and

precipitation of pigment complexes (Wrolstad et al., 1980). The change in the sample

fortified with acyl-pgd 3-soph 5-glu was not accurately detectable due to haze

interference.

The overall decrease in chroma indicates loss in color saturation as produced by

monomeric ACN degradation. The presence of haze in the concentrate fortified with

radish ACN made this measurement unreliable.

Changes in HPLC profile

Figure 4.5 is the HPLC chromatogram of the ACNS in the unfortified juice and

spiked juice samples before storage. Peaks have been previously assigned as: (1)

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Figure 4.5. HPLC profile of the anthocyanins in unfortified juice and juice spiked with pelargonidin derivatives before storage. Peak identification: (1) cyanidin 3-glucoside; (2) pelargonidin 3-glucoside; (3) pelargonidin 3-rutinoside; (4) pelargonidin 3- glucoside acylated with succinic acid (isomer 1); (5) pelargonidin 3-glucoside acylated with succinic acid (isomer 2); (6) pelargonidin 3-sophoroside; (7) pelargonidin 3-sophoroside 5-glucoside; (8) pelargonidin 3-sophoroside 5-glucoside acylated with cinnamic acids; (9) pelargonidin 3-sophoroside 5-glucoside acylated with cinnamic and malonic acids.

~0

78

cyanidin 3-glucoside (cyn 3-glu) (Timberlake and Bridle, 1982); (2) pgd 3-glu

(Harbome, 1958); (3) pgd 3-rutinoside (Bakker et al., 1994); (4) pgd 3-glu acylated w

succinic acid-isomer 1 (Garz6n and Wrolstad, 1997); (5) pgd 3-glu acylated with

succinic acid-isomer 2 (Garzdn and Wrolstad, 1997); (6) pgd 3-soph (Garz6n, 1997);

(8) pgd 3-soph 5-glu acylated with p-coumaric, ferulic and malonic acids (Guisti and

acids (Guisti and Wrolstad, 1996b) Wrolstad, 1996b); (9) pgd 3-soph 5-glu acylated

with p-coumaric and ferulic. Analysis of spectral characteristics shown in figure 4.6 and

comparison of retention time with a external standard made possible the identification

of peak 7 as pgd 3-soph 5-glu formed from hydrolysis of pgd 3-soph 5-glu acylated

with cinnamic acids. The ratio of absorbance at 440 nm to the absorbance at visible

maximum wavelength for this peak was about half of the ratio for pgd 3-

monoglucoside. Harbome (1967) reported that 3-glycosides exhibited ratios of

absorbance at 440 nan to absorbance at visible maximum wavelength that were about

twice as large as that for the 3,5-diglycosides.

DAD1, 8.389 (717 mAU.Apx) of FORT51A.D DADi. *.1S3 (*<5S rnAUApx; ol FOaVs ;A.D (a)

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100 ^^\^/-—

\\ C —

N**.^ ̂ ^-_ ■ i i i ... ,

300 400 500 ' 1

nm

nm

Figure 4.6. UV-visible spectra of (a) pelargonidin 3-glucoside, (b) pelargonidin 3- sophoroside 5-glucoside from strawberry juice fortified with radish anthocyanin.

79

Changes in relative peak area during storage of the juice and concentrate

samples spiked with non-acylated and acylated pgd derivatives are reported in figures

4.7 and 4.8, respectively. HPLC analysis confirmed the higher stability of the ACNS in

the juice samples as compared to the concentrates. The chromatographic profiles

indicated that the initial degradation of the samples was due to deacylation of succinyl-

pgd 3-glu. Deacylation occurred within the first 2 weeks of storage in all the samples,

except the juices fortified with pgd 3-glu and pgd 3-soph. For these samples 100%

hydrolysis of this acylated ACN only occurred until week 12 and week 8 of storage,

respectively. The pigment stability of the sample spiked with pgd 3-glu was likely due

to increase in concentration of this specific ACN. Markakis (1982) found that increased

concentration of ACNS in plant tissues intensifies their color stability through the

phenomena of intermolecular copigmentation in which a molecular interaction

occurring between the ACN colored form and the copigment (same ACN) controls the

extend of hydration reaction between the flavylium cation and the colorless pseudobase.

Likewise, Mazza and Miniati (1993) found that a small amount loss in analytical

pigment concentration can result in a proportionately great loss of color, especially at

high ACN concentrations.

The second major change observed in peak area was due to hydrolysis of

malonate from pgd 3-soph 5-glu acylated with malonic and cinnamic acids. This

hydrolysis occurred within the first two weeks of storage of the concentrate and within

8 weeks of storage of the juice, and was accompanied by increase in the peak area of

pgd 3-soph 5-glu acylated with cinnamic acids and pgd 3-soph 5-glu. Guisti (1996)

observed that during acid hydrolysis of radish ACNS with 2N HC160% of the malonic

acid was released after only 10 min of treatment resulting in formation of pgd 3-soph 5-

glu acylated with p-coumaric or ferulic acids as the major pgd derivative and small

concentration of pgd 3-soph 5-glu.

Pgd 3-soph was more stable in the juice than in the concentrate; the pigment was

undetectable after 2 weeks of storage of the concentrate suggesting hydrolysis of

sophorose. A small increase in the peak area of this compound in relation to pgd 3-glu

was observed in the juice. This seems to confirm earlier reports on increased ACN

80

•NSS ^ pgd 3-glu succinate T: pgd 3-glu /// PS^ 3-soph

(a)

2 ■

0

I I I I I 0 20 40 60 80 100

0 20 40 60 80 100

(c)

(d)

HVmmiFmmumffVmHiffffivii

I I I I 0 20 40 60 80 100

(e)

0 "1 1 \ 1 20 40 60 80 100

(f)

0 10 20 30 40 50 60 0 20 peak area (%)

40 60 -T 80 100

Figure 4.7. Changes in anthocyanin profile during storage of (a) juice, (b) juice fortified with pelargonidin 3-glucoside, (c) juice fortified with pelargonidin 3-sophoroside, (d) concentrate, (e) concentrate fortified with pelargonidin 3-glucoside, (f) concentrate fortified with pelargonidin 3-sophoroside.

81

i demalonated pgd 3-soph 5-glu + cinnamic acids

I ! pgd 3-soph 5-glu + cinnamic/malonic acids

pgd 3-glu succinate

pgd 3-glu

pgd 3-soph 5-glu

CO

(a)

16

12

0 jflyimm 3

i 1 1 1 1 1 r 0 10 20 30 40 50 60 (b)

16

12

0 533

—i 1 1 1 1 1 1 r 0 10 20 30 40 50 60 70 80

peak area (%)

70

90

Figure 4.8. Changes in anthocyanin profile during storage of (a) juice fortified with acyl- pelargonidin 3-sophoroside 5-glucoside, (b) concentrate fortified with acyl- pelargonidin 3-sophoroside 5-glucoside.

82

stability due to diglycosidic substitution as compared to monoglycosides (Rommel et

al., 1990; Rommel et al., 1992).

CONCLUSIONS

Fortification with non-acylated pgd derivatives increased the stability of the

ACNS when they were present in strawberry juice, but not in strawberry concentrate

whereas fortification with acylated pgd derivatives played a protective role in both

systems.

Color and pigment analysis showed that increased sugar substitution does not

seem to enhance ACN stability, however monitoring of changes in HPLC profile

showed higher stability of pgd 3-soph as compared to pgd 3-glu .

Fortification with pgd derivatives acylated with cinnamic acids increased the

tl/2 of the juice and concentrate, but the stability of the juice was comparable to that of

the sample spiked with pgd 3-glu. This suggests that concentration is an important

factor to take into consideration when evaluating stability of ACN with different

chemical pattern.

The lack of replication of this experiment does not allow statistical comparison

or conclusions on the stability of the pigments under study. This, in addition to our

observation of the need of more frequent readings during the two first weeks of storage

and elimination of additional steps like centrifugation of samples previous to analysis

lead us to new experiments on stability of pgd-based ACNS.

REFERENCES

Abers, J. and Wrolstad, R. E. 1979. Causative factors of color deterioration in strawberry preserves during processing and storage. J. Food Sci. 44 (1): 75-81.

Asen, Stewart, RN. and Norris, K.H. 1977. Anthocyanin and pH involved in the color of heavenly blue morning glory. Phytochem. 16:118.

Bakker, J., Bridle, P., and Bellworthy, S. J. 1994. Strawberry juice colour: A study of the quantitative and qualitative pigment composition of juices from 39 genotypes. J. Sci. Fd Agric. 64: 31-37.

83

Brouillard, R. 1981. Origin of the exceptional color stability of the Zebrina ACN. Phytochem. 20:143.


Erlandson, J. A. and Wrolstad, R.E. 1972. Degradation of anthocyanins at limited water concentration. J. Food Sci. 37:592.

Francis, F.J. 1982. Analysis of Anthocyanins. la Anthocyanins as food colors, p. 196, P. Markakis, (Ed.). Academic Press, New York.

Garcia-Viguera, C. and Zafrilla, P. 1998. Anthocyanins in processed fruit products, p. 134, In Papers Presented at the Third International Symposium on Natural Colorants for Food, Nutraceuticals, Beverages, Confectionery, and Cosmetics. Princeton, New Jersey.

Garz6n G. A. and Wrolstad, R. E. 1997. Characterization of two pelargonidin-based anthocyanins from strawberry (Fragaria anannassa) and nasturtium flowers (Tropaeolum majus). Unpublished data.

Gil, M. I., Garcia-Viguera, C, Artes, F., and Tom£s-Barber£n, F. A 1995. Changes in pomegranate juice pigmentation during ripening. J. Sci. Fd Agric. 68: 77-81.

Goto, T., Hoshino, J. and Takase, S. 1979. A proposed structure of commelin, a sky- blue anthocyanin complex obtained from the flower petals of commelina. Tetrahedron Lett., p 2905.

Guisti, M. M. and Wrolstad, R. E. 1996b. Characterization of red radish anthocyanins. J. Food Sci. 61 (2): 322-326.


Harbome J. B. 1967. Comparative Biochemistry of the Flavonoids. Academic Press, London, UK


Hoshino, T., Matsumoto, U. and Goto, T. 1980. The stabilizing effect of the acyl group on the copigmentation of acylated anthocyanins with C-glucosyl flavones. Phytochem. 19: 663.

Inami, O., Tamura, I., Kikuzaki, H. and Nakatani, N. 1996. Stability of anthocyanins in Sambucus canadiensis and Sambucus nigra. J. Agric. Food Chem. 44: 3090- 3096.

84

Kertesz, Z.I., and Sondheimer, E. 1948. To reduce color losses in strawberry preservi Food Inds. 20:106.

McGuire, R. G. 1992. Reporting of objective color measurements. HortSci. 27(12):1254-1255.

Mackinney, G and Chichester, CO. 1952. Color deterioration in strawberry preserves. Canner. 114(12): 13.

Markakis, P. 1982. Stability of anthocyanins in foods. InAnthocyanins as food colours. New York: Academic Press, pp. 163-180.


Pilando, L.S., Wrolstad, R.E. and Heatherbell, D. A. 1985. Influence of fruit composition, maturity and mold contamination on the color and appearance of strawberry wine. J. Food Sci. 50(4): 1121.

Rommel, A., Heatherbell, D. A. and Wrolstad, R.E. 1990. Red raspberry juice and wine: effect of processing and storage on anthocyanin pigment composition, color and appearance. J. Food Sci 55 (4): 1011-1017.

Rommel, A.,Wrolstad, R.E. and Heatherbell, D. A. 1992. Blackberry juice and wine: Processing and storage effects on anthocyanin composition, color and appearance. J. Food Sci. 57:385- 391,410.

Rwabahizi, S. and Wrolstad, R.E. 1988. Effects of mold contamination and ultrafiltration on the color stability of strawberry juice and concentrate. J. Food Sci. 53(3): 857-861.

Skrede, G. 1987. Evaluation of color quality in blackcurrant fruits grown for industrial juice and syrup production. Norwegian J. Agric. Sci. 1:67

Skrede, G., Wrolstad, R.E., Lea, P. and Enersen, G. 1992. Color stability of strawberry and blackcurrant syrups. J. Food Sci. 57 (1): 172-177.

Somers, T.C. and Evans, M. E. 1977. Spectral evaluation of young red wines: Anthocyanin equilibria, total phenolics, free and molecular SO2, "chemical age". J. Sci. Fd Agric. 28: 279-287.

Taylor, J. 1989. Colour stability of blackcurrant (Ribes nigrum) juice. J. Sci. Fd Agric. 49: 487.


85

Wrolstad, R.E. 1976. Color and pigment analysis in fruit products. Bull. 624, Oregon Agric. Exp. Stn., Corvallis, Ore.

Wrolstad, R. E., Putnam, T. P., And Varseveld, G. W. 1970. Color quality of frozen strawberries: effect of anthocyanin, pH, total acidity, and ascorbic acid availability. J. Food Sci. 35: 448.

Wrolstad, R.E., Lee, D. D., and Poei, M. S. 1980. Effect of microwave blanching on the color and composition of strawberry concentrate. J. Food Sci. 45:1573.

Wrolstad, R.E., Skrede, G., Lea, P. and Enersen, G. 1990. Influence of sugar on anthocyanin pigment stability in frozen strawberries. J. Food Sci. 55 (4): 1064- 1065.

86

CHAPTERS

COMPARISON OF THE STABILITY OF PELARGONIDIN-BASED ANTHOCYANINS IN STRAWBERRY JUICE AND CONCENTRATE



87

ABSTRACT

Pelargonidin 3-glucoside, pelargonidin 3-sophoroside, and acylated pelargonidin

3-sophoroside 5-glucoside were used to fortify strawberry juice (8 0brix) and concentrate

(65 0brix). Changes in pigment, color, and ascorbic acid were monitored during storage

at 25 0brix. Anthocyanin and ascorbic acid degradation followed first order kinetics.

Fortification increased the half life of the pigments from 3.5 to 5 days in concentrate and

from 5 to 12 days in juice. Half life of ascorbic acid was 2 days in juice samples and

ranged from 3 to 10 days in concentrate samples. Both systems showed variation in

chroma and hue angle, but maintained L* values.

Key words: Anthocyanin, pigment, strawberry juice, strawberry concentrate,

fortification, ascorbic acid, kinetics, stability.

INTRODUCTION

Pelargonidin 3-glucoside (pgd 3-glu) is the anthocyanin (ACN) responsible for

the appealing, bright, red color of fresh strawberries. Attractive color is a very important

organoleptic characteristic for the consumer of fruits and the processed food products

derived from them. Unfortunately, the attractive color of the fresh strawberries does not

prevail through processing and storage operations; instead, a dull, brownish pigment

often appears, which is perceived by the consumer as inferior quality and deterioration of

the fruit. This color instability has a negative effect on the market potential for processed

strawberry products.

The stability of the anthocyanins (ACNS) in food systems is influenced by factors

such as the chemical structure of the pigment. Hydroxyl, methoxyl, sugar and acylated

sugar substituent groups have pronounced effects on stability of the ACNS (Mazza and

Miniati, 1993). Diglycosidic substitution is reported to give more stability to the

molecule than monoglycosidic substitution (Markakis, 1982; Mazza and Miniati, 1993);

substitution with the disaccharide sophorose was proposed to be key factor contributing

to the better color stability of red raspberry products as compared to those processed

88

from strawberries and blackberries (Boyles and Wrolstad, 1993; Rommel et al., 1990;

Rommel et aL, 1992). Acylation of the molecule is believed to improve stability by

preventing it from hydration (Asen et aL, 1977; Brouillard, 1981; Goto et al., 1979;

Hoshino et al, 1980). Guisti and Wrolstad (1996a) reported high color and pigment

stability when coloring secondary bleached cherries with radish anthocyanin extract

(RAE). The high stability of RAE was attributed to the nature of its major ACN;

pelargonidin 3-sophoroside 5-glucoside acylated with malonic and ferulic acids.

Total ACN concentration and ascorbic acid are also believed to be major factors

influencing the stability of the ACNS (Meschter, 1953; Markalds et al., 1957; Sonheimer

and Kertesz, 1953 Poei-Langston and Wrolstad, 1979). Poei-Langston and Wrolstad,

(1981) observed that addition of ascorbic acid to model systems containing pgd 3-glu

produced loss of pigment stability. Skrede (1982) and Skrede et al.(1992) demonstrated

the effects of ACN and ascorbic acid content in strawberry jams and syrups. Fortification

of syrups with pgd 3-glu caused a decrease in the rate of pigment degradation while

fortification of syrups and jams with ascorbic acid caused a decrease in pigment stability.

Additional research has aimed to determine the factors responsible for color

degradation in other specific strawberry products such as preserves (Kertesz and

Sondheimer, 1948; Mackinney and Chichester, 1952; Abers and Wrolstad, 1979), frozen

strawberries (Wrolstad et aL, 1970), freeze-dried strawberries (Erlandson and Wrolstad,

1972), strawberry juice and concentrate (Wrolstad et aL, 1980) and strawberry wine

(Pilando et al., 1985).

The main objective of this study was to test the effect of ACN fortification on the

color and pigment stability of strawberry juice and concentrate. An additional objective

was to determine if increased glycosidic substitution or acylation of glycosides improved

pigment stability. A final aim was to study the changes in ascorbic acid during storage of

these systems and determine its relationship to ACN degradation.

89


Processing of juice and concentrate

Figure 5.1 shows the unit operations for the processing of strawberry juice and

concentrate. 30 Kg of individually quick frozen (I.Q.F) Totem strawberries obtained

from Conroy Packing Inc. (Salem, OR) were thawed overnight at room temperature.

The fruit was crushed using a hammermill (model D Comminuting machine, W. J

Fitzpatrick Co., Chicago, IL) equipped with a circular pore mesh, 1.27 cm in diameter at

a speed of 182 rpm. The pulp was depectinized in a steam jacketed kettle at 50 "C for 2

hr by adding 3 mL pectinase (RAPIDASE SUPER BE, Gist-brocades laboratories.

Charlotte, NC) per Kg. A negative alcohol precipitation test (1:1 juicerisopropanol +1%

HC1) was applied to monitor completion of depectination. Pressing of the pulp was done

at a final pressure of 3 bars for 30 min in a Willmes press bag (Type 60, Moffet Co., San

Jose, CA), one per cent rice hulls was used as press aid. Filtering was done at 2 PSI

with the addition of 2% diatomaceous earth in a multipad filtration unit (Herman

Strassburger K G. Westhofen BEP Worms, Germany). The unit was equipped with a

pad filter SWK supra 2600 (Scott Laboratories, Inc., San Rafael, CA).

The filtered juice was pasteurized by high temperature-short time treatment

(HTST) at 85-90 0C for 90 sec in a tubular heater with screws (Wingear type, model

200WU, Winsmith Co., Springville, NY) and divided into two portions. One portion was

used as single strength (8 0brix) and the other was concentrated to 65 0brix in a

Centritherm (Alfa Laval type CT-1B, Sweden), where the juice reached a final

temperature of approximately 80 0C. All processing steps were duplicated. Concentrate

samples were diluted to 8 0brix with distilled water before further analysis.

Compositional analysis

Total titratable acidity of the juice and concentrate was determined as grams

citric acid per 100 mL by titrating the juice with 0.1 N NaOH to pH 8.2. Measurement

of the pH was done with a Coming pH meter, model 340 (Coming Inc., NY, USA) and

soluble solids were determined on an Auto Abbe refractometer 10500 (Reichert-Jung,

90

Thaw 30 Kg I.Q.F. strawberries

Crush with hammermill

Depectinize with 'Rapidase Super BE' (30 mL)

Incubate (50 0C-2hr)

Alcohol test for pectin (incubate further if positive)

Add 1% rice hulls as press aid

Press at 3 bars for 30 min

Add 2% diatomaceous earth, pad-filter (SWK 'Supra 2600')

Filter at 4 PSI

HTST pasteurization (90 0C for 15-30 sec)

Divide in two portions

Portion 2-concentrate 650brix

Figure 5.1. Unit operations for the production of strawberry juice and concentrate.

91

Leica Inc., NY, USA). The instrument was used under the % soluble solids with

temperature compensate mode.

Pigment extraction, isolation, and purification

ACNS from strawberries (Fragaria anannassa cv, Totem), the petals of orange-

colored nasturtium flowers {Tropaeolum majus), and radish peel (Raphanus sativus L

cv, Fuego) were extracted and isolated as described by Guisti and Wrolstad (1996).

Frozen tissue was powdered in a stainless steel Waring blender upon addition of liquid

nitrogen. ACNS were then extracted with acetone (2 L/Kg powder) and separated from

the cake by filtration on a Buchner funnel; the cake residue was totally reextracted with

aqueous acetone (30:70 v/v). ACNS were partitioned with chloroform (1:2 acetone:

chloroform v/v). The aqueous portion was recovered and separated from the solvents by

rotoevaporation at 40 0C and the ACNS were purified subsequently using a C-18

cartridge (high load C-18 tube), 20 mL capacity (Alltech Assoc, Inc., IL). The cartridge

was washed with 0.01% HC1 upon loading with pigment, and the ACNS were eluted

with methanol acidified with 0.01% HC1 and then concentrated by rotoevaporation.

Purity of the three ACN fractions was checked by analytical HPLC.

Separation and collection of pelargonidin derivatives

Pgd 3-glu from strawberries, pgd 3-soph from nasturtium flowers and pgd 3-

soph 5-glu acylated with cinnamic and malonic acids from radish peel were separated via

preparatory HPLC using a Dynamax SD-300 (Raining Instrument Company, Inc.

Wobum, MA) chromatograph equipped with a Hewlett-Packard 1040A photodiode

array detector and a Hewlett-Packard 9000 computer. The column used for separation

was a Supelcosil PLC-18 (25 cm x 21.2 mm). Three systems were used for the mobile

phase as follows:

System I used for collection of Pgd 3-glu: (A) 12% acetonitrile, 10% acetic acid,

1% phosphoric acid. Isocratic conditions. Flow rate 20 mL / min, injection volume 1 mL.

Primary detection at 520 mn.

92

System II for collection of Pgd 3-soph: (A) 8% acetonitrile, 10% acetic acid, 1%

phosphoric acid. Isocratic conditions. Flow rate 20 mL / min, injection volume 1 mL.

Primary detection at 520 nm.

System III for collection of Pgd 3-soph 5-glu acylated: (A) 18% acetonitrile,

10% acetic acid, 1% phosphoric acid. Isocratic conditions. Flow rate 20 mL / min,

injection volume 1 mL. Primary detection at 520 nm.

Experimental design

Juice and concentrate fortification

Determination of the parameters characterizing the strawberry juice (8 "brix) and

concentrate (65 "brix) was performed before fortification; all samples were diluted to

8 "brix before measurements). Total monomeric ACN content (mg/L), titratable acidity

(% citric acid), pH, ascorbic acid content (ppm), color indices, and haze were

determined. Subsequently, fortification with the individual ACNS to a level equivalent to

twice the initial concentration of the pigment present in the strawberry juice and

concentrate respectively was done. The pH of the pigments was previously adjusted to

3.4 with IN NaOH to avoid changes in the pH of the juice or concentrate. Total

monomeric ACN, ascorbic acid content, pH, and color indices were measured again.

Each rephcate of juice and concentrate was divided into four samples: sample 1

was used as a control, sample 2 was spiked with pgd 3-glu, sample 3 was spiked with

pgd 3-soph and sample 4 was spiked with acylated pgd 3-soph 5-glu. Potassium sorbate

(0.1% w/w) and sodium benzoate (0.1% w/w) were added to each sample for

preservation purposes. For the juice samples, aliquots were transferred to 12-mL vials,

screwcapped, pasteurized at 90 0C for 1 min, and subsequently stored in the dark at

25 0C for 26 days. For the concentrate, aliquots were transferred to 2-mL vials,

screwcapped, and pasteurized under the same conditions before storage in the dark at

25 0C for 6 days.

93

Pigment analysis

The methods described by Somers and Evans (1977) were applied for

determination of color density (A 420nm + A noaJ* % polymeric color ((polymeric

color/color density) X 100), and browning (A420jm after bisulfite bleaching). Monomeric

ACN content was determined by the pH differential method as described by Wrolstad

(1976). The pigment concentration was expressed as mg pgd 3-glu/L with a molar

absorptivity of 22,400. All determinations were made on a Shimadzu UV visible

spectrophotometer, model UV 160 U, using 1-cm pathlength disposable cells.

Color and haze analysis

The method described by Guisti (1996a) was used. Hunter L*, a*, b* values, hue 2 2

angle (arctan b*/a*), chroma (a* + b* ), and % haze were measured in a ColorQUEST

Hunter colorimeter (Hunterlab, Hunter Associates Laboratories Inc., Reston, VA).

Samples diluted at 2 0brix with distilled water were placed in a 0.5 cm-path length optical

glass cell (Helma, Germany), and measurements were made in the transmittance mode

with a CIE observer angle of 10 degrees, illuminant C.

Analytical HPLC

ACN pigment composition was monitored by HPLC using a ODS C -18 column

(5 (i) 25 cm x 4.6 mm (PolyLC Inc., Columbia, MD). Conditions: Solvent A: 100%

acetonitrile, B: 5% acetonitrile, 10% acetic acid, 1% phosphoric acid. Linear gradient

from 2% to 13% A in 15 min, 13% to 15% A in 5 min, holding at 15% A for 15 min.

Elution at 1 mL/min, Injection volume 50 |iL, primary detection at 520 nm. UV-vis

spectra were taken directly from chromatographic runs.

Ascorbic add analysis

A 5 mL-sample at 8 0brix was loaded into an activated Sep-Pak C18 cartridge

(Alltech Assoc, Inc., EL). The first portion of eluate (2 mL) was discarded and the

94

remaining 3 mL collected. The collected portion of eluate was diluted to 6 ml with

deionized water and its pH was adjusted to 5-5.2 with NaOH 0.1N. 0.1% 1,4-

dithiothreitol (DTT) was added to each sample to keep ascorbic acid in the reduced

form. After two hour-reaction with DTT, samples were filtered through a 0.45-fim

millipore filter (type HA) and analyzed by HPLC.

HPLC analysis of total ascorbic acid was carried out using a Sperisorb ODS-2

column (5 micron) 250 mm x 4.6 mm (AHtech Associates, Inc., IL). Conditions: 0.5%

KH2P04 pH 2.5 with 0.1% DTT. Isocratic program for 20 min. Elution at 0.5

mL/min, Injection volume 50 [ih, detection at 260 nm.

For quantitation of ascorbic acid in the samples, a standard curve with three

concentrations of ascorbic acid was developed. Solutions with 10 ppm, 30 ppm and

50 ppm ascorbic acid to which 0.1% DTT had been added, were injected in the HPLC

following the same procedure applied to the samples. linear regression analysis was

applied to determine the ascorbic acid concentration in the juice and concentrate

samples.

Statistical analysis

Two replications of this study were performed. For statistical analysis

multifactor analysis of variance (ANOVA) was applied with sources of variance being

storage time, "brix and pigment. Significant (P < 0.05) differences between means

were identified using the least significant difference procedure (LSD). The analyses

were performed with Statgraphics plus, version 2.1.

The ACN and ascorbic acid concentrations were plotted against storage time. Linear

regression analysis was used to determine adequacy of the model describing kinetics

of destruction. Rate constants were calculated from slopes of the lines plotted, and

half lives (t1/2) were calculated from the equation: t1/2= Ln 0.5/k, where

k = rate constant.

95


Juice and concentrate prior to storage

The compositional data and color indices of the juice and concentrate

immediately after production are shown in table 5.1. The reported values are means of

three determinations and have been normalized to 8 "brix for comparative purposes

between juice and concentrate. The ascorbic acid content, which ranged from 34.2

mg/100 g to 48.8 mg/100 g juice was in agreement with previous findings. Pilando et aL

(1985) reported 43.8 mg ascorbic acid/100 g Totem strawberries. Concentration did not

have a pronounced effect on pH and acidity of the products; however, a reduction of

approximately 25% of the initial ascorbic acid was detected. This destruction of ascorbic

acid was expected as vitamin C is considerably affected during processing due to its

tendency to react with oxygen forming dehydroascorbic acid and further degradation

products. Such reaction is accelerated by temperature (Bode et al, 1990; Angberg et al,

1993). The changes observed during the concentration process are consistent with the

results reported by Rwabahizi (1988) for strawberry juice and concentrate made from

Benton strawberries.

The monomeric ACN content of the juice ranged from 268 to 290 mg/L. This

results are close to the ones reported by Pilando et al.(1985) and Bakker et al(1994).

They found a concentration of 258 mg/L and 333 mg/L of ACN in juice made of fully

ripe Totem strawberries. The concentration process caused reduction of the ACN, but

not a pronounced effect on Hunter L* value or percentage haze. A marked drop in

chroma was observed, which indicates reduction in color intensity. Hue angle was higher

after concentration indicating shift from red towards orange. Reduction of ACN content

in strawberry juice upon heating has been observed previously (Rwabahizi and Wrolstad,

1988; Bakker at al., 1992).

Additional changes due to pasteurization of the fortified samples are listed on

table 5.2. Decrease of ascorbic acid concentration ranged from 24 % to 44 % in fortified

juices and from 8 % to 22 % in fortified concentrates. No drop in ascorbic acid was

observed in the concentrate spiked with acylated pgd-soph 5-glu. A decrease in L* value

was observed upon fortification of all the samples, indicating a darkening effect.

96

Table 5.1. Compositional and color indices of strawberry juice and concentrate before fortification with pelargonidin derivatives.

Juice Concentrate-

Parameter Batch 1 Batch 2 Batch 1 Batch 2

Monomeric ACN (mg/L)a 268 290 210 227

T A (% citric acid) a 0.80 0.83 0.77 0.85

pH* 3.48 3.27 3.18 3.18 0Brix 8.06 8.25 75 69

Ascorbic acid (ppm) a 342 488 246 381

CIEL*b 87.9 88.3 90.2 90.0

CIEa*b 14.9 14.5 9.64 10.4

CIEb*b 11.7 11.4 9.19 9.39

Chroma b 18.0 18.4 13.3 14.0

Hue angle (degrees) 37.9 38.2 43.6 42.2

Haze (%) b 1.20 1.19 1.79 1.8

a Parameters measured on single-strength basis (8 °brix).

b Parameters measured on 2 "brix basis.

This effect was more evident in the juice samples spiked with pgd 3-glu and 3-soph. As

expected, chroma increased considerable due to spiking as the concentration of the

monomeric ACN was doubled, which is in accordance with Bakker et al. (1994) who found

good correlation between pigment content and color intensity of strawberry juice. Hue

angle remained almost constant and haze values were higher after fortification and

pasteurization.

Table 5.2. Compositional and color indices of strawberry juice and concentrate after fortification with pgd derivatives

Parameter

Sample Totala Ascorbic a CIE L* b CIE a* b CIE b* b Chroma b Hue angle b Haze b anthocyanin acid

(mg/L) (ppm)

Juice + pgd 3-glu

Juice + pgd 3-soph

Juice + acyl-pgd 3-soph 5-glu

Concentrate + pgd 3-glu

Concentrate + pgd 3-soph

Concentrate + acyl-pgd 3-soph 5-glu

516

(4.80)

486

(29.8)

473

(26.2)

453

(8.09)

477

(7.28)

502

(2.38)

275

(63.0)

322

(65.5)

315

(73.6)

246

(53.2)

289

(46.8)

314

(10.6)

69.1

(8.21)

80.3

(0.31)

87.1

(0.61)

84.3

(0.72)

81.6

(1.39)

86.3

(0.42)

24.9

(3.69)

25.8

(0.11) 16.0

(0.69)

17.2

(1.97)

23.4

(2.57)

16.4

(0.82)

19.2

(3.54)

19.1

(0.20)

8.80

(0.13)

18.0

(1.59)

21.2

(1.94)

13.3

(0.51)

31.5

(5.09)

32.1

(0.20)

18.6

(0.67)

24.9

(2.51)

31.6

(3.21)

21.1

(0.96)

37.5

(1.01)

36.3

(0.17)

28.6

(0.67)

46.3

(0.74)

42.3

(0.54)

39.1

(0.34)

7.47

(0.14)

1.65

(0.02)

1.97

(0.10)

7.02

(1.03)

2.01

(0.06)

1.95

(0.16)

Values in parenthesis represent standard errors of two replicates

a Parameters measured on single-strength basis (8 °brix)

b Parameters measured on 2 "brix basis

98

Changes of total anthocyanin with time

The initial ACN concentration in the fortified samples is presented in table 5.2.

Figures 5.2 and 5.3 report changes in pigment in strawberry juice and concentrate during

storage. In both systems, the monomeric pigment decreased, but this decrease was

significantly higher in the concentrate samples (p-value < 0.01).

Linear regression analysis confirmed that degradation of the juice and

concentrate followed first-order kinetics (p-values < 0.01). These results, which show

that the rate of the degradation is directly proportional to the concentration of the

pigments, agree with previous studies reporting first order kinetics for ACNS present in

black raspberry (Daravingas and Cain, 1968), sour cherry (Cemeroglu et al., 1994),

radish (Guisti and Wrolstad, 1996a), concord grape, red cabbage, and ajuga ACNS

(Baublis et al., 1994). Juice samples were significantly more stable than concentrate

samples (p-values < 0.01). The higher destruction of ACN in the concentrate is

confirmed in table 5.3 which shows the ACN degradation parameters found by linear

regression analysis. The half life (t^) of the juice samples was significantly higher than

the tm of the concentrate samples and, as expected, the rate of degradation of the juice

samples was lower (p-value < 0.01). The change in the ACN concentration is described

by the equation: Ln (C/C0) = -Bi (time) + Bo. Where C/Co = monomeric ACN

concentration at any time/initial monomeric ACN concentration. Bo = intercept, and Bi =

slope.

Fortification with ACN increased t1/2from 8 to 12 days in juice and from 3.5 to 5

days in concentrate. No difference in remaining ACN, t1/2 or rate of degradation was

found among the spiked juice samples or the spiked concentrate samples respectively (p-

values > 0.1). Our results for unfortified juice are in accordance with previous findings;

Wrolstad, et al. (1980) reported a t1/2 of 5 days for strawberry juice whose initial ACN

concentration was about half of the concentration in our juice. Similarly, fortification of

strawberry syrup with strawberry ACN to a level comparable to the one in blackcurrant

syrup improved ACN stability Skrede et al (1992); the tm of the strawberry syrup

increased from 27 days to 66 while the tj^ of the blackcurrant syrup was of 60 days.

They observed that although cyanidin 3-rutinoside is the major ACN present in

600

,^-,„

Juice

Juice + pgd 3-glu

Juice + pgd 3-soph


1 \ 1 1 1 1 1 1 1 1 1 r 0 2 4 6 8 10 12 14 16 18 20 22 24 26

Days

I* O O o o

•8 1 o

AH

i—I—I—i—i—i—i—r 10 12 14 16 18 20 22 24 26

Days

Figure 5.2. Changes in pigment during storage of juice systems

600

-*- Juice

-©- Juice + pgd 3-glu

-1^- Juice + pgd 3-soph

—♦— Juice + acyl-pgd 3-soph 5 -glu

-i—i—i—i—i—i—r 10 12 14 16 18 20 22 24 26

Days

i—i—i—i—i—i—i—i—i—i—r 0 2 4 6 8 10 12 14 16 18 20 22 24 26

Days

Figure 5.2. Changes in pigment during storage of juice systems

o o

101

Table 5.3 Linear regression parameters of monomeric anthocyanin degradation in juice and concentrate systems

System

Juice

Juice + pgd 3-glu

Juice + pgd 3-soph


Concentrate



Concentrate + acyl pgd 3-soph 5-glu -0.094 -0.15 " 0.94 5 (3.6 xlO"2) (1.2 xlO"2) (019)

Values in parenthesis are standard errors of 2 replicates.

blackcurrant, no difference in the stability of this pigment and the stability of pgd 3-glu

was observed after spiking. In that case, as well as in ours, the difference in stability that

has been observed for different types of ACNS (Markakis, 1982) showed to be less

important than the total ACN concentration.

The initial polymeric color was higher in the concentrate samples. This was

expected after manipulation and heating during the concentration process. It has been

Bo Bi r2

Rate of degradation t,/2

(days') (days)

-0.103 -0.079 0.95 8

(7.3 x 10-2) (4.7 x lO"3) (0.82)

-0.036 -0.057 0.96 12

(4.7 x 10-2) (3.1 x lO"3) (1-23)

-0.069 -0.054 0.97 12

(3.8 x 10-2) (2.4 x lO"3) (0.69)

-0.093 -0.052 0.94 12

(5 x 10-2) (3.2 x lO"3) (1.01)

-0.19 -0.21 0.87 3.5

(8 x lO-2) (2.6 x lO'2) (5.7 x lO'2)

-0.14 -0.20 0.93 4

(5.4 x lO"2) (1.8 x lO"2) (0.12)

-0.13 -0.17 0.92 5

(4.7 x lO'2) (1.6 x lO'2) (7.3 x lO'2)

102

demonstrated that temperature is the most important factor changing the kinetics of

color degradation in strawberry products (Meschter, 1953). Polymeric pigment (%)

increased with storage in all the samples and the % polymeric color at the end of storage

was not different when comparing the juice and the concentrate systems (p-value > 0.1).

In addition, no significant difference was detected between the control and the spiked

samples for any of the systems (p-values > 0.1). Our findings confirm reports of increase

in polymeric color during storage of strawberry juice (Rwabahizi and Wrolstad, 1988),

strawberry wine (Pilando et al, 1985), and strawberry preserves (Abers and Wrolstad,

1979)

Changes in color indices with storage

Figures 5.4 and 5.5 show changes in hue angle, chroma, and Hunter L* value

for the juice and concentrate samples respectively. All the samples presented loss of

redness during storage; this caused a change in hue angle. The juice samples showed a

significant increase of this parameter (p-value < 0.01), indicating a shift from red

towards orange. This change was more pronounced in the control and the sample

fortified with acylated pgd 3-soph 5-glu. Significant increase in hue angle was also

observed in the concentrate samples (p-value < 0.01); again, these changes were less

dramatic for the samples fortified with pgd 3-glu and pgd 3-soph. Significant decrease

in chroma was observed during storage of juice systems (p-value 0.03), but time did not

cause significant changes in the color intensity of concentrate systems (p-value 0.4).

These results are partially comparable with the results reported by previous researchers.

Skrede et al (1992) observed increase in hue angle during storage of strawberry and

blackcurrant syrups. The rate of change was similar in unmodified strawberry syrup and

blackcurrant syrup while strawberry syrup fortified with pgd 3-glu showed the smallest

increase. Rwabahizi and Wrolstad (1988) reported increase in hue angle and a

considerable drop in chroma after storage of strawberry concentrate. The degree of

lightness remained almost constant during storage of all samples. L* value increased

slightly, but not significantly (p-values 0.2).

Hue

ang

le (

degr

ees'

Chr

oma

CIE

L*

8 era B *>. n i era CD

on

B"

o o

1=

ts)

W

4^.

Lt

\

ON

-J

C

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o

o

o

o

o

o

(JO

o

w> -

p

&)

rra

CD

o t. o en

OS CD

M

to

S ©

M

_

O -

f

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: : <—(

+ 3.

3.

3.

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CD

m

o

CP

+

+ +

■a

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►o

era

era

era

CL

CL

a.

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en

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c*

CT

cr

C-ft

era

cf

p

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*<

1—

£

i

o

104

*

s o

o

Concentrate



Concentrate + acyl-pgd 3-soph 5-glu

00 c

Figure 5.5. Changes in color during storage of concentrate systems.

105

Changes in haze

All the samples showed an increase in haze through storage (figure 5.6), but

higher variation was observed in the juice samples and specially in the one fortified with

pgd 3-glu, which was statistically different from the others (p-value < 0.01). Similarly,

the concentrate fortified with pgd 3-glu showed a great increase in haze as compared to

the other spiked samples (p-value < 0.01).

Rwabahizi and Wrolstad, 1988; Rommel et al., 1990 have reported a strong

correlation between % polymeric color and haze formation for a number of ACN-

containing juices e.g., raspberry. An increase in % polymeric color was observed when

NaOH was added to purified pgd 3-glu before spiking; therefore, higher haze content

was detected in samples fortified with this ACN.

Changes in anthocyanin pigment profiles during storage

The HPLC profile of the ACNS in the unspiked juice and spiked juice samples

before storage is presented in figure 5.7. The peaks have been previously identified as (1)

cyanidin 3-glucoside (cyn 3-glu) (Timberlake and Bridle, 1982); (2) pgd 3-glu

(Harbome, 1958); (3) pgd 3-rutinoside (Bakker et al., 1994); (4) pgd 3-glu acylated with

succinic acid-isomer 1 (Garzdn and Wrolstad 1997); (5) pgd 3-glu acylated with succinic

acid-isomer 2 (Garzon and Wrolstad 1997) (6) pgd 3-soph (Garzdn and Wrolstad 1997);

(7) pgd 3-soph 5-glu acylated with/7-coumaric, ferulic and malonic acids (Guisti and

Wrolstad, 1996b); (8) pgd 3-soph 5-glu acylated withp-coumaric acid (Guisti and

Wrolstad, 1996b); (9) pgd 3-soph 5-glu acylated with ferulic acid (Guisti and Wrolstad,

1996). Changes in relative peak area during storage of the juice and concentrate samples

are reported in figures 5.8 and 5.9. Most of the samples presented decrease in the area of

peaks 4 and 5, which can be explained by the hydrolysis of the succinic acid that acylates

these ACNS. This change, along with an increase in the relative peak area of pgd 3-glu

was more noticeable in the controls. Exceptions were found on the juice spiked with pgd

3-glu in which no change in peaks 4 and 5 was detected. These findings are suggestive of

a protective effect of the ACN that takes place when the concentration of monomeric

pigment is high as in the case of the fortified juice (Skrede et al., 1992; Mazza and

Juice

Juice + pgd 3-glu

Juice + pgd 3-soph

Juice + acyl-pgd 3-soph 5- glu

0 12 Days

16 20 24

•••#•

Concentrate


■""&"" Concentrate + pgd 3-soph

—♦- Concentrate + acyl-pgd 3-soph 5-glu

Figure 5.6. Changes in haze during storage of (a) juice systems and (b) concentrate systems.

o

mWJ 2 6C0- Unfortified juice

500-

400-

300-

200-

1 L3A 5

A in ;'' i'--

A t?i ?!> ??'s ?'■ ?;'s n« - U

WO

6 2 1

Juice + pgd 3-soph

400-

300-

-.:

100-

0- L^ J vL4

5 A

.r,s. ...... .,,,. ■■■.■■■ ..^ It MS X 77i inn

m*U

1000- i Juice + pgd 3-glu

B0O-

800-

<00-

!O0- 4

KO

1 b A 5 /s i.j US S ITS ? Bi !S 17 S ™

2 Juice + acyl ated pgd 3-sop 5-glu

S00

M0-

100-

7

50- 1 I.3 1 J W/^v . | .............. &

mm mm

Figure 5.7. HPLC chromatograms of the anthocyanins in unfortified juice and juice spiked with pelargonidin derivatives before storage. Peak identification: (1) cyanidin 3-glucoside; (2) pelargonidin 3-glucoside; (3) pelargonidin 3-rutinoside; (4) pelargonidin 3-glucoside acylated with succinic acid (isomer 1); (5) pelargonidin 3-glucoside acylated with succinic acid (isomer 2); (6) pelargonidin 3-sophoroside; (7) pelargonidin 3-sophoroside 5-glucoside acylated with cinnamic acid; (8) pelargonidin 3- sophoroside 5-glucoside acylated with p-coumahc acid and malonic acids; (9) pelargonidin 3-sophoroside 5-glucoside acylated with ferulic and malonic acids.

o

pgd 3-glu

100-

80-

60-

..40-

^20-

I 60 50-|

40

30-

20-

10-

0

>>§ Pgd 3-glu + succinic acid

pgd 3-soph 5-glu

■zm

mm pgd 3-soph 5-glu +malonic/cinamic acids

pgd 3-soph 5-glu + cinnamic acids only

pgd 3-soph

0 14

Figure 5.8. Changes in peak area during storage of (a) juice, (b) juice + pelargonidin 3-glucoside, (c) juice + pelargonidin 3- sophoroside, (d) juice + acyl-pelargonidin 3-sophoroside 5-glucoside. o

oo

pgd 3-glu pgd 3-soph pgd 3-soph 5-glu + cinnamic acids

<U

•a <L>

PL,

100-

80-

60-

40 H

20

o- 60-

50-

40-

30-

20-

10

0 0

pgd 3-glu + succinic acid \y/^A pgd 3-soph 5-glu + cinnamic/malonic acids y/y

Fig 5.9. Changes in peak area during storage of (a) concentrate, (b) concentrate + pelargonidin 3-glucoside, (c) concentrate + pelargonidin 3-sophoroside, (d) concentrate + acyl- pelargonidin 3-sophoroside 5-glucoside.

o

110

Miniati, 1993). Such effect does not happen when the ACN is highly polymerized as in

the case of the concentrate

Relative stability of pgd 3-soph appeared to be slightly affected during storage.

The peak area of this ACN did not show changes in the juice sample, while in the

concentrate there was a small decrease suggesting hydrolysis of the diglycoside into the

monosaccharide glucose. Since the overall loss of pgd 3-soph was not significant, the

higher stability of pgd 3-soph seems to confirm earlier reports on increased ACN stability

due to diglycosidic substitution as compared to monoglycosides (Rommel et aL, 1990;

Rommel et aL, 1992).

Juice and concentrate samples fortified with acyl-pgd 3-soph 5-glu presented a

considerable increase in the area of peak 7 corresponding to acyl-pgd 3-soph 5-glu with

only cinnamic acids and a consequent decrease in the proportion of ACN acylated with

both cinnamic and malonic acids (peaks 8 and 9). Negligible amounts of pgd 3-soph 5-

glu originating from cleavage of cinnamic acids were detected in the juice samples.

Suggestion of higher stability of the pigments acylated with cinnamic acids are in good

agreement with results reported by Guisti and Wrolstad (1996a). In model systems

spiked with pgd 3-soph 5-glu acylated with cinnamic and malonic acids they observed

that peak 7 represented 20 % of the total ACNS at the beginning of the storage, but

after 10 weeks, release of malonic acid was evident, and peak 7 became the major ACN

present representing approximately 80%. The stability of ACNS acylated with

aromatic acids has been attributed to the stacking effects of acyl groups above

andbelow the anthocyanidin nucleus, which inhibits the access of water to form the

pseudobase. (Brouillard, 1981; Goto, 1987).

Changes in ascorbic add during storage

Figure 5.10 shows changes in ascorbic acid concentration during storage of juice

and concentrate systems. Storage resulted in a considerable decrease of this acid in all

the samples (p-values < 0.01), except the concentrate control (p-value > 0. 1). After five

days of storage, the degradation of the vitamin in the juice samples was much higher as

compared to the concentrate systems (p-values < 0.01).

400 T

Juice

juice + pgd 3-glu

juice + pgd 3-soph

juice + pgd 3-soph 5-glu acylated

8 10 12 14 16 18 20

Days

Concentrate



Concentrate + pgd 3-soph 5-glu acylated

400

Days

Figure 5.10. Changes in ascorbic acid concentration during storage of (a) juice systems and (b) concentrate systems.

112

Linear regression analysis provided evidence that ascorbic acid degradation

fitted first order kinetics (p-values < 0.01), in accordance with previous findings during

storage of strawberry juice and concentrate (Wrolstad et al, 1980) and model systems

containing pgd 3-glu and ascorbic acid (Markakis et al, 1957).

The linear parameters for the degradation of ascorbic acid in juice and

concentrate systems are reported in table 5.4, which shows the difference in behavior

between the two systems. Fortification did not have an effect on ascorbic acid

degradation in juice samples whose ti/2 was of 2 days. On the other hand, difference

was found in the remaining ascorbic acid and ti/2 of the ascorbic acid present in the

concentrate samples (p-values < 0.01). LSD analysis reported that the ascorbic acid was

more stable in the control (ti/210 days), followed by the concentrate samples fortified

with acylated pgd 3-soph 5-glu (ti/2 7 days), pgd 3-soph (ti/2 6 days), and pgd 3-glu (ti/2

3 days). One possible explanation for the higher stability of ascorbic acid in these

samples is that the portion of concentrate had less monomeric pigment available for

reaction. If the ascorbic acid does complex with ACNS, the concentration of

monomeric pigment was not high enough to cause such high degradation of ascorbic

acid. Jurd (1972) and Poei-Langston, (1981) have proposed a mechanism in which

ascorbic acid condenses directly with ACNS). Another suggestion of condensation

between ascorbic acid and monomeric ACN is the variation of the molar ratio between

these components in the two systems under study. As shown in figure 5.11, at time zero

the ratio values for the controls and the spiked samples, respectively, are very close.

However, ascorbic acid degradation was faster than ACN degradation in juice systems

while the concentrate spiked samples showed degradation rates close to 1:1. Markakis et

al, (1957) found similar results in model systems containing crystalline pgd 3-glu and

ascorbic acid in a citrate buffer. In that case, after 15 hours at 60 0C about 1 molecule of

pigment was destroyed for about every 50 molecules of ascorbic acid. On the contrary,

when ascorbic acid was oxidized with ascorbic acid oxidase, the molar ratio ascorbic

acid: ACN was closer to 1.

Ascorbic acid oxidase and peroxidase are enzymes capable of oxidizing ascorbic

acid. Inactivation of these enzymes from the additional heating process applied during

concentration of the juice may also account for the higher stability

113

Table 5.4. Linear regression parameters of ascorbic acid degradation in juice and concentrate systems

System 80 Bj r tm

Rate of degradation (days') (days)

Juice -0.28 -0.28 0.96 2

(O-1^ (1.8xl0-2) (7.72xl0-2) Juice + pgd 3-glu -0.54 -0.26 0.91 2

(0.28) (2.6 x lO-2) (6.33 x lO'2)

Juice + pgd 3-soph -0.51 -0.26 0.93 2

(0.23) (2.2x10-2) (1.19 xlO"1)

Juice + acyl-pgd 3-soph 5-glu -0.33 -0.29 0.95 2

(0.20) (2.2 x lO"2) (2.21 x lO"1)

Concentrate -0.0057 -0.071 0.58 10

(7.16x10-2) (1.88x10-2) (100)

Concentrate + pgd 3-glu -0.043 -0.27 0.63 3

(0.24) (6.4x10-2) (9.19 xlO"1)

Concentrate + pgd 3-soph -0.2 -0.10 0.68 7

(8.2 x lO'2) (2.1 x lO'2) (3.86 x iQ-l)

Concentrate + acyl-pgd 3-soph 5-glu -0.19 -0.11 0.70 6

(8.9x10-2) (2.3x10-2) (5.19x10-2)

Values in parenthesis are standard errors of 2 repHcates.

of ascorbic acid in the concentrate samples. Single strength juice reached a final

temperature close to 80 0C at which it remained in the collection vessel until it reached

room temperature (25 0C). Lopez-Serrano and Barcelo (1996) and L6pez-Serrano and

Barcel6 (1997) reported the presence of a partially-heat stable peroxidase isoenzyme

c o o

•4—• C

O a

o CO

o "•§ o o

—'®- Juice

•••#•••• Juice + pgd 3-glu

—jlf— Juice + pgd 3-soph

^ Juice + acyl-pgd 3-soph 5-glu

3.5-

31 1 (a)

2.5- li

2^ : \

"%v\ 0.5-

^^^fcs*^— CH 1 r 1 1 1-~-1 1 T 1— 1 » 1

e CO >-> O o

J3

"o S

o cd o

o u en nj

-2* "o

Concentrate

"'f*" Concentrate + pgd 3-glu

-~^r~ Concentrate + pgd 3-soph ^ Concentrate + acyl -pgd 3-soph 5-

g!2

8 10 12 14 16 18 20 days

Figure 5.11. Changes in molar ratios of ascorbic acid/anthocyanin during storage of (a) juice systems and (b) concentrate systems.

115

from strawberries. According to the authors this isoenzyme, remains active after

canning of strawberry products causing oxidative reactions at low H202 concentrations.

Regression analysis based on the Ln concentration was performed to determine

the extent of correlation between deteriorative ACN reaction and deteriorative ascorbic

acid reaction taking place during storage. The correlation coefficients are reported on

table 5.5.

Table 5.5. Correlation coefficients for Ln anthocyanin concentration and Ln ascorbic acid concentration during storage

System r

Juice 0.96 (< 0.01)

Juice + pgd 3-glu 0.51 (0.09)

Juice + pgd 3-soph 0.48 (0.1)

Juice + acyl pgd 3-soph 5-glu 0.48 (0.1)

Concentrate 0.50 (0.01)

Concentrate + pgd 3-glu 0.77 (<0.01)

Concentrate + pgd 3-soph 0.93 (< 0.01)

Concentrate + acyl pgd 3-soph 5-glu 0.91 (<0.01)

P values in parenthesis.

116

Significant correlation coefficients (p-values < 0.01) were obtained for the

relationships between the two variables in all the fortified concentrate samples and in the

control juice; yet, only suggestive evidence of correlation was obtained for the spiked

juice samples (p-values 0.09-0.1) and the concentrate control (p-value 0.1). These results

confirm previous reports on acceleration of ACN destruction when ascorbic acid is

present in model or natural systems (Markakis et aL, 1957; Sondheimer and Kertesz,

1953; Markakis, 1982; Poei-Langston and Wrolstad, 1981; Skrede et al, 1992).

CONCLUSIONS

This experiment has demonstrated the rate of degradation of ACNS is higher on

strawberry concentrate as compare to strawberry juice. It was also confirmed that

concentration of monomeric pigment increases stability, but there is no cause and effect

relationship between pigment structure and stability when strawberry juice or

concentrate is spiked with pgd-based ACNS. The stability of pgd 3-glu,(the main

pigment in strawberries) was similar to the stability of pgd 3-soph and pgd 3-soph 5-

glucoside acylated with cinnamic and malonic acids when ACN concentration was

adjusted to equivalent amounts. Therefore, the total ACN pigment content needs to be

considered when comparing color stability of different ACNS.

Ascorbic acid seems to be a factor contributing to ACN destruction in natural

systems. Since correlation between ascorbic acid degradation and ACN degradation was

found, further studies would be advised to determine the influence of ascorbic acid on

the degradation of the ACNS under investigation. This can be achieved through model

systems to determine the rate of degradation from logarithmic plots of each one of the

pigments at different concentrations of ascorbic acid.

REFERENCES


117

Angberg, M., Nystrom, C, Castenson, S. 1993. Evaluation of heat-conduction microcalorimetry in pharmaceutical stability studies VH Oxidation of ascorbic acid in aqueous solution. Int. J. Pharm. 90: 19-3.

Asen, Stewart, R.N. and Norris, K.H. 1977. Anthocyanin and pH involved in the color of heavenly blue morning glory. Phytochem. 16:118.

Bakker, J., Bridle, P. and Koopman A. 1992. Strawberry juice colour: The effect of some processing variables on the Stability of Anthocyanins. J. Sci. Fd Agric. 60: 471-476.

Bakker, J., Bridle, P., and Bellworthy, S. J. 1994. Strawberry juice colour: A study of the quantitative and qualitative pigment composition of juices from 39 genotypes. J. Sci Fd Agric. 64: 31-37.

Baublis, A., Spomer, A., and Berber-Jimenez, M. D. 1994. Anthocyanin pigments: comparison of extract stability. J. Food Sci. 59 (6): 1219-1221, 1233.

Bode, A. M., Cunningham, L., and Rose, R. S. 1990 Spontaneous decay of oxidized ascorbic acid (dehydro L-ascorbic acid) evaluated by high-pressure liquid chromatography. Clin. Chem. 36: 1807-1809.

Boyles, M. and Wrolstad, R. E. Anthocyanin composition of red raspberry juice: influences of cultivar, processing, and environmental factors. J. Food Sci. 58: 1135-1141.

Brouillard, R. 1981. Origin of the exceptional color stability of the Zebrina anthocyanin. Phytochem. 20: 143.



Erlandson, J. A. and Wrolstad, R. E. 1972. Degradation of anthocyanins at limited water concentration. J. Food Sci. 37: 592.

Garzon G. A. and Wrolstad, R. E. 1997. Characterization of two pelargonidin-based anthocyanins from strawberry (Fragaria anannassa) and nasturtium flowers (Tropaeolum majus). Unpublished data.

Goto, T. 1987. Structure, stability and color variation of natural anthocyanins. Progress Chem. Org. Nat. Prod. 52: 113-158.

118

Goto, T., Hoshino, J. and Takase, S. 1979. A proposed structure of commelin, a sky- blue anthocyanin complex obtained from the flower petals of commelina. Tetrahedron Lett., p. 2905.




Harbome J. B. Comparative Biochemistry of the Flavonoids. Academic Press, London, UK. 1967.

Harper, K. A Morton, AD., and Rolfe, E. J. 1969. The phenolic compounds of black currant juice and their protective effect on ascorbic acid. 3. The mechanism of ascorbic acid oxidation and its inhibition by flavonoids.. J. Food Tech. 4: 255.

Hoshino, T., Matsumoto, U. and Goto, T. 1980. The stabilizing effect of the acyl group on the copigmentation of acylated ACNS with C-glucosyl flavones. Phytochem. 19: 663.


Kertesz, Z. I., and Sondheimer, E. 1948. To reduce color losses in strawberry preserves. Food Inds. 20: 106.

Mackinney, G. and Chichester, C. O. 1952. Color deterioration in strawberry preserves. Cannerll4(12):13.

Markakis, P. 1982. Stability of anthocyanins in foods, pp. 163-180. In. Anthocyanins as Food Colors. P. Markakis,. (Ed.), Academic Press, New York.

Markakis, P., Livingston, G. E., and Fellers, C. R. 1957. Quantitative aspects of strawberry pigment degradation. Food Res. 22: 117.


Meschter, E. E. 1953. Effects of carbohydrates and other factors on strawberry products. J. Agr. Fd Chem. 1: 754.

119


Poei-Langston, M. S. and Wrolstad, R. E. 1981. Color degradation in an ascorbic acid- anthocyanin-flavanol model system. J. Food Sci. 46(4): 1218-1222, 1236.

Rommel, A., Heatherbell, D. A. and Wrolstad, R. E. 1990. Red raspberry juice and wine: effect of processing and storage on anthocyanin pigment composition, color, and appearance. J. Food Sci. 55 (4): 1011-1017.

Rommel, A., Wrolstad, R. E. and Heatherbell, D. A. 1992. Blackberry juice and wine: Processing and storage effects on anthocyanin composition, color and appearance. J. Food Sci. 57: 385- 391,410.

Rwabahizi, S. and Wrolstad, R. E. 1988. Effects of mold contamination and ultrafiltration on the color stability of strawberry juice and concentrate. J. Food Sci. 53(3) 857-861.


Skrede, G., Wrolstad, R. E., Lea, P., and Enersen, G. 1992. Color stability of strawberry and blackcurrant syrups. J. Food Sci. 57 (1): 172-177.

Somers, T.C. and Evans, M. E. 1977. Spectral evaluation of young red wines: Anthocyanin equilibria, total phenolics, free and molecular SO2, "chemical age".

Sondheimer, E. and Kertesz, Z. I. 1953. Participation of ascorbic acid in the destruction of anthocyanins in strawberry juice and model systems. Food Res. 18: 475.


Wrolstad, R. E. 1976. Color and pigment analysis in fruit products. Bull. 624, Oregon Agric. Exp. Stn., Corvallis, Ore.

Wrolstad, R. E., Lee, D. D., and Poei-Langston, M. S. 1980. Effect of microwave blanching on the color and composition of strawberry concentrate. J. Food Sci. 45: 1573.


120

CHAPTER 6

THE STABILITY OF PELARGONIDIN ANTHOCYANINS AT VARYING WATER ACTIVITY

G. Astrid Garzon and Ronald E. Wrolstad


121

ABSTRACT

The stability of pelargonidin 3-glucoside, pelargonidin 3-sophoroside and

pelargonidin 3-sophoroside 5-glucoside acylated with malonic and cinnamic acids at

varying water activities was determined. Model systems containing purified

anthocyanin in pH 3.4 citrate buffer and glycerol were stored at 25 0C in the dark for

242 days. Changes in pigment, degradation index, and anthocyanin profile as monitored

by HPLC were studied. In general, anthocyanin degradation followed first order

kinetics and the degree of anthocyanin degradation increased with water activity. Half

lives of the anthocyanins ranged from 56 to 934 days. Changes in the chromatographic

profile showed hydrolysis of pelargonidin 3-sophoroside to pelargonidin 3-glucoside

and hydrolysis of malonic acid from the acylated anthocyanin.

Key words: Anthocyanin, pigment, water activity, rate of degradation, half life.

INTRODUCTION

Pelargonidin 3-glucoside (pgd 3-glu), which is the major anthocyanin (ACN)

present in strawberries, is responsible for the attractive, bright red color of this fruit.

Color of food is an important factor influencing consumer's acceptability; consequently,

the loss of this quality reduces the marketability of strawberry products. The short half

life (t1/2) of pgd 3-glu and consequent appearance of undesirable brown substances upon

processing of the fruit has been well documented (Kertesz and Sondheimer, 1948;

Mackinney and Chichester, 1952; Wrolstad et al., 1970; Abers and Wrolstad, 1979;

Erlandson and Wrolstad, 1972; Wrolstad et al., 1980; Pilando et al., 1985; Garzon and

Wrolstad 1977b). It has been concluded from these studies that the prompt hydrolysis of

glucose from the ACN molecule is responsible for the pigment degradation during

processing; in addition, composition of the system can lead to Maillard browning,

enzymic browning, ascorbic acid degradation, and polymerization of reactive phenolics.

All the mentioned reactions can contribute to browning of strawberry products.

Garz6n and Wrolstad (1997b) observed that stability of pgd 3-glu also depends

to a big extend on the composition of the system in which they are present. Strawberry

juice and strawberry concentrate were spiked with the same pelargonidin derivatives at

122

the same concentration level; however, the tj^of the monomeric pigment in strawberry

concentrate ranged from 3.5 to 5 days while in strawberry juice ranged from 8 to 12

days.

The chemical structure of ACNS is hypothesized to be a major factor

influencing the stability of these pigments. ACNS may be glycosylated and acylated by

different sugars and acids. Increase in glycosidic substitution and the presence of

aromatic acyl groups are hypothesized to give more stability to the ACN molecule

(Markakis, 1982; Mazza and Miniati, 1993). The higher color stability of red raspberry

wine as compared to strawberry wine is believed to be due to the diglycosidic nature of

sophorose present in raspberry ACN (Rommel et al, 1990; Rommel et al., 1992).

Acylation of the ACN molecule is reported to improve stability to these pigments (Asen

et al., 1977; Brouillard, 1981; Goto et al., 1979; Hoshino et al., 1980). Studies on color

and pigment stability of cherries colored with radish ACN report a t1/2between 29 and

33 weeks for pelargonidin 3-sophoroside 5-glucoside acylated with cinnamic and

malonic acids (acyl-pgd 3-soph 5-glu), the main ACN present in radish (Guisti and

Wrolstad, 1996a).

Water activity (Aw) is another factor influencing the stability of the ACNS.

Markakis et al (1957) observed that ACNS were stable when stored in dry crystalline

form or on dry paper chromatograms, which resulted in the hypothesis that water was

involved in decoloration reactions. Erlandson and Wrolstad (1972) found that

degradation of ACNS in freeze-dried strawberry puree increased when stored at high

relative humidity conditions; however, Kearsley and Rodriguez (1981) reported that

ACN powder dissolved in different concentrations of glycerol-water solutions were

relatively stable to changes in Aw. Br0nnum, H and Flink (1985) showed that loss of

freeze-dried elderberry ACNS was significant only when Aw values were above 0.51

and Takur and Arya (1989) found that increase in Aw produced loss of grape ACNS

adsorbed onto micro-crystalline cellulose. Studies conducted by Sian and Soleha (1991)

resulted in a slight increase of retention of pineapple and papaya ACNS as the

percentage of glycerol increased from 10% to 20%, but a drop was observed when the

percentage increased from 20% to 30%.

123

Due to the complexity of chemical reactions taking place in natural systems e.g.,

juice, concentrate, jams and jellies, it is difficult to isolate a single factor that explains

changes in ACNS. Therefore, evaluation of a particular parameter requires model

systems in order to closely control the composition and factors influencing pigment

stability.

The objective of this project was to isolate and evaluate the effects of chemical

structure and Aw on the stability of purified pelargonidin ACNS. ACNS with mono,di

and acylated-triglycosidic substitution were evaluated in model systems in which

composition factors could be closely controlled. A second objective was to compare the

stability of the pelargonidin derivatives in model systems with previous findings on

stability in natural systems.


Plant Materials

Individually quick frozen (I.Q.F.) strawberries (Fragaria anannassa cv, Totem)

were supplied by Conroy Packing Inc. (Salem, OR). Orange nasturtimn flowers

(Tropaeolum majus) were collected from local gardens during the summer season

(Corvallis, OR). Stems and leaves were separated from the petals, which were then

frozen with liquid nitrogen. Red radishes {Raphanus sativus L cv, fuego) were obtained

from the local market in Corvallis, OR. The radishes were peeled manually, the peel

was washed and frozen under liquid nitrogen. All frozen materials were stored at -23 0C

until further processing.

Chemicals and reagents

Glycerol was supphed by Mallinckrodt Chemical, Inc (Paris, KY), citric acid

monohydrate was purchased form J. T. Baker (Phillipsburg, NT), sodium citrate was

obtained from Archer Daniels Midland (Faries Parkway, Decatur, IL), sodium benzoate

was purchased from Chemical Works (St Louis, MO), and potassium sorbate was

acquired from Monsanto Company (St Louis, MO).

124


Extraction and isolation of the ACNS from strawberries, nasturtium flowers and

radish peel were performed by the method described by Guisti and Wrolstad (1996a).

The amount of frozen material used for extraction was approximately 2 Kg of frozen

strawberries, 2 Kg of frozen petals, and 250 g of frozen radish peel. Individual samples

were ground to a powdery state with liquid nitrogen using a stainless steel Waring

blender. The powder was blended with acetone (2 L/Kg powder) for extraction of the

ACNS, which were then separated from the cake by filtration on a Buchner funnel.

Total extraction of the pigments from the cake residue was achieved with aqueous

acetone (30:70 v/v). Filtrates were combined and shook in a separatory funnel with

chloroform (1:2 acetone: chloroform v/v) and stored at 10 0C overnight for better

separation between the aqueous and the organic phases.

Anthocyanin purification

The aqueous extract was transferred to a Bucchi rotoevaporator to remove

residual acetone at 40 0C. The ACNS were purified subsequently using a C-18 cartridge

(high load C-18 tube), 20 mL capacity (Alltech Assoc, Inc., IL). The cartridge was

washed with 0.01% HC1 upon loading with pigment, and the ACNS were eluted with

methanol acidified with 0.01% HC1; elimination of methanol and concentration of

pigments were achieved by additional rotoevaporation at 40° C. In all cases, the aqueous

extract was diluted to a known volume with distilled water. Total monomeric ACN

content was determined as mg pgd 3-glu / L with a molar absorptivity of 22,400 and

molecular weight of 433.2 using the pH differential method as described by Wrolstad

(1976). Color density and polymeric color were calculated as the sum of the absorbance

at 420 nm and that at the absorbance maximum of an untreated or of a bisulfite treated

sample, respectively.

Semipreparative HPLC

Separation and collection of pelargonidin derivatives was done using

semipreparatory HPLC techniques. Pgd 3-glu from strawberries, pelargonidin

125

3-sophoroside (pgd 3-soph) from nasturtium flowers and acylated pelargonidin

3-sophoroside 5-gluccoside (acyl-pgd 3-soph 5-glu) from radish peel were obtained

using a Dynamax SD-300 chromatograph (Raining Instrument Company, Inc. Woburn,

MA) equipped with a Hewlett-Packard 1040A photodiode array detector and a Hewlett-

Packard 9000 computer. A reverse phase Supelcosil PLC-18 column (25 cm x 21.2

mm) (Supelco Inc., Bellefonte, PA) was used for preparative isolation. Three different

mobile phases were used for separation:

Mobile phase I used for collection of pgd 3-glu: 12% acetonitrile, 10% acetic

acid, 1% phosphoric acid. Isocratic conditions. Flow rate 20 mL / min., injection

volume 1 mL. Primary detection at 520 mn.

Mobile phase n for collection of pgd 3-soph: 8% acetonitrile, 10% acetic acid,

1% phosphoric acid. Isocratic conditions. Flow rate 20 mL / min., injection volume 1

mL. Primary detection at 520 nm.

Mobile phase m for collection of acyl-pgd 3-soph 5-glu: 18% acetonitrile, 10%

acetic acid, 1% phosphoric acid. Isocratic conditions. Flow rate 20 mL / min., injection

volume 1 mL. Primary detection at 520 nm.

Purity of the three ACN fractions was checked by analytical HPLC.

Analytical HPLC

The HPLC system consisted of a Perkin-Elmer Series 400 Liquid

Chromatograph (Perkin-Elmer Corp., Norwalk, CT) equipped with the detector and

computer described in the semipreparatory HPLC section. The analytical column was a

ODS C -18 column (25 cm x 4.6 mm), 5 |i particle size (PolyLC Inc., Columbia, MD).

The mobile phase composition was: solvent A: 100% acetonitrile, B: 5% acetonitrile,

10% acetic acid, 1% phosphoric acid. Separations were achieved with a linear gradient

from 2% to 13% A in 15 min, 13% to 15% A in 5 min, holding at 15% A for 15 min.

Elution at 1 mL/min., Injection volume 100 |jL, primary detection at 520 nm. UV-vis

spectra was taken directly from chromatographic runs.

126

Experimental design

Preparation of model systems

Model systems used for studies on ACN degradation were prepared at five Aw

levels, which were obtained using glycerol as liquid humectant according to the

proportions listed in table 6.1. Aw levels were calculated according to the method of

Nonish (1966):

LnAw -LnX2 = K2(X1)2

Where: Aw = water activity, X2 = mole fraction of water, Xj = mole fraction of

glycerol, and 1^ = a constant related to the free energy change in a binary system (for

water-glycerol = -0.38).

The calculated Aw values were confirmed by Food Products Laboratories

(Portland, OR) where measurements were carried out in a SC-3 thermocouple

psychrometer (Decagon Devices, Inc., Pullman, WA 99163). The experimental Aw

values were 1.0, 0.90 ± 0.009, 0.89 ± 0.009,0.66 ± 0.025 and 0.44 ± 0.08 as reported in

table 6.1. As described in figure 6.1, a randomized block design with two replicates was

used for each level of pigment; purified ACN ranging from 240-320 mg/L was added to

the buffer/glycerol solutions and 0.1 % (w/w) potassium sorbate and 0.1% (w/w)

sodium benzoate were used as antimicrobial agents. Twenty mL of each preparation

were placed into vials which together with the teflon-lined caps had been be previously

sterilized at 1250C for 15 min. After pasteurization for 30 sec at 850C, the tubes were

placed in a dark room at 250C for storage.

Pigment analysis

Total monomeric ACN was determined as described in the ACN purification

section. The methods described by Somers and Evans (1977) were used to determine

polymeric color, color density and degradation index. Polymeric color (%) was

calculated as the ratio between polymeric color and color density X 100. Degradation

Table 6.1. Composition of model systems used for evaluation of anthocyanin stability at varying Aw

(gm) Citrate buffer Aw

(gm)

ixperimental pH after pigment addition Monomeric anthocyanin Aw (m ig/L)

Model System

pgd 3-glu pgd 3-soph acyl-pgd 3-soph 5-glu


1.0 2.4 2.3 2.5 294 250 251 (0.23) (0.29) (0.12) (2.10) (0.30) (0.00)

0.90 ± 0.009 2.3 2.2 2.4 320 240 255 (0.11) (0.30) (0.09) (7.64) (0.60) (2.40)

0.89 ±0.009 2.5 2.6 2.6 288 251 263 (0.08) (0.04) (0.25) (5.10) (3.00) (2.25)

0.66 ± 0.025 2.4 2.3 2.4 319 241 267 (0.04) (0.23) 0.28) (8.54) (1.65) (7.49)

0.44 ± 0.08 2.3 2.2 2.4 298 263 271 (0.20) (0.07) (0.04) (2.10) (17.99) (1.05)

0 100 1.0

20 80 0.90

32.3 67.7 0.86

70 30 0.63

85 15 0.37

* The values reported are averages of two replicates.

Values in parenthesis are standard errors.

to

128

Prepare 0.1 M pH3.4 citrate buffer *

Portion 1 (replicate 1)

Portion 2 (replicate 2)

Add glycerol to obtain five water activity levels

Add purified pigment

Add 0.1% (w/w) potassium sorbate + 0.1% (w/w) sodium benzoate

Place 20 mL aliquot in presterilized vials

Pasteurize at 85 0C for 30 sec

Store in the dark at 25 0C

^Individual buffers were prepared for addition of individual pigments.

-^-The same procedure was followed for each replicate.

Figure 6.1. Flow chart of the preparation of model systems for evaluation of stability of pelargonidin derivatives at varying Aw-

129

index was calculated as the ratio of the absorbance maximum of the ACN/absorbance at

420 nm. Determinations were made on a Shimadzu UV visible spectrophotometer,

model UV 160 U, using 1cm disposable cells. Previously described analytical HPLC

techniques were applied periodically to monitor changes in chromatographic profile

during storage.

Statistical analysis

The pigment degradation was plotted to represent first order kinetics and linear

regression analysis was used to determine adequacy of the modeL Rate constants were

calculated from slopes of the lines plotted, and half lives (t1/2) were calculated from the

equation: t1/2= Ln 0.5/k, where k = rate constant.

Statistical analysis of variance (ANOVA) was applied. Significant (P < 0.05)

difference between means were identified using the least significant difference

procedure (LSD). The analyses were performed with Statgraphics plus, version 3.


Changes in total anthocyanin pigment during storage at different Aw

Linear regression analysis showed that ACN degradation for the systems

containing pgd 3-glu and pgd 3-soph followed first order kinetics for all Aw levels and

pigments tested. A first order reaction is described by the equation C/Co = exp. (-kt),

where C = pigment concentration at time t; Co = initial pigment concentration; k =

reaction rate constant; t = time. The linear regression model corresponding to this

equation is defined as: Ln (C/Co) = -Bi (time) + Bo. Where C/Co = monomeric ACN

concentration at any time/initial monomeric ACN concentration, Bo = intercept, and Bi

= slope. First order kinetics for degradation of ACNS has been reported on black

raspberry (Daravingas and Cain, 1986), sour cherry (Cemeroglu et al., 1994), concord

grape, red cabbage, and ajuga ACNS (Baublis et al., 1994), radish (Guisti and Wrolstad,

1996a), and strawberry (Garz6n and Wrolstad, 1997b).

130

When linear regression analysis was applied to the data from the samples at high

Aw (1, 0,90, 0.89) to which acyl-pgd 3-soph 5-glu had been added, high correlation

coefficients (r) were obtained. This indicates a relatively strong relationship between the

variables Ln of the ACN concentration and time; however, the lack of fit test was

significant (p-value < 0.01). This, along with some curvature observed on the plot

representing the two variables, suggested that linear regression model describing first

order kinetics was not an adequate model for the observed data. If t1/2 of the ACN

present in these particular samples were calculated based in linear regression analysis,

which involves extrapolation, the real t1/2 of the pigment could be underestimated.

Therefore, comparison of the stability at these particular Aw levels was based on the

time at which 15% of the initial ACN was degraded during storage; this calculation

involves interpolation where reasonable straight lines can be drawn and values are

known. The lack of linearity between the variables Ln ACN concentration and time of

some of our samples is in good agreement with results obtained by Erlandson and

Wrolstad (1972). During the study of degradation of freeze-dried strawberry ACNS

stored at six levels of RH, they found that none of the samples showed first order

kinetics throughout the entire storage time. Our findings along with the results from

Erlandson and Wrolstad (1972) seem to support the hypothesis by Labuza (1980). He

stated that a major effect of modification of Aw on reaction rates could be to change the

order of the reaction, but that unfortunately, most studies on food reactions did not

allow degradation to proceed far enough to discern what the true order was. This is

because most reactions are very slow or significant loss of quality occurs at a low extent

of reaction (Labuza, 1979).

Degradation parameters calculated from linear regression are listed in table 6.2,

6.3 and 6.4. Analysis of the three samples containing acyl-pgd 3-soph 5-glu that did not

follow linearity is reported on table 6.5a.

Table 6.2 shows that the extend of ACN degradation increased with Aw in

model systems containing pgd 3-glu. Reaction rate constants decreased from -3.74 x 10"

^ at Awl to -7.48 x 10"^ at Aw 0.66. In other words, reduction of the Aw from 1 to 0.66

caused a fivefold increase in tj^firom 186 to 934 days for pgd 3-glu. No significant

131

difference in rate of degradation was observed between the sample at Aw 0.44 and the

sample at Aw 0.66; likewise, no difference was detected between the sample at Aw 0.9

and the sample at Aw 0.89 (p- values > 0.1).

Table 6.2. Linear regression and first order reaction parameters for the degradation of model systems containing pelargonidin 3-glucoside

Aw Bo Bi r2 half life (intercept) (slope)

rate of degradation (K) days'

(t*4) days

1 -2.72 x 10-2 -3.74xl0-3a 0.97 186

(1.91 x lO"2) (1.62 xlO'4) (14.4)

0.90 -2.74 x 10-2 -2.08 x 10-3b 0.95 332

(1.37 x lO-2) (1.16 xlO"4) (2.44)

0.89 -1.28x10-2 -2.49 x 10-3b 0.93 278

(1.99 x lO'2) (1.68 x 10-4) (5.64)

0.66 3.41 x lO"3 -7.48 x 10-4d 0.97 934

(3.74 x lO"3) (3.15 xlO"5) (83.9)

0.44 1.27x10-2 -1.15xl0-3d 0.77 690

(1.84x10-2) (1.55 x lO"4) (234.9)

Values in parenthesis are standard errors of two duplicates.

Means in the same column with unlike superscripts indicate significant difference (p value < 0.05).

As shown in figure 6.2a, there seemed to be an increase in the rate of

degradation of monomeric ACN for the sample at Aw 0.44 at latter stages of the study.

Changes in monomeric pgd 3-glu presented in figure 6.2a are in accordance with

changes in polymeric color during storage (figure 2b). Model systems at Aw 1

developed more polymeric pigment as compared to samples with other Aw levels

132

350

0

-m~ Awl

~*~ Aw 0.90

Aw 0.89 -a- Aw 0.44

Aw 0.66

i 1 1 1 1 1 r 60 90 120 150 180 210 240

Days

Figure 6.2. Changes in (a) monomeric anthocyanin and (b) polymeric color (%) during storage of model systems containing pelargonidin 3-glucoside.

Means in the same column with unlike superscripts indicate significant difference (p-value < 0.05)

133

(p-values < 0.01). Although the sample at Aw 0.44 showed an increase in polymeric

pigment at the end of the storage this sample along with the one at Aw 0.66 presented

the lowest amount of polymers. No difference in % polymeric color was found among

samples at Aw < 1 (p- values > 0.1). Similar trends were observed with regard to effects

of Aw on the rate of ACN degradation for the model systems containing pgd 3-soph.

Increase in Aw caused increase in ACN, degradation. As reported on table 6.3, a

threefold increase in t1/2of pgd 3-soph from 232 to 733 days was observed. These

numbers correspond to rates of degradation ranging from -3.02 x 10"^ at Aw 1 to

9.46 x 10-^ at Aw 0.66. Samples at Aw 0.90 and Aw 0.89 did not present significant

variation (p-value > 0.1). Figure 6.3a shows an increased drop in monomeric ACN

concentration at the end of the storage of the sample at Aw 0.44. Consequently, no

significant difference was found between this sample and the one corresponding to Aw

0.9 (p-value > 0.1).

Changes in polymeric color in systems containing pgd 3-soph are shown in

figure 6.3b. Formation of polymers increased correspondingly with ACN pigment

degradation. The highest % of polymers was found in the sample at Aw 1, which was

significantly different from the others (p-value < 0.01). Samples with close levels of Aw

did not present differences in the amount of polymeric pigment e.g., Aw 0.89 and 0.9,

Aw 0.66 and 0.44 (p-values < 0.1). Under the conditions of our experiment, the model

systems containing pgd 3-glu and pgd 3-soph presented very similar trends during

storage. The highest retention of monomeric ACN for both samples was observed at Aw

0.66. However, there was no significant difference on the rate of degradation between

the samples at Aw 0.66 and Aw 0.44 (p-value > 0.1), which indicates higher stability at

lower Aw. The monomeric pigment in both systems presented the highest rate of

degradation at Aw 1 and significant variation was found when comparing all the

samples to the one at Aw 1 (p-value < 0.01). The similar behavior between these two

systems was attributed to their close molecular structure. Only one additional glucose

molecule attached to the aglycon is the difference between pgd 3-glu and pgd 3-soph.

134

Table 6.3. Linear regression and first order reaction parameters for the degradation of model systems containing pgd 3-soph

Aw Bo (intercept)

Bi (slope)

rate of degradation (K)

(days-i)

2 r ha

(half life) (days)

1 -2.66 x 10-2

(2.01 x 10-2)

-3.02 x 10-3a

(1.74 xlO"4)

0.95 232 (24.3)

0.90 -1.18x10-2

(9.63 x 10-3)

-1.67xl0-3b

(8.11x10-5)

0.96 416 (1.78)

0.89 -4.43 x 10-2 (1.56x10-2)

-2.02 x 10-3b

(1.31x lO'4)

0.94 345 (23.2)

0.66 8.78 x 10-3

(5.29 x 10-3) -9.46 x lO'40

(4.46 x lO'5)

0.97 733 (7.47)

0.44 -5.14x10-3

(2.28 x lO-2)

-1.39 x 10-3b c

(1.92K lO'4)

0.77 500 (40.9)

Values in parenthesis are standard errors of two replicates.


Figure 6.4 a illustrates changes in monomeric ACN in model systems to which

acyl-pgd 3-glu 5-soph had been added. A direct relationship was found between CAN

degradation and Aw, except for the sample at 0.44, which presented a high drop in

monomeric ACN during the entire period of storage. Table 6.4 reports the linear

regression parameters for the degradation of the samples at intermediate and low Aw

e.g., 0.66 and 0.44, which presented good fitness for the linear regression model. The

rate of ACN degradation between these two samples was significantly different ranging

from -2.49 x 10"3 at Aw 0.66 to -1.23 x 10"2 at Aw 0.44. This represents a 5 fold

135

300

6^

C o r \

o

c a.

~m~ Aw i -*- Aw 0.88 H:::- Aw 0.44

........ Aw 0.90 ~-«>~ Aw 0.66

242

Fi^ire 6.3. Changes in (a) monomenc anthocyanin and (b) polymeric color (%) during storage of* model systems containing pelargonidin 3-sophoroside. Means in the same column with unlike superscripts indicate significant difference (p-value < 0.05).

136

HUH Aw 1 -*- Aw 0.89

•"« Aw 0.90 ■~^~ Aw 0.66

Aw 0.44

120 150 Days

Figure 6.4. Changes in (a) monomeric anthocyanin and (b) polymeric color (%) during storage of model systems containing acyl-pelargonidin 3-sophoroside 5- glucoside.

Means in the same column with unlike superscripts indicate significant difference (p-value < 0.05).

137

Table 6.4. Linear regression and first order reaction parameters for the degradation of model systems at Aw 0.66 and 0.44 containing acyl-pelargonidin 3-sophoroside 5- glucoside.

Aw Bo (intercept)

Bi (slope)

rate of degradation (K) days"

2 r

(half life) days

0.66

0.44

2.21 x lO'3

(1.49x10-2)

8.23 x lO'2

(3.26 x lO"2)

-2.49 x 10-3a

(1.25 x lO"4)

-1.23xl0-2b

(2.75 x lO"4)

0.96

0.99

278 (9.05)

56 (1-76)



increase in t1/2from 56 to 278 days. Table 5a reports the time for 15% of the initial ACN

concentration to degrade in samples at high Aw (1,0.90,0.89); the times ranged from

10 days for the sample at Aw 0.90 to 15 days for the sample at Aw 1. No significant

difference in the time of degradation was detected among these samples (p-values >

0.1).

When examining figure 6.4b, a marked increase in polymeric ACN was noticed

in the sample at Aw 0.44 containing acylated pigment. This was the only sample

significantly different from the others (p-value < 0.01).

Parameters comparing the stability of the three pgd derivatives at the same Aw

level are summarized on tables 6.5a and 6.5b. The time needed for the model systems at

Aw 1, 0.90 and 0.89 to loose 15% of the initial pigment ranged between 15 and 74 days.

Statistical analysis showed that this time was significantly higher for the system

containing pgd 3-soph as compared to the other two systems (p-value < 0.01).

Comparison of the rate of degradation among samples with different pigments at Aw

138

Table 6.5a. Time for 15% of the initial anthocyanin to degrade during storage of model systems at high Aw levels (1, 0.90,0.89).

k.w Time (days)


1 28(a) 74(b) 15a (a) (7) (0) (0)

0.90 60(a) 49(a) 10ba(b) (10) (10) (2)

0.89 80(a) 50(b) llb(c)

(6) (0) (4)


Means within a column with different letters are significantly different (p < 0.05).

Means within a row with different letters in parenthesis for each Aw level are significantly different (p < 0.05).

0.66 and Aw 0.44 showed significant higher rate of degradation of the sample

containing acyl-pgd 3-glu 5-soph as compared to the other two (table 6.5b). Table 6.5b.

Rate constants for anthocyanin degradation during storage of model systems containing

acyl-pgd 3-soph 5-glu at intermediate and low Aw levels (0.66, 0.44). These results

differ from the ones obtained by Garz6n and Wrolstad (1977b) when comparing the

stability of the same pgd derivatives in strawberry juice and strawberry concentrate. No

difference in either k or t1/2 was found among these pigments; nevertheless, the ACNS

were more stable in juice; the t1/2 of all pgd derivatives was 12 days in juice as

compared to 4 days in the concentrate. From these results, it is clear that the

components of the environment in which ACNS are present is a definite factor involved

in the their degradation. The systems that contained acyl-pgd 3-glu 5-soph did not

present higher stability in spite of the acylated nature of the molecule. This was

139

Table 6.5b. Rate constants for anthocyanin degradation during storage of model systems containing acyl-pgd 3-soph 5-glu at intermediate and low Aw levels (0.66,0.44).

\w Rate of degradation constant (k)

days"


0.66 -2.49 x 10-3a -9.46 x 10-4a -2.49 x 10-3b

0.44 (1.25 x lO"4) -1.23 x 10-2a

(4.46 x lO'5) -1.39xl0-3a

(1.25 x lO'4) -1.23 x lO-2^

(2.75 x lO"4) (1.92x lO"4) (2.75 x lO"4)


Means within a column with different letters are significantly different (p < 0.05).

Means within a row with different letters in parenthesis for each Aw level are significantly different (p < 0.05).

especially true for the sample at Aw 0.44 whose tm was only 56 days. These results

suggest that the acylated ACN exhibits an unusual behavior when present in model

systems containing glycerol, which confirms the theory proposed by Tom£s-Barber£n

and Robins (1997) about color and stability of ACNS being deeply affected by the

nature of their physicochemical environment. Acylation of the ACN molecule lends

stability in most cases (Bassa and Francis, 1987). However, some model systems have

not shown increase in stability with increase in acylation. As an example, comparison of

the ACNS of grape, red cabbage, and Ajuga reptans in pH 3.5 citrate buffer solutions

did not present difference in the rate of degradation among the three pigments in spite

of their different chemical configuration (Baublis et al, 1994). ACNS from grapes

include mono and diglucosides of five different aglycons with the addition of

monoacylation (Lea, 1988), red cabbage contains diacylated triglucosides of cyanidin

(Mazza and Miniati, 1993), and Ajuga reptans contains glucosylated cyanidin, acylated

140

with p-hydroxycinnamic acid, ferulic acid, and malonic acid (Callebaut et al, 1990).

Similar findings were reported by previous studies comparing the stability of pgd 3-glu,

pgd 3-soph, and acyl-pgd 3-soph 5-glu showed no significant difference in remaining

ACN, t1/2 or rate of degradation in spiked strawberry juice or spiked strawberry

concentrate (Garz6n and Wrolstad, 1977b). The t1/2of the three pigments was 12 days in

strawberry juice and ranged from 4 to 5 days in strawberry concentrate.

The unusual behavior of the samples containing acylated ACN along with the

accelerated degradation of monomeric ACN at latter stages of the storage of model

systems at Aw 0.44 suggest some kind of interaction between the pigment and glycerol.

Labuza (1980) addressed that because glycerol is liquid, it has water-like properties that

produce a plasticizing effect and increase reactant mobility and/or solubility at Aw

levels below which most water soluble reactions occur very slowly. Previously reported

results on stability of ACNS at varying Aw using glycerol as humectant are comparable

to our general findings and support Labuza's theory. Soleha et aL (1990) lowered the

Aw of a tropical dried fruit mix with glycerol based on its humectant property. These

studies showed that the addition of glycerol increased the moisture content of the fruit

and that the increase was directly proportional to the glycerol concentration. Similarly,

evaluation of papaya and pineapple ACNS after fruit cubes were soaked overnight in

10%, 20% and 30% glycerol solutions by Sian and Soleha (1991) showed that the

concentration of ACN increased slightly as the % of glycerol increased from 10% to

20%, but dropped when 30% glycerol was added. This behavior was explained as a

result of hydrolysis and release of the sugar moeity of the ACN that takes place at high

water content such in a system with 30% glycerol. Kearsley and Rodriguez (1981)

compared absorbance readings at Xmax of model systems containing water, ACN

powder, and glycerol at Aw levels ranging from 0.37 to 1. After incubation of the

samples at 60 0C, 90 0C and 160 0C for 170 min, they found that in all cases there was

an inverse relationship between absorbance and that there was no difference in

absorbance of the ACNS at Aw 0.63 and Aw 0.47.

The importance of the physicochemical environment in which ACNS are

present, even at the same Aw level has been demonstrated. Systems using solutes other

141

than glycerol to decrease Aw have also found a direct relationship between Aw and

ACN degradation; however no accelerated degradation has been observed at low Aw

levels. Thakur and Arya (1989) tested the degradation of grape ACNS in isolated model

systems made of 80 % (v/v) aqueous methanol and micro-crystalline cellulose at 0,

0.33, and 0.73 Aw levels. Results showed that after 100 days of storage the % of ACN

retention was higher in the samples with lower Aw. When Br0nnum and Flink (1985)

stored freeze-dried elderberry ACNS in desiccators at different relative humidity levels,

they observed that loss of ACN was significant only at Aw levels above 0.51.

Storage did not cause significant changes in the degradation index (A510/A420) of

the samples. As reported in table 6.6, only a slight drop in this parameter was observed

except for systems containing pgd 3-glu and pgd 3-soph at Aw 0.89, and for the system

containing acyl-pgd 3-soph 5-glu at Aw 1 and 0.44 (p values < 0.01). Decrease in

degradation index indicates development of brown pigments.

Changes in HPLC profile during storage

Figures 6.5a and 6.5b are chromatograms of the model systems containing pgd

3-glu at day zero and day 242 of storage. Hydrolysis of glucose from the ACN aglycon

was not detected during storage; this peak represented 100% of the area in the HPLC

profile. Systems containing pgd 3-soph showed similar changes at Aw 1 and Aw 0.90.

Changes in chromatographic profile showed that at these levels of Aw pgd 3-soph

represented 100% of the total ACN at time zero of the storage (figure 6.6a). After 242

days a small amount (approximately 5%) of pgd 3-glu was detected (figure 6.6b), which

can be explained by the hydrolysis of the diglycoside sophorose to form pgd 3-glu.

Identification of pgd 3-glu was done by comparison of retention times between the new

peak and the purified ACN. Previous studies on stability of pgd-based ACNS by Garz6n

and Wrolstad (1997) produced similar results; the hydrolysis of sophoroside on

strawberry concentrate spiked with pgd 3-soph was not significant, which seemed to

confirm earlier reports on increased ACN stability due to diglycosidic substitution

(Rommel et al., 1990; Rommel et al., 1992). Changes in the HPLC profile of systems

142

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144

fortified with acyl-pgd 3-soph 5-glu, are in good agreement with changes in monomeric

pigment during storage. As shown in figure 6.7, the relative concentration of the ACN

acylated with malonic and cinnamic acids decreased with time in all the samples while

the area of the ACN acylated with only cinnamic acids increased. However, samples at

Aw 1 and Aw 0.44 showed the highest degradation. Immediately after addition of the

purified pigment to the system at Aw 1 acyl-pgd 3-soph 5-glu with both cinnamic and

malonic acids accounted for 57 % of the total area; partial hydrolysis of the malonic

acid is assumed to occur during removal of solvents by means of rotoevaporation. At

day 242 total hydrolysis of malonic acid was evident. Small amounts of pgd 3-soph 5-

glu and pgd 3-soph were found as shown in the chromatographic profile (figures 6.8b

(replicate 1), and 6.8bc (replicate 2). Spectral analysis reported in figure 6.9 and

comparison of retention times with the corresponding purified ACNS made

identification of these two peaks possible. Harbome 1969 and Timberlake et al. (1975)

found that at pH 3.5 glycosides have only 50 % of the absorption at 440 mn (when

compared to the color maximum absorbance) as do the 3-glycosides. When comparing

the spectra of these two peaks to that one of pgd 3-glu, we obtained the ratios described

by these authors.

ACN degradation was more pronounced in the sample at Aw 044 (figures 6.10a,

6.10b, 6.10c); at day 142 of storage (figure 6.10 b) total cleavage of malonic acid of the

acylated ACN accompanied by small amounts of pgd 3-soph 5-glu and pgd 3-soph from

hydrolysis of the latter was observed. At the end of the storage, pgd 3-soph 5-glu

acylated with cinnamic acids accounted for 100% of the pigment present (figure 6.10 c).

Guisti and Wrolstad (1996b) observed similar changes during acid hydrolysis of radish

ACN and storage of model systems containing acyl-pgd 3-soph 5-glu. After a short

period (10 min) of acid hydrolysis with 2N HC160% of the malonic acid was released

resulting in formation of pgd 3-soph 5-glu acylated with cinnamic acids as the major

pgd derivative and small amounts of pgd 3-soph 5-glu. In pH 3.5 model systems studied

by Guisti and Wrolstad (1996a), pgd 3-soph 5-glu acylated with cinnamic and malonic

acids represented 20 % of the total ACNS at the beginning of the storage, but after 10

weeks, release of malonic acid was evident, and pgd 3-soph 5-glu acylated with only

cinnamic acids became the major ACN present representing about 80%.

145

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sophoroside 5-glucoside.

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148

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149

During studies on stability of strawberry juice and concentrate fortified with

acyl-pgd 3-soph 5-glu Garz6n and Wrolstad (1977b) observed considerable increase in

the area corresponding to pgd 3-soph 5-glu acylated with only cinnamic acids and a

consequent decrease in the proportion of the ACN acylated with both cinnamic and

malonic acids. Negligible amounts of pgd 3-soph 5-glu originated from cleavage of

cinnamic acids were detected in the juice samples at the end of the storage.

CONCLUSIONS

The direct relationship between Aw and ACN degradation has been

demonstrated. Results suggest that degradation of ACNS may be accelerated when low

Aw levels are obtained by means of glycerol addition to the model system under study.

This degradation is more noticeable in model systems containing acylated pigments that

might be reacting with glycerol. It has also been demonstrated that the stability of the

three pgd derivatives under study is highly dependent on the components of the system

in which they are present; the t can be increased from 12 days in juice systems from

previous studies to 934 days in model systems in which Aw has been lowered. When

factors affecting ACN stability can be controlled, such as in the case of model systems,

it can be confirmed that parameters other than pigment structure account for pigment

degradation.

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Asen, Stewart, R.N. and Norris, K.H. 1977. Anthocyanin and pH involved in the color of heavenly blue morning glory. Phytochem. 16:118.

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152

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153

CHAPTER?

SUMMARY

The purpose of this project was to study and compare the stability of strawberry

ACNS in both natural (juice and concentrate) and model systems resembling strawberry

juice. The influence of three factors was evaluated: pigment concentration, chemical

structure of the ACN, and water activity (Aw). The experiment has demonstrated that,

ACN degradation in strawberry is a problem that depends on the nature and

physicochemical environment in which the pigments are present

Evaluation and comparison of the stability of pelargonidin derivatives in

strawberry juice and concentrate showed that the pigment degradation followed first

order kinetics. The rate of degradation of ACNS was higher when they were present in

the concentrate. It was also confirmed that higher monomeric pigment concentration

increases stability; ACN fortification increased the half life of the pigments from 3.5 to

5 days in concentrate and from 5 to 12 days in juice. On the other hand, there was no

cause and effect relationship between pigment structure and stability when strawberry

juice or concentrate was spiked with pgd-based ACNS. The stability of pgd 3-glu (the

main pigment in strawberries) was similar to the stability of pgd 3-soph and pgd 3-soph

5-glu acylated with cinnamic and malonic acids when ACN concentration was adjusted

to equivalent amounts. Therefore, the total ACN pigment content needs to be

considered when comparing color stability of different ACNS.

Transformations of individual ACNS as monitored by HPLC during storage of

natural systems demonstrated the lability of ACNS acylated with aliphatic acids, e.g.,

succinic and malonic, and the stability of cinnamic acid acylation. HPLC

chromatograms also showed evidence of greater stability of pgd 3-soph as compared to

pgd 3-glu.

Evaluation of ascorbic acid showed that the acid degradation also follows first

order kinetics and ascorbic acid seems to be contributing to ACN destruction in natural

systems. The shorter half life of ascorbic acid in juice samples (2 days) as compared to

the half life in concentrate samples (3 to 10 days) suggests condensation ascorbic acid-

monomeric ACN. Since correlation between ascorbic acid degradation and ACN

degradation was found, further studies would be advised to determine the influence of

154

ascorbic acid on the degradation of the ACNS under investigation. This can be achieved

through model systems to determine the rate of degradation from logarithmic plots of

each one of the pigments at different concentrations of ascorbic acid.

Evaluation of pigment stability in model systems confirmed the direct

relationship between Aw and ACN degradation. Results suggested that degradation of

ACNS does not follow first order kinetics during the entire time of storage and that it

may be accelerated when low Aw levels are obtained by means of glycerol; this was

specially evident for acylated pigments. Therefore, it is advised to evaluate and compare

ACN degradation using other substances to control Aw Changes in the

chromatographic profile were comparable to changes observed in natural systems.

Hydrolysis of pelargonidin 3-sophoroside to pelargonidin 3-glucoside and hydrolysis of

malonic acid from the acylated ACN were detected.

As a conclusion, It has been demonstrated that the stability of the three pgd

derivatives under study is highly dependent on the components of the system in which

they are present. The half life increased from 12 days in juice to 934 days in model

systems in which Aw had been lowered. When factors affecting ACN stability can be

controlled, such as in the case of model systems, it can be confirmed that parameters

other than pigment structure account for pigment degradation.

155

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an abstract of the thesis of · 2017-08-03 · members, especially dr. dan selivonchick, ... isomer...

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