DEVELOPMENT OF BETACYANIN PIGMENT EXTRACT AND MICROENCAPSULATED POWDER FROM RED PITAYA FRUIT
(HYLOCEREUS POLYRHIZUS)
RURI ADITYA SARI
UNIVERSITI SAINS MALAYSIA JUNE 2011
DEVELOPMENT OF BETACYANIN PIGMENT EXTRACT AND MICROENCAPSULATED POWDER FROM RED PITAYA FRUIT
(HYLOCEREUS POLYRHIZUS)
by
RURI ADITYA SARI
Thesis submitted in fulfillment of the requirements for the degree of Master of Science
UNIVERSITI SAINS MALAYSIA JUNE 2011
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ACKNOWLEDGEMENT
Praised be to Allah who aided me with grace, strength, and patience throughout my master studies.
I am deeply indebted to my beloved parents whom their love never stopped being a heaven of enlightment, serenity, and blessing.
I wish to express my sincere gratitude to my supervisor, Associate Professor Dr Norziah Mohd Hani for her patient guidance, invaluable advice and helpful discussion throughout the research. Without her perseverance and confidence in me, it is impossible for me to accomplish this task.
A special word of gratitude should be given to Pradana Putra and my sisters for being of unfailing support and encouragement, without which it would have been impossible to complete the project.
I would like to thank all the laboratory staffs from Food Technology division, School of Industrial Technology. Besides, special thanks are extended to Nizam, Kee Huey Ying and others lab-mates for their efforts and motivation when I encountered problems during the project.
Last but not least, greatest thanks to Universiti Sains Malaysia for their financial support under the USM Grant.
RURI ADITYA SARI JUNE 2011
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TABLE OF CONTENTS
PAGE
ACKNOWLEDGEMENT ii
TABLE OF CONTENTS iii
LIST OF TABLES vii
LIST OF FIGURES ix
LIST OF PLATES xi
LIST OF APPENDICES xii
ABSTRAK xiv
ABSTRACT xvi
CHAPTER 1 : INTRODUCTION 1
1.1 Background 1
1.2 Objectives 5
CHAPTER 2 : LITERATURE REVIEW 6
2.1 Red pitaya (Hylocereus polyrhizus) 6
2.1.1 Classification 6
2.1.2 Potential in food application 10
2.2 Food colourants 11
2.2.1 Food colourants in Malaysia 12
2.2.2 Synthetic colourant 13
2.2.3 Natural colourants 14
2.3 Betalains (Betacyanin and Betaxanthin) 20
2.3.1 Amaranth 24
2.3.2 Red beetroot 25
2.3.3 Extraction of pigment 26
2.3.4 Health benefits of betacyanin pigments 30
2.3.5 Betacyanin pigment stability 37
2.4 Microencapsulation 41
2.5 Colour measurements 47
CHAPTER 3 : PREPARATION OF BETACYANIN PIGMENT FROM RED PITAYA FRUIT (H.POLYRHIZUS)
3.1 INTRODUCTION 49
3.2 CHEMICALS AND REAGENTS 50
iv
3.3 MATERIALS AND METHODS 50
3.3.1 Treatment of fruit samples 50
3.3.2 Proximate analysis 51
3.3.3 Determination of dietary fibre composition of red pitaya 51 fruit peels
3.3.4 Extraction of pigment with different solvents 52 3.3.4.1 Determination of betacyanin content 53 3.3.4.2 Determination of phenolic content 53 3.3.4.3 Determination of flavonoid content 54 3.3.4.4 Determination of antioxidant activity 54 3.3.4.5 High Performance Liquid Chromatography 56 (HPLC) analysis
3.3.5 Pigment stability studies 57
3.3.5.1 Effect of pH, ascorbic acid, metal ions and 57 temperature
3.3.5.2 Effect of addition of ascorbic acid 58 3.3.6 Enzymatic depectination treatment and stability studies 58 3.3.6.1 Determination of yield and pH 59 3.3.6.2 Determination of betacyanin pigment 59 content and pigment retention
3.3.6.3 Colour measurement 60
3.3.6.4 Determination of phenolics, flavonoids and 60 antioxidant activity
3.3.6.5 Analysis of pigment composition with 60 Liquid Chromatography-Mass Spectrometer
(LC-MS) System
3.3.7 Statistical analysis 61
3.4 RESULTS AND DISCUSSIONS 62
3.4.1 Nutritional composition of red pitaya fruit flesh and peels 62 3.4.2 Dietary fibre composition of red pitaya fruit peels 63 3.4.3 Characteristic of pigment extracts from various solvent
extraction
66 3.4.3.1 Betacyanin content 66 3.4.3.2 Total phenolic and flavonoid content 68 3.4.3.3 Antioxidant activity 70 3.4.3.4 High Performance Liquid Chromatography 75 (HPLC) Analysis with UV detector
3.4.4 Pigment stability studies 80
3.4.4.1 Effect of pH on betacyanin pigment retention 80 3.4.4.2 Effect of ascorbic acid on betacyanin pigment 82 retention
3.4.4.3 Effect of metal ions on betacyanin pigment 84 retention
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3.4.4.4 Effect of temperature on betacyanin pigment 85 retention
3.4.4.5 Betacyanin pigment stabilization by addition 87 of ascorbic acid
3.4.5 Effects of enzyme treatment for clarification of pigment 91 extracts
3.4.5.1 Yield and pH 91
3.4.5.2 Pigment retention and betacyanin content 94
3.4.5.3 Colour measurement 98
3.4.5.4 Total phenolic and flavonoid content 101 pigment extract
3.4.5.5 Antioxidant activity of pigment extract 103 3.4.5.6 Pigment composition using LC-MS (Liquid 105 Chromatography - Mass Spectrometer) system
3.5 CONCLUSION 109
CHAPTER 4 : PREPARATION OF MICROENCAPSULATED
BETACYANIN PIGMENT FROM RED PITAYA FRUIT (H.POLYRHIZUS) BY SPRAY DRYING TECHNIQUE
4.1 INTRODUCTION 110
4.2 MATERIALS AND METHODS 111
4.2.1 Preparation of microencapsulated pitaya pigment 112 powder
4.2.2 Spray drying condition 112
4.2.3 Analysis on spray dried pitaya pigment powder 113 4.2.3.1 Determination of yield and pigment recovery 114 4.2.3.2 Determination of moisture and water activity 114
of pitaya pigment powder
4.2.3.3 Determination of hygroscopicity 115
4.2.3.4 Measurement of solubility 115
4.2.3.5 Colour measurement 115
4.2.3.6 Scanning Electron Microscope (SEM) analysis 115 4.2.3.7 Determination of pigment retention 116 4.2.4 Storage stability studies on the microcapsules under 116 different condition
4.2.5 Statistical analysis 117
4.3 RESULTS AND DISCUSSIONS 118
4.3.1 Evaluation of spray drying condition for production of 118 pitaya pigment powder
4.3.1.1 Yield and pigment recovery 118 4.3.1.2 Moisture content and water activity (aw) 123
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4.3.1.3 Hygroscopicity 128
4.3.1.4 Solubility 130
4.3.1.5 Colour measurement 133
4.3.1.6 Scanning Electron Microscope (SEM) 137 4.3.1.7 Pigment retention of spray dried pitaya pigment 139
powder
4.3.2 Storage stability analysis on microcapsules pitaya pigment 142 powders under different condition
4.3.2.1 Effect of light and relative humidity on 142 pigment retention
4.3.2.2 Colour measurement and colour retention 149 4.3.2.3 Morphology of spray dried pitaya pigment 153
powders upon storage
4.3.2.4 Kinetic degradation of spray dried pigment 155 powder
4.4 CONCLUSION 159
CHAPTER 5 : APPLICATION OF THE BETACYANIN PIGMENT POWDER FROM PITAYA FRUIT IN A MODEL FOOD
SYSTEM
5.1 INTRODUCTION 161
5.2 MATERIALS AND METHODS 162
5.2.1 Materials
5.2.2 Jelly preparation 162
5.2.3 Storage stability studies on jelly 163
5.2.4 Statistical analysis 164
5.3 RESULTS AND DISCUSSIONS 165
5.3.1 Colour measurement of jelly samples 165 5.3.2 Colour retention in jelly samples over storage time 169
5.4 CONCLUSION 175
CHAPTER 6 : SUMMARY AND RECOMMENDATION FOR 176 FURTHER STUDIES
REFERENCES 179
APPENDICES 200
LIST OF PUBLICATIONS 216
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LIST OF TABLES
Table 2.1 Colourants listed in Malaysian Food Acts and Regulations
12 Table 2.2 Comparisons of UK and USA food colour permitted 14 Table 2.3 Natural colours listed by the EU (European Union) 16 Table 2.4 Natural colourants listed by the FDA for food and
beverage use
17
Table 2.5 Distributed of Phenolic compounds 35
Table 2.6 Molecular weight and glass transition temperature (Tg) of anhydrous sugar
43 Table 3.1 Nutritional compositions of red pitaya fruit peels and
flesh (based on dry weight)
63 Table 3.2 Dietary Fibre (DF) composition (%, w/w) in fruits and
vegetables
65 Table 3.3 Concentration of betacyanin using different solvents for
extraction.
67 Table 3.4 Total phenolic and flavonoid contents using different
solvents for extraction.
69 Table 3.5 Inhibition activity (%) and antioxidant capacity (mg
VCEAC/ 100g) of peels/ flesh pigment extracts.
72
Table 3.6 EC50 of selected test samples. 74
Table 3.7 Retention time and relative concentration of respective identified betacyanins
79 Table 3.8 Colour properties of aqueous pigment extracts upon
storage.
90 Table 3.9 Betacyanin content and pH value of the clarified
pigment extract after extraction time 90 min
94 Table 3.10 Colour measurement of the clarified pigment extract 100 Table 3.11 Concentration of total phenolic compounds and
flavonoid content after extraction time 90 min
102 Table 3.12 Inhibition activity (%) and antioxidant capacity (mg
VCEAC/ 100g) after extraction time 90 min
104
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Table 3.13 Retention time and LC-MS data of clarified pigment extract.
108
Table 4.1 Formulation of feed mixtures. 113
Table 4.2 Pigment recovery (%) of spray dried pigment powders 123 Table 4.3 Means values of colour analysis of spray dried pitaya
pigment powders
135 Table 4.4 Pigment retention (%) of spray dried pitaya pigment
powder stored at 25 °C for 12 week (0 % RH)
140 Table 4.5 Colour analysis of spray dried pitaya pigment powder
after storage at 25 ˚C in the dark (D) and in presence of light (L) for 6 months
152
Table 4.6 Kinetic degradation data for spray dried pitaya pigment powder under 0% and 32 % RH (Relative Humidity) and stored at 25 ˚C for 6 months
157
Table 5.1 Colour measurement of jelly samples with added pitaya pigment powder
168 Table 5.2 Colour analysis of jelly with commercial colourant at
concentration 0.1 % (w/w)
169 Table 5.3 Lightness (L*) values and total colour difference (ΔE*ab)
of jelly at 4 ˚C in the dark over 12 weeks storage.
173 Table 5.4 Lightness (L* values) and total colour difference (ΔE*ab)
of jelly at 25 ˚C in the dark over 21 days storage
173
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LIST OF FIGURES
Figure 2.1 Anthocyanin molecule. 18
Figure 2.2 Structure of (A) betalamic acid; (B) Betaxanthins and (C) Betacyanins.
23
Figure 2.3 Sructure of Flavanoids. 36
Figure 2.4 Colour model. 48
Figure 3.1 Total phenolics correlated to antioxidant capacity (mg VCEAC/ 100g).
73 Figure 3.2 The flavonoids correlated to antioxidant capacity (mg
VCEAC/ 100g).
73 Figure 3.3 HPLC profile of pigment extracts from peels of red
pitaya fruit, Hylocereus Polyrhizus.
77 Figure 3.4 HPLC profile of aqueous pigment extracts from flesh
of red pitaya fruit, Hylocereus Polyrhizus.
78 Figure 3.5 Pigment retention (%) against pH at temperature 25
˚C.
81 Figure 3.6 Pigment retention (%) against ascorbic acid at
temperature 25 ˚C and pH 5.
83
Figure 3.7 Pigment retention (%) against metal ions concentration at temperature 25 ˚C and pH 5.
84 Figure 3.8 Pigment retention (%) against temperature at pH 5. 86 Figure 3.9 Pigment retention (%) upon storage days. 88 Figure 3.10 Colour retention (%) upon storage days. 89 Figure 3.11 Yield (%) of clarified pigment extract during
extraction time.
93
Figure 3.12 Pigment retention (%) of clarified pigment extract during extraction time.
95 Figure 4.1 Yield (%) of spray dried pitaya pigment powders. 120 Figure 4.2 Moisture contents (%) of spray dried pitaya pigment
powders.
125 Figure 4.3 Water activity (aw) of spray dried pitaya pigment
powders.
127
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Figure 4.4 Hygroscopicity (g/ 100g) of spray dried pitaya pigment powders.
129 Figure 4.5 Solubility (%) of spray dried pitaya pigment powders. 132 Figure 4.6 SEM micrographs of spray dried pitaya pigment
powders.
138 Figure 4.7 Pigment retention (%) of spray dried pitaya pigment
powders at different relative humidity (0 % RH and 32
% RH) and stored at 25 ˚C in presence of light condition upon 24 weeks storage.
145
Figure 4.8 Pigment retention (%) of spray dried pitaya pigment powders at different relative humidity (0 % RH and 32
% RH) and stored at 25 ˚C stored in dark condition upon 24 weeks storage.
148
Figure 4.9 Micrographs of spray dried pitaya pigment powders stored at 25 ˚C, in the dark condition.
154
Figure 4.10 Stability studies of spray dried pitaya pigment powders at 0% RH (Relative Humidity) and stored at 25 ˚C for 24 weeks.
155
Figure 4.11 Stability studies of spray dried pitaya pigment powders at 32 % RH (Relative Humidity) and stored at 25 ˚C for 24 weeks.
156
Figure 5.1 Colour retention of jelly upon storage at 4 ˚C in dark condition.
171 Figure 5.2 Colour retention of jelly upon storage at 25 ˚C in dark
condition.
171
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LIST OF PLATES
Plate 2.1 Types of Pitaya fruits ((a) Selenecerius Megalanthus, (b) H. undatus, (c) H. polyrhizus)
7
Plate 2.2 Pitaya’s plant 9
Plate 2.3 Pitaya’s bloom 9
Plate 4.1 Spray dried pitaya pigment powders 136
Plate 5.1 Jelly made with pitaya pigment powder and commercial colourants at different concentrations.
167
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LIST OF APENDICES
Appendix A HPLC profile of betanin standard from red beetroot. 200 Appendix B Clarified pigment extract with Rohapect B1L
(2%, 4% and 6% v/w, respectively) after 90 min extraction time
201
Appendix C Clarified pigment extract with Pectinex Ultra SP-L (2%, 4% and 6% v/w respectively) after 90 min extraction time
201
Appendix D Vitamin C standard curve for antioxidant activity 202 Appendix E Reaction kinetics of vitamin C with DPPH radicals 202 Appendix F Reaction kinetics of catechin with DPPH radicals 203 Appendix G Reaction kinetics of clarified pigment extract (4 %
Rohapect B1L) with DPPH radicals
203 Appendix H Reaction kinetics of control clarified pigmetn extract
with DPPH radicals
204 Appendix I Chromatogram of betanin standard
(i) The Chromatogram of betanin standard at m/z 551
(ii) The spectrum of betanin and isobetanin (betanin standard)
205 205 206 Appendix J Chromatogram of clarified pigment extract
(i) The TIC (Total Ion Count) chromatogram of clarified pigment extract
(ii) The chromatogram and spectrum of peak at RT
= 1.15 min (m/z 551.18)
(iii) The chromatogram and spectrum of peak at RT
= 1.53 min (m/z 637.04)
(iv) The chromatogram and spectrum of peak at RT
= 1.61 min (m/z 483.12)
(v) The chromatogram and spectrum of peak at RT
= 2.87 min (m/z 733.58)
(vi) The chromatogram and spectrum of peak at RT
= 2.97 min (m/z 766.99)
207 207 208 209 210 211 212 (vii) The chromatogram and spectrum of peak at RT
= 3.14 min (m/z 760.79)
213
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Appendix K Preparation of McIlvaine’s buffer (pH 5.6) 214 Appendix L Color of resolution pitaya pigment powder and red
beetroot
214 Appendix M Jelly with pigment powder and commercial colourants
over storage time
215
216
LIST OF PUBLICATIONS
Norziah, M.H., Fang, L.L., & Ruri, A.S. (2009). Physical and antimicrobial properties of enzyme-modified starch-based films incorporated with garlic oil. In Gums and Stabilisers for the Food Industry, Conference, 22-25 June 2009, Glywndr University, Wales, UK. Poster
Norziah, M.H., Ruri, A.S., & Tang, C.S. (2007). Evaluation of Colour Properties of Pitaya Betacyanin Pigment Powder for Application in Food. Paper presented in 12th Asian Chemical Congress (12ACC), p 254-255, 23-25 August 2007, Kuala Lumpur, Malaysia
Norziah, M.H., & Ruri, A.S. (2006). Compositional studies on high dietary fruit fibre obtained from local Malaysian fruits. Proceedings of the National Conference on Food Science & Nutrition. 13-14 December, 2006. Kota Kinabalu, Sabah, Malaysia, pp 249-255.
Norziah, M.H., Ruri, A.S., Tang, C.S., & Fazilah, A. (2008). Utilization of Red Pitaya (H. Polyrhizus) Fruit Peels for Value Added Food Ingredients. In proceedings of the International Conference on Environmental Research and Technology (ICERT 08), p 72-75, May 28-30, Penang, Malaysia
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PEMBANGUNAN EKSTRAK PIGMENT BETACYANIN DAN SERBUK MIKROENKAPSULASI DARIPADA BUAH PITAYA MERAH
(HYLOCEREUS POLYRHIZUS) ABSTRAK
Pigmen betacyanin dari buah naga merah (Hylocereus polyrhizus) boleh menjadi sumber menarik pewarna merah semula jadi untuk aplikasi makanan. Pigmen betacyanin telah diekstrak daripada isi dan kulit buah naga merah tempatan di Malaysia dengan menggunakan aseton, metanol dan air sebagai pelarut. Didapati bahawa kadar pigmen betacyanin di dalam isi lebih tinggi berbanding dengan kulit (10.14 0.57 dan 6.69 0.21 mg setara betanin, (BE)/ 100g, masing-masing).
Berdasarkan kandungan pigmen betacyanin, ekstrak pigmen daripada isi buah naga merah yang diekstrak menggunakan air dipilih untuk kajian seterusnya. Kestabilan larutan ekstrak pigmen dikaji pada pH yang berbeza (2 -10), suhu yang berbeza (25
˚C - 75 ˚C), kepekatan ion logam (Cu2+ dan Fe2+) yang berbeza (0 - 150 ppm) dan penambahan asid askorbik (0 - 1.6 %). Kandungan fenolik dan flavonoid daripada ekstrak pigmen didapati meningkat lebih banyak setelah mengalami proses klarifikasi. Dapatan kajian daripada analisis HPLC dan LC-MS mengesahkan kehadiran betanin dan phylocactin juga isoformnya termasuk hylocerenin dalam ekstrak pigmen betacyanin. Ekstrak pigmen buah naga telah dikeringkan menggunakan teknik pengeringan semburan untuk meningkatkan kestabilan dan jangkahayat ekstrak pigmen yang diperolehi. Dua jenis maltodekstrin (10 DE dan 25 DE) dan campuran (10 DE + 25 DE) digunakan sebagai agen lapisan dalaman bagi teknik pengeringan semburan untuk memberikan pepejal terlarut total (TSS) berjulat di antara 20 % - 30 %. Serbuk pigmen pitaya yang dihasilkan pada suhu pengeringan (200 ˚C) dicirikan kepada kandungan pigmen betacyanin, perolehan pigmen, warna, kandungan air, keterlarutan dan higroskopisiti. Pengaruh
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penambahan asid askorbik (0.1 % dan 1.0 % b/b, masing-masing) ke dalam ekstrak pigmen buah naga sebelum pengeringan semburan untuk mengkaji kestabilan ekstrak pigmen buah naga juga diselidiki dengan kehadiran cahaya dan kelembapan pada suhu bilik. Penggunaan campuran maltodekstrin (10 DE dan 25 DE) ke dalam serbuk pigmen buah naga sebagai agen pelapis dalaman meningkatkan higroskopisiti dan kestabilan selama penyimpanan berbanding dengan 25 DE dan 10 serbuk DE secara berasingan. Penggunaan campuran maltodextrin 10 DE dan 25 DE pada perbandingan 1:2 terpilih sebagai agen pelapis dalaman. Setelah pigmen disimpan pada relatif kelembapan 0 % RH (25 ˚C) selama 24 minggu dalam gelap, degradasi pigmen yang rendah (21.9 ± 0.26 %) diperhatikan pada serbuk pigmen buah naga yang ditambah dengan 0.1% (b/b) asid askorbik. Dalam kajian ini, serbuk pigmen buah naga dan pewarna komersil diaplikasikan dalam sistem model makanan iaitu jeli untuk menentukan ciri-ciri warna dan kestabilan pada suhu yang berbeza.
Keputusan kajian menunjukkan bahawa betacyanin diperolehi daripada isi buah naga (Hylocereus polyrhizus) merupakan pewarna semula jadi yang berpotensi untuk digunakan dalam aplikasi makanan.
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DEVELOPMENT OF BETACYANIN PIGMENT EXTRACT AND MICROENCAPSULATED POWDER FROM RED PITAYA FRUIT
(HYLOCEREUS POLYRHIZUS)
ABSTRACT
Betacyanin pigments from red pitaya fruit (Hylocereus polyrhizus) could be an attractive source of natural red colourant for food application. The extraction of betacyanin pigment was extracted from the flesh and peels of red pitaya fruits grown locally in Malaysia by using acetone, methanol and water as the extracting solvents.
Both the flesh and peels were investigated and it was found that the flesh had higher of pigment contents compared to the peels (10.14 0.57 and 6.69 0.21 mg of betanin equivalents, (BE)/100 g, respectively). Based on the betacyanin content, the pigment extract with water extraction obtained from red pitaya fruit flesh was selected for further study. The stability of aqueous pigments extract was investigated at different pH (2 -10), different temperatures (25 ˚C - 75 ˚C) and in presence of varying concentrations (0 - 150 ppm) of metal ions (Cu2+ and Fe2+) and ascorbic acid (0 - 1.6 %). The phenolic and flavonoid contents were effectively increased by further optimising of the clarification process on the pigment extracts. The HPLC and LC-MS studies confirmed the presence of betanin and phyllocactin and their isoforms including hylocerenin in the betacyanin pigment extract. In order to increase the stability of pitaya pigment extract, Spray-drying technique was also performed on the pigment extracts to increase the stability and shelf life of the pigment extracts obtained. Two types of maltodextrin with different DE’s (10 DE and 25 DE) and mixtures (10 DE + 25 DE) were used as coating agents in the spray drying technique to give a total soluble solid (TSS) ranging from 20 % - 30 %. The quality attributes of the pitaya pigment powders produced at drying temperatures
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(200 ˚C) in the spray drying technique were characterized by their betacyanin pigment content, pigment recovery, colour, moisture content, solubility and hygroscopicity. The stabilising effect of addition ascorbic acid (0.1 % and 1.0 % w/w, respectively) into feed pitaya pigment extract prior to spray drying was also investigated under the presence of light and moisture at room temperature. The use of mixtures maltodextrin (10 DE and 25 DE) in pitaya pigment powders as coating agent enhanced the hygroscopicity and the stability during storage compared to the 25 DE and 10 DE powders separately. The use of mixtures 10 DE and 25 DE at ratio 1:2 was selected as coating agent. After pigment being stored at 0 % RH (25 ˚C) for 24 weeks in the dark, lower pigment degradation (21.9 ± 0.26 %) was observed in pitaya pigment powder supplemented with 0.1% (w/w) ascorbic acid. In this study, pitaya pigment powders and commercial colourants were evaluated to determine colour characteristics and stability at different temperatures in jelly. The results showed that betacyanin obtained from pitaya (Hylocereus polyrhizus) could be a potential natural colourant use in food applications.
1 CHAPTER 1 INTRODUCTION 1.1 BACKGROUND
Colour is the first characteristic of a food and often predetermines or
“colours” our expectation. We use colour as a way to identify a food and a way to judge the quality of a food. Now health-conscious consumers are taking the more seriously. They want appropriate colour, but they want it “natural.” Consumers are concerned about the foods and beverages they consume and how it affects their health and the health of their children.
According to Heller (2009), there is an ongoing review of colourants within Europe. The Food Standards Agency (FSA) in UK has published a list of food products that have been reformulated to remove six food colours associated with hyperactivity in young children. In 2008, the FSA proposed a voluntary ban to phase out six food colourants (Tartrazine (E102), Quinoline Yellow (E104), Sunset Yellow (E110), Carmoisine (E122), Ponceau 4R (E124) and Allura Red (E129)) from food products by 2009, which is in line with the action by the European Union (EU). This new legislation on food additives that states a requirement for food products in the market containing any of the six colours should carry additional labeling information that states “Consumption may have an adverse effect on activity and attention in children”.
Hylocereus polyrhizus mostly known as pitaya, pitahaya or dragon fruit were native to central South America. For many years efforts have been made to develop the cultivation of pitayas, vine species of the genus Hylocereus. The colour of the fruits depended on the species, some of them contained pulp of red and or/ purple colours in various hues (Mizrahi et al., 1997; Mizrahi & Nerd, 1999).
2
In pitayas the most important pigments are betalains which consist of the betacyanins and betaxanthins (Gibson & Nobel, 1986). The most important betalain sources for natural red colouring is from a variety of red beetroot, commercial preparation of which are mainly composed of the red-purple betanin and its C15-isomer isobetanin.
Red beetroot or Beta vulgaris which is commercially prepared in powder forms or juice concentrates and listed as E162 in Europe, is extensively used in the food industry worldwide as a red colourant. However, because of the unfavourable earthlike flavour characteristics caused by geosmin and pyrazine derivatives, as well as high nitrate concentrations associated with the formation of carcinogenic nitrosamines, there is a demand for alternative compounds (Castellar et al., 2006;
Stintzing & Carle, 2004). Red pitaya exhibits a pigment spectrum consisting of non- acylated (i.e., betanin = betanidin 5-O-β-glucoside) as well as acylated betacyanins (i.e., phylocactin = betanidin 5-O-β-malonyl-glucoside), (Stinzing et al., 2002b;
Wybraniec et al., 2001). Hence, fruits from the Cactaceae family have been proposed as a promising betalain source (Stintzing et al., 2001, 2003). Fruits from Hylocereus polyrhizus produce a deep purple-coloured flesh comparable to red beetroot (Stinzing et al., 2000) or amaranth (Cai et al., 1998a). It is known that Hylocereus cacti are the third richest betacyanin source for food colouring agents after Beta vulgaris and Amaranthus species. Betacyanin pigments from red purple pitaya fruit could be an attractive source of red colourant for food application.
In previous studies (Wybraniec et al., 2001; Stintzing et al., 2002b;
Wybraniec & Mizrahi, 2002; Wu et al., 2006), efforts have been made to quantify and determine the identity of betacyanin pigment in pitaya fruits cultivated in Taiwan and Israel. Wu et al. (2006) also studied on total phenolics and flavonoids in the fruits.
3
Though efforts have been made to characterize the pitaya fruits grown in Taiwan and Israel, both places are of different climate compared to Malaysia. Since the plants are able to tolerate drought, heat, poor soil and cold, they are able to grow under most of the climates in different parts of world. In Malaysia, there is an alternating wet and dry seasons of the tropical climate and this suitable for the growth of pitaya fruits. From a survey conducted by Department of Agriculture, Malaysia (Anonymous, 2006a), about 435.8 ha of land located separately in Malaysia has been established for the cultivation of pitaya fruits, particularly in parts of Kluang, Johore. Even though it was just introduced few years ago, the fruit cultivation had increased substantially over the years due to the high demand from local and overseas market.
Betalains show good stability in the pH range from 3 to 7, thus have a great potential in colouring a broad array of food. However, these pigments are generally considered heat-labile, and are also affected by pH, light, air and water activity (Jackman & Smith, 1996a) and are highly instable compared to synthetic food colourant like Amaranth and FD&C Red #3 (Cevallos-Casals & Cisneros-Zevallos, 2004). Thus alternatively these colour pigments could be encapsulated and spray dried to produce pigment powder to enhanced storage stability. There are many studies on spray drying of betacyanin pigments from Amaranthus (Cai & Corke, 2000), Opuntia ficus-indica (Saénz et al., 2009), Opuntia stricta (Obón et al., 2009) and Red beetroot (Azeredo et al., 2007). However, no reports were found on production of spray dried betacyanin pigment from red pitaya fruit (Hylocereus polyrhizus) grown in Malaysia. Thus the evaluation of spray drying condition to produce pigment powder needs to be studied.
4
The research study comprised of three phases. The first phase was extraction of betacyanin pigment from red pitaya (H. Polyrhizus) fruits with various solvents to obtain the most suitable solvent to be used. These pigment extracts were characterized further for the phenolic, flavonoid and antioxidant capacity. This phase included stability studies against selected factors (temperature, pH, metal ions and additive) and investigated the effects of two pectinase enzymes for clarification of the aqueous pigment extracts. The second phase involved microencapsulation study to produce pigment powder using spray drying technique. The third phase investigates the colour stability of pigment powder in food model system.
5 1.2 OBJECTIVES
The main objective of this research is to produce natural colourant from red pitaya fruit. The specific objectives of this research work are:
a) To produce and characterize the aqueous betacyanin pigment extract.
b) To prepare and characterize microencapsulated betacyanin in a powder form using spray dried technique.
c) To evaluate the colour stability in a model food system in comparison with commercial red colourants.
6 CHAPTER 2 LITERATURE REVIEW
2.1 Red pitaya (Hylocereus polyrhizus) 2.1.1 Classification
Red pitaya is native to Central South America and the tropical forest regions of Mexico (Mizrahi et al., 1997). The pitaya (Hylocereus sp.), known as strawberry pear, thany loy (in Vietnam), pitahaya roja (in Spanish) and la pitahaya rouge (in French), grows on tropical climbing cacti. In Malaysia, pitaya is alternatively known as dragon fruit, „huo long guo‟ or „buah naga‟ in various languages. Currently, they are being grown commercially in Taiwan, Nicaragua, Colombia, Vietnam, Israel, Australia and USA. For the past few years, extensive efforts have been made to develop the cultivation of pitayas commonly known as the „dragon fruit‟ in Malaysia.
The classification of pitaya (Hylocereus polyrhizus) is shown below (Danial, 2008);
Red pitaya (Hylocereus polyrhizus) Order : Caryophyllales
Family : Cactaceae Genus : Hylocereus Species: Polyrhizus
There are three types of pitaya, the normally white-fleshed (H. Undatus), red- purple fleshed (H. Polyrhizus) and white-fleshed with yellow skin (Selenicereus megalanthus) (Plate 2.1), varying degrees dependent upon variety. All of these species are sprinkled with tiny edible black seed. Species of Hylocereus included H.
7
Costaricensis, H. Purpusii (all red flesh species) and the white flesh species H.
Undatus (Wybraniec & Mizrahi, 2002) among which H. Polyrhizus having a glowing deep red-purple fruit flesh is more commercially cultivated in Malaysia.
Plate 2. 1 Types of Pitaya fruits ((a) Selenecerius Megalanthus, (b) H. Undatus, (c) H. Polyrhizus) (Source: Danial, 2008)
Nerd et al. (1999) studied the optimum date of harvest in relation to colour development of pulp and peel and postharvest behaviour of Hylocereus undatus (Haworth) Britton & Rose and H. Polyrhizus. Nerd and Mizrahi (1998) reported that yellow pitaya (Selenicereus megalanthus) showed the duration of fruit development depends on seasonal temperatures and that the fruits reach the optimal flavour close
a
b
c
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to full colour stage. However, little research has been done on red pitaya fruit development and on the behaviour of the fruit during or after storage.
Pitaya have a climbing growth habit, reaching 10 m or more in height if suitable supports available (Plate 2.2). Seeds grow well in compost or potting soil mix - even as a potted indoor plant. Pitaya cacti usually germinate between 10 and 14 days after shallow planting (Danial, 2008). As they are cacti, overwatering is a concern for home growers. As their growth continues, these climbing plants will find something to climb on, which can involve putting aerial roots down from the branches in addition to the basal roots. Once the plant reaches a mature 4.5 kg weight, the flower of the plant could be grown. The plants can flower between three and six times in a year depending especially on growing conditions. Pitaya cacti flower overnight, usually wilting by the morning (Plate 2.3). They rely on nocturnal creatures such as bats or moths for fertilization by other pitaya. Self fertilization will not produce fruit. This limits the capability of home growers to produce the fruit.
Like other cacti, if a healthy piece of the stem is broken off, it may take root in soil and become its own plant. This is a much shorter route to reproduction. The plants handle up to 40 oC and very short periods of frost, but do not survive long exposure to freezing temperatures (Danial, 2008). Pitaya is large in size, average of 300 - 500 g for each fruit. It is oblong in shape with a red peel and large green scales.
The scales turn yellow upon ripening. For the H. Polyrhizus, the colour of the fruit skin begins to change 25 to 35 days from flowering. At the same time, flesh firmness approaches a minimum and the eating quality approaches a maximum 33 to 37 days after flowering.
As the fruit matures, acidity reaches a peak just as the skin colour change occurs, then declines 25 to 30 days after flowering (Nerd et al., 1999). Fruits can be
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harvested from 25 to 45 days after flowering. The size of the fruit depends on seed number in fruit (Castellar et al., 2003). The recommended storage temperature for pitaya is around 10 ˚C since lower temperatures induce microbial growth and chilling injury. Fruit must be kept moist; either by misting, or by storage in a sealed box or plastic bags to prevent desiccation. If the skin removed, the inner flesh will keep well for up to a month in the refrigerator; or can be frozen for later use.
Plate 2.3 Pitaya‟s bloom (Source: Danial, 2008) Plate 2.2 Pitaya‟s plant (Source: Danial, 2008)
10 2.1.2 Potential in food application
There are many studies stated that pitaya is a good source of fibres, which gave the juice a favourable mouth feel and helps to reduce blood sugar and plasma cholesterol levels (Fernández et al., 1992; Munoz-de-Chavez et al., 1995; Trejo et al., 1995). Beside, pitaya has significantly high amount of vitamin C and have high nutritional value and medicinal value, it helps our body especially in digestion, preventing colon cancer and diabetes. Antioxidants in the diet reduce the risk of cardiovascular disease and cancer (Pedreno & Escribano, 2001; Esribano et el., 1998;
Kanner et al., 2001). Antioxidant activity and health improving capacity of pitaya has been reported in the previous studies (Lim et al., 2007; Wu et al., 2006;
Mahattanatawee et al., 2006). The pitaya fruit has a stronger antioxidant activity than most vegetables (Schliemann et al., 1999).
Pitaya is very particular for the presence of betalains, a widely used natural colourant in the food industry. Recently, Pitaberry Sdn. Bhd. was applied the pitaya fruit to beverages. They claimed that there are no artificial colouring added due to the natural occurring of red-purple colour impart provided by itself. In addition, it also can be made into a nutritious fermented “enzyme” drink which is sold in Malaysia as a health food supplement.
There are some studies conducted on mucilages of Cactaceae family fruit which consists of complex polysaccharides, mainly composed of arabinose, galactose, rhamnose, and galacturonic acid (Lee et al., 1998; Saénz et al., 1992). The fruit‟s mucilages have a high water-holding capacity, so they could serve as thickening or emulsifying agents and form viscous or gelatinous colloids, these properties are needed especially in food systems like jam, jellies and ice-cream production (Piga, 2004). Furthermore, Arriffin et al. (2009) and Rui et al. (2009)
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have been studied the extraction and characterization of seed oil obtained from pitaya fruit.
In addition, the consumers concern about side effect of artificial colourant applied in food system, thus there was increasing demand for natural colourant in drink, dairy products and many food productions. In cacti, the most important fruit pigments are the betacyanin and betaxanthins (Wybraniec et al., 2001). The known betacyanin pigments of Hylocereus polyrhizus are betanin, phylocactin (6'-O- malonylbetanin), and a recently discovered betacyanin, hylocerenin (5-O-[6'-O-(3"- hydroxyl-3"-methyl-glutaryl)-β-D-glucopyranoside) (Wybraniec & Mizrahi, 2002;
Wybraniec et al., 2001).
2.2 Food colourants
The first characteristic of food that is noticed is its colour and this predetermines our expectation of both flavour and quality. Food quality is first judged on the basis of colour and we avoid wilting vegetables, bruised fruit, rotten meat and overcooked food. Numerous tests have demonstrated how important colour is to our appreciation of food. When foods coloured, the colour and flavour are matched, i.e. green for lime, yellow for lemon and the flavour is correctly identified on most occasions. However, if the flavour does not correspond to the colour then it is unlike to be identified correctly (Henry, 1996).
Colour level also affects the apparent level of sweetness. The colour of a food will therefore influence not only the perception of flavour, but also that of sweetness and quality. The best food with perfect balance nutrients is useless if it is not consumed. Consequently, food needs to be attractive. During this century, the use of synthetic colour has steadily increased at the expense of these products of natural
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origin, due principally to their ready availability and lower relative price. Generally two types of organic food colours are recognized in the literature: synthetic colours and natural colours.
2.2.1 Food colourants in Malaysia
According to Malaysian Food Acts and Regulations (Anonymous, 2006b) as shown in Table 2.1, all the additives were added in food which gave colour are considered as colourants. Colourants which added in food should be contained more than 4 percent of approved colourants that listed in Food Act. Futhermore, liquid colourants could be contained benzoate acid (below 400 ppm) as the preservatives.
Table 2.1 Colourants listed in Malaysian Food Acts and Regulations
Colourant Index number
Allura Red AC 16035
Amaranth 16185
Brilliant Black PN 28440
Brilliant Blue FCF 42090
Carmoisine 14720
Chocolate Brown HT 20285
Erythrosine BS 45430
Fast Green FCF 42053
Green S 44090
Indigotine 73015
Ponceau 4R 16255
Red 2G 18050
Sunset Yellow FCF 15985
Tartrazine 19140
Quinoline Yellow 47005
Source: Anonymous (2006b)
13 2.2.2 Synthetic colours
Colour is a major contributor to flavour anticipation and is often perceived before aroma. It is known to play a major role in the acceptability of food products.
Indeed there are many product types which would be greatly curtailed without the ready availability of safe colourants. For example, soft drinks, puddings, gelatins, reconstituted and imitation fruits, candies, jelly, pastries, ice-cream, syrups, sauces and snacks. Some studies have been shown that food does not taste „right‟ when it does not have the proper colour (Johnson & Lichtenberger, 1980). Since the synthetic colours have high tinctorial power, very small amounts of them are adequate for practically all applications. Use level customarily needed range from 20 or 30 ppm to about 300 ppm in foods ready for consumption (Francis, 1996).
These are colourants that do not occur in nature and are produced by chemical synthesis. The development of food laws in the USA followed those of European are governed by the Food and Drug Act. The 1938 Act established the term Food Drug and Cosmetic (FD & C) Colours and stated that colourants, presumably held to higher specifications for purity, would be allowed in foods, drugs and cosmetics. Francis (1996) reported that the first group comprises seven synthetic FD
& C colour additives which are certified to comply with the purity specifications required by the FDA. The principles of food law are similar throughout the world but the specifics vary considerably between countries. The EU (European Union) has three main principles: protection of health of the consumer, prevention of fraud and removal of non-tariff barriers to intra-community trade. The EU Colours Directive lists colourants deemed to be suitable for food use and specifies the limits of impurities. Each colourant is identified by a specific E number. There are some
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synthetic colourants which are permitted in USA but not permitted in UK, and vice versa (Table 2.2).
Table 2.2 Comparisons of UK and USA food colour permitted
Colour E Number FDA Number
Tartrazine* E102 Yellow No.5
Quinoline Yellow* E104 -
Sunset Yellow FCF* E110 Yellow No.6
Ponceau 4R* E124 -
Carmoisine* E122 -
Amaranth E123 -
Erythrosine E127 Red No.3
Patent Blue V E131 -
Indigo Carmine E132 Blue No.2
Brilliant Blue FCF - Blue No.1
Green S E142 -
Black PN E151 -
Allura Red AC* E129 Red No.40
Fast Green FCF - Green No.3
Source: Walford (1968)
*Synthetic colourants which are still in question by Food Standard Agency, UK (Heller, 2009)
2.2.3 Natural colourants
Organic colourants are derived from natural edible sources using recognized food preparation methods, such as cucurmin (from turmeric), bixin (from annatto seeds), anthocyanin (from red fruits) and betalains (betacyanin and betaxanthin).
This description of a natural colour would exclude caramels manufactured using ammonia and its salts and also copper chlorophyllins, since both of these products involve chemical modification during processing using methods not normally associated with food preparation (Henry, 1996).
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Natural colours are widely permitted throughout the world. However, there is no universally accepted definition of this term and many countries exclude from their list of permitted colours those substances that have both flavouring and a colouring effect. The trend towards natural ingredients in foodstuffs is continuing and this is evidenced by consumer acceptance of „natural‟ foods and the various regulations which completely or selectively ban artificial colours from food.
The EU permits a wide range of colours, some of which are of natural origin and these are listed in Table 2.3. From table 2.3, lycopene is not widely available commercially and four of the colours are only available commercially as nature- identical products. Nature-identical colours are manufactured by chemical synthesis so as to be identical chemically to colourants found in nature (Henry, 1996). The USA has a different set of „natural‟ colours and those currently permitted by the Food and Drug Administration (FDA) which listed in Table 2.4; these not require certification and permanently listed.
One of the advantages of using natural colours is that they are generally more widely permitted in foodstuffs than synthetic colours. At present the use of natural colourants in food is limited, due to their instability, poor tinctorial power and the limited range of colours available. Natural colourants produced for use are crude extracts of pigments which are basically unstable, and strongly dependent on condition of storage and processing (Jeszka, 2007). The apparent stability of some food products owes more to the amount of pigment present than to the tinctorial power of the pigment itself. For example red beetroot, even after prolonged cooking, retains an attractive deep red colour, but the extracted pigment is unstable (Taylor, 1980).
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Table 2.3 Natural colourants listed by the EU (European Union)
Colour E Number
Curcumin E100
Riboflavin, riboflavin-5'-phosphate* E101 Cochineal, carminic acid, carmines E120 Chlorophylls and chlorophyllins E140 Copper complexes of chlorophylls and
chlorophyllins
E141
Plain caramel E150a
Vegetable carbon E153
Mixed carotenes and β-carotene E160a Annatto, bixin, norbixin E160b Paprika extract, capsanthin,
capsorubin
E160c
Lycopene E160d
β-Apo-8'-carotenal (C30)* E160e
Lutein E161b
Canthaxanthin* E161g
Beetroot red, betanin E162
Anthocyanins E163
Source: Henry (1996)
*available commercially as nature-identical products
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Table 2.4 Natural colourants listed by the FDA for food and beverage use Colour
Annatto extract β-Apo-8'-carotenal*
β-carotene*
Beet powder Canthaxanthin*
Caramel Carrot oil
Cochineal, carmine Cottonseed flour, toasted Fruit and vegetable juices Grape colour extract Grape skin extract
Paprika and paprika oleoresin Riboflavin*
Saffron
Turmeric and turmeric oleoresin
Source: Henry (1996)
*available commercially as nature-identical products
Carotenoids are the most widespread and the important group of pigments in nature. It comprises a group of structurally related colourants that are mainly found in plants, algae and several lower organisms. All carotenoid contains a system of conjugated double bonds that influence their physical, biochemical and chemical properties. In principal, each of the polyene chain double bond could exist in a cis or trans conformation, thus creating a number of isomers. It is relatively stable and there is sufficient demand to make complex chemical synthesis of „natural-identical‟
carotenoids worthwhile (Jeszka, 2007; Sikorski, 2007). Their colour range is limited to yellow/ orange/ red and they are naturally oil soluble although water-soluble forms are available.
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While, anthocyanins are among important groups of plant pigments which are present in almost all higher plants and are the dominant pigments in many fruits and flower, which performed in red, violet or blue colour. They play a definite role in attracting animals in pollination and seed dispersal. Anthocyanins are part of very large and widespread group of plant constituents known as flavonoids, which posses the same C6-C3-C6 basis skeleton. They a glycoside of polyhydroxyl and polymetoxy derivatives of 2-phenylbenzopyrilium salts or flavylium cation and are most commonly based on six anthocyanidins: pelargonidin (orange-red), cyaniding, peonidin, delphinidin (blue-violet), petonidin and malvidin. The sugar moiety present is most commonly one of the following: glucose, galactose, rhamnose and arabinose (Figure 2.1). Anthocyanin preparations have found use in some products, but their colour variation with pH has restricted their use, mainly to acidic products (Henry, 1996).
Figure 2.1 Anthocyanin molecule (Source: Francis, 1999).
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The degradation of anthocyanin is at pH value above 2, this explained by intramolecular copigmentation which is based on the stacking of the hydrophobic acyl moiety and the flavylium nucleus, thus reducing anthocyanin hydrolysis (Dangles et al., 1993; Stintzing & Carle, 2004). In addition, anthocyanin glucosides are affected by glucosidases resulting in the formation of the highly labile aglycones which in turn oxidize easily and resulted deterioration of colour accompanied by unwanted browning (Stintzing & Carle, 2004). Ascorbic acid, glucose and fructose may even accelerate anthocyanin colour loss catalyzed by high temperature, oxygen and metal ions. The stability of anthocyanin is depending on the co-pigment, high stability of anthocyanin was obtained in an aqueous environment, at pH value between 3.1 and 4.7 at low temperatures. During extraction process of anthocyanin, factors promoting colour loss is promoted by deactivation endogenous and microbial enzymes such as glycosidases, peroxidises and polyphenoloxidases released upon tissue maceration (Stintzing & Carle, 2004). Thus, producing anthocyanin in concentrates and powder form could enhanced and stabilize the colour.
Another important pigment in nature and is present in all plants capable of photosynthesis is chlorophyll. However, the addition of chlorophyll as a colour to foodstuffs is very limited, principally because of its poor stability. It is an oil-soluble colour that can be extracted from a range of green leaves, but usually grass, nettles or alfalfa is used. Chlorophyll degrades easily, particularly in acidic conditions, losing its magnesium ion to yield phaeophytin, which is yellow-brown in colour.
Chlorophyll colours tend to be rather dull in appearance and of an olive green-brown colour. Chlorophyll extracts can be standardized using vegetable oil for oil-soluble products or blended with a food solvent or permitted emulsifier to give a water- miscible form (Delgaldo-Vargas & Paredes-Lopéz, 2003).
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Cochineal is described both the dried insects themselves and also the colour derived from them. Coccid insects of many species have been used for thousands of years as a source of red colour. Each insect is associated with a specific host plant and each is the source of a particular colour such as Armenian red, kermes, Polish cochineal, American cochineal. Cochineal extract exhibits shade changes with changes in pH levels. At pH levels of 4.0 and below, it is orange; at 4.0-6.0, it is magenta red colour; and above 6.0 it is a blue-red shade (Henry, 1996).
2.3 Betalains (Betacyanin and Betaxanthin)
Betalains have a limited distribution in the plant world and it would appear that betalains and anthocyanin are mutually exclusive. Plants producing betalains do not contain anthocyanins. Betalains can be divided into two classes, the red betacyanins and yellow betaxanthins. Most varieties of red beetroot contain the red betacyanin, betanin as the predominant colouring compound and this represents 75%
to 90% of the total colour present (Henry, 1996).
Betalains are water-soluble nitrogenous pigments that replace the anthocyanins in a small number of taxonomically related plants families (Strack et al., 1993; Clement & Mabry, 1996; Herbach et al., 2006a). Structurally, betalains are immonium derivatives of betalamic acid (Figure 2.2), the lemon-yellow colour of the latter resulting from the 1, 7-diazaheptemethinium resonance system exhibiting three conjugated double bonds (Zrÿd & Christinet, 2004). The betalains comprise a quite modest number of about 55 structures including the red-violet betacyanins and the yellow-orange betaxanthins (Stintzing & Carle, 2007), while up to 550 anthocyanins have been identified in nature thus far (Andersen & Jordheim, 2006). Although not yet being clarified, the co-occuring betacyanin C15-stereoisomers are mainly
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considered isolation artifacts. In contrast, the analogous C11-isomers for the betaxanthins have not yet been detected as genuine compounds.
The uniqueness of betalains is their N-heterocyclic nature with betalamic acid being their common biosynthetic precursor. In comparison with the anthocyanins (Andersen & Francis, 2004), a much smaller number of substituent have been reported for the betalains: glucose, glucoronic acid and apiose are typical sugar monomers, while malonic and 3-hydroxy-3-methyl-glutaric acids as well as caffeic-, p-coumaric, and ferulic acids represent typical acid substituents (Strack &
Schliemenn, 2003). Noteworthy, sinapic acid has been rarely reported for betalains (Kugler et al., 2007; Wybraniec et al., 2007), while inversely 3-hydroxy-3-methyl- glutaric acid has never been found as a structural feature in anthocyanins. The yellowish counterparts to the acyanic flavonoids, the so-called anthoxanthins, are the betaxanthins (Kremer, 2002).
Betaxanthins are condensation products of betalamic acid and amino acids or amines respectively. Depending on the particular structure of the amino compound, maximum absorption of betaxanthins varies between 460 and 480 nm (Stintzing et al., 2002a). The most common and frequently addressed betaxanthins (Figure 2.2) are glutamine-betaxanthins (vulgaxanthin I), betaxanthin in red beetroot (Beta vulgaris L.) and indicaxanthin (proline-betaxanthin), the predominant pigment in yellow cactus pears (Opuntia sp.), respectively.
Condensation products of betalamic acid and cyclo-Dopa [cyclo-3-(3,4- dihydroxyphenylalanine)] are commonly referred as betacyanins due to their deep red violet colour. Their strong bathocromic shift of 50 to 70 nm as compared to betaxanthins is ascribed to the aromatic structure of cyclo-Dopa (Zrÿd & Christinet
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2004). By glycosylation with one or two monosacharides as well as acylation of the resulting 5-O- or 6-O-glucosides, a great variety of betacyanin structures is possible.
Nevertheless, betacyanin research has mainly focused on betanin (betanidin 5-O-β-glucoside; Figure 2.2), the most abundant betalains in red beetroot (Beta vulgaris L.). As already stated for betaxanthins, the absorption maximum of the betacyanins is influenced by the particular substitution pattern of the betanidin backbone. Generally, glycosylation of betanidin comes along with a hypsochromic shift of the resulting betacyanin, glucose attached at C6 being less effective than C5
glycosylation (Stintzing et al., 2004). While esterification with aliphatic acyl moieties was reported to have little impact on the maximum absorption of betacyanins (Wybraniec et al., 2001; Stintzing 2002b), acylation with aromatic acids leads to a bathochromic shift (Heuer et al., 1992). Schliemann & Strack (1998) was explained by copigmentation like intramolecular association. Moreover, C6
attachment of acyl-glucosides enhances the bathocromic shift, which possibly results from a more rigid conformation (Heuer et al., 1994).
Besides this biochemically related distinctions, the betalains are more water soluble than the anthocyanins and exhibit a tinctorial strength up to three times higher than the anthocyanins. Betalains possess higher molar absorption coefficients in the visible light spectrum than anthocyanins (Clement & Mabry, 1996), indicating their function in UV protection. Due to the high molar extinction coefficients, the colouring power of betacyanin is competitive to synthetic colourants (Henry, 1996).
Apart from that, contrast to anthocyanins, their appearance is maintained and stable over a wide pH range from 3 to 7, making them ideal pigments for colouring low pH acidic foodstuffs (Stintzing & Carle, 2004) also reported that betalains are not as susceptible to hydrolytic cleavage as the anthocyanins.
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Figure 2.2 Structure of (A) betalamic acid; (B) Betaxanthins and (C) Betacyanins.
Source: Herbach et al. (2006a)
24 2.3.1 Amaranth
Betalains occur only in the plants from 10 families of the order Caryophyllales (old name: Centrospermae), such as the family Amaranthaceae which includes several important genera, i.e. Amaranthus, Celosia, Gomphrena, and Iresine (Cai & Corke, 2005). Amaranth is still cultivated as a minor crop in Central Asia and Africa. Grain amaranth (Amaranthus) has developed worldwide as a new crop over the past 20 years, with good nutritional quality, strong tolerance to stress conditions (drought, salinity, alkalinity, acidic or poor soil), and high biomass yield.
For the last decade, some researchers have been conducting research and development of amaranth, including natural betalains from the plants in the Amaranthaceae (Cai et al., 1998a; 1999; 2005; Cai & Corke, 2000; Degaldo-Vagas et al., 2000). Red violet betacyanin pigments from Amaranthus genotypes produce particularly high biomass and contain high levels of pigment. Production commercial betalains depends not only on efficient processing techniques (i.e. enzymatic control, extraction, purification, concentration, and drying operations), but also on a continuous availability of highly pigmented sources (Degaldo-Vagas et al., 2000).
The betacyanins in Amaranthus tricolour were identified as amaranthin (the 5-O-[2-O-(β-D-glycopyranosyluronic acid)-β-D-glucopyranoside] of betanidine) and isoamaranthin (C-15 epimer) (Cai et al., 1998b). Amaranthine has the same basic structure-betanidin (aglycon) as the betacyanins from red beetroot. The distribution of betacyanins in 37 species of eight genera in the Amaranthaceae was investigated and the total of 16 kinds of betacyanins and three kinds of betaxanthins were isolated and characterized by Cai et al. (2001). They consist of six simple (nonacylated) betacyanins and 10 acylated betacyanins, including eight amaranthin-type pigments, six gomphrenin-type pigments and two betanin-type pigments. Acylated betanin
25
were identified as betanidin 5-O-(2'-O-(glucuronosyl) glucoside or betanidin 6-O-(- glucoside acylated with ferulic, p-coumaric or 3-hydroxy-3-methylglutaric acids.
Three new types of betaxanthins were isolated from three Celosia species in the Amaranthaceae and identified to be immonium conjugates of betalamic acid with dopa-mine, 3-methoxytyramine and (S)-tryptophan (Schliemann et al., 2001). In Amaranthus, the colour of red-violet was performed by betacyanins, like red beetroot betalains, are susceptible to temperature and also affected by pH, light, air and water activity, with better pigment stability at lower temperatures (<15 ˚C) in the dark and in the absence of air over the pH range 5-7, being more stable pH 5.6. The Amaranthus pigment powders were very stable at 25 ˚C, with longer half-life (t1/2 = 23.3 months) and higher pigment retention (78.2%) compared to aqueous pigment extracts (t1/2 = 1.04 months and 18.3%) after 43.5 weeks storage (Cai et al., 2005).
2.3.2 Red beetroot
In the first place, betalains are associated with red beetroot because it is not only rich in betacyanins but also the exclusive commercially exploited betalains crop. Red beetroot was proposed to be included in low-acid food items such as meat and dairy products. The main topics that needed to be addressed were the fast browning through polyphenoloxidase activities and the reduction of the naturally high nitrate content. While the first controlled by heat inactivation and oxygen removal, the latter were reduced by fermentation strategies (Stintzing et al., 2007).
Red beetroot contains abundant amounts of betalain pigments which include two main groups of compounds, namely red betacyanins and yellow betaxanthins.
The betacyanins, 75 - 95% consists of betanin together with smaller amounts of isobetanin and prebetanin; the main betaxanthin derivatives are vulgaxanthin I and II.
