LATVIA UNIVERSITY OF LIFE SCIENCES AND TECHNOLOGIES Latvijas Lauksaimniecības universitāte. Faculty of Food Technology Pārtikas tehnoloģijas fakultāte

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1 LATVIA UNIVERSITY OF LIFE SCIENCES AND TECHNOLOGIES Latvijas Lauksaimniecības universitāte Faculty of Food Technology Pārtikas tehnoloģijas fakultāte Mg.ing.so. Galina Zvaigzne Dissertation EFFECT OF UHT PROCESSING ON THE BIOACTIVE COMPOUNDS AND ANTIOXIDANT CAPACITY IN ORANGE AND SEA BUCKTHORN JUICES UHT APSTRĀDES IETEKME UZ BIOLOĢISKI AKTĪVĀM UN ANTIOKSIDANTU AKTIVITĀTI APELSĪNU UN SMILTSĒRKŠĶU SULĀS For acquiring of the scientific degree (Dr.sc.ing) in Engineering sciences in the field of Food science and subfield of Food quality Supervisor of doctoral dissertation prof. Dr.sc.ing. Daina Karklina Scientific adviser of the doctoral dissertation prof. Dr.habil.sc.ing. Lija Dukalska Dissertation autor Jelgava 2018

2 ANNOTATION The Doctoral dissertation by Galina Zvaigzne EFFECT OF UHT PROCESSING ON THE BIOACTIVE COMPOUNDS AND ANTIOXIDANT CAPACITY IN ORANGE AND SEA BUCKTHORN JUICES The doctoral dissertation was elaborated during the period from 2009 to 2015 in the Biofresh S.A. juice and concentrate production company, in Laconia, Greece, Investigate Consulting Research Laboratory UBF GmbH in Altlandsberg, Germany, Laboratory of the Faculty of Food Technology of the Latvia University of Agriculture, Jelgava and Laboratory of Institute of Horticulture, Latvia University of Agriculture, Dobele. The hypothesis of the doctoral dissertation: thermal processing of orange and sea buckthorn juices by Ultra Hight Temperature (UHT) treatment retains the bioactive compounds, antioxidant capacity and ensures unaffected sensory attributes. Object of the research orange fruits (Citrus sinensis L.), orange juices of Greek summer variety Valencia and winter variety Navel and sea buckthorn ( Leikora, Hergo, Botanicheskaya-Lubitelskaya ) juices. To prove the hypothesis, the aim of the doctoral dissertation was to investigate the influence of UHT processing on the chemical parameters, bioactive compounds and antioxidant capacity of orange and sea buckthorn juices. The following objectives of the research were set to reach the aim of the doctoral dissertation: 1. to determine chemical parameters and bioactive compounds in fresh orange juices from two varieties of orange fruits (winter s Navel and summer s Valencia) during maturation and harvesting time; 2. to evaluate the effect of High Temperature Short Time (HTST) processing on chemical parameters, bioactive compounds and antioxidant capacity in not from concentrate (NFC) and reconstituted from concentrate (OJFC) orange juices; 3. to study the changes of the chemical parameters, bioactive compounds and antioxidant capacity of pasteurized not from concentrate (NFC) orange juices in aseptic packaging during one year refrigerated storage; 4. to investigate the Ultra-High Temperature (UHT) processing impact on the stability of chemical parameters, bioactive compounds and antioxidant capacity in orange juice compared with HTST processing; 5. to find out the effect of UHT processing on chemical parameters, bioactive compounds and antioxidant capacity in sea buckthorn juices and blended orange-sea buckthorn juices; 6. to estimate the sensory attributes of orange juice and blended orange-sea buckthorn juices produced by UHT treatment. The doctoral thesis consists of three chapters. Chapter 1. Descriptionof the origin of orange fruits in the global, European and Latvian market; factors influencing the quality of orange juice, physicochemical parameters, bioactive compounds and antioxidant potential of fresh squeezed and pasteurised orange juices; orange juice categories and their characteristics, the description of orange juice production process, the effect of pasteurisation, packaging and the role of storage in retention of the orange juice quality. Chapter 2. Description of materials, methods and statistical processing of data used in the doctoral dissertation. Chapter 3. Summary of the results and discussion. Orange juice physicochemical parameters, bioactive compounds and antioxidant capacity importance in harvesting time of 2

3 two varieties of Greek oranges; the winter variety Navel and summer variety Valencia were studied for two years. The processing technology, storage temperature and time influence on chemical parameters, bioactive compounds and antioxidant capacity of NFC orange juice in industrial scale. The chemical parameters, bioactive compounds and antioxidant capacity of orange juice effect of UHT processing in comparison with HTST processing as well impact of the UHT processing on chemical parameters, bioactive compounds and antioxidant capacity in sea buckthorn and blended orange-sea buckthorn juices. The doctoral dissertation is written in English on 112 pages and includes 16 tables, 58 figures and additional materials, such as 16 appendices in the research process. 236 information sources were used in this research. 3

4 ANOTĀCIJA Doktorantes Gaļinas Zvaigznes promocijas darbs: UHT APSTRĀDES IETEKME UZ BIOLOĢISKI AKTĪVĀM VIELĀM UN ANTIOKSĪDANTU AKTIVITĀTI APELSĪNU UN SMILTSĒRKŠĶU SULĀS Promocijas darbs tika izstrādāts sulu un sulas koncentrāta rūpnīcā S.A 'Biofresh' Grieķijā, zinātniskajā laboratorijā UBF GmbH Vācijā, Latvijas Lauksaimniecības Universitātes Pārtikas Tehnoloģijas fakultātes laboratorijā, Jelgavā un Latvijas Valsts Augļkopības institūtā Dobelē laika posmā no 2009 līdz 2015 gadam. Promocijas darba hipotēze: ultrapasterizācijas temperatŗas (Ultra-High Temperature - UHT) apstrādes procesā apelsīnu un smiltsērkšķu sulās saglabājas bioloģiski aktīvās vielas, antioksidantu aktivitāte, kā arī sulu raksturojošās sensorās īpašības. Promocijas darba mērķis: izvērtēt ultrapasterizācijas temperatūras (UHT) apstrādes procesa ietekmi uz bioaktīvajām vielām un antioksidantu aktivitāti apelsīnu, smiltsērkšķu un jauktās apelsīnu-smiltsēršķu sulās. Darba mērķa sasniegšanai izvirzīti šādi uzdevumi: 1. noteikt ķīmiskos parametrus un bioaktīvās vielas svaigā sasaldētā pēc tam atkausētā apelsīnu sulā, kas iegūta no ziemas Navel un vasaras Valencia šķirņu apelsīnu augļiem novākšanas laikā dažādās gatavības stadijās; 2. analizēt augstas temperatūras īslaicīgas pasterizācijas (HTST) ietekmi uz ķīmiskajiem parametriem, bioaktīvo vielu un antioksidantu aktivitāti ne no koncentrāta (NFC) un no koncentrāta atjaunotās (OJFC) apelsīnu sulās; 3. izpētīt ne no koncentrāta (NFC) augstā temperatūrā īslaicīga laika (HTST) pasterizētas apelsīnu sulas ķīmisko parametru, bioloģiski aktīvo vielu un antioksidantu aktivitātes izmaiņas uzglabāšanas laikā aseptiskā iepakojumā atdzesētā vidē; 4. vērtēt UHT un HTST apstrādes metožu ietekmi uz ķīmisko parametru, bioloģiski aktīvo vielu un antioksidantu aktivitāti apelsīnu sulās; 5. skaidrot UHT procesa ietekmi uz bioaktīvām vielām un antioksidantu aktivitāti smiltsērkšķu sulās un apelsīnu-smiltsērkšķu sulu maisījumos; 6. sensori izvērtēt UHT režīmā apstrādātu apelsīnu un jauktu apelsīnu-smiltsērkšķu sulu īpašības. Promocijas darbs strukturēts 3 nodaļās: 1. nodaļa. Apelsīnu augļi un to izcelsmes apraksts, Eiropas un Latvijas sulu tirgus apskats, sulas kvalitāti ietekmējošie faktori, fizikāli ķīmiskās īpašības, bioaktīvo vielu un antioksidantu esamība svaigi spiestā un pasterizētā sulā, apelsīnu sulu iedalījums un to raksturojums, apelsīnu sulas ražošanas procesa apraksts, pasterizācijas ietekme, iepakojums un uzglabāšanas ietekme uz apelsīnu sulas kvalitāti. 2. nodaļa. Pētījuma materiāli un darbā lietotās metodes. 3. nodaļa. Pētījuma rezultāti un diskusija. Apelsīnu sulu fizikāli ķīmiskie parametri, bioaktīvie savienojumi un antioksidantu aktivitāte divu Grieķu apelsīnu šķirņu, vasaras Valencia un ziemas Navel divigadi tika pētītās apelsīnu novākšanas laikā; Apstrādes tehnoloģijas, uzglabāšanas temperatūres un laika ietekme uz ķīmiskājiem parametriem, bioaktīvajām savienojumiem apelsīnu sulā rūpnieciskā mērogā; UHT apstrades processa ietekme uz ķīmiskajiem parametriem, bioaktīvām vielam un antioksidantu aktivitāti salīdzinājumā ar HTST apstrādi, kā arī UHT apstrādes ietekme uz ķīmiskajiem parametriem, bioaktīvam vielam un antioksidantu aktivitāti smiltsērkšķu un sajauktajās apelsīnu smiltsērkšķu sulās. Promocijas darbs ir uzrakstīts angļu valodā un tas sastāv no 112 lappusēm, tajā iekļautas 16 tabulas un 58 attēli. Darbs papildināts ar 16 pielikumiem. Pētījumā izmantoti 236 literatūras avoti. 4

5 CONTENTS INTRODUCTION THE BIOCHEMISTRY OF ORANGE FRUIT AND OBTAINED JUICES, AND THEIR SUITABILITY FOR THE MODERN TECHNOLOGY The origin of orange in the global, European and Latvian market Factors influencing the quality of orange juice The physicochemical characteristics, bioactive compounds and the natural antioxidants in orange juice Orange juice categories and their characteristics Influence of the technological process on the orange juice quality Orange juice technology process description and flowchart Effect of pasteurisation on chemical characteristics and bioactive compounds of orange juice Advances in pasteurisation technology of orange juice Packaging and the role of storing in orange juice quality Possibility to improve orange juice by blending with other juices and expand the range MATERIALS AND METHODS Time and place of the research Characteristics of the material The structure of the research Evaluation of physicochemical parameters, bioactive compounds and antioxidant capacity in different maturity stage, processing and storage of two varieties of orange juice in industrial scale Evaluation of UHT processing on the chemical characteristics, bioactive compounds and antioxidant capacity of orange juice Evaluation of UHT processing on the chemical parameters bioactive compounds and antioxidant capacity of seabuckthorn juices and orange-seabuckthorn blended juices Methods for determination of chemical parameters, bioactive compounds and antioxidant capacity Method of sensory analysis Data mathematical analysis

6 3. RESULTS AND DISCUSSION Evaluation of the quality parameters in winter s Navel and summer s Valencia varieties of oranges, grown in Greece during harvesting in industrial scale Evaluation of physicochemical parameters of fresh orange juice during harvesting within two years Evaluation of chemical parameters, bioactive compounds and antioxidant capacity of fresh defrosted orange juice on different maturity stage Evaluation the influence of processing methods on thechemical parameters, bioactive compounds and antioxidant capacity of orange juice The dynamic of chemical parameters, bioactive compounds and antioxidant capacity of pasteurised NFC orange juice during refrigerated storage UHT processing effect on chemical parameters, bioactive compounds and antioxidant capacity of orange juice compared with HTST processing Impact of UHT processing on chemical parameters, bioactive compounds and antioxidant capacity in sea buckthorn juices and blended orange-sea buckthorn juices UHT processing effect on chemical parameters, bioactive compounds, and antioxidant capacity of sea buckthorn juices UHT processingeffect on chemical parameters, bioactive compounds and antioxidant capacity in blended orange-sea buckthorn juices The sensory evaluation of orange juice and orange seabuckthorn juices processed by UHT treatment CONCLUSION REFERENCES

7 APPROBATION OF THE SCIENTIFIC WORK Research results have been summarised and published in 11 peer reviewed scientific issues. Publications 1. Zvaigzne G., Karklina D., Moersel J.T., Kuehn S., Krasnova I., Seglina D. (2017) Impact of UHT on bioactive compounds and sensory attributes of orange juice comparison with traditional processing. Proceedings of the Latvian Academy of Sciences. Section B., Vol.71, No.6, p DOI: /plalas Zvaigzne G., Moersel J.T., Kuehn S., Karklina D., Krasnova I., Seglina D. (2015) Effect of Processing Techniques on Sea buckthorn Juice and Orange -Sea buckthorn Beverages. In: Singh V., et al. (eds) Sea buckthorn: Emerging Technologies for Health Protection and Environmental Conservation Sea buckthorn. Published by Dr. Virendra Singh, CSK Himachal Pradesh Agricultural University, Palampur , Himachal Pradesh, India, p ISBN (HB) 3. Zvaigzne G., Moersel J.T., Tsirenova E. (2014) Health promotion chemical components of Sea buckthorn and Orange. In: Moersel J.T. Zubarev Y., Eagle D. (eds) Sea buckthorn. Research for promising crop. BoD Books on Demand, Norderstedt, Ltd. Publishers, p ISBN Zvaigzne G., Karklina D., Papadimitrakopoulos C. (2013) Biochemical characteristic of orange in maturity time. In: Proceeding of the X International Conference Innovations in Science, Education and Business 2012, September 2013, Kaliningrad, Russia. Kaliningrad State Technical University. Kaliningrad, Калининград, Часть 1, с ISSN Zvaigzne G., Moersel J.T., Tsirenova E. (2013) Health promotion chemical components of sea buckthorn and orange. In: The 6 th Conference of the International Sea buckthorn Association SBT a fresh look at Technology, health and environment, October, Potsdam, Germany. Brandenburg: p Zvaigzne G., Karklina D. (2013) The effect of production and storage on the content of vitamin C in NFC orange juice. In: Proceeding of the19 th Annual InternationalScientific Conference Research for Rural Development 2013, May 2012, Jelgava. Latvia University of Agriculture. Jelgava: LLU, Latvia. Vol.1, p. ISSN Zvaigzne G., Karklina D., Papadimitrakopoulos C. (2012) Some biochemical characteristic of orange juice. In: Proceeding of the X International Conference Innovations in Science, Education and Business 2012, October 2012, Kaliningrad, Russia. Kaliningrad State Technical University. Kaliningrad.Известия КГТУ: научный журнал. - No 29 (2013), с ISSN Zvaigzne G., Karklina D. (2012) Health Promotion Chemical components of Orange Juice. Proceedings of the Latvian Academy of sciences. Section B, Vol.67. pp ISSN X, DOI: 9. Zvaigzne G., Karklina D. (2010) Orange in maturity effect on juice quality. In: Proceeding of the5 th Baltic Conference onfood Science and Technology, FOODBALT- 2010, October 2010 Tallinn, Estonia University of Technology. Tallinn: TTU, p ISSN Zvaigzne G., Karklina D., Seglina D., Krasnova I. (2009) C vitamin and polyphenol content in various citrus fruit juices In: Proceeding of the 8 th International Conference FOODBALT-2009, May 2009, Kaunas, Lithuania University of Technology. Kaunas: Chemine Technologija. p. 75. ISSN Zvaigzne G., Karklina D., Seglina D., Krasnova (2009) Antioxidants invarious citrus fruits I.Cheminė technologija, Vol. 3 (52), p

8 The results of the research work have been presented at 15 international scientific conferences, congresses and seminars in Latvia, Estonia, Lithuania, Germany, Russia and India. Presentations Zvaigzne G. (2016) Impact of UHT on bioactive compounds and sensory attributes of orange juice comparison with traditional processing // G. Zvaigzne, D. Karklina, J.T. Moersel, S. Kuehn, I. Krasnova, D. Seglina // 2 nd International Conference Nutrition and Health. October 5-7, Riga, Latvia (oral presentation). 2. Zvaigzne G. (2016) Chemical composition of seabucthorn leaves, branches and buts // I. Grad, S. Kuhn, J.T. Morsel, G. Zvaigzne // 4 th European Workshop on Seabuckthorn Euro Works Augusts 17-19, Riga, Latvia (oral presentation). 3. Zvaigzne G. (2015) Effect of Processing Techniques on Seabuckthorn Juice and Orange-Seabuckthorn Beverages. //G. Zvaigzne, J.T. Moersel, S. Kuehn, D.Karklina, D. Seglina, I. Krasnova // 7 th International Seabuckthorn Association conference Seabuckthornn - Emerging Technologies for Health Protection and Environmental Conservation. November 24-26, New Deli, India (oral presentation). 4. Zvaigzne G. (2013) Health promotion chemical components of seabuckthorn and orange // G. Zvaigzne, J.T. Moersel, E.Tsirenova // 6 th International Seabuckthorn Association conferences SBT a fresh look at Technology, health and environment. October 14-17, Potsdam, Germany (oral presentation). 5. Zvaigzne G. (2013) Biochemical characteristic of orange in maturity time. // G. Zvaigzne, D. Karklina, C. Papadimitrakopoulos // XI International Conference Innovations in Science, Education and Business September 25-27, Kaliningrad, Russia (oral presentation). 6. Zvaigzne G. (2013) The effect of production and storage on the content of vitamin C in NFC orange juice. // G. Zvaigzne, D. Karklina // 19 International Scientific conference Research for Rural Development. May 17-19, Jelgava, Latvia (oral presentation). 7. Zvaigzne G. (2012) Some biochemical characteristic of orange juice. // G. Zvaigzne, D. Karklina, C. Papadimitrakopoulos// X International Conference Innovations in Science, Education and Business 2012, October 17-19, Kaliningrad, Russia, (oral presentation). 8. Zvaigzne G. (2012) Health Promotion Chemical components of Orange Juice. // G. Zvaigzne, D. Karklina // International Conference Nutrition and Health, September 4-6, Riga, Latvia (oral presentation). 9. Zvaigzne G. (2012) Quality parameters of orange NFC in maturity time. //G. Zvaigzne, D. Karklina // 18 International Scientific conference Research for Rural Development. May 16-18, Jelgava, Latvia (oral presentation). 10. Zvaigzne G. (2012) Impact of processing in orange juice quality. // G. Zvaigzne, Karklina D., Papadimitrakopoulos C. 1 st North European Congress on Food NEFood April 22-24, Sankt Petersburg, Russia (oral presentation). 11. Zvaigzne G. (2011) Importance of citrus flavonoids for human nutrition. // G. Zvaigzne, D. Karklina, C. Papadimitrakopoulos// 7 th International Scientific Conference Students on their Way to Science, May 25, Jelgava, Latvia (oral presentation). 12. Zvaigzne G. (2011) Pectin chemistry and its commercial uses. // G. Zvaigzne, D. Karklina, C. Papadimitrakopoulos // 6 th International Scientific Conference Students on their Way to Science, May 27, Jelgava, Latvia (oral presentation). 13. Zvaigzne G. (2010) Orange in maturity effect on juice quality // G. Zvaigzne, D. Karklina // 5 th International Baltic Conference on Food Science and Technology Food Balt October 29-30, Tallinn, Estonia (poster presentation). 8

9 14. ZvaigzneG. (2010) Quality changes in orange harvesting time and process // G. Zvaigzne, D. Karklina, C. Papadimitrakopoulos // 5 th International Scientific Conference Students on their Way to Science. May 28 Jelgava, Latvia (oral presentation). 15. ZvaigzneG. (2009) Antioxidant various citrus fruit juices // G. Zvaigzne, D. Karklina, D. Seglina, I. Krasnova // 8 th International Conference Food Balt May 12-13, Kaunas, Lithuania (oral presentation). 9

10 ACKNOWLEDGEMENTS Completing this thesis would not have been possible without the time and expertise of the many people who helped me every step of the way. My sincere gratitude goes to all, who supported and guided me. First, I would like to thank the head of the project, Prof. Dr. Daina Karklina, whose profound understanding of the field was an invaluable asset in helping to construct the thesis. It was her guidance and genuine interest in the subject that served as a great source of encouragement for my progress. For their guidance, support and great contact, I would like to thank Faculty of Food Technology and lecturers of Latvian University of Agriculture. I would like to express my great appreciation to Dr. Joerg - Thomas Moersel, Associate Professor at Technical University of Berlin and at University of Applied Sciences in Neubrandenburg and the Director of Scientific Research Institute UBF GmbH. in Germany, who has provided a number of consultations and helped with so many experiments. And I am particularly grateful for the assistance given by the fantastic laboratory team, their cooperation and brilliant ideas and who made sure of the progress in the practical part of this thesis. I would like to thank the Institute of Horticulture of the Latvian Agricultural University Dr. Dalia Seglina the Senior Research Fellow the Director of Technology and Biochemistry and the Research Associate, Dr. Inta Krasnova. I would like to thank you for the excellent cooperation, helps and opportunities for research I wish to acknowledge the help provided by Cristos Papadimitrokopolos the owner of the Greek company BIOFRESH and my former colleagues. Without them there would be no data and it would be impossible to finish the research or the final project. Finally, I wish to thank my family and friends for their support and encouragement throughout my study. 10

11 LIST OF TABLES INCLUDED IN DOCTORAL THESIS Table number Table title P. 1.1 EU-28 Imports and exports of orange juice market year 2015/ Reducing and non-reducing and total sugar contain in citrus fruits Organic acid contain in juice commercial citrus fruits AIJN quality requirements for orange juice RSK* values for orange juice Requirements for USDA grade A orange juice Orange fruit characteristics Characteristic of industrial scale and laboratory pilot type equipment used 47 for experiments 2.3 Standards and analytical methods used for determination of orange juice Chemical parameters of fresh defrosted orange juice Valencia and Navel 62 in different maturity stage; early stage (A), mid (B, C) and end (D) of the seasons 3.2 Dynamics of total soluble solids (TSS) and total acidity (TA) content in 74 pasteurised (HTST) orange juices during storage at 5 C± 2 C 3.3 UHT and HTST processing methods effects on chemical parameters of 81 orange Navel juice 3.4 Chemical characteristics of fresh and UHT treated sea buckthorn juices Antioxidant capacity in fresh and UHT processed sea buckthorn juice Chemical characteristics of mixed orange - sea buckthorn juices processed by UHT 91 11

12 LIST OF THE FIGURES INCLUDED THE DOCTORAL THESIS Figure number The title of the figures P The structure of an orange Products derived from oranges Production share of single strength orange juice in the global market Fruit juice and nectars according to the flavour demand in Latvia 2015/ (A.I.J.N.,2016) 1.5. Fruit juice and nectars according to the flavour demand in Lithuania /2016 (A.I.J.N.,2016) 1.6. Fruit juice and nectars according to the flavour demand in Estonia /2016 (A.I.J.N.,2016) 1.7. Chemical antioxidant mode of action Chemical phytochemical mode of action Chemical structure of Hesperidin and Naringin Structure of ß-carotene Tocopherol structure Structure of pectin molecular Production steps for orange juice Flow diagram of a tubular evaporator Factors influencing juice quality Vitamin C degradation curves for different packages of orange juice stored 40 at 23 C Effect of temperature on vitamin C content in Orange juice during storage General scheme of orange juice production Evaluation of physicochemical and bioactive compounds and antioxidant 50 capacity of two varieties of orange juice during maturation, processing and refrigerated storage in industrial scale 2.3. UHT and HTST processing orange juice quality parameters determination UHT processing of sea buckthorn and blended orange-sea buckthorn juice 53 quality parameters determination 3.1. The fresh juice content in the Valencia and Navel orange fruits during 58 harvesting 3.2. The content of TSS in fresh Valencia and Navel orange juices during 59 harvesting 3.3. The content of total acidity in the Valencia and Navel orange juices during 60 harvesting 3.4. The content of vitamin C in the Valencia and Navel orange juices during 61 harvesting 3.5. The content of individual sugars in fresh defrosted Navel and Valencia varieties orange juices on maturity early stage (A), mid (B, C) and end (D) of the season 63 12

13 Continuation List of the figures 1 Figure number The title of the figures P The content of vitamin C in fresh defrosted Navel and Valencia varieties 64 orange juices on maturity early stage (A), mid (B, C) and end (D) of the season 3.7. The content of total phenolics compounds and hesperidin in fresh defrosted 65 Navel and Valencia varieties orange juices on maturity early stage (A), mid (B, C) and end (D) of the season 3.8. The content of total carotenoids in fresh defrosted Navel and Valencia 66 varieties orange juices on maturity early stage (A), mid (B, C) and end (D) of the season 3.9. The content of ß-carotene in fresh defrosted Navel and Valencia varieties 66 orange juices on maturity early stage (A), mid (B, C) and end (D) of the season The content of water-soluble pectin in fresh defrosted orange juices 67 Valencia and Navel orange varieties on maturity early stage (A), mid (B, C) and end (D) of the seasons The values of antioxidant capacity in fresh defrosted Navel and Valencia 68 varieties orange juices on maturity early stage (A), mid (B, C) and end (D) of the season The content of vitamin C in fresh defrosted (Control), pasteurised not from 70 concentrate (NFC) and reconstituted pasteurised from concentrate (OJFC) orange juices Navel and Valencia The content of total phenolics in fresh defrosted (Control), pasteurised not 71 from concentrate (NFC) and reconstituted pasteurised from concentrate (OJFC) Navel and Valencia orange juices The content of total carotenoids and ß-carotene in fresh defrosted (Control), 71 pasteurised not from concentrate (NFC) and reconstituted pasteurised from concentrate (OJFC) Navel and Valencia orange juices The content of WS pectin in fresh defrosted (Control), pasteurised not from 72 concentrate (NFC) and reconstituted pasteurised from concentrate (OJFC) Navel and Valencia orange juices The content of antioxidant capacity (ABTS) in fresh defrosted (Control), 73 pasteurised not from concentrate (NFC) and reconstituted pasteurised from concentrate (OJFC) Navel and Valencia orange juices The dynamics of vitamin C content in pasteurised orange Valencia and 75 Navel NFC juices during storage at 5 ± 2 C The dynamics of total phenolics compounds in pasteurised orange Valencia 76 and Navel NFC juices during storage at 5 ± 2 C The dynamics of hesperidin content in pasteurised orange Valencia and 77 Navel NFC juices during storage at 5 ± 2 C The dynamics of total carotenoids and ß-carotene in pasteurised orange 78 Valencia and Navel NFC juices during storage at 5 ± 2 C The dynamics of water-soluble pectin content in pasteurised orange 79 Valencia and Navel NFC juices during storage at 5 ± 2 C The dynamics of antioxidant capacity (ABTS) in pasteurised orange Valencia and Navel NFC juices during storage at 5 ± 2 C 79 13

14 Continuation List of the figures 2 Figure number The title of the figures P UHT and HTST processing effect on Vitamin C content in Navel orange juice UHT and HTST processing effect on total phenolics compounds and 83 hesperidin content in Navel orange juice UHT and HTST processing methods effect on total content of carotenoids in 84 Navel orange juice UHT and HTST processing methods effect on the antioxidant capacity 85 measured by means of ABTS in Navel orange juice UHT and HTST processing methods effect on antioxidant capacity 86 measured by FRAP and DPPH in Navel orange juice The content of vitamin C in fresh and UHT processed sea buckthorn juices The content of total phenolics compounds in fresh and UHT processed sea 88 buckthorn juices The content of total carotenoids and vitamin E in fresh and UHT processed 89 sea buckthorn juices Content of vitamin C in blended orange-sea buckthorn juices and orange 91 (control) juice processed by UHT Content of total carotenoids and vitamin E in blended orange-sea buckthorn 92 juices and orange (control) juice processed by UHT Content of total phenolics compounds in blended orange-sea buckthorn 92 juices and orange (control) juice processed by UHT Antioxidant capacity measured by means of ABTS in blended orange-sea 93 buckthorn juices and orange (control) juice processed by UHT Antioxidant capacity measured by means of and FRAP in blended orangesea 93 buckthorn juices and orange (control) juice processed by UHT Results of hedonic evaluation of fresh frozen then defrosted (Control), 95 HTST and UHT processed Navel orange juice Results of hedonic evaluation of blended orange-sea buckthorn juices processed by UHT 95 14

15 LIST OF THE ACRONYMS, TERMS AND ABBREVIATIONS AND INTERPRETATIONS USED IN THE DOCTORAL THESIS AIJN Association of the Industry of Juices and Nectars ANOVA analysis of variance AOAC Association of Official Analytical Chemists BC "before Christ" Bi B Bag-in-Box C degree of Celsius cis cis isomer cm Centimetre DF dietary fibre DM dry matter DNA deoxyribonucleic acid EBM extrusion blow-moulding et al./etc. and others EVON ethylene vinyl alcohol EU European Union FAO Food and Agricultural Organization FAOSTAT Food and Agricultural Organization Statistics Fapr. Fisher s criteria FCOJ Frozen Concentrated Orange Juice fig. Figure Fkrit. critical value of Fisher s criteria FRAP Ferric reducing antioxidant power g Gram GAE gallic acid equivalents h Hour HCl hydrochloric acid HDPE high-density polyethylene HPT high pressure technique HPLC high- performance liquid chromatography HTST High Temperature Short Time i.e. id est. Inc. including ISO International Standard Organization IUB International Union of Biochemistry IU International unit IUPAC International Union of Pure and applied Chemistry l Litre LDPE low-density polyethylene LLU Latvia University of Agriculture LSIFG Latvia State Institute of Fruit growing LVS Latvian State Standard µg Microgram mg 100 ml -1 mili grams per 100 millilitre min Minute ml Millilitre n number of samples NFC Not From Concentrate n.i. no information n.n. not detected 15

16 No. numbers in sequential order NSP non starch polysaccharide OJFC Orange Juce From Concentrate (reconstituted) p. Page p p-value PA Polyamide PET E polyethylene terephthalate PP Polypropylene ph the negative logarithm of hydrogen ion concentration ppm parts per million r correlation coefficient RDA Resource Description and Access RSK Richtwert Schwankungsbreiten und Kennzahlen von Fruchtsäften RTD Ready-to-drink PTF Faculty of Food Technology s Second IT the value of integrated or multicriterial evaluation (coefficient) for particular cultivar SP standard deviation SPSS statistical package for social science tab. Table trans trans isomer UHT Ultra-High Temperature UK United Kingdom USA United States of America UV ultraviolet lamp v/v proportion of volumes Vol. Volume w/w ratio of weight WHO World Health Organization 16

17 INTRODUCTION The consumption of fruit juices and nectars has increased in recent years, mainly because of the higher consumers awareness about the importance of choosing healthy foods in reducing the risks of developing diseases and improving quality of life (Carbonell-Capella et al., 2015). Many investigators made sure that orange fruits and orange juices have long been appreciated for their beneficial nutrients and antioxidant properties. The bioactive compounds has been studied in numerous studies and tests (Burns et al, 2003; Cassano et al., 2003; Gardner et al., 2000; Kurowska et al., 2000; Lichtenthaler, Marx, 2005; Topuz et al., 2005). Numerous studies have shown that not only biologically active substances but also such as soluble and insoluble dietary fibers in oranges are effective in reducing the risk of cancer, obesity and many other chronic diseases (Bazzano et al., 2002; Borradaile et al., 2002; Liu et al., 2001; Miyagi et al., 2000; Poulose et al., 2005; Slattery et al., 2000). In the food industry still use thermal processing methods such as pasteurisation, sterilisation and evaporation to guarantee microbial safety of food. Currently the food industry is looking for replacing the traditional well-established preservation techniques with advanced thermal and non-thermal technologies, which may produce high quality food products with improved energy efficiency and to be more environmentally friendly (Kulwant et al., 2012). Latvian producers of orange juice supply customers mainly with juices from Frozen Concentrated Orange Juice (FCOJ). However, in the recent years, consumers have increasingly sought for so-called ''fresh'' products such as fresh juice. Pasteurised orange juice not from concentrate (NFC) is preferable in taste to reconstituted juices, and consumers prefer orange juice NFC because of it organoleptic characteristics. With the development of modern technologies and packaging material, it is now becoming a reality to produce and deliver NFC juice to European countries and increase production volumes of orange juice to the levels of fresh juice. The Ultra-High Temperature (UHT) technology is an attractive technology to extend the shelf life and safety of orange juice while maintaining of fresh test of orange juice. Nevertheless, no researches have integrated the comparative study of the impact of UHT processing on the chemical parameters and bioactive compounds of orange juice. The review of the situation gives a great opportunity to formulate the doctoral dissertation hypothesis. The hypothesis of the doctoral dissertation: thermal processing of orange and sea buckthorn juices by Ultra Hight Temperature (UHT) treatment retains the bioactive compounds, antioxidant capacity and ensures unaffected sensory attributes. The hypothesis of the doctoral dissertation is supported by the following theses: 1. the physicochemical characteristics and bioactive compounds and antioxidant capacity of orange juice depend on orange variety, maturation and harvesting time; 2. the processing technology of orange juices influence on the chemical parameters, bioactive compounds and antioxidant capacity; 3. the storage conditions and packaging affect the chemical parameters, bioactive compounds and antioxidant capacity in orange juices; 4. UHT treatment better provides the chemical parameters, bioactive compounds and antioxidant capacity of orange juice compared with High Temperature Short Time (HTST) processing method; 5. bioactive compounds and antioxidant capacity in orange juice increase by blending it with sea buckthorn juice; 6. UHT processing provides organoleptic qualities of treated juices comparable to fresh juice. 17

18 The research object of the doctoral dissertation: orange fruits (Citrus sinensis L.) and orange juices of Greek winter s variety Navel and summer s variety Valencia variety and sea buckthorn (Hippophae rhamnoides L.) Leikora, Hergo, Botanicheskaya Lubitelskaya juices. The aim of the doctoral dissertation was to investigate the influence of UHT processing technology on the chemical parameters, bioactive compounds and antioxidant capacity of orange, sea buckthorn and blended orange-sea buckthorn juices. The following research objectives were set to reach the aim of the doctoral dissertation: 1. to determine chemical parameters and bioactive compounds in fresh orange juices from two varieties of orange fruits (winter s Navel and summer s Valencia) during maturation and harvesting time; 2. to evaluate the effect of High Temperature Short Time (HTST) processing on chemical parameters, bioactive compounds and antioxidant capacity in not from concentrate (NFC) and reconstituted from concentrate (OJFC) orange juices; 3. to study the chemical parameters, bioactive compounds and antioxidant capacity changes in pasteurized NFC orange juices in aseptic packaging during one year refrigerated storage; 4. to investigate the UHT processing impact on the stability of chemical parameters, bioactive compounds and antioxidant capacity in orange juice compared with HTST processing; 5. to find out the effect of UHT processing on chemical parameters, bioactive compounds and antioxidant capacity in sea buckthorn juice and blended orange-sea buckthorn juices; 6. to estimate the sensory attributes of orange juice and blended orange-sea buckthorn juices produced by UHT treatment. The novelty and scientific significance of the doctoral dissertation: 1. for the first time in Latvia the quality parameters of orange juice has been investigated during harvesting at maturation and degree of readiness time; 2. the influence of different processing technologies and refrigerating storage on the chemical parameters, bioactive compounds and antioxidant capacity of orange juice in industrial scale has been evaluated; 3. the possibility of UHT processing technology for orange and sea buckthorn juices are explored for the first time; 4. new products (orange-sea buckthorn juices) with high level of bioactive compounds content have been developed using UHT treatment. The economic significance of the doctoral dissertation: 1. the studies of ultra-high temperature (UHT) processing suitability for orange juice sterilization offers an opportunity to deliver orange juice not from concentrate (NFC) out of producer foreign countries to Latvia for industrial scale filling in consumer packaging; 2. Ultra-high temperature (UHT) processing can be an alternative to high temperature short term (HTST) pasteurization of orange, sea buckthorn and blended orange-sea buckthorn juices. Mentioned processing technology creates denaturation of enzymes and micro-organisms inactivates occurs juice with extended shelf life and good sensory properties (taste and odour) could be obtained; 3. Orange-sea buckthorn blended juices expands product range in the domestic juice market with high level of bioactive compounds and antioxidant capacity. The present study is a sign of a bright prospect in terms of processing blended fruit juices by UHT processing. 18

19 1. THE BIOCHEMISTRY OF ORANGE FRUIT AND OBTAINED JUICES AND THEIR SUITABILITY FOR THE MODERN TECHNOLOGY 1.1. The origin of orange in the global, European and Latvian market Citrus is botanically a large family, its principal members are the sweet orange (Citrus sinensis), mandarin or tangerine orange (Citrus reticulata), grapefruit (Citrus paradisi), lemon (Citrus limon) and lime (Citrus aurantifolia). Oranges consist of bitter (sour) orange (Citrus quarantium), sweet orange (C. sinensis), and mandarin orange (C. reticulata), and they are cultivated in most parts of the world with over 100 orange varieties of commercial importance (Ladaniya, 2008). Also in other literary publications is given the classification of citrus fruits (Braddock, 1999; Kulwant et al., 2012; Nagy et al., 1993). Citrus fruit growth and the physicochemical parameters, bioactive compounds and other fruit quality parameters depends on many factors as well as climatic conditions, vatietiy of fruits, water presence and cultural practices. Flowering in citrus, under subtropical climate, lasts for a month or so. Before the harvest fruit stores different metabolites which are later utilized and some more metabolites are formed after harvest as the development takes place towards maturity and senescence. The best quality fruits are grown in the Mediterranean characterised by its relatively dry climate, hot summers, and cool wet winters (winter rains) at fruit maturity. Citrus fruits are non-climacteric; they have already done most of their ripening on the plant and will slowly begin to rot after they are picked. Non-climacteric fruits also ripen without the release of ethylene and boost in cellular respiration accompanied by major changes in flavour and biochemical composition after harvest in relation to ripening. Citrus fruits progressively become edible on the tree and remain so (beyond normal harvest time) for 5-6 months (in case of oranges). This maturity period varies with the harvesting season (Ladaniya, 2008). It has long been known that in many regions of the world oranges (Citrus sinensis) are an important food source because they are delicious and nutritious fruits and them much liked the people. Oranges are generally available from winter through summer with seasonal variations depending on the varieties. For many varieties of citrus the fruit will mature from 7 months (lemon, limes) to 10 months (orange, grapefruit, tangerines) after tree flowering (Braddock, 1999; Ladanya, 2008). The sweet oranges are classified into four groups: common orange, acid less orange, pigmented orange and Navel orange. The acid less orange is of minor commercial importance and is mainly grown in Latin America. Among the common oranges there are Valencia, Pineapple, Hamlin, Parson Brown, Jaffa, and Shamouti which are cultivated in various parts of the world. The pigmented oranges are widely cultivated in the Mediterranean region. In Greece the Navel oranges are primary grown for fresh market because they normally develop a bitter taste in the processed products but sometimes take for processing. The Washington Navel is considered the most important cultivar of this group. The sweet orange varieties Blood Navel and the Common while fleshed oranges yield more juice and soluble solids when grown in conditions other than a dry Mediterranean climate. The Mediterranean region is a secondary centre of diversity in the sweet orange varieties. Another type of sweet orange called the "blood orange" or "sanguine orange" often has red marks on the skin and some parts of the inside look as if they have blood in them. Some blood oranges make juice that is ruby red (Ladaniya, 2008). From a biological point of view orange fruit is a modified berry or a specialized form of berry (hesperidium) resulting from a single ovary. Essentially orange is a ball of juice sacs protected by a waxy skin the peel. The peel consists of a thin outer layer called flavedo and a thicker fibrous inner layer called albedo. Orange coloured substances in the flavedo are called carotenoids; they give the fruit its characteristic colour. Vesicles (a small sac or cavity) containing peel oil are also present in the flavedo they contribute to the fruits fresh aroma. The white spongy albedo contains several substances including flavonoids D-limonin and pectin, which influence juice quality and if they find their way into extracted juice make a negative 19

20 impact (Kimbal, 1991). The deepest layer of the pericarp which surrounds a seed in a fruit and constitutes edible portion of the fruit is called endocarp. It consists of a central fibrous core, individual segments, segments walls and an outer membrane. The segments contain juice vesicles or juice sacs that are held together by a waxy substance. Seeds are also normally present inside the segments (The orange Book, 2004) (see Fig. 1.1). Flavedo Albedo Juice vesicles Oil sacs Central core Segment Seed Segment wall Fig The structure of an orange (Kimbal, 1999) The juice contains sugars, acids, vitamins, phenolics content, minerals, pectin and many other components. In theory the aim of the juice extraction process is to receive the maximum amount of juice from the fruits without any peel and pulp wash. In practice a compromise is made between the possible juice yield and the desired product quality. Depending on the fruit variety and local climate the orange juice yield varies from 40 to 60% by weigh. Beneficial oil from the peel is obtained during juice extraction as well as volatile flavours from the juice in the stage of processing. A variety of products that can be attained from oranges is brought in the figure below (Kimball, 1991; The Orange book, 2004) (Fig. 1.2). Pulp 30kg Peel oil 3kg Peel, rag and seeds: kg 553 kg Essence oil 0.1kg Essence aroma 1.1 kg 65 Brix concentrate 100kg Evaporated water 452kg Fig Products derived from oranges (Braddock, 1999; The Orange book, 2004) Citrus fruits are in great demand among consumers. Oranges make up to 50% of the world production of citrus fruits. According to statistics Brazil accounts for about 80% of the frozen concentrated orange juice export (FCOJ 65 Brix), followed by United States (FCOJ, 42 Brix). The estimated word production of oranges was thousand tonnes in 2016 (US FDA, 2017). Brazil ranked first in orange production ( thousand tonnes) followed by China, India, United States (Florida) (7000.0, thousand tonnes) respectively. EU orange production is concentrated in the Mediterranean region. Spain and Italy represent nearly 80 percent of the EU s total production of oranges. Market year 2016/17 EU-28 orange juice production is forecast at MT, an increase of 1.7 percent 20

21 compared to the previous year as more oranges are expected to be processed mainly due to Spanish growth (USDA, 2018). Table 1.1 shows the import and export of orange juice in the European Union. EU-28 Imports and exports of orange juice market year 2015/2016 Table 1.1 Imports of Orange Juice by Origin in MT (Brix 65) Exports of Orange Juice by destination in MT (Brix 65) Country of origin Market Year 2015/2016 Country of origin Market Year 2015/2016 Brazil Saudi Arabia Mexico Japan United States Switzerland South Africa Algeria Others Others Total imports Total exports USDA, 2016 estimate. The consumer market is currently demanding minimally processed juices with characteristics closer to fresh juice (Martınez et al., 1999; Oliveira, Oliveira, 1999). The top five countries for single strength juice production are Brazil, USA, Mexico, Spain, and Morocco (see Fig. 1.3). 7% 6% 6% Brazil U.S.A. 15% 66% Mexico Spain Morocco Fig Production share of single strength orange juice in the global market (FAOSTAT, 2014) Just like in most of the world in the Baltic States orange juice has been an important part of the people s diet for many years. Among EU countries Germany is the largest consumer of fruit juices and nectars; the volume of juice consumption amounts to 2391 million litres of which total fruit juice (100% juice content) was 1640 million litres, chilled juice constitutes 105 million litres, juice from concentrate constitutes 1295 million litres, juice not from concentrate makes up for 240 million litres, and 25-99% nectars 751 million litres. In Latvia, juice consumption amounts to 28 million litres of which: concentrate makes up for 11 million litres, not from concentrate juice constitutes 0.1 million litres, and nectars 17 million litres Association of the Industry of Juices and Nectars (AIJN) European Juice, 2016 market report). Despite the differences in tastes in the Baltic countries orange juice continues to occupy a leading position in the consumer market (see Fig ). 21

22 Latvia 25% 23% Apple Orange 7% 12% 14% 19% Tomato Grape Flavour mixes Other Fig Fruit juice and nectars according to the flavour demand in Latvia 2015/2016 (USDA, 2016) 5% 24% 11% 15% Lithuania 27% 18% Orange Flavour mixes Tomato Apple Vegetable Other Fig Fruit juice and nectars according to the flavour demand in Lithuania 2015/2016 (USDA, 2016) Estonia 8% 11% 21% 17% 25% 18% Orange Flavour mixes Apple Tomato Plum Other Fig Fruit juice and nectars according to the flavour demand in Estonia 2015/2016 (USDA, 2016) 22

23 1.2. Factors influencing the quality of orange juice Orange juice is known as a popular juice among many consumers for its pleasant taste, fresh flavour and nutritional value as an important source of bioactive compounds such as phenolics compounds (e.g. flavanone glycosides and hydroxycinnamic acids). Many studies have shown that biochemical compounds, proteins, amines, carbohydrates, organic acids, lipids, phenolics compounds, mineral elements, vitamins and other play an important role in the physiology and metabolism of citrus fruits and orange fruits. The content of biochemical compounds of orange juice depends on number of factors as orange fruit variety, place of growing, harvesting time and processing manner (Del-Caro et al., 2004; Gorinstein et al., 2001; Ladaniya, 2008; Wibovoet al., 2015a) The physicochemical characteristics, bioactive compounds and the natural antioxidants in orange juice The most important parameters of orange juice are its sugar content and ratio of sugar to acid content. The sugar content of juice is normally expressed as soluble solids or Brix (degree Brix) and it is nearly 75 to 85 percent of the total soluble solids (TSS). Sugars are distributed in ratio of sucrose, glucose, and fructose 2:1:1 in orange juice. Sucrose is a disaccharide (formed with two molecules of monosaccharides, D- fructose and D- glucose) and a major non-reducing sugar in citrus fruits (Table 1.2). In oranges, the amount of sucrose is smaller than that of monosaccharide glucose as compared to mandarin. Reducing, non-reducing, and total sugar contain of citrus fruits (Selvaraj, Raja, 2000; Ladaniya, 2008) Table 1.2 Sugars (% of fresh weight basis) Fruit Reducing Non-reducing Total Mandarin Valencia orange Navel orange Grapefruit Lemon Note: Sugar in the fresh edible portion of fruit The Association of the Industry of Juices and Nectars from Fruits and Vegetables of the European Economic Community has accepted several standards for orange juice. For direct orange juice the corresponding Brix is a minimum of 10.0 Brix and relative density minimum 1.040, although most single strength juices will show a relative density 20/20 of or higher. It has been recognized that single-strength juices from defined origins and varieties can show lower values but the lowest acceptable relative density is For juices from concentrate the corresponding Brix is a minimum of 11.2 Brix and relative minimum density is The sucrose, total sugar ratio and the total sugar content are naturally subject to large variations. The glucose/fructose ratio is practically constant and is max of 1. Glucose and fructose content ranges of grams per litre, sucrose content ranges of grams per litre. The average values for glucose and fructose are distinctly less than 30 grams per litre. Total sugar contents vary widely in different kinds of orange fruit and they are present mostly in free form as monosaccharides and disaccharides in the juices (Fresh Fruit and Vegetables Codex (2002). 23

24 Generally, the sugar content in the orange fruit varies from 9 to 13 percent. However, some orange fruit varieties from Cuba and Mexico have high content of sugar till 25% with low acidity content. The location of the fruit on the tree influences sugar content those receiving the most sunshine have the highest sugar concentration. Juice from the similar half of the fruit is sweeter than the half that is located near the stem and adjacent segments within the same fruit were reported to differ to 2.7 percent of the total soluble solids (TSS) (Ladaniya, 2008). Fruit ripening is assessed by TSS and total acidity (TA) of the juice. The maturity of fruits is assessed from TSS and total acidity (TA) of the juice. Immature oranges have high sucrose content but during maturation this decreases slightly (Anwar et al., 1999; Esteve et al., 2005). Maturity standards for oranges in Greece require a minimum Brix of 9.0 and minimum Brix acid ratio of 10. The different varieties of orange have been reported to produce of % TSS and % TA content. The total sugars are strongly affected by the growing season. Organic acids are also a vital part of the quality of citrus juices, second only to the soluble solids in importance. In combination with sugars, they are important attributes of the sensory quality of raw and processed fruits. Organic acids contribute to the particular flavour and palatability of orange juice and are found as a result of biochemical processes or in the case of fermentations through the development of certain spoilage microorganisms. To large extent organic acids of orange juice protect it against the development of pathogens. Acids in citrus fruits are formed from the energy releasing citric acid cycle common to all life form. This respiratory process also known as the Krebs cycle or tricarboxylic acid cycle breaks down stored carbohydrates to carbon dioxide and various organic acids. In orange juice citric acid is the most abundant followed by malic both being present mostly as free acids although in limited quantities they are also combined as citrates or malates which give orange juice its buffer effect. Other non-volatile free acids (oxalic, tartaric and galacturonic, quinic and many others) are found in much lower quantities (Ladanya, 2008). Major acids in some commercially important fruits are provided in the Table 1.3. Organic acids contain in juice commercial citrus fruits Table 1.3 Fruits Citric (%) Malic (%) Succinic (%) Clementine Tangerine Valencia orange Hamlin Washington Navel orange Bhalerao, Mulmuley (1992); Selvaraj, Raja (2000). There are many studies carried out on the free acids, total acids and combined acidity on the citrus juice and citrus peel. The TA values vary depending on the origin, climate, variety and degree of maturity within the limits of the range. The majority of orange juices acids show values over 1.1 grams per litre. The minimum value of 0.8 grams per litre is only achieved in rare cases in juices from Mediterranean area and from Californian Navels. The L- malic acid content range from 0.8 to 3.0 grams per litre and is primarily determined by the variety and origin (AIJN). Estevia et al. (2005) studied the total acidity in the four orange juices (juices A, B, C, D) and reported that total acidity was significantly different (0.78, 0.86, 0.93, 1.26 g 100g -1 respectively) but in all cases it was within the recommended values (0.6 to 1.6 g 100 g -1 ). Biologically active compounds of pectin s are based on its indigestibility and nondigestibility i.e. it is a soluble dietary fibre. To this end, it is used to enrich food products. An 24

25 antioxidant is a molecule that inhibits the oxidation of other molecules. Oxidation is a chemical reaction that transfers electrons or hydrogen from a substance to an oxidizing agent. Oxidation reactions can produce free radicals. In turn these radicals can start chain reactions. All this damage leads to a consequent aging of tissues and the appearance of degenerative diseases (Halliwell, 1996). Lampe (1999) describes various mechanisms by which fruits and their constituents can have a protective effect. Antioxidants terminate these chain reactions by removing free radical intermediates and inhibit other oxidation reactions. They do this by oxidizing themselves so antioxidants are often reducing agents such as thiols, ascorbic acid, or polyphenols (Singh, 2006). Antioxidant can be classified differently. They can be categorized as endogenous - living in the living system and exogenous - with food intake. They can be divided into lipophilic and hydrophilic, high molecular weight and low molecular weight, natural and synthetic. Antioxidant is also classified according to the mechanism of action. They can both react with free radicals and delay production. The most important of lipophilic antioxidants are tocopherols, carotenoids, and other lipid soluble compounds, which possess the ability to react with radicals. Hydrophilic representatives of this group are ascorbic acid (vitamin C) and plant phenolic compounds (Tirzitis, Skesteris, 2007). Recent studies have found connection between prevention of diseases like cancer and cardiovascular problems and intake of fruit and vegetables, which are rich in antioxidants like vitamin C, vitamin E and carotenoids (Attaway, 1992; Krinsky, Johnson, 2005; Nishino et al., 2009). Orange juice is an excellent dietary source of bioactive compounds that retains antioxidant properties. Consumption of fresh fruits and vegetables has been described in many researches as inversely correlated with stomach, pancreas, oral cavity and oesophagus cancer and to a lesser extent of the breast, cervix, rectum and lung cancer (Block et al., 2001, Manners, Hasegawa, 2000) where protective role of vitamin C has been emphasized. It was found that vitamin C inhibits the formation of carcinogenic N-nitroso compounds like N-nitrosamines (Liu et al., 2008). The high content in vitamin C, carotenoids and natural antioxidants is particularly appreciated (Abeysinghe et al., 2007; Bull et al., 2004; Rekha et al., 2012). In addition to vitamin C, orange juice is rich in provitamin A carotenoids (ß-carotene, α-carotene, ß- cryptoxanthin and cryptoxanthin) as well as in antioxidant carotenoids (β-carotene, α- carotene, β-cryptoxanthin, α-cryptoxanthin, lutein and zeaxanthin) and flavanones, among other phytochemicals. The vitamin C is also known as L-ascorbic acid and its chemical formula is 2.3- didehydro-l-threo-hexano-1.4-lactone (C 6 H 8 O 6 ). Chemical reactions require that equivalent amounts of the antioxidant and the protected compound be present. The ascorbic acid acts as an antioxidant by giving away electrons (see Fig. 1.7). Fig Chemical antioxidant mode of action (Belitz, et al., 2012; Eitenmiller, Landen, 1999) Vitamin C is considered to be the most important water-soluble antioxidant. It is sensitive to air, light, heat and easily destroyed by prolonged storage and over processing of food. As a water-soluble compound vitamin C is easily absorbed however, it is not accumulated in a body. Hence vitamin C has to be regularly consumed through diet or tablets 25

26 in order to support ascorbic acid level in the body. The recommended daily acceptance (RDA) for vitamin C is from 100 to 120 mg to achieve cellular saturation and optimum risk reduction of heart diseases, stroke and cancer in healthy individuals (Naidu, 2003). It protects compounds in extracellular and intracellular spaces in most biological systems and reduces tocopherol radicals back to their active form at the cellular membranes (Kaur, Kapoor, 2001). Vitamin C is also essential for the synthesis of collagen, the most abundant protein in mammals. Collagen is a major fibrous element of skin, bone, blood vessels and teeth. A lack of vitamin C may lead to scurvy, which causes loss of teeth, bleeding skin and ulcers (Zozulya et al., 2000). Some studies suggest that vitamin C has anticancer effect because of its inactivation reaction of free radicals in the body. The investigators describe orange juices are rich source of vitamin C which is an important antioxidant in these juices (Arena et al., 2001; Gardner et al., 2000; Glisczynska-Swiglo et al., 2004). The content of vitamin C is also important parameter for assessing the nutrition value of the food and beverages. Production conditions and equipment affect the content of vitamin C also play an important role the storage time and temperature. Different varieties and the place where grow oranges also have different levels of vitamin C. Early varieties as Hamlin and Navel fruits have more vitamin C than the late maturing Valencia. The average natural L-ascorbic acid content of fresh orange juice is between 400 and 500 mg per litre (AIJN). One glass of orange juice (200 ml) can deliver about 30 80% of recommended daily intake of vitamin C (Gliszczynska-Swiglo et al., 2004). Vitamin C is usually high in immature oranges. As fruit ripens and increases in size, the concentration of vitamin C decreases. Fruits at the top and on the outside of the tree contain more vitamin C than those in the inside and at the lower level. A significant correlation between ascorbic acid content and the contents of reducing sugars (hexose sugars) suggests that these constituents are associated in ascorbic acid synthesis. Concentration of ascorbic acid in the juice of oranges is one-fifth than that of the flavedo and one third of the albedo respectively. The peel of orange is rich of ascorbic acid (Braddock, 1999; Kulwant et al., 2012; Ladaniya, 2008). Flavonoids are a large family of compounds synthesised by plants that have common chemical structure. According to their molecular structures, flavonoids are divided into six classes: flavones, flavanones, flavanols, isoflavones, anthocyanidins and flavonols (Belitz et al., 2012; Tripoli et al., 2007). Some major pigments that give colour to citrus fruits are: chlorophylls (green), carotenoids (yellow, orange, and deep orange), anthocyanin (blood red) and lycopene s (pink or red) (Ladaniya, 2008). Flavonoids are the most important plant pigments for flower coloration. They are also powerful antioxidants against free radicals, because they act as radical-scavengers (Pietta, 2000; Rice-Evans, 2001). This activity is attributed to their hydrogen-donating ability. Indeed the phenolic groups of flavonoids serve as a source of a readily available H atom such that the subsequent radicals produced can be delocalized over the flavonoid structure (Burda, Oleszek, 2001;). Physiological properties of these flavonoids are attributed to their ability to inhibit cell proliferation, promote differentiation (Kuo, 1996), and function as antioxidants (Yu et al., 2005). Phytochemicals may interact directly with the cells and send a signal to modify cell s activity. Only very small amount is needed to achieve a result (see Fig. 1.8). Phytochemical antioxidant reactions are only one of many ways by which juice compounds can have a beneficial effect on human health (Bombardeli, Morazzoni, 1993). 26

27 Fig Chemical phytochemical mode of action (Bombardeli, Morazzoni, 1993) Flavonoids connected to one or more sugar molecules are known as flavonoid glycosides, while those that are not connected to a sugar molecule are called aglycones. With the exception of flavanols (catechins and proanthocyanidins), flavonoids occur in plants and most foods as glycosides (Hollman, 1999). Until recently, it was considered that vitamin C was the only chemopreventive agent in citrus because of its free radical scavenger ability. However recent studies suggest citrus contains several possible anti-cancer agents such as flavonoids and limonoids (Poulose et al., 2005; Tanaka et al., 2000; Tanaka et al. 2001; Tian et al., 2001). Total phenolics compounds content of citrus fruits as well as of orange fruits increases to а maximum during the early stage of fruit development and then remains constant. Houjin et al. (1990) and other researches found in orange fruit the concentration of flavonoid decreased with the fruit size increased and with time of maturity. Some literature on flavanone content in commercial orange juices is available (Babbar et al., 2011; Belajova, Suhaj, 2004; Gil-Izquierdo et al., 2002; Kanitsar et al., 2001; Tomas-Barberan, Clifford, 2000), but is marginal and differentiated because of differences in varieties of oranges their ripeness and the processes of technology used to obtained commercial juice. Vanamala et al. (2006) reported that total flavonoid content was significantly higher in made from concentrate orange juices compared to the NFC orange juices. Prominent glycosides in citrus are hesperidin, naringin, narutin, and poncirin (Mouly et al., 1998). Hesperidin and naringin are important glycosides of orange juice their chemical structure is shown in Figure 1.9. Fig Chemical structure of Hesperidin and Naringin (Belitz et al., 2012; Franke et al., 1998) Hesperidin and narirutin flavones are abundant in orange juice in a partially dissolved, partially suspended and partially colloidal form (Dauchet et al., 2008; Ghasemi et al., 2009; Xu et al., 2008). Hesperidin is the essential flavanone oranges contain. It is not bitter in taste. In hesperidin, rhamnose and glucose are in the form of rutinose as a disaccharide moiety and 27

28 because of rutinose they are not bitter. Hesperidin can be found in the segment membrane in the form of white spots crystals in frozen, damaged oranges and in the form of white specs in frozen concentrated orange juice. Hesperidin content in citrus fruits differs with the variety of fruit (Omidbaigi et al., 2004). According to Association of the Industry of Juices and Nectars from Fruits and Vegetables of the European Economic Community (AIJN), hesperidin content on average is about 800 mg per litre. The maximum value indicated of 1000 mg per litre may be exceeded slightly in the case of specific varieties of the Mediterranean area and especially in the case of soft fruits. In general, concentration of flavanones decreases as fruit matures. The principle of bitter (sour) oranges is naringin in place of hesperidin in sweet oranges. The bitter taste of naringin is considered due to the structure of the disaccharide moiety (Tripoli et al., 2007). Naringin and neohesperidin are flavonoids found only in certain citrus fruits, sweet orange cultivars do not contain these compounds and their presence in orange juice indicates adulteration with juice from certain other citrus fruits such as grapefruit (Widmer, 2000). The dihydrochalcone glycosides of naringin, neohesperidin and hesperidin are 300, 1100, and 300 times as sweet as sucrose respectively on a weight basis and are potential sweeteners without calories. These sweeteners have a great future in industrial production since the synthetic sweetener saccharin has side effects. Many epidemiological studies suggest that consumption of polyphenol-rich foods and beverages is associated with a reduced risk of cardiovascular diseases, stroke and certain forms of cancer. These protective effects have partly been ascribed to the antioxidant properties of flavonoids (Borradaile, 1999; Hollman, Katan, 1997; Kaur, Kapoor, 2001; Prior, Cao, 2000a, b). Recent research shows that all citrus including oranges flavonoids, especially hesperidin has shown a wide range of therapeutically properties such as anti-inflammatory, antihypertensive, diuretic, analgesic and hypolipidemic activities (Filatova, Kolesnova, 1999; Galati et al., 1994; Galati et al., 1996; Monforte et al., 1995), reviewed the antioxidant properties of phenolic compounds. The effects of the citrus flavonoid naringin were tested by using it as a supplement in a highcholesterol diet. The research team, from the University of East Anglia (UEA) UK reported that women consuming the highest amounts of flavanones a subclass of flavonoids that are found in especially high levels in citrus fruits were associated with up to a 19% reduction in stroke risk compared to those in the group who consumed the lowest amount (Cassidy et al., 2012). The intense colour of citrus juice is mainly due to compounds called carotenoids. Colour which should be bright yellow to orange-red and not too pale is an important quality parameter. Carotenoids are lipid-soluble pigments that play an important role in human health. Orange juice is rich in provitamin A carotenoids (β-carotene, α-carotene, β- cryptoxanthin and α-cryptoxanthin) as well as in antioxidant carotenoids (β-carotene, α- carotene, β-cryptoxanthin, α-cryptoxanthin, lutein and zeaxanthin). Over 600 carotenoids have been characterised. Nomenclature for the carotenoids specified by the International Union of Pure and Applied Chemistry (IUPAC) and the international Union of Biochemistry (IUB) was recently reviewed by Weedom, Moss (1995). Increased consumption of these compounds has been related to a decreased risk of developing several types of cancer, agedrelated macular degeneration and cardiovascular disease (Krinsky, Johnson, 2005; Nishino et al., 2009). Development of pigments and colour is mainly dependent on weather conditions and citrus variety. The main carotenoids responsible for the orange colour of orange juice are α-carotene, zeta-anther-axanthin (yellowish), violaxanthin (yellowish), β-citraurin (reddish orange) and β-cryptoxanthin (orange). The red colour in blood oranges and related varieties is due to the presence of anthocyanins. With ripening total carotenoids increase in the peel as well as in the pulp. Fruit rind is the place of higher carotenoid concentration and 50-70% of the total orange carotenoids exist in the peel (Ladanya, 2008). The chemical structures of carotenoids are based on a long zigzag chain of carbon and hydrogen atoms, frequently with a six-membered ring at one or both ends (see Fig. 1.10). 28

29 β-carotene Fig Structure of ß-carotene (Eitenmiller, Landen, 1999; Belitz et al., 2012) According to AIJN Reference Guideline orange juices contain the total of 2-5 mg per litre of carotenoids. Juices from early season contain less than juices from late season varieties. The maximum of total carotenoids content is 15 mg per litre of orange juice. The content of β-carotene is between 0.5 to 5% and the maximum percentage of total carotenoids is 5%. ß-carotene and others carotenoids have antioxidant properties in vitro and in animal models. It preserves the body from harmful molecules, which are called free radicals. Free radicals damage cells in a process known as oxidation. In a long time period this molecule deprivation can lead to a number of chronic illnesses. There is a proof that consuming more antioxidants in your diet helps boost your immune system protects against free radicals and may lower your risk of heart disease and cancer. However, it is not as simple when it comes to taking antioxidant supplements. Mixtures of carotenoids or a combination of others antioxidants (e.g. vitamin E) can increase their activity against free radicals (Paiva, Russell, 1999). The effect of externally added ascorbic acid on the deterioration of carotenoid pattern and colour of orange juice has been studied by Melendez-Martinez et al. (2007). Regard less of the addition of ascorbic acid the changes in the carotenoid profiles of orange juices were similar and involved mainly the epoxycarotenoids. The decrease in carotenoids was higher with the higher amounts of ascorbic acid addition. Vitamin E (α-tocopherol) was isolated from wheat germ oil by Evan s research group. These researches called vitamin E tocopherol in translation from Greek means bearing the off spring. As shown by special studies this vitamin is really required to prevent infertility, for normal course of pregnancy and the birth of a robust off spring. Other manifestations of vitamin E deficiency are muscle weakness and anemia or poor blood (Spirichev, 2000). Vitamin E is fat-soluble and soluble in organic fat solvents. All forms are colourless to pale yellow, viscous oils. Vitamin E refers to a group of compounds that include both tocopherols and tocotrienols. The National Research Council defined dietary vitamin E activity in terms of RRR-α-tocopherol equivalents (α-tes) (Eitenmiller, Landen, 1999). Structure of tocopherol is shown in Figure 1. Fig Tocopherol structure (Eitenmiller, Landen, 1999; Belitz et al., 2012) Tocopherols are the natural antioxidants synthesized at various levels and in different combinations by all plant tissues. They are amphipathic molecules with the polar chromanol ring and hydrophobic saturated side chain. The four homologues, α-, β-, γ-, and δ-tocopherol, differ in the number and position of methyl groups in the aromatic ring (Della Penna, Pogson, 2006). The physiological and biochemical functions of vitamin E are comprehensive. Tocopherols act as antioxidants by scavenging peroxy- radicals of polyunsaturated fatty acids or by reacting with singlet oxygen and other reactive oxygen species (ROS). One tocopherol 29

30 molecule can protect about polyunsaturated fatty acids at low peroxide values (Della Penna, Pogson, 2006; Kamal-Eldin, Appelgvist, 1996).The berries of sea buckthorn are rich in tocopherols and take the highest position between all fruit and berry plants by this parameter. The content of tocopherols in the berries of sea buckthorn significantly varies from 1.4 to 50% (Bekker, Glushenkova, 2001). Pectin can be described as a natural component of all edible plant material. It is available in the plant cell walls and in a layer between the cells named middle lamella. Pectin gives firmness to the plants and effect on growth and water storage. Citrus pectin is an important product gel forming in food industries and it is used as thickening agents (Sato et al., 2011; Ywassaki et al., 2011). The general term pectin indicates those water soluble pectinic acids of varying methyl ester content and degree of neutralization that are capable of forming gels with sugar and acid under suitable condition. All pectin substances are indicated as galacturonic acid anhydride (see Fig. 1.12). Fig Structure of pectin molecule (Belitz et al., 2012) These monosaccharide units comprise most of sugar units found in pectin (Wand et al., 2014). Citrus fruits also contain non-starch polysaccharides (NSP) also known as dietary fibre which is a complex carbohydrate with important health benefits. Residues from orange juice extraction are potentially an excellent source of dietary fibre because this material is rich in pectin and is usually available in large amounts. The predominant type of fibre in orange is pectin making up 65 to 70% of the total fibre. Larrauri et al. (1997) reported as Valencia oranges ripened the water soluble pectin in the albedo and the pulp increased to a peak and then decreased while the acid extracted pectin material continued to decrease. The total pectin showed only a slight decrease when the fruit was fully ripened. The total pectin and the various soluble pectin substances (water soluble, oxalate-soluble, alkali soluble) vary depending on the variety of oranges, maturity and juice extraction technologies. According to AIJN standards the total pectin is a maximum of 700 milligram per litre and water soluble pectin is a maximum of 500 milligram per litre. Orange pectin has also been considered in the class of dietary fibres which reportedly lower cholesterol, adjust serum glucose levels, and affect other digestive processes (Brown et al., 1999; Cipriani et al., 2009). Pre-clinical studies indicated that pectin had several biological and physiological functions such as delay of 30

31 gastric emptying and inducing apoptosis of colon cancer cells (Olano-Martin et al., 2003). Pectin in orange juice is important to the processing industry because of its function as a cloud formation and stabilization in the juice. Orange fruits have high content of pectin substances and they are used in commercial pectin production. Important pectin substances include proto-pectin, pectin acid, and pectin. These compounds have varying degrees of methyl ester content and neutralization and are capable to forming gels (jellies) with sugar and acid Orange juice categories and their characteristics Orange juice manufacturing is varied and complex. The quality of juices and concentrates depends on a number of factors as orange fruit varieties, places of growing, harvesting time and processing manner (Arena et al., 2001; Decio, Gherardi, 1992; Johnston, Bowling, 2002; Kimbal, 1999; Manso et al., 2001; Parish, 1996). Different types of orange juices are available in the market. In the world orange juice is commonly marketed in three forms: Chilled Orange Juice, or Single Strength or Not-From Concentrate (NFC); Frozen Concentrated Orange Juice (FCOJ); Concentrated Orange Juice (COJ). Freshly squeezed orange juice is packaged directly after extraction but without pasteurisation or any other physical or chemical treatment. The chilled single-strength orange juice has a rather short shelf life and requires installation of expensive refrigerated tanks. Freshly squeezed unpasteurised orange juice is very desirable for the consumer because of its fresh aroma and flavour but the shelf life is less than 20 days at 1 C as it is highly susceptible to microbial spoilage (Wicker et al., 2003). Not from concentrate (NFC) juice has undergone neither a concentration step nor dilution during production. Now the technology is available on a large scale to extract process and store single-strength juice in bulk aseptic refrigerated tanks, minimizing microbial spoilage and product quality deterioration. NFC orange juice is produced from rigorous fruit selection and is used as ready-to-drink juice without the need of reconstitution with the same freshness of the fresh-squeezed juice. This technology enables provision of blended juices to consumers on a year-round basis when the fruit is not in season (Wilke, 2002). Concentrated orange juice with soluble solids content of 65 Brix is now largely produced in the world. Concentration of fruit juices permits economic advantages in packaging, storage and distribution. The primary water removal technology is high temperature short-time evaporation, although freeze concentration and membrane processes are used as well. The concentration process is accompanied by aroma recovery. The juice quality characteristics vary with variety, rootstock, scion, fertilization, and frequency of irrigation, date of harvesting, age of tree, tree spacing and position of fruit on the tree, climatic conditions, and place of growing. The most significant juice quality indices for consumers are taste, aroma and colour as well as content of functional active substances in juice that can be characterised by nutritional content. The most important compounds that influence the quality of orange juice are sugars and acids, flavour and colour components, and vitamin C. These compounds plus cloud are analysed to define and grade juice. Guidelines for quality standards for fruit juices for European Union are specified in the Code of Practice for evaluation of fruit and vegetable juices published by AIJN (see Table 1.4) 31

32 AIJN quality requirements for orange juice Table 1.4 Orange juice NFC Juice from concentrate Relative density 20/20 min Brix min min L-ascorbic acid (vit.c) at the end of shelf life, mg/l min. 200 min. 200 Volatile oils, ml/l max. 0.3 max. 0.3 max. 0.4 Hydroxymethylfurfural (HMF), mg/l max. 10 max. 10 Volatile acid as acetic acid, g/l* max. 0.4 max. 0.4 Ethanol, g/l max. 3.0 max. 3.0 D-/ L- Lactic acid, g/l max. 0.2 max. 0.2 Arsenic and heavy metals, mg/l max max (various values) * Indication of hygiene not juice acidity. Source: AIJN Code of Practice Another source of guidelines is the German Guidelines RSK (Richtwerte, Schwankungsbreiten, Kennzahlen) values which have been developed by the association of the German Fruit Juice Industry. Some of the properties specified for orange juice are given in the Table 1.5. RSK* values for orange juice Parameters Reconstituted juice Relative density 20/20 min Brix min L-ascorbic acid (vit.c) at the end of shelf life, mg/l min. 200 Volatile acid as acetic acid, g/l max. 0.4 Ethanol, g/l max. 3 Lactic acid, g/l max. 0.5 Arsenic and heavy metals, mg/l max *RSK, Richtwerte, Schwankungsbreiten, Kennzahlen (guidance value, range, reference number) Table 1.5 In the USA the US Department of Agriculture USDA is responsible for specifying quality standards for orange juice. To be labelled USDA Grade A orange juice produced in Florida must meet the quality requirements shown in Table 1.6. The quality factors are measured on a 100-point scale. If the total score is above the limit but just one of the quality factors does not meet the Grade A requirements the juice still may not be labelled Grade A (The Orange Book, 2004). 32

33 Requirements for USDA grade A orange juice Table 1.6 Orange juice NFC FCOJ (when reconstituted to 11.8 Brix) Brix min min Ratio ( Brix: acid) Recoverable oil % v/v max max Appearance fresh orange juice fresh orange juice Reconstitution - reconstitutes properly Colour very good, min.36 points very good, min.36 points Flavour very good, min.36 points very good, min.36 points Defects practically free, min. 18 points practically free, min. 18 points Total score min. 90 points min. 90 points Source: USDA According to the CODEX General Standard for Fruit Juices and Nectars an authentic fruit juice product must maintain the essential physical, chemical, organoleptic, and nutritional characteristics of the fruits from which it comes. Juices are more convenient to consume and generally have a longer shelf life than fresh fruit (IFFP, 2005) Influence of the technological process on the orange juice quality Processing steps to stabilise extracted orange juice with respect to enzyme and microbial activity are indispensable before concentration, bulk storage, packaging and distribution. One exception is perhaps for the small amount of freshly squeezed, unpasteurised single-strength orange juice which is distributed, chilled and has a shelf life of up to 3 weeks often shorter. Heat treatment with respect to time and temperature settings should be designed to minimize unwanted chemical and flavour changes in the product. Never the less it should still give an adequate safety margin concerning the activation of enzymes and spoilage of microorganisms (The Orange Book, 2004) Orange juice technology process description and flowchart Orange processing plants are normally located near the fruit growing area. Fruits must be processed as soon as possible after harvesting because fruit decomposes quickly at high temperatures found in citrus growing areas. The production steps for orange juice are shown in Figure

34 Fig Production steps for orange juice (The Orange Book, 2004) After reaching the processing plant the laboratory draws a small portion of fruit at this stage for testing the total acidity, total soluble solids ( Brix), vitamin C and juice yield. The tests for fruits have been discussed by Miller, Hendrix (1996), Kimbal (1991), Kulwant et al. (2012), in the Orange Book (2004) and others. Fruits that have been certified are then transported along a conveyor belt where they are washed with the roller brushes. The fruits are rinsed and dried. Proper size of the fruit is critical for the extraction process. Individual oranges are directed to the most suitable extractor in order to achieve optimum juice yield. This process is necessary as it eliminates debris and dirt and reduces the number of microbes. 34

35 There are two types of extractor present in the orange processing industry: the squeezer type and the reamer type. There are two major brands for these plants: FMC (squeezer type) and Brown (reamer type). The aim of the juice extraction process is to obtain as much juice out of the fruit as possible while preventing rag, oil and other components of the fruit from entering the juice. Orange juice is being either squeezed or reamed out of either whole or halved oranges by means of mechanical pressure. As the extraction operation determines juice yield and quality the correct setting of extractor operating conditions is very important. Thus, in one stroke five oranges are separated into juice, pulp, peel, peel oil, seeds and rag. Various types of extractors and finishers including Rotary Juice Press, FMC In-Line Extractor, and various Brown Model extractors have been discussed by different workers (Antonio, 1992). Followed the extraction the pulpy juice (about 50% of the fruit) is clarified by separating juice from pulp with primary finishers. The process consists of the mechanical separation method based on sieving. The juice stream is additionally clarified by centrifugation. The pulp stream containing pieces of ruptured juice sacs and segment walls may then to go pulp recovery or to pulp washing. From this point the juice may either used as a freshly squeezed orange juice, go directly into a pasteuriser in the case of NFC or it goes on to the evaporators where most of the water is taken out of the juice by vacuum and heat then chilled to yield frozen concentrated orange juice (FCOJ) (The Orange Book, 2004). Freshly squeezed unpasteurised orange juice is desired because of its fresh aroma and flavour but the shelf life is less than 20 days at 1 C, as it is highly susceptible to microbiological spoilage. The manufacturing operations from fruit washing to packaging must be exceptionally clean to minimize product spoilage. Pectin esterase activity in unpasteurized juice results in loss of cloudiness (Wicker et al., 2003). Freshly squeezed unpasteurised orange juice is packaged directly after extraction without pasteurisation or any other physical or chemical treatment. After clarification the juice often undergoes some degree of blending with juice from other batches in order to balance its flavour, colour, and acidity and soluble solids levels before further processing. If intended for NFC production the juice leaving the clarification section should be cooled to 4 C to minimize the potential of microbiological activity before being passed into the buffer/blending tanks (Kulwant at al., 2012). The aim of NFC processing is to produce orange juice using the minimum of thermal processing. Although several methods have been tested pasteurisation is the only process used industrially to inactivate the enzyme. Pasteurisation of orange juice is necessary for inactivating enzymes and for destroying microorganisms capable of growing during storage. Enzyme activity leads to cloud loss in single-strength juice and gelation in orange juice concentrate. Diverse pasteurisation techniques are used commercially. One common method passes juice through a tube next to a plate heat exchanger, so the juice is heated without direct contact with the heating surface. Another method uses hot pasteurised juice to preheat incoming unpasteurised juice. The preheated juice is further heated with steam or hot water to the pasteurisation temperature. Typically reaching a temperature of C for about 30 seconds is adequate to reduce the microbe count and prepare the juice for filling (Pareek et al., 2011). Recent tendencies suggest the use of High Temperature Short Time (HTST) pasteurisation with either tubular or plate-type heat exchangers heated either by steam or hot water. Heating usually takes about 30 seconds or less and the juice is heated rapidly without local overheating. Modern heat exchangers and automatic controls are usually designed so that scorching or under heating of portions of the juice is prevented (Kulwant et al., 2012). HTST treatment can minimise those undesirable quality changes due to the much less duration of heat treatment. Currently HTST pasteurization is the most commonly used method for heat treatment of fruit juice (Braddock 1999). Deoiling sometimes is used to decrease oil levels in the juice and deaerating to remove oxygen is part of good practice. Several filling system for aseptic bag-in-box (BiB) containers evolved from conventional (no aseptic) BiB system. A sterile chamber surrounds the filling head and 35

36 chemical sterilisers are used for sterilisation. Other systems were subsequently developed later specifically for aseptic filling. It incorporates a simple filling system (spout and filling valve) and steam is used as the sterilising agent. NFC may be stored for up to one year (The Orange Book, 2004). The NFC juice is processed and stored in bulk under aseptic or frozen conditions for some months until it is reprocessed and packaged. For large volume NFC production such as found in Florida and Brazil aseptic tank farms is the most common form of NFC storage. Vitamin C degradation and changes in flavour during the storage period are minimized by freezing but the energy and warehouse costs of freezing and storing frozen NFC are high. The frozen product is usually kept at -18 C or lower (Smitt, Hui, 2004; Kulwant et al., 2012). As an alternative to frozen storage NFC may be stored chilled in aseptic tanks. The tanks are sterilised prior to filling by flooding them with a sterilising fluid (e.g. iodoform). The preferred storage temperature is about 1 C just above the freezing temperature of the juice. Pressurised nitrogen above the juice surface is often maintained to minimize risk of vitamin C loss through oxidation. Orange juice in aseptic bag-in-box (bags of 200 litre or typically 1000 litre) used for stored and transported. The bags placed in bins usually made from wood, and are then stored under refrigerated conditions. For long-term storage of juice (6 months or more) bag material with a very good oxygen barrier is recommended. Bags made with foil based aluminium laminate offer higher protection against oxygen compared with metallised laminates where the aluminium layer is much thinner (The Orange Book, 2004). Concentrated orange juice with soluble solids content of 65 produced mostly in Brazil and with soluble solids content of 45 Brix in United States in Florida Concentration of many fruits juices including orange juice provides economic advantages in packaging, storage and distribution. It also helps in the economic utilization of perishable fruits during the peak harvest periods thus stabilizing the market prices of fresh produce. For juice concentration from the blending tanks and after clarification juice goes to the evaporator. The most common type of tubular evaporator system used for orange juice is the TASTE evaporator. It is generally described as a continuous high- temperature short time evaporator of the long vertical tube falling-film type. Versions with as many as seven effects are installed (seven effects means basically that the steam is reused to evaporate water in seven steps). Such systems have very low specific steam consumption: only 1 kg of steam is used to evaporate 6 kg of water. Additional effects increase the residence time for the product in the evaporator accordingly (Smith and Hui, 2004). These evaporator systems are dedicated to citrus fruit. A flow diagram of an evaporator with seven products stages is presented in Figure

37 Fig Flow diagram of a tubular evaporator (Smith and Hui, 2004) The juice is first preheated to C. Holding at pasteurisation temperature stabilizes the juice by means of microbial and enzyme inactivation. The product then passes through a number of stages under vacuum until a concentration of up to 66 Brix is achieved. By this time the product temperature has fallen to about 40 C. The residence time in the evaporator is typically 5-7 minutes or longer. During the evaporation process volatile flavour components flash off and can be recovered in an essence recovery unit. Juice concentrate is cooled and blended with other production batches as required to level out fluctuations in quality (The Orange Book, 2004). After evaporation the Brix concentrate is chilled to -10 C (concentrate does not freeze solid at -10 C). It is then routed to frozen storage. Storage takes place in the bulk storage tanks or 200 litre drums with plastic linear. Drum storage is normally maintained at -20 to -25 C bulk storage in large tanks often at -10 C. The concentrate is blanketed with nitrogen and carefully monitored for quality characteristics. Under these conditions the concentrate can be stored for over a year with little loss in quality (The Orange Book, 2004; Smith, Hui, 2004). Concentrated juices are distributed in large containers as a base for the manufacture of a variety of soft drinks. The same is reconstituted to single-strength juice for direct consumption. Controlling viscosity is very important for the efficient evaporation and pumping of citrus concentrates. Alternatives to evaporation for concentrating orange juice have been developed and tested but so far none are in commercial operation on a large scale. Lower Brix levels of the concentrate and often high operational costs compared with evaporator system in common use have prevented the commercialisation of the new systems (Smith, Hui, 2004). Two methods which do not use heat for concentration are freeze-concentration and membrane filtration. Freeze concentration method is based on the fact that during the freezing of sugar solutions ice crystals are first formed which can be separated out from solution there by increasing the sugar concentration. When freeze concentration is applied to juice inactivation of enzymes is necessary. This may be carried out by pasteurising the juice before freezing or pasteurising the resulting concentrate. Membrane filtration is another method evaluated for concentrating orange juice without using heat but the resulting high viscosity of concentrate reduces filtration efficiency and limits the degree of concentration that can be achieved. To minimize viscosity the pulp is first separated from the juice e.g. by ultrafiltration to leave a clear liquid (serum) which is concentrated by reverse osmosis. The pulpy stream rich with enzymes is pasteurised before being recombined with the serum concentrate. Mixing back of the 37

38 insoluble solids stream, essentially at single-strength juice concentration reduces the Brix value of the concentrate. Concentration up to 42 Brix has been reported (Kulwant et al., 2012) Effect of pasteurisation on chemical characteristics and bioactive compounds of orange juice The heat treatment continues to be the most widely used method of preserving and extending the shelf life of foods (Awuah et al., 2007; Martin et al., 1995; Plaza et.al, 2011; Polydera et al., 2005; Uelgen, Oezilgen, 1993). The aim of juice processing is to produce orange juice using the minimum of thermal processing. Nevertheless the thermal treatment should be sufficient to ensure that the product is physically and microbiologically stable. Many important nutrients in orange juice including sugars, acids, minerals, flavonoids, and other components are quite heat stable under conditions of pasteurisation. Nevertheless orange juices are sensitive to heat and the impact of pasteurisation on quality is clearly appreciable. Their vitamin content, delicate fresh aroma and flavour are lost or damaged by exposure to heat so they are usually pasteurised as rapidly as possible. Pasteurisation of orange juice produces subtest threshold levels of p-vinylguaiacol (PVG) and ascorbic acid degradation but has little effect on browning. The ascorbic acid concentration, density, cloud, and fructose levels of the juice are significantly influenced by the processing method when unpasteurised juice is bottled and frozen at -18 C; pasteurised juice is either bottled or frozen at -18 C or stored (Farnworth et al., 2001). The natural ph of juices differs with the variety of oranges. Optimisation of microbial destruction enzyme inactivation and vitamin C retention during pasteurisation of ph-adjusted orange juice was reviewed by Uelgen, Oezilgen, (1993). For heat inactivation relationships of time-temperature are important of enzyme pectin esterase in orange juice under different conditions (Lee, Coates, 2003). Juice pasteurisation also have a major impact on the stability of juice and stability of juice cloud of orange juice concentrates because of the deactivation of pectin esterase enzymes. The chemical, physical, organoleptic properties and volatile components of juice are affected by pasteurisation to varying extents depending upon the technique of pasteurisation used (Gil-Izquierdo et al., 2002; Min et al., 2003). Thermal processing technology play important role in juice production and can often lead the changes in sensory attributes and content of nutritional value in products (Farnworth et al., 2001; Lee, Coates, 2003). Colour of orange juice is mainly due to carotenoid pigments (Lee, Castle, 2001; Melendez-Martınez et al., 2007; Vikram et al., 2005) and it bears a relation with the technological treatments: a relatively large loss of provitamin A (βcarotenoid, α-carotene, and β-cryptoxanthin) and carotenoids was found after thermal processing in orange juice. Hyoung and Coates (2003) and Lee and Coates (2003) reported that pigment loss and potential visual colour changes are associated with thermal processing of Valencia orange juice which is quantitatively the richest in carotenoids amongst sweet oranges Advances in pasteurisation technology of orange juice Currently the food industry is looking at replacing the traditional well-established preservation techniques with novel thermal and non-thermal technologies which may produce high quality food products with improved energy efficiency and to be more environmentally friendly. The new technologies, such as dielectric heating, ohmic heating, also pulsed electric fields (PEF), and high hydrostatic pressure (HHP) and processing, have been reviewed by Pereira and Vicente (2010). These technologies and also as high-pressure (HP) processing, are being introduced by the food industry as an alternative or complementary process to 38

39 traditional thermal treatment this technologies have potential to improve the quality and freshness character of processed food while assuring its microbiological safety (Bull et al., 2004; De Ancos et al., 2002; Elez-Martinez et al., 2005; Munoz et al., 2007; Odriozola- Serrano et al., 2008; Polydera et al., 2005; Sanchez-Moreno et al., 2005; Snir et al., 1996). Industrial applications are already known in Japan, United States, France and Spain. The European Commission includes products obtained by this technology in the Novel Foods group subject to Novel Food Legislation (Esteve, Frigola, 2007). Some studies have been carried out on the conventional and high-pressure technique (HPP) in the orange juice (Boff et al., 2003; Goodner et al., 1998; Nienaber, Shellhammer, 2001; Polydera et al., 2003; Polydera et al., 2005; Zhang et al., 2010). It was proved that application of HHP can lead to an extended shelf life of orange juice compared to that of untreated juice with minimal product quality loss and a good retention of fresh-like flavour (Donsi et al., 1996; Nienaber, Shellhammer, 2001). These studies suggest also that microwave heating could be used for continuous pasteurisation of citrus juice (Nikdel et al., 1993). Orange juice is pasteurised by pumping it through a coil of Teflon tubing in an oven heated with microwave energy (at 2450 MHz). The juice temperature (95 C) is controlled by varying the flow rate at 100% microwave power. Over 90% of the 570 W microwaves power is absorbed by the juice. Pectin methyl-esterase activity is reduced by more than 99.9% by pasteurising for seconds at 95 C. Bacteria are also rapidly inactivated at either 70 C or 90 C. Juice pasteurization using microwave does affect flavour compared with fresh unpasteurised juice (Kulwant et al., 2012). Ultra-high temperature processing or ultra-heat treatment sterilises food by heating it above 135 C - the temperature required to kill spores in juices in 1 to 2 seconds. This technique is commonly used in milk production but the same process is also used for fruit juices, cream, soya milk, yogurt, wine, soups, honey and stews. Tetra Pak recommended for juice nectar and still drinks process 138 C for 4 seconds 1. The technology by UHT processing is proven to be effective in eliminating of all microorganisms and in inactivating the thermoresistant spores including spores of Alicyclobacillus species. The reduction in process time due to higher temperature and the fast cooling leads to a higher quality of the product. UHT process guarantees long shelf life. The only complication using UHT treatment methods include the complexity of equipment and plants that need to maintain sterile atmosphere between processing and packaging (packaging materials, pipework, tanks, and pumps) also higher skilled operators and sterility must be maintained through aseptic packaging Packaging and the role of storing in orange juice quality One of the main aims of a packaging system and packages is to protect orange juice from microbial spoilage and chemical deterioration during distribution and storage. For orange juice measures should be taken to protect vitamin C and flavour compounds and prevent microbial growth and colour changes. The quality of orange juice at consumption depends on all of the processing and packaging steps from raw material intake up to the product consumption. Some important operating parameters which influence juice quality at difference steps are presented in Figure Raw materials Processing Bulk storage Packaging Shell storage Fig Factors influencing juice quality (Orange Book, 2004)

40 From the day of processing to the day of consumption orange juice undergoes various physical and chemical changes depending on the type of packaging and storage conditions (Ayhan et al., 2001; Bezman et al., 2001; Esteve et al., 2005; Ewaidah, 1992; Goyle, Roig et al., 1998; Polydera et al., 2005). Several studies have shown that quality and shelf life determination of an orange juice is strongly based on vitamin C evolution during storage as well as colour and flavour parameters (Esteve et al., 2005; Kabasakalis et al., 2000; Lee, Coates, 1999; Zerdin et al., 2003). From Tetra-pack researches degradation of vitamin C in orange juice stored at ambient temperature in two types of package is shown in Figure Fig Vitamin C degradation curves for different packages of orange juice stored at 23 C (Orange Book, 2004) The aluminium foil layer is a good oxygen barrier whereas packages with a polymer barrier layer like EVOH allow higher oxygen permeability. Some packages were stored in an oxygen-free atmosphere and in this case the vitamin C loss represents mainly the anaerobic degradation pathway (The Orange Book 2004). Oxygen plays a major role in the loss of quality in orange juice during storage mainly because of vitamin C degradation and colour changes (browning). High storage temperatures combined with oxygen are the main factors involved with quality deterioration over time. The adverse effects of dissolved oxygen on fruit juice quality have been analysed by many researchers and include degradation of ascorbic acid increased browning and growth of aerobic bacteria and moulds (Bull et al., 2004; Hyoung, Coates, 2003; Kennedy et al., 1990; Solomon et al., 1995). Manso et al. (1996) studied changes in dissolved oxygen concentration during storage of packaged oranges. Single-strength Valencia orange juice aseptically packaged and stored 5 month at 4-5 C was analysed for dissolved oxygen. Effect of packaging materials on colour, vitamin C and sensory quality of refrigerated mandarin juice in different containers was studied by Beltran- Gonzalez et al. (2008). They proved that the presence of oxygen in the headspace of mandarin juice decreased the ascorbic acid and darkening of colour during storage. Generally, vitamin C is lost through two different chemical pathways anaerobic and aerobic degradation. As its name implies, the anaerobic pathway is independent of oxygen and it s mainly driven by the storage temperature. Losses caused by this degradation pathway cannot be prevented by packaging, and they are the same in all types of package. The only possible countermeasure is to reduce the storage temperature. Both anaerobic and aerobic degradation take place simultaneously in orange juice. Which one predominates depends on 40

41 the storage temperature and the availability of oxygen. The changes of vitamin C content in orange juice during storage for 30 weeks at 4 C and 23 C respectively in the same package type (Tetra Brik Aseptic, TBA) is shown in Figure Fig Effect of temperature on vitamin C content in orange juice during storage (Orange Book, 2004) The difference in vitamin C retention between storing at 4 C and 23 C is obvious. During 30 weeks storage an increase in temperature from 4 C to 23 C results in increased losses of vitamin C of 28 mg per litre due to anaerobic degradation and 42 mg per litre due to aerobic degradation. For packages with good oxygen-barrier properties e.g. glass bottles anaerobic degradation plays the major role regarding the total loss of vitamin C. The rate of oxidative degradation of vitamin C slows down dramatically under chilled storage. Consequently, packages for chilled distribution do not need as high oxygen-barrier properties as are required for packages stored at ambient temperature (Choi et al., 2002; Plaza et al., 2006). High storage temperatures have been studied to observe the effects of temperature on certain factors as browning development of hydroxymethylfurfural loss of vitamin C, etc. (Cortes et al., 2008a; Cortes et al., 2008b; Hirsch et al., 2008; Manso, et al., 2001). The colour changes or rather the darkening of orange juice during storage are based on the appearance of brown-coloured compounds caused by the chemical reaction of orange juice components present in the juice matrix. The brown compounds are formed in the end phase of the Maillard Reaction (also known as non-enzymatic browning) it is a well-known reaction between sugars and amino acids. This reaction type is generally not dependant on oxygen but is clearly temperature driven. Browning in the food product can significantly reduce vitamin C and can participate in the development of browning via its degradation products resulting from both the aerobic and anaerobic pathways. Consequently the oxygen barrier of packages influences browning because it determines the supply of oxygen to the aerobic pathway of vitamin C degradation (Belitz et al., 2012). The impact of light on the quality of orange juice during storage has been proven in respect to its impact on the aerobic pathway of vitamin C degradation (and only this pathway). The light has an effect only when oxygen is present; consequently packages with high oxygen-barrier properties e.g. glass bottles and high-barrier PET bottles do not need a light barrier (The Orange Book, 2004). Moyssiadi et al. (2004) studied effect of light transmittance and oxygen permeability of various packaging materials. In cases where permeation of oxygen into the package is considerable headspace oxygen is present or oxygen is dissolved in the product the contribution of anaerobic degradation to the total vitamin C loss is small compared with aerobic degradation. 41

42 Orange juice aroma is a complex mixture of many volatile compounds. In chemical terms it is mainly a mixture of hydrocarbons, aldehydes, alcohols and esters. The predominant fraction consists of hydrocarbons of which one single compound D-limonene accounts for more diffusion coefficients with polar and nonpolar aroma compounds. This results in good barrier properties against both types of compounds. However, polar polymers are some times more difficult to heat seal than polyolefin. Aroma losses due to absorption into or permeation through polymer packaging primarily involve nonpolar aroma compounds (e.g. hydrocarbons like limonene) in contact with nonpolar polymers low density polyethylene (LDPE), highdensity polyethylene (HDPE) and polypropylene (PP) commonly used as sealing layers. There is little absorption of aroma compounds into polar polymers like polyesterpolyethylene terephthalate (PET), ethylene vinyl alcohol (EVOH) and polyamide (PA) (The Orange Book, 2004). Different types of packaging including can bottles, cartons, drums, barrels made up of glass, metal, plastic, or laminates are used for the packaging of orange juice and concentrates. The latest trends are forward aseptic packaging in flexible plastic films and laminates. Laminated carton packages are the predominant form of packaging in most countries for orange juice. One notable exception is Germany where glass bottles are mainly used. The use of oxygen scavengers with an appropriate packaging material can further reduce the presence of dissolved oxygen in the juice or present initially in the headspace. This technique has been investigated for the packaging of solid foods and orange juice in plastic bags (Zerdin et al., 2003). Glass bottles are the second most common orange juice package used worldwide. For shelf-stable orange juice in glass bottles the most common filling method is hot filling. Aseptic filling of glass bottles at ambient temperature is of minor importance compared with hot filling. Glass bottles are normally considered to have the best oxygen barrier properties from all the orange juice package types used today. Blow-moulded plastic bottles are an alternative to glass bottles for orange juice. The most common plastic bottles used are HDPE bottles and PET bottles. Losses in the aroma of orange juice from PET bottles can be lowered with (HDPE) closures (Berlinet et al., 2008). PET bottles for aseptic filling are sterilised prior to filling with product. High-pressure treatment (HPT) is also compatible with existing range of semi rigid and flexible packaging materials (Parish, 1998). Hot filling and storage at less than 15 C gives bottled citrus juices a shelf life of almost one year (Kulwant et al., 2012) Possibility to improve orange juice by blending with other juices and expand the range The progress of the juice and beverages industries towards production of the mixed fruit juices and nectars is a great way to develop unique drinks such as, for example, those with new flavours improved colour and consistency and moreover mixed fruit juices are an alternative for adding nutritional value (Sobhana et al., 2015). Mixed fruit juices can be prepared from different fruits and berries in order to combine all the nutrients present in the different kinds of fruits and berries. This usually gives better quality of juice and sensory attributes (Matsuura et al., 2004; Seglina et al., 2014; Torregrosa et al., 2006). According to Zotarelli et al. (2008) mixed fruit products join nutritional characteristics of two or more fruits and provide pleasant sensory characteristics in order to gradually gain prime space in the consumer market. The combination of fruit can also contribute to reducing costs of some products by the addition of cheaper fruits to high cost fruits fill shortages. Juice blending is one of the methods to improve the nutritional value of the juice (Ogbonna et al., 2013; Rathod et al., 2014; Sobhana et al., 2015). This claim for safer juice blends retaining great quality such as sensory freshness characteristics and biological properties forced researchers and manufacturers to produce new processing and conservation technologies (Hernandez-Carrion et al., 2014). Leahu et al. (2013) studied the changes of the physicochemical parameters of 42

43 orange, kiwi, apple and mix juices from these fruits during storage and they found that mix juices like kiwi and orange (70:30) has the highest content of vitamin C 96.8 mg per 100 g of product. Also Hashem et al., (2014) reported of changes in physicochemical quality and volatile compounds of orange carrot juice blend during storage. Chinese scientists believe that sea buckthorn and their products will become one of the most important products for health promotion in the 21st century. Sea buckthorn (Hippophae rhamnoides L.) from a little-known wild bush became a well-established fruit crops (Singh, 2003). Fresh sea buckthorn fruits contain significant amounts of vitamin C and vitamin E, carotenoids, organic acids, polyunsaturated fatty acids, phenolic compounds, and the basic elements (Singh, 2006; Abid et al., 2007). Fructose and glucose are the principal sugar types in sea buckthorn. All studies report that glucose content in sea buckthorn is the same or higher than that of fructose and the ratio of glucose to fructose varies from 1:1 to 10:1 (Bekker, Glushenkova, 2001; Kallio et al., 1999; Tiitinen et al., 2005; Tiitinen et al., 2006). The most obtained fruits acids in sea buckthorn are malic and quinic acids and citric acid is present to some extent (Bekker, Glushenkova, 2001; Kallio et al., 1999; Tiitinen et al., 2005). The fruit of the plant has high vitamin C content in a range from 114 to 1550 mg 100 g -1 (Zeb, 2004) with an average content (695 mg 100 g -1 ) about 15 times greater than oranges (45 mg 100 g -1 ), placing sea buckthorn fruit among the most enriched plant sources of vitamin C. The total content of tocopherols depends on species of berries (it varies from 100 mg to 700 mg 100g -1 ). Tocopherols and tocotrienols have strong antioxidative effects and therefore they have an indirect effect on the sensory quality of the berry (Kallio et al., 1999; Kallio et al., 2002; Zeb, 2004). The major constituents of phenolic compounds in sea buckthorn are flavonols, proanthocyanidins, catechins and phenolic acid. Free phenolic acid reported in sea buckthorn includes gallic acid, salicylic, pyrocatechuic, protocatechuic and further acids (Antonelli et al., 2006; Rosch et al., 2003; Zadernowski et al., 2005). There is great variation in the reports of content of phenolic compounds in sea buckthorn due to differences in the methods of analysis and calculations. Clinical studies have been conducted to investigate the physiological effects of the various fractions and products such as oil and juice from berries, particularly in China and Russia. Effects on the health seabuckthorn antioxidant, anti-inflammatory action, the development of cardiovascular disease, reducing risk of developing type two diabetes have been reported (Geetha et al., 2002;Geetha et al., 2008; Gupta et al., 2002; Singh et al., 2006; Suryakumar, 2011). The one of most popular sea buckthorn processed products is juice. Sea buckthorn juice is one of the most important products obtained from fresh berries and is commercially very much in demand. Sea buckthorn juice and drinks are assumed as the optimal food for use in any human diet (Zeb, 2004). Sea buckthorn beverages in trading are presented as a drink or nectar with added sugar or other sweeteners because they have low soluble solids and high content of acidity so they do not always provide the benefits expected by the consumer. Fruit beverages were among the earliest sea buckthorn products developed in China. Juice blending is one of the best methods to improve the nutritional quality of the juice. It can improve the vitamin and mineral content depending on the kind and quality of fruits and vegetables used (De Carvalho et al., 2007). Fresh orange juice and sea buckthorn juice is a product of high commercial and nutritional value due to its rich vitamin C content, vitamin E and ß- carotene, organic acids, polyunsatured fatty acids, phenolic compounds, essential elements and its desired sensory characteristics. Latvia consumers have access to some locally produced food products with sea buckthorn, natural juice nutritional supplements, herbal teas, and honey with sea buckthorn and some quantities of sea buckthorn oil. Sea buckthorn juices and beverages can be used as functional foods in our daily diet as their consumption is known to be beneficial for the human body. Sea buckthorn based juice is popular in Germany and Scandinavian countries. Therefore as an alternative to orange and sea buckthorn juice, a new product is proposed as a blend of orange and sea buckthorn juices. The UHT processing 43

44 technology can be an alternative to conventional thermal technology for the orange juices, sea buckthorn juices and blended juices and beverages. Such a treatment denatures enzymes and eliminates microorganisms responsible for spoilage of juice without detrimental effects on the sensory and nutritional quality of juice. Orange juice is the number one in the world for consumption and sea buckthorn juice provides great opportunities to supplement juices with important nutritional value. Analysis of the literature shows that the treatment of sea buckthorn in Latvia can bring economic interest to both breeders and processors which is an essential addition to the market of juices and beverages with new important and valuable food products. Summary of the biochemistry of orange fruit and obtained juices and their suitability for the modern technology Orange fruits and orange juices have long been appreciated for their beneficial nutrients and antioxidant properties. The biological activity of vitamin C, flavonoids and carotenoids was studied in many tests (Burns et al, 2003; Cassano et al., 2003; Gardner et al., 2000; Kurowska et al., 2000; Lichtenthaler, Marx, 2005; Topuz et al., 2005). Orange fruits are one of the largest world crops and its juices are one of the most appreciated by consumers. Orange fruits and orange juices have long been appreciated for their beneficial nutrients and antioxidant properties. The biological activity of total phenolics compounds, flavonoid, vitamin C and carotenoids was studied in many different tests including human. There is clinical and epidemiological evidence showing the bioactive compounds of orange fruits and orange juices have a great protective effect the risk of chronic diseases as cardiovascular diseases, cancer, obesity, diabetes, etc. Oranges are generally available from winter through summer with seasonal variations depending on the variety. The juice quality depends on various factors as orange fruit variety, place of growing, harvesting time and processing manner. The vitamin C concentration is a significant indicator for the quality of orange juice. Thermal processing continues to be the most widely used method of preserving and extending the shelf life of foods, nonetheless there is a growing interest in consuming minimally processed foods with characteristics closer to those of fresh juices. Currently the food industry is looking at replacing the traditional well-established preservation techniques with novel thermal and non-thermal technologies that may produce high quality food products with improved energy efficiency and to be more environmentally friendly. Several studies have shown that non-thermal processing technologies including high pressure processing (HPP) and pulsed electric fields (PEF) result in a higher retention level of ascorbic acid relative to thermally processed juices and the effect of conventional thermal pasteurisation and alternative HHP processing on post processing results in quality loss (Kulwant et al., 2012). Some researchers have investigated the effects of ultrasound or PEF on orange juice shelf life but up to date no published data are available on the combined impact of ultrasound and PEF. Many studies have been carried out on the quality and stability of pasteurised orange juices and juices obtained from concentrates. In this case the impact of pasteurisation on quality is clearly appreciable. Several studies show what happens during the season when the consumer buys oranges in the market in order to prepare freshly squeezed juice. Latvian producers supply customers mainly with juice from concentrate Frozen Concentrated Orange Juice (FCOJ) is surely a world "commodity" or concentrated orange juice (COJ) in aseptic bags in drums. Pasteurised orange juice not from concentrate (NFC) is superior in taste to reconstituted juices. Today s consumers prefer orange juice NFC because of it organoleptic characteristics and qualitative parameters. The Ultra-High-Temperature (UHT) processing presents itself as a great technique to extend the shelf life of the product and increase the consumer safety while maintaining the 44

45 fresh quality of orange juice. However, no researches have integrated the comparative study of the impact of UHT and heat treatments on other bioactive compounds of orange juice such as vitamin C, flavonoids, carotenoids, pectin and the property antioxidant activity. Finally, the progress of the beverage industries towards production of the mixed fruit juice or nectar is a great way to develop new tastes, improve colour and consistency of juices, as well as a great alternative to add nutritional value. Blended orange and sea buckthorn juices are products with a high content of bioactive compounds and excellent sensory properties. 45

46 2. MATERIALS AND METHODS 2.1. Time and place of the research The study was carried from 2009 until 2015 in: S.A. BIOFRESH, juice and concentrate Production Company, in Laconia, Greece. Physicochemical characteristics and biochemical compounds of orange fruits during maturation, juices processing process and refrigerated storage in industrial scale were studied; UBF GmbH, Investigative consulting Research Laboratory GmbH in Altlandsberg, Germany. Physicochemical characteristics, bioactive compounds and antioxidant capacity of orange juice during maturation processing technology and during refrigerated storage were studied; Latvian University of Agriculture in Jelgava, laboratory pilot a miniature-scale HTST/UHT processing system (Armfield FT74, HTST/UHT Processing Unit) UHT processing was studied; State Institute of Fruit-Growing in Dobele, Latvia. Laboratory equipment, sterilization autoclave with counter pressure and fast cooling (AES-110 RFG, Raypa, Spain). Physicochemical characteristics, bioactive compounds and antioxidant capacity of orange juice High Temperature Short Time (HTST) and UHT processing technology were studied Characteristics of the material The objects of the research are orange fruits (Citrus sinensis L.) and orange juices (see characteristics below). Orange fruits and orange juices of Greek summer variety Valencia and winter variety Navel were obtained from Manufacturing S.A. Biofresh production line of commercial juice plant in Laconia Southern Greece. The characteristics of orange varieties are provided in Table 2.1. The orange fruits were harvested during November, December, January and February for winter variety Navel and from March until September for summer variety Valencia. Orange fruit (Citrus sinensis L.) characteristics Table 2.1 Fruit variety Navel oranges (winter variety orange) Valencia oranges (summer variety orange) Characteristics The Navel orange actually grows a second twin fruit opposite its stem. The second fruit remains underdeveloped but from the outside it looks like a human navel-hence the name. In Greece this variety is available from November to April, with peak in January and December. They are seedless peel easily and are considered one of the world s best tastes oranges. The Valencia orange is typically available starting in March and continuing through September or so. After bloom it usually carries two crops on the tree, the old and the new ones. Valued for their high juice content and availability outside of the typical citrus season Valencia oranges are usually thin skinned and have a few seeds. They are considered one of the best oranges for juicing. 46

47 Additional, the following materials were used: Fresh frozen orange juice of variety Navel and Valencia fruits, from Manufacturing S.A. Biofresh production line, Greece; Pasteurised orange juice not from concentrate (NFC) Navel and Valencia varieties, from Manufacturing S.A. Biofresh production line, Greece; Orange juice reconstituted from concentrate (OJFC) of Navel and Valencia varieties, from Manufacturing S.A. Biofresh production line, Greece; Fresh seabuckthorn (Hippophae rhamnoides L.) juices of German cultivars Leikora and Hergo from Manufacturing Sanddorn GmbH production line Germany; Fresh seabuckthorn (Hippophae rhamnoides L.) juice of Latvian cultivar Botanicheskaya-Lubitelskaya from Latvian State Institute of Fruit-Growing experimental processing Laboratory; All samples of orange juices were delivered by plane from Manufacturing S.A. Biofresh production line, Laconia Southern Greece in aseptic bags. Frozen samples of orange juices were kept frozen in a forced circulation freezer and kept at -18 ± 2 C until using. Frozen orange juice samples were defrosted. Pasteurised orange juices in aseptic bags were kept at 5 ± 2 C in refrigerator. Fresh cooled sea buckthorn juices were transported from Germany and Latvia. Packaging materials Aseptic bag-box (volume 1000 ml and 5000 ml) used to store juice samples under production conditions; Glass jars with screw cap closures (volume 150 ml type JPG) used for HTST and UHT processing technology experimental samples storage before analyses. Used equipment Characteristics of industrial scale and laboratory pilot type equipment used for experiments are summarized in Table 2.2. Characteristic of industrial scale and laboratory pilot type equipment used for experiments Table 2.2 Equipment Brand Manufacturer Industrial extractors CENTENARIO Manufacturer Organization Industrial CENTENARIO, Brazil Plate-type heat exchanger Bertuzzi Bertuzzi Food Processing SRL, Italy Machine Aseptic system ROSSI and ROSSI and CATELLI, Italy CATELLI Tubular evaporator T.A.S.T.E. JBT FoodTec, Brazil Sterilisation Autoclave with AES-110 RFG Raypa, Spain counter pressure and fast cooling A miniature-scale HTST/UHT processing system Armfield FT74, HTST/UHT Hampshire, England 47

48 2.3. The structure of the research The study of doctoral thesis is divided into two steps: 1. The first stage of the study was carried out directly on the juice production plan in Greece in the production laboratory. Evaluation of physicochemical parameters as juice content (yield), total soluble solids (TSS), total acidity (TA), TSS/ TA ratio, and vitamin C were tested in orange juice (Navel and Valencia) in harvesting time (2 seasons of harvesting). For more in-depth studies of orange and its juices, the juices from production line were delivered to German UBF laboratory for determination of bioactive compounds (vitamin C, total phenolics compounds, hesperidin, total carotenoid, ß-carotene and pectin) and antioxidant capacity on different maturity stage, after processing and during refrigerated storage. 2. The second step of the study was undertaken to compare the impact of two treatment methods: High Temperature Short Time (HTST) and an alternative Ultra-high temperature processing (UTH) on chemical parameters, bioactive compounds and antioxidant capacity. Sensory attributes of fresh orange juice was compared with HTST and UHT treated orange juices. Also there was defined also the effect of UHT processing on chemical characteristics, bioactive compounds and antioxidant capacity of the sea buckthorn juice and blended juices of orange-sea buckthorn, as well as sensory attributes of blended orange-sea buckthorn juices. Orange juice production begins from fruit evolution in the maturation stage on the growing field till obtaining safe and quality juice in the production enterprises. General scheme of juice production from oranges in industrial scale and experimental UHT processing of orange juice, sea buckthorn juices and blended orange-sea buckthorn juices is provided in Figure

49 Fig. 2.1.General Scheme of orange juice production Evaluation of physicochemical parameters, bioactive compounds and antioxidant capacity in different maturity stage, processing and storage of two varieties of orange juice in industrial scale Scheme of juice production from oranges with indication to evaluation of physicochemical characteristics and bioactive compounds and antioxidant capacity of two varieties of oranges in different maturity stage, processing and juice refrigerated storage in industrial scale is provided in Figure

50 Fig. 2.2.Evaluation of chemical parameters, bioactive compounds and antioxidant capacity of two varieties of orange juice during maturation, processing and refrigerated storage in industrial scale The collected orange fruits are delivered to plant for juice processing, where they are unloaded to bin storage. The orange fruits are inspected and graded before they can be used. The analyst of laboratory takes 2 kg sample to analyse in order to make sure the fruits meet maturity requirements for processing. The certified fruits are then transported along a conveyor belt where they are washed as they pass over roller brushes. This process removes debris and dirt and reduces the number of microbes. The fruits are rinsed and dried. Graders remove bad fruits as they pass over the 50

51 rollers and the remaining quality pieces are automatically segregated by size prior to extraction. There are 13 of such size codes. Proper size is critical for the extraction process. There are normally 2 3 different size settings in an ex tractor line. For oranges, the size code 0 is for mm diameter of fruit, code 1 for mm, code 2 for mm, code 3 for mm, and code 4 for mm (Fresh Fruit and Vegetables Codex, 2002). Orange juice was prepared using CENTENARIO extractor (Manufacturer Organization Industrial CENTENARIO, Brazil). Freshly squeezed orange juice, after extraction without pasteurisation or any other physical or chemical treatment, was selected from manufacturing in laboratory flask and tested immediately. NFC orange juice and reconstituted from concentrate orange juice (OJFC) pasteurisation was carried out at 94 C temperature for 30 s using HTST (High Temperature Short Time) in plate-type heat exchanger Bertuzzi (Bertuzzi Food Processing SRL, Italy) by heating with hot water. Storage method of pasteurised NFC orange juices for experiments: fresh squeezed (unpasteurised) chilled orange juices from buffer/blending tank each variety were filled into two aseptic bags (1000 ml each) and then were immediately frozen in a forced circulation freezer and kept at -18 ± 2 C until usage; NFC pasteurised orange juices from both varieties were aseptically filled using ROSSI and CATELLI equipment. Four aseptic bags were frozen and kept at 5 ± 2 C; twelve aseptic bags of each variety of pasteurised NFC orange juices were selected from manufacturing line and were stored for four, eight and twelve months in refrigerated storage at 5 C to 2 C; OJFC from each variety was filled into four aseptic bags (1000 ml each) and kept at 5 C ± 2 C until use. For juice concentration, the tubular evaporator TASTE was used. The orange juice was first preheated to C for 30 seconds. The product then was passed through seven stages under vacuum, until the concentration of up to Brix was achieved. By this time the product was cooled to about 40 C. The residence time in the evaporator was from five to seven minutes. After evaporation, the concentrate was chilled to 10 ± 2 C (concentrate did not freeze solid at -10 C). Samples of the juices were evaluated in terms of physicochemical parameters: juice content, total soluble solids (TSS), total acid (TA), vitamin C, and calculated TSS/TA ratio in production laboratory (in industrial scale). The analysis was carried out at +20 ± 1 C, and was repeated 2 times. Pasteurised orange juice samples and frozen samples of orange juice were delivered by airplane to Germany into the UBF research laboratory and kept under controlled conditions until use. Such biochemical compounds as content of individual sugars, vitamin C, total phenols, hesperidin, total carotenoid, ß-carotene, antioxidant capacity and water-soluble pectin were analysed. Analyses were carried out at +20 ± 1 C, and were repeated 2 times Evaluation of UHT processing on the chemical characteristics, bioactive compounds and antioxidant capacity of orange juice The objective of the second step of this work was to comparatively evaluate the effect of HTST and an alternative UHT processing on the chemical parameters, bioactive compounds, antioxidant capacity and sensory attributes of fresh frozen and then defrosted Navel orange juice (see Fig. 2.3). 51

52 Fig. 2.3.UHT and HTST processing orange juice quality parameters determination Fresh orange juices from freshly harvested orange Navel variety fruits, grown in the south region of Greece were used. The orange juice was produced at the orange juice production plant S.A Biofresh Company. Four bags (5000 ml each) of freshly squeezed and chilled Navel orange juice were immediately frozen in a forced circulation freezer and kept at -18 C ± 2 C. After they were delivered by aircraft to Germany into UBF research laboratory and kept at -18 C ± 2 C until use. Samples with frozen orange juice were then defrosted and analysed. Processed by HTST methods juices were carried out on the laboratory equipment AES- 110 RFG (Raypa, Spain) at 94 C for 30 seconds. UHT processing of orange juice was carried out using a miniature-scale HTST/UHT processing system Armfield FT74 (Hampshire, England) at 130 C for 2 seconds. Both processed juices (orange and sea buckthorn) were filled in glass bottles (150 ml) with screw cap closures. Four samples of each juice were immediately sent to laboratory for the determination of total soluble solids, total acidity, vitamin C, total phenolics compounds, total carotenoids and antioxidant capacity. Ten bottles of orange juice from each processing method were kept for sensory evaluation. 52

53 Evaluation of UHT processing on the chemical parameters bioactive compounds and antioxidant capacity of sea buckthorn juices and orange-sea buckthorn blended juices Fresh chilled (0-5 C) sea buckthorn juices of Leikora and Hergo varieties were delivered from company Sanddorn GmbH, and Botanicheskaya Lubitelskaya variety was delivered from Latvia State Institute of Fruit Growing. UHT processing of sea buckthorn juices was carried out using the miniature-scale HTST/UHT processing system Armfield FT74 (Hampshire, England) at 130 C for 2 seconds and was filled in 150 ml glass bottles with screw cap closures (see Fig. 2.4). Fig UHT processing of sea buckthorn and blended orange-sea buckthorn juice quality parameters determination Two samples of each sea buckthorn juice variety were immediately sent to laboratory for the determination of total soluble solids, titration acidity, vitamin C, total phenols, total carotene and antioxidant capacity. Six bottles of each juice were kept for sensory evaluation. Sensory analyses were accomplished for orange-sea buckthorn juice blend. Both (orange and sea buckthorn) juices processed by UHT were blended in proportion 90: Methods for determination of chemical parameters, bioactive compounds and antioxidant capacity Methods for determination of the chemical parameters, bioactive compounds and antioxidant capacity of orange juice are summarised in Table

54 Table 2.3 Standards and analytical methods used for determination of orange juice No. Parameters Methods 1. Total soluble solids, Brix AOAC ; ISO 2173: Total acid content, % AOAC ; ISO 750: Yield,g Gravimetric method, electronic weigh (± 0.001g) (Lacey et al., 2009) 4. Glycose, fructose, sucrose content, g 100 ml L- ascorbic acid, mg 100 ml Total content of total phenolics compounds, mg 100 ml Total carotenoids, mg 100 ml -1 Enzymatic method, r - Biopharm Cat. No Cat. No Iodine method (Moor et al., 2005) - production method 2. Enzymatic calorimetric method, r Biopharm Cat. No ; LVS EN 14130:2003 Spectrophotometric method, (Singleton et al., 1999) Spectrophotometric method, DGF Einheitsmethoden F-II 2a (1975)/Unit methods (Germany) 7. β-carotene, mg 100 ml -1 Spectrophotometric method, DGF Einheitsmethoden F-II 2b (1975)/Unit methods (Germany) 8. Hesperidin, mg 100 ml -1 High-Performance Liquid Chromatographic Method, DIN EN ISO/IEC 17025:2005; IFU No Antioxidant capacity (ABTS), mmol TE 100 ml Antiradical activity, (DPPH), mmol TE 100 ml Antioxidant activity Ferric reducing antioxidant power (FRAP), mmol TE 100 ml Water Soluble Pectin (Carbozole Method), mg 100 ml -1 Spectrophotometric method, TEAC Method AOCA intern Spectrophotometric method, (Brand-Williams et al.1995) Spectrophotometric method, (Benzie and Strain, 1996) Spectrophotometric method Carbazole method 21 (Amador et al., 2008) Chemical parameters and bioactive compounds analysis carried out in healthy oranges and orange juice during maturation time, processing and storage in industrial scale, as well as by the laboratory pilot line Method of sensory analysis The studies of sensory attributes of orange juice and blended orange-sea buckthorn juices were evaluated following treatment with a combination of technologies (HTST/UHT) and blended juices after UHT technology method. The sensory evaluation was conducted one week after the juice processing. Orange juices were evaluated using the hedonic rating scale according to ISO 4121:1987 (Sensory analysis Guidelines for the use of quantitative response scales). Consumer acceptability was evaluated by 14 trained panellists who were the analysts in the UBF GmbH Company, (10 female, 4 male), from 21 to 58 years of age. Prior to sensory evaluation orange juice samples were refrigerated, randomly coded and served (20 ml) at 54

55 18 C together with non-salted crackers and still water. The panellists were seated separately in booths, in order to allow an unbiased evaluation of the sensory attributes. Samples of orange juice processed by UHT and HTST pasteurisation were compared in terms of aroma, flavour, and overall acceptability on a 9-point hedonic scale, where one was the lowest and nine was the highest score Data mathematical analysis The mathematical processing of experimentally obtained data was performed by mathematical statistical methods by using the Microsoft Excel for Windows 7.0 and SPSS program SPSS 15 package. The obtained results were necessary to calculate the following indicators: the arithmetical mean value, standard deviation and standard error. For the interpretation of orange juice physical and biochemical parameters data were used univariate analysis of variance with replicates. The Brix / acid ratio was obtained by dividing the total soluble solids ( Brix corrected for acids and temperature) by the total titratable acid (% Acid, w/w) at 20 C (Amador, 2008). Hesperidin is identified by comparison of retention time with a standard (Lot 011M1865V, Sigma). Hesperidin concentration (mg/10 ml) in sample was calculated from sample absorbance based on a linear regression equation of the standard curve of absorbance peak are (PA) at 280 nm against concentration of hesperidin standards. Hesperidin where: PA peak of absorbance; DF sample dilution factor; β concentration in mg/10ml. mg 10 ml = PA sample β standard DF PA standard (2.1) The closeness of the relationship between the analysed parameters was determined according to the Pearson method (Arhipova, Balina, 2006). The Pearson correlation (r) measures the linear dependence between two variables (x and y). The correlation coefficient is a unitless measure which ranges between -1 and 1. The greater the absolute value of a correlation coefficient the stronger the linear relationship. The weakest linear relationship is indicated by a correlation coefficient equal to 0. n i=1(x i x ) (y i y ) r = ( (x i x ) 2 ) ( (y i y ) 2 ) (2.2) To evaluate the sensory analysis a One Sample T-Test using 2.4 formula, a Two- Sample-F using 2.3 formula and -T-Test using formula 2.4 and 2.5 (Ann. ). Those statistical parameter tests compare the variances and averages of the different samples and show if there is a significant difference. T = x μ 0 s n, (2.3) T t 1 α 2,m m = n 1 55

56 where: µ is the "Control"; s standard deviation; α significance level; n number of members of sample; x bar average. An F-test (Snedecor and Cochran, 1983) is used to test if the variances of two different juice samples are equal. F = s 1 2 s 2 2, with s 1 2 > s 2 2, F f 1 α,m1,m 2 m i = n i 1 where: s 1 and s 2 are the samples variances; α significance level; f 1-α,m1,m2 critical value of the f distribution with m degrees of freedom. (2.4) T = x 1 x 2 s pooled n 1 n 2 n 1 +n 2, (2.5) s pooled = (n 1 1)s (n 2 1)s 2 2 n 1 + n 2 2 T t 1 α 2,m m = n 1 + n 2 2 where: α significance level; S pooled pooled standard deviation; t α 1,m critical value of the t distribution with m degrees of freedom. 2 56

57 3. RESULTS AND DISCUSSION Fresh oranges fruits varieties of Navel and Valencia were tested during harvesting time for the juice content (yield), Brix, total acidity, and vitamin C (industrial test) at the direct processing enterprise in Laconia, Greece, from 2009 till For more in-depth studies of orange juices, bioactive compounds (vitamin C, total phenolics compounds, hesperidin, total carotenoids, ß-carotene and water soluble pectin) and antioxidant capacity were determined in fresh defrosted orange juice during harvesting, as well orange juice from concentrate (OJFC) and NFC orange juice processed in industrial scale. NFC orange juice was analysed at the production phase and in aseptic packaging during storage time. Fresh defrosted orange juice Navel and processed by HTST and UHT technology orange juice Navel, as well chilled sea buckthorn juices and processed by UHT technology sea buckhorn juices were tested for the bioactive compounds and antioxidant capacity determination. Blended orange-sea buckthorn juices were tested for the bioactive compounds, antioxidant capacity and sensory attributes Evaluation of the quality parameters in winter s Navel and summer s Valencia varieties of oranges, grown in Greece during harvesting in industrial scale The available literature on biochemical composition and metabolism of oranges is vast and has been reviewed time to time by various researchers. A number of studies suggest that biochemical composition of oranges depends on many factors: growth place (Braddock, 1999), climate conditions, maturity state, position on the tree, species and varieties of fruits (Ladanya, 2008). This study looks at the principles of processing orange juice that have to be taken into consideration in designing new technologies and process plants. It should be stressed that despite the long experience of the industry in processing orange fruit and the large amount of research that has been done on the subject, all the secrets of orange juice have not yet been revealed Evaluation of physicochemical parameters of fresh orange juice during harvesting within two years In Greece, most oranges bloom from March to May. The fruits of summer variety, such as Valencia, mature from March to September. The winter variety fruits, such as Navel, reach maturity in the end of October through February. In industrial scale, the most important components that influence the quality of orange juice are sugars acids and vitamin C. In addition several compounds such as flavonoids, carotenoids, and pectin are present in orange juice. The content of these functional active substances in the orange juice can be characterised by antioxidant capacity. Due to insufficient information on the date for the characterisation (bioactive compounds) of fresh orange juices obtained during season of processing, the purpose of this work was to study the main. Sampling: orange Navel and Valencia fruit samples were collected every production day in plant laboratory during harvesting of each season. For the purposes of this study, the average test results were selected directly from the plant's production laboratory (Ann.1. Ann. 2). Juice content is an important measurement of internal quality in production. The results, concerning the juice contents of orange, showed a significant difference (p < 0.05) in the stage of harvesting, as shown in Figure 3.1 (in the graph you can see average results for the month every day production). 57

58 60 Juice content, % Maturity time Valencia 2009 Valencia 2010 Navel Navel Fig. 3.1.The fresh juice content in thevalencia and Navel orange fruits during harvesting During two seasons ( ) Valencia oranges in early (March) stage with 42.2 and 40.5% and mid (June - July) stage with 48.5 and 46.8% showed significantly high (p < 0.05) juice percentage compared to late (August September stage (36.4 and 38.2%) respectively. In seasons, the juice content from Navel varieties was 35.5 and 36.1% in early seasons (November), and 44.2 and 42.7% during mid (December -January) season's also showed significantly high (p < 0.05) juice percentage compared to late season (35.0 and 36.8%) respectively. The difference between early and mid-stage was non-significant (p > 0.05) in both varieties of orange. These contents remain variable for all of the periods of the season in fruit harvesting. Decrease in juice contents reveals quality decline (Ladaniya, 1996). After mid-season the juice content decrease might be due to the onset of new growth and new fruit development, which sometimes sucks back the moisture and nutrients from the old fruits. These findings of juice contents are in agreement with Anwar et al. (1999). During two seasons, average juices content measurements in both varieties of orange were about 40%. Juice content reduction to the end of the season maximum has become 22%, with advancement of maturity, and it was in Valencia orange in 2009 season; the minimum of the juice content reduction (9%) was observed in Navel variety orange. These results are similar with the observed results by Anwar et al. (1999), Musaratet al. (2015). Total soluble solid (TSS) is a percentage of sugar or Brix. The statistical data of two seasons (from 2009 to 2011) of industrial production for two varieties (Valencia and Navel) orange juice showed that the TSS significantly increased during maturation. The Valencia oranges variety which harvest from March through October is the most popular variety for processing. In seasons 2009 and 2010 Valencia orange clearly observed TSS increase at the beginning of March (10.26 and Brix) with TSS/TA ratio of 9.1 and 9.5 by the end of the season, in October (12.31 and Brix) with TSS/TA ratio 15.8 and 17.5 respectively. Early variety of Navel orange matures from November until April. In the two seasons of Navel orange, the TSS increased from (10.87 and Brix) with TSS/TA ratio of 9.5 and 10.2 to (12.62 and Brix) with TSS/TA ratio of 18.6 and 16.9 respectively (Fig. 3.2). 58

59 TSS, Brix Maturity time Valencia 2009 Valencia 2010 Navel Navel Fig The content of TSS in fresh Valencia and Navel orange juices during harvesting The season of Valencia orange in 2010 showed a significant increase of TSS by 25% at the end of the season in comparison with the previous 2009 season where the increase of TSS was 20%. The seasonal increase of TSS oranges in Navel orange was of 18% and was higher in the season 2010/2011 compared to the 2009/2010 season, where the increase of TSS was only 16%. When the fruit remains on the tree, TSS continues to increase and acids decreases until the fruit becomes over ripe. Rekha et al. (2012) and Anwar et al. (1999) also found increase in TSS with time of mature in sweet orange. It should be noted that for orange varieties during maturation the amount of data available is still scarce. Many studies on TSS content in orange juices are available (Farnworthet al., 2001; Ywassaki et al., 2011; etc.), but they differentiated because of differences in varieties of oranges their ripeness and the technological processes used to obtain commercial juice. The organic acids of the orange fruits are of interest because of its important influence on the sensory properties of juices. In the observed seasons from 2009 to 2011, the acidity of Valencia and Navel orange varieties has decreased significantly with time. The maximum content of total acidity (TA) was 1.12 and 1.08% in March in Valencia orange juice, and maximum content of TA 1.15 and 1.05% in November in Navel orange juice respectively (see Fig. 3.3). 59

60 Total acidity, % Maturity time Valencia 2009 Valencia 2010 Navel Navel Fig The content of total acidity in thevalencia and Navel orange juices during harvesting In both seasons the average content of TA in orange juice Valencia variety was 0.90 % and 0.92 % in orange juice Navel variety. The decrease of TA content of both orange varieties with time was considered to be due to the dilution, as the fruit increased in size and the juice contain, and may be due to the accumulation of sugars in the juice. In the orange fruits the organic acids might be used for energy production through cellular respiration and the conversion of organic acids to sugar (glucose) through gluconeogenesis. Our results are in agreement with those found by Esteve et al. (2005). They reported a significant decrease in TA content during maturation. Redd et al., (1986) studied total acid in four orange juices and found significantly different contents between them (p 0.05) but in all cases it was within the recommended values (0.6 to 1.6 g per 100 g). The relative sweetness or sourness of citrus fruit is determined by its ratio of sugars to acids (TSS/TA). This is a maturity index and is used to determine the legal maturity of oranges and is also used as a guide of harvest indices in the citrus industry. A higher TSS/TA ratio indicates decreasing acid content. When the ratio reaches 20 or more the taste of citrus juice is too sweet and no acid is detected (Ladaniya, 2008). Maturity standards for oranges in Greece require a minimum Brix of 9.0 and minimum TSS/TA ratio of 10. The increase in TSS and the decrease in TA leads to a gradual increase in the TSS/TA ratio in both varieties of orange. The maximum average ratio (17.5) was obtained in October 2010 of Valencia orange juice and maximum average ratio (18.6) was obtained in April innavel orange juice. Ratio of oranges was unstable, because the content of sugar and acidity also fluctuated during maturity. Vitamin C (ascorbic acid) is the most important nutrient in orange juice and its content in different orange fruits varies considerably. In general the content of vitamin C in orange juices varied from 40 to 70 mg per 100 ml and this value depends of many factors reported above. The content of vitamin C usually is higher in immature orange fruit (Baldwin, 1993) and decreases during fruit maturation. The orange fruits in early season have more vitamin C than orange fruits from later season. According the study seasons of two years ( ) in the orange juice plant in Laconia were analysed two varieties of orange fruits and orange juices. The study results showed that the value of vitamin C content ranged from to mg 100 ml -1 in Navel orange juice and from to mg 100 ml -1 in Valencia orange juice (see Fig. 3.4). 60

61 Vitamin C, mg 100 ml ¹ Maturity time Valencia 2009 Valencia 2010 Navel Navel Fig The content of vitamin C in thevalencia and Navel orange juices during harvesting Vitamin C content in orange juice reduced in both varieties with maturity (about 25%). The maximum of the vitamin C content (59.90 mg 100 ml -1 ) was found in December of the season 2009/2010 in Navel orange juice and vitamin C content (45.80 mg 100 ml -1 ) was found in April of the season 2009 in Valencia orange juice. During seasons in Navel and Valencia oranges juices the average vitamin C content was 47 and 40 mg 100 ml -1 respectively. Decrease in the concentration of vitamin C was about 25% in both varieties of orange juices. Anwar et al. (1999), Cepeda et al. (1993), Lee, Coates (1999) also reported decrease in ascorbic acid content in orange fruit. Ywassaki, Canniatti-Brazaca (2011) studied ascorbic acid in different sizes and parts of citric fruits. Differences were found between sizes. Due to the fact that most of the production of citrus juices is in the immediate vicinity of plantations of oranges the juice is being produced year round. There can be a one to two months break it depends on the climatic conditions that affect the ripening of the fruits. In Laconia the break between seasons was one month. The aim of this part of research was to investigate the effect of maturation on the physicochemical properties of orange fruits Navel and Valencia widely cultivated in Greece. The obtained results indicated that stage of maturity had significant effect on the physicochemical parameters of both varieties of orange fruits. In the present study it was observed that TSS and TSS/TA ratio, juice content tended to increase toward maturity but at the end of the season there was a decrease in this parameter. On the other hand, TA and vitamin C contents were high at the early stage of the fruits maturation Evaluation of chemical parameters, bioactive compounds and antioxidant capacity of fresh defrosted orange juice on different maturity stage The research available in the literature is on biologically active compounds during storage at various temperatures and treated with different technologies. Not enough research has integrated the comparative study of the impact of maturity on bioactive compounds of orange such as vitamin C, total phenolic compounds, carotenoids and water soluble pectin. 61

62 Biochemical changes during fruit development and maturation are the key determinant of juice quality. Keeping in view the importance of the physicochemical parameters in orange fruits in industrial scale effects of harvesting on biochemical compounds was the next step of present study. The changes of the chemical parameters and bioactive compounds in fresh defrosted orange juices Navel and Valencia varieties were evaluated during fruits harvesting at different stages of maturity: early stage (A) in the beginning of the season, mid (B, C) of the season, when fruits were fully mature and at the end (D) of the season, when fruits were more mature (Ann. 3.) For advanced study of changes in the bioactive compounds of orange juice, during the seasons of each variety ( ) the samples in due indicated data were selected (fresh frozen orange juice in 16 bags in box) from production and taken to a laboratory in UBF, Germany and then defrosted till 18 ± 2 C for testing. Total soluble solids (TSS), total acidity (TA), TSS to TA ratio, sugars, vitamin C, total carotenoids and ß- carotene, total phenolics compounds, hesperidin, water-soluble (WS) pectin and antioxidant capacity of two orange juice were estimated. The chemical parameters of selected juices are shown in Table 3.1. Table 3.1 Chemical parameters of fresh defrosted orange juice Valencia and Navel in different maturity stage; early stage (A), mid (B, C) and end (D) of the seasons Total acidity, Date of Sample and TSS, Brix Ratio % sampling variety Average Navel A ± 0.01 a 0.96 ± 0.04 a 11.9 ± 0.01 a Navel B ± 0.01 b 0.88 ± 0.03 b 12.5 ± 0.01 b Navel C ± 0.01 c 0.79 ± 0.02 c 16.2 ± 0.01 c Navel D ± 0.01 d 0.75 ± 0.05 d 16.5 ± 0.01 c Valencia A ± 0.01 b* 0.98 ± 0.02 a 11.4 ± 0.01 d Valencia B ± 0.01 c 0.92 ± 0.04 e 13.8 ± 0.01 e Valencia C ± 0.01 d 0.80 ± 0.05 c 15.3 ± 0.01 f Valencia D ± 0.01 e 0.80 ± 0.05 c 17.2 ± 0.01 g *Values, marked with the same letter, are not significantly different (p 0.05) Nearly 75 to 85 percent of TSS of orange juice is sugars: sucrose, glucose, and fructose. The reducing, non - reducing, and total sugars increase as fruit ripens on the tree (Ladaniya, 2008). There is generally an increase in reducing and non- reducing sugars with maturity in both varieties of orange juice. The study results show that at the beginning of the season the content of sucrose increased from 4.51 to 5.43 g 100 ml -1, and from 4.80 to 5.51 g 100 ml -1 in Navel and Valencia orange juices respectively. Similarly, contents of fructose and glucose increased from 2.36 to 2.86 g 100 ml -1, from 2.92 to 3.44 g 100 ml -1, from 2.3 to 2.8 g 100 ml -1 and from 2.65 to 3.12 g 100 ml -1 in Navel and Valencia orange juice respectively (see Figure 3.5). Increase of sugar content in orange juice during maturity was: sucrose 17%, fructose 17.3% and glucose 16.1% in both varieties of orange. 62

63 Fig. 3.5.The content of individual sugars in fresh defrosted Navel and Valencia varieties orange juices on maturity; early stage (A), mid (B, C) and end (D) of the season The glucose-fructose ratio is practically constant and does not exceed the value of 1.00; these results are in agreement with Association of the Industry of Juices and nectars (AIJN) Code of Practice. The average values for reducing sugars (glucose and fructose) are under 3 g 100 ml -1. Hollman et al. (1999) also found an increase in sugars content during growing in Okitsu Wase and Silverhill orange juices. There are several reports previously presented for immature orange fruits and sugars content during maturation (Anwar, 1999; Esteve et al., 2005; Kimball, 1991; Ladanya, 2008). The investigators found that total sugars are strongly affected by the growing season. Farnworth et al. (2001) studied the concentration of sugars (glucose, sucrose, and fructose) in orange juice during storage, and found that the concentration of sugars did not vary during storage, but the total soluble solids increased with time. When harvesting in different parts of the plantation could be fruits with a different content of vitamin C. Although it needs to be considered that the content of the vitamin C can differ from different factors such as variety, climatic conditions, cultural practices, maturity, harvesting methods and postharvest handling procedures (Braddock, 1999; Lee, Kader, 2000; Ladanya, 2008). During season 2013/2014, in both Navel and Valencia varieties of oranges the amount of vitamin C decreased from 67.0 to 56.3 mg 100 ml -1 and from 51.9 to 43.5 mg 100 ml -1 respectively (see Fig. 3.6). 63

64 Fig. 3.6.The content of vitamin C in fresh defrosted Navel and Valencia varieties orange juices on maturity early stage (A), mid (B, C) and end (D) of the season The study results also showed significant differences in vitamin C amount. The Navel early maturing orange fruit had higher amount of vitamin C compared with later maturing fruit Valencia. The 16 percent decrease in amount of vitamin C was the same for both varieties of oranges during season. This is due to an increase in fruit size and correlation between vitamin C content and the contents of reducing sugars suggests that constituents are associated with ascorbic acid synthesis. Decreases of vitamin C are lower in acidic media. The phenolic antioxidants of fruit juices protect the vitamin C content from oxidative degradation. In general, the total content of phenolics compounds decreases as fruit matures. The study results showed that the content of total phenolics compounds decreased from mg 100 ml -1 to mg 100 ml -1 and to mg 100 ml -1 in Navel and Valencia oranges juice respectively during maturation (see Fig. 3.7). The study results are in a good agreement with those reported in the literature (Gardner et al., 2000; Houjin et al., 1990, Rapisarda et al., 1999). Rekha et al., (2012) estimated total phenolics content of fresh juices, from ripe and unripe oranges and found that total content of phenolic compounds ranged from 532 to 960 µg GAE/mL of fruit juice and high content of phenolics was observed in unripe fruits. Gorinshein et al. (2001) found that the total content of polyphenols was 154 mg 100 g -1 in fresh peeled orange fruits and their peels contain 179 mg 100 g -1 of total polyphenols. Hesperidin content of citrus fruit varies with species (Omidbaigi et al., 2002). Hesperidin is a main flavonoid of orange (Gorinstein et al., 2006). During maturation in the Navel and Valencia oranges their concentrations ranged from to and to mg 100 ml -1 respectively (see Fig. 3.7). 64

65 Content, mg 100 ml Nav-A Nav - B Nav- C Nav- D Val -A Val - B Val -C Val- D Dec.13 Jan.14 Feb.14 March.14 Apr.13 May.13 Jun.13 Aug.13 Sample of orange juice and date Total phenolics Hesperidin Fig. 3.7.The total content of phenolics compounds and hesperidin in fresh defrosted Navel and Valencia varieties orange juices on maturity early stage (A), mid (B, C) and end (D) of the season In this study the results of the hesperidin content are in agreement with results previously reported by Mouly et al. (1998), Vanamala et al. (2006). The study results showed that hesperidin level within the fruit generally decreases (26% and 20%) with maturity in both orange juices respectively. This is because, as the fruit matures, it accumulates moisture, which dilutes the hesperidin concentration. It is interesting that hesperidin content was greater (up 20%) in the Valencia variety orange juice compared to Navel orange juice. In spite of this, the appearance of hesperidin flakes in citrus juices increases with fruit maturity and may become higher in the late season, especially in Valencia juice. This is probably due to lower acid levels which reduce the solubility of hesperidin. All citrus fruits and oranges also are complex source of carotenoids (Gama J.J.T., Sylos C.M. 2005; Melendez-Martinez et al., 2007 and 2008). Carotenes are localized in subcellular organelles (plastids) in chloroplast and chromoplast mainly associated with macromolecules such as proteins and membrane lipids (Schieber, Carle, 2005). With ripening, total carotenoids increase in the peel as well as in the pulp and the juice. Juices from early season have less content of carotenoids if compared with the juices from late season varieties. Valencia orange is quantitatively the richest juice in carotenoids, known to have the most complex pigment among oranges (Lee, Coates, 2003). The study results during season 2013/2014 showed that in both orange varieties (Navel and Valencia) the total carotenoids content increases (1.98 to 2.29 mg 100 ml -1 and 3.11 to 3.44 mg 100 ml -1 ) respectively (Fig. 3.8). The content of total carotenoids is comparable to values found by De Ancos et.al (2002), Lee, Coates, (2003). During the season, the content of total carotenoids in Navel orange juice had a more significant increase (up to 16%), compared with content of total carotenoids in Valencia orange juice (up to 10%). However, the average content of total carotenoids in Valencia variety of orange juice was higher by 50% compared with total carotenoids content of Navel orange juice. 65

66 Carotenoid content, mg 100 ml Nav- A Nav - B Nav- C Nav- D Val -A Val - B Val-C Val- D Dec.13 Jan.14 Feb.14 March.14Apr.13 May.13 Jun.13 Aug.13 Sample of juice and date Fig. 3.8.The content of total carotenoids in fresh defrosted Navel and Valencia varieties orange juices on maturity early stage (A), mid (B, C) and end (D) of the season The content of ß-carotene increased from 0.05 to 0.07 mg 100 ml -1 and from 0.06 to 0.13 mg 100 ml -1 in Navel and Valencia variety of orange juice respectively (see Fig. 3.9.). Fig The content of ß- carotene in fresh defrosted Navel and Valencia varieties orange juices on maturity early stage (A), mid (B, C) and end (D) of the season The obtained results indicated that there was a significant effect on the total carotenoids and ß-carotene content in both orange juices during maturation. As showed study results, the average content of ß-carotene was 3% from the total content of carotenoids in Navel orange juice and average content of ß-carotene 4% was in the juice of Valencia. These results correspond to the AIJN standard where the maximum content of ß-carotene 5% is established of total carotenoids content. 66

67 Orange fruits are a source of pectin which is used in the food industry and is a dietary fibre. Water soluble (WS) pectin was evaluated in two varieties of orange juice. The results of the study showed that the content of WS pectin decreases as the fruit ripens. As can be seen from the graph (see Fig. 3.10) the WS pectin increased to a peak (3.35 to 4.91 mg 100 ml -1 and 3.14 to 3.42 mg 100 ml -1 ) respectively in the orange juices Navel and Valencia and decreased by the end of the season (from 4.91 to 4.01 mg of 100 ml -1 and 3.42 to 3.18 mg of 100 ml -1 ) respectively. WS pectin content, mg 100 ml Nav- A Nav - B Nav- C Nav- D Val -A Val - B Val-C Val- D Dec.13 Jan.14 Feb.14 March.14 Apr.13 May.13 Jun.13 Aug.13 Sample of juice and date Fig The content of water-soluble pectin in fresh defrosted orange juicesvalencia and Navel orange varieties on maturity early stage (A), mid (B, C) and end (D) of the seasons The results were in agreement with results of Ywassaki, Ganniati-Brazaca, (2011). They studied pectin and soluble pectin in different sizes of citrus (orange and mandarin) fruits and concluded that the total and soluble pectin contents increased in juice as fruits size decreased. They found 2.91 mg 100 g -1 of soluble pectin in medium size Valencia fruits and mg 100 g -1 in medium size Bahia (cousin to the Washington Navel) variety of orange. The antioxidant capacity in both varieties of orange juices (Navel and Valencia) was determined using ABTS radical-cation method trolox equivalent antioxidant capacity (TEAC) during season. The results of the experiment showed that antioxidant capacity of the orange juice depends of the orange juice variety, the content of bioactive compounds in them and the time of fruit harvesting (see Fig. 3.11). The higher values of antioxidant capacity was 0.95 mmol TE 100 ml -1 in Navel orange juice in December and for the orange juice Valencia in April where antioxidant capacity value has reached 0.92 mmol TE 100 ml -1. According to the results of this study it can be seen that changes in values of antioxidant capacity in the orange juice samples during season are related to changes in the content of vitamin C, phenolics compounds and hesperidin in them. 67

68 Antioxidant capacity (ABTS) mmol TE 100 ml Nav- A Nav - B Nav- C Nav- D Val -A Val - B Val-C Val- D Dec.13 Jan.14 Feb.14 March.14 Apr.13 May.13 Jun.13 Aug.13 Sample of juice and date Fig The values of antioxidant capacity in fresh defrosted Navel and Valencia varieties orange juices on maturity early stage (A), mid (B, C) and end (D) of the season The highest positive correlation has been observed in the case of antioxidant capacity with vitamin C (r = 0.938), total phenolics (r = 0.924) and hesperidin (r = 0.997) in orange juice Navel variety (Ann. 4.) and significant positive correlation of antioxidant capacity with vitamin C (r = 0.998), total phenolics compounds (r = 0.998) and hesperidin (r = 0.965) in orange juice Valencia variety during season (Ann. 5.). These parameters mostly influence the total antioxidant capacity. The positive correlations were also observed between TSS and sucrose, fructose, glucose, total carotenoids and ß-carotene and between TA and vitamin C, total phenolics compounds, hesperidin and antioxidant capacity in orange juice Navel and Valencia respectively during seasons. The results indicate that orange juices have high content of antioxidant compounds and are a good source of nutritional ingredients. Data correlates with that observed in L- ascorbic acid discussed above confirming the good correspondence between the vitamin C content and the water-soluble ant-oxidative potential of orange juice (Miller, Rice-Evans, 1997). They found that L -ascorbic acid is responsible for at least 87% of the water-soluble antioxidative capacity of commercial orange juice. Remaining capacity could be attributed mainly to soluble fractions of hesperidin and narirutin. The obtained results indicated that stage of maturity had significant effect on the chemical parameters and bioactive compounds of orange fruits and juices. Sugars content, total soluble solids, TSS/ acidity ratio and juice content tended to increase towards maturity. On the other hand acidity and ascorbic acid, total phenolics compounds, carotenoids contents were high at the early stage when fruits were immature. This conclusion was in agreement with Riaz et al., (2015) who studied effect of maturity stage and harvesting time of citrus fruit on physicochemical parameters. Summary of chapter 3.1 Two commercial orange varieties Navel and Valencia grown in Greece have been evaluated during maturation in industrial scale in Laconia area, S.A. Biofresh, in Greece. The physicochemical parameters of orange and orange juice were determined during two years, and bioactive compounds and antioxidant capacity during one year. The study results showed the physicochemical parameters and bioactive compounds of orange juice summer variety Valencia and winter variety Navel fruits was different and they changes during maturation: 68

69 the content of total acidity (TA) and yield in both varieties of orange juice decreases, while total soluble solids (TSS) and their ratio (TSS/TA), as well an individual sugars content significantly increased (p < 0.05). Content of vitamin C, total phenolics compounds, hesperidin and WS pectin in fresh orange juices decreased insignificant (p > 0.05) however, the total carotenoids and ß- carotene content significantly increased (p < 0.05) during maturation. The content of vitamin C and antioxidant capacity was higher in variety of orange Navel juice. The antioxidant capacity significantly decreased (p < 0.05) in orange juices of both varieties of oranges during maturation. The obtained results indicated that stage of maturity have effect on the physicochemical parameters and bioactive compounds of orange fruits and juices Evaluation the influence of processing methods on the chemical parameters, bioactive compounds and antioxidant capacity of orange juice Orange juice is a rich source of bioactive compounds such as vitamins; flavonoids including hesperidin, total carotenoids and pectin. To compare the effect of the HTST pasteurization process on fresh defrosted orange juice (Control) Navel and Valencia varieties in production, orange juice not from concentrate (NFC) and reconstituted orange juice (OJFC) from orange juice concentrate were used. The content of vitamin C, content of total phenolics compounds, total carotenoids, ß-carotene, and water soluble (WS) pectin and antioxidant capacity were quantified. Often results show that orange juices made from frozen concentrate orange juice (FCOJ) have higher content of vitamin C compared to freshly squeeze or NFC juices. This is probably due to the fact that vitamin C degrades over time in fresh and also in NFC orange juices but not degrades as much in FCOJ due to its being concentrated and frozen until reconstitution. Another thing to consider was if the FCOJ is reconstituted to the same strength as fresh or NFC orange juices. If enough water hasn t been added then the vitamin C (and other compounds) would be more concentrated. Fresh frozen then defrosted orange juice (Control sample), pasteurised orange juice not from concentrate (NFC) and pasteurised orange juice reconstituted from concentrate (OJFC) of Navel and Valencia variety oranges were delivered from commercial production company to UBF laboratory for analysis. The orange juice samples (Control) of Navel and Valencia orange varieties (10.92 and Brix, TA 0.86 and 0.82%, ratio 12.7 and 14.4) respectively from the same batch NFC orange juice (11.00 and Brix, TA 0.88 and 0.80%, ratio 12.5 and 14.9) respectively, and orange juices from concentrate (OJFC) (11.21 and Brix, TA 0.86 and 0.79%, ratio and 14.2) respectively (Ann.6.). OJFC reconstituted from concentrate Navel (65 Brix) and Valencia (65 Brix) with water (1:5, w/w). NFC and OJFC orange juices were pasteurised (HTST, 94 C 30 s) followed by aseptic filling into aseptic bags (1L). Vitamin C. The results of the study showed that the content of vitamin C in fresh defrosted orange juices (Control sample) in Valencia and Navel varieties were significantly higher (43.35 and mg 100 ml -1 ) than the content of vitamin C in these orange juices NFC (37.06 and mg 100 ml -1 ) and OJFC (36.51 and mg 100 ml -1 ) respectively after pasteurisation (94 C 30 s) (see Fig. 3.12). 69

70 Vitamin C, mg 100 ml ¹ Control NFC OJFC Control NFC OJFC Valencia Navel Juice type Fig The content of vitamin C in fresh defrosted (Control), pasteurised not from concentrate (NFC) and reconstituted pasteurised from concentrate (OJFC) orange juices Navel and Valencia As it was said above the different varieties of orange juices have a different content of vitamin C however, the process used for obtaining juice has an effect on the content of vitamin C in final product. The content of vitamin C in pasteurised NFC and OJFC orange juices was significantly decreased (p < 0.05). The losses of vitamin C was around 16% in both NFC orange juices and 19% in the orange juices made from concentrate. These results are in agreement with those found in our previous study about vitamin C in different citrus juices (Zvaigzne et al., 2009). Farnworth et al. (2001) reported that the concentration of ascorbic acid was affected by the method of production. Vikram et al. (2005) studied the status of vitamin C during thermal treatment of orange juice, heated by different methods (conventional heating, electromagnetic processing including infrared, ohmic heating, and microwave heating) at different treatment temperatures (50, 60, 75, and 90 C) from 0 to 15 minutes. The degradation was highest during microwave heating due to uncontrolled temperature generated during processing and ohmic heating gave the best result vitamin retention at all temperatures. Total phenolics compounds. Higher content of total phenolics compounds was found in the control sample of Valencia orange juice, mg 100 ml -1 compared with content of total phenolics compounds (84.57 mg 100 ml -1 ) in control sample of Navel orange juice. These results are comparable with the result by Pala, Toklucu (2012), they found that the total phenolics content of untreated orange juice was mg 100 ml -1. In our study the pasteurised NFC orange juices Valencia and Nave, the content of total phenolics compounds was and mg 100 ml -1 respectively and it was and mg 100 ml -1 of Valencia and Navel OJFC orange juices respectively (see Fig. 3.13). 70

71 Total phenolics, mg 100 ml ¹ Control NFC OJFC Control NFC OJFC Valencia Navel Juice type Fig The content of total phenolics compounds in fresh defrosted (Control), pasteurised not from concentrate (NFC) and reconstituted pasteurised from concentrate (OJFC) Navel and Valencia orange juices The results of the study showed that content of total phenolics compounds was higher 8 and 10% in control samples compared with NFC samples and 13 and 22% compared with samples OJFC of Navel and Valencia orange juices respectively. In both varieties of orange juices after pasteurisation the content of total phenolics compounds was higher in NFC orange juices compared with OJFC. The study results are in a good agreement with those reported in the literature (Rapisarda et al., 1999; Gardner et al., 2000). Total carotenoids and ß-carotene. The changes of total carotenoids content and ß-carotene are reflected in Fig and The total content of carotenoids were 0.85 and 1.23 mg 100 ml -1 and ß-carotene were 0.03 and 0.05 mg 100 ml -1 in fresh defrosted (Control) samples of Valencia and Navel orange juices respectively. These results are in agreement with those found by Plaza et al. (2011). They found in orange juice the content of total carotenoids of 1.31mg 100 ml -1 and ß-carotene of 0.03 mg 100 ml -1. Total carotenoids, mg 100 ml ¹ Control NFC OJFC Control NFC OJFC Navel Valencia Juice type β-carotene, mg 100 ml ¹ Fig The content of total carotenoids and ß-carotene in fresh defrosted (Control), pasteurised not from concentrate (NFC) and reconstituted pasteurised from concentrate (OJFC) Navel and Valencia orange juices 71

72 After pasteurization, the total content of carotenoids showed an increase in both varieties of orange juice by 10 % in Navel NFC and by 7 % in Valencia NFC orange juice. The content of ß-carotene increased by 15 % in both processed Navel orange juices however, in Valencia orange juices the content of ß-carotene show not siginificant differences (p> 0.05) after pasteurization. These results are in agreement with those found by Sanchez -Moreno et al. (2005) and Plaza et al. (2011) they reported about carotenoid stability in orange juice subjected to different non-thermal and thermal technologies. Researchers Lee, Coates (2003) also reported insignificant losses in provitamin A activity after thermal pasteurisation (90 C, 30 s) although, they detected losses in the content of the xanthophylls. Water-soluble (WS) pectin. The study results showed that content of water-soluble pectin was 3.29 and 3.37 mg 100 ml -1 in control samples of Navel and Valencia orange juices respectively (Fig. 3.15). WS pectin mg 100 ml ¹ Control NFC OJFC Control NFC OJFC Navel Valencia Juice type Fig The content of WS pectin in fresh defrosted (Control), pasteurised not from concentrate (NFC) and reconstituted pasteurised from concentrate (OJFC) Navel and Valencia orange juices After the pasteurisation NFC and OJFC orange juices showed slight increase (about 2%) of water-soluble pectin with regard to control sample in both varieties of orange juices. This increase during pasteurisation may increase the water-soluble pectin from the pulp and insoluble particles in the juice. Bioactive compounds and antioxidant capacity. Thermal processing influences the bioactive compounds and as a result the antioxidant capacity in orange juice and mostly it is due to the degradation of the vitamin C and changes of total phenolics compounds in orange juice. In Figure 3.1 can see how the technological process affects the antioxidant capacity in orange juice. 72

73 Antioxidant capacity (ABTS) mmol TE 100 mi ¹ Control NFC OJFC Control NFC OJFC Navel Valencia Juice type Fig The content of antioxidant capacity (ABTS) in fresh defrosted (Control), pasteurised not from concentrate (NFC) and reconstituted pasteurised from concentrate (OJFC) Navel and Valencia orange juices In the control samples of orange juices Navel and Valencia the values of antioxidant capacity measured by ABTS [2.2 -Azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) method were 1.08 and 0.97 mmol TE 100 ml -1 respectively. After pasteurization in both varieties of orange juice values ABTS decreased to 0.86 and 0.78 mmol TE 100 ml -1 in NFC and OJFC orange juices Navel respectively, and to 0.77 and 0.74 mmol TE 100 ml -1 in NFC and OJFC orange juices Valencia respectively. As can be seen from the graph, antioxidant capacity values in orange juices decreases after thermal treatment, especially in the juice obtained from OJFC. In this study, a decrease in the content of vitamin C and total phenolics compounds following both processing methods was observed as well. Those changes created the decrease in antioxidant capacity in both varieties of orange juice. Summary of chapter 3.2 The effect of the high temperature short time (HTST) process on the chemical, bioactive compounds and antioxidant capacity in orange juice not from concentrate (NFC) and reconstituted from concentrate orange juice (OJFC) was compared with fresh frozen following defrosted orange juices (Control) of Navel and Valencia varieties produced in industrial scale. The changes in TSS and TA contents were not significant (p > 0.05) in both varieties of orange juices processed by HTST. The content of vitamin C, total phenolics compounds and antioxidant capacity decreased in pasteurized NFC orange juices Navel and Valencia in comparison with control samples. The content of WS pectin slight increase in both HTST processed orange juice samples but the increases were not significant (p > 0.05). The total content of carotenoids increased in both varieties of orange juices: by 10 % in Navel NFC and by 7 % in Valencia NFC orange juice. The content of ß-carotene increased by 15 % in both varieties of treated Navel orange juice samples, on the other hand in both Valencia orange juice samples the ß-carotene showed not changes influenced by pasteurisation. 73

74 3.3. The dynamic of chemical parameters, bioactive compounds and antioxidant capacity of pasteurised NFC orange juice during refrigerated storage Quality and shelf life determination of an orange juice is strongly based on vitamin C evaluation during storage. Although, there are other quality parameters that are also very important (Alwazeer et al., 2003; Esteve et al., 1996; Kabasakalis et al., 2000; Lee, Coates, 1999; Plaza et al., 2006; Plaza et al., 2011; Polydera et al., 2003; Zerdin et al., 2003). The modern manufacturers use a properly balanced mixture of freshly squeezed and mildly pasteurised juices stored aseptically, with known characteristics. In literature most of the available studies with regard to the effect of refrigerated storage on bioactive compounds of treated orange juice have focused on the vitamin C (Polydera et al., 2005). Evaluation of the effect of time and temperature on the content of vitamin C, total phenolics compounds, hesperidin, total carotenoids, ß-carotene and WS pectin in pasteurised NFC orange juices in aseptic bags during refrigerator storage temperature 5 ± 2 C was carried out for a period of one year (Ann.7.). Every four months the chemical parameters, bioactive compounds and antioxidant capacity were analysed. Total soluble solids (TSS). At the beginning of storage the content of TSS in orange Valencia and Navel juices not from concentrate (NFC) was 11.3 and 10.8 Brix respectively. During first 4 month the TSS slightly increased in both NFC orange juices (11.4 and 10.9 Brix) then remained constant overall remaining storage time (12 month) (see Tab. 3.2). Orange juice samples NFC- Not from concentrate Valencia NFC - Not from concentrate Navel Table 3.2 Dynamics of total soluble solids (TSS) and total acidity (TA) content in pasteurised (HTST) orange juices during storage at 5 C± 2 C Storage, Temperature, Parameters month C TSS, Brix TA, % 0 5 ± ± 0.03 a 0.94 ± 0.01 a 4 5 ± ± 0.02 a 0.94 ± 0.01 a 8 5 ± ± 0.01 a 0.94 ± 0.01 a 12 5 ± ± 0.02 a 0.96 ± 0.01 b 0 5 ± ± 0.03 b 0.96 ± 0.02 b 4 5 ± ± 0.01 b 0.96 ± 0.01 b 8 5 ± ± 0.01 b 0.97 ± 0.01 c 12 5 ± ± 0.01 b 0.97 ± 0.01 c Column values with different online letters (a, b, c) differ (p < 0.05). Results were presented as means ± standard error (n = 5). Those results are within the average TSS content of orange juice reported by Farnworth et al., 2001 during refrigerated storage and at different temperatures (Esteve et al., 2005; Kelebek et al., 2009; Leahu et al., 2013). On the other hand some studies found a significant change in the TSS content during the storage of orange juice (Cortes et al., 2008 a). During storage time the sugars may break down producing carboxylic intermediates which further react to form brown polymers (Robertson, Samaniego, 1986). The relationship between Brix and browning reactions was investigated by Buedo et al. (2000). Total acidity (TA). As it was previously stated organic acids play a significant role in browning reactions and the taste of the orange juice. Also organic acids are the main factor affecting the stability of vitamin C. Acids together with sugars give a balance of sweetness and sourness which makes orange juice refreshing to consumers. In this study TA is expressed as citric acid. The TA value ranged from 0.94% to 0.96% in pasteurised NFC (Valencia)orange juice and from 0.96% to 0.97 % in pasteurised NFC (Navel) orange juice during storage (see Table 3.2.). The changes in TA content during the entire storage were not 74

75 significant (p > 0.05) in the both varieties of orange juice. The same conclusion was reported by several authors (Manso et al., 2001; Farnworth et al., 2001; Esteve et al., 2005; Kelebek et al., 2009; Leahu et al., 2013). Vitamin C. Reportedly, orange juice is a rich source of vitamin C which is a powerful antioxidant in this juice. Therefore it is very important to choose the optimum storage temperature for orange juice to save vitamin C. Oxygen is the reactive element which can cause changes in the chemical composition of orange juice during processing of orange juice and during storage. The most important change is the loss of vitamin C. During orange juice storage both anaerobic and aerobic degradation take place simultaneously in orange juice therefore in packaged products vitamin C degradation cannot usually be attributed to solely one pathway. Which process predominates depends on the storage temperature and the availability of oxygen. According to the available study results and literature the content of vitamin C decrease during storage and the losses depend on processing, storage temperature and kind of packaging (Del Caro et al., 2004; Kabasakalis et al., 2000; Polydera et al., 2003; Polydera et al., 2005; Zerdin et al., 2003;). The investigations showed that there were some similarities but also differences between the both Valencia and Navel NFC orange juices. Initial vitamin C content in orange Valencia and Navel NFC juices was and mg 100 ml -1 respectively. These results are within the range of vitamin C content in orange juice reported by Kabasakalis et al. (2000), Solomon et al. (1995), Vervoort et al. (2011), and Wiboro et al. (2015a). The degradation dynamics of vitamin C in orange Valencia and Navel NFC juices showed in the figure Vitamin C, mg 100 ml ¹ R² = R² = Storage time, month NFC Valencia NFC Navel Fig The dynamics of vitamin C content in pasteurised orange Valencia and Navel NFC juices during storage at 5 ± 2 C In the first 4 month both juices showed the largest loss of vitamin C content (NFCV 7.5%; NFCN 8.4%). Over the twelve month storage period, the decrease in vitamin C was about 1.3% per week for both varieties of NFC orange juices and in general the loss of vitamin C was by 15% and 16% respectively. The losses of vitamin C were caused by anaerobic decomposition of vitamin C, which cannot be prevented by packaging. Presented results are in agreement with data obtained by Choi et al. (2002), Fan et al. (2002), Roig et al. (1999) and Rodrigo et al. (2003). Also Kennedy et al. (1992), Zerdin et al. (2003) noted reduction of vitamin C in the investigated commercial orange juices. Roig et al. (1995) reported that low temperature storage is imperative in order to ensure L-ascorbic acid retention. However, the degradation of vitamin C in pasteurised orange juice was observed by several authors (Arena et al., 2001; Kabasakalis et al., 2000; Klimczak et al., 2006). 75

76 Total content of phenolics compounds. Some information in scientific literature is available the changes in total content of phenolics compounds and individual flavonoids of orange juices during storage. The results of our studies showed that during storage time at 5 ± 2 C temperature the content of total phenolics compounds went down. At the beginning of the storage the content of total phenolics compounds in Valencia and Navel NFC orange juices was and mg 100 ml -1 respectively which is relatively similar to results that found Esteve, Frigola (2007). A significant decrease (p < 0.05) in the content of total phenolics compounds (15 and 10 %) was observed during eight months storage (see Fig. 3.18). Total phenolics, mg 100 ml ¹ Storage time, month NFC Valencia NFC Navel Fig The dynamics of total phenolicscontent in pasteurised orange Valencia and Navel NFC juices during storage at 5 ± 2 C The peculiarity the total content of phenolics compounds the both orange juices was slight increase in the amount at the last four month but this increase (p > 0.05) was not significant. Especially in Navel NFC orange juice the total content of the phenolics compounds increased from to mg 100 ml -1 it is 4%. Total content of phenolics compounds in Valencia NFC orange juice also increased from to mg 100 ml -1 but it was only 2.3% in total. Therefore, the assumption is that after eight month a mechanism which leads to a regenerative process of phenolic compounds starts. This mechanism may represent a reducing reaction of the before oxidized phenols. The degradation of total phenolics compounds during storage also mainly related to the residual activity of polyphenol oxidase and peroxidase. Hesperidin. Content of the hesperidin decreased during the refrigerated storage time. Initially the content of hesperidin in the orange Valencia and Navel NFC juices was and mg 100 ml -1 and during storage it decreased to 6.79 and 8.08 mg 100 ml -1 respectively (Fig. 3.19). 76

77 Hesperedin, mg 100 ml Storage time, month NFC Valencia NFC Navel Fig The dynamics of hesperidin content in pasteurised orange Valencia and Navel NFC juices during storage at 5 ± 2 C At the end of storage juices of both orange varieties showed a significant (p < 0.05) decrease of hesperidin content. That means there is a decline of 41.4% in Valencia and 42.6% in Navel NFC orange juices. It is possible that after the pasteurisation the enzymes which lead to a degradation of hesperidin still exist. Probably the glycosides are be cleaved to the aglycon its corresponding sugar molecule (rhamnose). These findings were shown in different references (Del Caro et al., 2004; Klimczak et al., 2007; Sanchez- Moreno et al., 2003). The effect of refrigerated storage (4 C over days) of freshly squeezed orange juice on flavonoid content has been reported (Del Caro et al., 2004; Sanchez Moreno et al., 2003; Wibovo et al., 2015b). Some research data are available on flavonoid content in orange juice during storage (Choi et al., 2002; Galati et al., 1994; Klimczak et al., 2007; Plaza et al., 2011). Carotenoids. Processing and storage may cause the instability of the polyene chain of carotenoids as well of their highly saturated conformation; carotenoids are prone to oxidation and isomerization during processing and storage (Dutta et al., 2005). Many investigators reported of the impact of thermal treatment and not thermal treatment on the total and individual carotenoids of orange juice, followed by refrigerated storage (Choi et al., 2006; Cortes et al., 2008a; Cortes et al., 2008 b; Esteve et al., 2009). At the beginning of storage the total carotenoids content was 1.36 mg 100 ml -1 in pasteurised Valencia NFC orange juice and 1.04 mg 100 ml -1 in Navel NFC orange juice with the content of ß-carotene and mg 100 ml -1 respectively (see Fig. 3.20). These results are comparable to values by Esteve, Frigola (2007) they found the content of total carotenoids 1.19 mg 100 g -1 in Navel variety pasteurised orange juice. Plaza et al., (2011) reported that content of total carotenoids was 1.3 mg 100 ml -1 and ß-carotene mg 100 ml -1 in low temperature pasteurised (70 C 30 s) orange juice of Valencia variety. 77

78 Total carotenoids, mg 100 ml ¹ Storage time, month NFC Valencia carotenoid NFC Navel carotenoid NFC Valencia β carotene NFC Navel β carotene β-carotene, mg 100 ml -1 Fig The dynamics of total carotenoids and ß-carotene in pasteurised orange Valencia and Navel NFC juices during storage at 5 ± 2 C The initial content of total carotenoids was by 24% higher in Valencia NFC orange juice compared with content of Navel NFC orange juice. The orange juice Valencia is one of richest in carotenoids as reported in literature. It has the most complex pigment pattern among sweet oranges. In literature most of available studies are related to the effect of bioactive compounds in treated for short time refrigerated orange juice (Esteve, Frigola, 2007; Plaza et al., 2011; Wibowo et al., 2015) they have observed an insignificant decrease of initial values of total carotenoid and individual carotenoids. In our study has been determined that during refrigerated storage (5 ± 2 C) the total carotenoids and ß-carotene content showed less than 20% decrease in both varieties of orange juice. During storage in Valencia and Navel NFC juices the total carotenoids content decreased by 17% and 14% and ß-carotene by 12% and 13% respectively. Plaza et al., (2011) reported loss (< 11%) of total carotenoids compared after treatment and at the end of storage (40 days). Results according ß-carotene is in agreement with those reported by Bull et al. (2004) they found that content of ß-carotene insignificantly decrease during storage at 4 C after thermal processing (85 C, 25 s) of orange juice. A slight decrease of total carotenoids content during refrigerated storage could be probably related to the protection from oxidation that ascorbic acid offers to them and the lack of oxygen in the aseptic bag. This limited degree of degradation during refrigerated storage also was noticed by Cortes et al. (2006), Plaza et al. (2011), Vervoort et al. (2011), and Wibovo et al. (2014). Moreover Choi et al. (2002) reported a lower loss of total carotenoids in juice fortified with ascorbic acid if compared to control orange juice during 7 weeks storage. Pectin. As it was previously stated pectin is the major component in orange juice cloud and has important role in juice stabilisation. In the presence of active pectin methylesterase (PME) enzyme pectin forms calcium pectate complex which causes precipitation of cloud particles (Croak, Corredig, 2006). Considering the general amount of water soluble (WS) pectin in both varieties of orange juices the decrease of WS pectin content was insignificant (see Fig. 3.21). 78

79 Water soluble pectin, mg 100 ml Storage time, month NFC Valencia carotenoid Fig The dynamics of water-soluble pectin content in pasteurised orange Valencia and Navel NFC juices during storage at 5 ± 2 C The loss of WS pectin was from 3.36 mg 100 ml -1 to 3.12 mg 100 ml -1 (7 %) in Valencia NFC orange juice and from 3.12 mg 100 ml -1 to 2.96 mg 100 ml - 1 (5 %) in Navel NFC orange juice. Maybe the reaction starts. Sugar consumes part of the hydrated water from the sol of the pectin, and in result the stability of pectin decreases. Antioxidant capacity. In this study the antioxidant capacity in orange juice was evaluated by using ABTS radical cation assay method. The antioxidant capacity in orange juice NFC Valencia and Navel NFC orange juice after pasteurisation was 1.01 and 1.05 mmol TE 100 ml -1 respectively (see Fig. 3.22). As can be seen, antioxidant capacity decreased significantly (p< 0.05) during refrigerated storage in both varieties of pasteurized orange juices. Antioxidant capacity (ABTS), mmol TE 100 ml Storage time, month NFC Valencia NFC Navel Fig The dynamics of antioxidant capacity (ABTS) in pasteurised orange Valencia and Navel NFC juices during storage at 5 ± 2 C The decrease of values of antioxidant capacity in both NFC orange juices Valencia and Navel were 20 % and 22 % respectively. Small difference between both NFC juices in the same storage temperature may be due to the fact that initially in the orangenavel NFC juice the total acid content was higher and this contributed to retention of vitamin C and as a result - the antioxidant capacity during refrigerated storage. Klimczak et al. (2007) obtained close results in orange juice stored for six month at different temperature. They found that at higher storage temperature antioxidant values were decreased more quickly. Polydera et al (2005) studied the antioxidant activity in orange juice processed by different methods (high-pressure 79

80 compared with thermal pasteurised) and they found that antioxidant activity decreased in both processed juices during storage mainly of ascorbic acid loss. The total antioxidant capacity during storage also was studied by Del Caro et al. (2004). They found a slight decrease in the antioxidant capacity values for orange juice stored 15 days at 4 C. The antioxidant activity studied Arena et al. (2001) of freshy squeezed and processed different orange juices (blood and blond oranges).they reported during storage 60 days at 2 C the antioxidant activity didn't show changes in both orange juices NFC and reconstituted from concentrate however, show changes at 20 C accordance with the vitamic C losses. The research data showed that antioxidant capacity value is depending on the content of antioxidants in orange juice such as vitamin C, total phenols, hesperidin, total carotenoids, ß-carotene and pectin and closely correlated with them. The highest positive correlation has been observed in the case of antioxidant capacity with vitamin C (r = 0.991), total phenolics compounds (r = 0.854), total carotenoid (r = 0.958), ß-carotene (r = 0.905), hesperidin (r = 0.973) and WS pectin (r = 0.982) in Valencia NFC orange juice (Ann. 8.) In Navel orange juice antioxidant values correlated positively with vitamin C (r = 0.986), total phenolics compounds (r = 0.804), total carotenoids (r = 0.925), ß-carotene (r = 0.991), hesperidin (r = 0.979), and WS pectin (r = 0.942) (Ann.9.). The content of this compound influence the total antioxidant capacity. Despite the fact that in the Navel NFC orange juice the content of vitamin C and hesperidin was higher than in Navel NFC orange juice the difference in antioxidant capacity value was not significant (p > 0.05) compared with the Valencia NFC orange juice containing higher values of total carotenoids, ß-carotene and WS pectin. It s means that the juices with a high content of carotenoids and pectin also have a high antioxidant value. Summary of chapter 3.3 Dynamics of the chemical characteristics and bioactive compounds in pasteurised (HTST) NFC orange juices Navel and Valencia varieties oranges were carried out during storage for one year in aseptic bags and refrigerated storage at 5 C ± 2 C. The changes in TSS and TA during the entire storage time were not significant in both NFC orange juices. On the other hand vitamin C, total phenolics compounds, hesperidin, total carotenoids, ß-carotene and WS pectin showed significant changes in content as a function of time and temperature. A significant decrease of vitamin C content can be attributed to the ascorbic acid degradation reaction, which is known as a potential precursor of brown pigments in orange juice. The results showed a significant decrease in content of total phenolics compounds during eight months in refrigerated storage. This degradation of total phenolics compounds has been related to the residual activity of polyphenol oxidase and peroxidase also this mechanism may represent a reducing reaction in the phenols oxidised before. The same pattern was observed for hesperidin. However, at the end of the storage both varieties of orange juices showed a significant decrease of hesperidin content (41.4% and 42.6%) in NFC Valencia and Navel orange juices respectively. It is possible that even after the pasteurisation enzymes still exist which leads to a degradation of hesperidin. That means that possibly the glycoside are cleaved to aglycon- its corresponding sugar molecule (rhamnose).the antioxidant capacity during refrigerated storage in both pasteurized Valencia and Navel orange juices decreased significantly (p < 0.05). The research data showed that antioxidant capacity value is depending on the content of bioactive compounds in orange juice such as vitamin C, total phenolics compounds, hesperidin, total carotenoids, ß-carotene and pectin and closely correlated with them. 80

81 3.4. UHT processing effect on chemical parameters, bioactive compounds and antioxidant capacity of orange juice compared with HTST processing As it was previously stated the primary purpose of pasteurisation in food processing is to destroy pathogenic organisms and inactivate enzymes. Thermal processing continues to be the most widely used method of preserving and extending the shelf life of foods (Awuah et al., 2007) The UHT processing differs from the classical sterilisation because it rapidly reaches higher temperatures with very short duration (direct UHT system) as a result organoleptic and nutritional properties of treated foods should suffer a few variations only. The technology of UHT processing also applies to bulk products (heat penetration is extremely rapid) and is associated with aseptic packaging techniques (Micali, Florino 2016). There are not many reports in literature comparing UHT processing on juices mainly studies in terms of microbial inactivation changes in ph, suspended solids and colour (Gallardo-Reyes, et al., 2008; Zhang et al., 2015). There are not plenty studies found in scientific literature reporting the effects of UHT processing on bioactive compounds and antioxidant capacity of orange juices and generally are not studies of the effect of adding sea buckthorn juices to orange juice. However, there are several studies on the effects of UHT processing on the biochemical compounds of apple, pomegranate and sugarcane juices (Jittanit, 2011; Lewis et al., 2000; Qu et al., 2014; Sanchez-Vega et al., 2009). Chemical and bioactive compounds of orange juice processed by UHT technology compared by HTST processed the results of the study are given in the annex 10. In this research the TSS, TA, and TSS/TA ratio was tested by UHT and HTST processed Navel variety orange juice and results compared with fresh frozen and then defrosted (control) orange juice, the results are provided in the Table 3.3. Table 3.3 UHT and HTST processing effects on chemical parameters in the Navel variety orange juice Samples Total soluble solids, Brix Total acidity, % Ratio Fresh frozen and then ± 0.05 a 0.79 ± 0.04 a defrosted (control) HTST pasteurized ± 0.04 b 0.80 ± 0.00 b UHT processed ± 0.04 b 0.80 ± 0.01 b Column values with different online letters (a, b) not differ (p > 0.05) Results were presented as means ± standard error (n = 4) There were no significant differences in these values after HTST and UHT treatments in comparison with fresh frozen and then defrosted (control) orange juice (p < 0.05). Gallardo- Reyes et al. (2008) also did not find any significant difference in ph and soluble solids in the treated by pulsed electric fields and by UHT treatment orange juice compared with control sample. Zhang et al. (2015) studied the effect of UHT processing technology at different temperatures (110, 120, 135 C) of TSS content in watermelon juice, and the results showed no effect of TSS in watermelon juice. Jittanit et al. (2011) have found similar results in their study in the UHT processing technology at the temperatures 135 and 140 C didn t effected on the TSS content in the sugarcane juice. There are other studies showing not significant changes of TSS and TA content in orange juices processed by different treatment technologies (Pala, Toklucu, 2013; Vervoort et al., 2011). Vitamin C. It is known that thermal treatments may reduce antioxidant activity and content of bioactive compounds in the juices. Vitamin C is one of the important indicators of 81

82 all citrus juice quality including orange juice and serve as an indicator that all process steps of production were respected (Lee, Coates, 1997; Lee, Chen, 1998; Manso et al., 1996; Polydera et al., 2005). As described above the vitamin C is an unstable compound and therefore the juice processing technology plays a key role in the stability of this compound. Gil-Izquierdo et al. (2002) evaluated vitamin C in orange juices processed by different techniques and found that mild and standard pasteurisation slightly increased the content of vitamin C. Vikram et al. (2005) studied effect of thermal treatment on vitamin C in orange juice heated by different methods, temperature and time and they found that destruction of vitamin C during juice storage was influenced by the thermal method and the processing temperature. Polydera et al. (2003, 2005) also studied the effect of different treatment methods of orange juice. They reported that in all cases, the ascorbic acid degradation rates were lower for the pressurised juice and this is leading to an extension of its shelf life compared with conventionally pasteurised juice. In this study the results of the HTST and UHT processing influence on vitamin C changes in Navel orange juice is presented in the Figure Fig UHT and HTST processing effect on Vitamin C content in Navel orange juice OJ Fresh Fresh frozen and then defrosted orange juice (control); OJ HTST high temperature short time pasteurised fresh frozen and then defrosted orange juice; OJ UHT ultra high treatment treated fresh frozen and then defrosted orange juice. The content of vitamin C in the fresh frozen then defrosted orange juice (OJ Fresh) variety was mg 100 ml -1 this value is comparable to the values indicated in other studies (Esteve, Frigola, 2007). In orange juice processed by HTST (OJ HTST) and UHT (OJ UHT) methods the retention of the vitamin C was 92 % and 93 % respectively. The results showed that both treatment methods influenced vitamin C degradation however this degradation was insignificant (p > 0.05). There are not found results in scientific literature reporting effects of UHT processing on vitamin C in orange juice. Lee, Coates (1999) carried out several studies to quantify the kinetic destruction of vitamin C at different processing temperatures and found differences in kinetic parameters. They attributed these variations to the fact that nutrient destruction is a complex function of many variables such as ph, oxygen, salts, sugar, enzymes, amino acids and metal catalysts. Total phenolics compounds and hesperidin. As it was previously stated orange juice is a rich of bioactive compounds such as phenolics compounds including hesperidin. The results of the content of total phenolics compounds and hesperidin content in orange juice are shown in Figure

83 Content, mg 100 ml ¹ OJ Fresh OJ HTST OJ UHT Treatment type Total phenolics Hesperidin Fig UHT and HTST processing effect on total phenolic compounds and hesperidin content in Navel orange juice OJ Fresh Fresh frozen and then defrosted orange juice (control); OJ HTST high temperature short time pasteurised fresh frozen and then defrosted orange juice; OJ UHT ultra high treatment treated fresh frozen and then defrosted orange juice The value of total phenolics compounds is higher in OJ Fresh was mg 100 ml -1 followed OJ HTST processed was mg 100 ml -1 and OJ UHT was mg 100 ml -1 in processed orange juices however the difference is not significant (p < 0.05). Gorinshein et al. (2001) reported that total polyphenols content in peeled oranges was 154 ± 10.2 mg 100 g -1 of fresh fruits. Vanamala et al. (2006) studied phenolics compounds in orange juice made from concentrate (MFC) and orange juice NFC, and they found that phenolics compounds in MFC (53.2 mg 100 ml -1 ) orange juice was significantly higher compared to phenolics compounds in NFC (36.49 mg 100 ml -1 ) orange juice. Hesperidin is a main flavonoid of oranges and its content varies with variety of oranges (Omidbaigi et al., 2002). In our study in fresh frozen and then defrosted Navel orange juice the content of hesperidin was of mg 100 ml -1. These results are in agreement with previously reported values (Mouly et al., 1998; Vanamala et al., 2006). Also it is a good agreement with results reported by Kanitsar et al. (2001) they found the content of mg 100 ml -1, and by Tomas-Barberan and Clifford (2000) the content of hesperidin was mg 100 ml -1. However Belajova and Suhaj (2004) stated a lower content of hesperidin level of mg 100 ml -1 in commercial orange juices. The content of hesperidin increased by 14% by UHT processing compared with the sample of fresh frozen and then defrosted Navel orange juice (Fig. 3.24). This may be due to hydrolysis of the glycoside at high temperature it also could be due to changes in the structure of vesicles in the orange, permitting a greater extraction of flavanones as pasteurisation generally reduces it. However, HTST processed orange juice did not show significant changes (p > 0.05 ) on hesperidin content compared to the fresh frozen and then defrosted orange juice. Robards et al. (1997) and Tomas-Barberan, Clifford (2000) reported that hesperidin level in the orange juice depends on technological treatment. Carotenoids. As it was previously stated carotenoids are important quality indicators (for the colour and nutritional value) of orange juice some of them are known for their antioxidant capacity (e.g. β- carotene and lutein) (Rao, Rao, 2007). The total carotenoid content in OJ Fresh Navel was 2.26 mg 100 ml -1 which is comparable to values found by Lee, Coates, (2003) although higher concentration of total carotenoids is reported by others (Esteve et al., 2009; Gama, Sylos, 2007; Sanchez-Moreno et al., 2005). Both processing methods considerably decreased the total carotenoid content to 83

84 1.82 mg 100 ml -1 by HTST processed orange juice, thus resulting in a 19 % loss and to 1.95 mg 100 ml -1 by UHT processed juice, thus resulting in 14% in comparison with total carotenoid content in OJ Fresh (see Fig. 3.25). Comparing both methods of processing, it can be stated that in this study the method of UHT processing was more effective for the retention of carotenoids. Fig UHT and HTST processing effect on total content of carotenoids in Navel orange juice OJ Fresh Fresh frozen and then defrosted orange juice (control); OJ HTST high temperature short time pasteurised fresh frozen and then defrosted orange juice; OJ UHT ultra high treatment treated fresh frozen and then defrosted orange juice Similar results on carotenoid content after thermal processing were found in orange juice (Cortes et al., 2006; Esteve et al., 2009; Hyoung, Coates, 2003; Lee, Coates, 2003) they also noticed a significant processing impact. Some authors have reported that processing had not significant influence on the carotenoid profile (Lee, Coates, 2003; Vervoort et al., 2011). In study researches Sanchez-Moreno et al. (2005) in them study they didn t found significant changes in carotenoids after pulsed electric fields (PEF) treatment and Donsi et al. (1996) and Esteve et al. (2009) didn t found significant differences in value of carotenoids after high hydrostatic pressure (HPP) treatment of orange juices. Plaza et al. (2011) reported that low pasteurisation temperature of orange juice didn t show carotenoid degradation, but after the high-pressure (HP) treatment orange juice showed a significant increase on total carotenoids (45.19%), compared to untreated juice. They also found that HP juice showed the highest carotenoid content among all tested juices. No one of researchers has integrated the comparative study of the impact by UHT processing on total carotenoids content in orange juice. Crino et al. (2012) studied the stability of natural red and pink food colours in natural colour products and evaluated their stability during UHT processing. The results of experiment had a negative effect on the stability of the natural colourants. All coloured samples except fermented red rice showed significant colour loss following UHT processing (p < 0.05). Antioxidant capacity. In the literature no information is available on the changes in antioxidant capacity of orange juice processed by UHT. Ascorbic acid is one of the bioactive compounds that contribute to the antioxidant capacity in the juice it contributes from 56 to 77% of the antioxidant capacity of orange juice, and to 46 % of the tangerine juice, and from 66 to 77% of grapefruit juice (Vinson et al., 2002). In the present study the antioxidant capacity of orange Navel juice was evaluated by using ABTS radical cation assay using DPPH free radical-scavenging and ferric reducing 84

85 antioxidant power (FRAP) assays. Figure 3.26 presents the results of the antioxidant capacity in orange juice measured by ABTS radical cation assay. Fig UHT and HTST processing effect on the antioxidant capacity measured by ABTS in Navel orange juice OJ Fresh Fresh frozen and then defrosted orange juice (control); OJ HTST high temperature short time pasteurised fresh frozen and then defrosted orange juice; OJ UHT ultra high treatment treated fresh frozen and then defrosted orange juice. In the fresh frozen and then defrosted orange juice the antioxidant capacity values was 0.95 mmol Trolox equivalent 100 ml -1 and 0.87 and 0.94 mmol Trolox equivalent 100 ml -1 in orange juices processed by HTST and UHT respectively. As can be seen from the graph the antioxidant capacity values decreased insignificantly (p > 0.05) in orange juice by both processed methods. Arena et al. (2001) studied the total antioxidant activities of freshly squeezed and processed orange juices and measured them using the ABTS radical-cation method. They reported that an antioxidant activity value was higher in freshly-squeezed juices compared with processed orange juices. Fiore et al. (2005) didn t find differences in antioxidant activity of pasteurised and sterilised red orange juices. In the DPPH assay, the antioxidant values were of , and mmol Trolox equivalent 100 ml -1 in fresh frozen and then defrosted orange juice UHT and HTST processed respectively. The antioxidant was also determined using FRAP assay. The FRAP values of antioxidants were of 55.22, and mmol Trolox equivalent 100 ml -1 for fresh frozen and then defrosted UHT and HTST processed Navel orange juices respectively. Chosen methods for antioxidant capacity determination didn t show significant differences (p > 0.05) in orange juice samples processed by HTST and UHT methods (Fig. 3.27). 85

86 Antioxidant capacity (DPPH, FRAP) mmol TE 100 ml ¹ OJ Fresh OJ HTST OJ UHT Treatment type DPPH FRAP Fig UHT and HTST processing effect on the antioxidant capacity measured by FRAP and DPPH in Navel orange juice OJ Fresh Fresh frozen and then defrosted orange juice (control); OJ HTST high temperature short time pasteurised fresh frozen and then defrosted orange juice; OJ UHT ultra high treatment treated fresh frozen and then defrosted orange juice The results showed that juice contained higher concentration of vitamin C and phenolics compounds have a higher antioxidant capacity. Literature dates suggest changes in individual antioxidants. Davidov-Pardo et al. (2011) studied some individual antioxidant in grape seed extract using different treatment methods. The results showed that the individual antioxidants behaved differently during heating but they not showed significant changes on total antioxidant capacity after thermal treatment. Grouped statistics show that ABTS correlates directly with the vitamin C, total phenolics compounds, total carotenoids and hesperidin (r = 0.688; r = 0.563; r = 0.802; r = respectively). In the test with DPPH radical and by FRAP clearly correlated (r = 0.993; r = 0.999; r = and r = 0.899; r = 0.961; and r = 0.817) with the vitamin C, total carotenoids and total phenolics compounds respectively (Ann. 11.). Summary of chapter 3.4 The effect of HTST (94 C 30 s) and UHT (130 C 2 s) processing methods on chemical parameters, bioactive compounds and antioxidant capacity were studied in orange juice Navel variety.the changes of TSS, TA and TSS/TA ratio in Navel orange juice affected by thermal treatment (HTST and UHT) were not significant (p > 0.05). Degradation of vitamin C and total phenolics compounds influenced by both thermal treatment methods was insignificant as well (p > 0.05). The antioxidant capacity in orange juice was evaluated by means of ABTS, DPPH and FRAP assay the changes during processing were unsubstantial (p > 0.05) although the retention of antioxidant capacity values by UHT processed juice measured by all used methods was higher than by HTST processed juices and its value nearly did not differs from value in fresh frozen then defrosted juice. However in orange juice processed by UHT methods the content of hesperidin increased significantly (p < 0.05) while total content of carotenoids decreased significantly as a result of both processing methods (p < 0.05) compared with fresh frozen and then defrosted juice. 86

87 3.5. Impact of UHT processing on chemical parameters, bioactive compounds and antioxidant capacity in sea buckthorn juices and blended orange-sea buckthorn juices UHT processing effect on chemical parameters, bioactive compounds, and antioxidant capacity of sea buckthorn juices Fresh sea buckthorn fruits contain significant amounts of bioactive compounds such as vitamin C and greatly high vitamin E content, carotenoids, phenolics compounds and so on (Sinha et al., 2012). These characteristics were used to prepare blended juices with high antioxidant value. Sea buckthorn juices same as orange juices is sensitive to heat and content of bioactive compounds and fresh aroma may be lost or damaged by exposure to heat. In this study the experimental results are given in the Annex 12. The chemical parameters of UHT processed sea buckthorn juices Leikora, Hergo and Botanicheskaya- Lubitelskaya are shown in the Table 3.4. Chemical parameters of fresh and UHT treated sea buckthorn juices Table 3.4 Samples Total Soluble Solids (TSS), Brix Total acidity (TA), % Ratio Leikora Fresh 7.16 ± 0.15 a 3.64 ± 0.04 a 1.97 UHT (130 C 2 s) 7.29 ± 0.15 a 3.66 ± 0.04 a 1.99 Hergo Fresh 5.98 ± 0.15 b 2.72 ± 0.04 b 2.20 UHT (130 C 2 s) 6.11 ± 0.15 b 2.77 ± 0.04 b 2.21 Botanicheskaya Lubitelskaya Fresh 8.65 ± 0.15 c 3.12 ± 0.04 c 2.77 UHT (130 C 2 s) 8.98 ± 0.15 c 3.18 ± 0.04 c 2.82 Column values with different online letters (a, b, c) differ (p < 0.05) Results were presented as means ± standard error (n=4) TSS in fresh sea buckthorn juices of different varieties was found within the range of 5.98 to 8.65 Brix. Out of these sea buckthorn samples maximum TSS was found in the sample Botanicheskaya-Lubitelskaya sea buckthorn juice. The highest content of TA was ascertained in sea buckthorn Leikora juice (3.64%). After UHT processing the content of TSS and TA slightly increased in all analysed samples of sea buckthorn juices but changes was not significant (p > 0.05). Vitamin C content. The primary vitamin in sea buckthorn berries is vitamin C. The berries are a rich source of vitamin C, which in the species of European origin can be from 28 to 310 mg 100 g -1, subspecies fluviatis from 460 to 1330 mg 100 g -1, but subspecies sinensis from 200 to 2500 mg 100 g -1 (Antonelli et al., 2006; Yao et al., 1992; Tang, 2002;). The effect of UHT processing on stability of vitamin C is shown in Figure

88 Fig The content of vitamin C in fresh and UHT processed sea buckthorn juices Vitamin C content in fresh Leikora sea buckthorn juice was significantly higher ( mg 100 ml -1 ) (p 0.05) than in juices of Hergo (94.28 mg 100 ml -1 ) and Botanicheskaya-Lubitelskaya (97.28mg 100 ml -1 ). UHT processing slightly cut down the content of vitamin C in all processed sea buckthorn juices if compared to its content in fresh juices. The study results showed that retention of vitamin C after UHT treatment was 93, 92 to 91% in Leikora, Hergo and Botanicheskaya-Lubitelskaya species of sea buckthorn juices respectively. The retention of vitamin C in the Leikora juice was a little higher but not significant (p > 0.05) and this may be explained due to the fact that the total acidity in Leikora juice was higher. Total content of phenolics compounds. The results of study demonstrated highest content of total phenolics compounds ( mg 100 ml -1 ) in Leikora sea buckthorn juice, but in the juices Hergo and Botanicheskaya-Lubitelskaya it was considerably lower and mg 100 ml -1 respectively (see Fig. 3.29). Total phenolics, mg 100 ml ¹ Fresh UHT Fresh UHT Fresh UHT Leikora Hergo Botan-Lubit Samples Fig The content of total phenolics compounds in fresh and UHT processed sea buckthorn juices 88

89 The content of total phenolics compounds in all varieties of sea buckthorn juices processed by UHT has decreased a little although it was not significant (p 0.05). Total carotenoids. Sea buckthorn juice contains large amount of carotenoids and vitamin E, which ensure the colour intensity of sea buckthorn berries from yellow to red. UHT processing effect on the carotenoid and vitamin E content in different sea buckthorn juices was quantified in our studies (Zvaigzne, et al., 2014). The results are presented in Figure Fig The content of total carotenoids and vitamin E in fresh and UHT processed sea buckthorn juices The highest content of total carotenoids was found in the sea buckthorn Leikora juice (9.70 mg 100 ml -1 )while in Hergo and Botanicheskaya-Lubitelskaya juices it was lower 5.46 and 7.03 mg 100 ml -1 respectively. In all analysed sea buckthorn juices processed by UHT processing the content of total carotenoids slightly decreased about 10 % although it was not significant (p 0.05). Vitamin E. An important compound in the sea buckthorn juices is vitamin E. The highest content of vitamin E was found in the sea buckthorn Leikora juice (12.38 mg 100 ml -1 ) while in Hergo and Botanicheskaya-Lubitelskaya juices it was significantly lower 6.32 and 6.74 mg 100 ml -1 respectively (see Fig ) After treatment by UHT the content of vitamin E slightly increased but the increase was insignificant (p > 0.05). Antioxidant capacity. The antioxidant capacity in different species of sea buckthorn juices was determined using three measurement methods: DPPH, FRAP and ABTS. The results are shown in the Table

90 Leikora Hergo Table 3.5 Antioxidant capacity in fresh and UHT processed sea buckthorn juices Samples Antioxidant capacity Treatment DPPH FRAP ABTS mmol Trolox equivalent 100 ml -1 Fresh ± b ± 0.88 c 0.37 ± 0.02 a UHT ± a ± 0.76 c 0.34 ± 0.04 a Fresh ± a ± 1.00 b 0.21 ± 0.01 a UHT ± b ± 2.15 a 0.19 ± 0.03 a Fresh ± 4.86 c ± 0.50 c 0.13 ± 0.05 a UHT ± b ± 2.53 a 0.11 ± 0.01 a Botanicheskaya- Lubitelskaya Column with different online letters (a, b, c) differ (p < 0.05) Results were presented as means ± standard deviation (n=2) The results of three used antioxidant capacity measuring methods showed similar values in all analysed juice samples. The highest antioxidant values measured by DPPH method was observed in fresh Leikora sea buckthorn juice ( ± mmol Trolox equivalent 100 ml -1 ); while in Hergo and Botanicheskaya-Lubitelskaya juices it was near two to three times lower ( ± and ± 4.86 respectively). After UHT processing the values of antioxidant capacity in all analysed juices decreased, which is linked to vitamin C and total phenolics compounds decrease in the sea buckthorn juices after treatment by UHT, however this decrease was not significant (p 0.05). Bioactive compounds and antioxidant capacity changes in sea buckthorn juices under the influence of UHT processing currently had not been tested. The effect of treatment by UHT on the quality of watermelon juice was studied by Zhang et al. (2015) and they found that the temperature of 120 and 135 C was effective to reduce the total count of microflora in watermelon juice. Several studies on the effects of UHT treatment on chemical content changes in apple, pomegranate and sugarcane juices are carried out (Lewis et al., 2000) UHT processing effect on chemical parameters, bioactive compounds and antioxidant capacity in blended orange-sea buckthorn juices The objective of this part of the study was to evaluate the chemical parameters, bioactive compounds and antioxidant capacity in blended orange-sea buckthorn juices, processed by UHT method as well as to evaluate the consumer acceptance of those new kinds of products. As an alternative for the production UHT processed Navel orange juice was blended with Leikora, Hergo and Botanicheskaya-Lubitelskaya varieties of sea buckthorn juices also UHT processed. The content of sea buckthorn juice was 10% and juices were prepared without any addition of sugar or other sweeteners. Blended juices immediately chilled and kept until use. All blended juices were compared with Navel orange juice as a control sample in terms of chemical parameters, bioactive compounds and antioxidant capacity (Ann.13). The results of the chemical parameters of the three mixed juice samples are shown in the Table

91 Table 3.6 Chemical parameters of blended orange - sea buckthorn juices processed by UHT Parameters Control (Navel juice) Navel-Leikora Navel-Hergo Navel- Botanicheskaya- Lubitelskaya TSS, Brix ± 0.10 a ± 0.05 b ± 0.03 c ± 0.35 c TA, % 0.80 ± 0.04 a 1.09 ± 0.00 b 0.99 ± 0.03 c 0.96 ± 0.00 c Ratio Column values with different online letters (a, b, c) differ (p < 0.05). d Results were presented as mean ± SD (n=2) In table 3.6 changes in chemical parameters of the blended orange-sea buckthorn juices are visible. As can be seen from the table, addition of sea buckthorn juices to orange juice has decreased the TSS values in blended juices compared with control sample; decreases were not significant by an average of 5%. In terms of total acid, in blended juices the acidity increased significantly in mixed juices of Navel- Leikora up to 25%, Navel- Hergo 23% and Navel- Botanicheskaya-Lubitelskaya 20%. The TSS acid ratio decrease was in average 20 % in all blended juices. The content of vitamin C in the blends orange-sea buckthorn juice Navel- Leikora and Navel- Hergo increased more than two times and in Navel- Botanicheskaya-Lubitelskaya blend content of vitamin C increased per 26% (see Fig. 3.31) Vitamin C, mg 100 ml ¹ Navel control Navel-Leikora Navel -Hergo Navel - Bot.Lub Samples Fig Content of vitamin C in blended orange-sea buckthorn juices and orange (control) juice processed by UHT Due to a considerable extent of total carotenoid content in all sea buckthorn juices the content of total carotenoids in the blended juices of Navel- Leikora, Navel- Hergo and Navel- Botanicheskaya-Lubitelskaya greatly 2.30, 1.87 and 2.01 mg 100 ml -1 (p < 0.05) respectively and 1.59 mg 100 ml -1 in orange Navel juice (see Fig. 3.32). Vitamin E is a fat-soluble vitamin, which is an important antioxidant and important component of the sea buckthorn juice. So, blended orange-sea buckthorn juices received additionally high vitamin E content (see Fig. 3.32). 91

92 Content, mg 100 ml ¹ Navel control Navel-Leikora Navel -Hergo Navel - Bot.Lub Samples Carotenoids Vitamin E Fig Content of total carotenoids and vitamin E in blended orange-sea buckthorn juices and orange (control) juice processed by UHT Vitamin E content in the Navel- Leikora juice was significantly higher (1.23 mg 100 ml -1 ) than its content in Navel- Hergo and Navel- Botanicheskaya Lubitelskaya blended samples (0.64 and 0.78 mg 100 ml -1 ) respectively. At the same time vitamin E content was significantly higher (p < 0.05) in all blended juices compared with control sample containing minimal vitamin E amount. In the control sample the total content of phenolics compounds was mg 100 ml -1 (see Fig. 3.33) and this amount differed significantly (p < 0.05) in two mixed orange-sea buckthorn juices. Total phenolics, mg 100 ml ¹ Navel control Navel-Leikora Navel -Hergo Navel - Bot.Lub Samples Fig Content of total phenolics compounds in blended orange-sea buckthorn juices and orange (control) juice processed by UHT In blended sea buckthorn juices of Navel-Leikora and Navel- Hergo samples total phenolics compounds content increased by 20% and 16 % respectively compared with the control sample. The blended juice sample of Navel- Botanicheskaya-Lubitelskaya show insignificant difference in the content of total phenolics compounds compared with control 92

93 sample. The content of total phenolic compounds in Botanicheskaya-Lubitelskaya sea buckthorn juice was lower than in other sea buckthorn juices but not significant (p > 0.05). Fig Antioxidant capacity measured by ABTS in blended orange-sea buckthorn juices and orange (control) juice processed by UHT Values of antioxidant capacity in the blended orange-sea buckthorn juices were significantly higher (p < 0.05) if compared with control sample of orange juice (see Fig and Fig. 3.35). The sample of Navel- Leikora blended juice showed a higher antioxidant capacity value measured by all analysed methods. As can be seen from the results described above Navel- Leikora blended juice contained higher content of vitamin C, total phenolics compounds, total carotenoids and vitamin E (109.67, , 2.30 and 1.23 mg 100 ml -1 ) respectively. Fig Antioxidant capacity measured by DPPH and FRAP in blended orange-sea buckthorn juices and orange (control) juice processed by UHT No data were found in the literature about the antioxidant capacity of Navel-Leikora, Navel-Hergo or Navel-Botanicheskaya Lubitelskaya blended juices. Xu et al. (2015) studied 93