Aus dem Institut für Humanernährung und Lebensmittelkunde der Christian-Albrechts-Universität zu Kiel

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1 Aus dem Institut für Humanernährung und Lebensmittelkunde der Christian-Albrechts-Universität zu Kiel Auswirkung von Trägermatrix und verfahrenstechnischen Parametern auf Struktur und Stabilität von mikroverkapselten langkettigen mehrfach ungesättigten Fettsäuren Dissertation zur Erlangung des Doktorgrades der Agrar- und Ernährungswissenschaftlichen Fakultät der Christian-Albrechts-Universität zu Kiel vorgelegt von M.Sc. Yvonne Serfert aus Annaberg-Buchholz Kiel, 2009 Dekan: Prof. Dr. Uwe Latacz-Lohmann 1. Berichterstatter: Prof. Dr. Karin Schwarz 2. Berichterstatter: Prof. Dr. Stephan Drusch Tag der mündlichen Prüfung:

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3 JEDER AUFSTIEG IN GROßE HÖHEN GESCHIEHT AUF EINER WENDELTREPPE FRANCIS BACON ( ) DANKE Prof. Dr. Karin Schwarz & Prof. Dr. Stephan Drusch Forschungskreis Ernährungsindustrie e.v. Arbeitsgemeinschaft industrieller Forschungsvereinigungen e.v. AiF Projekt 14591N Bundesministerium für Wirtschaft und Technologie Institut für thermische Verfahrenstechnik, Universität Karlsruhe (TH) Abteilung für pharmazeutische Technologie und Biopharmazie, Christian-Albrechts-Universität zu Kiel Abteilung Lebensmitteltechnologie & Ehemalige, Christian-Albrechts-Universität zu Kiel Rolf & Sigrid Serfert Alexander Ludwig, Barbara Hindel, Beate Schulze, Bernhard Trierweiler, Carsten Mehlfeld, Diana Würfel, Elli Serfert, Elmar Zinsius, Erwin Hindel, Eva Maria Hubbermann, Gabriele Kranz, Ina Wlodasch, Jeannette Vetter, Jérôme Hindel, Julia Lange-Grumfeld, Kathleen Oehlke, Katja Borschel, Lotti, Mandy Dimbat, Monika Kühne, Ortwin Meinicke, Petra Mehlfeld, Ralf Lindauer, Rigo Serfert, Simone Haftendorn, Sonja Berg, Stefan Esser, Susann Lorenz, Thomas Serfert, Wolfgang Würfel

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5 Zusammenfassung Ziel der vorliegenden Arbeit war es, die komplexen Mechanismen und Interaktionen aufzuklären, welche die oxidative Stabilität von Fischöl während der Mikroverkapselung, Sprühtrocknung und anschließender Lagerung bestimmen. Die Notwendigkeit einer chemischen Stabilisierung zeigte sich während des Mikroverkapselungsprozesses und der anschließenden Lagerung. Aufgrund der verschiedenen Phasenübergänge war der Zusatz lipophiler Antioxidantien und Synergisten sowie hydrophiler Chelatbildner notwendig. Weiterhin konnten durch Variation des emulgierenden Bestandteils und der Kohlenhydratkomponente der Trägermatrix physikalische Partikelcharakteristika ermittelt werden, welche die Stabilität des mikroverkapselten Fischöls beeinflussen. Hierzu zählen mikrostrukturelle Parameter wie die wahre Dichte und nanostrukturelle Aspekte wie freie Volumenelemente. Am Beispiel verschiedener n-octenylsuccinatderivatisierter Stärken (nosa-stärke) wurde der Einfluss der Sprühtrocknungsbedingungen (Trocknungstemperatur, Sauerstoffgehalt des Trocknungsgases) auf die oxidative Stabilität der Mikrokapseln untersucht. Dabei zeigte sich, dass der Anteil diskreter Lufteinschlüsse die oxidative Stabilität während der Lagerung determinierte. Das Sprühtrocknen unter inerten Bedingungen hatte keinen Einfluss auf den Verlauf der Lipidoxidation von mikroverkapseltem Fischöl. Im Hinblick auf die Sensorik wurden Attribute identifiziert, mit denen die Geruchsqualität von Bulkfischöl und mikroverkapseltem Fischöl charakterisiert werden kann. Die für Bulkfischöl gewählten Attribute trafen ebenso für das mikroverkapselte Fischöl zu, in Abhängigkeit der Trägermatrix wurden außerdem weitere Attribute identifiziert. Im Vergleich zu nosa-stärke basierten Mikrokapseln war die Intensität einzelner Geruchsattribute der Natrimcaseinat basierten Mikrokapseln signifikant niedriger. Dies konnte eher auf eine höhere Oxidationsstabilität des Fischöls als auf eine aromenbindende Wirkung des Proteins zurückgeführt werden. Eine aktive Modifizierung der Geruchsqualität von mikroverkapseltem Fischöl war durch Zusatz von β- Cylodextrin und Aromen möglich. v

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7 Abstract Aim of the present study was to investigate the complex mechanisms and interactions which affect the oxidative stability of fish oil during microencapsulation and storage. A chemical stabilisation was necessary during the microencapsulation process and subsequent storage. Due to different multiphase systems and processing steps, the addition of lipophilic antioxidants and synergists as well as hydrophobic chelating agents was efficient. To identify physicochemical characteristics which influence the oxidative stability of microencapsulated fish oil, microcapsules were prepared by utilising different emulsifying constituents and carbohydrate sources as carrier matrices. Particularly, among these characteristics, the micro- and nanostructure like apparent density and hole size of free volume elements, respectively, determined the oxidative stability during storage of the microcapsules. To study the effect of the process parameters (drying temperature, oxygen content of the drying gas) on the oxidative stability of the microcapsules, different n-octenylsuccinate-derivatised starches (nosastarch) were utilised for spray drying. During storage, the oxidative stability of the fish oil was determined by the amount of discrete air inclusions inside the microcapsules. Spray drying under inert conditions did not affect the course of lipid oxidation during storage. With regard to sensory aspects of bulk fish oil and microencapsulated fish oil, attributes were identified which characterise the odour quality. Data showed that the most important odour attributes describing the sensory quality of fish oil can also be utilised for microencapsulated fish oil. In addition, other odour attributes as affected by the carrier matrix may occur. Differences in the sensory profile between the oil encapsulated in the OSA-starch and sodium caseinate could not clearly be associated to flavour binding by the protein and were rather attributed to the lipid oxidation status of the encapsulated oil. An active modification of the odour quality of microencapsulated fish oil was achieved by the addition of β-cyclodextrin or flavours. vii

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9 Inhaltsverzeichnis Abkürzungsverzeichnis... xi 1 Einleitung und Zielsetzung... 1 Literaturverzeichnis CHEMICAL STABILISATION OF OILS RICH IN LONG CHAIN POLYUN- SATURATED FATTY ACIDS DURING HOMOGENISATION, MICROENCAP- SULATION AND STORAGE Abstract Introduction Materials and Methods Results and Discussion References THE IMPACT OF PHYSICOCHEMICAL CHARACTERISTICS ON THE OXIDATIVE STABILITY OF FISH OIL MICROENCAPSULATED BY SPRAY- DRYING Summary DIFFERENCES IN FREE VOLUME ELEMENTS OF THE CARRIER MATRIX AFFECT THE STABILITY OF MICROENCAPSULATED LIPOPHILIC FOOD INGREDIENTS Abstract Introduction Materials and Methods Results and Discussion References ix

10 5 PROCESS ENGINEERING PARAMETERS AND TYPE OF n-octenyl- SUCCINATE-DERIVATISED STARCH AFFECT OXIDATIVE STABILITY OF MICROENCAPSULATED LONG CHAIN POLYUNSATURATED FATTY ACIDS Abstract Introduction Materials and Methods Results and Discussion Conclusions References SENSORY ODOUR PROFILING AND LIPID OXIDATION STATUS OF FISH OIL AND MICROENCAPSULATED FISH OIL Abstract Introduction Materials and Methods Results and Discussion Conclusions References Zusammenfassende Diskussion Literaturverzeichnis x

11 Abkürzungsverzeichnis BET Citrem DHA DE EDTA EPA HS-SPME-GC-O/GC-MS nosa-stärke o/w Emulsion o-ps PALS Brunauer Emmett Teller Citronensäureester von Mono- und Diglyceriden Docosahexaensäure Dextroseäquivalent Ethylendiamintetraessigsäure Eicosapentaensäure Headspace-Festphasenmikroextraktion- Gaschromatographie-Olfaktometrie und Gaschromatographie-Massenspektroskopie n-octenylsuccinatderivatisierte Stärke Öl-in-Wasser Emulsion ortho-positronium Positronenannihilationlebensdauer- spektroskopie PLS-Analyse REM RH TBARS Partial Least Square Analyse Rasterelektronenmikroskopie Relative Luftfeuchte Thiobarbitursäure-reaktive Substanzen xi

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13 1 Einleitung und Zielsetzung EINLEITUND UND ZIELSETZUNG Die Mikroverkapselung ist eine häufig angewandte Technologie, um Öle, Aromen, Probiotika und andere empfindliche Inhaltsstoffe zu verkapseln. Es existiert eine Vielzahl von Verfahren zur Mikroverkapselung, wobei die Sprühtrocknung das am weitesten verbreitete Verfahren ist (Gouin, 2004; 1998). Bei der Mikroverkapselung lipophiler Inhaltsstoffe durch Sprühtrocknung wird der zu verkapselnde Stoff nach Herstellung einer Emulsion in eine amorphe Trägermatrix eingeschlossen und damit in ein pulverförmiges Produkt überführt, wodurch es anderen, vorzugsweise pulverförmigen Lebensmitteln zugesetzt werden kann. Durch die Mikroverkapselung kann die Freisetzungsrate kontrolliert und die Oxidationsstabilität von lipophilen Inhaltsstoffen erhöht werden. Im Hinblick auf die Mikroverkapselung von funktionellen Inhaltsstoffen nehmen omega-3 fettsäurereiche Öle wie Fischöl eine Sonderstellung ein, da die langkettigen, mehrfach ungesättigten Fettsäuren Eicosapentaensäure (EPA) und Docosahexaensäure (DHA) sehr oxidationsanfällig sind. Durch den Zerfall primärer Lipidoxidationsprodukte werden über die Entstehung flüchtiger Oxidationsprodukte sensorische Abweichungen (off-flavor) hervorgerufen (Frankel, 2005). Die Fettsäuren EPA und DHA üben zahlreiche positive Effekte auf die menschliche Gesundheit aus, besonders in Bezug auf die Prävention kardiovaskulärer Erkrankungen (Kris-Etherton et al., 2003; Reiffel & McDonald, 2006; Ruxton et al., 2007). Die Fachorganisation ISSFAL (International Society for the Study of Fatty Acids and Lipids) empfiehlt gesunden Menschen zur Erhaltung der kardiovaskulären Gesundheit eine tägliche Aufnahme von insgesamt 500 mg EPA und DHA (ISSFAL, 2004). Jedoch ist in den westlichen Ländern im Allgemeinen die Aufnahme an diesen Fettsäuren unzureichend (Bauch et al., 2006; Meyer et al., 2003; Sanders, 2000), sodass der Verzehr von omega-3 Fettsäuren angereicherten Lebensmitteln empfohlen wird, da so keine drastische Änderung der Ernährungsgewohnheiten vollzogen werden muss. Die Bioverfügbarkeit von omega-3-fettsäuren aus angereicherten Lebensmitteln wurde in zahlreichen Studien nachgewiesen (Arterburn et al., 2007; Higgins et al., 1999; L. E. Lent, 1998; Lovegrove et al., 1997; Maki et al., 2003). Wie im Folgenden weiter ausgeführt wird, existieren zahlreiche Einflussfaktoren, die die Oxidationsstabilität des Fischöls während der Mikroverkapselung, der Anreicherung von Lebensmitteln und deren Lagerung bestimmen. Ziel der vorliegenden Arbeit ist es, die komplexen Mechanismen und Interaktionen, 1

14 EINLEITUND UND ZIELSETZUNG welche die Stabilität des Fischöls beeinflussen, aufzuklären. Dabei steht die Aufklärung des Zusammenhangs zwischen der chemischen Stabilisierung des Fischöls bzw. der physikochemischen Partikeleigenschaften und der oxidativen Stabilität von mikroverkapseltem Fischöl während des Mikroverkapselungsprozesses selbst und der Lagerung des mikroverkapselten Öls im Vordergrund. Darauf aufbauend wurde die Sensorik von mikroverkapseltem Fischöl und eine mögliche Beeinflussung des Aromaprofils untersucht. Chemische Stabilisierung von Fischöl während des Mikroverkapselungsprozesses und der Lagerung Im Hinblick auf die chemische Stabilisierung ist zu berücksichtigen, dass während des Mikroverkapselungsprozesses verschiedene physikalische Zustände durchlaufen werden. Zunächst liegt das Öl als Bulköl, danach als Öl dispergiert in der wässrigen Phase und schließlich dispers verteilt in der sprühgetrockneten Matrix vor. Die Wirksamkeit der Antioxidantien wird durch deren Verteilung zwischen den verschiedenen Phasen beeinflusst. Die chemische Stabilisierung von Bulkfischöl ist weitreichend untersucht worden (Drusch et al., 2008; Hamilton et al., 1998; Koschinski & MacFarlane, 1993; Kulås & Ackman, 2001a; Kulås & Ackman, 2001b). Eine Kombination bestehend aus einem hohen Anteil δ-tocopherol bei gleichzeitig niedrigem α- Tocopherol-Anteil, Ascorbylpalmitat sowie Zitronensäureester von Mono- und Diglyceriden (Citrem) oder alternativ Lecithin wurde in vorangehenden Arbeiten der Arbeitsgruppe als effektive chemische Stabilisierung von Bulkfischöl herausgestellt (Drusch et al., 2008). In Bezug auf den Prozess der Mikroverkapselung konnte sowohl in eigenen vorhergehenden Arbeiten (Drusch et al., 2006) und Arbeiten anderer Autoren (Baik et al., 2004; Hogan et al., 2003) bereits während des Emulgierens bzw. Sprühtrocknens eine oxidative Schädigung von Fischöl nachgewiesen werden. Zahlreiche Studien zur oxidativen Stabilität von Fischölemulsionen sind vorhanden (Frankel, 2002; Hu et al., 2004; Jakobsson & Sivik, 1994; Klinkesorn et al., 2005a; Let et al., 2003; Let et al., 2005a). Dabei wird der Zusatz von chelierenden Substanzen als besonders wichtig erachtet, um die Metallionen katalysierte Lipidoxidation des Fischöls zu verhindern. Emulsionen zur Mikroverkapselung mittels Sprühtrocknung zeichnen sich im Gegensatz zu den in den aufgeführten Studien untersuchten Emulsionen jedoch durch eine komplexere Zusammensetzung aus: sie enthalten zusätzlich die gelösten Komponenten der Trägermatrix wie z.b. eine Kohlenhydratquelle und zumeist einen höheren Anteil an Emulgator und Öl. Verfügbare Studien zur chemischen Stabilisierung von sprüh- 2

15 EINLEITUND UND ZIELSETZUNG getrockneten Fischölemulsionen sind in Tabelle 1.1 aufgeführt. Eine zusammenfassende Analyse der Studien ist nicht möglich, da sich die aufgeführten Studien hinsichtlich der eingesetzten Verkapselungsmaterialien, analysierten Lipidoxidationsprodukte und Lagerungsbedingungen unterscheiden. Zum einen können von der Trägermatrix selbst antioxidative Effekte ausgehen wie zum Beispiel durch Natriumcaseinat. Zudem sind teilweise die angewandten Methoden zur Beurteilung der oxidativen Stabilität des mikroverkapselten Fischöls wie Oxipres, Bestimmung des Polyenverhältnisses oder der Thiobarbitursäurereaktiven Substanzen (TBARS) als ungeeignet einzuschätzen (Frankel, 2005). Weiterhin kann durch Lagerung der mikroverkapselten Fischöle bei erhöhten Temperaturen (37 C) die Glas übergangstemperatur der Trägermatrizes erreicht werden, in Folge dessen strukturelle Veränderungen auftreten können, welche die Lipidoxidation zusätzlich beeinflussen (Grattard et al., 2002; Partanen et al., 2005). Im Hinblick auf den Prozess der Mikroverkapselung wurde in den in Tabelle 1 aufgeführten Studien mit Ausnahme der Arbeiten von Hogan et al. (2003) und Baik et al. (2004) weder der Einfluss des Emulgierens noch des Sprühtrocknens auf die oxidative Stabilität des Fischöls untersucht. Hypothese Basierend auf den aufeinanderfolgenden Prozessschritten und den damit einhergehenden physikalischen Zustandsänderungen wurde unterstellt, dass während der Emulsionsherstellung aufgrund der vorhandenen Wasserphase der Einsatz von hydrophilen Chelatbildnern über Komplexierung von Metallionen die Oxidation des Fischöls inhibiert, wohingegen im mikroverkapselten Öl analog zum Bulköl das Vorhandensein lipophiler Antioxidantien und Synergisten effektiv ist. Ziel war es daher, die Effizienz von Antioxidantien (Tocopherolen), Synergisten (Ascorbyl-palmitat, Lecithin, Carnosolsäure) und chelierenden Substanzen (Citronensäure, Citrem) mit unterschiedlicher Polarität während der aufeinanderfolgenden Phasen des Mikroverkapselungsprozesses zu untersuchen. Experimenteller Ansatz Das Fischöl wurde zunächst standardisiert, indem es chromatographisch zu einem Triglyceridgemisch aufgereinigt wurde. Dabei wurden vorhandene Antioxidantien, freie Fettsäuren, Pigmente sowie Mono- und Diglyceride entfernt. Zur Abbildung der Kernprozesse wurden zuerst Homogenisier- und Sprühtrocknungsversuche unter alleinigem Zusatz von Tocopherolen durch- 3

16 EINLEITUND UND ZIELSETZUNG geführt und anschließend deren Kombination mit den oben aufgeführten Additiven. Die Emulsionen wurden verschlossen 2 Tage bei 20 C, das mikroverkapselte Fischöl wurde 8 Wochen bei 20 C und 33 % relativer Luftfeuchte (RH) unter Lichtausschluss gelagert. Die Beurteilung der antioxidativen Aktivität der eingesetzten Additive wurde durch Bestimmung des Hydroperoxid- und Propanalgehaltes vorgenommen (Kapitel 2). 4

17 Tabelle 1.1: Verfügbare Studien anderer Autoren zur chemischen Stabilisierung von mikroverkapseltem Fischöl Quelle Trägermatrix Antioxidantien Konzentration Beurteilung der antioxidativen Aktivität Schlussfolgerung Baik et al. (2004) Natriumcaseinat, Glucosesirup (DE 36) Lecithin α-tocopherol oder Ascorbylpalmitat 250/1000 mg/kg 250 mg/kg Lagerung: 0,11, 33, 43 % RH, 30 C; Hydroperoxidgehalt, TBARS a, Sensorik Zusatz von Tocopherolen verminderte die Lipidoxidation des verkapselten und nicht verkapselten Öls (Hydroperoxidgehalt nach 7 Tagen Lagerung: 40 mmol/kg Öl) Hogan et al. (2003) Natriumcaseinat, Glucosesirup (DE 28) α-tocopherol oder Trolox 100/500 mg/kg Lagerung: 4 C ; Hydroperoxidgehalt, Anisidingehalt Hydroperoxidgehalt war in Proben mit Tocopherolen niedriger als in Proben, die Trolox enthielten; 100 mg Tocopherol/kg Öl waren effizienter als 500 mg/kg Öl. Jonsdottir et al. (2005) Natriumcaseinat, Lactose, Lecithin α-tocopherol 1000 mg/kg Lagerung: Vakuum, 4 C, 20 Wochen; Oxipress, TBARS, Sensorik, HS- SPME-GC-O/GC-MS b Zusatz von Tocopherol bewirkte eine verminderte Intensität der Ranzigkeit Klinkesorn et al. (2005b) Chitosan, Lecithin, Glucosesirup (DE 36) Mischung von Tocopherolen, EDTA c 500 mg/kg 12 µm Lagerung: 11, 33, 52 % RH, 37 C; Hydroperoxidgehalt, TBARS, Sensorik Verminderte Lipidoxidationsrate bei allen Bedingungen Shaw et al. (2007) Natriumcaseinat, Glucosesirup (DE 24), Lecithin EDTA 25 µm Lagerung: 33 % rh, 37 C; Hydroperoxidgehalt, TBARS, Propanalgehalt in rekonstituierten Emulsionen Starke Inhibierung der Hydroperoxid- und Propanalbildung Marquez-Ruiz et al. (2000) Natriumcaseinat, Lactose Ascorbinsäure, Lecithin δ-tocopherol 300 mg/kg 500 mg/kg 200 mg/kg Lagerung: Licht, 30 C; TBARS, Polyen-Verhältnis, oxidierte und polymerisierte Triglyceride Die Lagerstabilität verdoppelte sich durch den Zusatz von Antioxidantien Velasco et al. (2000) Natriumcaseinat, Lactose Ascorbic acid Lecithin δ -Tocopherol 300 mg/kg 500 mg/kg 300 mg/kg Lagerung: Licht, 30 C, begrenzte Sauerstoffverfügbarkeit, 25 C vakuumverpackt oder offen; oxidierte und polymerisierte Triglyceride Die Lagerstabilität wurde durch den Zusatz von Antioxidantien besonders im nicht verkapselten Öl erhöht a Thiobarbitursäure-reaktive Substanzen, b Headspace-Festphasenmikroextraktion-Gaschromatographie-Olfaktometrie und Gaschromatographie- Massenspektroskopie, c Ethylendiamintetraessigsäure

18 EINLEITUNG UND ZIELSETZUNG Der Einfluss der Trägermatrix auf physikalische Partikelcharakteristika und die Lipidoxidation von mikroverkapseltem Fischöl Die Trägermatrix von Mikrokapseln besteht im Allgemeinen aus einem emulgierenden Bestandteil und einem Füllmaterial wie Kohlenhydrate. Zur Mikroverkapselung von lipophilen Inhaltsstoffen mittels Sprühtrocknung eignen sich eine Vielzahl von emulgierenden Materialien: Gummi arabicum (Fang et al., 2005; McNamee et al., 1998; Turchiuli et al., 2005), Gummi arabicum/noctenylsuccinat-derivatisierte (nosa-) Stärke (Kanakdande et al., 2007; Krishnan et al., 2005; Soottitantawat et al., 2005; Soottitantawat et al., 2004), nosa-stärke (Drusch & Schwarz, 2006; Drusch et al., 2006), Zuckerrübenpektin (Drusch, 2007) und Milchproteine (Hogan et al., 2001; Kagami et al., 2003; Keogh et al., 2001). In der Mehrzahl dieser Studien erfolgte die Charakterisierung der Proben lediglich über die Partikelgröße und die Mikroverkapselungseffizienz. Hypothese Es kann unterstellt werden, dass die Stabilität von mikroverkapselten lipophilen Inhaltsstoffen durch deren physikalischen Charakteristika beeinflusst wird. Dieser Zusammenhang wurde in den bislang verfügbaren Studien zur Mikroverkapselung von Fischöl (Hogan et al., 2003; Klinkesorn et al., 2005b; Kolanowski et al., 2004; Kolanowski et al., 2005; Marquez-Ruiz et al., 2000; Velasco et al., 2000) kaum untersucht. Experimenteller Ansatz Durch eine physikochemische Charakterisierung von sprühgetrockneten Fischölemulsionen sollen Parameter abgeleitet werden, welche die oxidative Stabilität des mikroverkapselten Fischöls bestimmen. Dazu wurde zunächst der emulgierende Anteil variiert, um den Zusammenhang zwischen physikalischen Eigenschaften der Emulsion und den Mikrokapseln aufzuklären. Es wurden nosa-stärke, Gummi arabicum, Zuckerrübenpektin sowie Natriumcaseinat und ausgewählte Kombinationen der aufgeführten Trägermatrizes eingesetzt. Die Partikelcharakterisierung erfolgte über die Bestimmung der Partikelgröße, der scheinbaren und der wahren Dichte, der Partikeloberfläche, der Porengröße und volumen, des Gehaltes an extrahierbarem Öl sowie der Partikelmorphologie mittels rasterelektronischer Mikroskopie. Zur Abschätzung des Einflusses der physikochemischen Eigen- 6

19 EINLEITUNG UND ZIELSETZUNG schaften wurde eine Hauptkomponentenanalyse durchgeführt und über PLS- Analyse mit der Lipidoxidation korreliert. Die Lipidoxidation wurde 6 Wochen bei 20 C und 33 % RH verfolgt (Kapitel 3). Neben dem emulgierenden Anteil hat die Kohlenhydratkomponente in der Kapselmatrix einen Einfluss auf die oxidative Stabilität des verkapselten Materials. Die Permeabilität von Sauerstoff durch die glasartige Kapselmatrix ist als einer der bestimmenden Faktoren der Lipidoxidationsgeschwindigkeit anzusehen (Andersen et al., 2000; Orlien et al., 2000). Dabei hat die molekulare Packung der Kohlenhydratmatrix einen deutlichen Einfluss auf das Diffusionsvermögen von Sauerstoff (Townrow et al., 2007). Nach Reineccius (1991) soll Glucosesirup mit einem hohen Dextroseäquivalent (DE) aufgrund verringerter Sauerstoffpermeabilität einen besseren Schutz bewirken als Glucosesirup mit einem niedrigen DE. Beispielsweise erreichten Hogan et al. (2003) unter Verwendung eines Glucosesirups DE 38 die höchste oxidative Stabilität von in Natriumcaseinat mikroverkapselten Fischöl, welches unter Zusatz von Maltodextrinen bzw. Glucosesirup unterschiedlicher DE hergestellt wurde. Weiterhin konnten Desobry et al. (1999) für mikroverkapseltes Carotin zeigen, dass dessen Stabilität in Matrizes mit gleichem DE (DE 25) durch das Molekulargewichtsprofil beeinflusst wurde. Hypothese Basierend auf der o.a. Literatur wurde vermutet, dass die oxidative Stabilität des mikroverkapselten Fischöls über Variation des Molekulargewichtsprofils der Kohlenhydratkomponente und damit über die molekulare Packung der Mikrokapsel beeinflusst werden kann. 7

20 Experimenteller Ansatz EINLEITUNG UND ZIELSETZUNG Um dies zu untersuchen, wurde Fischöl unter Verwendung von nosa-stärke sowie Mischungen aus Maltodextrin, Glucosesirup und einem niedermolekularem Kohlenhydrat (Maltose) mikroverkapselt. Der Verlauf der Lipidoxidation wurde über 8 Wochen Lagerung bei 20 C und 33 % RH beobachtet. Die Charakterisierung der Proben erfolgte über Bestimmung der wahren Dichte und der Partikeloberfläche, um strukturelle Unterschiede festzustellen. Die molekulare Packung der Mikrokapseln und Trägermatrizes wurde über die Bestimmung der freien Volumenelemente 1 mittels Positronenannihilationslebensdauerspektroskopie 2 (PALS) bestimmt (Kapitel 4). Einfluss der Prozessparameter auf physikalische Partikelcharakteristika und die Lipidoxidation von mikroverkapseltem Fischöl Neben der Trägermatrix beeinflussen auch die Prozessbedingungen physikochemische Parameter wie die Mikroverkapselungseffizienz, die Partikelgröße und dichte sowie die Stabilität verkapselter Inhaltsstoffe (Danviriyakul et al., 2002; Fang et al., 2005; Finney et al., 2002; Kelly et al., 2002). Walton (2000) klassifizierte das Trocknungsverhalten verschiedener Stoffe und definierte eine Gruppe filmbildender Substanzen wie Milch, welche stellvertretend für Öl-in-Wasser Emulsionen (o/w Emulsionen ) mit einem Überschuss an hochmolekularen Emulgatoren angesehen werden können. Hecht & King (2000) wiesen nach, dass in Abhängigkeit von der Viskosität und der Oberflächenspannung der zu sprühtrocknenden Lösung der Trocknungsverlauf, die Partikelmorphologie und die Partikeleigenschaften wie Lufteinschluss aufgrund von Vakuolenbildung beeinflusst werden. Die Bildung von dünnwandigen Hohlpartikeln (Ballooning) resultiert aus der Dampfbildung im Inneren des versprühten Tröpfchens durch Interaktion zwischen Trägermatrix (filmbildend, hohe Viskosität) und Prozessbedingungen (hohe Einlasstemperatur ( 200 C). Keogh et al. (2001) zeigten, dass sich Luf teinschlüsse in mikroverkapseltem Fischöl negativ auf dessen Sensorik auswirken. Ähnlich führten 1 Das freie Volumen beschreibt den Platz zur Ausführung thermisch aktivierter Bewegungen von Molekülen und Molekülgruppen. 2 Die Positronenannihilationslebenszeitspektroskopie dient zur Messung der lokalen Elektronendichte, welche direkt der Materiedichte folgt und Rückschlüsse auf das freie Volumen zulässt. Bei der Messung reagieren in die Materie eindringende Positronen mit Elektronen u.a. zu einem dem Wasserstoffatom ähnlichen gebundenen Zustand, dem ortho-postronium. Über die Annihilationszeit des ortho-positroniums, die über Messung emittierter gamma-quanten detektiert wird, kann mittels des Tao-Eldrup-Modells die mittlere Größe der freien Volumenelemente errechnet werden. 8

21 EINLEITUNG UND ZIELSETZUNG Velasco et al. (2006) die Oxidation mikroverkapselten Fischöls auf intrapartikuläre Lufteinschlüsse zurück. In beiden Arbeiten wurde jedoch der Einfluss der Prozessbedingungen auf intrapartikuläre Lufteinschlüsse nur teilweise bzw. gar nicht untersucht. Es sind keine Daten zum Einfluss der Prozessparameter auf die Lipidoxidation von mikroverkapselten Fischöl während der Lagerung vorhanden, hierzu zählen die Sprühtrocknungsparameter und der Sauerstoffgehalt des Trocknungsgases. Hypothese Es wurde unterstellt, dass sich diskrete Lufteinschlüsse ebenso wie Hohlpartikelbildung auch auf die Lipidoxidation von mikroverkapseltem Fischöl während der Lagerung auswirken. Eine unzureichende Entgasung wird dabei als Ursache für diskrete Lufteinschlüsse angesehen. Der über die Trocknungsluft eingebrachte Sauerstoffgehalt ist hingegen nur während der Sprühtrocknung hinsichtlich der Lipidoxidation von Bedeutung. Experimenteller Ansatz Um gezielt Mikrokapseln herzustellen, die sich in stabilitätsrelevanten Partikelcharakteristika unterscheiden, wurden sowohl die Trocknungsbedingungen als auch der emulgierende Trägermatrixbestandteil variiert. Es wurden vier nosa-stärken mit unterschiedlichem Hydrolysegrad und damit unterschiedlichem mittlerem Molekulargewicht eingesetzt. Um die Interaktionen zwischen dem nosa-stärke-typ und den Sprühtrocknungsbedingungen zu untersuchen, wurden die Fischölemulsionen bei zwei verschiedenen Einlass- /Auslasstemperatur Kombinationen (160/70 C und 2 10/90 C), die Matrixlösungen bei vier verschiedenen Einlass-/Auslasstemperatur Kombinationen (160/70 C, 160/90 C, 210/60 C und 210/90 C) spr ühgetrocknet. Die Mikrokapseln wurden anhand der scheinbaren Dichte, der Partikelgröße und oberfläche sowie des Gehaltes an extrahierbarem Öl charakterisiert. Die Lagerung des mikroverkapselten Fischöls erfolgte 8 Wochen bei 20 C und 33 % RH. Die Partikelcharakteristika der Mikrokapseln wurden auf Basis eines faktoriellen Designs statistisch ausgewertet. Um den Einfluss des Sauerstoffgehalts des Trocknungsgases zu untersuchen, wurden Fischölemulsionen mit Stickstoff begast und anschließend mit Luft oder Stickstoff als Trocknungsgas sprühgetrocknet. Die Lagerung des mikroverkapselten Fischöls erfolgte über einen Zeitraum von 5 Wochen bei 20 C und 33 % RH (Kapitel 5). 9

22 EINLEITUNG UND ZIELSETZUNG Sensorische Bewertung von mikroverkapseltem Fischöl dessen Beeinflussung über die Trägermatrix und Die sensorische Beurteilung von lipidhaltigen Systemen ist neben der Analytik von Lipidoxidationsprodukten von besonderer Bedeutung, da die Produktsensorik die Verbraucherakzeptanz entscheidet (Frankel, 2005). Dennoch werden sensorische Untersuchungen nur selten in die Analytik lipidhaltiger Systeme einbezogen (Jacobsen, 1999). Die Freisetzung und damit Wahrnehmung von sekundären Lipidoxidationsprodukten wird durch deren Löslichkeit in der vorliegenden Matrix und deren Flüchtigkeit bestimmt, sodass sich die Freisetzung aus Ölen grundsätzlich von der Freisetzung aus Wasser unterscheiden kann (Jacobsen, 1999). Zusätzlich wird die Freisetzung flüchtiger Verbindungen durch andere Inhaltsstoffe wie Proteine und Hydrokolloide beeinflusst (Guichard, 2002; Roberts et al., 1996; Shen et al., 2007). So können beispielsweise Proteine die Freisetzung von erwünschten Aromen hemmen (Kühn et al., 2006; Saint-Eve et al., 2006; W. Chobpattana, 2002). Weiterhin kann durch Zusatz von Cyclodextrinen, speziell β-cyclodextrin, die Qualität eines Lebensmittels über Maskierung eines unerwünschten Geruchs oder Geschmacks beeinflusst werden (Reineccius et al., 2003; Szejtli & Szente, 2005; Szente & Szejtli, 2004). Hinsichtlich der sensorischen Beurteilung von Fischöl liegen bereits einige Arbeiten vor (Hsieh et al., 1989; Karahadian & Lindsay, 1989a; 1989b; Schnepf et al., 1991). Auch fischölhaltige Emulsionen wurden sensorisch bewertet (Let et al., 2003; Let et al., 2005a; Let et al., 2005b; Nielsen et al., 2004; Venkateshwarlu et al., 2004), wohingegen zu mikroverkapseltem Fischöl und zu mit mikroverkapseltem Fischöl angereicherten Produkten kaum Arbeiten vorhanden sind. Im Hinblick auf mikroverkapseltes Fischöl wurden unabhängig von der Trägermatrix ausschließlich die Geruchsattribute ranzig (Baik et al., 2004; Jonsdottir et al., 2005) sowie metallisch/lackartig (Keogh et al., 2001) beschrieben. Hypothese Da der Einfluss von Lipidoxidationsprodukten auf die Sensorik komplexer Lebensmittelsysteme von deren Zusammensetzung abhängig ist, wurde unterstellt, dass das Geruchsprofil von mikroverkapseltem Fischöl durch die Trägermatrix modifiziert wird. Weiterhin wurde angenommen, dass die Intensität der Geruchsattribute aktiv durch den Zusatz von Aromen bzw. Aromenbindern beeinflusst werden kann. 10

23 Experimenteller Ansatz EINLEITUNG UND ZIELSETZUNG Um dies zu untersuchen, wurden zunächst Prüfer für die sensorische Beurteilung von Fischöl spezifisch ausgebildet, wobei zu Beginn unter Verwendung von unterschiedlich gelagerten Fischölen Geruchsattribute identifiziert wurden, die dessen Qualität charakterisieren. Daran schloss sich die Identifizierung der häufigsten Geruchsattribute von nosa-stärke basierten Mikrokapseln und die Erstellung eines konventionelles Geruchsprofil in Abstimmung mit den Prüfern an. Für die sensorische Prüfung wurde das mikroverkapselte Fischöl mit Wasser aufgeschlämmt, da zum einen davon auszugehen ist, dass ein angereichertes Produktes vor dem Verzehr rekonstituiert wird und zum anderen die Freisetzung von flüchtigen Verbindungen durch den Zusatz von Wasser erhöht wird. Über einen Zeitraum von 4 Wochen bei 20 C wurde wöchentlich die Intensität von Geruchsattributen des mikroverkapselten Fischöls mittels bewertender Prüfung mit Skala (0-5) bewertet. Zur Feststellung von Abweichungen im Geruchsprofil im Vergleich zu der bei 18 C gelagerten Probe wurde zusätzlich ein Dreieckstest durchgeführt. Als Trägermatrizes wurden nosa-stärke und Natriumcaseinat eingesetzt. Darüber hinaus wurde der Einfluss von Aromen und eines aromenbindenden Wirkstoffs (β- Cyclodextrin) auf die Wahrnehmbarkeit und Intensität der Geruchsattribute von nosa-stärke basierten Mikrokapseln während der Lagerung überprüft. Um die Proben hinsichtlich des Lipidoxidationsstatus` zu charakterisieren, erfolgte parallel dazu die Analyse des Hydroperoxidgehaltes und der sekundären Lipidoxidationsprodukte (Kapitel 6). 11

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30 EINLEITUNG UND ZIELSETZUNG Velasco, J., Marmesat, S., Dobarganes, C., & Márquez-Ruiz, G Heterogeneous Aspects of Lipid Oxidation in Dried Microencapsulated Oils. Journal of Agricultural and Food Chemistry, 54(5), Venkateshwarlu, G., Let, M.B., Meyer, A.S., & Jacobsen, C Chemical and Olfactometric Characterization of Volatile Flavor Compounds in a Fish Oil Enriched Milk Emulsion. Journal of Agricultural and Food Chemistry, 52, W. Chobpattana, I.J.J.J.S.S.T.M.L Mechanisms of Interaction Between Vanillin and Milk Proteins in Model Systems. Journal of Food Science, 67(3), Walton, D.E The morphology of spray-dried particles a qualtiative view. Drying Technology, 18(9),

31 2 CHEMICAL STABILISATION OF OILS RICH IN LONG CHAIN POLYUNSATURATED FATTY ACIDS DURING HOMOGENISATION, MICROENCAPSULATION AND STORAGE Y. Serfert, S. Drusch & K. Schwarz Food Chemistry 2009, 113(4), DOI: /j.foodchem Institute of Human Nutrition and Food Science, University of Kiel, Germany 19

32 CHEMICAL STABILISATION 2.1 Abstract Aim of the present study was to systematically investigate the impact of chemical stabilisation on the autoxidation of microencapsulated oils rich in long chain polyunsaturated fatty acids. During the microencapsulation process the fish oil undergoes multiple changes of its physical properties: bulk oil, dispersed oil in aqueous phase, dispersed oil in dry matrix. Autoxidation already occurred in the first stages of the microencapsulation process itself during emulsification and spray-drying. An efficient stabilisation was achieved using a ternary combination of lipophilic antioxidants, synergistic compounds and a trace metal chelator, e.g. a combination of tocopherols, rich in the δ-derivative and low in the α-derivative, with ascorbyl palmitate and lecithin. Trace metal chelation by e.g. Citrem or lecithin in combination with ascorbyl palmitate proved to be of particular importance in the emulsion, but not during storage of the microencapsulated oil. In the microencapsulated oil, the addition of rosemary extract rich in carnosic acid to ternary blends of tocopherols, ascorbyl palmitate and lecithin or Citrem significantly retarded autoxidation. 20

33 CHEMICAL STABILISATION 2.2 Introduction Among the different techniques available for encapsulation of bioactive food ingredients, spray-drying is the most important technique (Desai & Park, 2005). The process involves four stages: preparation of a dispersion or emulsion; homogenisation of the dispersion; atomisation of the in-feed emulsion; and dehydration of the atomised particles (Shahidi & Han, 1993). Encapsulation in an amorphous glassy matrix thereby offers protection against light and oxygen (Ubbink & Krüger, 2006). In the amorphous glassy state mobility of molecules as well as oxygen permeation, a key process in diffusion-controlled reactions like Maillard reaction and lipid oxidation, is reduced, but nevertheless, oxygen permeation through glassy carbohydrate matrices still occurs (Andersen, Risbo, Andersen & Skibsted, 2000; Orlien, Andersen, Sinkko & Skibsted, 2000). Although encapsulation itself prevents lipid oxidation, additional stabilisation with antioxidants is required to ensure maximum protection during processing and subsequent storage of microencapsulated bioactive ingredients. As reviewed by Chaiyasit, Elias, McClements & Decker (2007), research over the past few decades has shown that the physical properties of food systems are extremely important to the chemistry of lipid oxidation, but research mainly focussed on emulsions as e.g. reviewed by Jacobsen, Let, Nielsen & Meyer (2008). It is generally accepted in this context, that the multiple physical environments and different operation steps during microencapsulation and subsequent storage require a balanced antioxidant composition different from the blends utilised for stabilisation of bulk oils. Available studies on the stabilisation of microencapsulated fish oil are summarised in Table 2.1. As can be seen, a systematic approach on the stabilisation of microencapsulated fish oil is missing. A combined analysis of the available studies is not possible due to differences in the storage conditions and the methods utilised for monitoring lipid oxidation. Furthermore the studies do not consider the process steps of emulsification and differ in the type of wall material utilised, which e.g. in the case of caseinate and lecithin wall materials itself exhibit an antioxidative property (Drusch, Groß & Schwarz, 2008; Hamilton, Kalu, McNeill, Padley & Pierce, 1998; Hu, McClements & Decker, 2003). In different studies high levels of α-tocopherol have been added to the product, which are known to promote hydroperoxide formation via accumulation of tocopheroxy-radicals and are therefore not considered to be efficient for the stabilisation of oils rich in polyunsaturated fatty acids (Frankel, Cooney, 21

34 CHEMICAL STABILISATION Moser, Cowan & Evans, 1959; Heimann & Pezold, 1957; Kulås & Ackman, 2001a). 22

35 Table 2.1: Available studies on the stabilisation of microencapsulated oils rich in long-chain polyunsaturated fatty acids Reference Wall material Antioxidants Content Evaluation of antioxidant activity Main conclusion Storage at 0,11, 33, 43 % rh, 30 C; Sodium caseinate, Baik et al. α-tocopherol or 250/1000 mg/kg hydroperoxide content, thiobarbituric glucose syrup (DE 36) (2004) ascorbyl palmitate 250 mg/kg acid active substances (TBARS), sensory evaluation 7 days of storage: 40 mmol/kg lecithin oil) Hogan et al. (2003) Jónsdóttir et al. (2005) Klinkesorn et al. (2005) Shaw et al. (2007) Márquez- Ruiz (2000) Velasco et al. (2000b) Watanabe et al. (2002) Fang et al. (2006) Sodium caseinate, glucose syrup (DE 28) Sodium caseinate, lactose, lecithin Chitosan, lecithin, glucose syrup (DE 36) Sodium caseinate, glucose syrup (DE 24), lecithin Sodium caseinate, lactose Sodium caseinate, lactose Gum arabic or maltodextrin Gum arabic or maltodextrin (DE 2-5) α-tocopherol or Trolox α-tocopherol Mixed tocopherols, EDTA 2 100/500 mg/kg 1000 mg/kg 500 mg/kg 12 µm EDTA 25 µm Ascorbic acid, lecithin δ-tocopherol Ascorbic acid lecithin δ-tocopherol Acyl-L-ascorbate (acyl chain: C8, C10, C12, C16) Ferulic acid, 1-pentylferulate 1-hexylferulate 1-heptylferulate 300 mg/kg 500 mg/kg 200 mg/kg 300 mg/kg 500 mg/kg 300 mg/kg molar ratio to linoleic acid: 0.1 molar ratio to linoleic acid: 1*10-4, 1*10-3, 3*10-3, 1*10-2 Storage: 4 C ; hydroperoxide content, anisidine value Storage: vacuum, 4 C, 20 weeks; Oxipress, TBARS, sensory, HS- SPME-GC-O/GC-MS 1 Storage: 11, 33, 52 % rh, 37 C; hydroperoxide content, TBARS, sensory evaluation Storage: 33 % rh, 37 C; hydroperoxide content, TBARS, propanal content in the reconstituted emulsions Storage: under light, 30 C; TBARS, Polyene-ratio, oxidised and polymerised triglycerides Storage: under light, 30 C, limited oxygen availability, 25 C vacuumsealed or open; oxidised and polymerised triglycerides Storage:12 % rh; 37 C non-oxidised linoleic acid Storage:12 % rh; 37 C non-oxidised linoleic acid Addditon of tocopherols reduced lipid oxidation in the encapsulated and nonencapsulated oil (Hydroperoxide content after Hydroperoxide content was lower in samples containing tocopherols compared to samples containing trolox. 100mg tocopherol/kg were more efficient than 500 mg/kg. Decreased rate of lipid oxidation at all storage conditions Strong inhibition of hydroperoxide and propanal formation Storage stability doubled upon addition of antioxidants Storage stability increased upon addition of antioxidants particularly in the fraction of nonencapsulated oil Highest stability upon addition of caprinoylascorbate Highest stability when using alkyl-ferulates (due to their solubility in oil) 1 HS-SPME-GC-O/GC-MS: Headspace-Solid Phase Microextraction Gas Chromatography-Olfactometry and Gas Chromatography-Mass Spectrometry, 2 EDTA: ethylendiamintetraacetic acid

36 CHEMICAL STABILISATION In a recent publication, efficient combinations of antioxidants for stabilisation of bulk fish oil high in δ-tocopherol and low- α-tocopherol in combination with lecithin or citric acid esters from monoglycerides (Citrem) and ascorbyl palmitate have been reported (Drusch et al., 2008). Aim of the present study was to systemically investigate the impact of different antioxidant combinations on the stability of an oil rich in long-chain polyunsaturated fatty acids during the process steps of microencapsulation (emulsification, spray-drying) and subsequent storage. In addition to stabilisation of the bulk oil, antioxidants must provide efficient protection of the oil in environments with multiple phases like the parent emulsion and the dispersed oil in the amorphous carrier matrix. Based on the partitioning of the individual compounds in the feed emulsion, it was assumed, that the most efficient stabilisation during homogenisation may be achieved by combining lipophilic antioxidants and synergistic compounds with hydrophilic metal chelators. 2.3 Materials and Methods Refined fish oil was provided by Cognis Deutschland GmbH & Co. KG, Illertissen, Germany. The fish oil contained in sum approximately 27 % eicosapentaenoic and docosahexaenoic acid. α- and δ-tocopherol as well as ascorbyl palmitate were purchased from Sigma Chemicals, citric acid from Carl Roth GmbH & Co. KG. National starch and Chemicals GmbH & Co. KG provided n- octenyl-succinate-derivatised starch (HiCap 100). Citric acid esters from monoglycerides (Citrem) were provided by Danisco A/S, lecithin (Epikuron 110) and glucose syrup (C*Dry 01934; DE38) were a kind gift of Cargill Deutschland GmbH. Stabiloton OS Ultra, a rosemary extract with a content of at least 40 % carnosic acid, was provided by Raps GmbH, Kulmbach, Germany Stabilisation of the fish oil Naturally occurring antioxidants, free fatty acids, pigments and mono- or diglycerides in the oil were removed by column chromatography as described by Lampi & Kamal-Eldin (1998). Stock solutions of the different antioxidants were prepared in ethanol and an aliquot was added to the individual samples. Table 2.2 gives an overview on the antioxidant combinations tested in the homogenisation and spray-drying trials. 24

37 CHEMICAL STABILISATION Table 2.2: Antioxidant systems tested for stabilisation of the fish oil during homogenisation and microencapsulation Sample code α-tocopherol (mg/kg oil) δ-tocopherol (mg/kg oil) Ascorbyl palmitate (mg/kg oil) Citrem (mg/kg oil) Lecithin (mg/kg oil) Rosemary extract (mg/kg oil) T1* T2* ALT(1) ALT(2)* ALTR(1) ALTR(2)* ACT(1) ACT(2)* ACTR(1) ACTR(2)* *Combination of antioxidants used in microencapsulation trials Emulsion preparation and storage The basic composition of the emulsions for homogenisation was 18 % fish oil, 4.5 % n-octenylsuccinate-derivatised starch and 22.5 % glucose syrup. Generally, after dissolution of the water soluble compounds a coarse dispersion was prepared using a hand-blender. The emulsion was prepared by subsequent homogenisation at 50 bar in a high pressure homogeniser (Panda 2K, Niro Soavi Deutschland, Lübeck, Germany) followed by a final two-step homogenisation at 250/50 bar with two passes. For monitoring lipid oxidation, samples were stored in duplicate in sealed glass bottles at 20 C in the dark. Headspace to sample ratio in the glass bottles was approximately 2: Microencapsulation by spray-drying For microencapsulation, emulsions were prepared a second time as outlined above. The antioxidant combinations high in α-tocopherol and low in δ- tocopherol were not included in the experiment. Spray drying was carried out at 180/70 C on a laboratory scale spray dryer (1-7 kg/h water evaporative capacity, Mobile Minor, Niro A/S, Denmark) equipped with a rotating disk for atomisation. Selected antioxidant combinations were tested in the spray-drying trials (marked with an asterisk in Tab. 2.2). 25

38 CHEMICAL STABILISATION For monitoring lipid oxidation, samples were stored in duplicate for eight weeks in glass bottles at 20 C over a saturated so lution of magnesium chloride resulting in a relative humidity of 33 % Determination of the oil droplet size Oil droplet size was determined using a laser-diffraction sensor (Helos, Sympatec GmbH, Clausthal-Zellerfeld, Germany) equipped with a cuvette following the instruments standard procedure. For analysis of the oil droplet size of the microcapsules, an aliquot of powder was reconstituted in water. Results are reported as 50th and 90th percentile of the distribution. All analyses were performed with four replicates Determination of the extractable oil content The amount of extractable oil was determined according to the method described by Westergaard (2004). Briefly, 10 g of sample are weighed into a screw-capped flask. 50 ml petrol ether are added and the sample is mixed slowly for 15 min. The dispersion is filtered and 25 ml of the filtrate are evaporated to dryness. The residual oil is weighed and the percentage of extractable oil is calculated Analysis of the hydroperoxide content For extraction of the oil either the emulsion itself or 300 mg microencapsulated oil re-dissolved in 2.5 ml water were used and 7.5 ml of an isooctane/2- propanol mixture (1:1 v/v) was added to the sample. Contents were mixed, centrifuged and the organic phase was removed for lipid isolation (adapted from Hu, McClements & Decker (2004). The organic solvent was evaporated under a stream of nitrogen. Hydroperoxide content was determined using the IDF standard method 74A:1991 for the determination of the peroxide value in anhydrous milk fat (International Dairy Federation, 1991) with slight modifications as reported (Drusch, Serfert, Scampicchio, Schmidt-Hansberg & Schwarz, 2007). All analyses of hydroperoxides were performed with three replicates Determination of propanal by static headspace gas chromatography The major volatile aldehyde resulting from the degradation of omega-3 fatty acids is propanal. In several studies, propanal proved to be a suitable marker for the evaluation of the oxidative status of fish oil (Faraji & Lindsay, 2004; Horiuchi, Umano & Shibamoto, 1998; Satué-Gracia, Frankel, Rangavajhyala 26

39 CHEMICAL STABILISATION & German, 2000; Shahidi, 1998). For the analysis of the propanal content, 1 g of sample was re-dissolved in 2 ml EDTA solution (0.5%). Samples were equilibrated at 70 C for 15 min. An aliquot of the headspace (1 ml) was injected into a Agilent 6890 gas chromatograph equipped with a HP-Innowax column (60m x 0.32 mm x 0.5 µm) and a Agilent 5975 inert mass selective detector. The injector was operated in the split mode (5.3:1). Injector and detector temperature was set at 270 and 250 C, respe ctively. Initially, the oven temperature was set at 50 C and was held for 1.5 m in. Temperature was increased to 240 C at a rate of 20 C min -1, where it was held for 3 min. The mass spectrometer was operated in electron ionisation mode (70 ev), and data were acquired in the full-scan mode for the range m/z The temperature of the ion source and the detector were 150 and 230 C, respectively. Propanal was identified using the retention time of an external standard and by reference to the NIST library (NIST/EPA/NIH Mass Spectral Library Version 2.0d, National Institute of Standards and Technology, Manchester, UK). Quantification of propanal was done after calibration with known amounts of propanal standard Statistical analysis Statistical analysis of the emulsification experiments was performed using two-way ANOVA and one-way ANOVA with post-hoc testing according to Tukey to detect significant differences between the lipid oxidation parameters in the oil, after emulsification and after storage for 2 days using Minitab 14 (Minitab Inc.). The development of hydroperoxide and propanal content in the microencapsulated oil during storage was analysed via a comparison of multiple linear regression using Matlab R13 (Mathworks Inc.). Significant differences are reported based on a probability value of p <

40 2.4 Results and Discussion CHEMICAL STABILISATION Lipid oxidation during homogenisation The process of emulsification led to a significant development of lipid hydroperoxides when using fish oil without added antioxidants (control sample). The hydroperoxide content increased from 4.6 mmol/kg oil to 17.8 mmol/kg oil (Table 2.3). Intense mechanical stress and agitation due to shear and turbulence during the preparation of the coarse emulsion lead to oxygen inclusion. In addition, droplet disruption by cavitation and subsequent rearrangement of oil droplets during homogenisation promotes distribution of oxygen, catalysts and lipid oxidation products among the newly-arranged oil droplets and may thus accelerate the lipid oxidation. Also, droplet disruption and shearing during homogenisation result in large intermediate surface area, which may increase the oxidation rate. Let et al. recently highlighted that in terms of prolonged storage of the emulsion the increase in surface area determines the course of lipid oxidation in oil-in-water emulsions next to the interfacial thickness and its composition (Let, Jacobsen, Pham & Meyer, 2005; Let, Jacobsen, Soerensen & Meyer, 2007). Also more generally, mean hydroperoxide content increased during emulsification and after storage of the emulsions for two days, but only the latter was statistically significant (p < 0.05; Table 2.4). To investigate the role of the chelating agent with respect to its polarity, citric acid was compared to Citrem during emulsion processing in the presence of tocopherol and a mixture of tocopherol and ascorbyl palmitate. During homogenisation the difference between the two chelating agents was low and not significant, but the increase in hydroperoxide formation after two days of storage was markedly lower when Citrem (0.9 mmol/kg oil) was added instead of citric acid (7.4 mmol/kg oil). It is likely that Citrem chelates metal ions more effectively in the interface due to its polarity compared to citric acid. Hence, Citrem was utilized for further processing experiments as chelating agent. 28

41 CHEMICAL STABILISATION Table 2.3: Development of the hydroperoxide and propanal content during the emulsification process and subsequent storage of the emulsions for two days at room temperature in the dark Hydroperoxide content [mmol/kg oil] Propanal content [µmol/kg oil] Sample code Oil Emulsion Emulsion (after 2 days) Oil Emulsion Emulsion (after 2 days) Control T(1) T(2) ALT(1) ALT(2) ALTR(1) ALTR(2) ACT(1) ACT(2) ACTR(1) ACTR(2) Data are mean data from replicate analysis. At a hydroperoxide content above 1 mmol/kg oil, the coefficient of variation between independent samples ranges from 15 to 20 %. Concerning the mean propanal content an increase after both, emulsification and storage for two days, could be observed (Table 2.4). Data from the oneway-anova for the processing step show that only the latter was statistically significant. When removing the control sample from the data set, which causes a high variation in the statistical analysis, more distinct differences become evident and all processing steps significantly differed. 29

42 CHEMICAL STABILISATION Table 2.4: Results of the statistical analysis of the hydroperoxide and propanal content of the homogenisation experiments Code Hydroperoxide content Propanal content Mean Response vs. Processing step Standard deviation Mean Standard deviation Oil 2.44 a a 3.57 Emulsion 3.71 a,b b Emulsion after storage 6.87 b c a,b Values within the same column flowed by different letters are significantly different (p < 0.05) Antioxidant combination in emulsions The effect of antioxidants combination on lipid oxidation (hydroperoxide and propanal formation) during homogenisation and subsequent storage of the emulsion for 2 days is shown in Tab Generally, stabilisation with antioxidants led to a lower increase in the hydroperoxide content during emulsification compared to the control sample. Depending on the combination of tocopherol derivatives, the hydroperoxide content after emulsification amounted to 10.8 mmol/kg oil and 3.1 mmol/kg oil for the oil stabilised with 1000 mg α- tocopherol/100 mg δ-tocopherol (sample T1) and 100 mg α-tocopherol/1000 mg δ-tocopherol (sample T2), respectively. The antioxidant effect of α- tocopherol is well described (2001b; Kulås & Ackman, 2001c) and involves stabilisation of peroxyl radicals by hydrogen donation. As a consequence, hydroperoxide formation again is accelerated, on the other hand levels of secondary lipid oxidation products usually developing during decomposition of lipid hydroperoxides may be reduced. Differences in polarity may also contribute to the different efficacy of the tocopherol derivatives. δ-tocopherol is more polar than α-tocopherol and may therefore accumulate more rapidly and efficiently at the oil-water-interface and suppresses the development of lipid hydroperoxides (Chaiyasit, McClements & Decker, 2005). However, we hypothesise that the polarity effects of δ-tocopherol and α-tocopherol are superimposed by the intense blending during the homogenisation process itself and only gain importance during further storage of emulsions. 30

43 CHEMICAL STABILISATION When using ternary combinations of tocopherols, lecithin or Citrem and ascorbyl palmitate, no increase in the hydroperoxide content during emulsion preparation could be observed. The low hydroperoxide content of oils containing ascorbyl palmitate (Table 3) can be attributed to an accelerated decomposition of hydroperoxides. Mäkinen and Hopia showed that ascorbyl palmitate can reduce cis,trans-hydroperoxides to the corresponding hydroxy compounds (Mäkinen & Hopia, 2000). Thereafter, in the present study, an increase in hydroperoxide content during two days of storage was observed, but only in samples stabilised with combinations including 1000 mg α-tocopherol and 100 mg δ-tocopherol/kg (ALT(1), ALT(2)). Generally, the metal-chelating activity of Citrem and lecithin also contributed to the stabilisation of the oil during emulsification and storage. Shaw, McClements & Decker (2007) also observed a protective effect of trace metal chelation in reconstituted microencapsulated oil and therefore it must be considered to be an important part of the stabilising system with respect to the emulsified aqueous state. Finally, when using a ternary blend of antioxidants in combination with a rosemary extract rich in carnosic acid no increase in hydroperoxide content during emulsification and storage could be detected in samples ALTR(1) and (2), whereas a slight decrease of the hydroperoxide content was observed in ACTR(1) and ACTR(2). Degradation of hydroperoxides led to a concurrent increase in the propanal content. A strong increase in stability when using tocopherol and rosemary extract rich in carnosic acid in bulk fish oil further stabilised by lecithin was also reported by Drusch et al. (2008). The efficiency of carnosic acid in bulk corn oil has been investigated by Hopia, Huang, Schwarz, German & Frankel (1996). The authors explained the synergistic effect between carnosic acid and tocopherols by the reduction of tocopheroxy radicals to tocopherol, i.e. the similar mechanism as described for ascorbyl palmitate. Furthermore, the improved stability of the oil in the presence of carnosic acid is caused by an antioxidative activity of the intermediates in the oxidation cascade of carnosic acid. In contrast to other antioxidants, which loose their antioxidative activity upon oxidation, oxidation products developing from the carnosic acid quinone themselves possess antioxidative activity (Masuda, Inaba, Maekawa, Takeda, Tamura & Yamaguchi, 2002; Richheimer, Bailey, Bernart, Kent, Vininski & Anderson, 1999). In a study by Frankel & Huang (1996) a significant antioxidative activity of a water soluble rosemary extract in bulk fish could be shown, whereas the rosemary extract was less effective in emulsified fish oil. The authors explained their observation by the affinity of the rosemary extract towards the water-oil interface where lipid oxi- 31

44 CHEMICAL STABILISATION dation takes place. As the principal compounds of hydrophilic rosemary extract remain in the water phase it may have a less inhibitory effect on lipid oxidation. However, in the present study a lipid soluble rosemary extract was utilised. Main active compounds carnosic acid and carnosol are considered to partition into the interface of the oil droplet (Hopia et al., 1996) and therefore showed the highest antioxidative activity in emulsified oil further stabilised by tocopherols, ascorbyl palmitate and lecithin or Citrem in the present study. In conclusion, data from the homogenisation experiments clearly showed that the process of emulsion preparation itself may lead to formation of significant amounts of lipid oxidation products. Efficient stabilisation during emulsion preparation can already be achieved using a combination of tocopherols (high in the δ-tocopherol derivative and low in α-tocopherol), lecithin or Citrem and ascorbyl palmitate. To stabilise the emulsion during storage addition of rosemary extract is required. Table 2.5: Hydroperoxide content in fish oil, in-feed emulsions and after microencapsulation of the oil as well as physicochemical characteristics of the microencapsulated fish oil Sample Hydroperoxide content oil droplet size, 50th percentile Oil Emulsion Microcapsules oil droplet size, 90th percentile extractable oil [mmol/kg oil] µm µm % oil T ± ± ± 0.00 T ± ± ± 0.30 ALT(2) ± ± ± 0.18 ALTR(2) ± ± ± 0.07 ACT(2) ± ± ± 0.33 ACTR(2) ± ± ±

45 CHEMICAL STABILISATION Lipid oxidation during microencapsulation All samples showed similar values for the 50 th percentile of the oil droplet size of the microencapsulated oils, which amounted to approximately 1.5 µm (Table 2.5). Oil droplet size of the emulsion partially determines the amount of non-encapsulated oil and thus a difference in the oil droplet size leads to a higher amount of extractable oil. Consequently in the present study, extractable oil was similar for all samples and ranged from 2.6 and 3.8 % of the total oil load. I.e., those differences in oxidative stability can be mainly attributed to antioxidant additives. During the spray-drying process the increase in lipid oxidation products was low in samples, in which an effective chemical stabilisation during the homogenisation experiments could be shown (samples ACT(2), ACTR(2); Table 2.5), whereas a moderate increase in the hydroperoxide content was observed for the samples T1 and T2 after spray-drying. The hydroperoxide content of the feed emulsion samples T1 and T2 increased from 8.0 and 4.0 mmol/kg oil to 16.4 and 8.2 mmol/kg oil after spray-drying, respectively. In samples containing lecithin in combination with 100 mg α-tocopherol/1000 mg δ-tocopherol, ascorbyl palmitate (ALT(2)) and rosemary extract (ALTR(2)), the hydroperoxide content, which was not affected during the homogenisation trials increased after the spray-drying process. The hydroperoxide content amounted to 1.0 and 3.1 mmol/kg oil after emulsification of the samples ALT(2) and ALTR(2), respectively, and increased to 4.4 and 7.4 mmol/kg oil after spray-drying, respectively Storage of microencapsulated fish oil During storage of the microencapsulated oils, a significantly faster increase in hydroperoxide content in the sample T1 stabilised with 1000 mg α-tocopherol and 100 mg δ-tocopherol compared to the sample T2 occurred (Fig. 2.1). The development of the propanal content was similar in both samples until day 42. Thereafter a fast increase in propanal content of T2 occurred. It seems likely that hydroperoxides, which accumulate in the presence of a high α-tocopherol content (Kamal-Eldin & Appelqvist, 1996), decomposed and caused this increase. 33

46 CHEMICAL STABILISATION Samples additionally stabilised with lecithin or Citrem and ascorbyl palmitate (samples ALT(2) and ACT(2)) showed no significant retardation of the hydroperoxide and propanal formation compared to the sample T2. The lower combinatory effects due to the addition of Citrem, lecithin and ascorbyl palmitate in the dried emulsion (microcapsule) can be associated with a lower interaction between antioxidants (lecithin/tocopherol) lower catalysing effect of metal ions as the mobility is strongly decreased with the low water activity. Similarly, Velasco, Dobarganes & Márquez-Ruiz (2000a) observed no synergistic effect for ascorbyl palmitate, lecithin and tocopherols in fish oil encapsulated in a matrix of sodium caseinate and lactose. However, the results were obtained by using a Rancimat operated at 100 C. Li pid oxidation mechanism differs at 100 C significantly from ambient temper ature and furthermore, significant changes in the physical state of the capsule with subsequent release of the encapsulated oil from the matrix and chemical changes in terms of the development of Maillard reaction products may occur (Bandarra, Campos, Batista, Leonor Nunes & Empis, 1999). 34

47 CHEMICAL STABILISATION 180 Hydroperoxides [mmol/kg oil] T1 T2 ALT(2) ALTR(2) ACT(2) ACTR(2) Propanal [µmol/kg oil] T1 T2 ALT(2) ALTR(2) ACT(2) ACTR(2) Storage at 20 C and 33 % rh [days] Figure 2.1: Development of hydroperoxide and propanal content in microencapsulated fish oil stabilised with different combinations of tocopherols, ascorbyl palmitate, lecithin, citrem and rosemary extract The lowest content of both, hydroperoxides and propanal was observed in samples, in which the antioxidant system contained rosemary extract. After 56 days of storage the hydroperoxide and propanal content amounted to approximately 20 mmol/kg oil and a maximum of 47 µmol/kg oil in the sample ALTR(2) and ACTR(2), respectively. The slope of the regression line of the development of the hydroperoxide content in these two samples was significantly lower compared to the slope of the regression lines for all other samples (Table 2.6). Ahn, Kim, Seo, Choi & Kim (2008) showed also a significant reduction in the peroxide and anisidine value of the extractable oil fraction in microencapsulated high oleic sunflower oil stabilised by rosemary extract. 35

48 CHEMICAL STABILISATION However, these results have to be interpreted with caution, since extractable oil, but not the microencapsulated oil was analysed and furthermore, storage at 60 C for 30 days may have led to significant ch anges in the physical structure as discussed above. Finally, in the present study the development of propanal correlated well with the development of other secondary volatile compounds. E.g. correlation with 1-penten-3-ol ranged from r = in ALT(2) to r = in T1. Table 2.6: Slope, intercept and regression coefficients from the linear regression analysis of the course of lipid oxidation in microencapsulated fish oil over a period of eight weeks stored at 20 C and 33 % rh Sample Hydroperoxide content Propanal content Slope Intercept Regression coefficient Slope Intercept Regression coefficient T a a T b a ALT(2) b a ALTR(2) c b ACT(2) b a ACTR(2) c b a,b Values within the same column flowed by different letters are significantly different (p < 0.05) In conclusion, chemical stabilisation of microencapsulated oils rich in long chain polyunsaturated fatty acids can only be achieved taking into consideration the heterogeneous characteristics of the different multiphase systems during microencapsulation, storage and final application. Results from the present study clearly show that autoxidation can already occur in the very first stages of the microencapsulation process itself. Chemical stabilisation of the oil during the process of encapsulation and storage can not be achieved by tocopherols alone. The antioxidant combination should be based on a combination of tocopherols high in the δ-derivative and low in the α-derivative. An efficient stabilisation can be achieved when using a ternary combination of lipophilic antioxidants, synergistic compounds and a trace metal chelator, e.g. a combination of tocopherols rich in the δ-derivative and low in the α- derivative with ascorbyl palmitate and lecithin. Trace metal chelation by hydrophobic agents such as Citrem or lecithin in combination with ascorbyl palmitate is of particular importance in liquid emulsions (i.e. oil dispersed in the aqueous phase) during storage, but not during storage of the dried emulsion (i.e. microencapsulated oil). Addition of rosemary extract rich in 36

49 CHEMICAL STABILISATION carnosic acid to ternary blends significantly retarded autoxidation during storage in the microencapsulated system. Acknowledgments This research project was supported by the FEI (Forschungskreis der Ernährungsindustrie e.v., Bonn), the AiF and the Ministry of Economics and Technology. AiF Project No N. The authors gratefully acknowledge the skilful help of Sonja Berg, Jörg Knipp and Melanie Dörling as well as Matteo Scampicchio from the Università degli Studi di Milan, Italy. References Ahn, J.-H., Kim, Y.-P., Seo, E.-M., Choi, Y.-K., & Kim, H.-S. (2008). Antioxidant effect of natural plant extracts on the microencapsulated high oleic sunflower oil. Journal of Food Engineering, 84, Andersen, A. B., Risbo, J., Andersen, M. L., & Skibsted, L. H. (2000). Oxygen permeation through oil-encapsulating glassy food matrix studied by ESR line broadening using a nitroxyl spin probe. Food Chemistry, 70, Baik, M.-Y., Suhendro, E. L., Nawar, W. W., McClements, D. J., Decker, E. A., & Chinachoti, P. (2004). Effects of antioxidants and humidity on the oxidative stability of microencapsulated fish oil. Journal of the American Oil Chemists Society, 81(4), Bandarra, N. M., Campos, R. M., Batista, I., Leonor Nunes, M., & Empis, J. M. (1999). Antioxidant synergy of a-tocopherol and phospholipids. Journal of the American Oil Chemists Society, 76(8), Chaiyasit, W., Elias, R. Y., McClements, D. J., & Decker, E. (2007). Role of physical structures in bulk oils on lipid oxidation. Critical Reviews in Food Science and Nutrition, 47, Chaiyasit, W., McClements, D. J., & Decker, E. (2005). The relationship between the physicochemical properties of antioxidants and their ability to inhibit lipid oxidation in bulk oil and oil-in-water emulsions. Journal of Agricultural and Food Chemistry, 53, Desai, K. G. H., & Park, H. J. (2005). Recent developments in microencapsulation of food ingredients. Drying Technology, 23, Drusch, S., Groß, N., & Schwarz, K. (2008). Efficient stabilisation of bulk fish oil rich in long chain polyunsaturated fatty acids. European Journal of Lipid Science and Technology, in press. Drusch, S., Serfert, Y., Scampicchio, M., Schmidt-Hansberg, B., & Schwarz, K. (2007). Impact of physicochemical characteristics on the oxidative stability of fish oil microencapsulated by spray-drying. Journal of Agricultural and Food Chemistry, 55, Fang, X., Kikuchi, S., Shima, M., Kadata, M., Tsuno, T., & Adachi, S. (2006). Suppressive effect of alkyl ferulate on the oxidation of microencapsulated linoleic acid. European Journal of Lipid Science and Technology, 108,

50 CHEMICAL STABILISATION Faraji, H., & Lindsay, R. C. (2004). Characterization of the antioxidant activity of sugars and polyhydric alcohols in fish oil emulsions. Journal of Agricultural and Food Chemistry, 52, Frankel, E. N., Cooney, P. M., Moser, H. A., Cowan, J. C., & Evans, C. D. (1959). Effect of Antioxidants and Metal Inactivators in Tocopherol-Free Soybean Oil. Fette, Seifen Anstrichmittel, 61, Frankel, E. N., & Huang, S.-W. (1996). Evaluation of antioxidative activity of rosemary extracts, carnosol and carnosic acidin bulkvegetable oils and fishoil and their emulsions. Journal of the Science of Food and Agriculture, 72, Hamilton, R. J., Kalu, C., McNeill, G. P., Padley, F. B., & Pierce, J. H. (1998). Effects of tocopherols, ascorbyl palmitate, and lecithin on autoxidation of fish oil. Journal of the American Oil Chemists Society, 75(7), Heimann, W., & Pezold, H. v. (1957). Über die prooxygene Wirkung von Antioxygenen. Fette, Seifen Anstrichmittel, 59, 330. Hogan, S. A., O'Riordan, E. D., & O'Sullivan, M. (2003). Microencapsulation and oxidative stability of spray-dried fish oil emulsions. Journal of Microencapsulation, 20(5), Hopia, A. I., Huang, S.-W., Schwarz, K., German, J. B., & Frankel, E. N. (1996). Effect of different lipid systems on antioxidant activity of rosemary constituents carnosol and carnosic acid with and without a-tocopherol. Journal of Agricultural and Food Chemistry, 44, Horiuchi, M., Umano, K., & Shibamoto, T. (1998). Analysis of volatile compounds formed from fish oil heated with cysteine and trimethylamine oxide. Journal of Agricultural and Food Chemistry, 46, Hu, M., McClements, D. J., & Decker, E. (2004). Impact of chelators on the oxidative stability of whey protein isolate-stabilized oil-in-water emulsions containing omega-3 fatty acids. Food Chemistry, 88, Hu, M., McClements, D. J., & Decker, E. A. (2003). Lipid oxidation in corn oil-in-water emulsions stabilized by casein, whey protein isolate, and soy protein isolate. Journal of Agricultural and Food Chemistry, 51, International Dairy Federation (1991). Anhydrous milk fat. Determination of peroxide value. International IDF Standards, Square Vergot 41, Brussels, Belgium, sec. 74A:1991. Jacobsen, C., Let, M. B., Nielsen, N. S., & Meyer, A. S. (2008). Antioxidant strategies for preventing oxidative flavour deterioration of foods enriched with n-3 polyunsaturated lipids: a comparative evaluation. Trends in Food Science and Technology, 19, Jónsdóttir, R., Bragadóttir, M., & Arnason, G. Ö. (2005). Oxidatively derived volatile compounds in microencapsulated fish oil monitored by solid-phase microextraction (SPME). Journal of Food Science, 70(7), C433-C440. Kamal-Eldin, A., & Appelqvist, L.-Å. (1996). The chemistry and antioxidant properties of tocopherols and tocotrienols. Lipids, 31(7), Klinkesorn, U., Sophanodora, P., Chinachoti, P., McClements, D. J., & Decker, E. (2005). Stability of spray-dried tuna oil emulsions encapsulated with twolayered interfacial membranes. Journal of Agricultural and Food Chemistry, 53,

51 CHEMICAL STABILISATION Kulås, E., & Ackman, R. G. (2001a). Different tocopherols and the relationship between two methods for determination of primary oxidation products in fish oil. Journal of Agricultural and Food Chemistry, 49, Kulås, E., & Ackman, R. G. (2001b). Properties of a-, g-, and d-tocopherol in purified fish oil triacylglycerols. Journal of the American Oil Chemists Society, 78(4), Kulås, E., & Ackman, R. G. (2001c). Protection of a-tocopherol in nonpurified and purified fish oil. Journal of the American Oil Chemists Society, 78(2), Lampi, A.-M., & Kamal-Eldin, A. (1998). Effect of a and g-tocopherols on thermal polymerization of purified high oleic sunflower triacylglycerols. Journal of the American Oil Chemists Society, 75(12), Let, M. B., Jacobsen, C., Pham, K. A., & Meyer, A. S. (2005). Protection against oxidation of fish-oil-enriched milk emulsions through addition of rapeseed oil or antioxidants. Journal of Agricultural and Food Chemistry, 53, Let, M. B., Jacobsen, C., Soerensen, A.-D. M., & Meyer, A. S. (2007). Homogenization conditions affect the oxidative stability of fish oil enriched milk emulsions: Lipid oxidation. Journal of Agricultural and Food Chemistry, 55, Mäkinen, M., & Hopia, A. (2000). Effects of a-tocopherol and ascorbyl palmitate on the isomerization and decomposition of methyl linoleate hydroperoxides. Lipids, 35(11), Márquez-Ruiz, G., Velasco, J., & Dobarganes, M. C. (2000). Evaluation of oxidation in dried microencapsulated fish oils by a combination of adsorption and size exclusion chromatography. European Food Research and Technology, 211, Masuda, T., Inaba, Y., Maekawa, T., Takeda, Y., Tamura, H., & Yamaguchi, H. (2002). Recovery mechanism of the antioxidant activity from carnosic acid quinone, an oxidized sage and rosemary antioxidant. Journal of Agricultural and Food Chemistry, 50, Orlien, V., Andersen, A. B., Sinkko, T., & Skibsted, L. H. (2000). Hydroperoxide formation in rapeseed oil encapsulated in a glassy food model as influenced by hydrophilic and lipophilic radicals. Food Chemistry, 68, Richheimer, S., Bailey, D., Bernart, M., Kent, M., Vininski, J., & Anderson, L. (1999). Antioxidative activity and oxidative degradation of phenolic compounds isolated from rosemary. Recent Research Developments in Oil Chemistry, 3, Satué-Gracia, T., Frankel, E. N., Rangavajhyala, N., & German, J. B. (2000). Lactoferrin in infant formulas: Effect on oxidation. Journal of Agricultural and Food Chemistry, 48, Shahidi, F. (1998). Indicators for evaluation of lipid oxidation and off-flavor development in food. In: E. T. Contis, C. T. Ho, C. J. Parliment, F. Shahidi, & A. M. Spanier, Food Flavors: Formation, Analysis, and Packaging Influences: Proceedings of the 9th International Flavor Conference, the George Charalambous Memorial Symposium., vol. 40 (pp ): Elsevier. Shahidi, F., & Han, X. Q. (1993). Encapsulation of food ingredients. Critical Reviews in Food Science and Nutrition, 33,

52 CHEMICAL STABILISATION Shaw, L. A., McClements, D. J., & Decker, E. (2007). Spray-dried multilayered emulsions as a delivery method for w-3 fatty acids into food systems. Journal of Agricultural and Food Chemistry, 55, Ubbink, J., & Krüger, J. (2006). Physical approaches for the delivery of active ingredients in foods. Trends in Food Science and Technology, 17, Velasco, J., Dobarganes, M. C., & Márquez-Ruiz, G. (2000a). Application of the accelerated test Rancimat to evaluate oxidative stability of dried microencapsulated oils. Grasas y Aceites, 51(4), Velasco, J., Dobarganes, M. C., & Márquez-Ruiz, G. (2000b). Oxidation of free and encapsulated oil fractions in dried microencapsulated fish oils. Grasas y Aceites, 51(6), Watanabe, Y., Fang, X., Minemoto, Y., Adachi, S., & Matsuno, R. (2002). Suppressive effect of saturated acyl L-ascorbate on the oxidation of linoleic acid encapsulated with maltodextrin or gum arabic by spray-drying. Journal of Agricultural and Food Chemistry, 50, Westergaard, V. (2004). Milk powder technology. Evaporation and spray-drying. Kopenhagen: Niro A/S. 40

53 3 THE IMPACT OF PHYSICOCHEMICAL CHARACTERISTICS ON THE OXIDATIVE STABILITY OF FISH OIL MICROENCAPSULATED BY SPRAY-DRYING S. Drusch 1, Y. Serfert 1, M. Scampicchio 2, B. Schmidt-Hansberg 3 & K. Schwarz 1 Journal of Agricultural and Food Chemistry 2007, 55(26), DOI: /jf072536a 1 Institute of Human Nutrition and Food Science, University of Kiel, Heinrich-Hecht-Platz 10, Kiel, Germany 2 Dipartimento di Scienze Tecnologie Alimentari e Microbiologiche, Università degli Studi di Milano, Milan, Italy 3 Institute of Thermal Process Engineering, University of Karlsruhe, Kaiserstraße 12, Karlsruhe, Germany 41

54 Summary PHYSICOCHEMICAL CHARACTERISTICS & OXIDATIVE STABILITY The present study aimed at identifying the principal parameters determining the oxidative stability of microencapsulated fish oil. The microcapsules were prepared by spray-drying using different types of n-octenylsuccinatederivatized starch (nosa-starch), gum Arabic, sugar beet pectin, sodium caseinate and/or glucose syrup. By principal component analysis, two principal components to classify the different microcapsules accounting for up to 79 % of the variance were identified. The principal components were determined by physicochemical parameters reflecting the emulsifying ability of the encapsulant, for instance oil droplet size and extractable oil content, and the drying behaviour of the parent emulsion, which is influenced by the viscosity of the feed emulsion. The microcapsules containing solely gum Arabic, sugar beet pectin or nosa-starch with a low degree of hydrolysis were identified by principal component analysis to be significantly different and also exhibited a low stability upon storage. Therefore, the principal components and, thus the underlying physicochemical parameters analysed in the present study, are correlated with core material stability. Keywords: Wall material, matrix, polyunsaturated fatty acids, particle properties, lipid oxidation 42

55 4 DIFFERENCES IN FREE VOLUME ELEMENTS OF THE CARRIER MATRIX AFFECT THE STABILITY OF MICROENCAPSU- LATED LIPOPHILIC FOOD INGREDIENTS S. Drusch 1, K. Rätzke 2, M.Q. Shaikh 2, Y. Serfert 3, H. Steckel 4, M. Scampicchio 1, I. Voigt, K. Schwarz 3 & S. Mannino 1 Journal of Food Biophysics 2009, 4(1), DOI: /s DISTAM, Università degli Studi di Milan, Italy 2 Institute for Materials Science, Chair for Multicomponent Materials, University of Kiel Germany 3 Institute of Human Nutrition and Food Science, University of Kiel, Germany 4 Pharmaceutical Institute, University of Kiel, Germany 5 National Starch and Chemical GmbH, Germany 43

56 4.1 Abstract FREE VOLUME ELEMENTS OF THE CARRIER MATRIX Fish oil was encapsulated by spray-drying into different matrices based on n- octenylsuccinate-derivatised starch (nosa starch) and carbohydrate blends varying in dextrose equivalent and molecular weight profile. Based on the development of the hydroperoxide and propanal content upon storage significant differences in the stability of the microencapsulated oil were observed. With 40 mmol/kg oil the hydroperoxide content after eight weeks of storage at 20 C and 33 % relative humidity was lowest in fish oil encapsulated in a matrix containing nosa starch and maltose. In contrast, the lowest stability was observed in fish oil encapsulated in a matrix based on nosa-starch and maltodextrin with a dextrose equivalent of 18. Physical characteristics like viscosity of the feed emulsion and oil droplet size, which influence drying behaviour as well as particle characteristics like particle size, density or surface area did not differ and thus cannot explain the differences in the rate of autoxidation. Positron annihilation lifetime spectroscopy clearly showed distinct differences in the ortho-positronium lifetime, and thus in the size of free volume elements between the carrier matrices. It is suggested that as a consequence the matrices differed in oxygen diffusivity, which must be considered to be a key determinant in autoxidation of fish oil encapsulated in glassy carbohydrate matrices. 44

57 4.2 Introduction FREE VOLUME ELEMENTS OF THE CARRIER MATRIX Encapsulation of sensitive food ingredients in an amorphous carrier matrix of carbohydrates is the basic principle of microencapsulation technologies like spray-drying, freeze-drying or extrusion. The amorphous carrier matrix is described as a rigid solid with an extremely high viscosity 1 and thus, molecular mobility of the carbohydrate molecules within the matrix is significantly reduced. As reviewed by Le Meste 2 motions are mainly local, not involving the surrounding atoms or molecules and are limited to vibration of atoms and reorientation of small groups of atoms like rotation of lateral groups or to local conformation changes of a main chain. This local movement of chains is the basis for gas transport and separation in glassy polymers 3. As summarised by Orlien et al. 4, in the 1990s a theory of diffusants travelling in cavities in amorphous matrices has been developed. An increase in diffusion coefficients for small molecules has been observed by the same authors with an increase in molecular weight of the carbohydrates forming the glassy matrix 4. With respect to autoxidation of microencapsulated lipophilic food ingredients, oxygen diffusion through the glassy matrix must be considered to be one of the key factors of the reaction rate. Orlien et al. 4 investigated hydroperoxide formation in microencapsulated rapeseed oil prepared by freeze drying. Using electron spin resonance, the authors showed that hydroperoxide formation was enhanced by radicals generated in the oil phase, but not by hydrophilic radicals, which were trapped in the glassy matrix. In a later study, the authors specify this result as they could show that 2 2-azobis(2,4- dimethylpentanenitrile), a rather bulky molecule is immobilized in the glassy matrix, but 4 4-azobis(4-cyanopentanoic acid), a more linear radical initiator, was not completely immobilized 5. The study of Orlien et al. 5 also showed that at temperatures of 25 and 45 C oxygen transport th rough a maltodextrin matrix was the rate-limiting step for hydroperoxide formation. Andersen et al. 6 concluded that the rate of oxidation of microencapsulated oils is governed by the surface to volume ratio of the particle, the permeability coefficient of the matrix material and the thickness of the encapsulating layer. Permeability, diffusivity of guest molecules, glass transition and mechanical flexibility of polymers are influenced by the free volume, which can be characterized by the size and the size distribution of micro- and nanocavities or socalled free volume elements (FVEs). 45

58 FREE VOLUME ELEMENTS OF THE CARRIER MATRIX This quantity, to a first approach is defined as the difference in density between the amorphous structure and the respective ideal crystal reference structure, is hence of major importance for most of the polymeric properties. Common experimental techniques for free volume investigations are PVT (pressure-volume-temperature) measurements, where the equation of state is measured in a closed system 7, or probe methods like Xe-129 spectroscopy and positron annihilation lifetime spectroscopy (PALS) 8. Whereas the first technique only gives information about the total free volume and its temperature dependence, the latter two give information about the average microscopic size of free volume entities. The information is limited to the part of the size distribution of the free volume, which corresponds to holes larger than the probe molecules. With approximately 0.1 nm the diameter of the orthopositronium (o-ps) as a probe is very small and allows the analysis of the free volume in a magnitude similar to the van der Waals radius of oxygen. This is a major advantage compared to other experimental probe methods like inverse gas chromatography and spin probe methods. E.g. in the case of inverse gas chromatography this means holes larger than 0.5 nm for solvent molecules with more than three carbon atoms or 10 nm for nitroxyl-based probes in electron spin resonance 8. Decreasing the molecular weight of the carbohydrate matrix led to an increase in oxidative stability of the encapsulated core material as it was e.g. described by Anandaraman and Reineccius for epoxide formation in orange peel oil encapsulated in matrices of maltodextrin with increasing dextrose equivalent 9. Furthermore, there is experimental evidence available from the literature that the combination of sugars and high molecular weight saccharides improves the oxidative stability of microencapsulated lipophilic food ingredients. Desobry et al. 10 reported a significant increase in the kinetic half-life of β- carotene encapsulated in a carbohydrate based carrier matrix with a dextrose equivalent (DE) of 25 prepared from maltodextrin with DE 4 and sugars compared to a matrix with DE 25 prepared from maltodextrin with DE 15 and sugars or a commercially available glucose syrup with DE 25. Recently, Drusch et al. presented results on the stability of microencapsulated fish oil by spray drying, in which a significant retardation of oxidation was achieved when changing the molecular weight profile of n-octenylsuccinate-derivatized starch 11 or when combining caseinate with different carbohydrate sources as carrier matrix

59 FREE VOLUME ELEMENTS OF THE CARRIER MATRIX Therefore the aim of the present study was to investigate the impact of the molecular weight profile of the carrier matrix on the structural characteristics of a microencapsulated lipophilic food ingredient and its stability. Gas displacement and adsorption techniques using helium and nitrogen were used to characterize the samples in order to detect structural differences. Positron annihilation lifetime spectroscopy, a technique, which is generally accepted to investigate free volume in gas separation membranes 8, 13, 14, was chosen to investigate differences in the free volume elements of the amorphous carrier matrices and microcapsules. Fish oil rich in polyunsaturated fatty acids was used as core material and the development of hydroperoxide content and the formation of propanal as a secondary volatile lipid oxidation product was monitored over time. 4.3 Materials and Methods Fish oil containing 33 % of long-chain polyunsaturated fatty acids was provided by Cognis Deutschland GmbH & Co. KG. Maltose (Sunmalt S, Hayashibara Inc.) was a kind gift of Georg Breuer GmbH, glucose syrup with a dextrose equivalent of 38 (C*Dry 01934) was provided by Cargill Deutschland GmbH and maltodextrin with DE 18 (M3) as well as n-octenylsuccinatederivatised starch (HiCap100) by National Starch and Chemical GmbH Preparation of the microcapsules Microencapsulation of the fish oil was performed by spray-drying. The final composition of all microcapsules was 40 % fish oil, 10 % nosa starch and 50 % of the individual bulk carbohydrate constituent. The latter consisted either of maltodextrin with a dextrose equivalent of 18, commercially available glucose syrup with a dextrose equivalent of 38, blends of maltodextrin with either maltose or glucose with a dextrose equivalent of 38 or maltose (Table 4.1). For preparation of the feed emulsion (45 % dry matter) all water-soluble constituents were dissolved in distilled water. The oil was dispersed into the aqueous solution using a hand blender and the dispersion was subsequently homogenized using a high pressure homogenizer (Panda 2K, Niro Soavi Deutschland, Lübeck, Germany) at 50 bar and at 200/50 bar with two passes. The emulsions were spray-dried in a Mobile Minor spray-drier (Niro A/S, Denmark) at an inlet air temperature of 180 C and an outlet ai r temperature of 70 C. 47

60 FREE VOLUME ELEMENTS OF THE CARRIER MATRIX Table 4.1: Composition of the carbohydrate blends used as bulk carrier matrix Sample acronym Carbohydrate source Estimated DE value MD 18 Maltodextrin (DE18) 18 GLCS38 Glucose syrup (DE38) 38 MD/G38 Maltodextrin (DE18)/ Glucose* 38 MD/M38 Maltodextrin (DE18)/ Maltose** 38 M50 Maltose 50 *17 parts of maltodextrin and 5.5 parts of glucose = 22,5 % carbohydrate blend ** 11.5 parts of maltodextrin and 11 parts of maltose = 22.5 % carbohydrate blend Characterisation of the carbohydrate blends For characterisation of the carrier matrix, the dextrose equivalent of the carbohydrate blend and the molecular weight profile of the final carrier matrix (consisting of the carbohydrate blend and the nosa starch) were analyzed. Determination of the dextrose equivalent was based on the Lane and Eynon method, in which the amount of reducing sugars is determined by titration with copper sulphate in alkaline tartrate 15. The determination of the molecular weight profile was performed by gel permeation chromatography. Samples were heated at 100 C for one hour prior to injecti on with constant stirring at 1mg/ml. The mobile phase consisted of dimethyl sulfoxide (BDH Chemicals Ltd, England) with 30 mm NaNO 3 (Mallinckrodt Beker, Phillipsburg NJ, USA). Flow rate was 1 ml/min, the column compartment temperature was adjusted to 80 C. The columns used were a Phenogel 10u 100A, a Phenogel 10u 10 3 A, a Phenogel 10u 10 5 A (all 300*7.8mm) and a Phenogel 10u guard, 50*7.8mm Physical characterisation of the emulsions and microcapsules Viscosity of the feed emulsion was analysed using a rotary viscosimeter (Haake Viscotester 7L, Thermo Electron Corporation, Dreiech, Germany). A laser diffraction sensor (Helos, Sympatec GmbH, Clausthal-Zellerfeld, Germany) equipped with a cuvette was used to determine the oil droplet size of the feed emulsion, the re-dissolved microcapsules and the particle size distribution of the microcapsules. For the latter analysis the spray-dried particles were dispersed in an inert oil (MCT oil, Gustav Hees Oleochemische Erzeugnisse GmbH). Results are reported as 50th and 90th percentile of the particle size. 48

61 FREE VOLUME ELEMENTS OF THE CARRIER MATRIX Furthermore, physical characterisation of the microcapsules included the determination of the true density of the microcapsules using a Pycnomatic helium pycnometer (Thermo Electron Corporation, Dreiech, Germany) and the surface area according to the BET method. BET analyses were performed using a Gemini 2360 (Micromeritics, Mönchengladbach, Germany). Samples were degassed under vacuum in a Vacprep 061 (Micromeritics, Mönchengladbach, Germany) at a temperature of 25 C Positron annihilation lifetime spectroscopy (PALS) PALS measurements were either performed on the spray-dried carrier matrix without oil or on the spray-dried microcapsules with 40 % fish oil. Samples were equilibrated at 20 C and 33 % relative humidi ty prior to analysis. Once injected from a radioactive source, in most of the polymers, positrons form hydrogen-like positronium (Ps) states. The pick-off lifetime of orthopositronium, (τ o-ps), is well correlated to the free-volume hole size in polymers. The success of PALS in polymer research is largely due to the so-called standard model developed by Tao and Eldrup 16, 17. This simple quantum mechanical model assumes the Ps to be confined to spherical holes with infinitely high walls and gives a direct relationship between τ o-ps and the size of the free volume holes. This can quantitatively be expressed as τ o Ps = 1 R 1 2 R + R0 1 2πR + sin 2 π R + R 0 1 (4.1) where R is the average of holes (free volume element) radius, R 0=R+ R and R=0.166 nm. One should note that this relation includes a linear increase of lifetime with hole diameter in the experimentally relevant range. Since there is a relatively broad distribution of hole sizes in amorphous polymers, the discrete τ o-ps obtained by fitting to the lifetime spectra and, hence, the calculated hole radius from it, has to be regarded as an average value 18. On the other hand, the interpretation of the o-ps intensity, which has often been used as a measure for the hole concentration, is questionable, as the intensity is also affected by the positronium formation probability 19. Therefore, it was not considered in the present paper. 49

62 FREE VOLUME ELEMENTS OF THE CARRIER MATRIX Positron annihilation experiments were performed in a fast-fast coincidence setup with a home made temperature-controllable sample holder under high vacuum conditions as described elsewhere 20. In a Cu-pan the powdery samples were sandwiched around a 1 MBq Na 22 source, with approximately 1mm of material on either side of the radioactive source to ensure complete absorption of the positrons in the sample. The Cu-pan was sealed with a Cu-lid and the whole container was mounted into a sample holder in vacuum (10-4 mbar). Positron annihilation spectra were recorded with ~5*10 6 annihilation events, typically within several hours. Evaluation was performed with the LT9.0 routine program 21, using the common background subtraction and the final resolution function, which was determined as a sum of two Gaussians with full widths at half maximum of approx. 282 ps and 490 ps, with a fixed ratio of 80 % and 20 % respectively. Three life time components τ p-ps<τ e+<τ o-ps, the latter one with dispersion 3, were assumed. In the first series of fit the τ p-ps was kept fixed at 125 ps (lifetime of p-ps in vacuum) and later freed Monitoring of lipid oxidation in the microencapsulated fish oil Lipid oxidation was monitored over a period of eight weeks. Individual samples for each analysis were kept in a desiccator over a saturated solution of magnesium chloride to obtain a relative humidity of 33 % at a temperature of 20 C. Hydroperoxide content was determined followi ng the test procedure of the International Dairy Federation 22 with modifications as described 11. Propanal was analysed as a marker for the development of secondary volatile lipid oxidation products using static headspace gas chromatography as previously described 23. Statistical analysis of the development of hydroperoxide and propanal content in the microencapsulated oil during storage was analysed via a comparison of multiple linear regression using Matlab R13 (Mathworks Inc.). For linear regression analysis only data points from the onset of lipid oxidation were included in the calculation. Significant differences are reported based on a probability value of p <

63 FREE VOLUME ELEMENTS OF THE CARRIER MATRIX 4.4 Results and Discussion The dextrose equivalent of the different bulk carbohydrate constituents ranged from 21 to 57 for the samples MD18 and M50, respectively. The dextrose equivalent for the two blends of maltodextrin with glucose (MD/G38) or maltose (MD/M38) were similar to the dextrose equivalent of the commercial glucose syrup (GLCS38), but showed a different molecular weight profile (Figure 4.1). 10*6 dri *6 dri MD Minutes GLCS Minutes MD/G38 M Minutes Minutes MD/M Minutes Figure 4.1: Molecular weight profile of the different carrier matrices M50 showed an intense peak at the end of the chromatographic run corresponding to the high proportion of maltose. In contrast, MD 18 showed a twin peak of oligosaccharide units and maltose at 26 to 28 min and another broad peak at 22 to 23 min, which was close to the retention time of the pullulan standard with a molecular weight of Da. Also, the three carbohydrate matrices with a dextrose equivalent of 38 showed different molecular weight profiles reflecting the proportions of the individual components of the blend. The course of autoxidation significantly differed between the oil encapsulated in M50, the three carbohydrate blends with a DE of 38 (GLCS38, MD/G38, MD/M38) and MD18 (Table 4.2). 51

64 FREE VOLUME ELEMENTS OF THE CARRIER MATRIX Table 4.2: Regression analysis for the course of lipid oxidation parameters Sample Hydroperoxide content Propanal content Slope Regression coefficient Slope Regression coefficient MD a a GLCS b b MD/G b b MD/M b b M c c Values within the same column followed by different letters are significantly different (p < 0.05) In the maltose containing system M50, the hydroperoxide and the propanal content was lowest throughout the storage period and increased to 41 mmol/kg oil and 9.7 µmol/kg oil, respectively, after eight weeks of storage at 20 C at 33 % relative humidity (Figure 4.2). No si gnificant difference in the course of lipid oxidation was observed for the samples GLCS38, MD/G38 and MD/M38. Finally, the lowest stability against autoxidation was found in the sample MD18. The hydroperoxide and propanal content amounted to 262 mmol/kg oil and 277 µmmol/kg oil, respectively. Drusch and co-workers 11, 24 have shown that the molecular weight profile of the carrier matrix and the viscosity of the feed emulsion can significantly influence the drying behaviour, the resulting physicochemical characteristics of the microcapsules and the course of lipid oxidation. In the present study, the viscosity of the feed emulsion ranged from 14 to 19 mpa s and also emulsification properties as determined by the oil droplet size distribution were similar (Table 4.3). The 50 th percentile of the oil droplet size ranged from 1.16 to 1.30 µm. Based on these data a similar drying behaviour for all feed emulsions and similar particle characteristics on the micro-scale were expected and confirmed later by physical analyses. 52

65 FREE VOLUME ELEMENTS OF THE CARRIER MATRIX Hydroperoxide content [mmol/kg oil] MD18 GLCS38 MD/G38 MD/M38 M Storage at 20 C and 33 % rh [days] Propanal content [µmol/kg oil] MD18 GLCS38 MD/G38 MD/M38 M Storage at 20 C and 33 % rh [days] Figure 4.2: Development of the hydroperoxide and propanal content of fish oil microencapsulated into matrices with different molecular weight profile upon storage at 20 C and 33 % relative humidity 53

66 FREE VOLUME ELEMENTS OF THE CARRIER MATRIX Table 4.3: Physicochemical characterisation of the carbohydrate blends and the feed emulsion Sample Dextrose Equivalent Viscosity Oil droplet size 50. percentile Oil droplet size 90. percentile [mpas] [µm] [µm] MD GLCS MD/G MD/M M The particle size of the microcapsules was not affected by the different bulk carbohydrate constituents (Table 4.4). Furthermore, helium-restricted volume did not differ between the microcapsules. This parameter can be derived from data obtained by gas displacement techniques like the determination of the true density by helium pycnometry and has been used by Moreau and Rosenberg for characterisation of the porosity of whey protein-based microcapsules 25. In the present study, the true density varied from to g/cm 3 for MD18 and M50, respectively. The specific surface area according to Brunauer, Emmet and Teller ranged from 0.18 to 0.22 m 2 /g for GLCS38 and MD/G38, respectively. The content of extractable oil decreased with an increasing amount of low molecular weight compounds (Table 4.4). Drusch and Berg 26 have recently shown that the extractable oil is heterogeneously distributed in microencapsulated fish oil prepared by spray-drying. The authors postulated a similar distribution for the fraction of extractable oil as it was described for encapsulated milk fat 27 consisting of surface oil, surface-near encapsulated oil, oil covering pores within the particle and inner fractions, which are accessible to the solvent. It can be assumed that the solvent permeability of the carrier matrix differed in the samples of the present study and led to differences in the amount of extractable oil. E.g. Hogan et al. 28 reported an increase in microencapsulation efficiency when increasing the DE value of the carbohydrate used for encapsulation of fish oil in caseinate-carbohydrate systems. However, in the present study these differences could not explain the course of lipid oxidation, since the development of hydroperoxide and propanal content significantly differed between MD18 and GLCS38 as well as between MD/G38 and M50 although the samples had almost the same content of extractable oil. 54

67 FREE VOLUME ELEMENTS OF THE CARRIER MATRIX Table 4.4: Physicochemical characterisation of the spray-dried microcapsule Sample Particle size 50th percentile Particle size 90th percentile Oil droplet size 50th percentile Oil droplet size 90th percentile Extractable oil True density Surface area [µm] [µm] [µm] [µm] [% of total oil] [g/cm 3 ] [m 2 /g] MD GLCS MD/G MD/M M The evaluation of free volume of all samples with respect to o-ps lifetime τ 3 (τ o- Ps) and its intensity I 3 (I o-ps) at room temperature (30 C) is summarized in Table 4.5. For the interpretation of the results, it is useful to consider the reliability of the results with respect to known artefacts of positron annihilation. As the o-ps intensity was high and did not vary for different samples, quenching or inhibition effects were not expected to take place. In the present study, intensity and width of the distribution were similar in all samples, hence the difference can be mainly attributed to τ o-ps and therefore to the average hole size. Typical relative errors in τ o-ps taking into account statistics of the fit, reproducibility of the experiments and stability of the system are approximately ± 3 %. When comparing τ o-ps in spray-dried carrier matrices and spray dried microcapsules with fish oil it is obvious that the oil affected the average lifetime. τ o-ps ranges from 1.14 to 1.33 ns in the spraydried carrier particles compared to 2.17 to 2.27 ns in the spray-dried microcapsules. Thus the average lifetime in the latter samples was a mixture of annihilation in the oil and in the carrier matrix. Comparing the average τ o-ps in the spray-dried carrier matrices, a decrease of the lifetime from 1.33 ns in MD18 to 1.14 ns in M50 was evident. 55

68 FREE VOLUME ELEMENTS OF THE CARRIER MATRIX Table 4.5: Average (Τ o-ps) and width (Τ o-ps) of the o-ps lifetime for the spray-dried carrier matrix with and without encapsulated fish oil Sample Spray-dried carrier matrix without fish oil Spray-dried microcapsules (with fish oil) Τ o-ps [ns] Τ o-ps [ns] Τ o-ps [ns] Τ o-ps [ns] MD GLCS MD/G MD/M M According to equation (4.1) the lifetime of 1.25 ns corresponds to an average hole radius of 0.2 nm and an average hole volume of nm 3. As discussed above, the average hole size increases linearly with τ o-ps. The values presented in the present study are similar to values obtained in the literature 29. Although the difference within the measured series of samples was small, it is still significant with respect to lipid oxidation especially when taking into consideration that the diffusivity is exponentially related to the radius of the free volume elements. The differences in free volume elements and their impact on lipid oxidation are supported by the data from the determination of true density of the fish oil-containing microcapsules, which increased with increasing dextrose equivalent. In addition to the difference in the average hole size, oxygen solubility within the matrices also affects diffusivity. This might be a reason, why a difference in the course of lipid oxidation might occur between MD18 and MD/G18, which show a similar τ o-ps. In conclusion, the main result of the present investigation is the increase in τ o- Ps and hence an increase in average free volume with increasing molecular weight of the carbohydrate blend, which partially explains the significant differences in the course of lipid oxidation during storage. Due to the exponential dependence according to the free volume theory even small changes in free volume affect the diffusivity drastically. The data therefore suggest that oxygen diffusivity is one of the key determinants of autoxidation of nutritional oils encapsulated into glassy carbohydrate matrices. 56

69 Acknowledgements FREE VOLUME ELEMENTS OF THE CARRIER MATRIX The present project was funded by the Cariplo foundation. The authors gratefully acknowledge the skilful help of Jörg Knipp and Sonja Berg at the Food Technology Division, University of Kiel. References 1. Y. Roos, M. Karel, Biotechnology Progress 7(1), (1991) 2. M. Le Meste, D. Champion, G. Roudat, D. Blond, D. Simatos, J Food Sci 67(7), (2002) 3. P. H. Pfromm, ed. by Y. Yampolskii, I. Pinnau,B. D. Freeman. Materials Science of Membranes for Gas and Vapour Separation (John Wiley & Sons Ltd., 2006), pp V. Orlien, A. B. Andersen, T. Sinkko, L. H. Skibsted, Food Chem 68(2), (2000) 5. V. Orlien, J. Risbo, H. Rantanen, L. H. Skibsted, Food Chem 94(1), (2006) 6. A. B. Andersen, J. Risbo, M. L. Andersen, L. H. Skibsted, Food Chem 70(4), (2000) 7. G. Dlubek, J. Wawryszczuk, J. Pionteck, T. Goworek, H. Kaspar, K. H. Lochhaas, Macromolecules 38(2), (2005) 8. Y. Yampolskii, V. Shantarovich, ed. by Y. Yampolskii, I. Pinnau,B. D. Freeman. Materials science of membranes for gas and vapor separation. (John Wiley & Sons Ltd., 2006), pp S. Anandaraman, G. A. Reineccius, Food Technol 11), (1986) 10. S. Desobry, F. M. Netto, T. P. Labuza, Journal of Food Processing Preservation 23(1), (1999) 11. S. Drusch, Y. Serfert, M. Scampicchio, B. Schmidt-Hansberg, K. Schwarz, J Agric Food Chem 55(26), (2007) 12. S. Drusch, S. Berg, M. Scampicchio, et al., Food Hydrocolloids 23( (2009) 13. M. Rudel, J. Kruse, K. Rätzke, et al., Macromolecules 41(788 (2008) 14. D. M. Sterescu, D. F. Stamatialis, E. Mendes, et al., Macromolecules 40(5400 (2007) 15. Corn Refiners Association, Dextrose Equivalent pdf, visited: M. Eldrup, D. Lightbody, J. N. Sherwood, Chemical Physics Letters 63( (1981) 17. S. J. Tao, Applied Physics A: Materials Science & Processing 10(1), (1976) 18. C. Nagel, E. Schmidtke, K. Günther-Schade, et al., Macromolecules 33(6), (2000) 19. Y. C. Jean, P. E. Mallon, D. Schrader, Principles and Application of Positron & Positronium Chemistry (World Scientific Publishing Co. Pte. Ltd., 2003) 20. J. Kruse, J. Kanzow, K. Rätzke, et al., Macromolecules 38(23), (2005) 57

70 FREE VOLUME ELEMENTS OF THE CARRIER MATRIX 21. J. Kansy, Nuclear Instruments and Methods in Physics Research Section A: Accelerators, Spectrometers, Detectors and Associated Equipment 374(2), (1996) 22. International Dairy Federation, International IDF Standards Square Vergot 41, Brussels, Belgium, sec. 74A:1991 (1991) 23. Y. Serfert, S. Drusch, K. Schwarz, Food Chem 113( (2009) 24. S. Drusch, K. Schwarz, Eur Food Res Technol 222(1), (2006) 25. D. L. Moreau, M. Rosenberg, J Food Sci 64(3), 405 (1999) 26. S. Drusch, S. Berg, Food Chem 109(1), (2008) 27. T. J. Buma, Netherlands Milk & Dairy Journal 25(2), (1971) 28. S. A. Hogan, B. F. McNamee, E. D. O'Riordan, M. O'Sullivan, Int Dairy J 11(3), (2001) 29. S. Townrow, D. Kilburn, A. Alam, J. Ubbink, Journal of Physical Chemistry B 111(44), (2007) 58

71 5 PROCESS ENGINEERING PARAMETERS AND TYPE OF n-octenylsuccinate-derivatised STARCH AFFECT OXIDATIVE STABILITY OF MICROENCAPSULATED LONG CHAIN POLYUNSATURATED FATTY ACIDS Y. Serfert 1, S. Drusch 1, B. Schmidt-Hansberg 2, M. Kind 2 & K. Schwarz 1 Journal of Food Engineering 2009, 95(3), DOI: /j.foodeng Institute of Human Nutrition and Food Science, University of Kiel, Germany 2 Institute of Thermal Process Engineering, University of Karlsruhe, Germany 59

72 PROCESS ENGINEERING PARAMETERS & TYPE OF nosa-starch 5.1 Abstract Aim of the present study was to investigate the impact of the emulsifying carrier matrix constituent, n-octenylsuccinate-derivatised (OSA)-starch, and process conditions on physical characteristics and oxidative stability of microencapsulated fish oil. Furthermore, the effect of the drying medium, i.e. air or nitrogen, on lipid oxidation during spray-drying and subsequent storage was investigated. Particle characteristics and lipid oxidation of microencapsulated fish oil were both influenced by the type of OSA-starch and the drying conditions. The highest oxidative stability was observed for fish oil microencapsulated in OSA-starch with the lowest average molecular weight and glucose syrup spray-dried at a moderate temperature setting. Particle characteristics of the microcapsules were not attributable for differences in lipid oxidation during storage. In spray-dried carrier matrix particles, the particle size increased with increasing average molecular weight of the OSA-starch and was attributed to an increase in air inclusion. Thus differences in lipid oxidation of the microencapsulated fish oil were attributed to differences in air inclusion as affected by the type of OSA-starch. In terms of spray-drying under inert conditions and in the presence of air, lipid oxidation of microencapsulated fish oil was rather attributed to oxygen availability in the feed emulsion than in the drying gas. Keywords: n-octenylsuccinate-derivatised starch, spray-drying, microencapsulation, fish oil, lipid oxidation, inert spray-drying 60

73 PROCESS ENGINEERING PARAMETERS & TYPE OF nosa-starch 5.2 Introduction Among various methods for microencapsulation of lipophilic food ingredients in amorphous carrier matrices, spray-drying of an oil-in-water emulsion is the most widely utilised technique (Gouin, 2004; Ré, 1998). Different emulsifying constituents are available for their use as encapsulants including e.g. milk proteins (Hogan et al., 2003; Keogh & O'Kennedy, 1999), gum Arabic (McNamee et al., 1998) as well as n-octenylsuccinate-derivatised (OSA)- starch (Drusch & Schwarz, 2006; Drusch et al., 2006a; Drusch et al., 2006b) and their selection affects physicochemical particle characteristics and lipid oxidation during storage. It is well described that particle characteristics, such as microencapsulation efficiency, particle size or density are affected by process engineering parameters and the type of encapsulant as it was reported e.g. for microencapsulated orange oil (Finney et al., 2002), milk fat (Danviriyakul et al., 2002) and linoleic acid (Fang et al., 2005). Walton and Mumford (1999) classified the drying behaviour of different substances and defined one class of film forming substances including milk powder, which also reflects the drying behaviour of oil-in-water emulsions prepared with an excess amount of a high molecular weight emulsifier. One concern when drying these emulsions is particle ballooning with the formation of vacuoles due to incomplete water evaporation and internal steam formation. Hecht and King (2000) showed that particle morphology and particle characteristics, namely air inclusion by formation of voids (vacuoles) are influenced by the viscosity and the surface tension of the spray-drying solution. For microencapsulation of fish oil it has been shown, that lipid oxidation already occurs in the microencapsulation process itself during emulsification and spray-drying (Drusch & Schwarz, 2006; Serfert et al., 2009). With respect to OSA-starch, a strong influence of the degree of hydrolysis and drying conditions on particle morphology and lipid oxidation during processing has been shown. Drusch and Schwarz (2006) observed particle ballooning and higher oxidative deterioration in the microencapsulated fish oil at high spraydrying temperatures in comparison to low spray-drying temperatures when OSA-starch with a low degree of hydrolysis and thus high average molecular weight, was utilised. In the cited study, no data on long term oxidative stability of the fish oil microcapsules were provided, but it is known from emulsions, that the course of lipid oxidation is significantly affected by the initial state of autoxidation after processing (Let et al., 2003; Let et al., 2005). 61

74 PROCESS ENGINEERING PARAMETERS & TYPE OF nosa-starch In another study investigating the influence of different milk proteins on stability of encapsulated fish oil, the importance of air inclusion inside the microcapsules with regard to sensory characteristics during long term storage was highlighted (Keogh et al., 2001), but chemical analysis of lipid oxidation parameters was not performed and the impact of the conditions of the spraydrying process on air inclusion was not investigated. Drusch et al.(2007) derived critical determinants of the oxidative stability of microcapsules on the basis of carrier matrices chosen from a wide range of biopolymers and proteins, but the influence of process conditions was not investigated. Aim of the present study was to investigate the impact of the process conditions and the emulsifying carrier matrix constituent, namely OSA starch, on the physical characteristics of the microcapsule and the oxidative stability of the microencapsulated oil during storage. 5.3 Materials and Methods Four types of OSA starch, which differed in degree of hydrolysis and thus average molecular weight in combination with glucose syrup (C*Dry 01934, Cargill Deutschland GmbH, Krefeld, Germany) were utilised as carrier matrices. The type of OSA starch is reflected in the sample acronym throughout the experiments as depicted in Table 5.1. Fish oil (Omevital 18/12 TG Gold, Cognis Deutschland GmbH & Co. KG, Illertissen, Germany) contained 10.9 and 16.5% docosahexaenoic acid and eicosapentaenoic acid, respectively. Solutions of carrier matrix solutions were prepared at 33% solids and blends of OSA-starch and glucose syrup were prepared in a 1:5 ratio. 62

75 PROCESS ENGINEERING PARAMETERS & TYPE OF nosa-starch Table 5.1: Test factors for the general factorial design for the spray-drying experiments on carrier matrix solutions with OSA starch or OSA starch/glucose syrup blends Factor A: Drying conditions Factor B: Average molecular weight of the of OSA-starch Characterisation Acronym 160/60 C >80 % Maltose OSA1 OSA1/Gl 160/90 C Da OSA2 OSA2/Gl 210/60 C Da OSA3 OSA3/Gl 210/90 C Da OSA4 OSA4/Gl Microencapsulation of fish oil Fish oil was microencapsulated at an oil load of 40% of the dry matter. The acronyms MFO1, MFO2, MFO3 and MFO4 were applied. Increasing number in the acronym indicates increasing average molecular weight of the OSAstarch utilised. Feed emulsions were prepared at 45% total solids by dissolution of the carrier matrices (OSA-starch and glucose syrup in a ratio of 1:5) and subsequent dispersion of the fish oil. The ph of solutions containing OSA2, OSA3 and OSA4 was adjusted to 4.5 prior to emulsification of the fish oil to improve emulsion stability. The emulsion was homogenised in a highpressure homogeniser by a two-step homogenisation at 250/50 bar with two passes (Panda 2K; Niro Soavi Deutschland, Lübeck, Germany). Spray-drying was performed on a laboratory spray dryer (Mobile Minor, Niro A/S, Denmark) with rotating disc for atomisation. Two different spray-drying conditions were tested varying in outlet and inlet temperature (160/70 C and 210/90 C) Microencapsulation of fish oil under inert conditions To investigate both, the influence of oxygen dissolved in the feed emulsion and oxygen present in the drying gas, two subsequent spray-drying trials were performed. Microcapsules prepared from emulsions flushed with N 2 to remove oxygen and spray-dried with N 2 were compared against microcapsules prepared from emulsions with and without N 2-flushing and drying with air. OSA1 and glucose syrup were utilised as carrier matrices. Emulsion preparation was performed as described in subsection 2.1. N 2 flushing (5 min/500 ml emulsion) was carried out in a closed flask equipped with 2 tubes for gas inlet and outlet. Spray-drying was performed by two-fluid nozzle atomisation utilising a laboratory spray-dryer (Construction of the Institute of 63

76 PROCESS ENGINEERING PARAMETERS & TYPE OF nosa-starch Thermal Process Engineering, University of Karlsruhe, Germany) at 180/70 C. The spray dryer was composed of a cylind rical chamber with an 400 mm I.D. and 1320 mm height, followed by a conical chamber. Two-fluid nozzles (Series 970, form 3, spray pattern: circular full-cone) were obtained from Düsen-Schlick GmbH, Untersiemau, Germany. The gas stream was held constantly at 37 m³/h, the outlet temperature was adjusted by the mass flow rate of the carrier matrix solution (between 600 and 2000 g/h). For inert spraydrying, N 2 was utilised and contained 0.14% residual oxygen Spray-drying of carrier matrix solutions Solutions of the carrier matrix compounds were prepared at 33% solids and blends of OSA-starch and glucose syrup were prepared in a 1:5 ratio. Spraydrying of the carrier matrix solutions was performed as described in 2.2. To separately evaluate the influence of inlet and outlet temperature on the particle characteristics, all samples were spray dried at 210 and 160 C inlet temperature and at 90 and 60 C outlet temperature Physicochemical characterisation of spray-dried carrier matrix particles and microencapsulated fish oil Viscosity of the OSA solutions and emulsions was determined utilising a rotational viscometer (Haake Viscotester 7L, Thermo Electron Corporation, Dreieich, Germany). Scanning electron microscopy was carried out with a CamScan 44 REM/EDX (CamScan USA Inc, Cranberry Township PA, USA). Surface tension measurements were performed on a Krüss K12 tensiometer (Krüss, Hamburg, Germany) utilising the platinum plate method (19.9 mm x 0.2 mm) according to Wilhelmy. The equipment was tested by the determination of the surface tension of σ = mn/m of bidistilled water at 25 C. The carrier matrix solutions of OSA-starch as well as the emulsions containing OSA1 to OSA4 were prepared as described above. Surface tension was determined by recording 5 data points in the measuring interval of 5 sec. The sensitivity was set to g for all measurements performed at 25 C. Between different measurements, the beaker wa s purged with bidistilled water and with the new sample. The platinum plate was also purged and subsequently annealed to prevent carry over of emulsifier molecules. The apparent density and particle surface area were determined with the Brunauer-Emmett-Teller (BET)- method utilising a Nova 2200 high-speed gas sorption analyser (Quantachrome GmbH, Odelzhausen, Germany. Nitrogen (at 77K) served as adsorbate, considering a multilayer sorption. The particle 64

77 PROCESS ENGINEERING PARAMETERS & TYPE OF nosa-starch size of the spray-dried carrier matrix particles was analysed by a laserdiffraction sensor (Mastersizer S, Malvern Instruments Ltd, Worcestershire, United Kingdom) in the dry dispersion mode and expressed as volume related 50 th percentile of the particle size (X 50.3,µm). Analyses were performed in triplicate. The size of the oil droplets and the particle size of the spray dried microencapsulated fish oils was determined using a laser diffraction sensor (Helos; Sympatec GmbH, Clausthal-Zellerfeld, Germany) equipped with a cuvette. Oil droplet size of the feed emulsions was determined after dilution of the sample with water; for analysis of the oil droplet size of redissolved emulsions, an aliquot of powder was dissolved in water. Particle size of the microcapsules was determined after dispersing an aliquot of the powder in an inert oil (MCT oil, Gustav Hees Oleochemische Erzeugnisse GmbH, Stuttgart, Germany). Analyses were performed with four replicates. Extractable fat was measured gravimetrically after extraction with petrol ether as described by Westergaard (2004) Analysis of lipid oxidation parameters Microencapsulated fish oil was stored at 20 C and 3 3% relative humidity in the dark for a period of 56 days. Microencapsulated fish oils spray-dried under inert conditions vs. spray-drying with air were stored for 35 days. Lipid oxidation was monitored through the analysis of the hydroperoxide content and propanal, which is one of the major volatile secondary oxidation products of n-3 fatty acids, via static headspace gas chromatography with mass selective detection. The hydroperoxide content (mmol/kg oil) was measured by the ferric thiocyanate hydroperoxide value method with slight modifications after extraction of the oil as described by the International Dairy Federation (1991). For determination of propanal with static headspace gas chromatography, 1 g of powder was weighed in 20 ml crimp-sealed glass vials and was redissolved by adding 2 ml of EDTA solution (0.5%). Samples were equilibrated at 70 C for 15 min. An aliquot of the headspace (1 ml) was injected into an Agilent 6890 gas chromatograph equipped with a VF-1701 ms column (60m x 0.32 mm x 1.0 µm) and an Agilent 5975 inert mass selective detector. The injector was operated in the split mode (5.3:1). Injector and detector temperature was set at 260 and 250 C, resp ectively. The oven temperature program was set as follows: 45 C, 5 C m in -1 to 100 C, 15 C min - 1 to 240 C and finally hold at 240 C for 4 min. The m ass spectrometer was operated in electron ionisation mode (70 ev), and data were acquired in the full-scan mode for the range m/z The temperature of the ion source 65

78 PROCESS ENGINEERING PARAMETERS & TYPE OF nosa-starch and the detector was 150 and 230 C, respectively. P ropanal was identified using the retention time of an external standard and by reference to the NIST library (NIST/EPA/NIH Mass Spectral Library Version 2.0d, National Institute of Standards and Technology, Manchester, UK). Quantification of propanal was done after calibration with known amounts of propanal standard. Analysis of the hydroperoxide content was done in triplicate. Headspace analysis was done in duplicate. Results were expressed as means and standard deviation Statistical analysis Physicochemical characteristics of the spray-dried microencapsulated fish oils as well as the spray-dried carrier matrix particles were analysed on the basis of a general factorial design using Design Expert, Version (Stat Ease Inc, Minneapolis, USA). Statistical analysis of the development of hydroperoxide and propanal content in the microencapsulated oil during storage was analysed via a comparison of multiple linear regression using Matlab R13 (Mathworks Inc.). Significant differences are reported based on a probability value of p < Slopes and regression coefficients are reported. Data on the development of the propanal content were transformed by extraction of the square root before statistical analysis was carried out. 66

79 PROCESS ENGINEERING PARAMETERS & TYPE OF nosa-starch 5.4 Results and Discussion Physicochemical characteristics of microencapsulated fish oil All microcapsules were smooth and spherical with some wrinkles or scars on the surface irrespective of the drying conditions (Figure 5.1). Figure 5.1: Scanning electron micrograph of microencapsulated fish oil (MFO3) spray-dried at 210/90 C Regarding the inner morphology, local vacuole formation was observed. The physicochemical characteristics of the microencapsulated fish oils (MFO) prepared with different types of OSA-starch are summarised in Table

80 Table 5.2: Physicochemical properties of the feed emulsions and microcapsules spray-dried with different types of OSA-starch and glucose syrup at 160/70 C and 210/90 C Drying conditions 160/70 C 210/90 C Carrier matrix OSA starch/glucose syrup OSA starch/glucose syrup Type of OSA starch OSA1 OSA2 OSA3 OSA4 OSA1 OSA2 OSA3 OSA4 oil droplet size, feed emulsion, 50 th percentile [µm] oil droplet size, feed emulsion, 90 th percentile [µm] extractable oil [% of total oil] oil droplet size, re-dissolved emulsion, 50 th percentile [µm] oil droplet size, re-dissolved emulsion, 90 th percentile [µm] particle size, 50 th percentile [µm] particle size, 90 th percentile [µm] apparent density [g/cm³] BET-surface [m 2 /g]

81 PROCESS ENGINEERING PARAMETERS & TYPE OF nosa-starch The particle size (50 th percentile) of the microencapsulated fish oil was significantly influenced by the OSA-starch component (p<0.001) and increased from MFO1 to MFO4. Since the viscosity of the feed emulsion was similar for all samples, the increase in particle size cannot be attributed to the formation of larger droplets during atomisation, but may reflect discrete air inclusion in the particles. The content of extractable oil ranged from 3.6 to 6.0% and from 4.4 to 7.5% in samples spray-dried at 160/70 C and 210/90 C, respectively. The higher contents of extractable oil in samples spray-dried at 210/90 C in comparison to the samples spray-dried a t 160/70 C can be attributed to a higher porosity and the formation of vacuoles at high inlet temperatures as it was reviewed by Vignolles (2007). Drusch and Berg (2008) showed a heterogeneous distribution of the extractable oil throughout the microcapsule with one fraction covering pores within the particle explaining why the differences in porosity are not reflected in the analysis of the BET surface and the apparent density. The significant influence of OSA starch on the microencapsulation efficiency reflects the importance of the physical characteristics of the emulsion and its stability to shear and thermal stress Lipid oxidation as affected by drying conditions and type of OSAstarch The hydroperoxide contents of the oil and the feed emulsions were below the detection limit. The hydroperoxide content after spray-drying was significantly affected by the drying conditions (p<0.0001) and amounted to 0.5 mmol/kg oil and 1.5 mmol/kg at 160/70 C and 210/90 C, respect ively. During storage, the rate of lipid oxidation in the microencapsulated fish oil was both affected by the type of OSA-starch and the spray-drying conditions. At both temperature settings, the course of lipid oxidation in samples MFO1 measured as hydroperoxide content was significantly different from the samples MFO2, MFO3 and MFO4 (p<0.05; Table 5.3) and exhibited the highest oxidative stability. These data on lipid oxidation are consistent with the data obtained by Drusch et al. (2007) who utilised OSA1 and OSA3 and their combination with glucose syrup for microencapsulation of fish oil when comparing different polymers and proteins for microencapsulation. 69

82 PROCESS ENGINEERING PARAMETERS & TYPE OF nosa-starch Table 5.3.: Hydroperoxide and propanal content in microencapsulated fish oil spray-dried at 160/70 and 210/90 C at the end of t he storage period (56 days) and data from regression analyses on the course of lipid oxidation Sample Hydroperoxide content Propanal content [mmol/kg oil] Slope Regression coefficient [µmol/kg oil] Slope Regression coefficient 160/70 C MFO ± a ± a MFO ± b ± a MFO ± b ± a MFO ± b ± a /90 C MFO ± a ± a MFO ± b ± a 0.97 MFO ± b ± a MFO ± b ± a Values within the same column followed by different letters are significantly different (p<0.05) In terms of drying conditions, the samples MFO1 and MFO2 oxidised faster when spray-dried at 210/90 C than at 160/70 C. Howe ver, in samples MFO3 and MFO4 no distinct differences between the drying conditions as measured by the hydroperoxide content could be observed. In all samples, the propanal contents were higher when spray-dried at 210/90 C i n comparison to 160/70 C throughout the whole storage period. With r between 0.92 and 0.97, the development of hydroperoxide content correlated well with the propanal content. Several authors observed a correlation between the content of nonencapsulated core material and oxidative stability of the microencapsulated core material (Baik et al., 2004; Hardas et al., 2002; Ponginebbi et al., 1999). In the present study, differences in oxidation behaviour between the microcapsules prepared could not mandatory be related to the comparably small differences in the amount of extractable oil. This is in agreement with the previous studies on microencapsulated fish oil, in which the amount of extractable oil showed not to be a suitable characteristic to predict the shelf life of microencapsulated oils (Drusch & Berg, 2008). The differences in the oxidation behaviour observed in the present study cannot be attributed to the 70

83 PROCESS ENGINEERING PARAMETERS & TYPE OF nosa-starch physicochemical characteristics of the microcapsules measured in the present study. The oxidative stability of the microencapsulated fish oil may be affected by air inclusion inside the microcapsules which was reported by (Keogh et al., 2001; Velasco et al., 2006). In a previous study, Drusch and Schwarz (2006) showed particle ballooning and suggested that it is disadvantageous in terms of the oxidative stability of microencapsulated fish oil during storage. Based on these results, two experiments were performed in order to (1) further investigate the influence of air occlusion in the microcapsules on lipid oxidation and (2) to elucidate the structural characteristics of spray-dried matrix solutions without the core material Impact of oxygen availability on lipid oxidation of microencapsulated fish oil To investigate both the influence of oxygen dissolved in the feed emulsion and oxygen present in the drying gas, two subsequent spray-drying trials were performed. Microcapsules prepared from emulsions flushed with N 2 to remove oxygen and spray-dried with N 2 as drying gas were compared against microcapsules prepared from emulsions with and without N 2-flushing and drying with air. Spray-drying of emulsions flushed with N 2 and dried under inert conditions enhanced the oxidative stability in comparison to microcapsules prepared from emulsions without N 2-flushing and spray dried with air (Figure 5.2A). 71

84 PROCESS ENGINEERING PARAMETERS & TYPE OF nosa-starch N 2 -dried, flushed Air-dried, not flushed A N 2 -dried, flushed Air-dried, flushed B Figure 5.2: Development of hydroperoxide content in microencapsulated fish oil as affected by N 2-flushing of the emulsions and spray-drying medium during storage at 20 C and 33% rh At the end of the storage period, the hydroperoxide contents amounted to 51 and 67 mmol/kg oil, respectively. However, when both feed emulsions were flushed with N 2 prior to spray-drying under inert conditions and with air, the differences in the development of the hydroperoxide content were diminished (Figure 5.2B). These data suggest that lipid oxidation during spray-drying and subsequent storage is rather determined by the oxygen content in the feed emulsion than by the oxygen in the drying gas itself. 72

85 PROCESS ENGINEERING PARAMETERS & TYPE OF nosa-starch Characterisation of the spray-dried carrier matrix particles To precisely evaluate the interaction between the drying conditions and the type of OSA starch on the properties of the carrier matrix, OSA/glucose syrup blends (OSA/Gl) as they were utilised in the microencapsulation trial were spray-dried. Without the presence of oil, the type of OSA-starch significantly influenced the particle size (p<0.05) and the apparent density (p<0.01) of OSA/Gl particles, the later being also influenced by the drying conditions. The particle size (X 50.3) of the OSA/Gl particles increased with increasing average molecular weight of the OSA-starch component from 12.7 µm (OSA1/Gl) to 18.3 µm (OSA4/Gl) (Figure 5.3) and could not be related to the viscosities of the OSA/Gl solutions, since their viscosities ranged between 13 and 18 mpa s and thus were considered as similar. The apparent density of OSA/Gl particles decreased from OSA1/Gl (1.308 g/cm³) to OSA4/Gl (1.229 g/cm³). An decrease in apparent density is usually associated with air inclusion (Dijkink et al., 2007; Drusch et al., 2007). Thus, the data indicate inclusion of air in the form of discrete air bubbles. With respect to the drying conditions, the apparent density of the particles was lowest when spray-dried at 210/90 C (1.213 g/cm³) and highest in particles spray-dried at 160/60 C (1.308 g/cm³). 73

86 PROCESS ENGINEERING PARAMETERS & TYPE OF nosa-starch Particle size x 50 [µm] True density [g/cm 3 ] OSA4/Glc OSA3/Glc OSA2/Glc OSA1/Glc 160/60 160/90 210/60 210/90 Type of nosa starch Drying temperature [ C] Apparent density [g/cm 3 ] BET surface [m 2 /g] /60 160/90 210/60 210/90 OSA4/Glc OSA3 OSA2 OSA1 Drying temperature [ C] Type of nosa starch Figure 5.3: Model graphs on the influence of type of OSA-starch on the particle size, apparent density and BET-surface of OSA-starch/glucose syrup and OSAstarch particles Data points represent the average outcome of each sample spray-dried at different drying temperatures; the LSD-bars are set to provide 95% confidence No differences in the BET-surface of OSA/Gl particles were observed, i.e. the BET-surface was not affected by the type of OSA-starch. Also no differences in surface morphology between the different types of OSA-starch in OSA/Gl particles could be observed. All OSA/Gl particles showed a structured surface (Figure 5.4A), the proportion of dented, shrivelled particles was high in comparison to spherical smooth particles. 74

87 PROCESS ENGINEERING PARAMETERS & TYPE OF nosa-starch To elucidate differences of the OSA-starch type in more detail, pure OSAstarch solutions (OSA) were also spray-dried at different drying conditions. In pure OSA-starch solutions, the viscosity of the matrix solutions OSA1 to OSA4 strongly increased with increasing average molecular weight from mpa s (OSA1 and OSA2) to 126 mpa s (OSA3) and 4220 mpa s (OSA4) according to the differences in the degree of hydrolysis, whereas the surface tension of the OSA solutions was in a narrow range (33 41 mn/m). Particles of OSA4 could not be recovered from the spray-dryer since they stuck to the spray dryer wall. Figure 5.4: Scanning electron micrographs of a spray-dried OSA/glucose syrup particle (OSA3/Gl) spray-dried at 210/60 C (A) and spray-dried OSA-starch particles spray-dried at 160 C inlet temperature (B, OSA1; C, OSA2; D, OSA3) 75