Development of a membrane chromatographic method for the isolation of anthocyanins from bilberries and other fruits

Es wurde eine HPLC-Methode zur Charakterisierung und Quantifizierung von Heidelbeerpolyphenolen entwickelt. Mit dieser Methode erfolgte die Analytik von frischen und behandelten Heidelbeerproben, Heidelbeerprodukten und verkapselten Heidelbeerinhaltsstoffen sowie eines kommerziell erhältlichen Heidelbeerextraktes. Der Heidelbeerextrakt zeigte im Gegensatz zu den weiteren Heidelbeerprodukten während einer zweijährigen Lagerperiode bei Kühllagerung (-18°C) und bei Raumtemperatur (25°C), unter Feuchtigkeits-, Licht- und Sauerstoff-Ausschluss, keine Veränderung im Anthocyanprofil, Gesamtanthocyangehalt (27,6%), Gesamtpolyphenolgehalt (53,5%, Folin-Ciocalteu) sowie der antioxidativen Aktivität (3540 µmol/g, TEAC). Ein Screening frischer Heidelbeerproben aus Deutschland zeigte keine signifikanten Unterschiede hinsichtlich der Anthocyanprofile und gehalte in unterschiedlichen deutschen Regionen. Durch den Vergleich der Anthocyanprofile von frischen Heidelbeeren mit kommerziellen und im Labormaßstab hergestellten Extrakten konnte die Authentizität der Extrakte sowie eine schonende Herstellungsweise sichergestellt werden. Zur Klärung möglicher synergistischer Effekte der bioaktiven Komponenten des Heidelbeerextraktes wurde dieser in die drei Inhaltsstoffgruppen Anthocyane, Copigmente und Polymere mittels High-Speed-Countercurrent-Chromatographie (HSCCC) aufgetrennt. Zur vollständigen und TFA-rückstandsfreien Fraktionierung des Extraktes wurde eine membranchromatographische Methode entwickelt und erfolgreich eingesetzt. Mittels dieser Methode sowie der eingesetzten Kationenaustauschermembran gelang es, 99,8% der im Extrakt enthaltenen Anthocyane zu isolieren und als hochreine Anthocyanfraktion zu gewinnen. Im Labormaßstab ließ sich die membranchromatographische Methode neben der Heidelbeere auch auf weitere anthocyanhaltige Früchte und Extrakte, wie z.B. Brombeeren, Cranberry, Granatapfel und Hibiskus übertragen, so dass erstmals eine neuartige Methode zur vollständigen Isolierung von Anthocyanen aus anthocyanhaltigen Früchten zur Verfügung stand, die vollständig auf giftige Lösungsmittel und Reagenzien verzichtet. Nach Abtrennung der Anthocyane war es erstmals möglich, hochreine Anthocyan-, Polymer- und Copigmentfraktionen im präparativen Maßstab zu isolieren und diese Fraktionen zu Verkapselungszwecken sowie für biologische Studien und Testungen zur Verfügung zu stellen. In den verschiedenen in vitro-Testsystemen zeigte vor allem die Copigmentfraktion hohe Aktivitäten und positive Effekte auf die untersuchten Biomarker in Caco-2-Zellen (Darmkrebszellen). Aufgrund dieser Aktivitäten wurde auch die Copigmentfraktion detailliert untersucht. Es konnten neben Quercetin und Chlorogensäure (5-CQA) als Hauptkomponenten unter den Copigmenten, durch Anreicherung mittels LSRCCC und weitere Trennungen mittels HSCCC, erstmals 3 neue Verbindungen aus einem Heidelbeerextrakt isoliert und mittels NMR-Spektroskopie strukturell aufgeklärt werden. Ein weiterer wichtiger Punkt war die Erfassung und Isolierung der wertgebenden Komponenten des bei der Saftherstellung anfallenden Heidelbeertresters. Dabei konnte gezeigt werden, dass unter den lipophilen Inhaltsstoffen hauptsächlich Triglyceride sowie einige bioaktive Minorkomponenten, darunter z.B. β-Amyrin, β-Sitosterol oder Oleanolsäure, enthalten sind. Außerdem ließen sich durch Kombination der entwickelten membranchromatographischen Methode mit der methanolischen Extraktion und Abtrennung der lipophilen und semipolaren Komponenten 2,6 g einer hochreinen Anthocyanfraktion aus insgesamt 120 g gefriergetrocknetem Trester gewinnen.A HPLC method for the characterization and quantification of bilberry polyphenols was developed. This method was used for the analysis of fresh and treated bilberry samples, bilberry products, encapsulated bilberry constituents and commercially available bilberry extracts. The bilberry extract showed, in contrast to the other bilberry products, during a two year period of cold storage (-18 ° C) and storage at room temperature (25 ° C), with exclusion of humidity, light and oxygen, no change in the anthocyanin profile, total anthocyanin content (27,6%), total polyphenolic content (53.5%, Folin-Ciocalteu) and the antioxidant activity (3540 µmol/g, TEAC). A screening of fresh bilberry samples showed no significant differences in the anthocyanin profiles and contents in the different regions of Germany. By comparing the anthocyanin profile of fresh bilberries with commercial extracts and self-prepared extracts in laboratory-scale, the authenticity of the extracts as well as a gentle way of production could be guaranteed. To clarify possible synergistic effects under the bioactive components of the bilberry, the extract was separated into three groups of constituents, anthocyanins, copigments and polymers by using high-speed countercurrent chromatography (HSCCC). For complete and TFA-residue-free fractionation of the extract, a membrane chromatographic method was developed and used successfully. By using this method with a cation exchange membrane, it was possible to isolate 99.8% of the contained anthocyanins from the extract and to win as a highly purified anthocyanin fraction. In laboratory scale, the membrane chromatographic method could be transferred from blueberry to other anthocyanin containing fruits and extracts such as blackberry, cranberry, pomegranate and hibiscus. Thus, for the first time, a novel method for total isolation of anthocyanins from anthocyanin containing fruits was available which completely dispensed with toxic solvents and reagents. After separation of anthocyanins, it was possible for the first time to isolate high purity anthocyanin, polymer and copigment fractions on a preparative scale, in order to provide these fractions for encapsulation purposes as well as for biological studies and tests. In various in vitro test systems, especially the copigment fraction showed high activities and positive effects on the studied biomarkers in Caco-2 cells (cancer cells). Due to this activity, the copigment fraction was also investigated in detail. Besides quercetin and chlorogenic acid (5-CQA) as main components amongst the bilberry copigments, for the first time three novel compounds could be isolated from a bilberry extract by enrichment with LSRCCC and additional separations using HSCCC as well as confirmed by NMR spectroscopy. Another important point was the detection and isolation of the value-adding components of the resulting pomace in bilberry juice production. It could be shown that amongst the lipophilic substances primarily triglycerides are contained and some bioactive minor components like β-amyrin, β-sitosterol and oleanolic acid are included. Also 2.6 g of a high purity anthocyanin fraction could be obtained from a total of 120 g of freeze-dried pomace by combining the developed membrane chromatographic method with methanol extraction and partitioning of the lipophilic and semi polar components

1H-NMR-Spektroskopie als Authentizitätsnachweis bei Eiern

Eine Methode zur Überprüfung der Haltungsform und Qualitätsparameter wäre für den Authentizitätsnachweis von Eiern nützlich. Dieses Projekt zeigt, dass die Haltungsformen (Kleingruppen- und Bodenhaltung) von Hühnern durch 1H-NMR-Spektroskopie des Eigelbs analysiert und unterschieden werden können

Analyse des Einflusses einer nachhaltigen und ökologischen Haltung von Legehennen auf die Eiqualität mittels 1H-NMR-Spektroskopie

Die Haltungsform von Legehennen wird in Deutschland anhand eines Stempels auf jedem einzelnen Hühnerei vermerkt. Dieser Stempel soll die Rückverfolgung der Herkunft des Eies zulassen, gewährleistet allerdings nicht immer die tatsächliche Authentizität der Eier. Um einen Authentizitätsnachweis zu ermöglichen, wurde im Rahmen dieses Projekts eine 1H-NMR-Methode entwickelt und validiert, mit der 4.453 authentische Eier von Legehennen bekannten Ursprungs aus vier Haltungsformen (Kleingruppe-, Boden-, Freiland- und ökologischer Haltung) und vier Rassen (Lohmann Selected Leghorn, Dekalb, Lohmann Brown, Sandys) aufgearbeitet und dessen Eigelbextrakte gemessen wurden. Auf Basis dieser 1H-NMR-Spektren wurden mit Hilfe multivariater Datenanalyseverfahren, sog. lineare Diskriminanzanalysen (LDA), berechnet, um die Haltungsform von Legehennen anhand des 1H-NMR-Spektrums des Eigelbextraktes vorhersagen zu können. Das LDA-Modell für die Klassifizierung von Eiern aus konventioneller und ökologischer Haltung zeigt eine Modellgenauigkeit von 99,9 %, während das LDA-Modell zur Klassifizierung von Eiern aus den vier Haltungsformen eine Modellgenauigkeit von 97,1 % aufweist. Zusätzlich konnte ein LDA-Modell für die Vorhersage der Rasse der Legehennen (Lohmann Selected Leghorn + Dekalb, Lohmann Brown, Sandys) mit einer Modellgenauigkeit von 98,4 % berechnet werden. Die entwickelten statistischen Modelle ermöglichen somit die Vorhersage der Haltungsform und der Rasse von Legehennen anhand eines 1H-NMR-Spektrums eines Eigelbextraktes und dadurch die Sicherstellung der Authentizität der Eier

In Silico-Assisted Isolation of <i>trans</i>-Resveratrol and <i>trans</i>-ε-Viniferin from Grapevine Canes and Their Sustainable Extraction Using Natural Deep Eutectic Solvents (NADES)

Grapevine canes are an important source of bioactive compounds, such as stilbenoids. This study aimed to evaluate an in silico method, based on the Conductor-like Screening Model for Real Solvents (COSMO-RS) to isolate stilbenoids from a grapevine cane extract by offline heart-cut high-performance countercurrent chromatography (HPCCC). For the following extraction of resveratrol and ε-viniferin from grapevine canes, natural deep eutectic solvents (NADES) were used as an environmentally friendly alternative to the traditionally used organic solvents. In order to evaluate a variety of combinations of hydrogen bond acceptors (HBAs) and hydrogen bond donors (HBDs) for the targeted extraction of stilbenoids, COSMO-RS was applied. In particular, ultrasonic-assisted extraction using a solvent mixture of choline chloride/1,2-propanediol leads to higher extraction yields of resveratrol and ε-viniferin. COSMO-RS calculations for NADES extraction combined with HPCCC biphasic solvent system calculations are a powerful combination for the sustainable extraction, recovery, and isolation of natural products. This in silico-supported workflow enables the reduction of preliminary experimental tests required for the extraction and isolation of natural compounds

Matrix- and Technology-Dependent Stability and Bioaccessibility of Strawberry Anthocyanins during Storage

Anthocyanins are often associated with health benefits. They readily degrade during processing and storage but are also dependent on the matrix conditions. This study investigated how strawberry anthocyanins are affected by preservation technologies and a relatively protein-rich kale juice addition during storage. A strawberry&ndash;kale mix was compared to a strawberry&ndash;water mix (1:2 wt; pH 4), untreated, thermally, pulsed electric fields (PEF) and high-pressure processing (HPP) treated, and evaluated for anthocyanin stability and bioaccessibility during refrigerated storage. The degradation of strawberry anthocyanins during storage followed first-order kinetics and was dependent on the juice system, preservation technology and anthocyanin structure. Generally, the degradation rate was higher for the strawberry&ndash;kale mix compared to the strawberry&ndash;water mix. The untreated sample showed the highest degradation rate, followed by HPP, PEF and, then thermal. The relative anthocyanin bioaccessibility after gastric digestion was 10% higher for the thermally and PEF treated samples. Anthocyanin bioaccessibility after intestinal digestion was low due to instability at a neutral pH, especially for the strawberry&ndash;kale mix, and after thermal treatment. The storage period did not influence the relative bioaccessibility; yet, the absolute content of bioaccessible anthocyanins was decreased after storage. This research further presents that processing and formulation strongly affect the stability and bioaccessibility of anthocyanins during storage

Semisynthetic Preparation and Isolation of Dimeric Procyanidins B1–B8 from Roasted Hazelnut Skins (Corylus avellana L.) on a Large Scale Using Countercurrent Chromatography

Dimeric procyanidins B1–B8 were produced via semisynthesis from a polymeric proanthocyanidin fraction of hazelnut skins (Corylus avellana L.). This polymeric fraction was found to consist mostly of (+)-catechin and (−)-epicatechin as upper units. Therefore, according to the choice of nucleophile agent, it is possible to semisynthesize dimeric procyanidins B1, B3, B6, and B7 with (+)-catechin and B2, B4, B5, and B8 with (−)-epicatechin. The semisynthetic mixtures were separated on a preparative scale using high-speed countercurrent chromatography (HSCCC) and low-speed rotary countercurrent chromatography (LSRCCC). C4 → C8 linked dimeric procyanidins B1–B4 were isolated in amounts of 350–740 mg. To the best of the authors’ knowledge this is the first study isolating dimeric procyanidins B1–B8 in large amounts with countercurrent chromatography. Moreover, the dimeric prodelphinidins B1, B2, and B3 and their structural elucidation by ¹H NMR spectroscopy without derivatization are described for hazelnuts as natural compounds for the first time

Formation of amyloid fibrils from ovalbumin under Ohmic heating

Ohmic heating (OH) is an alternative sustainable heating technology that has demonstrated its potential to modify protein structures and aggregates. Furthermore, certain protein aggregates, namely amyloid fibrils (AF), are associated with an enhanced protein functionality, such as gelation. This study evaluates how Ohmic heating (OH) influences the formation of AF structures from ovalbumin source under two electric field strength levels, 8.5 to 10.5 and 24.0–31.0 V/cm, respectively. Hence, AF aggregate formation was assessed over holding times ranging from 30 to 1200 sunder various environmental conditions (3.45 and 67.95 mM NaCl, 80, 85 and 90 °C, pH = 7). AF were formed under all conditions. SDS-PAGE revealed that OH had a higher tendency to preserve native ovalbumin molecules. Furthermore, Congo Red and Thioflavin T stainings indicated that OH reduces the amount of AF structures. This finding was supported by FTIR measurements, which showed OH samples to contain lower amounts of beta-sheets. Field flow fractioning revealed smaller-sized aggregates or aggregate clusters occurred after OH treatment. In contrast, prolonged holding time or higher treatment temperatures increased ThT fluorescence, beta-sheet structures and aggregate as well as cluster sizes. Ionic strength was found to dominate the effects of electric field strength under different environmental conditions

Ohmic vs. conventional heating: Influence of moderate electric fields on properties of potato protein isolate gels

The influence of moderate electric fields (MEF) on thermally induced gelation and network structures of patatin enriched potato protein (PPI) was investigated. PPI solutions with 9 wt% protein (pH 7) and 25 mM NaCl were heated from 25 to 65 °C via OH (3–24 V/cm) or conventional heating (COV) at various come-up (240 s and 1200 s) and holding times (30 s and 600 s). Self-standing gels were produced but less proteins denatured when heated via OH. Further, SDS-PAGE and GPC measurements revealed more native patatin remaining after OH treatment. Scanning electron microscopy showed OH gels to have more gap-like structures and frayed areas than COV treated gels which resulted in lower water holding capacity. On molecular scale, less hydrophobic interactions were measured within the protein network and FTIR trials showed the MEF to affect beta-sheet structures. OH gels further showed lower rigidity and higher flexibility, thus, gelling functionality was affected via OH

Matrix- and Technology-Dependent Stability and Bioaccessibility of Strawberry Anthocyanins during Storage

Anthocyanins are often associated with health benefits. They readily degrade during processing and storage but are also dependent on the matrix conditions. This study investigated how strawberry anthocyanins are affected by preservation technologies and a relatively protein-rich kale juice addition during storage. A strawberry–kale mix was compared to a strawberry–water mix (1:2 wt; pH 4), untreated, thermally, pulsed electric fields (PEF) and high-pressure processing (HPP) treated, and evaluated for anthocyanin stability and bioaccessibility during refrigerated storage. The degradation of strawberry anthocyanins during storage followed first-order kinetics and was dependent on the juice system, preservation technology and anthocyanin structure. Generally, the degradation rate was higher for the strawberry–kale mix compared to the strawberry–water mix. The untreated sample showed the highest degradation rate, followed by HPP, PEF and, then thermal. The relative anthocyanin bioaccessibility after gastric digestion was 10% higher for the thermally and PEF treated samples. Anthocyanin bioaccessibility after intestinal digestion was low due to instability at a neutral pH, especially for the strawberry–kale mix, and after thermal treatment. The storage period did not influence the relative bioaccessibility; yet, the absolute content of bioaccessible anthocyanins was decreased after storage. This research further presents that processing and formulation strongly affect the stability and bioaccessibility of anthocyanins during storage.TU Berlin, Open-Access-Mittel – 202