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Food and Beverage Chemistry/Biochemistry
Interactions of anthocyanins with pectin and pectin fragments in model solutions Lena Rebecca Larsen, Julia Buerschaper, Andreas Schieber, and Fabian Weber J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b03108 • Publication Date (Web): 30 Jul 2019 Downloaded from pubs.acs.org on August 4, 2019
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Journal of Agricultural and Food Chemistry
Interactions of anthocyanins with pectin and pectin fragments in model solutions Lena Rebecca Larsen, Julia Buerschaper, Andreas Schieber, Fabian Weber*
Institute of Nutritional and Food Sciences, Molecular Food Technology, University of Bonn, Endenicher Allee 19b, D-53111 Bonn, Germany
*E-mail:
[email protected]. Phone: +49-228-734462. Fax: +49-228-734429.
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ABSTRACT
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Anthocyanins determine the color and potential health promoting properties of red fruit juices, but
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the juices contain remarkably less anthocyanins than the fruits, which is partly caused by the
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interactions of anthocyanins with the residues of cell wall polysaccharides like pectin. In this study,
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pectin was modified by ultrasound and enzyme treatments to residues of polysaccharides and
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oligosaccharides widely differing in their molecular weight. Modifications decreased viscosity and
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degree of acetylation and methylation and released smooth and hairy region fragments. Native and
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modified pectin induced different effects on the concentrations of individual anthocyanins after short-
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term and long-term incubation caused by both hydrophobic and hydrophilic interactions. Results
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indicate that both pectin and anthocyanin structure influence these interactions. Linear polymers
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generated by ultrasound formed insoluble anthocyanin complexes, whereas oligosaccharides produced
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by enzymes formed soluble complexes with protective properties. The structure of the anthocyanin
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aglycone apparently influenced interactions more than the sugar moiety.
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Keywords: anthocyanins, sugar beet pectin, pectinases, ultrasound treatment, size exclusion
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chromatography
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Journal of Agricultural and Food Chemistry
INTRODUCTION
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Red berries and the derived juices are rich in anthocyanins that are primarily responsible for the
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appealing red to purple color and are also associated with numerous potential health benefits.1,2 Red
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juices produced from these fruits contain significantly less anthocyanins compared to the raw material
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due to several processing steps that entail degradation of these molecules.3 The highest proportion of
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anthocyanins is lost during maceration and pressing. Anthocyanins are either insufficiently extracted or
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complexed by matrix compounds such as cell wall polysaccharides and, thus, a high quantity of
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anthocyanins remains in the press cake.4–6 Despite being very important for anthocyanin yield in the
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eventual juice, these interactions are not well understood so far.
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Juice production requires efficient cell wall degradation, which is commonly achieved by the
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application of specific enzyme preparations containing various pectinases with further side activities.
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The advantages are an increased juice yield, lower juice viscosity, and increased extraction of bound
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anthocyanins.7,8 Ultrasound technology provides another method to degrade plant cell walls by shear
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forces and cavitation.9 While this technique is mainly used for pasteurization, it also shows a high
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potential for gentle extraction of anthocyanins.10
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Plant cell walls consist of numerous polysaccharides and those of red berries contain a notable
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higher proportion of pectin.11 Pectin contains two main structural elements: homogalacturonan (HG,
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approx. 60%) and rhamnogalacturonan I (RG I, approx. 20‑35%). HG is composed of a linear chain of
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1,4‑linked galacturonic acid (GalAc, minimum 72‑100 residues) that can be methylated at C‑6 and
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acetylated at positions O‑2 and O‑3, expressed as the degree of methylation (DM) and acetylation (DA),
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respectively.12 RG I has a backbone of alternating rhamnose and GalAc residues, while 20‑80% of
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GalAc is attached to neutral sugar side chains like galactans, arabinans and arabinogalactans type I and ACS Paragon Plus Environment
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II.13 Rhamnogalacturonan II (RG II) is a minor component of pectin (0.5‑8%) attached to HG. Its
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backbone consists of 8‑10 GalAcs with four complex side chains consisting of 12 different monomers
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including rare sugars like fucose.14
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Pectin polysaccharides have been demonstrated to interact with anthocyanins by weak bonds like
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hydrophobic forces and hydrogen bonds.15 The latter are formed between hydroxy groups of
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anthocyanins and non‑esterified GalAc in the pectin structure. The pH of juices reinforces these
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interactions, due to the pH‑dependent equilibria of anthocyanins forming flavylium cations and the
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negatively charged dissociated carboxylic acid groups of pectin.16,17
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During fruit juice production, several pectin fragments are generated that may interact with extracted
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anthocyanins. Like the enzyme preparations used for juice production, ultrasound treatment degrades
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cell walls and produces several polymers with varying molecular weight (MW), sugar composition, or
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DM and DA. Here, treatment type, dosage or energy input, treatment time, and cell wall structure
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influence the resulting blend of numerous different polysaccharides and oligosaccharides.18,19 Due to the
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modification of pectin polysaccharides by these different treatments, the interactions toward
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anthocyanins will be changed, which has not been investigated so far.
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Previous studies have focused on the interaction of native pectins from various sources and selected
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anthocyanins16,20,21, but generally lack information on the effects of juice processing on these
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interactions. The present work investigates similar interactions observed between modified pectin
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fragments, which are produced in the maceration step, and individual anthocyanins. These effects on a
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broad profile of anthocyanins were studied in a model solution to obtain a better understanding of the
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molecular drivers of anthocyanin-pectin interactions, which can lead to the mentioned anthocyanin
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losses during juice production. Sugar beet pectin resembles berry pectin rather than citrus or apple
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pectin regarding composition and characteristics.5,20 Pectin was modified by two different approaches to
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generate distinct pectin fragments. Ultrasound modified pectin (UMP) and enzyme modified pectin ACS Paragon Plus Environment
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(EMP) were incubated for two hours or two weeks with two anthocyanin mixtures varying in their
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aglycone and glycoside composition.
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MATERIALS AND METHODS
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Chemicals, reagents, and standards. Ultrapure water was obtained from a PURELAB flex 2 water
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purification system (ELGA LabWater, Paris, France). Acetonitrile (HPLC grade), ethanol (99.7%), and
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acetic acid were obtained from VWR (Mannheim, Germany). Ethanol (HPLC grade), methyl tert-butyl
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ether (MTBE, HPLC grade) and citric acid monohydrate were purchased from Carl Roth GmbH & Co.
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KG (Karlsruhe, Germany). Methanol (HPLC grade) and sulphuric acid (95%) were from Th. Geyer
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(Renningen, Germany) and cyanidin‑3‑O‑glucoside (>97.0%) from Phytoplan (Heidelberg, Germany).
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Formic acid (99.9%) was obtained from Sigma‑Aldrich (St. Louis, MO). The compounds
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m‑phenylphenol, n‑propanol, propionic acid, and sodium azide were purchased from Merck (Darmstadt,
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Germany) and sodium hydroxide from Honeywell (Morris Plains, NJ). Potassium sorbate (>99%),
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sodium tetraborate decahydrate, and D‑(+)‑GalAc monohydrate (99%) were obtained from Fluka
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(Munich, Germany), sodium nitrate (99%) from Acros Organics (Geel, Belgium) and trisodium citrate
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dihydrate and n‑butanol (99%) from Alpha Aesar (Ward Hill, MA).
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Enzymes and assay kit. Enzyme preparation Klerzym®150 and Rohapect®MA Plus were kindly
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provided by DSM Food Specialities B.V. (Heerlen, The Netherlands) and AB Enzymes GmbH
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(Darmstadt, Germany), respectively. L‑Fucose assay kit, D-glucuronic acid/D‑GalAc assay kit, and
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L‑rhamnose assay kit were purchased from Megazyme (Wicklow, Ireland).
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Preparation of pectin model solution and modified pectin residues. Sugar beet pectin (SBP)
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Betapec RU 301 was kindly provided by Herbstreith & Fox (Neuenbürg, Germany). SBP was dissolved ACS Paragon Plus Environment
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in a 0.05 M sodium citrate buffer (pH 3.5) at a concentration of 0.75% (w/v) by stirring the suspension
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overnight at 60 °C. SBP was modified by an enzyme or ultrasound treatment to produce the pectin
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residues EMP and UMP, respectively. The two enzyme preparations are commonly applied for berry
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juice production and were used at a dosage of 100 ppm. Incubation was performed in sealed flasks in a
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shaking water bath (80 rpm, 40 °C) for 1 h, 2 h, and 4 h. Enzymes were inactivated at 100 °C for 3 min.
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The experiments were carried out in triplicate. Ultrasound treatments were performed with an
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ultrasound probe processor (UIP 1000hdT, 1000 W, 20 kHz, Hielscher, Teltow, Germany) equipped
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with sonotrode (9.0 cm2) and booster horn (100% amplitude: 53 µm). To generate UMP, the ultrasound
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probe was immersed 2 cm below the liquid level in pectin solution (60 mL). Treatment was run at 60%
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amplitude (pulse duration 2 s) for 40 min or 150 min, cooled on ice to keep the temperature below
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40 °C. Specific energy input did not exceed 4.9 W·s-1·mL-1 and maximum energy density was
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33 W·cm‑2. The experiments were carried out in triplicate.
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Pectin characterization. The total uronic acid content was determined by the m‑hydroxydiphenyl assay.22
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DM and DA were determined as described in the literature with slight modifications.23,24 Methanol
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and acetic acid were quantified by headspace solid‑phase dynamic extraction gas chromatography (HS
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SPDE GC) with flame ionization detection (FID) after hydrolysis with 2 M sodium hydroxide. The
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SPDE equipment (Chromtech, Idstein, Germany) was installed in a CTC‑Combi‑PAL‑Autosampler
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(Bender and Hobein, Zurich, Switzerland) to a GC FID system (Agilent Technologies model 6890). A
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SPDE needle (PDMS/AC/DVB coating, 50 mm × 0.8 mm, 0.53 mm) attached to a 2.5 mL gas‑tight
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syringe (Hamilton, Darmstadt, Germany) pumping 50 cycles (100 µL·s‑1) at a vial temperature of 55 °C
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was used for extraction. Splitless injection was performed at 250 °C onto an OPTIMA®WAXplus
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column (30 m × 0.25 mm, 0.25 µL, Marcherey-Nagel, Düren, Germany) using nitrogen as the carrier
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gas (flow rate 0.7 mL·min-1). For the determination of methanol the oven temperature gradient profile ACS Paragon Plus Environment
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was 45 °C (4 min) to 180 °C (5 min) at 20 °C/min and a final step of 250 °C (4 min), for acetic acid it
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was 45 °C (4 min) to 180 °C (3 min) at 20 °C/min and to 250 °C (6 min) at 30 °C/min, respectively. For
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the release of bound methanol, 1.3 mL sample (0.75% pectin solution), 100 µL n-propanol (0.1%) and
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600 µL 2 M NaOH were filled into a 10 mL GC-Vial, sealed, and kept for 1 h at 40 °C. External
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calibration was done using methanol in a range of 0.01% ‑ 0.04% (w/v) with n-propanol as the internal
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standard (0.01% w/v). For the release of bound acetic acid, 0.75% pectin solution (2.7 mL) was
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combined with 900 µL 2 M NaOH in a 5 mL volumetric flask for 1 h at room temperature. 60 µL
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propionic acid (1%) and 1110 µL sulphuric acid (1 M) were added to ensure a pH
