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Antioxidant activity and interactions protein-polyphenol in a pomegranate (Punica granatum L.) yogurt Lorena Trigueros, Aneta Wojdylo, and Esther Sendra J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 18 Jun 2014 Downloaded from http://pubs.acs.org on June 19, 2014
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Journal of Agricultural and Food Chemistry
Antioxidant activity and interactions protein-polyphenol in a pomegranate (Punica granatum L.) yogurt
Lorena Trigueros1, Aneta Wojdyło2, and Esther Sendra*,1
*Corresponding autor, (Tel: 34966749735, Fax: 966749677,
[email protected])
1
IPOA Research Group (UMH-1 and REVIV_Generalitat Valenciana), Departamento de
Tecnología Agroalimentaria, Universidad Miguel Hernández, Ctra. de Beniel km 3.2, 03300, Orihuela (Alicante), Spain 2
Department of Fruit and Vegetable Processing, Wroclaw University of Environmental
and Life Science, 37/41 Chełmońskiego Street, 51-630, Wroclaw, Poland
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ABSTRACT
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Pomegranate juice (PGJ) is rich in phenolics which are potent antioxidants but also
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prone to interact with proteins. A yogurt rich in PGJ (40%) made from arils was
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elaborated (PGY) to determine the antioxidant activity and to estimate the interaction
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phenolics-proteins during 28 days of cold storage. Juice, yogurts and protein free
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permeates were analyzed for phenolic composition. Yogurt fermentation modified the
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anthocyanin profile of the initial PGJ, especially the content in cyanidin-3-O-glucoside.
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During storage, individual anthocyanin content in PGY decreased but it did not modify
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yogurt color. The analysis of permeates revealed that the degree of interaction phenol-
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protein depends on the type of phenolic, being ellagic acid and dephinidin-3,5-O-
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diglucoside the least bound phenolic compounds. The presence of PGJ in yogurt
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enhanced radical scavenging performance, whereas all the observed ferric reducing
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power ability of PGY was strictly due to the PGJ present. The 84.73% of total
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anthocyanins remained bound to proteins at 1st day of storage and 90.06% after 28
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days of cold storage revealing the high affinity of anthocyanins for milk proteins.
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KEY WORDS: pomegranate, fermented milk, phenolic compounds, protein-polyphenol
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interaction
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Journal of Agricultural and Food Chemistry
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INTRODUCTION
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The pomegranate (PG, Punica granatum L.), a fruit extended throughout the
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Mediterranean region, in Southeast Asia, California and Arizona in USA is one of the
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oldest known edible fruit tree species.1 In terms of crop yield, although having a very
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little area (2000ha), Spain ranks first (18.5 t/ha) followed by the USA (18.3 t/ha). Due
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to its immense potential for health benefits, PG has achieved the title of “super-food”.2
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The health effects of whole pomegranate, its juices and extracts, have been studied in
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relation to a variety of chronic diseases.3,4 The in vitro antioxidant activity has been
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attributed to its high polyphenolic content, specifically punicalagins, punicalins, gallagic
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acid, and ellagic acid.5
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The edible part of the PG fruit (50%) consists of 40% arils (which are used to
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obtain juice) and 10% seeds. PG juice polyphenols are: hydrolysable tannins (mainly
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ellagitannins), anthocyanins, non-colored flavonoids and phenolic acids.6 In arils juice,
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anthocyanins are the major group of phenolics and are responsible for its red color.
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PGs are eaten fresh, and also used to obtain juice (PGJ), grenadine syrup (a
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reduced juice from fresh PG seeds), “anardana” (dried PG raisins) and extracts. Also,
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PGJ and seeds are used to prepare toppings, sauces and dips for many types of food.2
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PGJ concentrate is currently used in the production of commercial PG Greek style
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yogurts, especially in the USA and UK. The attractive red color provided by PG depends
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on pigment concentration and it is one of the most important quality factors in fruit
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yogurts.
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The binding of polyphenols to milk proteins has been suggested to reduce their
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bioavailability and functionality and, thus, to reduce their antioxidant potential.7,8
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There are two main groups of proteins in milk, usually defined depending on their 3 ACS Paragon Plus Environment
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solubility at pH 4.6 at 25 oC: caseins, constituting about 80% of the total proteins in
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milk, and whey proteins.9 Due to its relatively high charge, caseins show a tendency to
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associate with other proteins according to the hydrophobic character of the micelle.8
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Furthermore, caseins are proline-rich proteins which in turns have a strong affinity for
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the hydroxyl (-OH) group of phenolic compounds.8 We previously developed a new
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dairy product formulated with PGJ rich in phenolic compounds10 but the degree of
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interaction protein-phenols and its effect on the antioxidant properties were still
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unsolved. Juices solely made from arils are typically characterized by low phenolic
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contents with the predominance of anthocyanins, gallotannins, hydroxybenzoic acids,
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hydroxycinnamic acids and di-hydroflavonols.11 Anthocyanins have been associated
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with the prevention of cardiovascular disease, obesity, and diabetes.12 Anthocyanins
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are the major group of phenolic compounds in pomegranate juice, so their stability as
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well as their interaction with milk proteins during yogurt shelf-life is of great interest.
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The specific functionality of phenolic compounds in dairy products is based on their
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ability to interact with milk proteins. Furthermore, the protein-polyphenol interaction
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is maximal at the isoelectric point of the protein,13 i.e. when yogurt is produced.
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The aim of this work was to determine the antioxidant activity of a yogurt rich
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in PGJ made from arils during 28 days of refrigerated storage as well as to estimate the
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interaction between the phenolic compounds presents in the PGJ and milk proteins
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and its impact on the antioxidant activity of the yogurt.
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MATERIALS AND METHODS
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Materials and chemicals
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Commercial starter cultures of Streptococcus thermophilus and Lactobacillus
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delbrueckii subsp. bulgaricus (Ezal© MY900, Rhodia Food-Danisco A/S, Sassenage, 4 ACS Paragon Plus Environment
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Journal of Agricultural and Food Chemistry
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France) were used at the concentrations recommended by the suppliers. For all the
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study the same batch of skim milk powder was used (34% protein, 52% lactose, 1% fat,
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6.8% ash, 5.2% moisture) (Central Lechera Asturiana, CAPSA, Granada-Siero, Spain).
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2,2-diphenyl-1-picrylhydrazyl (DPPH•), Folin-Ciocalteu reagent, gallic acid, iron
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(III) chloride, trichloroacetic acid (TCA), aluminium chloride, Trolox and formic acid
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were from Sigma Chemical Company (Germany). Methanol of HPLC ultra-gradient
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grade, HCl, potassium chloride, sodium acetate, dibasic potassium phosphate, sodium
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nitrite (II), sodium hydroxide, sodium carbonate and dibasic sodium phosphate were
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from Merck (Darmstadt, Germany). Potassium hexacyanoferrate was from Fluka
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BioChemika (Germany). Ellagic acid was from Tocris Bioscience (Ellisville, MO, USA) and
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pure punicalagins were from Chengdu Biopurity Phytochemicals Ltd. (Sischuan, China).
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Cyanidin-3,5-O-diglucoside and -glucoside, pelargonidin-3-O-glucoside and delphinidin-
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3-O-glucoside, were from Extrasynthese (Genay, France). Acetonitrile for HPLC-DAD
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and LC/MS (Gradient grade) was from Merck (Darmstadt, Germany). LC grade water,
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prepared by using an HLP SMART 1000s system (Hydrolab, Gdańsk, Poland), was
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additionally filtered through a 0.22 μm membrane filter immediately before use.
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Plant material
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Mature PG fruit cultivar ‘Mollar de Elche’, with no visible external cuts or
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spoilage was purchased from a local market. The PG fruits were peeled manually and
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the arils were introduced in a blender Ju2000 Vitae (Moulinex, Barcelona, Spain) to
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obtain PGJ. PGJ was filtered through cotton gauze to remove particles and was kept
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frozen until use.
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Yogurt manufacture
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Set-type yogurt was produced following the manufacture method developed by
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Trigueros et al.,10. Briefly, skim milk powder (SMP) was reconstituted with deionised
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water at 15 % w/v total solids to serve as control. SMP was reconstituted at 25% w/v
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total solids to be further completed with PGJ to have a final solid content of 15% w/v
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after pasteurization. PGJ, 15% reconstituted skim milk (RSM) and 25% RSM were
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separately pasteurized at 80 oC for 30 min. After cooling to 43 oC, PGJ was added to the
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RSM to a final concentration of 40 % v/v. At this point the starter culture was added
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and the inoculated mix was incubated at 43 oC for 4h, and then cooled and stored at 4
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o
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Colorimetric analysis
C for 28 days.
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The CIE LAB color space of yogurts was studied, the following color coordinates
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were determined: lightness (L*), redness (a*, +/- red-green), and yellowness (b*, +/-
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yellow-blue). Color determinations were made at 12 ± 2 oC by means of a Minolta CM-
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2002 (Minolta Camera Co., Osaka, Japan) spectrophotometer, with a liquid accessory
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CR-A70 (Minolta Camera Co., Osaka, Japan), with illuminant D65 and an observer of
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10o.
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Preparation of the free-protein phase (permeate)
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In order to quantitatively estimate the interaction of phenolic compounds with
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proteins, a protein free phase (permeate) was obtained with a laboratory scale
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ultrafiltration (UF) cartridge (Amicon® Ultra-15; Merck Millipore Ltd., Ireland), with a
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nominal cut-off of 10,000 Da and a nominal area of 7.6 cm2 was used. To remove
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caseins 30 g of yogurt were centrifuged at 7,000 rpm for 30 min, at to 4 oC. The
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supernatant was ultrafiltrated through the cartridge at 5,500 rpm for 30 min, at 4 oC,
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and permeate was collected for further analysis. 6 ACS Paragon Plus Environment
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Journal of Agricultural and Food Chemistry
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Extraction of phenolics from PJ and yogurt permeate
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PGJ and permeates (10 mL) were extracted with 30 mL of acidified methanol
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(methanol containing 100 µL conc. HCl) for 30s at 12,000 rpm (IKA® T25 digital ULTRA-
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TURRAX®, IKA® Werke Staufen, Germany). Then, the mixtures were centrifuged at
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5,000 rpm for 10 min at 4oC. 5 mL of the extract was separated for determining
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anthocyanin content. The remaining portions were evaporated to dryness using a
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rotary evaporator R-205 (Büchi, Flawil, Switzerland) under reduced pressure (
