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Phytochemical Composition and Effects of Commercial Enzymes on the Hydrolysis of Gallic Acid Glycosides in Mango (Mangifera indica cv. Keitt) Pulp Kimberley A. Krenek, Ryan Crispen Barnes, and Stephen T. Talcott J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf5031554 • Publication Date (Web): 04 Sep 2014 Downloaded from http://pubs.acs.org on September 9, 2014
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Journal of Agricultural and Food Chemistry
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Phytochemical Composition and Effects of Commercial Enzymes on the Hydrolysis of
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Gallic Acid Glycosides in Mango (Mangifera indica L. cv. Keitt) Pulp
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Kimberley A. Krenek, Ryan C. Barnes, and Stephen T. Talcott*
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Department of Nutrition and Food Science, Texas A&M University, College Station, Texas
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77843-2254
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Running Title Header: Characterization of Phytochemicals in Keitt Mango Pulp
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* Author to whom correspondence should be addressed (phone (979) 862-4056; fax (979) 458-
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3704; e-mail:
[email protected])
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ABSTRACT
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A detailed characterization of mango pulp polyphenols and other minor phytochemicals was
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accomplished for the first time in the cultivar ‘Keitt’, whereby the identification and semi-
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quantification of five hydroxybenzoic acids, four cinnamic acids, two flavonoids, and six
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apocarotenoids was accomplished. Among the most abundant compounds were two mono-
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galloyl glucosides (MGG) identified as having an ester or ether-linked glucose, with the ester
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linked moiety present in the highest concentration among non-tannin polyphenolics.
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Additionally, the impact of side activities of three commercial cell-wall degrading enzymes
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during Keitt mango pulp processing was evaluated to determine their role on the hydrolysis of
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ester and ether-linked phenolic acids. The use of Crystalzyme 200XL reduced the concentration
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of ester-linked MGG by 66% and the use of Rapidase AR 2000 and Validase TRL completely
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hydrolyzed ether-linked MGG after 4 hrs of treatment at 50°C. Fruit quality, in-vivo absorption
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rate, and bioactivity of mango phytochemicals rely on their chemical characterization and
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characterizing changes in composition is critical for a complete understanding of in-vivo
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mechanisms.
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Keywords: Mango pulp polyphenolics; gallic acid glycosides; enzymatic phytochemical
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hydrolysis
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INTRODUCTION
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Mangoes are enjoyed world-wide for their exotic flavor and delicious taste. Over 1000 mango
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varieties have been identified and commercial production is reported in 87 countries.1,2 The bark,
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peel, leaves, and seed kernel of the mango contain high concentrations of phytochemicals that
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comprise the formulas of many traditional medicines to treat dysentery, asthma, and a host of
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other ailments.3-5 However mango pulp, the most commonly consumed portion of the fruit, has
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not been extensively chemically characterized in part due to the differences in chemistry among
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varieties and their relatively low concentrations.6 Studies have partially characterized
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polyphenols in mango pulp for several varieties, notably the cultivars Tommy Atkins, Úba,
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Haden, Kent, Atualfo, and Francis. Most notably was the identification of free gallic acid (GA),
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gallotannins, and mangiferin while many other minor compounds were uncharacterized.7,8
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Phytochemical extracts of mango pulp have been shown to have potential anticarcinogenic
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properties, 9,10 and to understand underlying in-vivo mechanisms and absorption rates for future
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studies a complete phytochemical characterization is required.
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The amount of fresh mangoes imported in the United States has increased by over 50% since
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200011 with larger volumes of processed mango products utilized in the form of juices and
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purees. Use of cell-wall degrading enzymes is a common processing practice in these products
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to aid in filtration and increase juice yield. Use rates of enzymes (0.05-1.0%) and processing
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conditions may vary among the enzymes used as processing aids, and may contain hydrolytic
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side activities that may impact the phytochemical composition of mango.12-13 Since mango pulp
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contains many glycosylated polyphenolics, the presence of hydrolytic side-activities can impact
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the composition, stability, and resultant bioactivity of the fruit. Therefore, the purpose of this
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study was to characterize the polyphenolics and minor water soluble phytochemicals in Keitt
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mango pulp, and to evaluate changes of the predominant phenolic acids found in Keitt after
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incubation with commercially available enzymes.
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MATERIALS AND METHODS
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Mango Fruit
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Fresh mangoes (cv. Keitt) were sourced from Mexico and imported through Frontera Produce
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(Edinburg, TX), and shipped refrigerated to the Department of Nutrition and Food Science at 3 ACS Paragon Plus Environment
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Texas A&M University. Fruit were allowed to ripen at ambient conditions, and 10 kg of fruit that
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exhibited uniform ripeness based on skin color and manual texture determination were manually
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peeled, deseeded, and stored at -20°C in vacuum sealed bags until analysis within 30 days. No
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samples were tested at different stages of ripeness due to previous results showing no change in
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phytochemical composition with ripening.14 Prior to extraction and analysis the pulp was thawed
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and thoroughly homogenized.
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Chemicals
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Standards of gallic acid, p-hydroxybenzoic acid, sinapic acid, ferulic acid, and p-coumaric acid
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were purchased from Sigma Aldrich (St. Louis, MO), and a standard of abscisic acid was
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purchased from Chromadex (Irvine, CA). Methanol, ethanol, acetone, and ethyl acetate were
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purchased from Fisher Scientific (Hampton, NH), and pre-prepared HPLC solutions were
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purchased from Sigma Aldrich. Enzymes, Validase TRL, Crystalzyme 200XL, and Rapidase AR
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2000 were kindly provided by DSM (Herleen, Netherlands).
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Phytochemical Extractions
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Compounds for characterization and semi-quantification were extracted from 10 g aliquots of the
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homogenous mango puree with 30 mL of a solvent mixture containing 1:1:1 methanol, acetone,
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and ethanol. The mixture was stirred for 30 min, filtered through cheesecloth, and the remaining
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solids re-extracted twice under the same conditions. Following a final filtration through #4
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Whatman filter paper, the solvents were evaporated under reduced pressure at 45°C and the
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concentrate brought up to 15 mL in water acidified with 0.01% HCl. Insoluble solids were
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removed by centrifugation prior to filtration through a Whatman 0.45µm PTFE membrane for
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HLPC-MS characterization.
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Additional isolation and concentration was deemed necessary to characterize minor compounds
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in mango pulp and was accomplished by extracting 1 kg of fruit as previously described.9
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Phytochemicals were partitioned from 10 g C18 Sep Pak cartridges. Columns were
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preconditioned with 10 column volumes of methanol followed by 10 column volumes of water.
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Extracts were loaded onto the column, and eluted with 100% methanol. C18 non-retained
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compounds were further extracted by liquid-liquid partition with two volumes of ethyl acetate 4 ACS Paragon Plus Environment
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with a solvent to sample ratio of 1:1. The solvent fractions were pooled for evaporation and
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likewise dissolution in 0.01% HCl water.
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Characterization and Quantitation of Phytochemicals with HPLC-MS
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Polyphenolics were characterized and quantified using a Thermo Finnigan LCQ Deca XP Max
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MSn ion trap mass spectrometer equipped with an ESI source. Separations were in reversed-
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phase using a Finnigan Surveyor HPLC coupled to a Surveyor PDA detector and gradient
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separations were performed using a Dionex Acclaim™ (Bannockburn, Il) C18 column, (250 x 4.6
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mm, 5 µm) at room temperature. Injections were made into the column by use of a 25 µL sample
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loop. Mobile phase A was 0.1% formic acid in water and mobile phase B was 0.1% formic acid
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in methanol run at 0.4 mL/min. A gradient was run of 0% Phase B for 3 min and changed to 21%
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Phase B in 20 min, from 21 to 35% Phase B in 30 min, 35 to 49% Phase B in 50 min, and 49% to
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70% Phase B in 70 min before returning to initial conditions. The electrospray interface worked
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in negative ionization mode. Source and capillary temperatures were set at 300°C, source voltage
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was 3.50 kV, capillary voltage was set at -42 V, and collision energy for MS/MS analysis was set
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at 35 eV. The instrument operated with sheath gas and auxillary gas (N2) flow rates set at 40
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units/min and 5 units/min, respectively. In addition to chromatographic separations, the mango
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concentrate was infused to capture MSn fragmentation patterns of poorly ionized compounds
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using instrument tuning specific to gallic acid, methyl gallate, and pentagalloylglucose.
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All compounds were quantified at 280 nm with their corresponding aglycones. If an aglycone
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standard was lacking concentrations were measured with the use of gallic acid as a standard. As
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the majority of the compounds were tentatively identified and lacked standards results of this
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study represent a relative concentration of polyphenols in Keitt mango pulp.
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Commercial Enzyme Hydrolysis
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Cell-wall active enzymes were applied to a Keitt mango puree at a uniform rate of 0.05% v/w to
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simulate an average commercial application rate of the enzyme and to evaluate their effects on
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mango chemistry. Enzymes included Validase TRL (Val) a mixed function enzyme with
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predominantly cellulase activity (6,800 U/g) with side activities as a pectinase and hemicellulase,
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Crystalzyme 200XL (Cz) an enzyme with predominantly pectinase activity (200,000 5 ACS Paragon Plus Environment
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depectinizing units/g), and Rapidase AR 2000 (Rap) a pectinase with approximately 25% ß-
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glucosidase side activity (>4000 U/g). Enzyme and mango puree mixtures were incubated at
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50°C for up to 4 hrs under a blanket of nitrogen. Following incubation, an aliquot of the fruit
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pulp was placed in boiling water for 3 min to inactivate the enzyme and immediately cooled on
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ice. Prior to analysis both control and enzyme treated pulps were centrifuged at 2000 x g to
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separate solids from the supernatant, which was further filtered through a 0.45 µm PTFE filter
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and quantified under the same conditions as the characterized mango.
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Statistics
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Semi-quantifications were evaluated in triplicate in independent reaction vessels. Data for
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commercial enzyme application represents the mean triplicate analyses using ANOVA (analysis
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of variance) followed by Each Pair Student’s t- test using the JMP statistics software package
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which provided corresponding probability (p
