The Cyclic Diarylheptanoid Asadanin as the Main Contributor to the

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The Cyclic Diarylheptanoid Asadanin as the Main Contributor to the Bitter Off-Taste in Hazelnuts (Corylus avellana L.) Barbara Singldinger, Andreas Dunkel, and Thomas Hofmann J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00026 • Publication Date (Web): 07 Feb 2017 Downloaded from http://pubs.acs.org on February 11, 2017

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Journal of Agricultural and Food Chemistry

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The Cyclic Diarylheptanoid Asadanin As the Main

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Contributor to the Bitter Off-Taste in Hazelnuts (Corylus

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avellana L.)

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Barbara Singldinger†, Andreas Dunkel†,‡ and Thomas Hofmann†,‡*

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†

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Chair of Food Chemistry and Molecular and Sensory Science, Technische

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Universität München, Lise-Meitner-Str. 34, D-85354 Freising, Germany, and ‡

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Bavarian Center for Biomolecular Mass Spectrometry, Gregor-Mendel-Straße 4, D-

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85354 Freising, Germany.

11 12 13 14 15

*

16

PHONE

+49-8161/71-2902

17

FAX

+49-8161/71-2949

18

E-MAIL

[email protected]

To whom correspondence should be addressed

19 20 21

ACS Paragon Plus Environment

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ABSTRACT

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Activity-guided fractionation and taste dilution analysis (TDA), followed by LC-

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MS/MS, LC-TOF-MS and 1D/2D-NMR spectroscopy led to the identification of the

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cyclic diarylheptanoid asadanin, 1, exhibiting a human bitter recognition threshold of

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13 µmol/kg, as the major inducer of the sporadic bitter off-taste of hazelnut kernels

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(Corylus avellana L.). Sensory analysis of hazelnut samples from two origins

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(Ordu/2013 and Akçakoca/2014) and from Cimiciato-infected hazelnut kernels,

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followed by LC-MS/MS quantitation of 1 and calculation of dose-over-threshold

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(DoT)-factors showed established evidence for the Cimiciato infection as the major

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inductor of asadanin biosynthesis in hazelnut kernels and, in consequence, as the

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reason for bitter off-taste development.

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KEYWORDS: taste, bitter, hazelnuts, Corylus avellana L., diarylheptanoids,

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asadanin

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INTRODUCTION

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Due to their appealing aroma and attractive taste profile, roasted hazelnuts (Corylus

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avellana L.) are used worldwide as a key ingredient for the manufacturing of

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confectionary, chocolate, and snack products. With a volume of about 580.000 metric

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tons per year (~85% of world market), Turkey is by far the largest producer of

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hazelnuts, followed by Italy with an annual production volume of about 90.000 metric

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tons. Plant growing and harvesting of hazelnuts differ largely between both countries

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of origin.1 While hazelnuts in Turkey are harvested manually by more than 320.000

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farmers and dried under the sun without moisture control, harvest in Italy is

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performed mechanically by a small number of highly professionalized farming

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operations, followed by highly controlled drying regimes to deliver rather

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homogeneous quality of hazelnuts.2,3

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Hazelnuts, in particular when originating from Turkey, have been reported to

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develop a sporadic bitter off-taste upon storage that is maintained throughout

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roasting to exhibit a flavor defect in final products, thus resulting in consumer

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complaints and causing a problem for the hazelnut producers as well as for the

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manufacturing industries.4,5 The reason for the sporadic off-taste development has

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not yet been identified and may be further complicated by the high biological

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heterogeneity of hazelnuts cultivated.6,7 Moreover, it has been reported that the

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attack of raw hazelnuts by a bug, named “Cimiciato” (cimici nocciolaie, Gonocerus

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acuteangulatus), induces tissue necrosis and a decrease in flavor quality

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accompanied by an increase in bitter taste.8,9 The molecules evoking the off-flavor in

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stored raw hazelnuts have, however, not yet been elucidated.

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Great advances have been achieved in the past 20 years to identify the key

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volatiles creating the typical aroma of foods.10 Bioresponse-guided identification of ACS Paragon Plus Environment

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most intense odorants by means of GC-olfactometry, their quantitation by means of a

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stable isotope dilution analysis, followed by recombination and sensory experiments

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revealed 3-methyl-4-heptanone, 5-methyl-(E)-2-hepten-4-one, 2-acetyl-1-pyrroline, 2-

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propionyl-1-pyrroline, 2,3-diethyl-5-methylpyrazine, 3,5-dimethyl-2-ethylpyrazine, 3,6-

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dimethyl-2-ethylpyrazine and 2-furfurylthiol as the key odorants creating the typical

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aroma profile of roasted hazelnuts.11 In comparison, the knowledge on taste-active

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non-volatiles in hazelnuts is rather scarce and, in particular, the components evoking

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the sporadic bitter off-taste of hazelnuts are not yet known.

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Preliminary studies on non-volatile taste compounds in hazelnuts revealed

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sucrose with amounts of 1.8 to 4.4 g/100 g, followed by stachyose und raffinose with

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amounts of up to 0.4 g/100 g as sweet-tasting compounds in hazelnuts. With total

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amount of 0.96-2.72 g/100 g, citric acid, malic acid, oxalic acid, lactic acid, succinic

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acid and acetic acid were identified as sour tasting organic acids in hazelnuts.12,13

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The levels of condensed polyphenols were reported to range between 3.99 and

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40.56 mg/g (catechin equivalents) and are decreased by more than 90% with

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removal of the testa to give about 0.3 to 3.0 mg/g upon hazelnut processing. As

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catechin oligomers show rather high bitter taste thresholds (~0.3 mg/g water),14 a

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contribution of polyphenols to the bitter taste still needs to be clarified.

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As bitter tasting dipeptides and oligopeptides are known to be produced upon

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enzymatic hydrolysis of animal proteins like milk proteins and plant-derived proteins

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like soy proteins, the generation of candidate bitter peptides has been studied during

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proteolytic hydrolysis of powdered hazelnuts.15 However, the development of a bitter

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taste upon proteolysis could not be observed,15 thus excluding bitter peptides as key

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molecules responsible for the off-taste of stored hazelnuts.

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While the storage-induced bitter taste development in lipid-rich plant seeds like

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poppy seeds has been shown to be caused by the lipolytic release of free fatty acids ACS Paragon Plus Environment

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as well as their autoxidation to give hydroxy fatty acids,16 storage experiments with

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hazelnuts only revealed marginal changes in amounts and composition of free fatty

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acids.17 Comparative storage of raw hazelnuts under oxygen, carbon dioxide, or

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nitrogen atmosphere did not reveal any significant role of oxygen in the alteration of

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the taste profile.5 Taking all these literature data into consideration, it may be

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concluded that the key molecules inducing the bitter taste defect in hazelnuts have

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still not been identified.

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In recent years, application of a taste-guided fractionation approach enabled

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the identification of the key taste and off-taste compounds in carbohydrate/amino

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acid mixtures,18 hops,19 carrots,20,21 coffee,22 asparagus,23 black tea infusions,24

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cocoa nibs,25 and spinach.26 The aim of the present investigation was, therefore, to

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identify the key molecules contributing to the bitter off-taste of hazelnuts, to

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determine their human recognition thresholds, and to evaluate its sensory

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contribution by means of concentration/activity considerations.

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MATERIALS AND METHODS

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Chemicals.

The

following

compounds

were

obtained

commercially:

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acetonitrile, methanol (J.T. Baker, Deventer, Netherlands), ethyl acetate, n-pentane

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AnalaR NORMAPUR (BDH Prolabo, Briare, France), formic acid, (Merck, Darmstadt,

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Germany); Solvents used for HPLC-MS/MS analysis were of LC-MS grade

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(Honeywell, Seelze, Germany), n-pentane and ethyl acetate were distilled before

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using, all other solvents were of HPLC grade (Merck). Deuterated solvents were

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obtained from Sigma Aldrich (St. Louis, MO). L-Tyrosine for qNMR was purchased

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from Sigma-Aldrich (Steinheim, Germany). Water for chromatography was purified by ACS Paragon Plus Environment

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the use of a Milli-Q water advantage A 10 water system (Millipore, Molsheim,

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France). Dry-stored raw hazelnut kernels from two origins in Turkey (Ordu, 2013, and

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Akçakoca, 2014) and cimiciato-infected hazelnut kernels, characterized and sorted

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by a visual inspection by a trained expert panel, were provided by the German food

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industry.

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Sequential Solvent Extraction. An aliquot of powdered hazelnuts (300 g),

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obtained by grinding deep-frozen kernels using a GM 300 type mill (Retsch, Haan,

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Germany) at 4000 U/min for 40 s, was extracted three times with methanol/water

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(70:30, v/v; 1 L) by stirring for 30 min at room temperature, the extracts were

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combined, methanol removed in vacuum at 39 °C, and the aqueous solution was

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then sequentially extracted with n-pentane (4 x 0.5 L), followed by ethyl acetate (3 x

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0.8 L). The corresponding extracts were combined and separated from solvent in

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vacuum at 39 °C, followed by lyophilization to obtain the pentane-solubles (fraction I),

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the ethyl acetate extractables (fraction II), and the water solubles (fraction III),

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respectively (Table 1). The residual hazelnut material was freeze-dried twice to result

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in the insoluble fraction IV, which did not show any taste activity. Aqueous solutions

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of fractions I-III were evaluated by means of a comparative taste profile analysis.

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Separation of Fraction II by Means of Medium Pressure Liquid

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Chromatography (MPLC). An aliquot (350 mg) of hazelnut fraction II was dissolved

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in acetonitrile/water (12:88, v/v; 3.5 mL) and separated on a 150 x 40 mm i.d

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polypropylene cartridge filled with 25-40 µm LiChroprep RP-18 material (Merck).

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MPLC was performed at a flow rate of 40 mL/min to give 12 fractions, namely II-1 to

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II-12. Using 0.1% formic acid in water (v/v) as solvent A and acetonitrile as solvent B,

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MPLC was performed with the effluent monitored using a Sedex LT-ELSD detector

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Model 80 (Sedere, Alfortville, France) at Gain 6 and the following gradient: 3 min

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100% A, within 2 min to 95% A, held 1 min at 95% A isocratically, within 2 min to ACS Paragon Plus Environment

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90% A, increased in 8 min to 85% A, held 5 min 85% A isocratically, increased in

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7 min to 80% A, within 10 min to 75% A, held 3 min 75% A isocratically, increased in

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6 min to 70% A, maintained 5 min at 70% A isocratically, within 8 min to 100% B,

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held 10 min at 100% B isocratically, decreased in 7 min at 0% B, and finally held

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10 min at 0% B. Each of the 12 fractions, collected by means of a fraction collector,

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was separated from solvent (vacuum, 39 °C), the residues were dissolved in water,

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freeze-dried twice, and then kept at -20 °C until further used.

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Identification of the Bitter Key Compound in Fraction II-8. Fraction II-8,

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evaluated with the highest bitter impact, was dissolved in acetonitrile/water (12:88,

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v/v; 25.5 mg/mL) and, after membrane filtration, separated by preparative RP-HPLC

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on a 250 × 21.2 mm i.d, 5 µm, Luna Phenyl-Hexyl column (Phenomenex,

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Aschaffenburg, Germany). Using a flow rate of 20 mL/min with 0.1% formic acid in

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water (v/v; solvent A) and acetonitrile (solvent B), chromatography was performed

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with the effluent monitored at 254 nm: starting with a mixture 25% B and 75% A, held

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at 25% B for 3 min, increasing the acetonitrile content to 30% B over 5 min, held

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isocratically with 30% B for 9 min, decrease in 8 min to 25% B and finally held at 25%

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B for 5 min. The effluent was separated to give 9 subfractions, namely II-8/1 to II-8/9.

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The individually collected fractions were freed from solvent in vacuum at 39 °C,

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freeze-dried twice, and the residues obtained were used for the taste dilution analysis

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(TDA) as well as for structural analysis. On the basis of UV/Vis, LC-MS/MS, TOF-MS

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and 1D/2D-NMR, the key compound in the most bitter fraction II-8-7 was identified as

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asadanin, 1, that has been isolated earlier from the heartwood of Ostrya japonica27

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and, subsequently, from hazelnut leaves.28

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Asadanin, 1, Figure 3: LC-MS (ESI-): m/z 343,13 [M-H]-; LC-MS/MS (DP = -

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35 V): m/z 343.0, 283.0, 270.2, 211.0, 193.0; LC-MS-TOF: m/z 343.1217

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(measured), m/z 343.1182 (calcd. for [C19H19O6]-); 1H NMR (500 MHz; DMSO-d6) δ ACS Paragon Plus Environment

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2.72-2.82 [m, 2H, H-C(7)], 2.72-3.01 [m, 2H, H-C(13)], 2.87-3.01 [d, 1H, J=6.5 Hz, H-

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C(11β)], 3.37-3.45 [m, 1H, H-C(11α)], 3.65 [dd, 1H, J=10.15, 6.85 Hz, H-C(8)], 4.06

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[dd, 1H, J=10.12, 3.93 Hz, H-C(9)], 4.46 [m, 1H, H-C(12)], 6.27 [d, 1H, J=1.65 Hz, H-

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C(18)], 6.51 [d, 1H, J=1.65 Hz, H-C(19)], 6.77 [d, 1H, J=8.14 Hz, H-C(4)], 6.81 [d,

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1H, J=8.18 Hz, H-C(16)], 6.99 [dd, 1H, J=8.35, 2.06 Hz, H-C(5)], 7.06 [dd, 1H,

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J=8.35, 2.24 Hz, H-C(15)];

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36,2 [C-11], 66.7 [C-12], 67.9 [C-8], 77.2 [C-9], 115,8 [C-4], 115,9 [C-16], 125.0 [C-1],

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125,8 [C-2], 128.3 [C-15], 129.3 [C-5], 129.5 [C-6], 129.6 [C-14], 133.7 [C-18, C-19],

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150.7 [C-17], 151.4 [C-3], 214.4 [C-10].

13

C NMR (126 MHz; DMSO-d6) δ 23.6 [C-13], 35.9 [C-7],

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Sensory Analyses. Sensory panel training and sample pretreatment. 17

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panelists (9 female, 8 male; 23-40 years in age), who had given informed consent to

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participate in the present sensory tests, were trained weekly for at least two years in

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order to became familiar with the sensory methodologies used and to evaluate

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aqueous reference solutions of taste compounds.23-25 Sensory analyses were

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performed in a sensory panel room at 22-25 °C while the panelist wore nose clips to

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prevent cross-model interactions with olfactory cues. Prior to sensory analysis,

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isolated fractions and compounds were confirmed to be effectively free of solvent

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traces.23

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Taste Profile Analysis. Aliquots of the lyophilized hazelnut fractions I-III were

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dissolved in their “natural” concentrations (~1.5 g powdered hazelnuts per mL) in

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bottled water. The sensory panel was asked to evaluate these solutions and to rate

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the intensity of the taste qualities “bitter”, “astringent”, and “sweet” on a scale from 0

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(not detectable) to 5 (strongly detectable).

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Taste Dilution Analysis (TDA). Aliquots of MPLC fractions and HPLC

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subfractions obtained from fraction II-8 were dissolved in “natural” ratios in bottled

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water (35 mL), aqueous serial 1:1 dilutions of each of these fractions were evaluated ACS Paragon Plus Environment

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by the sensory panel in order of ascending concentration, and the taste dilution (TD)

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factor for bitterness was determined.18 The TD-factors for each MPLC- and HPLC-

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fraction, evaluated in two independent sessions each were averaged.

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Determination of Human Taste Recognition Thresholds. The threshold

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concentration, at which the bitter taste quality of the compound was just detectable,

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was determined in bottled water using a two-alternative forced choice test (2-AFC)

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with increasing levels of the purified substance 1 and followed the procedure as

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mentioned. The values between individuals and between the independent sessions

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differed by not more than plus or minus one dilution step, meaning that an average

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threshold value of 13.0 µmol/L for 1 represents a range from 7.5-26.0 µmol/L.

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Sensory Confirmation of Quantitative Data. Methanol/water extracts (70:30, v/v)

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were prepared from three hazelnut samples: (A) Cimiciato-infected (Ordu), 2013, (B)

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Ordu, 2013, and (C) Akçakoca, 2014. After separating the solvent in vacuum at 39°C,

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the extracts obtained from these three samples were dissolved in water in their

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“natural” concentration and sensorially evaluated for bitterness on a 5-point scale.

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Quantitation of Asadanin 1. External Calibration Curve and Linear Range. A

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stock solution of purified asadanin 1 was prepared in acetonitrile/water (20:80; v/v)

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and its exact concentration (0.72 mg/mL) was verified by means of quantitative NMR

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(qNMR).28 This stock solution was diluted 1:20; 1:50; 1:100, 1:200, 1:500, 1:1000,

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1:2000 and 1:5000 with acetonitrile/water (20:80; v/v), the dilutions were analyzed by

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means of UHPLC-MS/MS using the MRM transition Q1/Q3 of m/z 343.1/211.1 as

216

quantifier and optimized instrument settings (DP = - 35 V, EP = - 10 V, CE = - 38 V,

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CXP = - 25 V). An external calibration curve (y = 1E+09x+14787, R² = 0.9998) was

218

calculated by plotting the peak area ratios of the analyte against its given

219

concentration.


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Solvent Extraction for Quantitation. Hazelnut samples (300 g) were frozen in

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liquid nitrogen, ground by using a GM 300 type mill (Retsch GmbH) at 4000 U/min for

222

40 s, extracted with methanol/water (70:30, v/v; 3 x 1L), separated from solvent in

223

vacuum at 39 °C, and lyophilization twice. The extracts were taken up in

224

acetonitrile/water (30:70, v/v; 1 mL/10 mg), membrane filtered and aliquots (1 µL)

225

were analyzed by means of UHPLC-MS/MS on a 100 x 2.1 mm, 100 Å, Kinetex

226

1.7µm Luna Phenyl-Hexyl column (Phenomenex, Aschaffenburg, Deutschland) at a

227

flow rate of 0.4 mL/min. Using 1% formic acid in water (v/v; solvent A) and 1% formic

228

acid in acetonitrile (v/v; solvent B), the following gradient was applied for UHPLC:

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start with 5% B, held at 5% for 1 min, increase in 2 min to 30% B, in 9 min to 70% B,

230

increase in 1 min to 100% B, held 0.5 min isocratically at 100%, decrease in 1 min to

231

5% B, held 5 min at 5% B. Using the MRM transition Q1/Q3 of m/z 343.1/211.1 as

232

quantifier, asadanin was quantitated in hazelnut samples by means of external

233

calibration.

234

Medium Pressure Liquid Chromatography (MPLC). For separation of

235

fraction II, an MPLC apparatus (Büchi, Flawil, Swiss) consisting of a binary pump

236

module C-605, a control unit C-620, a fraction collector C-660, and a Sedex LT-

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ELSD detector Model 80 (Sedere, Alfortville, France) was used. Chromatography

238

(40 mL/min) was done on a 150x40 mm polypropylene cartridge filled with 25-40 µm

239

LiChroprep RP18 material (Merck).

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High Performance Liquid Chromatography (HPLC). The HPLC apparatus

241

(Jasco, Gross-Umstadt, Germany) used comprised a binary high pressure HPLC

242

pump system PU-2080 Plus, a AS-2055 Plus autosampler, a DG-2080-53 degasser,

243

a MD-2010 Plus type diode array detector, and a Rh 7725i type Rheodyne injection

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valve (Rheodyne, Bensheim, Germany). Analytical separations (1 mL/min) were

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performed on an analytical 250 x 4.6 mm i.d., 5 µm, Luna Phenyl-Hexyl column ACS Paragon Plus Environment

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(Phenomenex). Data acquisition was done by means of Chrompass Chromatography

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Data System, Version 1.9 (Jasco). Preparative separation (20.0 mL/min) of fraction

248

II-8 was performed on a preparative 250 x 21.2 mm i.d., 5 µm, Luna Phenyl-Hexyl

249

column (Phenomenex).

250

Liquid Chromatography Mass spectrometry (LC-MS). A QTRAP 6500 mass

251

spectrometer (Sciex, Darmstadt, Germany), controlled by the Analyst 1.6.2 software

252

(Sciex), was used and operated in the full-scan mode (ion spray voltage: -4500 V):

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curtain gas (35 V), temperature (450 °C), gas1 (55 V), gas2 (65 V), collision activated

254

dissociation (-2 V) and entrance potential (-10 V). The samples were separated by

255

means of a Nexera UHPLC (Shimadzu Europa GmbH, Duisburg, Germany)

256

consisting of two LC pump systems 30AD, a DGU-20A5 degasser, a SIL-30AC

257

autosampler, a CTO-30A column oven and a CBM-20A controller, and equipped with

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a 100 x 2.1 mm, 100 Å, Kinetex 1.7 µm Luna Phenyl-Hexyl column (Phenomenex). A

259

gradient of 1% formic acid in water (v/v; solvent A) and 1% formic acid in acetonitrile

260

(v/v; solvent B) was used (0.4 mL/min).

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UPLC/Time-of-Flight Mass Spectrometry (UPLC/TOF-MS). An aliquot (1-

262

5 µL) of the analyte in acetonitrile/water (20:80, v/v; 1mL) was injected into an

263

Acquity UPLC core system (Waters) equipped with a 2.1 x 150 mm, 1.7 µm, BEH

264

C18 column (45 °C) and connected to a SYNAPT G2 HDMS spectrometer (Waters

265

UK Ltd., Manchester, UK) operating with the instrument settings reported earlier.23

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Nuclear Magnetic Resonance Spectroscopy (NMR). 1D- and 2D-NMR

267

spectra were recorded on a 500 MHz Avance III spectrometer (Bruker, Rheinstetten,

268

Germany) equipped with a cryo-TCI Probe (300 K). DMSO-d6 (600 µL) was used as

269

solvent and chemical shifts are reported in parts per million relative to the DMSO-d6

270

solvent signals:

271

processing was performed by using Topspin NMR software vers. 3.2 (Bruker) and

1

H-NMR: 2.50 ppm and 3.33 ppm;

13


C-NMR: 39.52 ppm. Data

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MestReNova 10.0 (Mestrelab Research, Santiago de Compostela, Spain). For

273

quantitative NMR spectroscopy (qNMR), the spectrometer was calibrated by using

274

the ERETIC 2 tool using the PULCON methodology as reported earlier.24 The

275

isolated signal at 6.27 ppm (d, J=1.65, 1H) was used for absolute quantitation of

276

asadanin, 1, using a defined sample of L-tyrosine as the external standard and its

277

specific resonance signal at 7.10 ppm (m, 2H) for analyses.29

278 279

RESULTS AND DISCUSSION

280 281

Aimed at identifying the key molecule evoking the bitter taste of raw hazelnut kernels,

282

freshly ground hazelnuts were extracted with methanol/water and, after removing the

283

methanol in vacuum, the aqueous extract was extracted with n-pentane (fraction I),

284

followed by ethyl acetate (fraction II) and the remaining aqueous fraction III,

285

respectively, followed by freeze-drying. To locate the key bitter compounds, the

286

fractions I-III were dissolved water in their “natural” concentrations and then used for

287

taste profile analysis. While fraction I exhibited only weak taste activity, high scores

288

for bitterness (4.0) were reported in fraction II (Table 1). In comparison, the aqueous

289

fraction III was evaluated mainly as astringent and sweet with intensity scores of 3.8

290

and 3.2. As fraction II showed the most intense bitter taste, the following fractionation

291

was focused on the identification of key bitter molecules in fraction II.

292

Activity-Guided Discovery of the Key Bitter Compound in Hazelnut

293

Fraction II. Aimed at locating the key bitter compounds, fraction II was further

294

separated by means of MPLC-ELSD using RP-18 material as the stationary phase

295

(Figure 1). The effluent was collected in twelve fractions, namely II-1 to II-12, which

296

were freed from solvent, taken up in equal amounts of water, then, sequentially

297

diluted 1:1 with water and, finally, used for taste dilution analysis (TDA) in order of ACS Paragon Plus Environment

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ascending concentrations. Among the 12 fractions, bitter taste was detectable in

299

fractions II-5 to II-10 with fraction II-8 evaluated with the highest TD-factor of 8

300

(Figure 1).

301

To identify the key molecule causing the bitter taste of fraction II-8, this fraction

302

was separated by means of preparative RP18-HPLC with UV detection at 254 nm

303

(Figure 2). A total of nine subfractions were collected individually, namely II-8-1 to II-

304

8-9, the solvent was separated in vacuum, each subfraction was dissolved in

305

“natural” concentration ratios in water and then used for TDA (Figure 2). Among the

306

nine subfractions, fraction II-8-7 was judged with the highest TD-factor of 8, followed

307

by subfractions II-8-6 and II-8-8 (TD-factor: 4).

308

As LC-MS/MS analysis revealed more quantitative, rather than qualitative

309

differences in these bitter subfractions II-8-6 to II-8-8, the major component 1 eluting

310

in the most intense bitter tasting fraction II-8-7 was purified by re-chromatography

311

and then analyzed by LC-MS/MS, UHPLC-TOF-MS, and 1D/2D-NMR experiments.

312

LC-MS (ESI-) analysis of 1 revealed m/z 343.1 as the pseudo molecular ion ([M-H]-),

313

thus indicating a molecular mass of 344.1 Da. This was confirmed by UHPLC-TOF-

314

MS as well as 1H and

315

C19H20O6.

316

The

13

13

C NMR spectroscopy showing an empirical formula of

C and 1H NMR spectra of bitter compound 1 demonstrated a total of 19

317

carbon atoms resonating between 20 and 210 ppm and 20 proton signals detected

318

between 2.5 and 7.5 ppm. Heteronuclear single-quantum correlation spectroscopy

319

(HSQC), optimized for 1JC,H couplings, helped to visualize direct connectivity of

320

carbons and protons and revealed three methylene groups, nine methine groups,

321

and seven quaternary C-atoms. Six protons resonating between 7.1 and 6.81 ppm

322

were correlated to the carbons of two connected phenyl ring systems. This was

323

confirmed using heteronuclear multiple-bond correlation spectroscopy (HMBC) ACS Paragon Plus Environment

13


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324

optimized for 2JC,H and 3JC,H couplings, e.g. couplings between C(2) at 125.8 ppm and

325

H-C(18) and H-C(4) resonating at 6.27 and 6.77 ppm, respectively, as well as

326

couplings between C(1) at 125.0 ppm and H-C(16) and H-C(19) with chemical shifts

327

of 6.81 and 6.51 ppm could be detected (Figure 3A). In addition, a C7-alkyl chain

328

connecting the biphenyl ring was assigned by the heteronuclear couplings between

329

carbon atom C(7) and the ring proton H-C(5) as well as carbon atom C(13) and the

330

ring proton H-C(15), thus indicating the presence of a cyclic diarylheptanoid structure

331

for 1 (Figure 3B). Finally, the

332

keto group within the C7-alkyl chain as demonstrated by the heteronuclear coupling

333

between the carbonyl carbon C(10) and the protons H-C(8) at 3.65 ppm, H-C(11α) at

334

3.45-3.37 ppm, H-C(11β) at 3.01-2.87 ppm, and the hydroxy proton HO-C(9) at

335

5.27 ppm (Figure 3C).

336

13

C-signal detected at 214.4 ppm was deduced as a

Taking all the analytical data into account, the key bitter compound in fraction

337

II-8-7

could

be

unequivocally

identified

as

3,8,9,12,17-pentahydroxy-

338

(8CI,9CI),tricyclo[12.3.1.12,6]nonadeca-1(18),2,4,6(19),14,16-hexaen-10-one,

339

(Figure 4). Although this phytochemical has been earlier isolated from the wood of

340

Ostrya japonica and named asdanin,27 and subsequently from leaves of Corylus

341

avellana L, ‘Tonda di Giffoni’,28 the occurrence of compound 1 in hazelnut kernels as

342

well as its bitter taste activity has not previously been reported in literature.

1

343

Bitter Taste Activity of Asadanin 1. Prior to the determination of a human

344

recognition taste threshold, the purity of asadanin 1 as well as the concentration of a

345

stock solution were checked by HPLC-MS and quantitative 1H NMR spectroscopy.29

346

A 2-alternative forced-choice test revealed a long-lasting bitter taste induced by the

347

cyclic diarylheptanoid 1 with a recognition threshold of 13 µmol/L.

348

Quantitation of Asadanin and Dose-Activity Considerations in Hazelnut

349

Kernels. To enable quantitation of asadanin 1 in hazelnuts by means of LC-MS/MS, ACS Paragon Plus Environment

14

Page 15 of 30


350

the MS/MS parameters were tuned to optimize the generation of specific product ions

351

through fragmentation of the pseudo molecular ion of 1 by infusing the pure

352

reference compound with a syringe pump into the mass spectrometer operating in

353

the ESI- mode. By means of LC-MS/MS with external calibration, asadanin 1 was

354

then quantitated in methanol/water extracts prepared from hazelnut samples from

355

two origins (Ordu/2013 and Akçakoca/2014) and from Cimiciato-infected hazelnut

356

kernels (Table 2). The quantitative data showed very high levels of 44.8 µmol/kg for

357

asadanin in Cimiciato-infected hazelnuts, whereas the non-infected hazelnuts from

358

Akçakoca and Ordu contained 9 to 32-times lower amounts of 5.0 and 1.4 µmol/kg,

359

respectively.

360

To correlate these quantitative data with sensory impact, extracts from all

361

three hazelnut samples were sensorially evaluated in their bitter taste intensity. By far

362

the highest bitter taste score of 4.0 was reported for the Cimiciato-infected hazelnuts

363

(Table 2). In comparison, the hazelnut samples from Akçakoca and Ordu were both

364

judged with a low bitterness score of 2.0 and 1.0, respectively, thus being well in line

365

with the lower levels of asadanin in these samples (Table 2). In order to assess the

366

bitter taste activity of asadanin 1 in the different hazelnut samples, dose over

367

threshold (DoT)-factors were determined as ratio of the concentration to the taste

368

threshold of the respective tastant.30–35 The DoT-factor for asadanin in Cimiciato-

369

infected hazelnuts was determined to be 3.5, thus indicating that the concentration of

370

1 exceeded its bitter taste threshold by a factor of 3.5 (Table 2).

371

On the basis of these data, asadanin, 1, may be concluded as the key inducer

372

of the sporadic bitter off-taste in hazelnuts. Quantitative analysis of 1 shows first

373

evidence for the Cimiciato infection as the major inductor of asadanin biosynthesis in

374

hazelnut kernels. The development of high-throughput quantitation methods and

375

analysis of asadanin generation in hazelnut kernels artificially infected with the ACS Paragon Plus Environment

15


Page 16 of 30

376

Cimiciato bug are currently in progress to further understand the mechanisms leading

377

to the induction of bitter taste development.

378 379

Acknowledgment

380

This IGF Project of the FEI is supported via AiF within the programme for promoting

381

the Industrial Collective Research (IGF) of the German Ministry of Economic Affairs

382

and Energy (BMWi), based on a resolution of the German Parliament. We are also

383

grateful to Prof. Lieberei, University of Hamburg, as well as the industry partners of

384

this project for providing us with authentic samples.

385


16

Page 17 of 30


386

LITERATURE CITED

387

(1) International Nut and Dried Food Foundation (INC). Global Statistical Review

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2014-2015. Hazelnuts. 2014, pp. 26–29.

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(2) Hütz-Adams, F. Hazelnuts from turkey. Ecological and social challenges in

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production (in German), Südwind e.V-Institut für Ökonomie und Ökumene. 2012,

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pp. 7–11.

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(3) Demir, I. The firm size, farm size, and transaction costs: the case of hazelnut

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farms in Turkey, J. Agric. Econ. 2016, 47, 81–90.

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(4) Özdemir, M.; Devres, O. Turkish hazelnuts: Properties and effect of

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microbiological and chemical changes on quality, Food Rev. Int. 1999, 15, 309–333.

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(5) Moscetti, R.; Frangipane, M. T.; Monarca, D.; Cecchini, M.; Massantini, R.

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atmospheres, Postharv. Biol. Technol. 2012, 65, 33-38.

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(6) Açkurt, F.; Özdemir, M.; Biringen, G.; Löker, M. Effects of geographical origin and

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variety on vitamin and mineral composition of hazelnut (Corylus avellana L.) varieties

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cultivated in Turkey, Food Chem. 1999, 65, 309–313.

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perspective for future biotechnology, Angew. Chem. Int. Ed. 2014, 53, 7124–7143.

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chromatography in combination with time-of-flight mass spectrometry (GC×GC-TOF-

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MS), J. Agric. Food Chem. 2013, 61, 5226–5235.

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Compounds of Turkish Hazelnut Varieties (Corylus avellana L.), J. Agric. Food

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Chem. 2010, 58, 8674–8679.

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Hazelnut (Corylus avellana L.). 1. Compositional Characteristics, J. Agric. Food

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Chem. 2003, 51, 3790–3796.

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cocoa nibs (Theobroma cacao) by means of quantitative studies and sensory

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taste in enzymic hydrolyzates of different proteins, Lebensm.-Wiss. Technol. 1972, 5,

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bioassay for the screening and identification of taste-active compounds in foods, J.

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Agric. Food Chem. 2001, 49, 231–238.

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contributing to the bitter off-taste of carrots (Daucus carota L.) and carrot puree, J.

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roast coffee beverage and identification of key bitter taste compounds, Eur. Food

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steroidal saponins from Asparagus apears (Asparagus officinalis L.), J. Agric. Food

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activity of N-phenylpropenoyl-L-amino acids from cocoa (Theobroma cacao), J.

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related compounds, Ringyo Shikenjo Kenkyu Hokoku. 1968, 209, 77–168.

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reference materials and natural isolates by means of quantitative

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Pizza, C.; Bifulco, G.; Piacente, S. Giffonins J-P, highly hydroxylated cyclized

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diarylheptanoids from the leaves of Corylus avellana cultivar "Tonda di Giffoni", J.

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1

H NMR

20

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492 493


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494

Page 22 of 30

FIGURE LEGEND

495 Figure 1.

(A) RP18-MPLC separation of fraction II isolated from bitter tasting hazelnut kernels and (B) taste dilution (TD)-factors of MPLC fractions II-1 to II-12.

Figure 2.

(A) RP-HPLC chromatogram of MPLC fraction II-8 and (B) taste dilution (TD)-factor of subfractions II-8-1 to II-8-9.

Figure 3.

Excerpts of the HMBC spectrum (500 MHz, DMSO-d6) of purified asadanin 1 showing (A) 2J and 3J coupling of C(1) and C(2), (B) 3J coupling of C(7) and C(13), and (C) 2J and 3J coupling of the carbonyl atom C(10).

Figure 4.

Chemical structure of the cyclic diarylheptanoid asadanin 1.

496


22

Page 23 of 30


Table 1. Taste Profile Analysis of Fractions I-III isolated from Hazelnut Kernels Showing a Bitter Off-Taste. Fractiona

Intensityb perceived for bitterness

astringency

sweetness

I

0.8

0.7

0.6

II

4.0

2.1

0.3

III

1.4

3.8

3.2

a

The n-pentane soluble fraction I, the ethyl acetate soluble fraction II, and the

aqueous fraction III were dissolved in water in their “natural” concentrations and then used for taste profile analysis. bA trained sensory panel was asked to rate the intensity of the given taste descriptors on a scale from 0 (not detectable) to 5 (intensely detectable).


23


Page 24 of 30

Table 2. Concentration, Dose-over-Threshold (DoT)-factor and Sensory Bitterness Evaluation of Different Hazelnut Samples. Conc. [µmol/kg] of

DoT-Factor of

Bitter taste

asadanin 1a

asadanin 1b

intensityc

Ordu (2013)

1.4

0.1

1.0

Akçakoca (2014)

5.0

0.4

2.0

Cimiciato (2013)

44.8

3.5

4.0

Sample

a

Concentrations were determined by LC-MS/MS and external calibration and are given as the means of triplicates. bDose over threshold (DoT)-factors were determined as ratio of the concentration to the taste threshold (13.0 µmol/kg) of 1. c A trained sensory panel was asked to rate the bitterness intensity of an aqueous solution of the methanol/water extractables prepared from hazelnut samples on a scale from 0 (not detectable) to 5 (intensely detectable).


24

Page 25 of 30


Figure 1 (Singldinger et al.)

A

B


25


Page 26 of 30

Figure 2(Singldinger et al.)

A

B


26

Page 27 of 30



A

B

C


27


Page 28 of 30



28

Page 29 of 30


Table of Content Graphic


29


TOC graphic 240x157mm (115 x 115 DPI)


Page 30 of 30

The Cyclic Diarylheptanoid Asadanin as the Main Contributor to the

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