Analysis and Sensory Evaluation of Volatile Constituents of Fresh

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Analysis and Sensory Evaluation of Volatile Constituents of Fresh Blackcurrant (Ribes nigrum L.) Fruits Kathrin Jung, Oxana Fastowski, Iulia Poplacean, and Karl-Heinz Engel J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03778 • Publication Date (Web): 09 Oct 2017 Downloaded from http://pubs.acs.org on October 10, 2017

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Journal of Agricultural and Food Chemistry

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Analysis and Sensory Evaluation of Volatile Constituents of

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Fresh Blackcurrant (Ribes nigrum L.) Fruits

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Kathrin Jung, Oxana Fastowski, Iulia Poplacean and Karl-Heinz Engel *

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Lehrstuhl für Allgemeine Lebensmitteltechnologie, Technische Universität München,

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Maximus-von-Imhof-Forum 2, D-85354 Freising-Weihenstephan, Germany

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* Corresponding Author (telephone: +49 8161 714250; fax +49 8161 714259; e-mail:

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[email protected])

1 ACS Paragon Plus Environment


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ABSTRACT

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Volatile constituents of fresh blackcurrant (Ribes nigrum L.) berries were isolated via

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vacuum-headspace extraction and analyzed by capillary gas chromatography-mass

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spectrometry. In agreement with previous studies with frozen fruits, short-chain

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esters and terpenes were major compound classes. However, rather high

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concentrations of C6-compounds (e.g. (E)-hex-2-enal, (Z)-hex-3-enal) constituted a

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striking difference to data reported for frozen fruits. Frozen storage of blackcurrant

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berries was shown to result in drastically reduced concentrations of C6-compounds

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and a shift of the volatile profile in favor of terpenes. The time-dependent enzymatic

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formation and isomerization of C6-compounds adds an additional element of

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variability to the spectrum of fresh blackcurrant volatiles. Nevertheless, blackcurrant

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cultivars can be classified according to the major classes of the volatiles of the fresh

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fruits, if prerequisites, such as the same growing location and the same state of

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ripeness are met. The sensory contributions of volatiles of blackcurrant berries were

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assessed by gas chromatography-olfactometry in combination with aroma extract

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dilution analysis. 4-Methoxy-2-methyl-2-butanethiol, (Z)-3-hexenal, ethyl butanoate,

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1,8-cineole, oct-1-en-3-one and alkyl-substituted 3-methoxypyrazines were among

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the volatiles showing the highest aroma activity values.

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KEYWORDS: Ribes nigrum L., blackcurrant, GC/O, volatiles, aroma, C6-compounds.

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INTRODUCTION

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Berries of blackcurrants (Ribes nigrum L.) are commonly used for the production of

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jam, juice, wine, liqueur, and spirits.1 In Germany, for example, in 2016 the

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production volume amounted to 6808 t.2 Investigations of the volatile constituents of

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blackcurrant fruits date back to the 1960s. A broad spectrum of terpenes, esters, and

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alcohols has been identified.3-10 In the following decades several studies on the

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variability of the volatile profiles of blackcurrant berries have been carried out.11-21

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However, in most of the investigations frozen berries have been analyzed.3-12,15,18-21

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Latrasse et al.11 demonstrated that methyl and ethyl butanoate, 1,8-cineole, diacetyl,

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and a ‘catty urine odor’ are important to the typical blackcurrant aroma. This ‘catty

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urine odor’ note was first identified in essential oils of blackcurrant buds as

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4-methoxy-2-methyl-2-butanethiol.22-23 Recently, unambiguous evidence has been

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provided that this sulfur-containing volatile also occurs in blackcurrant fruits.24 The

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determination of odor thresholds in water and in a blackcurrant-type matrix and the

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calculation of the odor activity values (OAV) indicated that 4-methoxy-2-methyl-2-

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butanethiol contributes to the aroma of fresh blackcurrant berries.24 Two further

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investigations identified odor-active compounds in frozen blackcurrant berries via

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capillary gas chromatography/olfactometry (GC/O).18-19 However, the sensory

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contributions of these odor-active compounds have not been evaluated by OAV, and

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their importance to the aroma of fresh fruits remained unclear.

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Therefore, the aims of the present study were: (i) to identify and to quantitate volatile

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compounds in fresh blackcurrant berries; (ii) to illustrate the effect of freezing and the

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state of ripeness on the contents of volatile constituents; (iii) to investigate the

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potential to classify blackcurrants based on the volatile profiles of fresh fruits and (iv)



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to assess the contributions of volatile constituents to the overall aroma by GC/O and

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aroma profile testing.

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MATERIALS AND METHODS

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Blackcurrant Material. Blackcurrant berries of the cultivar 'Andega' were harvested

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at the Allotment Garden of the University of Applied Sciences Weihenstephan-

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Triesdorf in Freising, Germany in two seasons (1 July, 2014; 13 July, 2015; 16 July,

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2015). The following cultivars were hand-picked in two seasons at Lehr- und

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Beispielsbetrieb für Obstbau in Deutenkofen, Germany: '8 Bona' (20 June, 2014; 29

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June, 2015), 'Ben Sarek' (20 June, 2014; 3 July, 2015), 'Rosenthals' (20 June, 2014;

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17 June, 2015; 3 July, 2015), 'Silvergieters' (20 June, 2014; 29 June 2015),

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'Supernova' (20 June, 2014; 29 June, 2015; 3 July, 2015), 'Titania' (20 June, 2014; 3

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July, 2014; 11 June, 2015; 17 June, 2015; 23 June, 2015; 29 June, 2015; 3 July,

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2015; 14 July, 2015), 'Tsema' (20 June, 2014; 29 June, 2015), and 'Ometa' (20 June,

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2014; 3 July, 2015). Except for three batches of 'Titania' (11 June, 2015; 17 June,

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2015; 23 June, 2015) and one batch of 'Rosenthals' (17 June, 2015), the berries

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were harvested at the ripe state. The degree of ripeness was evaluated on the basis

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of color and firmness. The color of unripe berries is green to light-red and changes to

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black at the ripe stage. Unripe berries are very hard and are getting softer during the

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maturation. In addition, commercially available blackcurrant berries were purchased

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at different time points ('Supernova': 22 July, 2014; 20 July, 2015; 4 July, 2016;

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'Tenah': 24 July 2014; 29 July, 2014; 28 July 2015; 'Tsema': 4 August, 2015; an

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undefined cultivar: 10 July, 2014; 12 July, 2014). The fruits had been declared to

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originate from locations in Southern Germany (Bühl, Oberkirch, and Tettnang) and

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Austria (Gleinstätten); no information on the date of harvest was available. 4 ACS Paragon Plus Environment

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Chemicals. Authentic reference chemicals were purchased from Alfa Aesar

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(Karlsruhe, Germany), Fluka (Steinheim, Germany), Merck (Darmstadt, Germany),

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SAFC (Steinheim, Germany), and Sigma-Aldrich (Steinheim, Germany) or provided

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by Frey+Lau GmbH (Henstedt-Ulzburg, Germany) and Silesia (Neuss, Germany).

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Heptan-2-ol and malic acid were purchased from Fluka (Steinheim, Germany), citric

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acid from Bernd Kraft (Duisburg, Germany), calcium chloride dihydrate, ethanol,

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fructose, glucose, and sucrose from Sigma-Aldrich (Steinheim, Germany), and

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ascorbic acid, sodium sulfate, and tartaric acid from VWR (Leuven, Belgium). The

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solvents diethyl ether (Merck, Darmstadt, Germany) and pentane (VWR, Fontenay-

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sous-Bois, France) were distilled before use.

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Isolation of Volatiles by Vacuum-Headspace Extraction (VHS). After removal of

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the peduncles, berries that had been stored at 4 °C were brought to room

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temperature. Frozen berries that had been stored at -20 °C were thawed overnight.

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Five hundred grams of blackcurrant fruits were homogenized (30 s) in a laboratory

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blender with 400 mL of water and heptan-2-ol as internal standard (150 µg). The

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homogenate was transferred into a 2 L round bottom flask, and the laboratory

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blender was rinsed with 150 mL of water. The flask was placed into a water bath at

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approximately 35 °C, and after connecting to a vacuum pump the isolation was

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performed at 1-10 mbar for 2 h. The aqueous distillate was condensed in three

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cooling traps which were cooled by a water-ice-mixture (I and II) and liquid nitrogen

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(III). The aqueous condensates were thawed, pooled, and extracted three times with

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50 mL of a mixture of diethyl ether and pentane (1:1; v/v). After drying over

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anhydrous sodium sulfate, the extract was concentrated at 40 °C to 1 mL using a

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Vigreux column. The extracts were further concentrated to a final volume of 0.5 mL

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under a gentle nitrogen flow and analyzed by capillary gas chromatography 5 ACS Paragon Plus Environment


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(HRGC/FID) and gas chromatography-mass spectrometry (GC-MS). This isolation of

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volatiles via VHS was performed in triplicate for each batch of blackcurrants.

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For isolations involving the inhibition of enzymes, calcium chloride solution instead of

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water was added after homogenization of the berries for 30, 60, 90 and 180 s,

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respectively.

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Capillary gas chromatography (HRGC/FID). The separations were performed on a

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Carlo Erba HRGC Mega II 8575 series gas chromatograph (Thermo Fisher Scientific,

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Dreieich, Germany) equipped with a flame ionization detector (FID) and a flame

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photometric detector (FPD) operating at 235 °C. Injections were performed with a

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split/splitless injector (215 °C, split ratio 1:10) using the capillary column DB-Wax

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(60 m x 0.32 mm i.d., 0.25 µm film thickness; J&W Scientific). The inlet oven

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temperature was programmed from 40 °C (5 min hold) at 4 °C/min to 240 °C (25 min

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hold). Hydrogen (5.0) was used as the carrier gas at a constant inlet pressure of

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110 kPa. Data acquisition was done with Chromcard software, version 2.5 (Thermo

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Fisher Scientific).

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Quantitation. Quantitation was based on heptan-2-ol as internal standard; 1 mL of a

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1:5 diluted stock solution (0.150 g/200 mL water) was added to blackcurrant berries.

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FID-response factors were determined with solutions of authentic reference

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compounds relative to heptan-2-ol (0.1 µg/µL in diethyl ether). Recoveries by VHS

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were determined in triplicates from aqueous solutions; 100 µL of stock solution

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(3 mg/mL of reference and heptan-2-ol in ethanol) was added to 1 L water and

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isolated via VHS. The limits of detection and the limits of quantitation were

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determined using the method of Hädrich and Vogelgesang.25 Five concentrations of

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octanal, (E)-oct-2-enal, ethyl hexanoate, methyl 3-hydroxybutanoate, and pent-1-en-

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3-ol between 780 and 12500 ng/mL were analyzed in triplicate, and by determining a 6 ACS Paragon Plus Environment

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calibration curve, the limits of detection and the limits of quantitation were calculated

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(assumption: recovery rate and response factor = 1).

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The quantitation of aroma-active compounds which were present below the limits of

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quantitation using GC/FID was performed via GC-MS in the selected ion monitoring

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(SIM) mode. Ten extracts obtained via VHS were pooled, concentrated to a volume

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of 0.25 mL and analyzed using 740 ng heptan-2-ol and 51 ng 2-methyl-5,6-

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diethylypyrazine (for the pyrazines), respectively, as internal standards.

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Gas chromatography-mass spectrometry (GC-MS). Mass spectral data were

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obtained with a GC 8000TOP interfaced with a Voyager GC-MS (Thermo Fisher

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Scientific, Dreieich, Germany). Injections were performed with a split/splitless injector

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(220 °C, split ratio 1:50) using the capillary column DB-WaxEtr (30 m x 0.25 mm i.d.,

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0.5 µm film thickness; J&W Scientific). The inlet oven temperature was programmed

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from 40 °C (5 min hold) at 4 °C/min to 240 °C (35 min hold). Helium (5.0) was used

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as the carrier gas at a constant inlet pressure of 75 kPa and at a flow rate of

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1 mL/min. Ionization was set at 70 eV, the source was kept at 200 °C and the

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interface temperature at 240 °C. Detection was based on scanning a mass range

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from m/z 25 to 250 .

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For quantitations in the SIM mode (dwell time 0.133) characteristic fragment ions

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were used. Data acquisition was done with Xcalibur software, version 1.4 (Thermo

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Fisher Scientific).

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Statistical data analysis. Each experiment was performed in triplicate and results

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were presented as mean ± standard deviation, if the data were normally distributed or

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as median (minimum-maximum), if the data were not normally distributed. Normal

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distribution was tested with Shapiro-Wilk test and equality of variances with Fisher´s

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F-Test. If normal distribution and equality of variances were shown, unpaired 7 ACS Paragon Plus Environment


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Student’s t-test was used to test for equality of means. Welch’s t-test was performed,

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if the data were normally distributed but equality of variances was not shown. Non-

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parametric Wilcoxon-Mann-Whitney U-test was used to compare medians, if data

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were not normally distributed. All tests were two-tailed and differences were

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considered as statistically significant at p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***).

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Statistical analyses were performed using XLSTAT 2017 (Addinsoft, Paris, France).

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Gas chromatography-olfactometry (GC/O). The separations were performed on a

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Trace GC Ultra gas chromatograph (Thermo Fisher Scientific, Dreieich, Germany)

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equipped with an FID (250 °C) and a sniffing port (200 °C). Injections were performed

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on a cold-on-column injector (injection volume 0.5 µL) using the capillary column DB-

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Wax (60 m x 0.32 mm i.d., 0.25 µm film thickness; J&W Scientific). The inlet oven

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temperature was programmed from 35 °C (1 min hold) at 30 °C/min to 40 °C (4 min

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hold) and was then increased at 4 °C/min to 240 °C (25 min hold). For the purity

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check of reference substances the inlet oven temperature was programmed from

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35 °C (1 min hold) at 10 °C/min to 240 °C and held for 15 min. Hydrogen (5.0) was

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used as carrier gas at a constant inlet pressure of 75 kPa. The GC effluent was split

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1:1 among FID and sniffing port; no humidified air or nitrogen was used.

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Aroma extract dilution analysis (AEDA). Twelve extracts (each 500 µL) obtained

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by VHS from ripe blackcurrant berries (cultivar: 'Titania'; dates of harvest: 29 June,

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2015; 3 July, 2015; 14 July, 2015; location: Deutenkofen) were pooled. One half of

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this combined extract was concentrated to 1.5 µL under a gentle nitrogen flow and

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subsequently used for AEDA. The concentrated extract was diluted gradually with a

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mixture of diethyl ether and pentane (1:1; v/v) and analyzed by two panelists by

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GC/O until no odor was detectable.


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Reconstitution experiments. The reconstitution model was prepared based on the

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following concentrations of aroma-active compounds determined in the cultivar

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'Tsema' (purchased: 4 August, 2015): methyl butanoate (795 µg/L), (E)-hex-2-enal

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(632 µg/L), (Z)-hex-3-enal (285 µg/L), ethyl butanoate (240 µg/L), (Z)-hex-3-en-1-ol

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(90 µg/L), α-pinene (85 µg/L), hexanal (70 µg/L), citronellol (52 µg/L), 1,8-cineole

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(44 µg/L), decanal (6 µg/L), linalool (5 µg/L), and pent-1-en-3-one (4 µg/L). The

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concentration of 4-methoxy-2-methyl-2-butanethiol (0.35 µg/L) was based on the

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quantitation in this batch after enrichment on mercurated agarose gel, as previously

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reported.24 Aroma-active compounds, which were present in this batch at

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concentrations below the limits of quantitation were added based on the

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concentrations determined in a pooled extract from ten batches of the cultivar

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'Supernova' (4 July, 2016): (Z)-rose oxide (1.59 µg/L), (E)-non-2-enal (0.57 µg/L),

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methional (0.29 µg/L), oct-1-en-3-one (0.24 µg/L), 2-isobutyl-3-methoxypyrazine

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(0.06 µg/L),

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methoxypyrazine (0.04 µg/L). In a second reconstitution experiment a lower

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concentration of (E)-hex-2-enal (22 µg/L) was used to assess the influence of this

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odorant on the aroma profile. All reference substances were checked for purity by

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GC/O. The odorants were dissolved in a blackcurrant-type matrix (49 g/L fructose,

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37 g/L glucose, 10 g/L sucrose, 2.5 g/L ascorbic acid, 22 g/L citric acid, 3 g/L malic

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acid, 0.9 g/L tartaric acid).

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Aroma profile test. The aroma profile test was performed by a panel of 15

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participants (10 females and 5 males; age from 21 to 76 years; median: 29 years).

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They had been trained in pre-sessions to assess the odorants used as descriptors

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and had given informed consent to participate. Samples (15 mL) were placed into

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glasses with lids and were orthonasally evaluated by the panel. The odorants

2-isopropyl-3-methoxypyrazine

(0.05 µg/L),


and

2-sec-butyl-3-


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(descriptors) ethyl butanoate (pineapple-like), methyl butanoate (cheesy-fruity), 1,8-

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cineole (eucalyptus-like), (Z)-hex-3-enal (grassy), (E)-hex-2-enal (apple-like), 4-

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methoxy-2-methyl-2-butanethiol (catty), 2-isopropyl-3-methoxypyrazine (musty), and

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pent-1-en-3-one (pungent-solvent-like) were dissolved in water at concentrations 50

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times above their odor thresholds. The panelists assessed each descriptor on a

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seven-point discontinuous scale from 0 (not detectable) to 3 (strong). The sensory

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evaluation of the blackcurrant was performed with 15 g of freshly mashed berries.

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RESULTS AND DISCUSSION

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Isolation of volatiles from fresh blackcurrant berries. Volatile constituents were

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isolated from fresh blackcurrant berries by VHS, a method previously applied to

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various foods such as passion fruits, rhubarb, gooseberries, and jostaberries.26-30

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Commercially available blackcurrant berries as well as berries hand-picked at two

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locations in Southern Germany were investigated. The obtained VHS extracts were

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analyzed via GC/FID and GC-MS. In total, 156 compounds (30 tentatively) were

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identified in 35 batches. Fifty-seven of these compounds (22 tentatively) were

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reported for the first time in blackcurrant berries. The distributions of volatiles

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determined in three cultivars are shown as examples in Table 1.

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In agreement with existing data, the major classes of volatiles isolated from fresh

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blackcurrant fruits were terpenes and esters. However, the rather high concentrations

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of C6-compounds, a typical class of so-called ‘secondary’ flavor compounds,

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constituted a striking difference compared to previous studies.3-12,15,18-21 In all these

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investigations frozen blackcurrant berries had been analyzed; this prompted us to

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specifically address the impact of freezing on the spectrum of blackcurrant volatiles.


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Impact of freezing on the volatile composition of blackcurrant berries.

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Blackcurrant berries of two cultivars were investigated directly after purchase and

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after 3, 6, 9, and 12 months of storage at -20 °C. The frozen berries showed a less

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intensive odor and were reminiscent of cooked berries. The distributions of the major

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classes of volatile compounds determined upon storage in frozen state up to 12

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months are depicted in Figure 1. In addition, the concentrations of individual volatiles

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analyzed directly after purchase and after 9 months of storage at -20°C are listed in

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Table 2. The most pronounced influence of the freezing step was observed for the

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C6-compounds. The

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corresponding alcohols, typical representatives of enzymatically formed degradation

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products of unsaturated fatty acids, decreased upon storage in frozen state; except

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for (Z)-hex-3-en-1-ol, they were all below their limits of detection (Table 2). On the

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other hand, the concentrations of hexanal, heptanal, (E)-hept-2-enal, (E)-oct-2-enal,

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decanal, and nonanal, known autoxidation products of linoleic acid and oleic acid31

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were increased after freezing. These results are in agreement with the low

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concentrations or the absence of C6-compounds in nearly all studies in which frozen

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blackcurrant fruits have been investigated.3-4,6-8,18 The increased concentration of

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hexanal upon storage in frozen state for 9 months is in agreement with the fact that

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this C6-compound has been quantitated at concentrations between 192 and

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1180 µg/kg in studies with frozen blackcurrant berries.20-21 Only one study performed

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with frozen fruits also reported the presence of (E)-hex-2-enal at high concentrations

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(up to 201 µg/kg).21 Otherwise, the C6-compounds (E)-hex-2-enal, (Z)-hex-3-enal,

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(E)-hexen-2-en-1-ol and (Z)-hex-3-en-1-ol have only been reported as volatile

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constituents in one of the few studies dealing with fresh blackcurrant fruits.28

concentrations

of

unsaturated C6-aldehydes


and their


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For all detected esters, except for ethyl butanoate in cultivar 'Tenah', the mean

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contents after storage at -20°C for 9 months were lower than those in the fresh fruits.

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However, owing to the lack of normal distribution and equality of variances,

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respectively, the differences in concentrations could only be shown to be statistically

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significant for ethyl butanoate in the cultivar 'Supernova' and for ethyl and methyl

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octanoate.

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For the terpenes, there were sporadic statistically significant differences, i.e.

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decreases of the concentrations of hydrocarbons and increases of the concentrations

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of 1,8-cineole and the terpene alcohols sabinene hydrate and α-terpineol, in frozen

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compared to fresh berries. However, these differences were only observed in one of

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the two investigated cultivars, and there was no consistent trend. This is in

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agreement with a previous study demonstrating that the relative distribution of

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terpenes was not affected by freezing of blackcurrant berries and that there were no

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significant changes in the enantiomeric distributions of chiral terpenes.15

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In conclusion, the data show that storage of blackcurrants in frozen state results in

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drastically reduced concentrations of C6-compounds. In combination with lowered

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concentrations of esters, this leads to a shift of the distribution of volatile constituents

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in favor of the terpenes in the frozen material.

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Impact of enzymatic reactions on the spectrum of C6-compounds. In addition to

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the effect of freezing, the influence of enzymatic reactions on the formation of

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C6-compounds was followed. Enzymatic activities were inhibited by addition of

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saturated aqueous calcium chloride solution to crushed berries of three batches after

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30, 60, 90, and 180 s. As shown in Figure 2, the total amounts of C6-compounds

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increased over time in the three investigated cultivars. However, the spectrum of

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C6-compounds was changing, that is, the content of (Z)-hex-3-enal decreased in 12 ACS Paragon Plus Environment

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favor of the isomer (E)-hex-2-enal and the corresponding alcohols (E)-hex-2-en-1-ol

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and (Z)-hex-3-en-1-ol. The compounds generated from linoleic acid, hexanal and

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hexan-1-ol, also increased. Similar changes have been observed in rhubarb.27 The

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extent of the isomerization of (Z)-hex-3-enal to (E)-hex-2-enal was different in the

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three cultivars; different proportions were observed upon enzyme-inhibition after

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180 s and these differences were even more pronounced after work-up of the berries

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without inhibition of enzymes.

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Impact of ripeness on the volatile composition of blackcurrant berries. Two

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blackcurrant cultivars were analyzed at the unripe and the ripe state (Table 3). The

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classification was based on color and firmness of the fruits. The unripe berries were

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green to light-red and very hard; in the ripe state they were soft and had turned to

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black color. The most pronounced impact of the state of ripeness was seen for the

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esters. In unripe berries of the cultivar 'Titania' none of the investigated short-chained

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esters was present above the limit of the detection; in the cultivar 'Rosenthals' there

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were statistically significant increases in the concentrations of ethyl butanoate and

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methyl hexanoate. The increase of the concentrations of esters during ripening is in

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agreement with data reported for other fruits such as gooseberries, jostaberries,

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guava and kiwi.28-29,32-34

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In the class of terpenes, there were statistically significant decreases of the

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concentrations of several monoterpene hydrocarbons upon ripening in the cultivar

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'Titania'. However, due to the lack of normal distribution and equality of variance,

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respectively, they could hardly be confirmed in the cultivar 'Rosenthals'. Decreasing

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concentrations of terpene hydrocarbons upon ripening of blackcurrants have been

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observed in two other studies.13,14 On the other hand, there have also been reports of

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increasing concentrations of ∆-3-carene, terpinolene and β-caryophyllene during 13 ACS Paragon Plus Environment


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ripening.5 The concentrations of the monoterpene alcohols terpinen-4-ol, sabinene

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hydrate, β-linalool and citronellol decreased during ripening, whereas the data

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observed for 1,8-cineole and α-terpineol were inconsistent.

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The total amounts of C6-compounds decreased upon ripening; this was mainly due to

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decreasing concentrations of (Z)-hex-3-enal and the corresponding alcohol (Z)-hex-

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3-en-1-ol. In unripe berries (Z)-hex-3-enal is the main compound, whereas in ripe

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berries (E)-hex-2-enal becomes the dominating compound. Similar results have been

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shown for gooseberries and jostaberries, two other representatives of the family

312

Grossulariaceae.28-29 Decreases of the concentrations of C6-compounds during

313

ripening have also been detected in guava and nectarine.33-35 On the other hand, in

314

kiwis no changes have been observed and in cherries an increase of C6-compounds

315

has been shown.32,36

316

Variability of the volatile compositions of blackcurrant berries. The distributions

317

of volatile constituents were analyzed in nine blackcurrant cultivars. All investigated

318

cultivars met the following conditions: (i) In contrast to previous studies on varietal

319

differences,12,20-21 fresh rather than frozen berries were analyzed; (ii) the fruits were

320

grown at the same location in Southern Germany (except for cultivar Andega); (iii) in

321

order to minimize the effect of the stage of ripeness, the fruits were hand-picked at

322

similar states of color and firmness; (iv) to confirm the consistencies of the observed

323

differences, fruits were harvested and analyzed in two consecutive seasons (2014

324

and 2015).

325

The distributions of the concentrations of selected representatives of the three

326

compound classes esters, terpenes and C6-compounds are depicted in Figure 3.

327

Large variations were observed for the esters methyl butanoate (37 - 33300 µg/kg),

328

ethyl butanoate (31 - 2827 µg/kg) and methyl hexanoate (5 – 634 µg/kg), as well as 14 ACS Paragon Plus Environment

Page 14 of 43

Page 15 of 43


329

for the main terpene-hydrocarbons β-phellandrene (56 - 3787 µg/kg), sabinene (8 -

330

2436 µg/kg), ∆-3-carene (44 - 1945 µg/kg) and the terpene-alcohol terpinen-4-ol (26 -

331

1588 µg/kg). This variability is in agreement with data previously reported for frozen

332

blackcurrant fruits.13,15,20-21 Regarding the esters, a quantitative preponderance of

333

methyl butanoate compared to ethyl butanoate was observed in six of the nine

334

investigated cultivars. The same trend could be observed for ethyl and methyl

335

octanoate (data not shown). For Tsema a predominance of methyl butanoate has

336

also been observed in a previous study20, but in most cultivars investigated the ethyl

337

ester showed higher concentrations than the methyl ester.20-21 In most other fruits

338

ethyl and methyl esters were in the same order of magnitude or ethyl esters

339

predominated.30,37-38 Higher concentrations of methyl esters have been previously

340

observed in gooseberries and jostaberries, two other representatives of the family

341

Grossulariaceae, and in pineapple.28-29,39

342

In accordance with the literature, main representatives in the group of terpenes were

343

sabinene, β-phellandrene and ∆-3-carene. Data on varietal differences in the spectra

344

of C6-compounds have not been available, owing to the nearly exclusive investigation

345

of frozen berries.3-12,15,18-21 The aldehyde (E)-hex-2-enal was identified in all batches,

346

except in one of 'Tsema', as the quantitatively dominating C6-compound (Figure 3C).

347

(E)-Hex-2-enal has also been found as dominating C6-compound in other fruits, like

348

jostaberries, kiwis, and nectarines.28,35,38,40

349

Despite the large variability of individual volatile constituents, the investigated

350

cultivars could be divided into three main categories, depending on their contents of

351

esters, terpenes, and C6-compounds (Figure 4A). Except for 'Silvergieters' and

352

'Tsema', these classifications were consistent for the two investigated years.

353

In addition to the fruits grown at the same location and picked at the same state of

354

ripeness, blackcurrant berries purchased at a local store were analyzed (Figure 4B). 15 ACS Paragon Plus Environment


355

For these fruits no consistent classification was possible. For example, the batch of

356

cultivar 'Supernova' purchased in 2014 was an ester-type, whereas the batch

357

purchased in 2015 was a terpene-type. Two batches of cultivar 'Tenah' purchased in

358

2014 were very different in composition; on the other hand, a batch bought in 2015

359

showed nearly the same distribution of compound classes as one of the batches from

360

2014. The actual dates of harvest of the purchased blackcurrant berries and their

361

exact growing locations were not known. Assuming that the cultivars were correctly

362

identified by the vendor, the impact of these parameters seems to be so decisive that

363

without this information a varietal classification of blackcurrant fruits based on the

364

volatiles is not possible.

365 366

Screening of the sensory contributions of aroma compounds. A pooled and

367

concentrated extract resulting from the isolation of volatiles via VHS from 3 kg of

368

fresh blackcurrant berries of the cultivar 'Titania' was subjected to AEDA and

369

assessed via GC/O. A total of 36 odor-active constituents were consistently detected

370

by two panelists. Twenty-six were identified by comparison of chromatographic, mass

371

spectrometric and sensory data with those of authentic reference compounds (Table

372

4).

373

2-isobutyl-3-methoxypyrazine, (Z)-octa-1,5-octadien-3-one and 4-methoxy-2-methyl-

374

2-butanethiol as the volatiles with the highest flavor dilution (FD) factors. As a second

375

step, odor activity values (OAV), that is, the ratios of the concentrations of the

376

individual substances and their odor thresholds were calculated. In a previous study,

377

4-methoxy-2-methyl-2-butanethiol has been shown to contribute to the aroma of

378

blackcurrant berries, and its concentration has been determined in seven cultivars.

379

Except for the cultivar 'Andega' which showed an outstandingly high concentration of

380

this sulfur-containing aroma compound, the concentration in the other investigated

Both panelists

detected ethyl

butanoate,

2-isopropyl-3-methoxypyrazine,


Page 16 of 43

Page 17 of 43


381

cultivars ranged from 0.16 to 0.72 µg/kg.24 The concentration of 4-methoxy-2-methyl-

382

2-butanethiol determined in the cultivar 'Tsema' (0.35 µg/kg) was nearly identical to

383

the

384

previously determined in six cultivars.24 Therefore, the concentrations of aroma-

385

active compounds determined in this cultivar were used as basis for the calculation of

386

the OAVs. However, several compounds with high FD factors, such as 2-isobutyl-3-

387

methoxypyrazine, 2-isopropyl-3-methoxypyrazine and oct-1-en-3-one, were present

388

in the extract below the respective limits of quantitation using GC/FID as detection

389

mode. Therefore, ten extracts obtained from a total of 5 kg of blackcurrant berries of

390

the cultivar 'Tsema' via VHS were pooled and quantitations were performed in the

391

SIM-mode.

392

The 20 compounds listed in Table 4 with an OAV ≥ 1 were used as a basis for

393

reconstitution experiments to confirm their importance to the blackcurrant aroma. To

394

imitate blackcurrant berries, these compounds were dissolved in a blackcurrant-type

395

matrix containing organic acids and sugars. According to the panelists, the

396

recombinate was reminiscent of blackcurrant berries. The aroma profile was also in

397

fairly good agreement with that of fresh blackcurrant berries; however, the descriptor

398

apple-like was rated higher in the recombinate (Figure 5). Therefore, in a second

399

recombinate the concentration of this apple/marzipan-like smelling compound was

400

reduced to 22 µg/L. This corresponded to the concentration determined in the

401

experiment with the cultivar 'Tenah' involving the inhibition of enzymes after 30 s

402

(Figure 2). Although this concentration was lower than the odor threshold of 77 µg/L

403

reported for (E)-hex-2-enal in water28, the difference in perception of this descriptor

404

between the fresh fruits and the recombinate remained. This indicates that there

405

might be rather narrow ranges regarding the concentrations and/or specific

406

proportions of aroma-active C6-compounds that are required for the blackcurrant

average

concentration

of

4-methoxy-2-methyl-2-butanethiol


(0.34 µg/kg)


407

berry aroma. The demonstrated time-dependent dynamics in the enzymatic formation

408

of C6-compounds upon crushing of blackcurrant berries renders the elucidation of this

409

phenomenon even more difficult.

410

In conclusion, the study demonstrated that freezing of blackcurrant berries prior to

411

the investigation of volatile constituents results in overlooking an important class of

412

compounds, that is, C6-compounds formed upon crushing of fresh berries. Their time-

413

dependent enzymatic formation and isomerization adds an additional element of

414

variability to the spectrum of fresh blackcurrant volatiles. Nevertheless, data obtained

415

from two seasons indicate that blackcurrant cultivars can be classified according to

416

their contents of C6-compounds, esters and terpenes in the fresh fruits, if certain

417

prerequisites, such as the same growing location and the same state of ripeness are

418

met.

419

The occurrence of the sulfur-containing compound 4-methoxy-2-methyl-2-butanthiol,

420

of the terpenoid 1,8-cineole and of three alkyl-substituted 3-methoxypyrazines at

421

concentrations above their odor thresholds, constitutes a decisive difference between

422

blackcurrants and the botanically related gooseberries (Ribes uva crispa L.) or

423

jostaberries (Ribes x nidigrolaria Bauer), a hybrid of blackcurrant and gooseberry.28-29

424

A recombination of the aroma of fresh blackcurrants fruits could not be fully achieved

425

in the present study. Further investigations of the dynamics of the enzymatic

426

formations and isomerizations of the C6-compounds might be the key to solve this

427

issue.

428 429 430

SUPPORTING INFORMATION


Page 18 of 43

Page 19 of 43


431

Compounds identified in blackcurrant in addition to those listed in Table 1, recovery

432

rates for the isolation of volatiles via VHS, characteristic fragment ions for

433

quantitations in SIM mode. This material is available free of charge via the Internet at

434

http://pubs.acs.org.

435



Page 20 of 43

436

REFERENCES

437

1.

438

Thieme Verlag: Stuttgart, 2006.

439

2.

440

Strauchbeerenanbau und -ernte, Fachserie 3 Reihe 3.1.9. Statistisches Bundesamt:

441

Wiesbaden, 2017.

442

3.

443

compounds. Acta Chem. Scand. 1964, 18, 1105-1114.

444

4.

445

compounds. Acta Chem. Scand. 1966, 20, 522-528.

446

5.

447

characterization of different varieties and stages of ripeness by gas chromatography.

448

Acta Chem. Scand. 1966, 20, 529-532.

449

6.

450

nigrum L. I. A commercial black‐currant distillate. J. Sci. Food Agric. 1969, 20, 91-98.

451

7.

452

nigrum L. II.—The fresh fruit. J. Sci. Food Agric. 1969, 20, 613-619.

453

8.

454

measured by instrumental methods. Lebensm. Wiss. Technol. 1971, 4, 54-58.

455

9.

456

measured by odour quality assessment techniques. Lebensm. Wiss. Technol. 1971,

457

4, 152-157.

458

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between instrumental and sensory data from unheated and heated black currants.

460

Lebensm. Wiss. Technol. 1973, 6, 86-89.

Eisenbrand, G.; Schreier, P., Römpp Lexikon Lebensmittelchemie. Georg

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und

Forstwirtschaft,

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Andersson, J.; von Sydow, E., The aroma of black currants I. Higher boiling

Andersson, J.; von Sydow, E., The aroma of black currants II. Lower boiling

Andersson, J.; von Sydow, E., The aroma of black currants III. Chemical

Nursten, H. E.; Williams, A. A., Volatile constituents of the black currant, Ribes

Nursten, H. E.; Williams, A. A., Volatile constituents of the black currant, Ribes

von Sydow, E.; Karlsson, G., Aroma of black currants IV. The influence of heat

von Sydow, E.; Karlsson, G., Aroma of black currants V. The influence of heat

Karlsson-Ekstrom, G.; von Sydow, E., Aroma of black currants VI. Correlations


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11.

Latrasse, A.; Rigaud, J.; Sarris, J., Aroma of the blackcurrant berry (Ribes

462

nigrum L.). Main odour and secondary notes. Sci. Aliments 1982, 2, 145-162.

463

12.

464

analytiques de l'arôme de différentes variétés de cassis (Ribes Nigrum L.). In

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Advances in fruit and vegetable juice industry: processes, quality evaluation, product

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development Juris-Verlag: Zürich, 1985; Vol. 18, pp 113-121.

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Biogeneration of aromas, Parliment, T. H.; Croteau, R., Eds. American Chemical

469

Society: Washington, 1986; Vol. 317, pp 184-192.

470

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nigrum L.) berries - their distribution and changes during ripening. In Flavour science

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Sons: New York, 1987; pp 57-62.

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composition

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microextraction/gas chromatography. J. Sci. Food Agric. 2002, 82, 1510-1515.

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J. Food Sci. 2002, 67, 3284-3288.

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isolated from leaves, buds, and berries of Ribes nigrum L. Proc. Estonian Acad. Sci.

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Chem. 2002, 51, 225-234.

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compounds and anthocyanins during black currant (Ribes nigrum L.) juice

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processing. J. Food Sci. 2002, 67, 3447-3455.

Bricout, J.; Clauzure, A.; Desmarest, P.; Menoret, Y., Caractéristiques

Marriott, R. J., Biogenesis of blackcurrant (Ribes nigrum) aroma. In

Marriott, R. J., Monoterpenes and sesquiterpenes in blackcurrant (Ribes

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Tiitinen, K.; Hakala, M.; Pohjanheimo, T.; Tahvonen, R.; Kallio, H., Flavour

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State-of-the-art in flavour chemistry and biology, Hofmann, T.; Rothe, M.; Schieberle,

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P., Eds. Deutsche Forschungsanstalt für Lebensmittelchemie Garching, 2004; pp

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518-522.

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compound profile of blackcurrant berries. In Developments in food science, Bredie,

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W. L. P.; Petersen, M. A., Eds. Elsevier Science: 2006; Vol. 43, pp 257-260.

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of black currant cultivars. In Acta Hortic. 777, P. Banados, A. D., Ed. 2008; pp 525-

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mercapto-butane, a major constituent of the aroma of the blackcurrant bud (Ribes

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nigrum L.). Sci. Aliments 1986, 6, 213-220.

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buds (Ribes nigrum L.). J. Agric. Food Chem. 1990, 38, 3-10.

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4‐methoxy‐2‐methyl‐2‐butanethiol in blackcurrant (Ribes nigrum L.) berries. Flavour

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Fragrance J. 2016, 31, 438-441.

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determination: a statistical approach for practitioners. Accred. Qual. Assur. 1998, 3,

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242-255.

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headspace method in aroma research: flavor chemistry of yellow passion fruits. J.

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Agric. Food Chem. 1998, 46, 1076-1093.

Christensen, L. P.; Pedersen, H. L., Varietal differences in the aroma

Kampuss, K.; Christensen, L. P.; Lindhard Pedersen, H., Volatile composition

Rigaud, J.; Etievant, P.; Henry, R.; Latrasse, A., 4-Methoxy 2-methyl 2-

Le Quéré, J.-L.; Latrasse, A., Composition of the essential oils of black currant

Jung,

K.;

Fastowski,

O.;

Engel,

K.

H.,

Occurrence

of

Vogelgesang, J.; Hädrich, J., Limits of detection, identification and

Werkhoff, P.; Güntert, M.; Krammer, G.; Sommer, H.; Kaulen, J., Vacuum


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27.

Dregus, M.; Engel, K.-H., Volatile constituents of uncooked rhubarb (Rheum

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rhabarbarum L.) stalks. J. Agric. Food Chem. 2003, 51, 6530-6536.

513

28.

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evaluation of jostaberry (Ribes x nidigrolaria Bauer) volatiles. J. Agric. Food Chem.

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2013, 61, 9067-9075.

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Analysis and sensory evaluation of gooseberry (Ribes uva crispa L.) volatiles. J.

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Agric. Food Chem. 2013, 61, 6240-6249.

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30.

520

vacuum headspace method for the analysis of fruit flavors. In Flavor analysis,

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developments in isolation and charecterization, Mussinan, C. J.; Morello, M. J., Eds.

522

American Chemical Society: Washington, 1998; Vol. 705, pp 38-60.

523

31.

524

Autoxidation of unsaturated lipids, Chan, H. W.-S., Ed. Academic Press London,

525

1987; pp 95-139.

526

32.

527

headspace solid-phase microextraction to volatile flavour profile development during

528

storage and ripening of kiwifruit. Food Res. Int. 1999, 32, 175-183.

529

33.

530

active compounds of two varieties of Colombian guava (Psidium guajava L.) during

531

ripening. Eur. Food Res. Technol. 2010, 230, 859-864.

532

34.

533

non-volatile chemical composition of the white guava fruit (Psidium guajava) at

534

different stages of maturity. Food Chem. 2007, 100, 15-21.

Hempfling, K.; Fastowski, O.; Celik, J.; Engel, K.-H., Analysis and sensory

Hempfling, K.; Fastowski, O.; Kopp, M.; Pour Nikfardjam, M.; Engel, K.-H.,

Güntert, M.; Krammer, G.; Sommer, H.; Werkhoff, P., The importance of the

Grosch, W., Reactions of hydroperoxides-products of low molecular weight. In

Wan, X. M.; Stevenson, R. J.; Chen, X. D.; Melton, L. D., Application of

Sinuco, D. C.; Steinhaus, M.; Schieberle, P.; Osorio, C., Changes in odour-

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35.

Engel, K.-H.; Ramming, D. W.; Flath, R. A.; Teranishi, R., Investigation of

536

volatile constituents in nectarines. 2. Changes in aroma composition during nectarine

537

maturation. J. Agric. Food Chem. 1988, 36, 1003-1006.

538

36.

539

de Guía Córdoba, M. a., Effect of the commercial ripening stage and postharvest

540

storage on microbial and aroma changes of ‘Ambrunés’ sweet cherries. J. Agric.

541

Food Chem. 2010, 58, 9157-9163.

542

37.

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strawberries, Fragaria ananassa cv. Senga Sengana, Senga Litessa and Senga

544

Gourmella. J. Sci. Food Agric. 1980, 31, 487-494.

545

38.

546

affecting

547

authentication, Rouseff, R. L.; Leahy, M. M., Eds. Americal Chemical Society:

548

Washington, 1995; Vol. 596, pp 59-67.

549

39.

550

Wieczorek, R. L.; Guentert, M., Volatile constituents of pineapple (Ananas comosus

551

[L.] Merr.). In Flavor chemistry trends and developments, Teranishi, R.; Buttery, R.

552

G.; Shahidi, F., Eds. American Chemical Society: Washington, 1989; Vol. 388, pp

553

223-237.

554

40.

555

osmotic dehydration and freezing on the volatile profile of kiwi fruit. Food Res. Int.

556

2003, 36, 635-642.

557

41.

558

qualities and retention indices of key food odorants. Deutsche Forschungsanstalt für

559

Lebensmittelchemie; Institut für Lebensmittelchemie der Technischen Universität

560

München: Garching, 1998.

Serradilla, M. J.; Martín, A.; Hernandez, A.; López-Corrales, M.; Lozano, M.;

Schreier, P., Quantitative composition of volatile constituents in cultivated

Young, H.; Stec, M.; Paterson, V. J.; McMath, K.; Ball, R., Volatile compounds kiwifruit

flavor.

In Fruit

flavors,

biogenesis,

characterization

and

Takeoka, G.; Buttery, R. G.; Flath, R. A.; Teranishi, R.; Wheeler, E. L.;

Talens, P.; Escriche, I.; Martínez-Navarrete, N.; Chiralt, A., Influence of

Rychlik, M.; Schieberle, P.; Grosch, W., Compilation of odor thresholds, odor


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561

42.

Leffingwell & Associates. Services and Software for the Perfume, Flavor, Food

562

and Beverage Industries. http://www.leffingwell.com (Accessed: 1 June 2016).

563

43.

564

constituents in the peel and pulp of a green Thai mango, Khieo Sawoei cultivar

565

(Mangifera indica L.). Food Sci. Technol. Res. 2001, 7, 72-77.

566

44.

567

Characterization of some volatile constituents of carrots. J. Agric. Food Chem. 1968,

568

16, 1009-1015.

569

45.

570

flüchtiger Verbindungen in Stachelbeeren (Ribes uva crispa L.) und Jostabeeren

571

(Ribes x nidigrolaria Bauer). Dissertation, Technische Universität München, 2014.

572

46.

573

to the characteristic aroma of chinese green tea infusions by aroma extract dilution

574

analysis. J. Agric. Food Chem. 2014, 62, 8308-8313.

Tamura, H.; Boonbumrung, S.; Yoshizawa, T.; Varanyanond, W., The volatile

Buttery, R. G.; Seifert, R. M.; Guadagni, D. G.; Black, D. R.; Ling, L.,

Schrade, K. Kapillargaschromatographische und sensorische Untersuchungen

Baba, R.; Kumazawa, K., Characterization of the potent odorants contributing

575



576

FIGURE CAPTIONS

577

Figure 1. Concentrations of volatile compound classes in fresh and frozen

578

blackcurrant berries of the cultivars (A) 'Supernova' (Bühl, 22 July, 2014) and (B)

579

'Tenah' (Gleinstätten, 24 July, 2014).

580

Figure 2. C6-compounds isolated via VHS from blackcurrant berries of the cultivars

581

(A) 'Tsema' (Bühl, 4 August, 2015), (B) 'Tenah' (Oberkirch, 28 July, 2015), and (C) an

582

unknown cultivar (Tettnang, 10 July, 2014), after enzyme inactivations at defined

583

time points (30, 60, 90, and 180 s) and without inhibition (n.i.).

584

Figure 3. Concentrations of major volatiles: (A) esters; (B) terpenes; (C)

585

C6-compounds; isolated via VHS from ripe blackcurrant berries at the locations

586

Deutenkofen and Freising ('Andega') in 2014 (left bars) and 2015 (right bars).

587

Figure 4. Classification of blackcurrant cultivars (A) harvested in Deutenkofen,

588

except for 'Andega' (Freising) in 2014 and 2015 or (B) purchased at a local market

589

(origin: Bühl, Gleinstätten and Oberkirch) based on the distribution of classes of

590

volatile compounds.

591

Figure 5. Aroma profiles of fresh blackcurrant berries (continuous line) and of the

592

reconstitution model (broken line) on the basis of concentrations of odorants in the

593

cultivars 'Tsema' from Bühl (4 August, 2015) and 'Supernova' from Oberkirch (4 July,

594

2016).


Page 26 of 43

Page 27 of 43


Table 1. Volatile Compounds Isolated via VHS from Berries of Three Blackcurrant Cultivars (Deutenkofen, 2014) '8 Bona' compound

RI

a

'Ben Sarek' µg/kg

'Ometa'

b

remark

C6-compounds (E)-hex-2-enal

1199

5977±601

403±84 j

3074±881

c, e

786±124

c, e

(Z)-hex-3-enal

1128

591±496

n.d.

(E)-hex-2-en-1-ol

1394

478±155

169±29

749±57

c, e

hexanal

1067

335±59

15±4

111±38

c, e

(Z)-hex-3-en-1-ol

1373

135±14

26±1

508±30

c, e

(E)-hex-3-enal

1123

83±4

n.d.

55±2

c, g, i

hexan-1-ol

1346

73±18

16±2

127±24

c, e

k

(E)-hex-3-en-1-ol

1353

5±1

n.q.

14±3

d, h, i

(Z)-hex-2-en-1-ol

1404

2±1

n.d.

8±1

c, f

(Z)-hex-2-enal

1185

n.d.

3±1

n.d.

d, h, i

ethyl butanoate

1028

2827±1357

1066±705

295±145

c, e

methyl butanoate

973

445±199

26389±3491

990±234

c, e

bornyl acetate

1563

47±4

5±1

3±0

c, e

ethyl octanoate

1425

23±5

13±10

6±5

c, f

methyl hexanoate

1175

20±9

546±51

50±8

c, e

butyl acetate

1060

21±16

5±2

n.d.

c, e

ethyl 2-hydroxybutanoate

1389

12±5

2±1

n.d.

d, h

(E)-hex-2-enyl acetate

1322

7±3

4±2

2±0

c, e

hexyl acetate

1262

4±4

n.d.

n.q.

c, e

citronellyl acetate

1649

5±1

14±1

21±3

c, f

methyl benzoate

1597

4±2

66±18

22±7

c, e

ethyl 3-hydroxybutanoate

1499

4±1

n.d.

n.d.

c, f, i

(Z)-hex-3-enyl acetate

1302

3±1

8±1

10±8

c, e

methyl salicylate

1746

2±1

8±2

9±3

c, e

methyl octanoate

1378

2±2

59±9

6±2

c, e

linalyl acetate

1544

2±0

n.d.

6±1

c, f

ethyl benzoate

1642

n.q.

6±4

5±3

c, e

ethyl decanoate

1626

n.q.

n.d.

n.q.

c, e

methyl 2-hydroxybutanoate

1362

n.q.

48±4

3±1

d, h, i

methyl decanoate

1584

n.q.

8±3

2±0

c, f

propyl butanoate

1111

n.q.

n.d.

n.d.

d, h, i

geranyl acetate

1742

n.d.

n.d.

5±2

c, f

methyl 3-hydroxybutanoate

1463

n.d.

7±1

n.d.

c, f, i

esters



Page 28 of 43

Table 1. continued '8 Bona' compound

RI

a

'Ben Sarek' µg/kg

'Ometa'

b

remark

1634

n.d.

4±1

n.d.

d, h, i

1040

n.d.

4±1

n.d.

d, h, i

1072

n.d.

4±1

n.d.

c, e, i

1706

n.d.

9±0

5±1

c, f

1680

n.d.

n.d.

9±1

c, f

2-methylbut-3-en-2-ol (Z)-pent-2-en-1-ol

1034

541±153

1530±294

899±166

c, e

1311

67±7

28±10

59±2

c, e, i

butan-1-ol

1136

33±40

18±9

n.d.

c, e

pentan-1-ol

1242

2±1

2±1

4±0

c, f

heptan-1-ol

1448

2±1

2±0

3±1

c, e

propan-1,2-diol

1572

n.c.

l

n.c.

n.c.

c, i

2-ethylhexan-1-ol

1482

n.q.

n.q.

n.q.

c, f

(E)-pent-2-en-1-ol

1301

n.q.

n.d.

2±0

d, h, i

benzyl alcohol

1852

n.q.

3±0

2±0

c, f

nonan-1-ol

1651

n.q.

2±1

2±1

c, f

oct-1-en-3-ol

1442

n.q.

n.q.

3±0

c, e

octan-1-ol

1549

n.q.

n.d.

n.q.

c, e

2-methylpropan-1-ol

1083

n.d.

4±1

3±2

c, e

hept-2-en-1-ol

1500

n.d.

n.q.

n.q.

d, h, i

allylphenol

2478

n.d.

10±1

n.q.

d, h

(E)-pent-2-enal

1109

35±9

7±1

46±2

c, e, i

(E)-hept-2-enal

1304

8±3

2±0

5±1

c, e

nonanal

1379

6±0

6±0

7±3

c, f

neral

1658

2±2

n.d.

7±3

c, e, i

(E)-oct-2-enal

1411

2±0

n.d.

2±0

c, f

(E)-undec-2-enal

1736

n.d.

n.q.

2±1

c, f, i

decanal

1485

n.d.

4±1

4±0

c, e

octanal

1274

n.d.

n.d.

2±0

c, f

pent-1-en-3-one

1008

47±9

4±1

19±4

c, e, i

heptan-2-one

1165

2±0

n.q.

2±0

c, f

3-methyl-3-cyclohexen-1-one

1427

n.d. 28

n.q.

n.q.

d, h, i

methyl 3-hydroxyoctanoate methyl (Z)-but-2-enoate + methyl but-3-enoate methyl pentanoate neryl acetate + benzyl acetate α-terpinyl acetate alcohols

aldehydes

ketones

ACS Paragon Plus Environment

Page 29 of 43



RI

a

'Ben Sarek' µg/kg

'Ometa'

b

remark

terpenes (Z)-β-ocimene

1224

147±18

202±198

340±192

c, e

β-phellandrene

1189

166±46

74±5

283±57

c, e

α-phellandrene

1148

111±57

15±3

30±3

c, e

β-pinene

1090

92±15

98±7

54±14

c, e

limonene

1181

65±15

40±1

147±35

c, e

∆-3-carene

1133

66±16

231±16

1140±258

c, e

myrcene

1151

86±35

15±2

30±3

c, e

(E)-β-ocimene

1239

37±19

52±3

206±110

c, e

terpinolene

1266

48±14

212±11

797±231

c, e

sabinene

1106

44±13

285±84

2436±308

c, e

1,8-cineole

1194

68±36

357±153

222±63

c, e

α-pinene

1010

33±9

17±2

74±11

c, e

terpinen-4-ol

1588

26±12

459±116

878±138

c, e

γ-terpinene

1229

11±3

161±5

249±78

c, e

α-terpineol

1683

14±9

77±29

42±10

c, e

caryophyllene

1577

13±7

14±1

66±18

c, e

α-terpinene

1162

9±2

113±4

186±58

c, e

sabinene hydrate

1456

9±2

56±14

207±57

c, f

spathulenol

2105

10±2

n.d.

n.d.

d, h, i

camphene

1047

9±2

n.q.

n.q.

c, f

citronellol

1755

9±1

5±1

23±0

c, e

linalool

1538

7±0

27±6

86±18

c, e

borneol

1685

3±1

3±1

4±2

c, f

p-cymen-8-ol

1830

2±0

3±0

5±1

c, f

β-phellandren-8-ol

1711

2±0

4±0

5±1

d, h, i

p-cymene

1251

2±0

6±0

8±2

c, e

α-phellandren-8-ol

1703

2±1

2±0

5±1

d, h, i

2-hydroxy 1,8-cineole

1712

n.q.

n.d.

n.q.

d, h, i

2-hydroxy 1,8-cineole

1844

n.q.

6±1

3±1

d, h, i

o/m-cymene

1250

n.q.

n.q.

3±1

d, h

α-thujene

1015

n.q.

3±1

11±2

d, h

2-carene

1116

n.d.

3±0

5±1

c, f, i

alloocimene

1358

n.d.

n.d.

n.q.

c, f

fenchene

1040

n.d.

n.d.

3±1

d, h

geraniol

1834

n.d.

2±1

2±1

c, e



Page 30 of 43


RI

a

'Ben Sarek' µg/kg

'Ometa'

b

remark

germacrene D

1689

n.d.

4±0

12±3

c, e

(Z)-piperitol

1665

n.d.

2±0

3±1

d, h, i

(E)-piperitol

1732

n.d.

3±1

4±1

d, h

β-terpinene

1226

n.d.

n.q.

7±2

d, h, i

sylvestrene

1179

n.d.

n.d.

3±1

d, h, i

acetic acid

1430

n.c.

n.c.

n.c.

c, i

propanoic acid

1515

n.d.

n.c.

n.d.

c, i

butanoic acid

1607

n.c.

n.c.

n.c.

c, i

hexanoic acid

1825

n.c.

n.c.

n.c.

c, i

(E)-hex-2-enoic acid

1943

n.c.

n.c.

n.c.

c, i

(E)-hex-3-enoic acid

1914

n.d.

n.c.

n.d.

c, i

hexadecanoic acid

2855

n.c.

n.c.

n.c.

c

octadecanoic acid

3097

n.c.

n.d.

n.c.

c, i

caryophyllene oxide

1961

3±0

7±1

23±2

c, f

p-α-dimethyl styrene

1416

n.d.

n.q.

2±1

c, f

acids

others

a

Linear retention indices on DB-Wax.

b

Triplicate analysis: means ± standard deviation.

c

Identification based on comparison of GC and mass spectral data with those of authentic reference compounds.

d

Tentatively identified by comparison of mass spectral data with those from database (NIST).

e

Quantitation on the basis of recovery rate and response factor.

f

Quantitation on the basis of response factor, no recovery rate considered.

g

Quantitation on the basis of recovery rate, no response factor considered.

h

No recovery rate and response factor were considered.

i

Identified for the first time in blackcurrant berries. 30 ACS Paragon Plus Environment

Page 31 of 43


j

Below limit of detection (0.5 µg/kg).

k

Below limit of quantification (1.5 µg/kg).

l

Detected but concentration not calculated because of too low recovery via VHS.



Page 32 of 43

Table 2. Concentrations of Main Volatiles in Fresh and Frozen Blackcurrant Berries 'Supernova'a

'Tenah'b frozenc

fresh µg/kgd

compound

frozenc

fresh µg/kgd

C6-compounds (E)-hex-2-enal

423±147

n.d.e

295±67

n.d.

(E)-hex-2-en-1-ol

197±55

n.d.

32±8

n.d.

(Z)-hex-3-en-1-ol

46±13

3±0 (*)

147 (113-160)

3 (3-3)

(Z)-hex-3-enal

41±58

n.d.

1292±329

n.d.

hexan-1-ol

35±4

28±5

21±6

17±2

hexanal

14±11

62±23 (*)

47±9

80±8 (**)

(E)-hex-3-en-1-ol

2±0

n.d.

2±0

n.d.

(Z)-hex-2-en-1-ol

2±0

n.d.

n.d.

n.d.

(E)-hex-3-enal

n.d.

n.d.

18±3

n.d.

10219±3959

2761±1074 (*)

46±45

60±17

methyl butanoate

1633 (710-6752)

1395 (923-1658)

770±481

133±50

methyl hexanoate

45 (20-133)

24 (21-24)

51±18

10±2

esters ethyl butanoate

f

ethyl octanoate

22±5

4±0 (*)

n.q.

methyl octanoate

4±3

n.q.

7±1

3±0 (**)

2-methylbut-3-en-2-ol

3888±141

2448±55 (*)

99 (62-231)

46 (38-77)

butan-1-ol

355±406

428±92

n.d.

11±4

pentan-1-ol

4±1

30±8 (*)

2±0

26±9 (*)

2 (2-2)

5 (5-5)

2 (2-2)

7 (6-7)

(E)-hept-2-enal

3±0

6±1 (**)

4±1

9±1 (**)

nonanal

4±1

7±1 (*)

8±1

8±1

decanal

n.d.

8±1

5±0

8±1 (**)

heptanal

n.d.

n.d.

2±1

4±1 (*)

(E)-oct-2-enal

n.d.

n.d.

2±0

5±0 (***)

∆-3-carene

620±32

669±127

1255±274

978±74

terpinolene

479±36

464±71

904±55

706±34 (**)

(Z)-β-ocimene

373 (212-395)

196 (180-205)

370±41

238±16 (**)

β-phellandrene

224±177

525±163

1261±200

1058±86

n.d.

alcohols

oct-1-en-3-ol aldehydes

terpenes


Page 33 of 43


Table 2. continued 'Supernova'a frozenc

fresh

frozenc

fresh

µg/kgd

compound α-phellandrene

'Tenah'b µg/kgd

162 (161-186)

13 (13-19)

33±5

25±2

myrcene

114±29

149±17

257±47

207±14

terpinen-4-ol

98±38

69±6

49±8

37±6

1,8-cineole

92±39

183±11 (*)

32±13

11±4

limonene

86±51

163±40

383±61

311±21

(E)-β-ocimene

85±16

81±9

247±21

158±10 (**)

sabinene

74±10

102±13 (*)

42±15

35±5

α-pinene

61±5

57±12

98±32

75±11

β-pinene

39±3

42±8

27±8

16±2

α-terpinene

39±4

32±4

49±5

38±3 (*)

caryophyllene

33±6

15±5 (*)

67±32

40±11

γ-terpinene

25±6

21±3

22±2

16±1 (*)

sabinene hydrate

17±5

21±2

12±1

15±2 (*)

α-terpineol

16±4

34±7 (*)

9±5

8±0

a

Bühl (July 22, 2014). b Gleinstätten (July 22, 2014).

c

Blackcurrant berries were stored for 9 months at -20 °C.

d

Triplicate analysis of fresh and frozen fruits; if data were normally distributed, values are mean ± standard deviation; if data were not normally distributed, values are median (minimum-maximum). Unpaired Student’s t-test was used to test for equality of means between fresh and frozen blackcurrant berries of the same variety, if the prerequisites of normal distribution (Shapiro-Wilk test) and equality of variances (Fisher´s F-test) were met; Welch’s t-test was performed, if data were normally distributed but equality of variances was not shown. Non-parametric WilcoxonMann-Whitney U-test was used to compare medians, if normal distribution was shown. All tests were two-tailed. (***), p < 0.001; (**), p < 0.01; (*), p < 0.05.

e

Below limit of detection (0.5 µg/kg). f Below limit of quantification (1.5 µg/kg).



Page 34 of 43

Table 3. Concentrations of Main Volatiles in Unripe and Ripe Blackcurrant Berries 'Rosenthals'a unripe

'Titania'b ripe

compound

unripe

ripe

c

µg/kg

C6-compounds (Z)-hex-3-enal

1520 (487-1635)

11 (9-13)

1121 (1095-1200)

7 (6-8)

(E)-hex-2-enal

866±381

1240±258

1668±288

1317±210

(Z)-hex-3-en-1-ol

371±116

29±4 (*)

1059±329

37±2 (*)

(E)-hex-2-en-1-ol

333±46

358±38

1122±300

288±52 (**)

hexan-1-ol

36±3

40±4

118±28

84±13

hexanal

27±3

47±13

66±8

137±39 (*)

(E)-hex-3-enal

7±3

14±3 (*)

11±1

14±3

(Z)-hex-2-enal

8 (2-8)

8 (7-9)

12±4

10±2

(E)-hex-3-en-1-ol

2±1

2±0

7±2

2±0 (*)

(Z)-hex-2-en-1-ol

2±0

2±1

4±2

3±0

esters methyl butanoate

6±2

31380±13468

n.d.d

4064±1027

ethyl butanoate

3±1

1331±478 (*)

n.d.

1921±259

methyl hexanoate

5±2

634±163 (*)

n.d.

155±10

methyl octanoate

3 (3-4)

59 (52-68)

n.d.

21±1

n.d.

3±1

n.d.

9±1

4 (1-6)

1216 (635-1252)

2 (1-5)

214 (210-244)

sabinene

2048±872

480±179 (*)

42±17

36±3

terpinen-4-ol

1273±177

944±218

106±23

56±7 (*)

∆-3-carene

453 (381-1067)

296 (287-360)

2137±558

725±336 (*)

terpinolene

608±271

277±42

1676±282

617±260 (**)

sabinene hydrate

369±98

106±18 (*)

28±11

11±5 (*)

255 (231-524)

169 (165-189)

475±94

261±81 (*)

240±89

123±15

329±50

120±43 (**)

(E)-β-ocimene

178 (165-368)

96 (90-117)

340±63

125±52 (*)

γ-terpinene

149 (130-374)

92 (89-174)

33±7

18±8

β-phellandrene

134 (121-323)

86 (83-105)

217±51

174±68

99 (88-268)

70 (67-105)

79±18

34±15 (*)

β-linalool

151±32

58±4 (*)

26±4

9±2 (**)

limonene

69 (65-156)

42 (39-53)

109±24

65±26

ethyl octanoate alcohols 2-methylbut-3-en-2-ol terpenes

(Z)-β-ocimene myrcene

α-terpinene


Page 35 of 43


Table 3. continued. 'Rosenthals'a

'Titania'b ripe

unripe

ripe

µg/kg

compound citronellol

unripe c

79±9

35±6 (**)

159±27

36±12 (**)

caryophyllene

34 (23-90)

29 (22-32)

132±34

37±12 (*)

α-pinene

29 (26-64)

29 (24-31)

56±15

30±9

β-pinene

17 (16-38)

14 (11-16)

7±3

18±2 (**)

23±3

157±45 (*)

35±3

24±3 (*)

12 (12-40)

10 (9-11)

32±8

13±6 (*)

15±2

31±7 (*)

16±5

5±1 (*)

1,8-cineole α-phellandrene α-terpineol a

Deutenkofen (17 June, 2015 and 3 July, 2015)

b

Deutenkofen (11 June, 2015 and 14 July, 2015)

c

Triplicate analysis of unripe and ripe fruits; values are mean ± standard deviation where data are normally distributed; values are median (minimum-maximum) where data are not normally distributed. Unpaired Student’s t-test was used to test for equality of means between unripe and ripe blackcurrant berries of the same variety when normal distribution and equality of variances were assumed; Welch’s t-test was performed when data were normally distributed but equality of variances was not assumed. Non-parametric Wilcoxon-Mann-Whitney U-test was used to compare medians when normality was not assumed. All tests were two tailed. (***), p < 0.001; (**), p < 0.01; (*), p < 0.05.

d

Below limit of detection (0.5 µg/kg).



Page 36 of 43

Table 4. Concentrations and Sensory Data of Odorants of Blackcurrant Berries

odorant (Z)-hex-3-enal 4-methoxy-2-methyl-2-butanethiol ethyl butanoate oct-1-en-3-one 1,8-cineole hexanal α-pinene 2-isopropyl-3-methoxypyrazine methyl butanoate 2-isobutyl-3-methoxypyrazine 2-sec-butyl-3-methoxypyrazine (E)-hex-2-enal pent-1-en-3-one (E)-non-2-enal (Z)-rose oxide (Z)-hex-3-en-1-ol decanal citronellol linalool methional methyl pentanoate (E)-hex-3-enal bornyl acetate geraniol α-terpineol (Z)-octa-1,5-dien-3-one

a

RI

1128 1199 1028 1289 1194 1067 1010 1416 973 1512 1482 1200 1008 1517 1351 1373 1485 1755 1538 1426 1072 1123 1563 1834 1683 1354

FD factorc A B

b

odor quality

grassy sulfury, sweat pineapple, fruity mushroom, metallic terpenic, forest grassy, green forest, wood musty sweat, buttery sweet green pepper, mushroom musty, earthy apple, marzipan solvent cucumber flowery green citrus flowery flowery, citrus, sweet, potato musty grassy, green citrus, flowery, fruity citrus fruity, sweet geranium, metallic, green

64 1024 64 32 32 1 256 32 512 4 32 32 1 1 2 8 8 32 32 32 32 64 4 256

4 32 256 16 8 2 32 8 32 2 2 2 8 4 2 8 8 8 16

36

ACS Paragon Plus Environment

odor threshold in water ref. µg/L

µg/kgd

0.6 0.001 2.5 0.005 2 4 6 0.004 63 0.005 0.004 77 1 0.15 0.5 28 5 40 5 0.2 20 160 75 5 182 0.0012

285 0.35f 240 0.24g 44 70 85 0.05g 795 0.06g 0.04g 632 4 0.57g 1.59g 90 6 52 5 0.29g 0.19g 14 8 3g 11 n.d.h

[28] [24] [28] [41] [28] [28] [42] [41] [28] [41] [41] [28] [28] [41] [42] [29] [41] [41] [41] [41] [39] [43] [44] [41] [45] [41]

conc.

OAVe

475 350 96 48 22 18 14 13 13 12 10 8 4 4 3 3 1 1 1 1

Analysis and Sensory Evaluation of Volatile Constituents of Fresh

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