The authors thank the Servicios Centrales de Apoyo a la Investigaci&#243;n from University of Malaga for HS-SPME/GC-MS measurements. We acknowledge the assistance of Sara Fern&#225;ndez-Palacios Campos in volatile quantification. We also thanks GoodBerry&#180;s consortium members for providing the fruit material.

1. Fruit harvesting
<ol>
	<li>Grow between 4 to 6 plants per genotype and/or treatment to ensure sufficient fruit material and variability.</li>
	<li>If possible, harvest the samples on the same date; if there is not enough fruit material, pool together samples harvested on different dates. 
	NOTE: It is recommended that the harvest time (morning, noon, afternoon) remains approximately identical as VOC profiles are affected by daytime/circadian rhythm28,29...

<ol>
	<li>Klee, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+the+flavor+of+fresh+fruits:+Genomics,+biochemistry,+and+biotechnology.">Improving the flavor of fresh fruits: Genomics, biochemistry, and biotechnology.</a> New Phytologist. 187 (1), 44-56 (2010).</li><li>Ferr&#227;o, L. F. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+association+of+volatiles+reveals+candidate+loci+for+blueberry+flavor.">Genome-wide association of volatiles reveals candidate loci for blueberry flavor.</a> New Phytologist. 226 (6), 1725-1737 (2020).</li><li>Klee, H. J., Tieman, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+of+fruit+flavour+preferences.">The genetics of fruit flavour preferences.</a> Nature Reviews Genetics. 19, 347-356 (2018).</li><li>Vallarino, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+quantitative+trait+loci+and+candidate+genes+for+primary+metabolite+content+in+strawberry+fruit.">Identification of quantitative trait loci and candidate genes for primary metabolite content in strawberry fruit.</a> Horticulture Research. 6, 4 (2019).</li><li>Jung, K., Fastowski, O., Poplacean, I., Engel, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+and+sensory+evaluation+of+volatile+constituents+of+fresh+blackcurrant+(Ribes+nigrum+L.)+fruits.">Analysis and sensory evaluation of volatile constituents of fresh blackcurrant (Ribes nigrum L.) fruits.</a> Journal of Agricultural and Food Chemistry. 65 (43), 9475-9487 (2017).</li><li>Vallarino, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+diversity+of+strawberry+germplasm+using+metabolomic+biomarkers.">Genetic diversity of strawberry germplasm using metabolomic biomarkers.</a> Scientific Reports. 8, 14386 (2018).</li><li>Zhang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+the+major+aroma-active+compounds+in+clear+red+raspberry+juice+(Rubus+idaeus+L.+cv.+Heritage)+by+molecular+sensory+science+approaches.">Insights into the major aroma-active compounds in clear red raspberry juice (Rubus idaeus L. cv. Heritage) by molecular sensory science approaches.</a> Food Chemistry. 336, 127721 (2021).</li><li>Farneti, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+blueberry+aroma+complexity+by+chromatographic+and+direct-injection+spectrometric+techniques.">Exploring blueberry aroma complexity by chromatographic and direct-injection spectrometric techniques.</a> Frontiers in Plant Science. 8, 617 (2017).</li><li>Tikunov, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetic+and+functional+analysis+of+flavor+in+commercial+tomato:+the+FLORAL4+gene+underlies+a+QTL+for+floral+aroma+volatiles+in+tomato+fruit.">The genetic and functional analysis of flavor in commercial tomato: the FLORAL4 gene underlies a QTL for floral aroma volatiles in tomato fruit.</a> The Plant Journal. 103 (3), 1189-1204 (2020).</li><li>Tieman, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chemical+genetic+roadmap+to+improved+tomato+flavor.">A chemical genetic roadmap to improved tomato flavor.</a> Science. 355 (6323), 391-394 (2017).</li><li>S&#225;nchez-Sevilla, J. F., Cruz-Rus, E., Valpuesta, V., Botella, M. 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E., Dale, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Horticulture+of+Ribes.">Horticulture of Ribes.</a> Forest Pathology. 40 (3-4), 251-263 (2010).</li><li>Vagiri, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenols+and+ascorbic+acid+in+black+currants+(Ribes+nigrum+L.):+Variation+due+to+genotype,+location,+and+year.">Phenols and ascorbic acid in black currants (Ribes nigrum L.): Variation due to genotype, location, and year.</a> Journal of Agricultural and Food Chemistry. 61 (39), 9298-9306 (2013).</li><li>Marsol-Vall, A., Kortesniemi, M., Karhu, S. 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A., Poll, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+isolation+methods+for+the+determination+of+important+aroma+compounds+in+black+currant+(Ribes+nigrum+L.)+juice,+using+nasal+impact+frequency+profiling.">Comparison of isolation methods for the determination of important aroma compounds in black currant (Ribes nigrum L.) juice, using nasal impact frequency profiling.</a> Journal of Agricultural and Food Chemistry. 52 (6), 1647-1652 (2004).</li><li>Andersson, J., von Sydow, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+aroma+of+black+currants+I.+Higher+boiling+compounds.">The aroma of black currants I. 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The authors declare no conflict of interest.

Breeding for fruit aroma has long been hindered by the complex genetics and biochemistry underlying the synthesis of volatile compounds and the lack of technologies for proper phenotyping. However, recent advances in metabolomic platforms, combined with genomic tools, are finally allowing the identification of the metabolites responsible for consumer preferences and to breed crops with improved flavor3. While most progress has been achieved in the model fruit, tomato9,...

Flavor is an essential quality trait for any fruit, impacting consumer acceptance and thus significantly affecting marketability. Flavor perception involves a combination of the taste and olfactory systems and depends chemically on the presence and concentration of a wide range of compounds that accumulate in edible plant parts, or in case of VOCs, are emitted by the ripe fruit1,2. While traditional breeding has focused on agronomic traits such as yield and pest resistance, fruit quality trait improvement, including flavor, has long been neglected due to the genetic complexity and the difficulty to properly phenotype these characteristics, leading to consumer discontent3,4. Recent advances in metabolomic platforms have been successful in identifying and quantifying key compounds responsible for fruit taste and aroma5,6,7,8. Furthermore, the combination of metabolite profiling with genomic or transcriptomic tools allows the elucidation of the genetics underlying fruit flavor, which in turn will help breeding programs develop new varieties with enhanced organoleptic characteristics2,4,9,10,11,12,13,14.
Blackcurrant (Ribes nigrum) berries are highly appreciated for their flavor and nutritional properties, being widely cultivated across the temperate zones of Europe, Asia, and New Zealand15. Most of the production is processed for food products and beverages, which are very popular in the Nordic countries, mainly due to the berries&#39; organoleptic properties. The intense color and flavor of the fruit are the result of a combination of anthocyanins, sugars, acids, and VOCs present in the ripe fruits16,17,18. The analysis of blackcurrant volatiles goes back to the 1960s19,20,21. More recently, several studies have focused on blackcurrant VOCs, identifying important compounds for fruit aroma perception and assessing the impact of genotype, environment, or storage and processing conditions on VOC content5,17,18,22,23.
Because of its numerous advantages, the technique of choice for high-throughput volatile profiling is HS-SPME/GC-MS24,25. A silica fiber, coated with a polymeric phase, is mounted on a syringe device, allowing the adsorption of the volatiles in the fiber until an equilibrium phase is reached. Headspace extraction protects the fiber from the nonvolatile compounds present in the matrix24. SPME can successfully isolate a high number of VOCs present at highly variable concentrations (parts per billion to parts per million)25. In addition, it is a solvent-free technique that requires limited sample processing. Other advantages of HS-SPME are the ease of automation and its relatively low cost.
However, its success can be limited, depending on the chemical nature of the VOCs, the extraction protocol (including time, temperature, and salt concentration), sample stability, and the availability of sufficient fruit tissue26,27. This paper presents a protocol for blackcurrant VOCs isolated by HS-SPME and analyzed by gas chromatography coupled with an ion trap mass spectrometer. A balance between the quantity of plant material, sample stability, and duration of extraction and chromatography was achieved to be able to process high numbers of blackcurrant samples, some of them presented in this study. In particular, VOC profiles and/or chromatograms of five cultivars (&#39;Andega&#39;, &#39;Ben Tron&#39;, &#39;Ben Gairn&#39;, &#39;Ben Tirran&#39;, and &#39;Tihope&#39;) will be presented and discussed as example data. Furthermore, the same protocol has been successfully put into practice for VOC measurement in other fruit berry species such as strawberry (Fragaria x ananassa), raspberry (Rubusidaeus), and blueberry (Vaccinium spp.).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 mL screw top headspace vials</td>
			<td>Thermo Scientific</td>
			<td>10-HSV</td>
			<td></td>
		</tr>
		<tr>
			<td>18 mm screw cap Silicone/PTFE</td>
			<td>Thermo Scientific</td>
			<td>18-MSC</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Tube with HDPE screw cap</td>
			<td>VWR</td>
			<td>216-0153</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>75002415</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol for HPLC</td>
			<td>Merck</td>
			<td>34860-1L-R</td>
			<td></td>
		</tr>
		<tr>
			<td>N-pentadecane (D32, 98%)</td>
			<td>Cambridge Isotope Laboratories</td>
			<td>DLM-1283-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Merck</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>SPME fiber PDMS/DVB</td>
			<td>Merck</td>
			<td>57345-U</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless grinding jars for TissueLyser</td>
			<td>Qiagen</td>
			<td>69985</td>
			<td></td>
		</tr>
		<tr>
			<td>TissueLyser II</td>
			<td>Qiagen</td>
			<td>85300</td>
			<td>Can be subsituted by mortar and pestle or cryogenic mill</td>
		</tr>
		<tr>
			<td>Trace GC gas chromatograph-ITQ900 ion trap mass spectrometer</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triplus RSH autosampler with automated SPME device</td>
			<td>Thermo Scientific</td>
			<td>1R77010-0450</td>
			<td></td>
		</tr>
		<tr>
			<td>Water for HPLC</td>
			<td>Merck</td>
			<td>270733-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Xcalibur 4.2 SP1</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td>software</td>
		</tr>
	</tbody>
</table>

profiling volatile compounds in blackcurrant fruit using headspace solid-phase microextraction coupled to gas chromatography-mass spectrometry

There is an increasing interest in measuring volatile organic compounds (VOCs) emitted by ripe fruits for the purpose of breeding varieties or cultivars with enhanced organoleptic characteristics and thus, to increase consumer acceptance. High-throughput metabolomic platforms have been recently developed to quantify a wide range of metabolites in different plant tissues, including key compounds responsible for fruit taste and aroma quality (volatilomics). A method using headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS) is described here for the identification and quantification of VOCs emitted by ripe blackcurrant fruits, a berry highly appreciated for its flavor and health benefits.
Ripe fruits of blackcurrant plants (Ribes nigrum) were harvested and directly frozen in liquid nitrogen. After tissue homogenization to produce a fine powder, samples were thawed and immediately mixed with sodium chloride solution. Following centrifugation, the supernatant was transferred into a headspace glass vial containing sodium chloride. VOCs were then extracted using a solid-phase microextraction (SPME) fiber and a gas chromatograph coupled to an ion trap mass spectrometer. Volatile quantification was performed on the resulting ion chromatograms by integrating peak area, using a specific m/z ion for each VOC. Correct VOC annotation was confirmed by comparing retention times and mass spectra of pure commercial standards run under the same conditions as the samples. More than 60 VOCs were identified in ripe blackcurrant fruits grown in contrasting European locations. Among the identified VOCs, key aroma compounds, such as terpenoids and C6 volatiles, can be used as biomarkers for blackcurrant fruit quality. In addition, advantages and disadvantages of the method are discussed, including prospective improvements. Furthermore, the use of controls for batch correction and minimization of drift intensity have been emphasized.

High-throughput VOC profiling in a large set of fruit crops grown under different conditions or locations or belonging to distinct genotypes is necessary for accurate aroma phenotyping. Here, a fast and semi-automated HS-SPME/GC-MS platform for relative VOC quantification in blackcurrant cultivars is presented. VOC detection and identification were based on a library that was developed to profile berry fruit species (Table 1). A typical ripe blackcurrant fruit volatile profile (total ion chromatogram) ob...

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Profiling Volatile Compounds in Blackcurrant Fruit using Headspace Solid-Phase Microextraction Coupled to Gas Chromatography-Mass Spectrometry

A headspace solid-phase microextraction-gas-chromatography platform is described here for fast, reliable, and semi-automated volatile identification and quantification in ripe blackcurrant fruits. This technique can be used to increase knowledge about fruit aroma and to select cultivars with enhanced flavor for the purpose of breeding.

There is an increasing interest in measuring volatile organic compounds (VOCs) emitted by ripe fruits for the purpose of breeding varieties or cultivars ...

Improving fruit aroma is one of the main objective in breeding programs. For this, we need a reliable technique which allow us to measure volatile components in fruits. This technique is fast and semi-automated, allowing to measure up to 22 samples per days.In addition, it is relatively cheap and requires minimal sample processing. This method can be easily applied for all the fruit species, such as economically important berry crops. In addition, the library size of the detected compound can be easily increased.To begin, add one milliliter of sodium chloride solution to a five milliliter tube containing the wade frozen sample. Shake the tube until the sample is completely thawed and homogenized. Then centrifuge at 5, 000 times g for five minutes at room temperature.Cut the end of the 1, 000 microliter pipette tip and use it to transfer to supernatant to the sodium chloride containing headspace file. Add five microliters of internal standard to each sample containing headspace file. Place the closed headspace file in a GC-MS auto sampler, at room temperature, for an automated HS-SPME/GC-MS run, making sure that biological replicates are not placed in successive positions in the auto sampler.Pre-incubate the headspace files for 10 minutes at 52 degrees Celsius with agitation at 17 times G.Insert an SPME device into the vial to expose the fiber to the headspace and perform VOC extraction for 30 minutes at 50 degree Celsius with agitation at 17 times G.Introduce the fiber into the injection port for one minute at 250 degree Celsius in splitless mode for volatile desorption. Then clean the fiber in an SPME cleaning station with nitrogen for five minutes at 250 degrees Celsius. Analyze VOCs with a gas chromatograph coupled to an ion trap mass spectrometer as described in the detects manuscript.Open raw GC-MS profile files. To identify compounds, compare the retention times, mass spectra and Kovats linear retention indexes with retention indexes obtained from authentic standards. For each commercial standard, annotate the retention time in the most abundant mass to charge ions.Then select a specific M by Z ion for each VOC. Calculate the peak area of each VOC relative to that of the internal standard to minimize instrumental variation and intensity drift. For batch effect correction, normalize the VOC peak area of each sample to the corresponding peak area in the control sample analyzed in the same run.A ripe blackcurrant fruit volatile total ion chromatogram profile obtained by HS-SPME/GC-MS identified 63 VOCs belonging to esters, aldehydes, alcohols, ketones, terpenes and furans based on a library that was developed to profile berry fruit species. Some of the most abundant peaks observed correspond to two monoterpenes, linalool and terpineol, and two C6 compounds, 2-Hexenal and 3-Hexenal. Mass spectra obtained from black current profiles in their comparison with spectra of pure commercial standards are shown for 2-Hexenal and terpineol.PCA of the VOC profiles of four different black current cultivar, showed that the environment strongly impacts volatile content, as PC1 separates samples based on their location. The effective genotype can be observed with PC2, as Ben Tirran is clearly separated from the remaining cultivars. The relative content of linalool and 2-Hexenal in the four assessed black current cultivar confirms that linalool content was generally higher in Poland than in Scotland, whereas 2-Hexenal showed the opposite trend.The proportion of linalool was highest in Ben Tirran cultivars, in that of 2-Hexenal was highest in Ben Tron cultivars. It is important to start with frozen material, ground into a fine powder to assure a proper volatile extraction. Once extracted, sample should be placed into the auto sampler, as soon as possible.This method can be combined with other metabolic platform to identify other important metabolic, for food taste or nutritional value, to breed varieties with enhanced organoleptic characteristic.

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Research

JoVE Journal

Biochemistry

3.5K Views.  Instituto de Hortofruticultura Subtropical y Mediterránea “La Mayora”, Universidad de Málaga-Consejo Superior de Investigaciones Científicas. Improving fruit aroma is one of the main objective in breeding programs. For this, we need a reliable technique which allow us to measure volatile components in fruits. This technique is fast and semi-automated, allowing to measure up to 22 samples per days.In addition, it is relatively cheap and requires minimal sample processing. This method can be easily applied for all the fruit species, such as economically important berry crops. In addition, the library size of the detected compo...