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.

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.