The Use of Enzyme Systems of the Genus Prunus for the Production of Benzaldehyde

The plants we investigated originated from Southwestern Slovakia. The agricultural cooperative Vajnory (near Bratislava) offered twigs with leaves of peach and apricot, namely: three varieties of “Haven” peach group: Firehaven, Groshaven, Redhaven (Owens, 2019); one Slovak peach variety, Flamingo (Benediková & Zetochová, 2018): and one apricot variety, Bergeval (Benediková, 2017). Young twigs were harvested by hand at the time, in August 2021.

The Research Institute of Plant Production in Piešťany offered a variety of cherry laurel that is one of the most popular and versatile evergreen hedging species. Young twigs with leaves were harvested by hand at the time of in the period from October 2021 to January 2022. The fresh plant material was stored at 4°C until further uses (no longer than 24h).

Apricot kernels were obtained from the Pezinok distillery.

The organic solvent ethyl acetate was p.a. grade (Sigma-Aldrich, USA). All other the synthetic standards were 99% purity (Sigma-Aldrich, USA).

Essential oils of Prunus species were extracted using a Clevenger apparatus as follows: 100 g of fresh leaves cut into 2 cm pieces were put into a 2,000 ml round-bottom flask and 1,000 ml of distilled water was added. The mixture was heated in a bath of stirred oil with a temperature of 130°C. The hydrodistillation was carried out during 2 hours from the first drop of distillate appeared in condenser. The yield of essential oil (ml/100 g) was determined on fresh weight basis. The isolated oils were stored in tightly closed vials at 4°C until analysis.

Pilot-scale experiments were performed under batch regime, processing 200 kg of twigs with leaves in a mixer after the addition of 1,000 litres of water, followed by distillation for 45 minutes. The obtained distillate was transferred into a separatory funnel, where essential oil was separated from aqueous phase. The hydrosol (aqueous phase below the layer of essential oil) was extracted by ethyl acetate. The extract was distilled in a vacuum, and organic phases were collected and dried with anhydrous magnesium sulfate. The samples of recovered essential oil were analysed by GC-FID and GC-MS.

Apricot kernel essential oil was prepared under laboratory conditions as follows: after grinding the apricot kernel using Philips blender, type HR2860, 100 g of flour (obtained from 390 g of crushed kernels) was homogenised with 1,000 ml of water and hydrodistilled in a Clevenger apparatus for 2 hours, followed by GC-FID analysis.

The essential oil was analysed by gas chromatography with flame-ionisation detection (GC-FID) using Agilent 7,890N apparatus with a column DB WAX (methyl dodecanoate PEG film, length 30 m, thickness 0.15 μm, diameter 0.25 mm).

The constituents of essential oil were identified using gas chromatography-mass spectrometry (GC-MS) by means of the Agilent 5,977C single quadrupole GC/MSD instrument, using the same column (DB WAX), as for the GC-FID analyses. Mass spectra were compared with the NIST Spectral Database. The operating conditions of GC-FID analysis were as follows: hydrogen was used as a carrier gas (1.5 ml/min); split mode (20:1); 1 μl of injection volume; oven temperature program: 95°C for 6 min, 10°C/min up to 120°C, 20°C/min up to 230°C, 230°C for 17 min; an injector temperature of 250°C; a detector temperature of 300°C.

The operating conditions of GC-MS analysis were as follows: helium was used as a carrier gas (1.5 ml/min), with split mode (50:1); 0.2 μl of injection volume; oven temperature 40°C for 5 min, 10°C/min up to 220°C, with 220°C for 5 min; an injector temperature of 250°C; and a detector temperature of 270°C. Electron impact mass spectra were acquired in scan mode in m/z range 27–330.

At the beginning of the testing, we processed wild peach leaves grown in vineyards on the Carpathian slopes near Bratislava, Slovakia. Given the preliminary positive results (92%–96% benzaldehyde content in essential oil), we decided to perform a series of experiments using leaves of cultivated peach varieties. We evaluated four peach varieties (Flamingo, Redhaven, Groshaven, and Firehaven) and one apricot variety (Bergeval), to compare the percentage of representation of benzaldehyde in the essential oils studied. The summary of experiments is summarized in the Table 1. No essential oils appeared during distillation of the apricot leaves of the Bergeval variety or while processing peach leaves of all varieties; the average yield of essential oil was 0.326% V/w and with 94.99% benzaldehyde content. GC-MS identification of volatile organic compounds in the essential oil of processed peach leaves is illustrated in the Table 2 and Figure 2.

Figure 2

The results confirmed that benzaldehyde is the most abundant volatile organic compound of peach leaf essential oil, while the content of the other compounds is significantly lower (individual peaks of the chromatogram are less than 1%). Based on the results reached, it was calculated that approximately 300–400 kg of fresh peach leaves are needed to produce 1 kg of benzaldehyde.

As no separation of essential oil was observed during the hydrodistillation of apricot Bergeval variety leaves, the hydrosol (condensed water after hydrodistillation in Clevenger apparatus) was extracted with ethyl acetate in the feed to solvent volume ratio of 3:1. In this way, benzaldehyde dissolved in water (benzaldehyde solubility is 0.3%–0.7% in water) and other components, as (E)-2-hexenal, (Z)-3-hexenol, (E)-2-hexenol were extracted as shown in Figure 3.

Figure 3

The essential oil obtained from the apricot and plum seeds can also be the alternative source of benzaldehyde. The process of benzaldehyde releasing from apricot seeds consist in the biochemical transformation (Figure 1) of amygdalin by β-glucosidase hydrolysis during milling (Tunçel et al., 1998).

The results of the hydrodistillation of apricot kernels are presented in Figure 4. The concentration of benzaldehyde reached only about 10%, and various unidentified polyunsaturated fatty acids (22%) can be seen in the chromatogram. In addition, the yield of the essential oil was only 0.03% w/w, which is about ten times less compared to the hydrodistillation of peach leaves.

Figure 4

Hydrodistillation of fresh peach twigs with leaves (Figure 5) in pilot conditions using SCC (spinning cone column) resulted in lower yields compared with those of laboratory experiments. Up to about 600 kg of twigs with leaves were needed to obtain 1 kg of benzaldehyde. The purity of the benzaldehyde reached about 96%, which is in good agreement with the laboratory experiments. The large losses of benzaldehyde (up to 42% of the feed material) were observed during the subsequent long-term evaporation of the ethyl acetate extract of distillate on a laboratory evaporator after 2 hours (55°C–60°C/30–35 kPa). Thus, the crude concentrate containing benzaldehyde will have to be purified by rectification in an inert atmosphere to avoid losses and spontaneous oxidation of benzaldehyde to benzoic acid. Pilot experiments also showed that no essential oil and targeted product – benzaldehyde – occurred in the stems or occurred only in insignificant quantities.

Figure 5

Cherry laurel leaves (Figure 6) have been tested as another promising source of benzaldehyde. The advantage of this plant is the rapid growth of its green phytomass, because up to 30% of pruned green leaf mass can regrow again within 3 months. Shrubs can be grown, machine cut, or rowed, which facilitates the collection of green material for industrial processing. According to the literature (Stanisavljevic et al., 2010), up to 99.7% of cherry laurel leaf essential oil is made up of benzaldehyde and insignificant quantities of (Z)-ocimenone (0. %) and (E)-2-hexenal (0.1%).

Figure 6

In Table 3 the results are presented of the essential oil extraction from cherry laurel leaves by hydrodistillation in the Clevenger apparatus. The isolated essential oil was of high purity, that is, 99.7% benzaldehyde. The results obtained are in good agreement with the data of Stanisavljevic et al. (2010). Cherry laurel leaves have been shown to be a more effective source of essential oil because they have a high benzaldehyde content compared with peach leaves. The yield of cherry laurel leaf essential oil reached about 0.463%, which means that approximately 200 kg of cherry laurel leaves are sufficient to obtain 1 kg of benzaldehyde. Moreover, peach trees are leafless in winter, while cherry laurel is an evergreen shrub, resistant at temperatures as low as minus 20°C. The harvesting of laurel leaves at minus 5°C in January 2022, followed by processing in a Clevenger apparatus gave lower benzaldehyde yields, which were still of relatively high purity.