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Downloaded by UNIV LAVAL on May 5, 2016 | http://pubs.acs.org Publication Date: August 25, 1986 | doi: 10.1021/bk-1986-0317.ch015
Biogenesis of Blackcurrant (Ribes nigrum) Aroma R.J.Marriott Barnett & Foster Ltd., Wellingborough, Northants NN8 2QJ, United Kingdom The terpene fraction of blackcurrants is largely responsible for its characteristic flavour and aroma. The biogenesis of terpenes present in blackcurrant fruit has been studied by analysis of the volatile oils during ripening of the fruit and by feeding experiments using (2-13C) mevalonate. Three blackcurrant cultivars, Ben Lomond, Baldwin and Wellington XXX were studied, Ben Lomond and Baldwin fruit showed very similar composition of terpene compounds, whereas in Wellington XXX fruit, the terpenes derived from the thujane skeleton were at greatly reduced levels. Changes in concentration of monoterpene hydrocarbons, alcohols and esters occurred mainly during the period of sugar accumulation, when a sharp decrease in the level of terpene hydrocarbons and an increase in monoterpene alcohols and esters was observed. Feeding experiments with (2-13C) mevalonate indicated parallel incorporation into all monoterpene hydrocarbons and monoterpene alcohols. The presence of monoterpene and aromatic glycosides have also been tentatively identified in blackcurrants for the first time. The analysis of the terpene fraction in ripening fruit was carried out by extraction with dichloromethane followed by gas chromatography/mass spectrometry using single ion monitoring, allowing detection of terpene compounds down to 1 μg/kg wet weight. The aqueous residue from the dichloromethane extract was treated with mixed glycosidases to hydrolyse non-volatile terpene glycosides. After hydrolysis, the free terpenes were extracted with dichloro methane and identified by GC/MS/SIM. Baldwin and Wellington XXX fruits were picked from the same bushes at two week intervals between the end of May and the end of August, and the volatile oils extracted immediately. Feeding experiments were carried out by immersing freshly cut, defoliated stems in a solution containing (2-1 C) mevalonate and glucose, followed by a water chase and then a nutrient solution supplemented by glucose. After feeding, fruits 0097-6156/86/0317-0184S06.00/ 0 © 1986 American Chemical Society Parliment and Croteau; Biogeneration of Aromas ACS Symposium Series; American Chemical Society: Washington, DC, 1986.
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185
Biogenesis of Blackcurrant
were picked at regular intervals and extracted with dichloromethane followed by GC/MS/SIM. The labelled terpenes were detected by monitoring m/z 138 and m/z 9h the molecular ion and M-hh fragment respectively. Although significant work has been carried out on the identification of individual terpenes in blackcurrant leaves, buds and fruit , no studies have been carried out on their bio synthesis, although J. has been assumed to be similar to that reported in other plants. Non-volatile terpene glycosides have been reported in a number of fruits and plants (2~M , but this is the f i r s t time they have been tentatively identified in blackcurrants. 9
Downloaded by UNIV LAVAL on May 5, 2016 | http://pubs.acs.org Publication Date: August 25, 1986 | doi: 10.1021/bk-1986-0317.ch015
Experimental Reagents. Analar dichloromethane (B.D.H. Chemicals) was r e d i s t i l l e d just prior to use. Pure terpene standards were obtained variously from Aldrich Chemicals Co., Fluka A.G., S.C.M. Organics, Bush Boake Allen Ltd and International Flavours and Fragrances. 3-Terpineol and γ-terpineol were generous gifts from Firmenich & Co. The purity of a l l standards was greater than 9&% and were used as supplied. A l l standards were stored under nitrogen at -25°C. The glycosidic enzyme used was Pectinol C obtained from Rohm and Haas. (2- C) MVA lactone (99 atom %) was obtained from MSD Isotopes. A l l other reagents were at least analar purity and used as supplied. 13
Fruit samples. Fruits were picked from 3 year old bushes grown on a commercial fruit farm, and were used immediately for aroma isolation. Stems used for labelling experiments were obtained from the same bushes. Isolation of aroma compounds. 20g of blackcurrants were homogenised with r e d i s t i l l e d dichloromethane (200ml) at -10°C to minimise enzyme reactions or terpene re-arrangements. After allowing the homogenate to separate, the dichloromethane layer was filtered through a previously dichloromethane washed silicone impregnated paper, and f i n a l l y dried over anhydrous sodium sulphate. The aroma isolate was stored at -25°C under nitrogen in hypovials. In order to detect some of the labelled compounds, J. was necessary to concentrate the dichloromethane extract by vacuum d i s t i l l a t i o n . Enzyme hydrolysis of non-volatile precursors. The aqueous residue from the dichloromethane extraction was washed twice with 10 volumes of r e d i s t i l l e d dichloromethane to remove any residual terpenes. The residue was suspended in pH 5·0 phosphate buffer and homogenised. The homogenate was rotary film evaporated briefly to remove any residual dichloromethane. Pectinol C (0.1$) was added in pH5.0 buffer and the homogenate incubated at 30°C for 2h hours. After incubation, the terpene aglycons were extracted with 10 volumes of r e - d i s t i l l e d dichloromethane as previously described. Feeding methods. Stems (ca. 150g) with 20 to 30 fruits were defoliated, cut under sterile water and immediately immersed in a solution of (2- C) mevalonate (0.1 mmol), and glucose (0.3 mmol) in sterile water (10 ml). Immediately before use, MVA lactone was converted into the potassium salt of the acid by incubation with an
Parliment and Croteau; Biogeneration of Aromas ACS Symposium Series; American Chemical Society: Washington, DC, 1986.
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BIOGENERATION OF AROMAS
excess of aqueous potassium bicarbonate at 37°C for 1 hour. Water (0.5 ml) was then fed to the stems followed by feeding nutrient solution (6) supplemented with glucose (1 mg ml" ) and adjusted to pH 7·8· Fruit were picked at regular intervals and extracted as described previously.
Downloaded by UNIV LAVAL on May 5, 2016 | http://pubs.acs.org Publication Date: August 25, 1986 | doi: 10.1021/bk-1986-0317.ch015
Capillary Gas Chromatography - Mass Spectrometry. Capillary GC/MS was carried out using a 50 m fused s i l i c a capillary column (0.25 mm i.d.) coated with OV-101 or Carbowax 20M in a Pye Unicam series 10U chromatograph with linear temperature program from 50°C to 200°C at 2°C/min. The column was directly coupled to the ion source of a V.G. Micromass 12B mass spectrometer set to the following conditions: accelerating voltage 5 KV; ionization voltage 70 eV; ion source temperature 200°C; resolution 1000 (10$ valley). The mass spectro meter was calibrated manually using reference compounds. Comparison of cultivars. Fully, ripe fruit was picked from 3 year old bushes of Baldwin, Ben Lomond and Wellington XXX cultivars, and the aroma isolated as previously described. The aroma isolate was analysed by GC/MS/SIM at m/z 93 and m/z 136. Identification was carried out by comparison of retention times against authentic standards using both 0V-101 and Carbowax 20M columns. Quantification of identified compounds was carried out by comparison with standard solutions of known concentration. Comparison of the terpene fraction of the corresponding leaf o i l s was also carried out in the same way. Although J. was found that the terpene fraction of the leaf o i l s of the three cultivars was essentially identical, the terpene fraction of the fruit was found to be similar in Baldwin and Ben Lomond cultivars, but not Wellington XXX which had greatly reduced levels of α-thujene and sabinene. Baldwin and WellingtonXXX were thus chosen to study the changes in terpene composition during ripening. Changes in terpene composition during ripening. Fruits were picked at 2 week intervals from the end of May to the end of August from the same bushes grown at the same site. The aroma isolation was carried out within 6 hours of picking, and the aroma extracts analysed as previously described. Feeding experiments with (2- C) mevalonate were carried out at three stages of ripening using stems from the same bushes and using the method previously described. Non-volatile precursors. It was observed during routine processing of blackcurrants that the level of monoterpene alcohols increased after the addition of pectin hydrolysing enzymes. This suggested the presence of terpene glycosides, and the level of these compounds in a l l samples of fruit picked during ripening was determined in the aqueous residue after dichloromethane extraction of the volatile aroma compounds using the method previously described. Results and Discussion Comparison of cultivars. The analysis of the monoterpene fraction of the aroma isolated from the three cultivars is given in table I. The analysis was carried out using both OV-101 and Carbowax 20M
Parliment and Croteau; Biogeneration of Aromas ACS Symposium Series; American Chemical Society: Washington, DC, 1986.
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Table I.
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Biogenesis of Blackcurrant
Monoterpenes found in 3 blackcurrant cultivars (levels in
Downloaded by UNIV LAVAL on May 5, 2016 | http://pubs.acs.org Publication Date: August 25, 1986 | doi: 10.1021/bk-1986-0317.ch015
μβ/Kg)
Monoterpene α-thuj ene a-pinene camphene sabinene β-pinene myrcene a-phellandrene A- -carene a-terpinene d-(+)-limonene 3
cis-3-ocimene
trans-ft-oc imene γ-terpinene terpinolene alio ocimene linalol cis-3-terpineol
trans-$-t erpineol terpinene-U-ol a-terpineol γ-terpineol citronellol nerol geraniol
Ben Lomond h9 3 6 2
Baldwin
Wellington
3 7
h2 2
5
3 7 10
1 8 6 7
2 8
k6 kQ
5 9
21
7 2
5 0
2 9
2 8
5 3
6 6 6
1U5U
6k9 2
