Biotechnology Letters 24: 551–556, 2002.© 2002 Kluwer Academic Publishers. Printed in the Netherlands.

551

Characteristics of aroma-active compounds in the pectin-elicitedsuspension culture of Zanthoxylum piperitum (prickly ash)

Tae Hwan Kim1,2, Tae Ho Kim1, Joong Han Shin1, Eun Jeong Yu1, Young-Suk Kim3 & HyongJoo Lee1,∗1Department of Food Science and Technology, School of Agricultural Biotechnology, and 2Research Center forAgriculture and Life Sciences, Seoul National University, Suwon 441-744, Korea3Department of Foods and Nutrition, Ewha Womans University, Seoul 120-750, Korea∗Author for correspondence (Fax: +82-31-293-4789; E-mail: leehyjo@snu.ac.kr)

Received 21 December 2001; Revisions requested 11 January 2002; Revisions received 28 January 2002; Accepted 1 February 2002

Key words: aroma-active compounds, 2,3-butanedione, pectin elicitation, suspension culture, Zanthoxylumpiperitum

Abstract

Zanthoxylum piperitum (prickly ash) was grown as a suspension culture in Schenk and Hildebrandt medium supple-mented with 50 g sucrose l−1 and 0.5 mg 2,4-dichlorophenoxyacetic acid l−1 for 21 days with elicitation by pectinadded at day 15. Volatile compounds were extracted from the culture and 3-hydroxy-2-butanone was identifiedby GC-MS as the most abundant compound, followed by γ -butyrolactone and 2,3-butanedione. 2,3-Butanedione,ethyl 3-methylbutyrate, and ethyl 2-methylpropanoate were identified as the most intense aroma-active compoundsand represented the characteristic aroma of the culture.

Introduction

Various plant cell lines have been studied for the pro-duction of natural flavors (Collin 1988, Sahai 1994).However, plant cell culture systems have many limi-tations, including low production yields and changedcompositions of phytochemicals, which need to beovercome for commercial applications (Sahai 1994,Dönenburg & Knorr 1995).

Zanthoxylum piperitum (prickly ash), an aromaticplant of Rutaceae family native to Korea, China, andJapan, has been used as not only medicinal plantagainst colds, gastrisis, and neuralgia but also as foodspice due to its characteristic aroma (Kim et al. 1989).When Kim et al. (1989) analyzed the essential oilof Korean Z. piperitum, they found that 1,8-cineole,limonene, geranyl acetate, and myrcene in fruit peel,and citronellal, 1,8-cineole, and citronellol in leafwere the major flavor compounds. On the other hand,Kojima et al. (1997) identified 2-tridecanone, (Z)-3-hexenol, and 2-undecanone as the major volatiles inthe essential oil of Z. piperitum. They also showed

that citronellol and citronellal were the predominantaroma-active compounds through aroma extract dilu-tion analysis (AEDA). The embryogenic callus hasbeen effectively induced from shoot tip and leaf of Z.piperitum in MT medium (Murashige & Tucker 1969)supplemented with 30 g l−1 sucrose l−1 and 0.5–1 mg2,4-dichlorophenoxyaceticacid l−1 (Song et al. 1991).

Among the very diverse volatile compounds infoods, only a few compounds can be defined as‘aroma-active compounds’ responsible for the char-acteristic aroma of foods (Mistry et al. 1997). Gaschromatography-olfactometry (GC-O) analysis, suchas AEDA and CharmAnalysis, is a key analytical tool,which combines sensory techniques with instrumen-tal methods for the detection of aroma-active com-pounds. In AEDA, serial dilutions of a flavor extractare sniffed to provide flavor dilution factors, whichare proportional to the relative aroma intensity of eacharoma-active compound.

This study was conducted to identify volatile flavorcompounds and evaluate characteristic aroma-active

552

compounds produced from Z. piperitum suspensionculture through GC-MS-O.

Materials and methods

Suspension culture

Callus was induced from stems of Zanthoxylum piper-itum on MT solid medium (Murashige & Tucker 1969)supplemented with 30 g sucrose l−1, 0.5 mg 2,4-D l−1,and 10 g agar l−1. Suspension culture was initiated inMT liquid medium without agar. The medium pH wasadjusted to 5.7 with 0.5 M NaOH before autoclaving.Cells were cultivated in a rotary shaker (Vision Scien-tific Co.) at 27 ◦C and 110 rpm under white fluorescentlight (1600 lux, 16 h day−1). A 20 ml portion of theculture was subcultured every 15 days. Cell weightsof suspension cultures were measured as described byMills & Lee (1996).

To determine the optimal medium for cell growth,a 15-day-old suspension culture grown in MTmedium was inoculated into 50 ml of either SHmedium (Schenk & Hildebrandt 1972), MS medium(Murashige & Skoog 1962), B5 medium (Gamborget al. 1968), or LS medium (Lin & Staba 1961)each supplemented with sucrose (30 g l−1) and 2,4-D

(0.5 mg l−1).SH medium containing 50 g sucrose l−1 and

0.5 mg 2,4-D l−1 (SHS5 medium) was used for thegrowth of suspension culture. Fifteen ml cell suspen-sion was inoculated into 50 ml of fresh medium onceevery 15 days.

Elicitation

Elicitors, chitosan (from crab shells), nigeran (fromAsperigillus japonicus), lichenan (from Cetraria is-landica), pectin (from citrus fruit), and yeast extractwere purchased from Sigma. Chitosan was purified aspreviously described (Chang et al. 1998), and otherelicitors were directly added without further purifica-tion step. Each elicitor was dissolved in SHS5 mediumat 750 mg l−1 (for chitosan and nigeran) or 7.5 g l−1

(for lichenan, pectin, and yeast extract), and adjustedto pH 5.7. Subsequently, 10 ml of the elicitor-addedmedia was added to 65 ml of 15-day suspension cul-tures prior to measuring the cell weight. Six daysafter the addition of elicitor, the suspensions wereharvested.

Extraction of volatiles

Extracellular volatile compounds in a 21-day sus-pension culture, to which 1 g pectin l−1 had beenadded as an elicitor at day 15, were extracted directlyfor GC-MS and GC-olfactometry (GC-O): 500 mlcell-free suspension passed through a filter paper,48.8 µg 3-heptanol (an internal standard), and 200 mldichloromethane (solvent) were added and the mix-ture was shaken vigorously in a separatory funnel.The remaining water in the dichloromethane layer wasremoved by freezing out and dehydrating over anhy-drous Na2SO4. The dichloromethane extract contain-ing the volatile compounds was avaporated under N2to 100 µl. For preparing a positive control, volatilesof the pectin-added SH medium without the culturedcells were also extracted according to the procedurementioned above.

Gas chromatography-mass spectrometry

A GC-MS was employed using an HP 5890 SeriesII GC/HP 5972 mass selective detector (MSD) sys-tem (Hewlett-Packard Co.). One µl of the extractwas injected (splitless mode) on two different typesof column, i.e., a non-polar HP-5MS (30 m length× 0.25 mm i.d. × 0.25 µm film thickness; Hewlett-Packard Co.) and polar DB-Wax (30 m length ×0.25 mm i.d. × 0.25 µm film thickness; J&W Scien-tific). Helium was run as a carrier gas at 0.8 ml min−1.The oven was held at 40 ◦C for 5 min, then raised to200 ◦C at 3 ◦C min−1, and held at 200 ◦C for 20 min.The injector and detector were at 200 and 250 ◦C,respectively. MSD conditions were as follows: ioniza-tion energy, 70 eV; mass range, 33–550 amu; scanningrate, 1.4 scans s−1. Retention indices (RIs) were deter-mined using n-paraffins C8-C22 as external references(Van den Dool & Kratz 1963).

Gas chromatography-olfactometry (GC-O)

GC-O was carried out using a Younglin 680D GC(Younglin Instrument) equipped with a flame ion-ization detector (FID) and a sniffing port (AlltechAssociates). Effluent from the end of the FSOT col-umn was split into 1:1 between the FID and thesniffing port using deactivated capillary columns. Se-rial dilutions (1:2) of the extract were prepared usingdichloromethane as a diluent. From each dilution, 1 µlwas injected (split ratio, 4:1) into an FSOT column(DB-5MS or DB-Wax, 30 m length × 0.25 mm i.d. ×

553

0.25 µm film thickness; J&W Scientific). The FD fac-tor of each aroma-active compound that correspondsto the maximum dilution value (Mistry et al. 1997).Oven temperature was programmed from 40 to 200 ◦Cat a rate of 10 ◦C min−1, with initial and final holdtimes of 5 and 24 min, respectively. Other GC con-ditions for GC-O were the same as those of GC-MS.GC-O was performed by two experienced panelists.

Identification and quantification

Volatile compounds were positively identified by com-paring mass spectra and RIs of the unknowns withthose of the authentic compounds. When standardswere not available, compounds were tentatively iden-tified with the aid of Wiley 275 mass spectral database(Hewlett-Packard Co., 1995) or by manual interpre-tation. Identifications of the aroma-active compoundswere based on matching their mass spectra, RIs, andaroma properties with those of the authentic stan-dards. Relative concentrations of the identified flavorcompounds were determined by comparing their peakareas with that of the internal standard (3-heptanol) ontotal ion chromatogram of GC-MS.

Results and discussion

Growth of suspension culture

When the cells were cultivated in SH medium supple-mented with 30 g sucrose l−1 and 0.5 mg 2,4-D l−1

(SHS3 medium), cell growth was enhanced by 134%in fresh cell weight (FCW) and 93% in dry cell weight(DCW), respectively, compared to MT medium withthe same concentrations of sucrose and 2,4-D (MTS3medium) (Figure 1). We also investigated the growthof suspension in MS, B5, and LS media containing thesame concentrations of sucrose and 2,4-D. However,cell weights in these media were much lower than thatin SHS3 medium (data not shown).

For obtaining higher cell weight, cells were in-oculated into SH media supplemented with 0.5 mg2,4-D l−1, to which various concentrations of sucrose(10, 30, 50, 70 and 90 g l−1) were added. At day 15,cell growth of the suspension culture in SH mediumcontaining 50 g sucrose l−1 (SHS5 medium) wasenhanced by 15% (FCW) and 57% (DCW), respec-tively, over SHS3 medium (Figure 1). The increaserate of FCW was lower than that of DCW, becausethe cell size and the cellular water content could havedecreased due to the higher sugar concentration or

Fig. 1. Effects of media on the cell growth of Zanthoxylum piper-itum suspension culture. Cell weights were measured on day 15.Media: MTS3, MT medium supplemented with 30 g sucrose l−1;SHS3, SH medium supplemented with 30 g sucrose l−1; SHS5,SH medium supplemented with 50 g sucrose l−1. FCW, fresh cellweight; DCW, dry cell weight. Each medium contained the sameconcentration of 2,4-D (0.5 mg l−1) as a growth regulator. Re-sults are the average ± standard deviation of eight independentexperiments.

medium osmolarity (Park & Kim 1993). Both FCWand DCW of the media, where sucrose at 10, 70 and90 g l−1 was added, were lower than those of SHS3and SHS5 media (data not shown). Based on theseresults, SHS5 medium was revealed to be optimal forthe growth of suspension culture, and was thus usedfor further experiments.

Elicitation

We added chitosan, nigeran, lichenan, pectin, andyeast extract as elicitors to stimulate the productionof volatile compounds from the cultures. Amongthese elicited suspension cultures, pectin-treated cul-ture exhibited stronger buttery and apple-like aromasthan the other cultures in a preliminary sensory test(data not shown). Pectin and pectic substances havebeen reported to enhance the production of secondarymetabolites in various plant cultures (Dönenburg &Knorr 1995). Cell growth of the pectin-elicited Z.piperitum suspension culture was repressed slightlyby 5.4 ± 1.8% in FCW and 9.6 ± 2.5% in DCW(n = 8) during a 6-day elicitation period from days 15to 21. Inhibition of the cell growth has been commonlyfound in many elicited cultures because elicitation is adefense mechanism against the attack of exogenous orendogenous pathogens in plants (Dönenburg & Knorr1995).

554

Table 1. Volatile compounds produced by the pectin-elicited suspension culture of Zanthoxylumpiperitum.

No.a Compound nameb Amount (µg l−1)c RId IDe

HP-5MS DB-Wax

1 2,3-Butanedione 97 ± 36 <800 959 A

2 3-Hydroxy-2-butanone 16000 ± 4100 <800 1277 A

3 2,3-Butanediol 56 ± 16 <800 1542 A

4 Meso-2,3-butanediol 70 ± 23 807 1580 A

5 Methyl 3-hydroxybutyrate 48 ± 30 859 1478 A

6 3-Hydroxy-3-methylbutyrate 9 ± 3 880 1368 B

7 γ -Butyrolactone 270 ± 53 922 1615 A

8 Methyl 3-hydroxy-2-methylbutyrate 72 ± 25 930 1519 C

9 Ethyl 3-hydroxybutyrate 17 ± 5 938 1515 A

10 Unknown (55, 83, 41, 43, 101) 41 ± 23 1147 1998

11 Unknown (71, 43, 55, 83, 89) 110 ± 57 1376 >2200

12 Unknown (121, 150, 65, 93) 35 ± 11 1540 >2200

13 Unknown (96, 151, 152, 109) 31 ± 19 1573 >2200

14 Tryptophol 32 ± 24 1750 –f A

15 Unknown (43, 87, 130) 14 ± 5 – 1378

16 Unknown (85, 41, 42) 43 ± 15 – 2036

17 Unknown (41, 69, 55, 87) 62 ± 26 – >2200

aCompound number.bNumbers in the brackets indicate major mass fragmented ions in mass spectrum.cAverage amount ± standard deviation of four independent experiments.dAverage retention index determined using n-paraffins C8–C22 as references (n = 4).eIdentification: A, mass spectrum and retention index were consistent with those of an authentic stan-dard; B, mass spectrum was consistent with that of Wiley 275 mass spectrum database (tentativeidentification); C, mass spectrum was interpreted manually (tentative identification).fNot detected.

Table 2. Aroma-active compounds produced by the pectin-elicited suspension culture of Zanthoxylum piperi-tum.

Compound name RIa Aroma description log2FDb IDc

DB-5MS DB-Wax DB-5MS DB-Wax

2,3-Butanedione <800 956 Buttery 7 7 A

Ethyl 2-methylpropanoate <800 957 Fruity, apple 2 –d B

Ethyl 3-methylbutyrate 859 1067 Fruity, apple 3 2 B

Unknown I 1042 – Fruity 1 –

Unknown II 1082 – Sweet 1 –

Unknown III 1181 1682 Buttery 1 1

aRetention index determined using n-paraffins C8–C22 as references.bLog2 (flavor dilution factor).cIdentification: A, retention index, mass spectrum, and aroma property were consistent with those of an authen-tic standard; B, retention index and aroma property were consistent with those of an authentic standard.dNot detected.

555

Volatile compounds of the pectin-elicited suspensionculture

Table 1 lists 17 volatile flavor compounds producedby the pectin-elicited Z. piperitum suspension culture,their amounts, and average RIs on HP-5MS and DB-Wax columns. A total of 74 compounds were detectedin the extract of the pectin-elicited cultures by GC-MS. Among these, 57 were also detected in the extractof the medium used for the production of flavor com-pounds (data not shown) and these compounds wereexcluded from the list in Table 1. Overall pattern ofthe volatile production by the elicited suspension cul-ture was different from that of the intact plant. Majorvolatiles of Z. piperitum (Kim et al. 1989, Kojimaet al. 1997), were not detected in the elicited culture.Sahai (1994) elucidated differences in the volatile pro-files between the cell cultures and their parent plants invarious culture systems. These differences are obviousin the undifferentiated culture systems such as callusand suspension cultures, which have no specific tis-sue responsible for accumulating the volatiles (Collin1988).

Carbonyl compounds were abundant in the pectin-elicited culture. In particular, 3-hydroxy-2-butanone(No. 2) constituted approximately 94% of totalvolatiles. 2,3-Butanedione (No. 1) was also detectedin the culture but at much smaller amount than thatof 3-hydroxy-2-butanone. Even though 3-hydroxy-2-butanone was nearly odorless, it was considered as theprincipal volatile compound since it could be oxidizedinto 2,3-butanedione, the character impact compoundof various fermented dairy products (Molimard &Spinnler 1996).

Three alcohols were detected in the suspension,of which meso-2,3-butanediol (No. 4) was the mostabundant, followed by 2,3-butanediol (No. 3) andtryptophol (No. 14). Speranza et al. (1997) sug-gested that meso-2,3-butanediol could be formed from2,3-butanedione and 3-hydroxy-2-butanone by lacto-bacilli. Tryptophol (indole 3-ethanol) is known as ametabolite of tryptophan and trypamine in plant tis-sues and microorganisms, where it accumulates asa by-product of indole-3-acetic acid synthesis (Fennet al. 1977).

Four esters were also detected in the suspension,all with similar chemical structures. That is, theyare esters of butanoic acid, which have a hydroxylgroup at the carbon 3 position. Of these, methyl andethyl 3-hydroxybutyrates (Nos. 5 and 9) were posi-tively identified. 3-Hydroxy-3-methylbutyrate (No. 6)

and methyl 3-hydroxy-2-methylbutyrate (No. 8) weretentatively identified in this study.

γ -Butyrolactone (No. 7) and seven unknown com-pounds (Nos. 10, 11, 12, 13, 15, 16, and 17) were alsodetected in the elicited suspension. γ -Butyrolactone,a volatile compound with a sweet aroma in cheese(Molimard & Spinnler 1996), had the second largestpeak area in the total ion chromatogram of GC-MS.

Aroma-active compounds of the pectin-elicitedsuspension culture

Aroma-active compounds detected in the pectin-elicited Z. piperitum suspension culture are shownwith their RIs and aroma properties in Table 2. Al-though 12 and 9 compounds were determined tohave aroma properties using GC-M5 with DB-5MSand DB-Wax columns (data not shown), respectively,some of these were excluded from the list of Table 2,because they were also detected in the extract of theproduction medium (the pectin-added SH medium).As shown in Table 2, FD factors of aroma-activecompounds (except for 2,3-butanedione) were com-paratively low, and four compounds had different FDfactors on the two columns. However, three char-acteristic compounds representing buttery and applearomas were positively identified.

2,3-Butanedione was the most predominant aroma-active compound with the highest log2FD factors onboth columns. It contributes to the characteristic but-tery aroma of the culture based on its strong aromaintensity and low threshold value of 3 µg l−1 in water(Schieberle & Hofmann 1997). Although 3-hydroxy-2-butanone, exerts a buttery aroma and was the mostabundant compound in the FID chromatogram, itexhibited no aroma at the sniffing port.

Two esters, ethyl 3-methylbutyrate and ethyl 2-methylpropanoate, play important roles in the apple-like aroma of the suspension. Although the concentra-tions of these compounds were too low to be detectedin GC-MS (Table 1), they could be identified throughtheir RIs and aroma properties. Ethyl 3-methylbutyratecompound has been identified as a potent aroma-activecompound in fresh strawberry juice with considerablylow threshold value of 0.4 µg l−1 in water (Schieberle& Hofmann 1997). Ethyl 2-methylpropanoate wasalso detected with fruity and apple-like aroma onthe DB-5MS column, but not on the DB-Wax col-umn. This phenomenon could be explained throughthe ‘contrast effect’, in which the elution order, aromacharacteristics, and perceived intensity of one com-

556

pound directly influence the perceived intensity ofthe next eluted compound (Mistry et al. 1997). Onthe DB-Wax column, the apple-like aroma of ethyl2-methylpropanoate could have been hidden by thestrong buttery aroma of 2,3-butanedione. The thresh-old value of ethyl 2-methylpropanoate has been re-ported as 0.1 µg l−1 (Schieberle & Hofmann 1997),the lowest among the identified aroma-active com-pounds.

Besides the three important aroma-active com-pounds described above, three unknown compoundswere also detected via AEDA, and these had relativelylow FD factors.

Although the flavor profile of the suspension cul-ture of Z. piperitum was different from that of itsintact plant, a total of 17 flavor compounds wereformed in the pectin-elicited suspension. In addition,aroma-active compounds including the buttery and ap-ple aromas produced by the suspension culture couldbe positively identified by comparing the suspensionculture with the culture medium through GC-MS-O.

Acknowledgements

This study was supported by the Korean Science andEngineering Foundation (KOSEF) through the Re-search Center for New Bio-Materials in Agriculture atSeoul National University. Authors are grateful for thegraduate fellowship provided by the Korean Ministryof Education through the Brain Korea 21 Project.

References

Chang JH, Shin JH, Chung IS, Lee HJ (1998) Improved mentholproduction from chitosan-elicited suspension culture of Menthapiperita. Biotechnol. Lett. 20: 1097–1099.

Collin HA (1988) Flavors. In: Constabel F, Vasil IK, eds. Cell Cul-ture and Somatic Cell Genetics of Plants, Vol. 5. San Diego, CA:Academic Press, pp. 569–585.

Dönenburg H, Knorr D (1995) Strategies for the improvement ofsecondary metabolite production in plant cell cultures. EnzymeMicrob. Technol. 17: 674–684.

Fenn PF, Durbin RD, Kuntz JE (1977) Synthesis of tryptophol ando-acetyltryptophol from tryptophan by Ceratocystis fagacearum.Phytochemistry 16: 899–901.

Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirementsof suspension cultures of soybean root cells. Exp. Cell Res. 50:151–158.

Kim JH, Lee KS, Oh WT, Kim KR (1989) Flavor components of thefruit peel and leaf oil from Zanthoxylum piperitum DC. KoreanJ. Food Sci. Technol. 21: 562–568.

Kojima H, Kato A, Kubota K, Kobayashi A (1997) Aroma com-pounds in the leaves of Japanese pepper (Zanthoxylum piperitumDC) and their formation from glycosides. Biosci. Biotechnol.Biochem. 61: 491–494.

Lin ML, Staba EJ (1961) Peppermint and spearmint tissue cultures.I. Callus formation and submerged culture. Lloydia 24: 139–145.

Mills DR, Lee JM (1996) A simple, accurate method for deter-mining wet and dry weight concentration of plant cell suspen-sion cultures using microcentrifuge tubes. Plant Cell Rep. 15:634–636.

Mistry BS, Reineccius T, Olson LK (1997) Gas chromatography-olfactometry for the determination of key odorants in foods. In:Marsili R, ed. Techniques for Analyzing Food Aroma. New York:Marcel Dekker, pp. 265–292.

Molimard P, Spinnler HE (1996) Compounds involved in the flavorof surface mold-ripened cheeses: origins and properties. J. DairySci. 79: 169–184.

Murashige T, Skoog F (1962) A revised medium for rapid growthand bio-assays with tobacco tissue cultures. Physiol. Plant 15:473–497.

Murashige T, Tucker DPH (1969) Growth factor requirements ofcitrus tissue culture. In: Chapman HD, ed. Proceedings of the 1stInternational Citrus Symposium, Vol. 3. University of California,Reverside: Reverside Publication, pp. 1155–1161.

Park IS, Kim DI (1993) Significance of fresh weight to dry cellweight ratio in plant cell suspension cultures. Biotechnol. Tech.7: 627–630.

Sahai O (1994) Plant tissue culture. In: Gabelman A, ed. BioprocessProduction of Flavor, Fragrance, and Color Ingredients. NewYork: John Wiley & Sons, pp. 239–275.

Schenk RU, Hildebrandt AC (1972) Medium and techniques forinduction and growth of monocotyledonous and dicotyledonousplant cell cultures. Can. J. Bot. 50: 199–204.

Schieberle P, Hofmann T (1997) Evaluation of the character impactodorants in fresh strawberry juice by quantitative measurementsand sensory studies on model mixtures. J. Agric. Food Chem. 45:227–232.

Song WS, Oh SD, Park EH, Yu SO (1991) In vitro propagation ofZanthoxylum piperitum DC. I. Somatic embryogenesis and plantregeneration. Korean J. Plant Tiss. Cult. 18: 17–25.

Speranza G, Corti S, Fontana G, Manitto P (1997) Conversionof meso-2,3-butanediol into 2-butanol by lactobacilli. Stereo-chemical and enzymatic aspects. J. Agric. Food Chem. 45:3476–3480.

Van den Dool H, Kratz PD (1963) A generalisation of the re-tention index system including linear temperature programmedgas-liquid partition chromatography. J. Chromatogr. 11: 463–471.