Biotechnology Letters, Vol 20, No 12, December 1998, pp. 1097 1099
Improved menthol production from
chitosan-elicited suspension culture of
Mentha piperita
Jun Hyong Chang1, Joong Han Shin1, In Sik Chung2
and Hyong Joo Lee1*
1
Department of Food Science and Technology and Research Center for New-Biomaterials in Agriculture,
Seoul National University, Suwon, 441 744 Korea
2
Department of Genetic Engineering, Kyung Hee University, Suwon, 449 701 Korea
The optimum concentration of chitosan to menthol production by Mentha piperita cells cultured in shake flasks was
200 mg/l, which gave 166 mg menthol/l after 12 days. Chitosan elicitation may activate the conversion of pulegone to
menthol.
Keywords: Chitosan, elicitation, Mentha piperita, menthol
Introduction Elicitation with chitosan
In cell cultures of Mentha piperita, the effects of medium Chitosan was purchased from Sigma Chemical Co. and
purified by the method of Young et al. (1982). Briefly, it
composition and bioreactor operation on cell growth and
was dissolved in 90 ml of 0.1 M acetic acid and the
menthol formation have been investigated (Chung et al.,
solution was centrifuged for 30 min. The insoluble frac-
1994; Kim and Lee, 1992; Oh et al., 1993; Park et al.,
tions were then discarded. This procedure was performed
1993; Park and Chae, 1990). However, some problems still
four times. After centrifugation, the supernatant was pre-
remain unresolved in improving menthol production. A
cipitated by adjusting of its pH to 8.0 with 5 M NaOH.
wide variety of elicitors have been employed to alter cell
The precipitates were washed extensively with distilled
metabolism in order to enhance the production of second-
water and then freeze-dried. The purified chitosan was
ary metabolites in plant cell cultures (Eilert, 1987; Funk
dissolved in 0.1 M acetic acid (1 g chitosan/90 ml acetic
and Brodelius, 1990; Kohle et al., 1985; Cline and Coscia,
acid) and the pH of the solution was adjusted to 5.0. For
1988; Payne et al., 1991). Among these elicitors, chitosan
the determination of the optimum concentration, chitosan
( -1,4-linked glucosamine) proved to be very effective in a
concentration was varied from 50 to 300 mg/l. To deter-
suspension culture of Vanilla planifolia, Glycine max, and
mine the effect of growth regulators on elicitation, 2,4-D,
Polygonum tinctorium cells (Funk and Brodelius, 1990;
-naphthaleneacetic acid (NAA), or kinetin was added to
Kohle et al., 1985; Kim et al., 1997). This suggested that
the culture medium at a concentration of 2 mg/l. Suspen-
menthol production could be enhanced if such an elicitor
sion cultures of M. piperita were performed for 20 days
was used in a suspension culture of M. piperita cells. To the
under the conditions as described above except that chitosan
best of our knowledge, we first report the effect of chitosan
was added to cell cultures unless otherwise specified.
elicitation on a suspension culture of M. piperita.
Analysis
Materials and methods
The cell suspension was centrifuged in a 15 ml tube at
Cell line and suspension culture
1100 g for 20 min. Centrifuged cells were washed two
Peppermint cell line was derived from the leaves of Mentha
times with distilled water and dried at 80°C for 24 h to
piperita L. Suspension cultures of M. piperita were estab-
analyze dry cell weight (DCW). Essential oil analysis was
lished and maintained in Lin-Staba (LS) medium supple-
performed as follows. Culture medium was collected after
mented with 2 mg 2,4-dichlorophenoxyacetic acid (2,4-D)
centrifugation (2000 g, 20 min). Peppermint oleoresin
and 20 g sucrose per litre. Suspension cultures were grown
was extracted with a mixture solution of pentane and
in shaking incubators at 120 rpm, 27°C, with 16 h dichloromethane (2:1) for 8 h in a continuous liquid-liquid
illumination per day. extractor. Menthone, menthol and pulegone were analyzed
© 1998 Chapman & Hall Biotechnology Letters Å" Vol 20 Å" No 12 Å" 1998 1097
J.H. Chang et al.
using a gas chromatograph (Hewlett Packard 5890 series concentration reached 166.4 mg/l at 12 days of elicitation.
II) fitted with a FID detector and an UItra-1 capillary This value is a 40 fold increase compared to the control.
column (Hewlett-Packard) packed with 100% dimethyl This result indicates that the optimum period of elicitation
polysiloxane. The flow rate of the carrier gas was 2 ml/min. for menthol production is 12 days. In our experiments,
Samples were injected at a 25:l split ratio via an injection menthol concentration decreased after 15 days. This
port at 250°C with l l aliquot and a temperature program decrease is unlikely due to cell death caused by the
of 80 l50°C at 5°C/min and 150 210°C at 20°C/min. depletion of substrate in medium since the elicited cells at
All the data were represented as the average of duplicate 15 days are in the exponential phase of cell growth. Rather,
experiments. this could be due to metabolism by extracellular enzymes
such as peroxidases. Similar results have been reported for
the production of monoterpenes by shoot cultures of
Results and discussion
peppermint (Rhodes et al., 1991).
The effect of chitosan concentrations (0, 50, 100, 150, 200,
250 and 300 mg chitosan per litre) on cell growth and
menthol production was investigated for 5 days using a
Pulegone is metabolized to menthone and then to men-
suspension culture of M. piperita. The chitosan did not
thol. Fig. 2 shows a time course production for pulegone,
inhibit the growth of M. piperita cells and menthol reached
menthone, and menthol in terms of % fraction in total
a maximum value (20 mg/l) at 200 mg chitosan/1. This
oleoresin. In elicited cells, pulegone content reached a
result is in agreement with previous findings on the opti- maximum at 6 days and decreased after 6 days. Menthone
mum concentration of chitosan (200 mg/l) in indirubin
content reached a maximum at 9 days, and then declined
production using suspension cultures of P. tinctorium (Kim
in elicited cells. However, menthol content in elicited cells
et al., 1997).
reached a maximum at 12 days. This result indicated that
a decrease of pulegone at 9 days coincides with an increase
of menthone in elicited cells. Moreover, menthol content
To determine the optimum period of elicitation, 200 mg
chitosan/l was added to suspension cultures of M. piperita.
In elicited cells, the specific menthol content increased up
to 27.8 mg/g DCW at 12 days and then decreased, whereas
the control specific menthol content remained low through-
out the 15-day period (Fig. 1). The maximum menthol
Figure 1 The effect of chitosan elicitation on the pro- Figure 2 The effect of chitosan elicitation on the forma-
duction of menthol using suspension culture of M. piperita. tion of menthol, menthone and pulegone in suspension
The initial cell concentration was 1 g DCW/l. Chitosan was culture of M. piperita. The initial cell concentration was
added at a concentration of 200 mg/l. MC, menthol con- 1 g DCW/l. Chitosan was added at a concentration of
tent (mg/l); SMC, specific menthol content (mg/g DCW). 200 mg/l. , Elicited cells; , non-elicited cells (control).
1098 Biotechnology Letters Å" Vol 20 Å" No 12 Å" 1998
Menthol production by chitosan-elicited suspension culture
increased at 12 days in elicited cells at the expense of Acknowledgement
menthone. On the other hand, menthol and menthone This work was supported by grants from the Korea Science
and Engineering Foundation through the Research Center
were low in the control without elicitation although
for New Bio-Materials in Agriculture.
pulegone was high throughout 12-day period. This implies
that the biosynthetic pathway of pulegone to menthone
might be blocked in the control. We found that in elicited References
Chung, IS, Kang, YM, Oh, JH, Kim, T, Lee, HJ and Chae, YA
cell an increased concentration of menthol and menthone
(1994). Biotechnol Tech 8:789 792
were observed in accordance with a decreased concentration
Cline, SD and Coscia, CJ (1988). Plant Physiol 86:161 165
of pulegone during the elicitation period. This result
Eilert, U (1987). Elicitation: methodology and aspects of applica-
suggests that chitosan may activate the conversion of
tion. In: Cell Culture and Somatic Cell Genetics of Plants, F
pulegone into menthol via menthone.
Constabel and IK Vasil eds vol 4 pp 153 196, New York:
Academic Press
Funk, C and Brodelius, P (1990). Phytochemistry 29: 845 848
The type of growth regulators in the culture medium can
Kim, JH and Lee, HJ (1992). J Kor Agric Chem 35: 443 448
affect the induction of secondary metabolites in cultured
Kim, JH, Shin, JH, Lee, HJ. Chung, IS and Lee, HJ (1997). J
cells quite dramatically (Cline and Coscia, 1988). To
Ferment Bioeng 83:206 208
investigate the effects of growth regulators on elicitation,
Kohle, H, Jeblick, W, Poten, F, Blaschek, W and Kauss, H
LS medium containing no growth regulators, 2 mg/l (1985). Plant Physiol 77: 544 551
Oh, JH, Kang, YM, Chung, IS, Lee, HJ and Chae, YA (1993).
2,4-D, 2 mg/l NAA, 2 mg/l kinetin was tested using
Kor J Biotechnol Bioeng 8:295 299
suspension cultures of M. piperita at 200 mg/l of chitosan.
Park, SH and Chae, YA (1990). Kor J Breed 22:53 57
In the medium with 2,4-D, the specific menthol content
Park, SH, Chae, YA, Lee, HJ and Kim, SU (1993). J Kor Agric
was highest, indicating that 2,4-D is the best among the
Chem 36:358 363
regulators tested.
Payne, GF, Bringi, V, Prince, C and Shuler, ML (1991). Questions
and strategies for productivity improvements. In: Plant Cell
and Tissue Culture in Liquid Systems, pp 329 335, New York:
In summary, menthol production was improved by 40 fold
Hanser Publishers
due to the elicitation of M. piperita with 200 mg/l of
Rhodes, MJC, Spencer, A and Hamill, JD (1991). Trans London
chitosan. Our results also suggest that chitosan elicitation
Biochem Soc 19: 702 706
may activate conversion of pulegone to menthol in suspen-
Young, DH, Kohle, H and Kauss, H (1982). Plant Physiol 70:
sion cultures of M. piperita. 1449 1454
Received: 17 August 1998
Revisions requested: 11 September 1998
Revisions received: 16 October 1998
Accepted: 19 October 1998
Biotechnology Letters Å" Vol 20 Å" No 12 Å" 1998 1099
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