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Volatile compounds and long-chain aldehydes formation in conchocelis-filaments of a red alga, Porphyra tenera
Phytochemistry, Vol. 29, No. 7, pp. 2193-2195, 1990. Printed in Great Britain.
0
003l-9422/90 $3.00+0.00 1990 Pergamon Press plc
VOLATILE COMPOUNDS AND LONG-CHAIN ALDEHYDES FORMATION IN CONCHOCELIS-FILAMENTS OF A RED ALGA, PORPHYRA TENERA TADAHIKO
KAJIWARA,
Department
MASANORI
of Agricultural
KASHIBE,
Chemistry, (Received
KENJI
Yamaguchi
MATSUI
University,
and
AKIKAZU
Yamaguchi
HATANAKA
753, Japan
9 October 1989)
tenera; marine red alga; aldehydes; volatile compounds; fatty acids.
Key Word Index-Porphyra
Abstract-The essential oil in conchocelis-filaments of a red alga, Porphyra tenera was obtained by simultaneous distillation extraction. Volatile compounds in the oil were identified by GC and GC-MS. The major constituents were cubenol, phytol and long-chain aldehydes such as tetradecanal, pentadecanal, (82,l lZ)-heptadecadienal and (8Z)heptadecenal. The aldehydes were shown to be formed enzymatically in the filaments of the alga. The substrate specificity for the long-chain aldehyde forming activity from the conchocelis-filaments was different from the thalli of a green alga, Ulua pertusa, i.e. saturated fatty acids were better substrates than unsaturated fatty acids. The reactivity decreased with both an increase and a decrease in the chain length away from that of palmitic acid (16:O). The substrate specificity seemed to reflect the composition of the long-chain aldehydes present in the essential oil.
Enzymatic formation of long-chain aldehydes in conchocelis-jilaments
INTRODUCTION The
volatile
components
of
marine
algae
have
heen
studied
for a long time [l], however, these investigation have been on dried algae. Recently, we have reported on the volatile compounds in essential oils from fresh marine algae, Ecklonia caua [2], Laminaria sp. [Z], Undaria sp. [2], Ulva pertusa [3] and Dictyopteris sp. [4]. In addition we have studied the enzymatic formation of long-chain aldehydes in the oils and its physiological role [S]. We now report on the volatile compounds in the essential oil of conchocelis-filaments of a marine red alga, Porphyrn tenera, and the biogenesis of the major volatile compounds, saturated long-chain aldehydes. RESULTS AND DISCUSSION
Volatile compounds in conchocelis-filaments The great thalli of the red alga, P. tenera is used as a food (‘Asakusa nori’) in Japan. An essential oil of cultivated conchocelis-filaments of the alga was prepared by simultaneous distillation extraction (SDE). The yield of essential oil was 0.1%. The volatile compounds in the oil were identified by GC and GC-MS (Table 1). The major compounds in the oil were cubenol (4.2%), phytol (19.2%), palmitic acid (13.2%) and the long-chain fatty aldehydes (16.9%) tetradecanal (TD), pentadecanal (PD), (82,l lZ)-heptadecadienal (HDD) and (8Z)-heptadecenal (HD). Flament and Ohloff [6] reported on the identification of more than 100 volatile constituents of dried thalli of P. tenera: nor-carotenoids (cc-ionone, /?-ionone, dihydroactinidiolide etc.) and unsaturated short-chain aldehydes [(2E,4Z)-decadienal, (2EPE)-heptadienal etc.]. However, these compounds were detected only as minor volatiles in conchocelis-filaments (Table 1). The longchain aldehydes were responsible for the characteristic odour of the essential oil from the filaments.
Recently, the biogenesis of long-chain aldehydes in the marine green alga, U. pertusa has been studied in our laboratory. However, long-chain aldehyde forming activity (LCAA) has not been reported for marine red algae. The major long-chain aldehydes such as TD, PD, HDD and HD were shown to be formed enzymatically by using a preparation from the conchocelis-filaments of the red alga. The long-chain aldehyde forming activity (LCAA) in the preparation was enhanced ca 20% by the addition of 0.2% Triton X-100 (data not shown). The pH optimum for LCAA from linoleic acid (18 : 2) was 7.5. Under anaerobic conditions, i.e. in the presence of glucose and glucose oxidase, LCAA was negligible (only 5% of control) (Table 2). Certain metal ligands (such as KCN and NaN,) showed a strong inhibitory activity (93 and 94%, respectively), whereas EDTA had no effect (Table 2). These results suggest that LCAA is probably responsible for the generation of an active species of molecular oxygen required to initiate the cc-oxidative process [7]. The substrate specificity for LCAA was examined using a series of saturated long-chain fatty acids in which the chain length varied from C,, to C,,. Palmitic acid (16:0) was the best substrate of all acids tested (Fig. 1). Unsaturated acids such as oleic acid (18: l), linoleic acid (18 : 2), cc-linolenic acid (GI-18 : 3), y-linolenic acid (y-l 8 : 3) were moderate substrates (18:2 > 18: 1 > a-18:3=?18: 3). When the number of double bond in the C,,-acid series was greater or less than two, the reactivity decreased. These results were in agreement with the substrate-specificity reported for cucumber fruit [7] and peanut cotyledons [S]. With polyenoic acids, such as arachidonic acid (20 : 4) and eicosapentaenoci acid (20: 5) LCAA showed no activity. The substrate specificity observed for LCAA in P. tenera was different from that of LCAA from thalli of U. pertusa; the latter showed a high
2193
T. KAJIWARAet ul.
2194 Table
1. Composition of volatiles identified in celis-filaments of a red alga, P. tenera
Table 2. Inhibitors
concho-
of LCAA from P. fenera Cont.
Compounds
Peak area (X)
Inhibitor
Relative activity*
5
loo 57
I
5
%
RI
Aldehydes (2E,4E)-Octadienal (2E,62)-Nonadienal (2E,4Z)-Decadienal (2E.4E)-Decadienal Tridecanal Tetradecanal Pentadecanal (7Z,lOZ)-Hexadecadienal (7Z)-Hexadecenal (82,l lZ)-Heptadecadienal (RZ)-Heptadecenal
0.09 0.09 + 0.07 0.46 1.37 10.58 0.88 0.27 1.16 1.97
634 694 884 913 1160 1276 1390 1453 1463 1553 1562
Alcohols Benzyl alcohol y-Terpineol Cubenol r-Cadinol Tetradecanol Pentadecanol Phytol
+ + 4.20 0.21 0.78 0.15 19.16
521 763 1327 1338 1351 1457 1796
Ketones cc-Ionone P-Ionone 6,10,14-Trimethylpentadecan-2-one
0.07 0.07 0.37
1063 1193 1518
Lactone Dihydroactinidiolide
+
1173
0.11 0.06 0.37 2.68 1.98 13.21 2.88
864 989 1224 1440 1534 1646 1822
Carboxylic acids Nonanoic acid Decanoic acid Laurie acid Myristic acid Pentadecanoic acid Palmitic acid Oleic acid
(mM)
None Imidazole L-Cysteine KCN NaN, EDTA DTT GSH Mercaptoethanol Catalase Peroxidase Glucose oxidase
I
7
1 1 I
6 I03
8
1 I
1mgml-’ 250 btg ml .- ’ 1 mgml-’
20 4 8 34 5
*Enzyme activities are expressed relative in mole number to HDD obtained from 18 :2 with no additions.
Relative activiv
(8 )
13:o 14:o IS:0 II p_ mu _
16:O
18:2
Hydrocarbons Limonene Tridecane Tetradecane Pentadecane Hexadecane Heptadecane Octadecane Nonadecane Icosane
0.02 + 0.07 0.33 0.19 0.19 0.22 0.68 0. I6
538 924 1050 1169 1289 1392 1493 1588 I679
Sulphur compound Benzothiazole
0.91
793
+ Less than 0.02%. reactivity for 18 : 2 and a-18 : 3, and a low reactivity for the saturated acids. Furthermore, the major volatile aldehydes in the conchocelis-filaments of P. tenera was PD, whereas those in Cl. pertusa were HDD and (82.1 lZ,14Z)-heptadecatrienal (HDT) [2]. From these results, the substrate specificity for LCAA from the filaments was considered to reflect the composition of volatile compounds in the essential oil from the filaments. LCAA in the thallus stage was much lower (5%) than
1
17:o
Y 2 ZL 18:O. 3 cc IX:1 m
I I I
a-18:3 r-18:3 IX:4
Fig. 1. Comparison of substrate specificity of LCAA in P. fenera and U. pertusa. Activities are expressed relative in mole number to HDD obtained with 18:2 in U. pertuscz, to PD obtained with 16:0 in P. temw.
that in the conchocelis. This fact suggests that LCAA might play an physiological role in the transistion from the conchocelis stage to the thallus stage. EXPERIMEhlTAL
Materiuls. Conchocelis-filaments of Porphyrasp. were cultivated at Yamaguchi Prefectural Naikai Sea-farming Center, Yamaguchi City, Japan. The plants were cultured in filtered
Essential oil of red alga sea-water containing 0.5 mll-1 of Porufiran Konko (Kyowa Hakko Ltd, Japan) at 20-23 ° under fluorescent light (100-2000 Ix, continuous light) with aeration for 3-6 months. Fresh fronds of U. pertusa were collected at Aio bay in Yamaguchi, Japan in May 1989. Fatty acids (over 98% purity) (Wako Pure Chemical Industries Ltd) were used after purification, imidazole, L-cysteine, KCN, NAN3, GSH and mercaptoethanol (Wako), DTT (Nakalai Tesque, Inc), catalase, peroxidase and glucose oxidase (Sigma).
Identification of volatile component in conchocelis-filaments of P. tenera. Fresh filaments (10 g) were homogenized with dist. H 2 0 (500 ml), and the homogenate immediately subjected to SDE. Extraction of the distillate (700 ml) with pentane 42H2C12 (200 ml x 3, 1:1) gave an essential oil (10 mg: yield 0.1%). Volatile compounds in the oil were identified by Rts on GC and GC-MS. GC was performed with a Hewlett Packard 5840 A instrument equipped with a FID and a fused silica capillary column 0.28 mm (i.d.)× 50 m coated with SF-96. The column temp. was held at 70 ° for 5 min and programmed to increase at 2° min- 1 from 70 to 220 °. GC-MS was recorded on a Hitachi H-80A instrument equipped with a fused silica capillary column 0.28 mm (i.d.) x 40 m coated with SF-96. The column temp. was programmed to increase from 75 to 190 ° at 3° min- ~ (25 min hold at 190 °) and then 190 to 210 ° at 3 ° min- ~. The ionization energy was 20 eV to give a stronger molecular ion. Most peaks were identified by comparison with R~s and MS of authentic compounds. Assay of LCCA in conchocelis-filaments of P. tenera and thalli of U. pertusa. Fresh conchocelis-filaments or U. pertusa (1 g) were homogenized with 50 mM Na-Pi buffer (pH 7.5) containing 0.2% Triton X-100. The homogenate was filtered through four layers of cheese cloth. The fatty acid substrate (3.57 #tool) was added to the filtrate (20 ml) and the mixture incubated at 25 ° for
2195
60 min. Aldehydes formed during the incubation were analysed by HPLC as 2,4-dinitrophenylhydrazone (2,4-DNPH) derivatives. Each reaction mixture was added to an 0.1% E t O H - H O A c soln of 2,4-dinitrophenylhydrazine (3 ml, pH 4.0). The D N P H derivatives were extracted with hexane (20 ml x 3) and the combined hexane extracts were washed with satd NaCI soln and then dried over Na2SO 4. The extract was evapd in vacuo to leave the solid 2,4-DNPH derivatives. The derivatives were dissolved in CHCI 3 (0.5 ml) and an aliquot (5 #1) was quantitatively analysed by HPLC with a UV detector at 349 nm: Zorbax ODS column 4.6 mm (i.d.) × 150 mm; solvent: M e C N - H 2 0 - T H F (90:9:1) flow rate 1.0 ml min - 1, pressure 70 kg cm - z.
REFERENCES 1. Katayama, T. (1962) in Physiolooy and Biochemistry of Algae (Lewin, R. A., ed.) p. 467. Academic Press, New York. 2. Kajiwara, T., Hatanaka, A., Kawai, T., Ishihara, M. and Tsuneya, T. (1988) J. Food Sci. 53, 960. 3. Kajiwara, T., Hatanaka, A., Kawai, T. and Ishihara, M. (1987) Nippon Suisan Gakkaishi 53, 1901. 4. Kajiwara, T., Hatanaka, A., Tanaka, Y., Kawai, T., Ishihara, M., Tsuneya, T. and Fujimura, T. (1989) Phytochemistry 28, 636. 5. Kajiwara, T., Yoshikawa, H. Matsui, K., Hatanaka, A. and Kawai, T. (1989) Phytochemistry 28, 407. 6. Flament, I. and Ohloff, G. (1984) in Progress in Flaoour Research (Adda, J., ed.), p. 281. Elsevier, Amsterdam. 7. Galliard, T. and Mathew, J. A. (1976) Biochim. Biophys. Acta 424, 26. 8. Shine, W. E. and Stumpf, P. K. (1974) Arch. Biochem. Biophys. 162, 147.
0
003l-9422/90 $3.00+0.00 1990 Pergamon Press plc
VOLATILE COMPOUNDS AND LONG-CHAIN ALDEHYDES FORMATION IN CONCHOCELIS-FILAMENTS OF A RED ALGA, PORPHYRA TENERA TADAHIKO
KAJIWARA,
Department
MASANORI
of Agricultural
KASHIBE,
Chemistry, (Received
KENJI
Yamaguchi
MATSUI
University,
and
AKIKAZU
Yamaguchi
HATANAKA
753, Japan
9 October 1989)
tenera; marine red alga; aldehydes; volatile compounds; fatty acids.
Key Word Index-Porphyra
Abstract-The essential oil in conchocelis-filaments of a red alga, Porphyra tenera was obtained by simultaneous distillation extraction. Volatile compounds in the oil were identified by GC and GC-MS. The major constituents were cubenol, phytol and long-chain aldehydes such as tetradecanal, pentadecanal, (82,l lZ)-heptadecadienal and (8Z)heptadecenal. The aldehydes were shown to be formed enzymatically in the filaments of the alga. The substrate specificity for the long-chain aldehyde forming activity from the conchocelis-filaments was different from the thalli of a green alga, Ulua pertusa, i.e. saturated fatty acids were better substrates than unsaturated fatty acids. The reactivity decreased with both an increase and a decrease in the chain length away from that of palmitic acid (16:O). The substrate specificity seemed to reflect the composition of the long-chain aldehydes present in the essential oil.
Enzymatic formation of long-chain aldehydes in conchocelis-jilaments
INTRODUCTION The
volatile
components
of
marine
algae
have
heen
studied
for a long time [l], however, these investigation have been on dried algae. Recently, we have reported on the volatile compounds in essential oils from fresh marine algae, Ecklonia caua [2], Laminaria sp. [Z], Undaria sp. [2], Ulva pertusa [3] and Dictyopteris sp. [4]. In addition we have studied the enzymatic formation of long-chain aldehydes in the oils and its physiological role [S]. We now report on the volatile compounds in the essential oil of conchocelis-filaments of a marine red alga, Porphyrn tenera, and the biogenesis of the major volatile compounds, saturated long-chain aldehydes. RESULTS AND DISCUSSION
Volatile compounds in conchocelis-filaments The great thalli of the red alga, P. tenera is used as a food (‘Asakusa nori’) in Japan. An essential oil of cultivated conchocelis-filaments of the alga was prepared by simultaneous distillation extraction (SDE). The yield of essential oil was 0.1%. The volatile compounds in the oil were identified by GC and GC-MS (Table 1). The major compounds in the oil were cubenol (4.2%), phytol (19.2%), palmitic acid (13.2%) and the long-chain fatty aldehydes (16.9%) tetradecanal (TD), pentadecanal (PD), (82,l lZ)-heptadecadienal (HDD) and (8Z)-heptadecenal (HD). Flament and Ohloff [6] reported on the identification of more than 100 volatile constituents of dried thalli of P. tenera: nor-carotenoids (cc-ionone, /?-ionone, dihydroactinidiolide etc.) and unsaturated short-chain aldehydes [(2E,4Z)-decadienal, (2EPE)-heptadienal etc.]. However, these compounds were detected only as minor volatiles in conchocelis-filaments (Table 1). The longchain aldehydes were responsible for the characteristic odour of the essential oil from the filaments.
Recently, the biogenesis of long-chain aldehydes in the marine green alga, U. pertusa has been studied in our laboratory. However, long-chain aldehyde forming activity (LCAA) has not been reported for marine red algae. The major long-chain aldehydes such as TD, PD, HDD and HD were shown to be formed enzymatically by using a preparation from the conchocelis-filaments of the red alga. The long-chain aldehyde forming activity (LCAA) in the preparation was enhanced ca 20% by the addition of 0.2% Triton X-100 (data not shown). The pH optimum for LCAA from linoleic acid (18 : 2) was 7.5. Under anaerobic conditions, i.e. in the presence of glucose and glucose oxidase, LCAA was negligible (only 5% of control) (Table 2). Certain metal ligands (such as KCN and NaN,) showed a strong inhibitory activity (93 and 94%, respectively), whereas EDTA had no effect (Table 2). These results suggest that LCAA is probably responsible for the generation of an active species of molecular oxygen required to initiate the cc-oxidative process [7]. The substrate specificity for LCAA was examined using a series of saturated long-chain fatty acids in which the chain length varied from C,, to C,,. Palmitic acid (16:0) was the best substrate of all acids tested (Fig. 1). Unsaturated acids such as oleic acid (18: l), linoleic acid (18 : 2), cc-linolenic acid (GI-18 : 3), y-linolenic acid (y-l 8 : 3) were moderate substrates (18:2 > 18: 1 > a-18:3=?18: 3). When the number of double bond in the C,,-acid series was greater or less than two, the reactivity decreased. These results were in agreement with the substrate-specificity reported for cucumber fruit [7] and peanut cotyledons [S]. With polyenoic acids, such as arachidonic acid (20 : 4) and eicosapentaenoci acid (20: 5) LCAA showed no activity. The substrate specificity observed for LCAA in P. tenera was different from that of LCAA from thalli of U. pertusa; the latter showed a high
2193
T. KAJIWARAet ul.
2194 Table
1. Composition of volatiles identified in celis-filaments of a red alga, P. tenera
Table 2. Inhibitors
concho-
of LCAA from P. fenera Cont.
Compounds
Peak area (X)
Inhibitor
Relative activity*
5
loo 57
I
5
%
RI
Aldehydes (2E,4E)-Octadienal (2E,62)-Nonadienal (2E,4Z)-Decadienal (2E.4E)-Decadienal Tridecanal Tetradecanal Pentadecanal (7Z,lOZ)-Hexadecadienal (7Z)-Hexadecenal (82,l lZ)-Heptadecadienal (RZ)-Heptadecenal
0.09 0.09 + 0.07 0.46 1.37 10.58 0.88 0.27 1.16 1.97
634 694 884 913 1160 1276 1390 1453 1463 1553 1562
Alcohols Benzyl alcohol y-Terpineol Cubenol r-Cadinol Tetradecanol Pentadecanol Phytol
+ + 4.20 0.21 0.78 0.15 19.16
521 763 1327 1338 1351 1457 1796
Ketones cc-Ionone P-Ionone 6,10,14-Trimethylpentadecan-2-one
0.07 0.07 0.37
1063 1193 1518
Lactone Dihydroactinidiolide
+
1173
0.11 0.06 0.37 2.68 1.98 13.21 2.88
864 989 1224 1440 1534 1646 1822
Carboxylic acids Nonanoic acid Decanoic acid Laurie acid Myristic acid Pentadecanoic acid Palmitic acid Oleic acid
(mM)
None Imidazole L-Cysteine KCN NaN, EDTA DTT GSH Mercaptoethanol Catalase Peroxidase Glucose oxidase
I
7
1 1 I
6 I03
8
1 I
1mgml-’ 250 btg ml .- ’ 1 mgml-’
20 4 8 34 5
*Enzyme activities are expressed relative in mole number to HDD obtained from 18 :2 with no additions.
Relative activiv
(8 )
13:o 14:o IS:0 II p_ mu _
16:O
18:2
Hydrocarbons Limonene Tridecane Tetradecane Pentadecane Hexadecane Heptadecane Octadecane Nonadecane Icosane
0.02 + 0.07 0.33 0.19 0.19 0.22 0.68 0. I6
538 924 1050 1169 1289 1392 1493 1588 I679
Sulphur compound Benzothiazole
0.91
793
+ Less than 0.02%. reactivity for 18 : 2 and a-18 : 3, and a low reactivity for the saturated acids. Furthermore, the major volatile aldehydes in the conchocelis-filaments of P. tenera was PD, whereas those in Cl. pertusa were HDD and (82.1 lZ,14Z)-heptadecatrienal (HDT) [2]. From these results, the substrate specificity for LCAA from the filaments was considered to reflect the composition of volatile compounds in the essential oil from the filaments. LCAA in the thallus stage was much lower (5%) than
1
17:o
Y 2 ZL 18:O. 3 cc IX:1 m
I I I
a-18:3 r-18:3 IX:4
Fig. 1. Comparison of substrate specificity of LCAA in P. fenera and U. pertusa. Activities are expressed relative in mole number to HDD obtained with 18:2 in U. pertuscz, to PD obtained with 16:0 in P. temw.
that in the conchocelis. This fact suggests that LCAA might play an physiological role in the transistion from the conchocelis stage to the thallus stage. EXPERIMEhlTAL
Materiuls. Conchocelis-filaments of Porphyrasp. were cultivated at Yamaguchi Prefectural Naikai Sea-farming Center, Yamaguchi City, Japan. The plants were cultured in filtered
Essential oil of red alga sea-water containing 0.5 mll-1 of Porufiran Konko (Kyowa Hakko Ltd, Japan) at 20-23 ° under fluorescent light (100-2000 Ix, continuous light) with aeration for 3-6 months. Fresh fronds of U. pertusa were collected at Aio bay in Yamaguchi, Japan in May 1989. Fatty acids (over 98% purity) (Wako Pure Chemical Industries Ltd) were used after purification, imidazole, L-cysteine, KCN, NAN3, GSH and mercaptoethanol (Wako), DTT (Nakalai Tesque, Inc), catalase, peroxidase and glucose oxidase (Sigma).
Identification of volatile component in conchocelis-filaments of P. tenera. Fresh filaments (10 g) were homogenized with dist. H 2 0 (500 ml), and the homogenate immediately subjected to SDE. Extraction of the distillate (700 ml) with pentane 42H2C12 (200 ml x 3, 1:1) gave an essential oil (10 mg: yield 0.1%). Volatile compounds in the oil were identified by Rts on GC and GC-MS. GC was performed with a Hewlett Packard 5840 A instrument equipped with a FID and a fused silica capillary column 0.28 mm (i.d.)× 50 m coated with SF-96. The column temp. was held at 70 ° for 5 min and programmed to increase at 2° min- 1 from 70 to 220 °. GC-MS was recorded on a Hitachi H-80A instrument equipped with a fused silica capillary column 0.28 mm (i.d.) x 40 m coated with SF-96. The column temp. was programmed to increase from 75 to 190 ° at 3° min- ~ (25 min hold at 190 °) and then 190 to 210 ° at 3 ° min- ~. The ionization energy was 20 eV to give a stronger molecular ion. Most peaks were identified by comparison with R~s and MS of authentic compounds. Assay of LCCA in conchocelis-filaments of P. tenera and thalli of U. pertusa. Fresh conchocelis-filaments or U. pertusa (1 g) were homogenized with 50 mM Na-Pi buffer (pH 7.5) containing 0.2% Triton X-100. The homogenate was filtered through four layers of cheese cloth. The fatty acid substrate (3.57 #tool) was added to the filtrate (20 ml) and the mixture incubated at 25 ° for
2195
60 min. Aldehydes formed during the incubation were analysed by HPLC as 2,4-dinitrophenylhydrazone (2,4-DNPH) derivatives. Each reaction mixture was added to an 0.1% E t O H - H O A c soln of 2,4-dinitrophenylhydrazine (3 ml, pH 4.0). The D N P H derivatives were extracted with hexane (20 ml x 3) and the combined hexane extracts were washed with satd NaCI soln and then dried over Na2SO 4. The extract was evapd in vacuo to leave the solid 2,4-DNPH derivatives. The derivatives were dissolved in CHCI 3 (0.5 ml) and an aliquot (5 #1) was quantitatively analysed by HPLC with a UV detector at 349 nm: Zorbax ODS column 4.6 mm (i.d.) × 150 mm; solvent: M e C N - H 2 0 - T H F (90:9:1) flow rate 1.0 ml min - 1, pressure 70 kg cm - z.
REFERENCES 1. Katayama, T. (1962) in Physiolooy and Biochemistry of Algae (Lewin, R. A., ed.) p. 467. Academic Press, New York. 2. Kajiwara, T., Hatanaka, A., Kawai, T., Ishihara, M. and Tsuneya, T. (1988) J. Food Sci. 53, 960. 3. Kajiwara, T., Hatanaka, A., Kawai, T. and Ishihara, M. (1987) Nippon Suisan Gakkaishi 53, 1901. 4. Kajiwara, T., Hatanaka, A., Tanaka, Y., Kawai, T., Ishihara, M., Tsuneya, T. and Fujimura, T. (1989) Phytochemistry 28, 636. 5. Kajiwara, T., Yoshikawa, H. Matsui, K., Hatanaka, A. and Kawai, T. (1989) Phytochemistry 28, 407. 6. Flament, I. and Ohloff, G. (1984) in Progress in Flaoour Research (Adda, J., ed.), p. 281. Elsevier, Amsterdam. 7. Galliard, T. and Mathew, J. A. (1976) Biochim. Biophys. Acta 424, 26. 8. Shine, W. E. and Stumpf, P. K. (1974) Arch. Biochem. Biophys. 162, 147.