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Incorporation of n-3 polyunsaturated fatty acids into phospholipids of a marine bacterium Vibrio sp. cultivated with sardine oil
JOUR.NAL OF FERMENTATION AND BIOENGINEERING
Vol. 73, No. 2, 169-171. 1992
Incorporation of n-3 Polyunsaturated Fatty Acids into Phospholipids of a Marine Bacterium Vibrio sp. Cultivated with Sardine Oil SEIICHI ANDO, I* KAZUYA NAKAJIMA, 2 AND MUTSUO HATANO 2
Laboratory of Food Chemistry, Faculty of Fisheries, Kagoshima University, Kagoshima 890, ~and Laboratory of Food Chemistry, Faculty of Fisheries, Hokkaido University, Hakodate 041,2 Japan Received 27 April 1991/Accepted 9 November 1991 The fatty acid compositions of phosphatidylethanolamine (PE) and phosphatidylglyceroi (PG) were modified by cultivating the strain VB-5, a marine bacterium of genus Vibrio, with sardine oil triglyceride (TG). lcosapentaenoic and docosahexaenoic acids from sardine oil TG were clearly detected in both PE and PG of the strain VB-5.
n-3 Polyunsaturated fatty acids (n-3 PUFA) such as icosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are of considerable pharmaceutical interest due to their biomedical properties (1-3)..Most of n-3 PUFA usually occupies the sn-2 position of phospholipids which are quantitatively the most significant component of membrane lipids (4). Various bioactive actions such as antihypertension, lowering of stored adipose tissue weight, and low lipid levels in plasma and liver were observed when fed EPA-containing phospholipids obtained from an EPA-producing bacterium to spontaneously hypertensive rats (5, 6). Much attention thus is being devoted to the function of n-3 PUFA-containing phospholipids in connection with atherosclerosis. In our previous studies, a potent marine bacterium of genus Vibrio for lipase production was isolated and tentatively named VB-5 (7). The lipase activity of the strain VB-5 was enhanced significantly when cultivated with olive oil, suggesting the lipase was induced by olive oil. The olive oil-induced lipase from the strain VB-5 was capable of liberatirig EPA and DHA from sardine oil triglyceride (TG). In the course of the studies on lipase production from the strain VB-5, we noticed that the fatty acid composition of the lipids from the strain VB-5 cultivated with olive oil was modified greatly. Taking into account that the main lipids in bacteria are phospholipids (4), this observation suggests that n-3 PUFA-containing phospholipids are obtained from the strain VB-5 when cultivated with fish oil TG. The present study was designed to examine whether the strain VB-5 utilized n-3 PUFA-containing sardine oil TG as its own lipid. A cultivation of genus Vibrio, tentatively named VB-5, isolated from the red tides of Tanabe Bay in Wakayama Prefecture, Japan, was grown in a selected medium, CPY, at 15°C with rotary shaking (100rpm) for 2d. The CPY medium comprised 0.1% casitone, 0.05% proteose peptone, 0.1% yeast extract and 300 ml of 75% natural seawater. In order to examine whether the strain VB-5 utilized n-3 PUFA-containing fish oil TG as its own)ipid, 1 or 3% sardine oil TG was also added to the CPY medium. Sardine oil was extracted from the sardine muscle and sardine oil TG was purified from the sardine oil by silica column chromatography. After cultivation of the strain VB-5 at 15°C for 2 d, cells
were separated from the growth medium by centrifugation. Cells harvested were thoroughly washed with cold 75% natural seawater to remove the growth medium containing sardine oil. Lipid was extracted from 1 g of wet cells according to the method of Bligh and Dyer (8). Lipid extraction from the cells was done at 4°C to minimize lipid deterioration. The total lipid was analyzed quantitatively by thin-layer chromatography (TLC). The TLC plates (Kieselgel 60, Merck) were developed using n-hexane-diethyl ether-formic acid 7 5 : 2 5 : 2 (by volume) for non-phospholipids, and chloroform-methanol-water 6 5 : 2 5 : 4 (by volume) for phospholipids, respectively. The TLC plate was sprayed with a 3% copper acetate-8% phosphoric acid, heated at 150°C for 5 min, and quantitated using a Cosmo F-808 densitometer. Identification of each lipid class was accomplished by co-TLC with authentic specimens of oleic acid, phosphatidylethanolamine (PE) and phosphatidylglycerol (PG). A portion of total lipid was applied to preparative TLC. The TLC plate was developed using chloroform-methanolwater 65 : 25 : 4 (by volume). The TLC plate was sprayed with a Rhodamine 6G reagent, and PE and PG were detected under ultraviolet light. Each lipid class was eluted with chloroform and methanol, and their fatty acid compositions were analyzed by gas-liquid chromatography (GLC) after methylation with methanol containing anhydrous hydrogen chloride. GLC analysis was performed with a Hitachi 063 gas chromatograph equipped with a hydrogen flame ionization detector, using a glass column (3 mm i.d. and 4 m length) packed with Unisole 3000 on a Uniport C (80-100 mesh). Identification of individual fatty acids was accomplished by comparing with relative retention times and equivalent chain lengths of authentic standards. The strain VB-5 used in the present study was classified as a typical marine bacterium, which was Gram-negative, rod-shaped, motile by means of a polar flagellum, and essentially required seawater for growth (7). Table 1 shows the total lipid content and lipid composition of the strain VB-5 cultivated at 15°C in the medium with or without sardine oil TG. The total lipid content of the strain VB-5 cultivated with sardine oil TG free medium was 1.46% on a wet weight basis, and tended to increase when cultivated with sardine oil TG. Phospholipid and non-phospholipid classes from the strain VB-5 cultivated with sardine oil TG
* Corresponding author. 169
170
J. FEILM~NT. BIOI/NG.,
ANDO ET AL.
TABLE 1. Total lipid content and lipid composition of VB-5 cultivated at 15°C with or without sardine oil triglyceride Sardine oil added Total lipid (%) (mg/gwet cells) 0 1
14.6 14.7
3
25.4
Lipid composition (%) PE*
PG
FFA
46.9 22.7 30.4 35.0 17.0 31.5 (41.9) b (20.4) (37.7) 21.2 10.1 21.7 (40.0) (19.1) (40.9)
TG
FAE
-16.5
---
37.9
9.0
a Abbreviations: PE, phosphatidylethanolamine; PG, phosphatidylglycerol; FFA, free fatty acid; TG, triglyceride; FAE, fatty acid ester. b Values in parentheses are percentages, with the sum of PE, PG and FFA as 100%. free m e d i u m were P E a n d P G , a n d free f a t t y acid, respectively. It is u n c l e a r w h y t h e s t r a i n VB-5 h a d u n u s u a l l y h i g h levels o f free f a t t y acid; b u t this m i g h t b e a s s o c i a t e d w i t h t h e lipase activity o f t h e s t r a i n VB-5 as d e s c r i b e d a b o v e . P E a n d P G are f o u n d as t h e m a i n p h o s p h o l i p i d s o f terrestrial b a c t e r i a , w h e r e a s significant a m o u n t s o f free f a t t y acid as well as P E a n d P G are d e t e c t e d f r o m m a r i n e b a c t e r i a ( K u n i m o t o , M . et al., Bull. Fac. F i s h . H o k k a i d o U n i v . , 25, 332-341, 3 4 2 - 3 5 0 , 1975). T h e p o s s i b l e significance o f free f a t t y acid is u n c l e a r , b u t free f a t t y acid f o u n d in t h e s t r a i n VB-5 m i g h t b e a t t r i b u t e d t o a c h a r a c t e r i s t i c o f m a r i n e b a c t e r i a . T G a n d f a t t y acid ester, besides P E , P G a n d free f a t t y acid, were f o u n d in t h e s t r a i n VB-5 c u l t i v a t e d w i t h s a r d i n e oil T G . N e w l y d e t e c t e d c o m p o n e n t s o f T G a n d f a t t y acid ester s e e m e d to b e c o n t a m i n a t i o n d e r i v e d TABLE 2. ,
f r o m t h e g r o w t h m e d i u m c o n t a i n i n g s a r d i n e oil T G . A c o m p l e t e s e p a r a t i o n o f the g r o w t h m e d i u m c o n t a i n i n g s a r d i n e oil T G f r o m cells was very difficult, a l t h o u g h t h e cells were t h o r o u g h l y w a s h e d w i t h s e a w a t e r t o r e m o v e t h e g r o w t h m e d i u m . T h e lipid c o m p o s i t i o n s o f P E , P G a n d free f a t t y acid were a l m o s t t h e s a m e , i r r e s p e c t i v e o f cult i v a t i n g t h e s t r a i n VB-5 w i t h o r w i t h o u t s a r d i n e oil T G . T h e m a i n p h o s p h o l i p i d s in t h e s t r a i n VB-5 were P E a n d P G , as well as o t h e r b a c t e r i a (4, 6; K u n i m o t o , M . et al., Bull. F a c . Fish. H o k k a i d o U n i v . , 25, 3 3 2 - 3 4 1 , 3 4 2 - 3 5 0 , 1975; W a t a n a b e , K. et al., P r o c . J a p a n . C o n f . B i o c h e m . L i p i d s , 33, 2 9 - 3 2 , 1991). W e e x a m i n e d w h e t h e r t h e s t r a i n VB-5 utilized n-3 P U F A - c o n t a i n i n g s a r d i n e oil T G a n d if t h e f a t t y acid c o m p o s i t i o n s o f p h o s p h o l i p i d s were m o d i f i e d . T a b l e 2 s h o w s t h e f a t t y acid c o m p o s i t i o n s o f P E a n d P G f r o m t h e s t r a i n VB-5 c u l t i v a t e d at 15°C in t h e m e d i u m w i t h o r w i t h o u t s a r d i n e oil T G . T h e m a i n f a t t y acids o f P E a n d P G f r o m t h e s t r a i n VB-5 c u l t i v a t e d w i t h s a r d i n e oil T G free m e d i u m were p a l m i t o l e i c (16 : 1), oleic (18 : 1) a n d p a l m i t i c (16 : 0) acids, a c c o u n t i n g f o r o v e r 8 0 % o f t h e t o t a l f a t t y acids. P E c o n t a i n e d relatively m o r e m y r i s t i c ( 1 4 : 0 ) , p a l m i t i c a n d oleic acids, a n d less p a l m i t o l e i c acid t h a n P G . N o n-3 P U F A was f o u n d in e i t h e r P E a n d P G f r o m t h e s t r a i n VB5 c u l t i v a t e d w i t h s a r d i n e oil T G free m e d i u m . T h e f a t t y acid c o m p o s i t i o n s o f P E a n d P G were m o d i f i e d b y c u l t i v a t ing t h e s t r a i n VB-5 w i t h s a r d i n e oil T G , w h o s e m a i n f a t t y acids were E P A ( 2 0 : 5 ) , D H A ( 2 2 : 6 ) , p a l m i t i c , oleic, m y r i s t i c a n d p a l m i t o l e i c acids. T h e selective i n c o r p o r a t i o n s o f f a t t y acids i n t o P E a n d P G were f o u n d w h e n t h e s t r a i n VB-5 was c u l t i v a t e d w i t h s a r d i n e oil T G . W i t h
Fatty acid composition (%) of phosphatidylethanolamine and phosphatidylglycerol from VB-5 cultivated at 15°C with or without sardine oil triglyceride
Fatty acids
Sardine oil
1"2: 0 13 : 0 14 : 0 14 : 1 14 : 2 15 : 0 15 : 1 15 : 2 •16 : 0 16 : 1 16 : 2 17 : 0 17 : 1 17 : 2 18 • 0 18 : 1 18 : 2 19 : 1 19 : 2 20 : 1 20 : 3 20 : 4 20 : 5 22 : 1 22 : 5 22 : 6 Others Saturated Monoenoic Polyenoic
0.82 -8.10 -0.19 0.35 m -14.52 7.50 0.48 1.62 1.77 -4.06 12.74 0.90 0.70 1.72 3.07 0.25 4.51 22.54 2.59 2.73 6.09 2.75 29.47 28.37 42.16
Phosphatidylethanolamine Sardine oil added (%) 0 1 3 0.17 0.17 2.44 1.15 0.32 1.51 2.07 0.68 13.75 33.43 1.02 2.64 5.83 0.38 1.72 31.35 0.18 0.87 -0.29 ---. --0.03 22.41 75.00 2.58
0.42 0.14 5.96 0.98 0.30 0.82 0.56 0.47 14.58 31.55 0.97 2.19 3.06 0.32 1.98 31.06 0.16 0.79 0.34 0.27 0.10 0.28 2.15 .
. -0.30 0.25 26.28 68.09 5.62
0.61 0.14 6.33 0.85 0.27 0.86 0.50 0.42 12.90 22.97 0.67 2.37 3.18 0.30 1.69 30.73 1.34 0.87 0.69 0.30 0.43 1.10 7.11 . 0.36 1.91 1.10 25.44 59.42 15.15
0
Phosphatidylglycerol Sardine oil added (%) 1
3
0.14 -0.51 0.24 m 0.78 0.77 0.43 9.16 46.50 1.10 2.16 8.93 0.42 1.61 26.17 0.18 0.69 -0.20 -~ --
0.52 0.I0 1.63 0.39 0.08 0.39 0.29 0.32 12.05 38.63 0.83 2.10 5.17 0.33 2.36 27.10 0.78 0.80 0.48 0.57 0.06 0.50 2.47
0.46 -2.20 0.27 -0.56 0.16 0.14 16.19 29.23 0.65 2.29 4.49 -1.81 27.15 1.74 0.60 0.49 0.62 0.66 2.01 5.40
0.14 0.93 0.98 19.16 72.94 7.90
0.25 1.51 1.01 24.17 62.51 13.32
.
. --0.20 14.42 83.86 1.72
VOL. 73, 1992
NOTES
171
cultivating them with potassium salts of E P A and D H A (Watanabe, K. et al., Proc. Japan. Conf. Biochem. Lipids, 33, 29-32, 1991). These results suggest that the strain VB-5 might produce n-3 P U F A - c o n t a i n i n g phospholipids by hydrolyzing sardine oil T G by means of lipase. Further studies on the optimal conditions for the incorporation of n-3 P U F A - c o n t a i n i n g phospholipids by the strain VB-5 will be required. We are indebted to M. Hosokawa and A. Yoshida for their helpful discussion. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (S.A.). REFERENCES 1. Dyerberg, J. and Jorgensen, K. A.: Marine oils and thrombogene-
sis. Prog. Lipid Res., 21, 255-269 (1982). FIG. I. Appearance of growth medium from the strain VB-5 cultivated at 15°C with (A) or without (B) sardine oil triglyceride. respect to saturated fatty acids of PE and PG, relatively more myristic and less palmitic acids were incorporated into PE than PG. The percentage of palmitoleic acid decreased in both PE and P G from the strain VB-5 when cultivated with sardine oil TG, whereas that of oleic acid was almost unchanged. E P A and D H A from sardine oil TG were clearly detected in both PE and PG. Taking into account the distribution patterns of palmitoleic acid, E P A and DHA, palmitoleic acid seemed to be substituted for E P A and D H A in both PE and PG from the strain VB-5 cultivated with sardine oil TG. It is very interesting how the strain VB-5 utilizes n-3 P U F A - c o n t a i n i n g sardine oil TG. The strain VB-5 produced a lipase by which n-3 P U F A such as E P A and D H A were liberated from sardine oil T G (7). Sardine oil T G added to the growth medium turned oil bowl-like clot, whose main component was free fatty acid, by means of the lipase produced by the strain VB-5 (Fig. 1). The incorporation of exogeneous fatty acids into phospholipids was found in Escherichia coli (9, 10). Furthermore, Yazawa et al. have recently reported that the fatty acid compositions in some bacterial phospholipids were greatly modified by
2. Suzuki, H. and Wada, S.: Metabolism and function of icosapen-
taenoic and docosahexaenoic acids. Yukagaku, 37, 781-787 (1988). 3. Watanabe, S. and Okuyama, H.: Physiological significanceof n3 fatty acid-containing phospholipids. Protein, Nucleic Acid and Enzyme, 36, 584-588 (1991). 4. Cronan, J.E. Jr,: Molecular biology of bacterial membrane lipids. Ann. Rev. Biochem., 47, 163-189 (1978). 5. Yazawa, K., Araki, K., Watanabe, K., lshikawa, C., Inoue, A., Kondo, K., Watabe, S., and Hashimoto, K.: Eicosapentaenoic
acid productivity of bacteria isolated from fish intestines. Nippon Suisan Gakkaishi, 54, 1835-1838 0988). 6. Yazawa, K., Jiang, M.-C., Ishikawa, C., Watanabe, K., Akahoti, Y., Kimura, S., and Konda, K.: Bioactivity of phospho-
lipids from EPA-producing bacteria. Seikagaku, 61, 1090 (1989). 7. Ando, S., Yoshida, A., and Hatano, M.: Occurrence of marine bacterial lipase hydrolyzing fish oil. Agric. Biol. Chem., 55, 2657-2659 (1991). 8. Bligh, E. G. and Dyer, W.J.: A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol., 37, 911917 (1959). 9. Cronan, J.E. Jr.: Evidence that incorporation of exogeneous fatty acids into the phospholipids of Escherichia coli does not require acyl carrier protein. J. Bacteriol., 159, 773-775 0984). 10. Rock, C. O. and Jaekowski, S.: Pathways for the incorporation of exogeneous fatty acids into phosphatidylethanolamine in Escherichia coli. J. Biol. Chem., 260, 12720-12724 (1985).
Vol. 73, No. 2, 169-171. 1992
Incorporation of n-3 Polyunsaturated Fatty Acids into Phospholipids of a Marine Bacterium Vibrio sp. Cultivated with Sardine Oil SEIICHI ANDO, I* KAZUYA NAKAJIMA, 2 AND MUTSUO HATANO 2
Laboratory of Food Chemistry, Faculty of Fisheries, Kagoshima University, Kagoshima 890, ~and Laboratory of Food Chemistry, Faculty of Fisheries, Hokkaido University, Hakodate 041,2 Japan Received 27 April 1991/Accepted 9 November 1991 The fatty acid compositions of phosphatidylethanolamine (PE) and phosphatidylglyceroi (PG) were modified by cultivating the strain VB-5, a marine bacterium of genus Vibrio, with sardine oil triglyceride (TG). lcosapentaenoic and docosahexaenoic acids from sardine oil TG were clearly detected in both PE and PG of the strain VB-5.
n-3 Polyunsaturated fatty acids (n-3 PUFA) such as icosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are of considerable pharmaceutical interest due to their biomedical properties (1-3)..Most of n-3 PUFA usually occupies the sn-2 position of phospholipids which are quantitatively the most significant component of membrane lipids (4). Various bioactive actions such as antihypertension, lowering of stored adipose tissue weight, and low lipid levels in plasma and liver were observed when fed EPA-containing phospholipids obtained from an EPA-producing bacterium to spontaneously hypertensive rats (5, 6). Much attention thus is being devoted to the function of n-3 PUFA-containing phospholipids in connection with atherosclerosis. In our previous studies, a potent marine bacterium of genus Vibrio for lipase production was isolated and tentatively named VB-5 (7). The lipase activity of the strain VB-5 was enhanced significantly when cultivated with olive oil, suggesting the lipase was induced by olive oil. The olive oil-induced lipase from the strain VB-5 was capable of liberatirig EPA and DHA from sardine oil triglyceride (TG). In the course of the studies on lipase production from the strain VB-5, we noticed that the fatty acid composition of the lipids from the strain VB-5 cultivated with olive oil was modified greatly. Taking into account that the main lipids in bacteria are phospholipids (4), this observation suggests that n-3 PUFA-containing phospholipids are obtained from the strain VB-5 when cultivated with fish oil TG. The present study was designed to examine whether the strain VB-5 utilized n-3 PUFA-containing sardine oil TG as its own lipid. A cultivation of genus Vibrio, tentatively named VB-5, isolated from the red tides of Tanabe Bay in Wakayama Prefecture, Japan, was grown in a selected medium, CPY, at 15°C with rotary shaking (100rpm) for 2d. The CPY medium comprised 0.1% casitone, 0.05% proteose peptone, 0.1% yeast extract and 300 ml of 75% natural seawater. In order to examine whether the strain VB-5 utilized n-3 PUFA-containing fish oil TG as its own)ipid, 1 or 3% sardine oil TG was also added to the CPY medium. Sardine oil was extracted from the sardine muscle and sardine oil TG was purified from the sardine oil by silica column chromatography. After cultivation of the strain VB-5 at 15°C for 2 d, cells
were separated from the growth medium by centrifugation. Cells harvested were thoroughly washed with cold 75% natural seawater to remove the growth medium containing sardine oil. Lipid was extracted from 1 g of wet cells according to the method of Bligh and Dyer (8). Lipid extraction from the cells was done at 4°C to minimize lipid deterioration. The total lipid was analyzed quantitatively by thin-layer chromatography (TLC). The TLC plates (Kieselgel 60, Merck) were developed using n-hexane-diethyl ether-formic acid 7 5 : 2 5 : 2 (by volume) for non-phospholipids, and chloroform-methanol-water 6 5 : 2 5 : 4 (by volume) for phospholipids, respectively. The TLC plate was sprayed with a 3% copper acetate-8% phosphoric acid, heated at 150°C for 5 min, and quantitated using a Cosmo F-808 densitometer. Identification of each lipid class was accomplished by co-TLC with authentic specimens of oleic acid, phosphatidylethanolamine (PE) and phosphatidylglycerol (PG). A portion of total lipid was applied to preparative TLC. The TLC plate was developed using chloroform-methanolwater 65 : 25 : 4 (by volume). The TLC plate was sprayed with a Rhodamine 6G reagent, and PE and PG were detected under ultraviolet light. Each lipid class was eluted with chloroform and methanol, and their fatty acid compositions were analyzed by gas-liquid chromatography (GLC) after methylation with methanol containing anhydrous hydrogen chloride. GLC analysis was performed with a Hitachi 063 gas chromatograph equipped with a hydrogen flame ionization detector, using a glass column (3 mm i.d. and 4 m length) packed with Unisole 3000 on a Uniport C (80-100 mesh). Identification of individual fatty acids was accomplished by comparing with relative retention times and equivalent chain lengths of authentic standards. The strain VB-5 used in the present study was classified as a typical marine bacterium, which was Gram-negative, rod-shaped, motile by means of a polar flagellum, and essentially required seawater for growth (7). Table 1 shows the total lipid content and lipid composition of the strain VB-5 cultivated at 15°C in the medium with or without sardine oil TG. The total lipid content of the strain VB-5 cultivated with sardine oil TG free medium was 1.46% on a wet weight basis, and tended to increase when cultivated with sardine oil TG. Phospholipid and non-phospholipid classes from the strain VB-5 cultivated with sardine oil TG
* Corresponding author. 169
170
J. FEILM~NT. BIOI/NG.,
ANDO ET AL.
TABLE 1. Total lipid content and lipid composition of VB-5 cultivated at 15°C with or without sardine oil triglyceride Sardine oil added Total lipid (%) (mg/gwet cells) 0 1
14.6 14.7
3
25.4
Lipid composition (%) PE*
PG
FFA
46.9 22.7 30.4 35.0 17.0 31.5 (41.9) b (20.4) (37.7) 21.2 10.1 21.7 (40.0) (19.1) (40.9)
TG
FAE
-16.5
---
37.9
9.0
a Abbreviations: PE, phosphatidylethanolamine; PG, phosphatidylglycerol; FFA, free fatty acid; TG, triglyceride; FAE, fatty acid ester. b Values in parentheses are percentages, with the sum of PE, PG and FFA as 100%. free m e d i u m were P E a n d P G , a n d free f a t t y acid, respectively. It is u n c l e a r w h y t h e s t r a i n VB-5 h a d u n u s u a l l y h i g h levels o f free f a t t y acid; b u t this m i g h t b e a s s o c i a t e d w i t h t h e lipase activity o f t h e s t r a i n VB-5 as d e s c r i b e d a b o v e . P E a n d P G are f o u n d as t h e m a i n p h o s p h o l i p i d s o f terrestrial b a c t e r i a , w h e r e a s significant a m o u n t s o f free f a t t y acid as well as P E a n d P G are d e t e c t e d f r o m m a r i n e b a c t e r i a ( K u n i m o t o , M . et al., Bull. Fac. F i s h . H o k k a i d o U n i v . , 25, 332-341, 3 4 2 - 3 5 0 , 1975). T h e p o s s i b l e significance o f free f a t t y acid is u n c l e a r , b u t free f a t t y acid f o u n d in t h e s t r a i n VB-5 m i g h t b e a t t r i b u t e d t o a c h a r a c t e r i s t i c o f m a r i n e b a c t e r i a . T G a n d f a t t y acid ester, besides P E , P G a n d free f a t t y acid, were f o u n d in t h e s t r a i n VB-5 c u l t i v a t e d w i t h s a r d i n e oil T G . N e w l y d e t e c t e d c o m p o n e n t s o f T G a n d f a t t y acid ester s e e m e d to b e c o n t a m i n a t i o n d e r i v e d TABLE 2. ,
f r o m t h e g r o w t h m e d i u m c o n t a i n i n g s a r d i n e oil T G . A c o m p l e t e s e p a r a t i o n o f the g r o w t h m e d i u m c o n t a i n i n g s a r d i n e oil T G f r o m cells was very difficult, a l t h o u g h t h e cells were t h o r o u g h l y w a s h e d w i t h s e a w a t e r t o r e m o v e t h e g r o w t h m e d i u m . T h e lipid c o m p o s i t i o n s o f P E , P G a n d free f a t t y acid were a l m o s t t h e s a m e , i r r e s p e c t i v e o f cult i v a t i n g t h e s t r a i n VB-5 w i t h o r w i t h o u t s a r d i n e oil T G . T h e m a i n p h o s p h o l i p i d s in t h e s t r a i n VB-5 were P E a n d P G , as well as o t h e r b a c t e r i a (4, 6; K u n i m o t o , M . et al., Bull. F a c . Fish. H o k k a i d o U n i v . , 25, 3 3 2 - 3 4 1 , 3 4 2 - 3 5 0 , 1975; W a t a n a b e , K. et al., P r o c . J a p a n . C o n f . B i o c h e m . L i p i d s , 33, 2 9 - 3 2 , 1991). W e e x a m i n e d w h e t h e r t h e s t r a i n VB-5 utilized n-3 P U F A - c o n t a i n i n g s a r d i n e oil T G a n d if t h e f a t t y acid c o m p o s i t i o n s o f p h o s p h o l i p i d s were m o d i f i e d . T a b l e 2 s h o w s t h e f a t t y acid c o m p o s i t i o n s o f P E a n d P G f r o m t h e s t r a i n VB-5 c u l t i v a t e d at 15°C in t h e m e d i u m w i t h o r w i t h o u t s a r d i n e oil T G . T h e m a i n f a t t y acids o f P E a n d P G f r o m t h e s t r a i n VB-5 c u l t i v a t e d w i t h s a r d i n e oil T G free m e d i u m were p a l m i t o l e i c (16 : 1), oleic (18 : 1) a n d p a l m i t i c (16 : 0) acids, a c c o u n t i n g f o r o v e r 8 0 % o f t h e t o t a l f a t t y acids. P E c o n t a i n e d relatively m o r e m y r i s t i c ( 1 4 : 0 ) , p a l m i t i c a n d oleic acids, a n d less p a l m i t o l e i c acid t h a n P G . N o n-3 P U F A was f o u n d in e i t h e r P E a n d P G f r o m t h e s t r a i n VB5 c u l t i v a t e d w i t h s a r d i n e oil T G free m e d i u m . T h e f a t t y acid c o m p o s i t i o n s o f P E a n d P G were m o d i f i e d b y c u l t i v a t ing t h e s t r a i n VB-5 w i t h s a r d i n e oil T G , w h o s e m a i n f a t t y acids were E P A ( 2 0 : 5 ) , D H A ( 2 2 : 6 ) , p a l m i t i c , oleic, m y r i s t i c a n d p a l m i t o l e i c acids. T h e selective i n c o r p o r a t i o n s o f f a t t y acids i n t o P E a n d P G were f o u n d w h e n t h e s t r a i n VB-5 was c u l t i v a t e d w i t h s a r d i n e oil T G . W i t h
Fatty acid composition (%) of phosphatidylethanolamine and phosphatidylglycerol from VB-5 cultivated at 15°C with or without sardine oil triglyceride
Fatty acids
Sardine oil
1"2: 0 13 : 0 14 : 0 14 : 1 14 : 2 15 : 0 15 : 1 15 : 2 •16 : 0 16 : 1 16 : 2 17 : 0 17 : 1 17 : 2 18 • 0 18 : 1 18 : 2 19 : 1 19 : 2 20 : 1 20 : 3 20 : 4 20 : 5 22 : 1 22 : 5 22 : 6 Others Saturated Monoenoic Polyenoic
0.82 -8.10 -0.19 0.35 m -14.52 7.50 0.48 1.62 1.77 -4.06 12.74 0.90 0.70 1.72 3.07 0.25 4.51 22.54 2.59 2.73 6.09 2.75 29.47 28.37 42.16
Phosphatidylethanolamine Sardine oil added (%) 0 1 3 0.17 0.17 2.44 1.15 0.32 1.51 2.07 0.68 13.75 33.43 1.02 2.64 5.83 0.38 1.72 31.35 0.18 0.87 -0.29 ---. --0.03 22.41 75.00 2.58
0.42 0.14 5.96 0.98 0.30 0.82 0.56 0.47 14.58 31.55 0.97 2.19 3.06 0.32 1.98 31.06 0.16 0.79 0.34 0.27 0.10 0.28 2.15 .
. -0.30 0.25 26.28 68.09 5.62
0.61 0.14 6.33 0.85 0.27 0.86 0.50 0.42 12.90 22.97 0.67 2.37 3.18 0.30 1.69 30.73 1.34 0.87 0.69 0.30 0.43 1.10 7.11 . 0.36 1.91 1.10 25.44 59.42 15.15
0
Phosphatidylglycerol Sardine oil added (%) 1
3
0.14 -0.51 0.24 m 0.78 0.77 0.43 9.16 46.50 1.10 2.16 8.93 0.42 1.61 26.17 0.18 0.69 -0.20 -~ --
0.52 0.I0 1.63 0.39 0.08 0.39 0.29 0.32 12.05 38.63 0.83 2.10 5.17 0.33 2.36 27.10 0.78 0.80 0.48 0.57 0.06 0.50 2.47
0.46 -2.20 0.27 -0.56 0.16 0.14 16.19 29.23 0.65 2.29 4.49 -1.81 27.15 1.74 0.60 0.49 0.62 0.66 2.01 5.40
0.14 0.93 0.98 19.16 72.94 7.90
0.25 1.51 1.01 24.17 62.51 13.32
.
. --0.20 14.42 83.86 1.72
VOL. 73, 1992
NOTES
171
cultivating them with potassium salts of E P A and D H A (Watanabe, K. et al., Proc. Japan. Conf. Biochem. Lipids, 33, 29-32, 1991). These results suggest that the strain VB-5 might produce n-3 P U F A - c o n t a i n i n g phospholipids by hydrolyzing sardine oil T G by means of lipase. Further studies on the optimal conditions for the incorporation of n-3 P U F A - c o n t a i n i n g phospholipids by the strain VB-5 will be required. We are indebted to M. Hosokawa and A. Yoshida for their helpful discussion. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (S.A.). REFERENCES 1. Dyerberg, J. and Jorgensen, K. A.: Marine oils and thrombogene-
sis. Prog. Lipid Res., 21, 255-269 (1982). FIG. I. Appearance of growth medium from the strain VB-5 cultivated at 15°C with (A) or without (B) sardine oil triglyceride. respect to saturated fatty acids of PE and PG, relatively more myristic and less palmitic acids were incorporated into PE than PG. The percentage of palmitoleic acid decreased in both PE and P G from the strain VB-5 when cultivated with sardine oil TG, whereas that of oleic acid was almost unchanged. E P A and D H A from sardine oil TG were clearly detected in both PE and PG. Taking into account the distribution patterns of palmitoleic acid, E P A and DHA, palmitoleic acid seemed to be substituted for E P A and D H A in both PE and PG from the strain VB-5 cultivated with sardine oil TG. It is very interesting how the strain VB-5 utilizes n-3 P U F A - c o n t a i n i n g sardine oil TG. The strain VB-5 produced a lipase by which n-3 P U F A such as E P A and D H A were liberated from sardine oil T G (7). Sardine oil T G added to the growth medium turned oil bowl-like clot, whose main component was free fatty acid, by means of the lipase produced by the strain VB-5 (Fig. 1). The incorporation of exogeneous fatty acids into phospholipids was found in Escherichia coli (9, 10). Furthermore, Yazawa et al. have recently reported that the fatty acid compositions in some bacterial phospholipids were greatly modified by
2. Suzuki, H. and Wada, S.: Metabolism and function of icosapen-
taenoic and docosahexaenoic acids. Yukagaku, 37, 781-787 (1988). 3. Watanabe, S. and Okuyama, H.: Physiological significanceof n3 fatty acid-containing phospholipids. Protein, Nucleic Acid and Enzyme, 36, 584-588 (1991). 4. Cronan, J.E. Jr,: Molecular biology of bacterial membrane lipids. Ann. Rev. Biochem., 47, 163-189 (1978). 5. Yazawa, K., Araki, K., Watanabe, K., lshikawa, C., Inoue, A., Kondo, K., Watabe, S., and Hashimoto, K.: Eicosapentaenoic
acid productivity of bacteria isolated from fish intestines. Nippon Suisan Gakkaishi, 54, 1835-1838 0988). 6. Yazawa, K., Jiang, M.-C., Ishikawa, C., Watanabe, K., Akahoti, Y., Kimura, S., and Konda, K.: Bioactivity of phospho-
lipids from EPA-producing bacteria. Seikagaku, 61, 1090 (1989). 7. Ando, S., Yoshida, A., and Hatano, M.: Occurrence of marine bacterial lipase hydrolyzing fish oil. Agric. Biol. Chem., 55, 2657-2659 (1991). 8. Bligh, E. G. and Dyer, W.J.: A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol., 37, 911917 (1959). 9. Cronan, J.E. Jr.: Evidence that incorporation of exogeneous fatty acids into the phospholipids of Escherichia coli does not require acyl carrier protein. J. Bacteriol., 159, 773-775 0984). 10. Rock, C. O. and Jaekowski, S.: Pathways for the incorporation of exogeneous fatty acids into phosphatidylethanolamine in Escherichia coli. J. Biol. Chem., 260, 12720-12724 (1985).