J. Nutr.
FATTY AND
ACID LIVER
Sci. Vitaminol.,
22, 255-261,
1976
COMPOSITION OF ADIPOSE TISSUE FATS OF THE RATS FED YEAST GROWN ON n-ALKANES1
Makoto TAJIMA,2 Noriyuki OKADA,2Suzue TADOKORO-YASUI,2 Sei-ichi HAYAKAWA,2 and Seiji YOSHIKAWA2 National Food Research Institute, Ministry of Agriculture and Forestry, Koto-ku, Tokyo, Japan (Received December 23, 1975)
Summary n-Alkane assimilating yeast, Candida tropicalis YO-148, was grown on an n-alkane-containing medium. A synthetic diet containing 6.8% of dried yeast was fed to rats. The fatty acid composition of adipose tissue and liver fats was determined after a two week feeding period. The percentage of odd-numbered acids increased in the animals fed the yeast diet. Furthermore, it was shown that heptadecenoic acid, the major odd-numbered acid in yeast, was accumulated in neutral lipid fraction of adipose tissue fat. Fatty acid composition of protein isolate prepared from yeast cells had a profile similar to that of the original cells. The possible use of microbial single-cell proteins as new protein resources for animal feed or food for humans has received much attention in recent years. Among these proteins, yeast protein produced from n-alkanes was the first to be put into practical use in the world. The fatty acid composition of the yeast grown on n-alkanes has been studied by several workers (1-3), and they pointed out existence of large amounts of odd numbered acids. It is a well-known fact that fatty acid composition of animal fats is influenced by dietary fats. It is assumed that odd-numbered acids are accumulated in the fat of animals fed yeast grown on n-alkanes. YOKOYAMA and KANEDA(4) have reported on the amounts of odd-numbered acids in the liver fats of rats fed on diet containing 10% yeast lipids grown on n-alkanes. In the present study, we fed rats a diet containing 6.8% yeast (0.9% lipids) and analyzed the fatty acid composition of the liver and of adipose tissue fats in order to elucidate the accumulation of odd-numbered acids. The diet containing 6.8% yeast may 1 Evaluation
of Single-cell Protein . Part II. Part I, see Ref. 11). A part of this report was presented at the annual meeting of Agricultural Chemical Society of Japan, held at Sapporo July 25, 1975. 2田 島
眞 ,岡 田 憲 幸,安 井 鈴 江,早 川 清 一,吉 川 誠 次 255
256
M. TAJIMA
be an appropriate
simulation
of a model
feed for domestic
MATERIALS Yeast
cells.
taining:
0.1g;
(SO4)2•E6H2O,
0.2mg;
was
added
alkanes3
up
YO-148
KH2PO4,
0.7g;
vested
liter
(6).
Tween
containing
after
from
1
40-47
n-alkane
hr
(5)
1 liter
of
on
the
was
was
medium
from
on
medium
0.67mg;
7.6g
rotary
shaker.
medium
and
0.1g; Fe(NH4)2
distilled
of out
con
NaCl,
4.0g;
carried
a
glucose
a
0.4g;
extract,
source
Cultivation
incubation
grown
MnSO4•EH2O, yeast
carbon
85.
was
MgSO4•E7H2O,
0.04mg;
The
animals.
METHODS
0.71mg;
CaSO4•E5H2O,
0.2ml
conical flasks
water
D-glucose at
or
30•Ž
in
Cells 66-74
were
hr
n
5-liter har
incubation
medium.
Determination with
tropicalis
5.0g;
ZnSO4•E7H2O,
to
with
AND
Candida
NH4H2PO4,
CaC12•E2H2O,
et al.
of
methanol
at
lipid
80•Ž
fractionated
by
FOLCH'S
speed
liquid
chromatography
graphy
is
shown
in
component
according
of
to
method
the
(8). (9).
Fig.
cells.
Cellular
method
of
Lipid
The
lipids
SHIGYO
components
condition
for
were
and were
extracted
TAKEDCHI
(7)
and
analyzed
by
liquid
chromato
high-speed
high
1.
Determination of fatty acid composition. The fatty acid composition of cellular lipids and adipose tissue and liver fats of rats was estimated by gas chro matography. Adipose tissue and liver fats were extracted with chloroform methanol (3:1) and fractionated by Folch's method. These lipids were saponi fi ed by 1N potassium hydroxide in ethanol and methylated with 15% boron trifluoride in methanol. Gas chromatography was performed using two different columns; (a) a polar column, 20% DEGS on Chromosorb W (60-80 mesh) and (b) a nonpolar column, 5% SE-30 on Gaschrom Q(80-100 mesh). Eluted com pounds were identified by plotting the logarithm of the retention time against the carbon number. Double bond(s) of unsaturated acids were detected by reduction of the peak after hydrogenation using platinum oxide as a catalyst. The relative proportions of fatty acids were calculated from the peak areas estimated by multiplying peak height by peak width. Animals each
and
were
powder
(10)
sterilized
the
acid
as to
because end
Preparation composition
of
the
of
of
lipid
period,
Wister
diets
is
protein.
for
1hr.
In
sulfur-containing
feeding
the
the
control
120•Ž
of protein of
rats
composition
used
heating
supplemented
Young
The
was
by
At
diets.
used.
amino the
isolate
from
bound
to
rats
were yeast
protein,
strain
shown
Yeast the
test
cells diet,
acids
are
killed
and
cells. cell
weighing in
Table for
the
0.07% lacking
about 1. test
protein
order was
diet
methionine in
the
freeze-dried In
Whole
80g egg were was
protein.4 immediately.
to
determine
isolated
fatty by
weak
3 C110 .3%, C120.5%, C13 1.4%, C14 4.3%, C1573.8%, C16 16.1%, C17 2.9%, Cabove180.7%; sample provided by Idemitsu Co. 4 Contents of sulfur-containing amino acids in the yeast: Met, 1.01; Cys, 0.89 (g/16g N).
FATTY
ACIDS
OF
FATS
OF
RATS
FED
ON YEAST
257
alkali extraction followed by acid precipitation according to the method described in a previous paper (11). The protein isolate thus obtained was digested by pepsin and fatty acid composition of liberated lipid was determined. Table 1.
Composition of dicta (%).
a Nitrogen levels were adjusted to 1.0%. b C . tropicalis YO-148 grown on n-alkanes. c Johnson salt mixture. d A , D, E, K and water-soluble vitamines.
Fig. 1. High speed liquid chromatogram of lipids extracted from yeasts. C. tropicalis YO-148 grown on glucose (upper) and n-alkanes (lower). Total lipid content: glucose medium, 6.9%; n-alkane medium, 13.2%.
258
M. TAJIMA
et al.
RESULTS
1. Lipid component of yeast cells Lipid component of yeast cells grown on glucose and n-alkanes was analyzed by high-speed liquid chromatography. Chromatograms of the lipids are shown in Fig. 1. Total lipid content of yeast cells grown on n-alkanes was almost double that of yeast cells grown on glucose due to an increase of the triglyceride fraction. The result of this experiment did not agree with the reports of YoKOYAMA et al. (4) or SHIGYOet al. (7), which showed that 74-76% of the lipids in yeast grown on n-alkanes were phospholipids. This disagreement may be due to the difference of strain of yeast. 2. Fatty acid composition of yeast cells The fatty acid composition of the yeast is illustrated in Fig. 2. Large amounts of odd-numbered acids, especially C15=0and C17=1,were found in yeast grown on n-alkanes. 3.
Growth of rats The results of the animal feeding studies are shown in Table 2. No significant difference was observed between the animals in PER values and amounts of fat in livers. From this fact it can be concluded that the yeast diet has no significant deficiency as nutrient for rats.
Fig. 2. Fatty acids pattern of yeasts. n-alkanes (lower).
C. tropicalis YO-148 grown on glucose (upper) and
FATTY Table
2.
ACIDS
OF
FATS
Body weight gain, protein
OF
quality
RATS
FED
ON YEAST
259
and liver fat of the rats fed for 2 weeks.
4.
Fatty acid composition of adipose tissue fats Subcutaneous adipose tissue was removed from freeze-dried carcass with a spatura and the fatty acid composition was determined. Table 3 shows fatty acid composition of tissue fats of rats fed on yeast and control diet. The fatty acids were identified by the method described in MATERIALS and METHOD. Small amounts of odd-numbered acids found in the fats of rats fed on the control diet will be transported from whole egg powder used as a protein source. Higher animals contain a small amount of odd-numbered acids among their fatty acids as a result of direct or indirect microbial diets (12). However, the figures in the table show remarkable increases in the percentage of odd-numbered fatty acids in animals fed a yeast diet. The total amount of odd-numbered acids was six times as much as that in aminals fed the control diet. In order to know the fraction of lipid in which odd-numbered acids in yeast ac cumulated, the lipid extracted from adipose tissue was fractionated by silicic acid column chromatography. Table 4 shows distributions of odd-numbered acid between the chloroform fraction, that is the neutral lipid, and the methanol frac tion, that is the conjugated lipid. It was shown that C17=1,the major odd-num Table
3.
Fatty
acids compositor
of adipose
tissue fats.
260
M. Table
4.
Contents
of odd-numbered
The lipid extracted
Table
bered
acids
in yeast,
TAJIMA
acids in adipose
was fractionated
5.
Fatty
accumulated
et al.
by column
acids composition
in the neutral
C15=0 was increased after fractionation The reason for this was not apparent.
by silicic
tissue fats of rats fed yeast diet . on silicic acid .
chromatography
of liver fats.
lipid fraction. acid
column
The contents
of
chromatography.
5.
Fatty acid composition of liver fats The fatty acid composition of liver fats of rats fed on yeast and the control diet is shown in Table 5. The contents of the odd-numbered acids also increased in liver fats as a result of the yeast diet. But, the increase was not so high and total amount of odd-numbered acids in the liver fat of rats fed the yeast diet was about three times as much as that in rats fed the control diet. 6. Fatty acid composition of protein isolate The fatty acid composition of lipids bound to the protein isolate, which was prepared from yeast grown on n-alkanes, was determined. As shown in Figs. 2 and 3, a close similarity was found between the profile of the fatty acid composi tion of lipid bound to protein isolate and that of whole cells. From this fact, it is expected that odd-numbered fatty acids may accumulate
FATTY
Fig.
3.
Fatty
acid pattern
in the fat of an animal grown
ACIDS
OF FATS
of protein
fed large
OF RATS
isolate prepared
amounts
FED
ON YEAST
from yeast
of protein
isolate
grown
261
on n-alkanes.
prepared
from
yeast
on n-alkanes. DISCUSSION
The fatty acid composition of adipose tissue and liver fat of rats fed on yeast grown on n-alkanes was determined. As shown in Tables 3 and 5, the yeast diet caused an increase in the contents of odd-numbered fatty acids, especially C17=1. The total content of odd-numbered acids in adipose tissue fat of rat fed on diet containing 6.8% yeast, which may be an appropriate simulation of a model of feed for domestic animals, was six times as much as that of the control diet. Therefore,
it is necessary
acids derived from food resource. The authors Research
Institute,
a yeast
wish to express
to elucidate
the metabolism
diet in animals
their
for his encouragement
thanks
before
to Dr. T. Watanabe,
throughout
of odd-numbered
yeast protein
Director
fatty
can be utilized
of National
as
Food
the work.
REFERENCES
1) 2) 3) 4) S) 6) 7) 8) 9) 10) 11) 12)
THORPE,R. F. and RATLEDGE,C., J. Gen. Microbiol., 72,151 (1972). MISHINA,H., YANAGAWA,S., TANAKA,A., and FUKUI, S., Agr. Biol. Chem., 37,863 (1973). IWAMOTO,H. and OZAWA,M., Hakko Kyokaishi, 31,199 (1974). YOKOYAMA, M. and KANEDA,T., Yukagaku, 21,900 (1972). KOMAGATA,K., NAKASE,T., and KATSUYA,N., J. Gen. Appl. Microbiol., 10,323 (1964). MILLER,T. L. and JOHNSON,M. J., Biotechnol. Bioeng., 8,549 (1966). SHIGYO,F. and TAKEUCHI,M., Nippon Nogeikagaku Kaishi, 46,27 (1972). FOLCH,J., LEES, M., and SLOANE-STANLEG, G. H., J. Biol. Chem., 226,497 (1957). KIUCHI, K., OHTA, T., and EBINE, H., J. Chromatog. Sci., 13,461 (1975). YAMAGUCHI,M. and KANDATSU,M., Agr. Biol. Chem., 37,809 (1973). TAJIMA,M. and YOSHIKAWA,S., Agr. Biol. Chem., 39,611 (1975). SCHLENK,H., Fed. Proc., Fed. Am. Soc. Exp. Biol., 31,1430 (1972).
FATTY AND
ACID LIVER
Sci. Vitaminol.,
22, 255-261,
1976
COMPOSITION OF ADIPOSE TISSUE FATS OF THE RATS FED YEAST GROWN ON n-ALKANES1
Makoto TAJIMA,2 Noriyuki OKADA,2Suzue TADOKORO-YASUI,2 Sei-ichi HAYAKAWA,2 and Seiji YOSHIKAWA2 National Food Research Institute, Ministry of Agriculture and Forestry, Koto-ku, Tokyo, Japan (Received December 23, 1975)
Summary n-Alkane assimilating yeast, Candida tropicalis YO-148, was grown on an n-alkane-containing medium. A synthetic diet containing 6.8% of dried yeast was fed to rats. The fatty acid composition of adipose tissue and liver fats was determined after a two week feeding period. The percentage of odd-numbered acids increased in the animals fed the yeast diet. Furthermore, it was shown that heptadecenoic acid, the major odd-numbered acid in yeast, was accumulated in neutral lipid fraction of adipose tissue fat. Fatty acid composition of protein isolate prepared from yeast cells had a profile similar to that of the original cells. The possible use of microbial single-cell proteins as new protein resources for animal feed or food for humans has received much attention in recent years. Among these proteins, yeast protein produced from n-alkanes was the first to be put into practical use in the world. The fatty acid composition of the yeast grown on n-alkanes has been studied by several workers (1-3), and they pointed out existence of large amounts of odd numbered acids. It is a well-known fact that fatty acid composition of animal fats is influenced by dietary fats. It is assumed that odd-numbered acids are accumulated in the fat of animals fed yeast grown on n-alkanes. YOKOYAMA and KANEDA(4) have reported on the amounts of odd-numbered acids in the liver fats of rats fed on diet containing 10% yeast lipids grown on n-alkanes. In the present study, we fed rats a diet containing 6.8% yeast (0.9% lipids) and analyzed the fatty acid composition of the liver and of adipose tissue fats in order to elucidate the accumulation of odd-numbered acids. The diet containing 6.8% yeast may 1 Evaluation
of Single-cell Protein . Part II. Part I, see Ref. 11). A part of this report was presented at the annual meeting of Agricultural Chemical Society of Japan, held at Sapporo July 25, 1975. 2田 島
眞 ,岡 田 憲 幸,安 井 鈴 江,早 川 清 一,吉 川 誠 次 255
256
M. TAJIMA
be an appropriate
simulation
of a model
feed for domestic
MATERIALS Yeast
cells.
taining:
0.1g;
(SO4)2•E6H2O,
0.2mg;
was
added
alkanes3
up
YO-148
KH2PO4,
0.7g;
vested
liter
(6).
Tween
containing
after
from
1
40-47
n-alkane
hr
(5)
1 liter
of
on
the
was
was
medium
from
on
medium
0.67mg;
7.6g
rotary
shaker.
medium
and
0.1g; Fe(NH4)2
distilled
of out
con
NaCl,
4.0g;
carried
a
glucose
a
0.4g;
extract,
source
Cultivation
incubation
grown
MnSO4•EH2O, yeast
carbon
85.
was
MgSO4•E7H2O,
0.04mg;
The
animals.
METHODS
0.71mg;
CaSO4•E5H2O,
0.2ml
conical flasks
water
D-glucose at
or
30•Ž
in
Cells 66-74
were
hr
n
5-liter har
incubation
medium.
Determination with
tropicalis
5.0g;
ZnSO4•E7H2O,
to
with
AND
Candida
NH4H2PO4,
CaC12•E2H2O,
et al.
of
methanol
at
lipid
80•Ž
fractionated
by
FOLCH'S
speed
liquid
chromatography
graphy
is
shown
in
component
according
of
to
method
the
(8). (9).
Fig.
cells.
Cellular
method
of
Lipid
The
lipids
SHIGYO
components
condition
for
were
and were
extracted
TAKEDCHI
(7)
and
analyzed
by
liquid
chromato
high-speed
high
1.
Determination of fatty acid composition. The fatty acid composition of cellular lipids and adipose tissue and liver fats of rats was estimated by gas chro matography. Adipose tissue and liver fats were extracted with chloroform methanol (3:1) and fractionated by Folch's method. These lipids were saponi fi ed by 1N potassium hydroxide in ethanol and methylated with 15% boron trifluoride in methanol. Gas chromatography was performed using two different columns; (a) a polar column, 20% DEGS on Chromosorb W (60-80 mesh) and (b) a nonpolar column, 5% SE-30 on Gaschrom Q(80-100 mesh). Eluted com pounds were identified by plotting the logarithm of the retention time against the carbon number. Double bond(s) of unsaturated acids were detected by reduction of the peak after hydrogenation using platinum oxide as a catalyst. The relative proportions of fatty acids were calculated from the peak areas estimated by multiplying peak height by peak width. Animals each
and
were
powder
(10)
sterilized
the
acid
as to
because end
Preparation composition
of
the
of
of
lipid
period,
Wister
diets
is
protein.
for
1hr.
In
sulfur-containing
feeding
the
the
control
120•Ž
of protein of
rats
composition
used
heating
supplemented
Young
The
was
by
At
diets.
used.
amino the
isolate
from
bound
to
rats
were yeast
protein,
strain
shown
Yeast the
test
cells diet,
acids
are
killed
and
cells. cell
weighing in
Table for
the
0.07% lacking
about 1. test
protein
order was
diet
methionine in
the
freeze-dried In
Whole
80g egg were was
protein.4 immediately.
to
determine
isolated
fatty by
weak
3 C110 .3%, C120.5%, C13 1.4%, C14 4.3%, C1573.8%, C16 16.1%, C17 2.9%, Cabove180.7%; sample provided by Idemitsu Co. 4 Contents of sulfur-containing amino acids in the yeast: Met, 1.01; Cys, 0.89 (g/16g N).
FATTY
ACIDS
OF
FATS
OF
RATS
FED
ON YEAST
257
alkali extraction followed by acid precipitation according to the method described in a previous paper (11). The protein isolate thus obtained was digested by pepsin and fatty acid composition of liberated lipid was determined. Table 1.
Composition of dicta (%).
a Nitrogen levels were adjusted to 1.0%. b C . tropicalis YO-148 grown on n-alkanes. c Johnson salt mixture. d A , D, E, K and water-soluble vitamines.
Fig. 1. High speed liquid chromatogram of lipids extracted from yeasts. C. tropicalis YO-148 grown on glucose (upper) and n-alkanes (lower). Total lipid content: glucose medium, 6.9%; n-alkane medium, 13.2%.
258
M. TAJIMA
et al.
RESULTS
1. Lipid component of yeast cells Lipid component of yeast cells grown on glucose and n-alkanes was analyzed by high-speed liquid chromatography. Chromatograms of the lipids are shown in Fig. 1. Total lipid content of yeast cells grown on n-alkanes was almost double that of yeast cells grown on glucose due to an increase of the triglyceride fraction. The result of this experiment did not agree with the reports of YoKOYAMA et al. (4) or SHIGYOet al. (7), which showed that 74-76% of the lipids in yeast grown on n-alkanes were phospholipids. This disagreement may be due to the difference of strain of yeast. 2. Fatty acid composition of yeast cells The fatty acid composition of the yeast is illustrated in Fig. 2. Large amounts of odd-numbered acids, especially C15=0and C17=1,were found in yeast grown on n-alkanes. 3.
Growth of rats The results of the animal feeding studies are shown in Table 2. No significant difference was observed between the animals in PER values and amounts of fat in livers. From this fact it can be concluded that the yeast diet has no significant deficiency as nutrient for rats.
Fig. 2. Fatty acids pattern of yeasts. n-alkanes (lower).
C. tropicalis YO-148 grown on glucose (upper) and
FATTY Table
2.
ACIDS
OF
FATS
Body weight gain, protein
OF
quality
RATS
FED
ON YEAST
259
and liver fat of the rats fed for 2 weeks.
4.
Fatty acid composition of adipose tissue fats Subcutaneous adipose tissue was removed from freeze-dried carcass with a spatura and the fatty acid composition was determined. Table 3 shows fatty acid composition of tissue fats of rats fed on yeast and control diet. The fatty acids were identified by the method described in MATERIALS and METHOD. Small amounts of odd-numbered acids found in the fats of rats fed on the control diet will be transported from whole egg powder used as a protein source. Higher animals contain a small amount of odd-numbered acids among their fatty acids as a result of direct or indirect microbial diets (12). However, the figures in the table show remarkable increases in the percentage of odd-numbered fatty acids in animals fed a yeast diet. The total amount of odd-numbered acids was six times as much as that in aminals fed the control diet. In order to know the fraction of lipid in which odd-numbered acids in yeast ac cumulated, the lipid extracted from adipose tissue was fractionated by silicic acid column chromatography. Table 4 shows distributions of odd-numbered acid between the chloroform fraction, that is the neutral lipid, and the methanol frac tion, that is the conjugated lipid. It was shown that C17=1,the major odd-num Table
3.
Fatty
acids compositor
of adipose
tissue fats.
260
M. Table
4.
Contents
of odd-numbered
The lipid extracted
Table
bered
acids
in yeast,
TAJIMA
acids in adipose
was fractionated
5.
Fatty
accumulated
et al.
by column
acids composition
in the neutral
C15=0 was increased after fractionation The reason for this was not apparent.
by silicic
tissue fats of rats fed yeast diet . on silicic acid .
chromatography
of liver fats.
lipid fraction. acid
column
The contents
of
chromatography.
5.
Fatty acid composition of liver fats The fatty acid composition of liver fats of rats fed on yeast and the control diet is shown in Table 5. The contents of the odd-numbered acids also increased in liver fats as a result of the yeast diet. But, the increase was not so high and total amount of odd-numbered acids in the liver fat of rats fed the yeast diet was about three times as much as that in rats fed the control diet. 6. Fatty acid composition of protein isolate The fatty acid composition of lipids bound to the protein isolate, which was prepared from yeast grown on n-alkanes, was determined. As shown in Figs. 2 and 3, a close similarity was found between the profile of the fatty acid composi tion of lipid bound to protein isolate and that of whole cells. From this fact, it is expected that odd-numbered fatty acids may accumulate
FATTY
Fig.
3.
Fatty
acid pattern
in the fat of an animal grown
ACIDS
OF FATS
of protein
fed large
OF RATS
isolate prepared
amounts
FED
ON YEAST
from yeast
of protein
isolate
grown
261
on n-alkanes.
prepared
from
yeast
on n-alkanes. DISCUSSION
The fatty acid composition of adipose tissue and liver fat of rats fed on yeast grown on n-alkanes was determined. As shown in Tables 3 and 5, the yeast diet caused an increase in the contents of odd-numbered fatty acids, especially C17=1. The total content of odd-numbered acids in adipose tissue fat of rat fed on diet containing 6.8% yeast, which may be an appropriate simulation of a model of feed for domestic animals, was six times as much as that of the control diet. Therefore,
it is necessary
acids derived from food resource. The authors Research
Institute,
a yeast
wish to express
to elucidate
the metabolism
diet in animals
their
for his encouragement
thanks
before
to Dr. T. Watanabe,
throughout
of odd-numbered
yeast protein
Director
fatty
can be utilized
of National
as
Food
the work.
REFERENCES
1) 2) 3) 4) S) 6) 7) 8) 9) 10) 11) 12)
THORPE,R. F. and RATLEDGE,C., J. Gen. Microbiol., 72,151 (1972). MISHINA,H., YANAGAWA,S., TANAKA,A., and FUKUI, S., Agr. Biol. Chem., 37,863 (1973). IWAMOTO,H. and OZAWA,M., Hakko Kyokaishi, 31,199 (1974). YOKOYAMA, M. and KANEDA,T., Yukagaku, 21,900 (1972). KOMAGATA,K., NAKASE,T., and KATSUYA,N., J. Gen. Appl. Microbiol., 10,323 (1964). MILLER,T. L. and JOHNSON,M. J., Biotechnol. Bioeng., 8,549 (1966). SHIGYO,F. and TAKEUCHI,M., Nippon Nogeikagaku Kaishi, 46,27 (1972). FOLCH,J., LEES, M., and SLOANE-STANLEG, G. H., J. Biol. Chem., 226,497 (1957). KIUCHI, K., OHTA, T., and EBINE, H., J. Chromatog. Sci., 13,461 (1975). YAMAGUCHI,M. and KANDATSU,M., Agr. Biol. Chem., 37,809 (1973). TAJIMA,M. and YOSHIKAWA,S., Agr. Biol. Chem., 39,611 (1975). SCHLENK,H., Fed. Proc., Fed. Am. Soc. Exp. Biol., 31,1430 (1972).
