Conjugated linoleic acid production from linoleic acid by lactic acid bacteria

Article abstract of DOI:10.1007/s11746-002-0451-4

After screening 14 genera of lactic acid bacteria, Lactobacillus plantarum AKU 1009a was selected as a potential strain for CLA production from linoleic acid. Washed cells of L. plantarum with high levels of CLA production were obtained by cultivation in

Full text of DOI:10.1007/s11746-002-0451-4

Conjugated Linoleic Acid Production from Linoleic Acid
by Lactic Acid Bacteria
Shigenobu Kishino, Jun Ogawa, Yoriko Omura, Kenji Matsumura, and Sakayu Shimizu*
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
ABSTRACT: After screening 14 genera of lactic acid bacteria, isomers (11,12); (ii) CLA is accumulated in washed cells as the
Lactobacillus plantarum AKU 1009a was selected as a potential
strain for CLA production from linoleic acid. Washed cells of L.
plantarum with high levels of CLA production were obtained by
cultivation in a nutrient medium with 0.06% (wt/vol) linoleic acid
(cis-9,cis-12-octadecadienoic acid). Under the optimal reaction
conditions with the free form of linoleic acid as the substrate,
washed cells of L. plantarum produced 40 mg CLA/mL reaction
mixture (33% molar yield) from 12% (wt/vol) linoleic acid in 108
h. The resulting CLA was a mixture of two CLA isomers, cis-
9,trans-11 (or trans-9,cis-11)-octadecadienoic acid (CLA1, 38%
of total CLA) and trans-9,trans-11-octadecadienoic acid (CLA2,
62% of total CLA), and accounted for 50% of the total FA ob-
tained. A higher yield (80% molar yield to linoleic acid) was at-
tained with 2.6% (wt/vol) linoleic acid as the substrate in 96 h,
resulting in CLA production of 20 mg/mL reaction mixture [con-
sisting of CLA1 (2%) and CLA2 (98%)] and accounting for 80%
of total FA obtained. Most of the CLA produced was associated
with the cells (ca. 380 mg CLA/g dry cells), mainly as FFA.
FFA, making it easy to recover, and the cells themselves can be
used as the CLA source. These merits prompted us to search
additional strains for practical production of CLA. We report
here that L. plantarum AKU 1009a, which was selected
through screening a wide range of lactic acid bacteria, produces
large amounts of CLA even under aerobic conditions. Investi-
gation of culture conditions to obtain active catalysts and opti-
mization of reaction conditions for practical CLA production
using L. plantarum AKU 1009a are also described.
EXPERIMENTAL PROCEDURES
Chemicals. Standard samples of cis-9,trans-11(or trans-9,cis-
11)-18:2 (CLA1), trans-9,trans-11-18:2 (CLA2), 10-hydroxy-
trans-12-18:1 (HY1), and 10-hydroxy-cis-12-18:1 (HY2) were
prepared as described previously (10). Linoleic acid (cis-9,cis-
12-octadecadienoic acid) and FA-free (<0.02%) BSA were pur-
chased from Wako Pure Chemicals (Osaka, Japan) and Sigma
Chemicals (St. Louis, MO), respectively. All other chemicals
used were of analytical grade and were commercially available.
Microorganisms, cultivation, and preparation of washed
cells. Lactic acid bacteria preserved in our laboratory (AKU
Culture Collection, Faculty of Agriculture, Kyoto University,
Kyoto, Japan) and those obtained from other culture collec-
tions (IAM, Institute of Molecular and Cellular Bioscience,
The University of Tokyo, Tokyo, Japan; IFO, Institute for
Fermentation, Osaka, Japan; and JCM, Japan Collection of
Microorganisms, Wako, Japan) were subjected to screening.
For screening, strains were cultivated in MRS medium (10)
supplemented with 0.06% linoleic acid. Each strain was inoc-
ulated into 15 mL of medium in screw-capped tubes (16.5 ×
125 mm) and then incubated under O2-limited conditions in
sealed tubes for 24–72 h at 28°C with shaking (120
strokes/min). For optimization of culture conditions for L.
plantarum AKU 1009a, cultivation was carried out essen-
tially under the same conditions as described above. For opti-
mization of reaction conditions and preparative CLA produc-
tion, cultivation was carried out aerobically with 550 mL
MRS medium containing 0.06% linoleic acid in 600-mL
flasks for 24 h at 28°C with shaking (120 strokes/min). Cells
were harvested by centrifugation (8,000 × g, 10 min), washed
twice with 0.85% NaCl, and centrifuged again, then used as
the washed cells for the reactions.
Paper no. J10037 in JAOCS 79, 159–163 (February 2002).
KEY WORDS: Conjugated linoleic acid, CLA, lactic acid bac-
teria, Lactobacillus, Lactobacillus plantarum, linoleic acid.
Interest in conjugated linoleic acid (CLA), an octadeca-
dienoic acid (18:2) with conjugated double bonds, has in-
creased in the last two decades because of its unique physio-
logical effects. It was reported that dietary CLA reduced car-
cinogenesis (1–4), atherosclerosis (5), and body fat (6), and
had several other beneficial effects (7–9).
We investigated biological systems for CLA production and
found that the washed cells of Lactobacillus acidophilus AKU
1137 produced CLA isomers from linoleic acid (10). They ef-
ficiently produced CLA from linoleic acid with 10-hydroxy-
12-octadecenoic acid (HY), a hydroxylated octadecenoic acid
(18:1) as a possible intermediate under microaerobic reaction
conditions. Systems using lactic acid bacteria for CLA produc-
tion were found to be advantageous for the following reasons:
(i) specific isomers of CLA, i.e., cis-9,trans-11(or trans-9,cis-
11)-18:2 (CLA1) and trans-9,trans-11-18:2 (CLA2), are ob-
tained, whereas chemical synthesis produces a mixture of CLA
*To whom correspondence should be addressed at Division of Applied Life
Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-
oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan.
E-mail: sim@kais.kyoto-u.ac.jp
Copyright © 2002 by AOCS Press
159
JAOCS, Vol. 79, no. 2 (2002)

160
S. KISHINO ET AL.
Reaction conditions. For screening and optimization of investigated using washed cells of lactic acid bacteria. The
culture and reaction conditions, the reaction mixture, 1 mL, following observations obtained in our previous study using
in test tubes (16.5 × 125 mm) was composed of 0.4% (wt/vol) L. acidophilus AKU 1137 (10) were taken into consideration:
linoleic acid complexed with BSA (0.08%, wt/vol), 0.1 M (i) washed cells of L. acidophilus with high levels of CLA
potassium phosphate buffer (KPB, pH 6.5), and 22.5% (wet production were obtained by cultivation in a medium with
cell, wt/vol) washed cells (corresponding to 3.2%; dry cell, linoleic acid, and (ii) production of CLA was only observed
wt/vol). The reactions were carried out microaerobically in under microaerobic conditions (O2 concentration was less
an O2-adsorbed atmosphere in a sealed chamber with O2- than 1%). More than 250 strains were tested from the genera
absorbent (Aneropack “Kenki”; Mitsubishi Gas Chemical Co, of Lactobacillus, Streptococcus, Pediococcus, Leuconostoc,
Ltd., Tokyo, Japan), and gently shaken (120 strokes/min) at Propionibacterium, Bifidobacterium, Weissella, Aquaspiril-
37°C for 24 to 72 h. For investigation of the effects of linoleic lum, Enterococcus, Tetragenococcus, Aerococcus, Butyrivibrio,
acid concentration and cell concentration on the reaction, and and Lactococcus. Of these, strains belonging to the genera
for preparative CLA production, the reactions were carried Enterococcus, Pediococcus, Propionibacterium, and Lacto-
out essentially under the same conditions as described above bacillus produced considerable amounts of two CLA isomers,
except that the volume of the reaction mixtures was 5 mL. All i.e., cis-9,trans-11(or trans-9,cis-11)-18:2 (CLA1) and
experiments were carried out in triplicate, and the averages of trans-9,trans-11-18:2 (CLA2). Table 1 summarizes the results
three separate experiments, which were reproducible within with strains that produced more than 0.07 mg CLA/mL reac-
10%, are presented in figures and tables, except for Figure tion mixture, most of which were lactobacilli. Either 10-
3, where exact error limits are provided.
hydroxy-trans-12-18:1 (HY1) or 10-hydroxy-cis-12-18:1
Lipid analyses. Lipids were extracted from the reaction (HY2), possible intermediates of CLA biosynthesis from
mixture with chloroform/methanol (1:2, vol/vol) according to linoleic acid (10), were also found in all of these reaction mix-
the procedure of Bligh and Dyer (13), and transmethylated tures. Pediococcus acidilactici AKU 1059 and L. rhamnosus
with 10% methanolic HCl at 50°C for 20 min. The resultant AKU 1124 showed almost the same level of CLA production
FAME were extracted with n-hexane and analyzed by GLC as L. acidophilus AKU 1137 (about 1.5 mg/mL reaction mix-
as described previously (10). Extraction and fractionation into ture). Lactobacillus plantarum AKU 1009a and L. plantarum
lipid classes were carried out essentially as described previ- JCM 1551 were found to produce CLA (sum of CLA1 and
ously (14,15).
CLA2) at more than 1.5 mg/mL reaction mixture. Notably,
L. plantarum AKU 1009a produced the highest amounts of
CLA (3.41 mg/mL), and was used for further optimization of
culture and reaction conditions.
RESULTS AND DISCUSSION
Screening of lactic acid bacteria producing CLA from linoleic
Optimization of culture conditions for the preparation of
acid. The ability to produce CLA from linoleic acid was washed cells of L. plantarum with high CLA productivity.
TABLE 1
Potential Strains for CLA Production from Linoleic Acida
FA (mg/mL reaction mixture)
Other
Strain
Origin
FA
LA
Total CLA
(CLA1:CLA2)
HY1
HY2
Enterococcus faecium
Pediococcus acidilactici
Propionibacterium shermanii
Lactobacillus acidophilus
L. acidophilus
AKU 1021
AKU 1059
AKU 1254
AKU 1137
IAM 10074
AKU 1122
IAM 1082
IFO 12004
JCM 1109
AKU 1142
IFO 3533
AKU 1148
IFO 12011
AKU 1138
AKU 1009a
JCM 8341
JCM 1551
AKU 1124
0.09
0.14
0.11
0.14
0.25
0.09
0.10
0.18
0.17
1.08
0.32
0.10
0.09
0.11
0.07
0.18
0.36
0.10
0.72
1.29
1.42
0.24
0.22
0.91
0.16
0.83
0.76
0.90
0.93
1.24
0.89
0.10
0.06
0.43
0.02
0.22
0.10
1.40
0.11
1.50
0.60
0.12
0.55
0.20
0.07
0.07
0.09
0.08
0.13
0.45
3.41
0.19
2.02
1.41
(0.04:0.06)
(1.00:0.40)
(0.09:0.02)
(0.85:0.65)
(0.18:0.42)
(0.02:0.10)
(0.23:0.32)
(0.05:0.15)
(0.02:0.05)
(0.04:0.03)
(0.05:0.04)
(0.05:0.03)
(0.10:0.03)
(0.10:0.35)
(0.25:3.16)
(0.04:0.15)
(0.10:1.92)
(0.69:0.72)
0.02
0.30
—
0.11
0.60
—
0.79
0.22
—
0.05
0.06
0.08
0.13
1.21
0.11
0.27
0.02
0.13
0.06
0.43
0.07
0.07
0.18
0.02
—
0.45
0.57
1.00
0.68
0.05
L. acidophilus
L. brevis
L. paracasei subsp. paracasei
L. paracasei subsp. paracasei
L. paracasei subsp. paracasei
L. paracasei subsp. paracasei
L. pentosus
L. pentosus
L. plantarum
L. plantarum
L. plantarum
0.74
—
0.16
0.40
0.46
0.15
L. plantarum
L. rhamnosus
aReactions were carried out in 72 h as described in the Materials and Methods section. Other FA included myristic acid, palmitic acid, palmitoleic acid,
oleic acid, vaccenic acid, and 2-hexy-1-cyclopropane-octanoic acid. LA, linoleic acid; CLA1, cis-9,trans-11- or trans-9,cis-11-18:2; CLA2, trans- 9,trans-11-
18:2; HY1, 10-hydroxy-trans-12-18:1; HY2,10-hydroxy-cis-12 18:1; —, not detected.
JAOCS, Vol. 79, no. 2 (2002)

CLA PRODUCTION BY LACTIC ACID BACTERIA
161
FIG. 1. Effects of linoleic acid on CLA production by Lactobacillus plan-
tarum AKU 1009a. Cultivations were carried out in MRS medium with
or without linoleic acid at the indicated concentrations. Reactions were
carried out with linoleic acid as the substrate for 72 h as described in
the Materials and Methods section. ME, linoleic acid methyl ester; Free,
free linoleic acid; LA, linoleic acid; HY, hydroxylated octadecenoic
acid, HY1 + HY2.
FIG. 2. Effects of oxygen on CLA production. Reactions were carried
out in 24 h as described in the Materials and Methods section in an O2-
adsorbed atmosphere or under air. LA, linoleic acid; CLA1, cis-9,trans-
11- or trans-9,cis-11-18:2; CLA2, trans-9,trans-11-18:2: HY1, 10-
hydroxy-cis-12-18:1; HY2, 10-hydroxy-trans-12-18:1.
Lactobacillus plantarum AKU 1009a was easy to cultivate
and showed a high growth rate even under aerobic conditions.
To obtain washed cells with high CLA productivity, culture
conditions were examined using MRS medium under aerobic
conditions. When free linoleic acid was added to the MRS
medium, CLA production increased markedly (Fig. 1). On the
other hand, addition of linoleic acid methyl ester resulted in
accumulation of the intermediate, HY. CLA production was
the highest with addition of 0.06% linoleic acid to the
medium (Fig. 1). Below 0.06%, linoleic acid did not affect
cell growth, but higher concentrations of linoleic acid (0.2%)
inhibited growth and decreased CLA productivity. The
changes in CLA productivity during cultivation in MRS
medium supplemented with 0.06% linoleic acid were moni-
tored. The cells at late log phase showed significant produc-
tivity, but further cultivation resulted in a decrease in produc-
tivity. Washed cells obtained from late log-phase culture (24-
h cultivation) were used for further optimization of reaction
conditions.
Optimization of reaction conditions. (i) Effects of reaction
pH: Reactions were carried out for 72 h in buffer systems of
0.1, 0.5, or 1.0 M acetate/sodium acetate buffer (pH 5.0, 6.0)
or KPB (pH 6.5, 7.0, 7.5). CLA was most efficiently pro-
duced with 0.1 M KPB, pH 6.5. (ii) Effect of reaction temper-
ature: Reactions were carried out for 72 h at different temper-
atures in the range of 20 to 52°C. CLA production increased
with increasing temperature from 20 to 37°C, but decreased
with higher temperature. At 52°C, neither CLA nor HY were
produced. At 20°C, HY was produced in good yield, but CLA
was not. (iii) Effects of substrate form: Free or methyl ester
forms of linoleic acid were tested as substrates (0.5%, wt/vol)
after treatment with BSA (0.5% wt/vol). BSA is a FFA car-
rier, increasing the solubility of FA in the reaction mixture.
After 72-h reaction, the free form of linoleic acid was well
converted to CLA (2.59 mg/ml), while the methyl ester was
not (<0.05 mg/mL). The effect of the ratio of BSA to linoleic
acid was also examined. The amount of CLA produced in 72-
h reactions was not markedly changed with ratios of linoleic
acid/BSA (weight ratio) between 5:2.5 and 5:1, but decreased
with higher and lower ratios. (iv) Effects of oxygen: Reac-
tions were carried out in an O2-adsorbed atmosphere in test
tubes in a sealed chamber with O2-absorbent, or under air in
open test tubes. The amounts of CLA produced and the FA
compositions of the lipids produced were almost the same
under both conditions. The results of 24-h reactions are pre-
sented in Figure 2. In our previous study using L. acidophilus
AKU 1137, the presence of oxygen promoted oxidative me-
tabolism, e.g., β-oxidation, and resulted in lower CLA pro-
duction (10). Based on these findings, screening under mi-
croaerobic conditions was conducted in this study. However,
the presence of oxygen did not affect CLA production from
linoleic acid by L. plantarum AKU 1009a, resulting in easy
control of the reaction conditions. Lactobacillus plantarum
AKU 1009a may lack oxidative linoleic acid degradation ac-
tivity. Strains with such high CLA-producing activity are
promising for efficient production of CLA.
Effects of concentrations of linoleic acid and washed cells.
Reactions were carried out for 48 h with 20% (wet cells,
wt/vol) washed cells and different concentrations of linoleic
acid in 5-mL reaction mixtures with a fixed ratio of linoleic
acid/ BSA (weight ratio), 5:1. CLA production increased with
increasing concentration of linoleic acid up to 2% (wt/vol)
and reached a plateau (8.9 mg/mL) with higher concentra-
tions, while HY production increased up to 5% (wt/vol)
linoleic acid and reached a plateau (18.5 mg/mL) at higher
concentrations.
JAOCS, Vol. 79, no. 2 (2002)

162
S. KISHINO ET AL.
FIG. 3. Time course of preparative CLA production with 12 (A and B) or 2.6% (wt/vol) (C and D) linoleic acid as the
substrate with 33 or 23% (wet wt/vol) washed cells, respectively, under the conditions described in the Materials
and Methods section. (A) and (C): Time course of the reaction. (■), linoleic acid; (▲), CLA (cis-9,trans-11- or trans-
9,cis-11- and trans-9,trans-11-18:2). (B) and (D): FA composition (wt%) of the lipid produced in 108- or 96-h reac-
tion, respectively. For abbreviations, see Figure 2.
Reactions were carried out for 48 h with 6.9% (wt/vol) linoleic acid are currently underway to produce one of the
linoleic acid and different amounts of washed cells in 5-mL CLA isomers specifically.
reaction mixtures. CLA production increased to 23.9 mg/mL
Distribution and lipid classes of the FA produced by L.
with increasing amounts of washed cells up to 33% (wet plantarum. The reaction mixture with 2.6% (wt/vol) linoleic
wt/vol), which corresponded to 5% (dry wt/vol), but de- acid as the substrate and 23% (wet wt/vol) washed cells as the
creased slightly with greater amounts of washed cells.
catalyst was centrifuged after 108-h reaction and separated
Time course of preparative CLA production. The time into supernatant and cells. The distribution and lipid classes
course of CLA production from linoleic acid was monitored of the FA produced in both supernatant and cells were ana-
under two different conditions. With 12% (wt/vol) linoleic lyzed (Table 2). Most of the FA (98.9%) were found in the
acid as the substrate and 33% (wet wt/vol) washed cells as the cells (or associated with the cells), in which CLA was found
catalyst, the production of CLA reached a maximum (40 as the most abundant FA. Of the CLA in the cells (or associ-
mg/mL) at 108 h and then gradually decreased (Fig. 3A). On ated with cells), 53 and 41% were found in the FFA and non-
the other hand, with 2.6% (wt/vol) linoleic acid as the sub- polar lipid fractions, respectively.
strate and 23% (wet wt/vol) washed cells as the catalyst, 80%
Biological systems are promising for the selective prepa-
(mol%) of the linoleic acid added was converted to CLA (20 ration of CLA isomers. Natural dietary sources of CLA are
mg/mL) in 96 h (Fig. 3C). FA compositions of the produced the meat and milk of ruminants and products made from
lipids are also presented in Figure 3. With a high substrate them. In these materials, the predominant isomer is cis-
concentration (Fig. 3B), a large amount of CLA1 (15 mg/mL) 9,trans-11-18:2, which accounts for over 75% of the total
was found, while with low substrate concentration (Fig. 3D) CLA (16,17). The cis-9,trans-11-18:2 is also produced from
a significantly lesser amount of CLA1 (0.6 mg/mL) was ob- linoleic acid as an intermediate of biohydrogenation by rumen
served and CLA2 became dominant (19.5 mg/mL). The pro- bacteria (18) and dairy starter cultures (19). However, the
portions of CLA isomers changed depending on reaction con- amounts of CLA in these materials are very low. The results
ditions. Lower substrate concentration and longer reaction presented here clearly show that L. plantarum AKU 1009a is
tended to increase CLA2 production. Investigation of the a promising biocatalyst for CLA production. The produced
mechanism controlling the proportions of CLA isomers and CLA accumulated in the cells, reaching ca. 38% (w/w) of the
the detailed analysis of the pathway of CLA production from dry cells obtained after the reaction.
JAOCS, Vol. 79, no. 2 (2002)

CLA PRODUCTION BY LACTIC ACID BACTERIA
163
TABLE 2
Distribution and Lipid Classes of FA Produced from Linoleic Acid by Washed Cells of Lactobacillus plantaruma
Distribution of FA in indicated lipid class (mol%) in:
FA concentration
(mg/mL reaction mixture)
after reaction
Supernatant
NLc
Cells
NL
Fatty acid
FAb
PLd
FA
PL
Total
e
Linoleic acid
CLA1
CLA2
HY1
HY2
0.19
0.38
16.33
1.32
2.20
1.33
0.2
0.1
0.5
—
—
—
—
—
—
0.1
0.8
39.7
0.2
9.6
5.2
0.1
0.9
30.4
5.8
0.2
0.6
0.4
—
4.4
—
0.3
0.2
5.3
0.8
1.8
75.0
6.0
10.1
6.3
100
—
—
Trace
—
0.1
0.1
Tracef
Trace
0.1
Other FA
Total
0.1
0.1
21.75
0.9
55.6
38.0
aLactobacillus plantarum was cultivated in MRS medium with linoleic acid (0.06%, wt/vol) for 24 h. The reaction was carried out with 2.6% (wt/vol) linoleic
acid as the substrate for 108 h under the conditions described in the Materials and Methods section. For abbreviations, see Table 1. Other fatty acids (mg/mL
reaction mixture) were palmitic acid (0.02), oleic acid (0.75), vaccenic acid (0.43) and 2-hexy-1-cyclopropane-octanoic acid (0.13). NL, nonpolar lipids; PL,
polar lipids; —, not detected; Trace, <0.05 mol%.
mation of Linoleic Acid by Lactobacillus acidophilus, Appl. En-
REFERENCES
viron. Microbiol. 67:1246–1252 (2001).
1. Pariza, M.W., and Y.L. Ha, Newly Recognized Anticarcino-
11. Haas, M.H., J.K.G. Kramer, G. McNeill, K. Scott, T.A. Foglia,
genic Fatty Acids, in Antimutagenesis and Anticarcinogenesis
Mechanism II, edited by Y. Kuroda, D. Shankel, and M.D. Wa-
ters, Plenum Press, New York, 1990, pp.167–170.
N. Sehat, J. Fritsche, M.M. Mossoba, and M.P. Yurawecz, Li-
pase-Catalyzed Fractionation of Conjugated Linoleic Acid,
Lipids 34:979–987 (1999).
2. Ha, Y.L., N.K. Grimm, and M.W. Pariza, Anticarcinogens from
12. Mounts, T.L., H.J. Dutton, and D. Glover, Conjugation of
Polyunsaturated Acids, Ibid. 5:997–1005 (1970).
13. Bligh, E.G., and W.J. Dyer, A Rapid Method of Total Lipid Ex-
traction and Purification, Can. J. Biochem. Physiol. 37:911–917
(1959).
14. Jareonkitmongkol, S., S. Shimizu, and H. Yamada, Fatty Acid
Fried Ground Beef: Heat-Altered Derivatives of Linoleic Acid,
Carcinogenesis 8:1881–1887 (1987).
3. Ha, Y.L., J. Storkson, and M.W. Pariza, Inhibition of
Benzo(a)pyrene-induced Mouse Forestomach Neoplasia by
Conjugated Dienoic Derivatives of Linoleic Acid, Can. Res.
50:1097–1101 (1990).
Desaturation-Defective Mutants of an Arachidonic-Acid-Pro-
ducing Fungus, Mortierella alpina 1S-4, J. Gen. Microbiol.
138:997–1002 (1992).
4. Ip, C., S.F. Chin, J.A. Scimeca, and M.W. Pariza, Mammary
Cancer Prevention by Conjugated Dienoic Derivatives of
Linoleic Acid, Ibid. 51:6118–6124 (1991).
5. Lee, K.N., D. Kritchevsky, and M.W. Pariza, Conjugated
Linoleic Acid and Atherosclerosis in Rabbits, Atherosclerosis
108:19–25 (1994).
6. Park, Y., K.J. Albright, W. Liu, J.M. Storkson, M.E. Cook, and
15. Shimizu, S., S. Jareonkitmongkol, H. Kawashima, K. Akimoto,
and H. Yamada, Production of a Novel ω1-Eicosapentaenoic
Acid by Mortierella alpina 1S-4 Grown on 1-Hexadecene, Arch.
Microbiol. 156:163–166 (1991).
16. Chin, S.E., W. Liu, J.M. Storkson, Y.L. Ha, and M.W. Pariza,
M.W. Pariza, Effect of Conjugated Linoleic Acid on Body Com-
Dietary Sources of Conjugated Dienoic Isomers of Linolec
position in Mice, Lipids 32:853–858 (1997).
7. Belury, M.A., Conjugated Dienoic Linoleate: A Polyunsaturated
Fatty Acid with Unique Chemoprotective Properties, Nutr. Rev.
53:83–89 (1995).
8. Parodi, P.W., Cows’ Milk Fat Components as Potential Anticar-
cinogenic Agents, J. Nutr. 127:1055–1060 (1997).
9. Yurawecz, M.P., N. Sehat, M.M. Mossoba, J.A.G. Roach, and
Acid, a Newly Recognized Class of Anticarcinogens, J. Food
Comp. Anal. 5:185–197 (1992).
17. Lin, H., T.D. Boylston, M.J. Chang, L.O. Luedecke, and T.D.
Shultz, Survey of the Conjugated Linoleic Acid Contents of
Dairy Products, J. Dairy Sci. 78:2358–2365 (1995).
18. Kepler, C.R., K.P. Hirons, J.J. McNeill, and S.B. Tove, Inter-
mediates and Products of the Biohydrogenation of Linoleic Acid
by Butyrivibrio fibrisolvens, J. Biol. Chem. 241:1350–1354
(1966).
Y. Ku, Oxidation Products of Conjugated Linoleic Acid and
Furan Fatty Acids, in New Techniques and Applications in Lipid
Analysis, edited by R.E. McDonald, and M.M. Mossoba, AOCS
Press, Champaign, 1997, pp. 183–215.
19. Jiang, L.L., L. Bjorck, and R. Fonden, Production of Conjugated
Linoleic Acid by Dairy Starter Cultures, J. Appl. Microbiol.
85:95–102 (1998).
10. Ogawa, J., K. Matsumura, S. Kishino, Y. Omura, and S.
Shimizu, Conjugated Linoleic Acid Accumulation via 10-Hy-
droxy-12-octadecaenoic Acid During Microaerobic Transfor-
[Received July 16, 2001; accepted November 14, 2001]
JAOCS, Vol. 79, no. 2 (2002)