INTRODUCTION

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Conjugated linoleic acid (CLA), an octadecadienoic acid with conjugated double bonds, has a variety of positional and geometric isomers. In the last 2 decades, CLA has attracted considerable attention because of its potentially beneficial effects. Ha and colleagues identified a lipid fraction from beef fat that possessed anticarcinogenic properties and demonstrated that it was composed of CLA isomers (8). Subsequent studies with animal models showed that consumption of CLA inhibited the initiation of carcinogenesis (18) and tumorigenesis (9, 11), reduced body fat content and increased muscle mass (3), decreased atherosclerosis (17), improved hyperinsulinemia (10), controlled immune systems (4, 16), and altered the low-density lipoprotein/high-density lipoprotein cholesterol ratio (15), all of which further heightened interest in CLA. Of the individual isomers of CLA, cis-9, trans-11-octadecadienoic acid (c9, t11-18:2) has been suggested to be the most important in terms of biological activity because it is the major isomer and it is incorporated into the phospholipid fraction of tissues of animals who are fed a mixture of CLA isomers (9). However, the commercially available CLA synthesized by alkali isomerization of linoleic acid is a mixture of four (8,10-, 9,11-, 10,12-, and 11,13-18:2) cis/trans positional isomers (19). Therefore, there is interest in the development of methods for the selective production of CLA isomers.

Dairy products are among the major dietary sources of CLA, of which c9, t11-18:2 is the main isomer (3, 7). CLA has been shown to be produced from polyunsaturated fat by certain rumen microorganisms such as Butyrivibrio species (6). c9, t11-18:2 has been suggested to be the first intermediate in the biohydrogenation of linoleic acid by the anaerobic rumen bacterium Butyrivibrio fibrisolvens (14). More recently, it was reported that Propionibacterium freudenreichii, commonly used as a dairy starter culture, was able to produce CLA from free linoleic acid (13). These findings suggest that CLA is derived from linoleic acid, but little is known about the precise structures of the CLA produced or the mechanisms of CLA production, and the amounts of CLA produced under the conditions mentioned above are very low.

To establish efficient and selective methods for CLA production and to clarify the reactions involved in CLA formation from linoleic acid by microorganisms, we investigated the ability of lactic acid bacteria to produce CLA from linoleic acid and found that washed cells of Lactobacillus acidophilus AKU 1137 produce CLA under microaerobic conditions, with a preceding accumulation of hydroxy fatty acids (HY). Here, we report the chemical structures of CLA and HY produced by L. acidophilus and discuss the roles of HY as intermediates in the production of CLA.

	MATERIALS AND METHODS

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Chemicals. Linoleic acid and fatty acid-free (<0.02%) bovine serum albumin (BSA) were purchased from Wako Pure Chemicals (Osaka, Japan) and Nacalai Tesque (Kyoto, Japan), respectively. All other chemicals used were of analytical grade and are commercially available.

Microorganism cultivation and preparation of washed cells. L. acidophilus AKU 1137 (AKU Culture Collection, Faculty of Agriculture, Kyoto University, Kyoto, Japan) was aerobically cultivated in MRS medium comprised of 1.0% tryptone, 1.0% meat extract, 0.5% yeast extract, 2.0% glucose, 0.1% Tween 80, 0.2% K₂HPO₄, 0.5% sodium acetate, 0.2% diammonium citrate, 0.02% MgSO₄ · 7H₂O, and 0.005% MnSO₄ · H₂O (pH 6.5). The strain was inoculated in 15 ml of liquid medium in screw-cap tubes (16.5 by 125 mm). The liquid medium occupied approximately 80 to 90% of the volume of the tubes. Cultivations were carried out for 3 days at 28°C with shaking (120 strokes/min). The cells were harvested by centrifugation (14,000 × g, 30 min), washed twice with 0.85% NaCl, centrifuged again, and then used as the washed cells.

Reaction conditions. Reactions were carried out at 28°C with gentle shaking (120 strokes/min) in screw-cap tubes (16.5 by 125 mm). Reactions were carried out under aerobic or microaerobic conditions with or without replacement of the air in the tubes by pure nitrogen (99.999% pure; Sumitomo-Seika Chemicals Co. Ltd., Osaka, Japan), respectively. In the microaerobic assays, the oxygen concentrations, monitored by an oxygen indicator (Mitsubishi Gas Chemical Co. Ltd., Tokyo, Japan), were kept under 1%. The reaction mixture contained, in 1 ml of 100 mM potassium phosphate buffer (pH 6.5), 5 mg of linoleic acid in a complex with BSA (0.2 mg of BSA/mg of linoleic acid) and the washed cells from 15 ml of culture broth (approximately 20 mg of cells [dry weight]).

Lipid analyses. Lipids were extracted from the reaction mixture with chloroform-methanol (1:2, vol/vol) according to the procedure of Bligh and Dyer (2), methylated with diazomethane in diethylether for 15 min, and further methylated with 0.5 M sodium methoxide in methanol for 30 min at 50°C. The resultant fatty acid methyl esters were extracted with n-hexane and analyzed by gas-liquid chromatography (GC) using a Shimadzu (Kyoto, Japan) GC-17A gas chromatograph equipped with a flame ionization detector and a split injection system and fitted with a capillary column (HR-SS-10, 50 m by 0.25 mm inside diameter; Shinwa Kako, Kyoto, Japan). The column temperature, initially 180°C, was raised to 220°C at a rate of 2°C/min and maintained at that temperature for 5 min. The injector and detector were operated at 250°C. Helium was used as carrier gas at 225 kPa/cm². Extraction and fractionation into lipid classes were carried out essentially as described previously (12, 20).

Isolation of reaction products. The methyl esters of the reaction products were separated by reverse-phase high-performance liquid chromatography using a Shimadzu LC-10A system equipped with a Cosmosil column (5C₁₈-AR, 20 by 250 mm; Nacalai Tesque). The mobile phase was acetonitrile-H₂O (8:2, vol/vol) at a flow rate of 3.0 ml/min, and the effluent was monitored by UV detection (205 nm). The chemical structures of purified fatty acids were determined by mass spectroscopy (MS), infrared spectroscopy (IR), proton nuclear magnetic resonance (¹H-NMR), and ¹H-¹H correlation spectroscopy (COSY).

Preparation of free fatty acids. Free fatty acids were prepared by heating the fatty acid methyl esters (50 mg) in a mixture of 50 µl of 7.0 N sodium hydroxide and 50 µl of methanol in screw-cap tubes. After being heated in a boiling water bath for 1 h, the solution was acidified to pH 2.0 with 10% (wt/vol) sulfuric acid in water. The free fatty acids were extracted with diethylether. The organic extract was washed with water and dried over anhydrous Na₂SO₄, and the solvent was removed under vacuum in a rotary evaporator.

Preparation of pyrrolidide fatty acids. Pyrrolidide derivatives were prepared by direct treatment of the isolated methyl esters with pyrrolidine-acetic acid (10:1, vol/vol) in screw-cap tubes for 1 h at 115°C followed by extraction according to the method of Andersson and Holman (1). The organic extract was washed with water and dried over anhydrous Na₂SO₄, and then the solvent was removed under vacuum in a rotary evaporator.

GC-MS analysis. GC-MS QP5050 (Shimadzu) with a GC-17A gas chromatograph was used for mass spectral analyses. The GC separation of fatty acid methyl esters was performed on an HR-SS-10 column as described above at the same temperature. The GC separation of fatty acid pyrrolidide derivatives was performed on the HR-1 column (25 m by 0.5 mm inner diameter; Shinwa Kako) at 300°C. MS was used in the electron impact mode at 70 eV with a source temperature of 250°C. Split injection was employed with the injector port at 250°C.

MS-MS analysis. MS-MS analyses were performed on the free acids of the fatty acids with a JEOL HX110A/HX110A tandem mass spectrometer. The ionization method was fast atom bombardment (FAB) and the acceleration voltage was 3 kV. Glycerol was used for the matrix.

IR analysis. IR analysis of fatty acid methyl esters was performed with infrared spectrophotometer IR-420 (Shimadzu) in a chloroform solution.

¹H-NMR and ¹H-¹H COSY analyses. All NMR experiments were performed on a JEOL EX-400 (400 MHz at ¹H), and chemical shifts were assigned relative to the solvent signal. Fatty acid methyl esters were dissolved in dichloromethane-d₂, and the diameter of the tube was 5 mm.

	RESULTS

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Transformation of linoleic acid by washed cells of L. acidophilus. When the reaction was carried out under aerobic conditions, linoleic acid was decomposed by washed cells of L. acidophilus without generation of detectable amounts of fatty acids (Fig. 1A). On the other hand, when the reaction was carried out under microaerobic conditions, four major, newly generated fatty acids, designated CLA1, CLA2, HY1, and HY2, were found on the GC chromatograms of the methylated fatty acids (Fig. 1B). The peaks for CLA1 and CLA2, with retention times slightly greater than that of linoleic acid, were shown to have the same retention times as those from the CLA mixture purchased from Nu-Chek-Prep, Inc. (Elysian, Minn.) The peaks for HY1 and HY2 indicated that HY1 and HY2 were relatively polar fatty acids such as hydroxylated ones because of their far greater retention times.

View larger version (14K): [in this window] [in a new window]   FIG. 1.   GC chromatograms of fatty acid methyl esters produced by washed cells of L. acidophilus. (A) Reaction with linoleic acid as a substrate under aerobic conditions. (B) Reaction with linoleic acid as a substrate under microaerobic conditions. (C) Reaction with HY2 as a substrate under microaerobic conditions.

Identification of CLA1 and CLA2. Mass spectra of the isolated methyl esters of both CLA1 and CLA2 exhibited molecular weights of m/e 294, and those of pyrrolidide derivatives showed molecular weights of m/e 333. These results suggest that CLA1 and CLA2 are C₁₈ fatty acids containing two double bonds. FAB-MS data of the free fatty acids of both CLA1 and CLA2 exhibited molecular weights of m/e 280 ([M-H]⁺, 279). These peaks (m/e 279) were fragmented again by MS-MS. Typical fragments (m/e) for both CLA1 and CLA2 were 127, 141, 167, 193, 207, and 208. The m/e 141, 167 and 193 were derived from cleavage between single bonds 8-9, 10-11, and 12- 13, numbered from the carboxyl group. m/e 127 and 207, derived from the cleavage of a single bond between the  and  positions from the double bond, were clearly detected. Hence, CLA1 and CLA2 were identified as 9,11 positional isomers of octadecadienoic acid.

View larger version (39K): [in this window] [in a new window]   FIG. 2.   1H-NMR spectra and structures of CLA1 (A) and CLA2 (B). The letters indicate the positions of protons and their corresponding signals.

1

1

1

Identification of HY1 and HY2. FAB-MS analysis of the isolated methyl esters of HY1 and HY2 revealed molecular weights of m/e 312 ([M+H]⁺, 313). On MS analyses of methyl esters of both HY1 and HY2, typical fragments of m/e 169, 201, and 294 were found. The fragment m/e 294 (M-18) was thought to indicate cleavage between the hydroxyl group and carbon and the existence of one double bond in the hydrocarbon chain. Moreover, cleavage between the  and  positions from the hydroxyl group yielded m/e 201. This suggests that the hydroxyl group is located at carbon 10, numbered from the carboxyl group.

View larger version (36K): [in this window] [in a new window]   FIG. 3.   1H-NMR spectra and structures of HY1 (A) and HY2 (B). The letters indicate the positions of protons and their corresponding signals.

Time course of linoleic acid transformation by washed cells of L. acidophilus under microaerobic conditions. The time course of changes in fatty acid composition during linoleic acid transformation by washed cells of L. acidophilus under microaerobic conditions was studied using the washed cells obtained by cultivation in MRS medium with or without linoleic acid (0.1%, [wt/vol]). In comparison with the results shown in Fig. 4A and B, the CLA productivity of the washed cells obtained by cultivation with linoleic acid was much higher than that of the cells obtained by cultivation without linoleic acid. This may have been due to induction of the enzymes catalyzing CLA formation by linoleic acid during cultivation. The amounts of cellular fatty acids (myristic acid, palmitic acid, palmitoleic acid, oleic acid, vaccenic acid, and 2-hexyl-1-cyclopropane-octanoic acid) did not significantly change for 4 days after the reaction. With the washed cells obtained by cultivation with linoleic acid, CLA (sum of CLA1 and CLA2) levels reached 36% (wt/wt) of total fatty acids on the first day and exceeded 80% (wt/wt) on the fourth day (Fig. 4B). The ratio of HY (sum of HY1 and HY2) was 25% (wt/wt) on the first day and rapidly decreased, followed by an increase in CLA level (Fig. 4B). These results suggest that HY produced by the washed cells may be converted to CLA and that HY may be the intermediate in CLA formation. The time courses of CLA and HY production from linoleic acid are presented in Fig. 4C. The final level of CLA was 4.9 mg/ml (CLA1, 3.3 mg/ml; CLA2, 1.6 mg/ml; molar conversion yield from linoleic acid, 98%).

View larger version (25K): [in this window] [in a new window]   FIG. 4.   Time courses of changes in fatty acid composition during the reaction with washed cells obtained by cultivation in MRS medium without (A) or with (B) linoleic acid (0.1% [wt/vol]) and of changes in levels of CLA and HY production from linoleic acid (C). The results are averages of three separate determinations that were reproducible within ±10%.

Production of CLA from hydroxy fatty acid. HY2 was isolated by preparative high-performance liquid chromatography and was used as the substrate for the reaction instead of linoleic acid to determine whether HY is converted to CLA by washed cells of L. acidophilus. The GC chromatogram of the fatty acids obtained after reaction under microaerobic conditions is shown in Fig. 1C. No HY2 was detected after the reaction, but CLA1 and CLA2 were found. These observations suggest that HY2 may be a substrate for CLA and an intermediate in the formation of CLA from linoleic acid. Preparative isolation of HY1 resulted in contamination with a small amount of HY2, so that it was difficult to determine whether HY1 was indeed an intermediate. However, during linoleic acid transformation, HY1 accumulated, while CLA was being produced, until the third day; then the level of HY1 decreased, followed by an increase in the level of CLA (Fig. 4C), indicating that HY1 is also an intermediate of CLA formation.

Distribution and lipid classes of the fatty acids produced by washed cells of L. acidophilus. The reaction mixture of linoleic acid transformation was centrifuged after 3 days of reaction and separated into supernatant and cells. The distribution and lipid classes of the fatty acids produced in both supernatant and cells were analyzed (Table 1). Most of the fatty acids (92.0%), were found in cells or associated with cells as free fatty acids, with CLA the most abundant. The fatty acids found in cells (or associated with cells) consisted of free fatty acids (86.4%), acylglycerols (5.6%), and trace amounts of phospholipids. Most of the CLA produced was found as free fatty acid in cells (or associated with cells).

	DISCUSSION

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Some anaerobic bacteria have been reported to produce CLA. The rumen bacterium B. fibrisolvens produces cis-9, trans-11-octadecadienoic acid as an intermediate of the biohydrogenation of linoleic acid to oleic acid (14). The lyophilized cells of some lactobacilli have been found to contain small amounts of CLA (5). However, the mechanism of CLA formation has not been elucidated in detail. The results reported here indicate that the transformation of linoleic acid to CLA is not a one-step isomerization of a nonconjugated diene to a conjugated diene. Rather, the transformation involves the production of hydroxy fatty acids, i.e., 10-hydroxy-trans-12-octadecaenoic acid and 10-hydroxy-cis-12-octadecaenoic acid. The findings obtained here using washed cells of L. acidophilus under microaerobic conditions, i.e., (i) accumulation of HY prior to CLA formation and its decrease concomitant with increased formation of CLA and (ii) conversion of exogenously added 10-hydroxy-cis-12-octadecaenoic acid to CLA, strongly support the above suggestion. It is not yet clear whether the generation of geometric isomers of HY and CLA is a biological or chemical process occurring during analysis nor whether the trans isomer of HY is involved as an intermediate. However, the pathway involving hydroxylation at carbon 10, numbered from the carboxyl group as the first step in the reaction, could be proposed for isomerization of linoleic acid to CLA (Fig. 5). Further analyses of the enzyme systems for CLA production induced by linoleic acid are under way in our laboratory to clarify the transformation pathway and its physiological significance for lactic acid bacteria.

View larger version (41K): [in this window] [in a new window]   FIG. 5.   Proposed pathway of production of CLA from linoleic acid by washed cells of L. acidophilus under microaerobic conditions.

A study of the production of CLA was performed with P. freudenreichii, a bacterium commonly used in dairy starter cultures, and showed the extracellular production of CLA (265 µg/ml) mainly consisting of cis-9, trans-11- or trans-9, cis-11-octadecadienoic acid (13). This previous study revealed the potential ability of lactic acid bacteria to produce CLA. The transformation of linoleic acid into CLA with washed cells of L. acidophilus under microaerobic conditions is a promising system for the following reasons: (i) specific isomers of CLA, i.e., cis-9, trans-11- or trans-9, cis-11-octadecadienoic acid and trans-9, trans-11-octaecadienoic acid, are obtained as reaction products; (ii) CLA accumulates at high concentrations (nearly 5 mg/ml); (iii) CLA content in the recovered fatty acids reaches nearly 90% (wt/wt); (iv) CLA is accumulated as intracellular or cell-associated lipids in free form, making it easy to recover by centrifugation, and cells themselves could be used as sources of CLA; and (v) the reaction requires only microaerobic conditions and no energy input. Because of its beneficial physiological effects, there is a potential demand for CLA. The results presented here may lead to the development of industrial production of CLA by lactic acid bacteria.