Biohydrogenation of C₂₀ polyunsaturated fatty acids by anaerobic bacteria

Abstract

The PUFAs include many bioactive lipids. The microbial metabolism of C₁₈ PUFAs is known to produce their bioactive isomers, such as conjugated FAs and hydroxy FAs, but there is little information on that of C₂₀ PUFAs. In this study, we aimed to obtain anaerobic bacteria with the ability to produce novel PUFAs from C₂₀ PUFAs. Through the screening of ∼100 strains of anaerobic bacteria, Clostridium bifermentans JCM 1386 was selected as a strain with the ability to saturate PUFAs during anaerobic cultivation. This strain converted arachidonic acid (cis-5,cis-8,cis-11,cis-14-eicosatetraenoic acid) and EPA (cis-5,cis-8,cis-11,cis-14,cis-17-EPA) into cis-5,cis-8,trans-13-eicosatrienoic acid and cis-5,cis-8,trans-13,cis-17-eicosatetraenoic acid, giving yields of 57% and 67% against the added PUFAs, respectively. This is the first report of the isolation of a bacterium transforming C₂₀ PUFAs into corresponding non-methylene-interrupted FAs. We further investigated the substrate specificity of the biohydrogenation by this strain and revealed that it can convert two cis double bonds at the ω6 and ω9 positions in various C₁₈ and C₂₀ PUFAs into a trans double bond at the ω7 position. This study should serve to open up the development of novel potentially bioactive PUFAs.

The PUFAs include many bioactive lipids that play an important role in the maintenance of biological functions in mammals (1, 2). The vast majority of PUFAs have two or more cis double bonds that are separated from each other by a single methylene group (known as methylene-interrupted FAs). They include two major subgroups (the ω3 and ω6 PUFAs) that have different functions (1–3). Arachidonic acid [cis-5,cis-8,cis-11,cis-14-eicosatetraenoic acid (20:4, ω6), AA], which is the C₂₀ PUFA of the ω6 class and is made from linoleic acid [cis-9,cis-12-octadecadienoic acid (18:2, ω6), LA], is involved in many cellular signaling mechanisms and is also the precursor for the formation of the 2 series of prostaglandins. On the other hand, EPA [cis-5,cis-8,cis-11,cis-14,cis-17-EPA (20:5, ω3)], which is a C₂₀ PUFAs of the ω3 class and is made from α-linolenic acid [cis-9,cis-12,cis-15-octadecatrienoic acid (18:3, ω3)], is the precursor for the formation of the 3 series of prostaglandins and can compete with the effects of AA, such as the AA conversion to the prostaglandins. Unlike methylene-interrupted FAs, rare isomers of PUFAs, which have at least two double bonds that are separated by a single carbon-carbon bond (known as conjugated FAs) (4–7) or two or more methylene groups [known as non-methylene-interrupted FAs (NMIFAs)] (8–10), have been found in several materials including plant oil. These rare PUFAs have also been reported to show interesting physiological effects (9, 11–15). Therefore, they have gained considerable attention, but natural sources rich in them are limited.

The partial hydrogenation of PUFAs is the process of converting PUFAs into the more saturated FAs and can produce NMIFAs from more readily available PUFAs. This can be performed mainly by chemical hydrogenation in industry and by microbial biohydrogenation in living organisms (16). Chemical partial hydrogenation is widely used to convert vegetable oils into foods such as margarine. The partial hydrogenation of vegetable oils produces various hydrogenated vegetable oils, including several isomers of octadecenoic acid (18:1), depending on the reaction conditions. In contrast, microbial biohydrogenation can selectively produce specific isomers (4–7). Thus, microbial biohydrogenation has several advantages over chemical hydrogenation.

Recently, some studies, including ours, have found that many anaerobic bacteria, such as Lactobacillus species, can produce conjugated LAs from LA (4, 17–20). Further, we have revealed that lactic acid bacteria produce unique PUFAs from various C₁₈ PUFAs through partial biohydrogenation (21–23). Thus, the biohydrogenation of C₁₈ PUFAs has been widely studied. However, as far as we know, the biohydrogenation of other FAs, especially C₂₀ PUFAs, has not been extensively studied so far.

In this paper, we report about the screening of anaerobic bacteria for the ability to transform C₂₀ PUFAs through biohydrogenation. We found that Clostridium bifermentans JCM 1386 can specifically convert AA and EPA into their partially saturated FAs with a trans double bond at the ω7 position. We further found that other C₁₈ and C₂₀ PUFAs were also converted in a similar manner. Thus, we succeeded in the production of various C₁₈ and C₂₀ NMIFAs with a trans double bond at the ω7 position through the biohydrogenation by C. bifermentans JCM 1386, leading to the development of novel potentially bioactive PUFAs.

MATERIALS AND METHODS

Chemicals

LA and α-linolenic acid were purchased from Wako Pure Chemical (Osaka, Japan). γ-Linolenic acid (cis-6,cis-9,cis-12-18:3), dihomo-γ-linolenic acid [cis-8,cis-11,cis-14-eicosatrienoic acid (20:3)], AA, and EPA were purchased from Sigma (St. Louis, MO). DHA [cis-4,cis-7,cis-10,cis-13,cis-16,cis-19-DHA (22:6)] was purchased from Cayman Chemical (Ann Arbor, MI). All other chemicals used were of analytical grade and are commercially available.

Microorganism and cultivation

The identified anaerobic bacteria used for this study (supplementary Table I) were preserved in our laboratory (AKU Culture Collection, Division of Applied Life Science, Faculty of Agriculture, Kyoto University, Kyoto, Japan) and those obtained from other culture collections (JCM, Japan Collection of Microorganisms, Saitama, Japan; and ATCC, Manassas, VA). The unidentified anaerobic bacteria used for this study were isolated from ponds, wastewater, fish viscera, and so on. The medium was Gifu Anaerobic Medium (GAM) broth (pH 7.0) (Nissui Pharmaceutical Co. Ltd., Tokyo, Japan) supplemented with 0.03% (w/v) LA, AA, or 0.02% (w/v) EPA. Each strain was inoculated into 15 ml of the medium in screw-capped tubes (16.5 × 215 mm) and then incubated in an anaerobic chamber (98% nitrogen and 2% hydrogen) at 37°C for 2–3 days. After the cultivation, the culture medium was separated into supernatant and cells by centrifugation (8,000 g, 10 min), and the supernatant was used for lipid analysis.

Lipid analysis

Lipids were extracted from the supernatants with chloroform-methanol according to the procedure of Bligh-Dyer (24) and methylated with 4% methanolic-HCl at 50°C for 20 min. The resultant FA methyl esters were extracted with n-hexane and analyzed by GC using a Shimadzu (Kyoto, Japan) GC-1700 gas chromatograph equipped with a flame ionization detector and a split injection system and fitted with a capillary column (ULBON HR-SS-10, 50 m × 0.25 mm inner diameter, Shinwa Kako, Kyoto, Japan). The column temperature was initially 180°C and was raised to 220°C at a rate of 2°C/min and maintained at that temperature for 20 min. The injector and detector were operated at 250°C. Helium was used as a carrier gas at 0.97 ml/min.

Isolation, derivatization, and identification of products

For the isolation of the newly generated FA in a culture of C. bifermentans JCM 1386 with 0.03% (w/v) AA (Unknown FA 1, UK1), its methyl esters were purified by HPLC (monitored at 205 and 233 nm) using a Shimadzu LC-VP system fitted with a Cosmosil column 5C18-ARII (20 × 250 mm, Nacalai tesque, Kyoto, Japan). The mobile phase was acetonitrile-water (80:20, v/v) at a flow rate of 5.0 ml/min and the column temperature of 30°C. The fraction containing UK1 was further purified by HPLC on Inertsil ODS SQ5-1385 (4.6 × 250 mm, GL Science Inc., Torrance, CA) joined with Capcelpak C18 UG20 (4.6 × 250 mm, Shiseido, Tokyo, Japan). Acetonitrile-water (80:20, v/v) was used as the mobile phase at a flow rate of 1.2 ml/min. For the isolation of the newly generated FA in a culture of C. bifermentans JCM 1386 with 0.02% (w/v) EPA (Unknown FA 2, UK2), its methyl esters were purified using a same procedure as described for UK1 except that the mobile phase used for the latter step was acetonitrile-water (80:20, v/v) at a flow rate of 1.0 ml/min.

The chemical structures of purified FAs were determined by MS, proton NMR (¹H-NMR), ¹H-¹H-chemical shift correlation spectroscopy (COSY), two-dimensional nuclear Overhauser effect spectroscopy (NOESY), and ¹H clean-total correlation spectroscopy (TOCSY).

¹H-NMR, COSY, NOESY, and TOCSY analyses

All NMR experiments were performed on a BrukerBiospin DX-750 (750 MHz for ¹H), and chemical shifts were assigned relative to the solvent signal (dichloromethane-d₂).

Preparation of pyrrolidide FAs

Pyrrolidide derivatives were prepared by direct treatment of the isolated methyl esters with pyrrolidine-acetic acid (10:1, v/v) in screw-cap tubes for 1 h at 115°C followed by extraction according to the method of Andersson and Holman (25). The organic extract was washed with water and dried over anhydrous Na₂SO₄, and then the solvent was removed by a vacuum in a rotary evaporator.

GC-MS analysis

GC-MS QP5050 (Shimadzu) with a GC-17A gas chromatograph was used for mass spectral analysis. The GC separation of the methyl ester and the pyrrolidide derivatives was performed on an ULBON HR-1 column (25 m × 0.5 mm, 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 used with the injector port at 250°C.

MS-MS analysis

MS-MS analyses were performed on the free acids of the FAs 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.

RESULTS

Screening of anaerobic bacteria that have the ability to convert C₂₀ PUFAs

The ability of anaerobic bacteria to convert the C₂₀ PUFAs of EPA and AA during cultivation was investigated together with LA as a reference of C₁₈ PUFA. We tested ∼100 strains, including the identified bacteria, which belonged to genera such as Megasphaera, Bifidobacterium, Lactobacillus, Propionibacterium, Clostridium, Bacteroides, Eubacterium, and so on (supplementary Table I), and the unidentified bacteria. The peaks of the PUFAs were identified by comparison with the retention time of the reference standards on GC analysis.

Of these bacteria, two strains of C. bifermentans (JCM 1386 and JCM 7832) showed the activity to convert AA and EPA, while five strains (including the two C₂₀ PUFA-converting strains mentioned previously) belonging to the genera of Clostridium and Propionibacterium were found to have the ability to convert LA to vaccenic acid (trans-11-18:1, VA) (Table 1).

TABLE 1.

Screening results for the ability of transforming PUFAs

		Produced FA (mg/ml Culture Broth)
Strain	No.	VA from LA	UK1 from AA	UK2 from EPA
C. bifermentans	JCM 1386	0.06	0.12	0.11
C. bifermentans	JCM 7832	0.05	0.13	0.10
Clostridium sporogenes	JCM 7849	0.11	—	—
C. sporogenes	JCM 7850	0.16	—	—
Propionibacterium acnes	JCM 6473	0.12	—	—

Cultivations were carried out in GAM broth with 0.03% (w/v) of LA, AA, or 0.02% (w/v) of EPA for 3 days as described in Materials and Methods. —, not detected.

Figure 1 shows the GC chromatogram of methylated FAs produced by C. bifermentans JCM 1386 from AA, EPA, and LA as examples. When C. bifermentans JCM 1386 was cultured with AA or EPA, newly generated FAs [UK1 from AA (Fig. 1A) and UK2 from EPA (Fig. 1B)] were detected on the GC chromatogram of methylated FAs. The same reactions were observed when C. bifermentans JCM 7832 was cultured with AA or EPA (Table 1). However, C. sporogenes JCM 7849, C. sporogenes JCM 7850, and P. acnes JCM 6473 could not convert AA and EPA.

Fig. 1.

GC chromatograms of methyl esters of FAs produced by C. bifermentans JCM 1386. Cultivations were carried out in GAM broth with 0.03% (w/v) of AA (A), EPA (B), or LA (C) for 3 days. The lipid products were extracted from the supernatant and methylated as described in Materials and Methods. AA, EPA, and LA are converted to UK1, UK2, and VA, respectively.

As the concentration of the C₂₀ PUFAs added grew, C. bifermentans JCM1386 showed higher activity than C. bifermentans JCM 7832 (data not shown). C. bifermentans JCM1386 was used for further analyses.

Identification of the newly generated FA in a culture of C. bifermentans JCM 1386 with AA

When the lipids extracted from the medium after cultivation of C. bifermentans JCM 1386 with AA were analyzed by thin-layer chromatography, almost all lipids were present in the free form (data not shown). After complete esterification of the free-form FA products, the resulting methyl esters were isolated and used for structural analysis. The mass spectrum of the isolated methyl ester of UK1 exhibited a molecular mass of m/z 320, indicating that UK1 is C₂₀ PUFA containing three double bonds. The molecular ion peak ([M+Na]⁺, 343) obtained by FAB-MS analysis (FAB⁺) of the methyl ester of UK1 was fragmented again by MS-MS [m/z (FAB⁺, 8.00 kV), 328 (1), 314 (2), 300 (2), 299 (3), 286 (3), 285 (4), 272 (12), 258 (1), 257 (2), 232 (3), 218 (3), 217 (28), 204 (33), 190 (1), 189 (1), 164 (35), 163 (12), 150 (3), 149 (4), 124 (5), 110 (13), 109 (68), 96 (100), 82 (6), and 81 (40)]. The m/z values 124, 150, 164, 190, 232, and 258 were derived from cleavage between single bonds 4-5, 6-7, 7-8, 9-10, 12-13, and 14-15, as numbered from the carboxyl group. The m/z values 110, 150, 164, 204, 218, and 272 derived from the cleavage of single bonds between the α and β positions from the double bonds were detected. On the basis of the results of MS analyses, UK1 was identified as the geometric isomers of 5,8,13-20:3.

¹H-NMR analysis also suggested that UK1 is an isomer of 20:3 (see Fig. 2). The signal intensity of L (5.36 ppm, m, 6H) indicates the existence of three double bonds in UK1. The sequence of the protons from the methyl end of the molecule was deduced A, B, E, L, L, J, L, L, G, C, F, L, L, H, D, and I or A, B, E, L, L, F, C, G, L, L, J, L, L, H, D, and I based on the pattern of crosspeaks in COSY analysis (see Fig. 2B). The sequence was confirmed as the latter one by the appearance of a crosspeak between J and H, but not J and E, in TOCSY analysis (see Fig. 2C). Furthermore, NOESY analysis was carried out to identify the geometric configurations of double bonds. The positive crosspeaks appeared between G and J, and H and J, indicating that the two double bonds of the Δ5 and Δ8 positions are in the cis configuration, whereas no positive crosspeak appeared between E and F, indicating that the Δ13 position is in the trans configuration (see Fig. 3A). On the basis of the results of the above spectral analyses, UK1 was identified as cis5,cis-8,trans-13-20:3 (see Fig. 2A).

Fig. 2.

¹H-NMR analysis of UK1 and structure of UK1 identified. A: Structure of methyl ester of UK1. B: COSY spectrum of the methyl ester of UK1. C: TOCSY spectrum of the methyl ester of UK1.

Fig. 3.

NOESY spectra of the methyl esters of UK1 (A) and UK2 (B). The negative diagonal peaks are denoted in blue. The positive Nuclear Overhauser effect crosspeaks are denoted in red.

Identification of the newly generated FA in a culture of C. bifermentans JCM 1386 with EPA

When the lipids extracted from the medium after cultivation of C. bifermentans JCM 1386 with EPA were analyzed by thin-layer chromatography, almost all lipids were present in the free form (data not shown). After complete esterification of the free-form FA products, the resulting methyl esters were isolated and used for structural analysis. The mass spectrum of the isolated methyl ester of UK2 exhibited a molecular mass of m/z 318. This result suggested that UK2 is C₂₀ PUFA containing four double bonds. The molecular ion peak ([M+Na]⁺, 341) obtained by FAB-MS analysis (FAB⁺) of the methyl ester of UK2 was fragmented again by MS-MS [m/z (FAB⁺, 8.00 kV), 326 (4), 312 (1), 311 (1), 286 (2), 272 (6), 271 (11), 258 (1), 257 (1), 232 (2), 218 (2), 217 (22), 204 (18), 190 (1), 164 (22), 150 (2), 149 (7), 124 (5), 110 (10), 109 (57), 96 (100), 82 (4), and 81 (28)]. The m/z values 124, 150, 164, 190, 232, 258, 286, and 312 were derived from the cleavage of single bonds 4-5, 6-7, 7-8, 9-10, 12-13, 14-15, 16-17, and 18-19 as numbered from carboxyl group. The m/z values 110, 150, 164, 204, 218, 272, and 326 derived from the cleavage of single bonds between the α and β positions from the double bonds were detected. On the basis of the results of MS analyses, UK2 was identified as the geometric isomers of 5,8,13,17-20:4. ¹H-NMR analysis also suggested that UK2 is an isomer of 20:4 (Fig. 4). The signal intensities of J (5.35 ppm, m, 6H) and K (5.42 ppm, m, 2H) indicate the existence of four double bonds in UK2. The sequence of the protons from the methyl end of the molecule was deduced A, E, J, J, F, E, K, K, D, B, E, J, J, H, J, J, F, C, and G based on the integration of COSY and TOCSY analyses (see Fig. 4B, C). NOESY spectrum revealed that the positive crosspeaks appeared between F and H, H and E, and F and E, and no positive crosspeak appeared between D and E, indicating that the three double bonds of the Δ5, Δ8, and Δ17 position are all in cis configuration, and that the double bond of the Δ13 position is in the trans configuration (see Fig. 3B). On the basis of the results of the previous spectral analyses, UK2 was identified as cis5,cis-8,trans-13,cis-17-20:4 (see Fig. 4A).

Fig. 4.

¹H-NMR analysis of UK2 and structure of UK2 identified. A: Structure of methyl ester of UK2. B: COSY spectrum of the methyl ester of UK2. C: TOCSY spectrum of the methyl ester of UK2.

Effects of AA and EPA concentration in the medium on their transformation by C. bifermentans JCM 1386

Effects of AA and EPA concentration on their transformation by C. bifermentans JCM1386 were investigated (see Fig. 5). When various concentrations of AA were added to the medium, the amount of UK1 production increased with increasing concentration of AA up to 0.42 mg/ml, giving a yield of 57% (0.24 mg/ml) against the added AA (0.42 mg/ml) (see Fig. 5A). When various concentrations of EPA were added to the medium, the amount of UK2 production increased with increasing concentration of EPA up to 0.18 mg/ml, giving a yield of 67% (0.12 mg/ml) against the added EPA (0.18 mg/ml) (see Fig. 5B). However, C. bifermentans JCM 1386 no longer produced UK2 when more than 0.24 mg/ml EPA was added.

Fig. 5.

Effects of FA concentration for medium on FA transformation by C. bifermentans JCM 1386. AA (A) and EPA (B). Cultivations were carried out with different concentrations of AA or EPA.

Substrate specificity of PUFA transformation by C. bifermentans JCM 1386

To examine the substrate specificity of PUFA transformation during the cultivation of C. bifermentans JCM 1386, FFAs of LA, α-linolenic acid, γ-linolenic acid, dihomo-γ-linolenic acid, AA, EPA, and DHA were added to the medium (see Fig. 6). C. bifermentans JCM 1386 could convert LA, AA, EPA, α-linolenic acid, γ-linolenic acid, and dihomo-γ-linolenic acid, but not DHA.

Fig. 6.

Transformation of PUFAs by C. bifermentans JCM 1386.

The GC-MS analysis of the products obtained from α-linolenic acid, γ-linolenic acid, and dihomo-γ-linolenic acid

The products from α-linolenic acid, γ-linolenic acid, and dihomo-γ-linolenic acid were analyzed by GC-MS (see Fig. 7). The spectrum of pyrrolidide derivative of the product from α-linolenic acid showed a molecular mass of m/z 333 and gaps of 26 amu between m/z 224 and 250, and between m/z 278 and 304, indicating that this is a C₁₈ PUFA with double bonds at the ω3 and ω7 positions (11,15-18:2) (see Fig. 7A). The pyrrolidide derivative of the product from γ-linolenic acid showed a molecular mass of m/z 333 and gaps of 26 amu between m/z 154 and 180, and between m/z 222 and 248, indicating that the product is a C₁₈ PUFA with double bonds at the ω7 and ω12 positions (6,11-18:2) (Fig. 7B). The pyrrolidide derivative of the product from dihomo-γ-linolenic acid showed a molecular mass of m/z 361 and gaps of 26 amu between m/z 182 and 208, and between m/z 250 and 276, indicating that the product is a C₂₀ PUFA with double bonds at the ω7 and ω12 positions (8,13-20:2) (Fig. 7C). Thus, C. bifermentans JCM 1386 could convert C₁₈ and C₂₀ PUFAs with double bonds at the ω6 and ω9 positions into their corresponding NMIFAs by C. bifermentans JCM 1386 (see Fig. 8).

Fig. 7.

GC-MS spectra of pyrrolidide derivatives of the products from α-linolenic acid (A), γ-linolenic acid (B), and dihomo-γ-linolenic acid (C). The deduced structures are shown above the spectra.

Fig. 8.

Pathway of PUFA transformation during cultivation of C. bifermentans JCM 1386.

DISCUSSION

The studies on PUFA conversion by anaerobic bacteria have been done with the primary aim to improve the quality of ruminant products such as milk or meat. In the course of these studies, numerous PUFA-transforming bacteria, such as Butyrivibrio fibrisolvens (4), Lactobacillus plantarum (20–23), and Bifidobacterium breve (19), have been isolated, and their metabolic pathways of C₁₈ PUFAs, such as LA and α-linolenic acid, have been revealed. However, the ability to transform C₂₀ and C₂₂ PUFAs has not been studied in detail, although there have been several reports that EPA and DHA are hydrogenated in the rumen in vivo (26) and disappear during incubations in vitro with mixed ruminal microorganisms (27, 28).

In this study, we found that C. bifermentans JCM 1386 could convert AA and EPA into cis-5,cis-8,trans-13-20:3 and cis-5,cis-8,trans-13,cis-17-20:4, respectively, which are NMIFAs with a trans double bond at the ω7 position (see Figs. 5, 8). This is the first report of the isolation of the bacterium transforming C₂₀ PUFAs into corresponding NMIFAs. Considering that similar reactions were observed with LA (see Fig. 1C), this strain can convert two cis double bonds at the ω6 and ω9 positions in PUFAs into a trans double bond at the ω7 position to generate the trans FAs regardless of the existence of double bonds at other positions. In addition, similar reactions were also observed of other C₁₈ and C₂₀ free PUFAs (α-linolenic acid, γ-linolenic acid, and dihomo-γ-linolenic acid) (see Fig. 6). They might be converted into the corresponding NMIFAs with a trans double bond at the ω7 position. However, C. bifermentans JCM 1386 could not convert DHA, indicating that C₂₂ PUFAs might not be a substrate for this strain. Thus, we succeeded in the production of various C₁₈ and C₂₀ NMIFAs with a trans double bond at the ω7 position through the biohydrogenation by C. bifermentans JCM 1386.

NMIFAs are a class of PUFAs that has received attention because of their unique structure and physiological activity, and they have often been found in plant oils. Pinolenic acid (cis-5,cis-9,cis-12-18:3) and columbinic acid (trans-5,cis-9,cis-12-18:3) are C₁₈ NMIFAs that were found in Pinus koraiensis and Aquilegia hybrida, respectively (8, 9). They are isomers of γ-linolenic acid and show various effects, such as the reduction of platelet aggregation by prostacyclin production, attenuation of the elevation of blood pressure, LDL lowering, and essential FA activity (9, 11, 12). Podocarpic acid (cis-5,cis-11,cis-14-20:3) is a C₂₀ NMIFA that was found in Platycladus orientalis oil (10). It has been reported to show a reduction in the AA concentration in the phosphatidylinositol fraction of rat liver (13), which functions in signal transduction, such as in the phospholipase C-signaling pathway (13, 29). Considering that PUFAs often show an isomer-specific function, novel NMIFAs are expected to show novel interesting physiological effects. Interestingly, several natural plant oils have a high content of PUFAs with a double bond at the ω7 position (30), and the biohydrogenation of PUFAs often produces PUFAs with a double bond at the ω7 position, such as VA (6, 7). These observations enable us to consider that a double bond at the ω7 position may become a key factor for a biological function. In this context, various C₁₈ and C₂₀ NMIFAs obtained in this study could be worthwhile. It is also noted that these NMIFAs were obtained in high yield (∼60%). Therefore, this study could serve to open up the development of novel methods in the preparation of these rare possibly bioactive PUFAs.

Lipid metabolism by anaerobic bacteria is an attractive research area from the viewpoint of the role of the gut microbiota in relation to health of the host. Interestingly, obesity induced by a high-fat diet has been suggested to be associated with alterations of gut microbiota composition (31, 32). Dietary fats are metabolized by gut microbiota as well as by the host. It is noted that the biohydrogenation of FAs might function as a detoxification mechanism in bacteria, and PUFAs especially are more toxic than saturated FAs (18, 33). This suggests that the capacity for the biohydrogenation of PUFAs might relate to the survival of gut bacteria when dietary intake of PUFAs is high. In addition, our recent research suggested the possibility that lipid metabolism by gut microbiota affects the health of the host by modifying FA composition (23). Therefore, our evidence-based studies on lipid metabolism by gut bacteria, including C. bifermentans (in this study) and Lactobacillus (21–23, 34), should serve to maintain and improve the health of the host.