CLA biosynthesis by bifidobacteria |
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| 申请号 | EP01113962.3 | 申请日 | 2001-06-08 | 公开(公告)号 | EP1264893A1 | 公开(公告)日 | 2002-12-11 |
| 申请人 | Teagasc Dairy Products Research Centre; | 发明人 | The designation of the inventor has not yet been filed; | ||||
| 摘要 | Nineteen strains of Lactobacillus , 2 strains of Lactococcus , 1 strain of Pediococcus, 4 strains of Propionibacterium and 23 strains of Bifidobacterium were screened for their ability to produce conjugated linoleic acid (CLA) from linoleic acid. Of these, 7 strains of Bifidobacterium , as well as 2 strains of Propionibacterium produced the cis-9, trans -11 CLA isomer from linoleic acid. In contrast, strains of Lactobacillus, Lactococcus and Pediococcus lacked the ability to synthesise CLA. CLA ( cis -9, trans- 11 isomer) production by the genus Bifidobacterium was shown to exhibit considerable interspecies variation, with B . breve and B . dentium being the most efficient producers among the strains tested, yielding up to 65 % conversion of linoleic acid to CLA at linoleic acid concentrations of 0.2-1.0 mg/ml in MRS medium. The growth of B. breve strains was inhibited by increasing concentrations of linoleic acid. Viability of B. breve 2257 was unaffected in the presence of up to 0.5 mg/ml linoleic acid for 48 h but was dramatically reduced to 1.5% survival at 1 mg/ml linoleic acid. However, viability of the B . breve strains NCFB2258, NCTC 11815, NCIMB 8815 and NCIMB 8807 was reduced to | ||||||
| 权利要求 | |||||||
| 说明书全文 | Conjugated linoleic acid (CLA) refers to a mixture of positional and geometric isomers of linoleic acid, and has gained considerable attention in recent years because of the many beneficial effects attributed to the cis-9, trans-11 and trans-10, cis-12 isomers, in particular. These include anticarcinogenic activity, antiatherogenic activity, the ability to reduce the catabolic effects of immune stimulation, the ability to enhance growth promotion and the ability to reduce body fat (Martin and Banni, 1998 for review, and references therein). Of the individual isomers of CLA, cis-9, trans-11-octadecadienoic acid has been implicated as the most biologically active because it is the predominant isomer incorporated into the phospholipids of cell membranes, liver phospholipids and triglycerides (Kramer et al., 1998). This isomer is also the predominant dietary form of CLA, obtained from fats derived from ruminant animals, including milk, dairy products and meat (Chin et al., 1992, O'Shea et al., 2000). In addition to the ability of certain rumen-derived strains, including Butyrivibrio fibrisolvens to form CLA from dietary linoleic acid (Kepler and Tove, 1967), it has also been shown that certain cultures used in food fermentations possess the ability to generate cis-9, trans-11 CLA. Strains of the intestinal flora in rats (Chin et al., 1994), two strains of Propionibacterium freudenreichii spp.. freudenreichii and one strain of P. freudenreichii subsp. shermanii (Jiang et al., 1998), and six lactic cultures, including L. acidophilus (Lin et al., 1999) have been shown to possess this capability. In this study, we assessed a collection of strains, many which are human intestinal isolates (previously isolated from the human GIT) with probiotic potential, for ability to form the cis-9, trans-11 CLA isomer, using linoleic acid as the substrate. The 19 strains of Lactobacillus, 2 strains of Lactococcus, 1 strain of Pediococcus, 4 strains of Propionibacterium and 19 strains of Bifidobacterium used in this study are listed in Table 1. The UCC strains used in the study were previously isolated from the human gastrointestinal tract (GIT) and obtained from University College Cork, Ireland under a restricted materials transfer agreement. The Lactobacilli, Pediococci and Bifidobacterium strains were cultured in MRS (Difco Laboratories, Detroit, MI, USA) under anaerobic conditions (anaerobic jars with 'Anaerocult A' gas packs; Merck, Darmstadt, Germany) and 1.5 % (w/v) agar (Oxoid Ltd. Basingstoke, Hampshire, UK) was included for plating. Pediococci, Lb.reuteri NCIMB 702655, Lb. reuteri NCIMB 7025656 and Lb. reuteri DSM 20016 were routinely cultured at 30°C and the remaining Lactobacillus strains were cultured at 37°C for 24 h. For Bifidobacterium, 0.05 % (w/v) L-cysteine hydrochloride (98 % pure, Sigma Chemical Co. St. Louis, MO, USA) was added to the medium and cultures were grown for 48 h at 37°C under anaerobic conditions. Lactococcus strains were cultured in MRS under aerobic conditions at 30°C for 24 h. The Propionibacterium strains were cultured in sodium lactate medium (SLM, Malik et al. 1968) at 20°C for 72 h under anaerobic conditions. Total viable counts were determined by pour plating of 10-fold serial dilutions in Maximum Recovery Diluent (Oxoid), using MRS agar for lactobacilli and MRS agar with 0.05 % (w/v) cysteine for bifidobacteria. Prior to examination of the strains for CLA production, each was subcultured twice in MRS broth (supplemented with cysteine, 0.05 % w/v for Bifidobacterium) for 48 h, using a 1 % innoculum. All strains were then cultured in MRS broth (supplemented with cysteine, 0.05 % w/v for Bifidobacterium), spiked with different concentrations of free linoleic acid (LA: cis-9, cis-12-octadecadienoic acid, 99 % pure, Sigma Chemical Co.). This was added as a 30 mg/ml stock solution of linoleic acid in 2 % (v/v) Tween 80 (polyoxyethylene sorbitan mono-oleate; Merck-Schuchardt, Germany), which was previously sterile-filtered through a 0.45 µm Minisart filter (Sartφrius AG, Germany). The strains were inoculated to a density of 106 cfu/ml in free linoleic acid-containing MRS media and incubated for their respective times and temperatures (described above). Following incubation, 5 ml of the cultures were centrifuged at 960 × g for 5 min at room temperature (Sanyo MISTRAL 2000R Centrifuge). The fatty acid composition of the resulting supernatant was analysed as follows. Initially, C13:0 (tridecanoic acid, 99 % pure, Sigma Chemical Co.) was added to 4 ml of the resulting supernatant, as an internal standard at a concentration of 0.25 x the initial linoleic acid concentration and lipid extraction was performed as follows. Two milliliters of isopropanol (99 % purity, Alkem Chemicals Ltd., Cork, Ireland) was added to the supernatant and the samples were vortexed for 30 sec. A total of 4.5 ml hexane (99 % purity, LabScan Ltd., Dublin, Ireland) was added to this and the mixture plased on a shaking platform for 3 min before centrifugation at 960 × g for 5 min at room temperature. The supernatant (the hexane layer containing the lipids) was removed and the procedure was repeated twice. The hexane layers were pooled and stored at -20 °C prior to preparation of fatty acid methyl esters (FAME) for gas liquid chromatographic (GLC) analysis. The lipid extracts in hexane were analysed by GLC following acid-catalyzed methylation as described previously (Stanton et al., 1997). Free fatty acids in oils such as sunflower and soybean oils were calculated as the difference between fatty acid concentrations obtained following acid and base catalyzed methylation, performed using 2 N methanolic KOH (Sigma Chemical Co.) at room temperature. The GLC was performed with reference to the internal standard C13:0. Separation of the FAME was performed on a Chrompack CP Sil 88 column (Chrompack, Middleburg, The Netherlands, 100 m x 0.25 mm i.d., 0.20 µm film thickness), using helium as carrier gas at a pressure of 37 psi. The injector temperature was held isothermally at 225°C for 10 min and the detector temperature was 250°C. The column oven was held at an initial temperature of 140°C for 8 min and then programmed at an increase of 8.5°C/min to a final temperature of 200°C, which was held for 41 min. Collected data were recorded and analyzed on a Minichrom PC system (VG Data System, Manchester, UK). The cis-9, trans-11 CLA isomer was identified by retention time with reference to a CLA mix (Nu-Chek- Prep. Inc., Elysian, MN). The percentage conversion to CLA and the remaining linoleic acid in the broth were calculated by dividing the amount of CLA and linoleic acid present in the broth after inoculation and incubation with the various cultures used with the amount of linoleic acid present in the spiked broth before incubation. B. breve NCFB 2258 was further investigated for ability to utilise triglyceride bound linoleic acid as substrate for CLA production. B. breve NCFB 2258 was inoculated from a fully grown culture into MRS broth with added cysteine (0.05 %) and trilinolein (C18:2, cis-9, cis-12, 99 % pure, Sigma Chemical Co.), soybean oil and sunflower oil (purchased from a local grocery store) containing known linoleic acid concentrations. The triglyceride mixtures were sterile-filtered through 0.45 µm Minisart filters and added as 5 mg/ml aqueous solutions in 2.5 % (v/v) Tween 80. Substantial vortexing was required to dissolve the fat particles. The volume of the triglyceride stock solutions added was calculated to give a final concentration of 0.2 mg linoleic acid/ml of broth. B. breve 2258 was inoculated into MRS broth in the presence of the triglyceride substrates under anaerobic conditions at 37°C and incubated for 48 h. Throughout the screening programme, the two Propionibacterium strains, Propionibacterium freudenreichii subsp. freudenreichii Propioni 6 (PFF-6) and Propionibacterium freudenreichii spp. shermanii 9093 (PFS), previously reported to synthesise CLA from linoleic acid (Jiang et al., 1998) were used as positive controls. The CLA biosynthetic assay was set up, with the positive controls in SLM broth, using similar incubation conditions as described previously (Jiang et al., 1998). GLC analysis confirmed that the two strains did convert free linoleic acid to the cis-9, trans-11 CLA isomer following incubation at 20 ºC for 72 h, using CRM (certified reference material) 164 and CLA standards for fatty acid identification (data not shown). However, the levels of CLA produced by the two strains of Propionibacterium were lower than that reported previously by Jiang et al. (1998), producing ∼ 60 µg/ml of CLA in comparison with 111.8 µg/ ml previously reported by Jiang et al. (1998), using 0.5 mg/ml linoleic acid as substrate. In addition, we found that the amount of linoleic acid remaining in the media following incubation with the PFS strain was ∼ 50 µg/ml, compared with 289.5 µg/ml reported previously (Jiang et al., 1998). The variation in these data may be a result of differences in the numbers of cells present during incubation, and possibly as a result of the different procedures used for fatty acid extraction and methylation. Three strains of Propionibacterium were then examined for their ability to produce CLA. These were Propionibacterium acidi propionici NCFB 5633, Propionibacterium freudenreichii spp. shermanii LMG 16424 and Propionibacterium freudenreichii spp. shermanii JS (Laboratorium Visby, Tonder, Denmark). The strains were incubated in the presence of 0.5 mg/ml linoleic acid using the same growth conditions and media as described above. The two Propionibacterium shermanii strains synthesized CLA in MRS media while Propionibacterium acidi propionici did not produce any detectable CLA product (Table 1). The amounts of CLA produced by the two Propionibacterium shermanii strains (12-14 µg/ml) were low however, compared with 60 mg/ml produced by PFS strain in this study. A variety of different strains of lactobacilli, lactococci, and pediococci, obtained from various sources (Table 2) were tested for ability to produce CLA from linoleic acid. These strains included a number of probiotic strains including Lactobacillus salivarius UCC 43310, Lactobacillus salivarius UCC 43348, Lactobacillus paracasei UCC 43338, Lactobacillus paracasei UCC 43364, Lactobacillus paracasei UCC 42319, five strains of Lb. reuteri and the bacteriocin producing Lactcoccus lactis DPC 3147 strain (Ryan et al., 1996). The strains were inoculated into MRS to a density of ∼106 cfu/ml and incubated under respective conditions as described above, in the presence of linoleic acid concentrations of 0.5 to 3.0 mg/ml. The ability of the strains to grow in the different linoleic acid concentrations varied considerably. Good growth of all five Lb. reuterii strains occurred at linoleic acid concentrations up to 1 mg/ml, while at 3 mg/ml, the growth of strains NCIMB 701359 and NCIMB 70256 was completely inhibited (data not shown). At all linoleic acid concentrations investigated, none of the Lb. reuteri strains investigated produced CLA in detectable quantities. Lactobacillus helveticus NCDO 1244 exhibited no growth in 0.5 mg/ml linoleic acid while Lactobacillus leichmanii NCDO 302 showed good growth in the presence of high linoleic acid concetrations (3 mg/ml). However, none of the strains produced CLA from linoleic acid (between 0.5 to 3 mg/ml) under the conditions used. A variety of bifidobacteria obtained from a number of sources (Table 3) were screened for CLA production. Since free linoleic acid was found to be inhibitory to the growth of bifidobacteria strains, the minimum inhibitory concentration of linoleic acid for the B. breve strains was initially determined. This involved inoculation (1 % from grown cultures) of the Bifidobacterium strains into MRS containing free linoleic acid concentrations ranging from 0.2 to 1.5 mg/ml and incubation under anaerobic conditions at 37°C for 48 h. The pH of the media remained unchanged following the addition of the linoleic acid substrate in this concentration range at pH ∼ 6.1. Viable bifidobacteria were enumerated at time zero and following 48 h incubation in the presence of the linoleic acid substrate. Viability of B. breve 2257 was unaffecetd in the presence of linoleic acid at concentrations up to 0.5 mg/ml. However, viability was dramatically reduced at 1.0 mg/ml and only 1.5 % survival was observed. In contrast the survival of strains B. breve 2258, 8807, 8815 and 11815 was reduced to <60 % at linoleic acid concentrations of 0.2 mg/ml and higher. The bifidobacteria strains were then screened for CLA production from linoleic acid substrate at a concentration of 0.5 mg/ml, using the incubation conditions described above. A number of the Bifidobacterium strains investigated produced CLA following incubation in MRS containing 0.5 mg/ml linoleic acid, and the results from this screening program showed that there was considerable inter-species variation in the ability of bifidobacteria to produce CLA (Table 3). All 5 strains of Bifidobacterium breve species examined tested positive for CLA production with four of these strains producing more than 60 µg/ml CLA, while strain B. breve NCFB 2257 produced 15 µg/ml under these conditions. In addition, B. dentium NCFB 2243 was an efficient CLA producer, also yielding >60 µg/mg CLA (Table 3), while B. pseudocatenulatum NCIMB 8811 produced >15 µg/ml under the experimental conditions employed. Among the other bifidobacteria species investigated, 3 strains of B .adolescentis, 2 strains of B. longum and 1 strain each of B. angulatum, B. bifidum and B. lactis were all negative for CLA production (Table 3). The exact role of biohydrogenation in the metabolic environment of the bacterial cell is unclear. In the study by Jiang et al. (1998), strains which were able to produce CLA were those inhibited by the presence of free linoleic acid, but a positive correlation between CLA production and tolerance to linoleic acid was observe within the three CLA producing strains of propionibacteria. This suggests that the conversion of linoleic acid to CLA is a detoxification mechanism for the bacterial cell. This is supported by the fact that the antimicrobial activity of fatty acids with double bonds of cis configuration is stronger than that of trans (Kabara, 1983). The most efficient CLA producers were strains B. breve 8815 and 2258 at linoleic acid concentration of 1.0 mg/ml. The strains B. breve 8815, 2258 and 2257 were present at less than 104 cells/ml at the highest linoleic acid concentration (1.5 mg/ml) and were not analysed for CLA conversion. B. breve NCFB 2258 converted ∼50 % of the added linoleic acid to CLA at 0.2 and 0.5 mg/ml linoleic acid concentrations. The ability of B. breve NCFB 2258 strain to utilise triglyceride bound linoleic acid as substrate for CLA production, using trilinolein (C18:2, cis-9, cis-12), sunflower and soybean oils was also investigated. The B. breve NCFB 2258 strain was found to be negative for ability to utilise triglyceride bound linoleic acid as substrate at 0.2 mg/ml linoleic acid for CLA production (data not shown), from trilinolein, sunflower and soybean oils. These data are in agreement with a previous study which showed that of 61 rumen isolates with ability to produce CLA, none utilised triglyceride bound linoleic acid (Fujimoto et al., 1993). In addition, trilinolein did not inhibit the growth of B. breve 2258 to the same extent as similar concentrations of free linoleic acid (data not shown). This indicates that linoleic acid in the free fatty acid form is more toxic to bifidobacteria than triglyceride-bound linoleic acid. B. breve 2258 and P. freudenreichii shermanii 9093 were screened for their ability to produce CLA. The strains were incubated in duplicate in the presence of 0.5 mg/ml linoleic acid (LA) and CLA (the pure cis-9, trans-11 isomer), respectively, using the same growth conditions and media as described above. In order to compare the fatty acid composition, control cultures without added LA and CLA were also incubated using the same conditions. The propionibacteria strain used in this study was previously reported to synthezise CLA from LA (Jiang et al., 1998) and therefore used as a positive control. However, in this study, that result was not reproducible since the strain was clearly inhibited in the presence of LA and CLA and hence grew very poorly. No CLA production from that strain was therefore detected and the results from the GLC analysis are not presented here. After separation (by centrifugation) of B. breve 2258 cells from the supernatant following incubation for 48 h, followed by lipid extraction and methylation, the fatty acid composition of both the cells (pellets) and the supernatant were analysed using GLC . GLC analysis confirmed that B. breve 2258 converted LA to CLA. Of the added 0.5 mg/ml, only 0.27 mg/ml (54 %) remained in the supernatant (Fig. 5), while the remainder (46 %) was converted to other fatty acids, preferentially the cis-9, trans-11 CLA isomer followed by cis-9-C 18:1 (oleic acid) and a peak of unidentified fatty acids, which most likely is another CLA isomer, although confirmation of this requires further study. The amount of cis-9, trans-11 CLA produced was 0.136 mg/ml, and the unidentified fatty acids accounted for 0.03 mg/ml. The amount of these two fatty acids present in the control supernatant was negligible (Fig. 5) There was also a substantial increase of cis-9-C 18:1 (oleic acid) (64.8 % compared with the control supernatant), which indicates that B. breve 2258 harbours a CLA reductase enzyme that hydrogenates the trans-11 double bond of cis-9, trans-11 CLA. Compared to the control culture there was 64,8 % more stearic acid in the LA added supernatant. Smaller increases were observed also in the concentrations of trans-11 C 18:1 (vaccenic acid) (30.3 %) and C 18:0 (stearic acid) (17.5 %) compared with the control supernatant, suggesting that other hydrogenating enzymes may be involved. The fatty acid composition of the membranes from the cultures (pellet) grown in MRS medium with 0.5 mg/ml LA was also analysed and compared with the control cultures (Fig. 9). Results are expressed as mg fatty acids from cells/ml fully grown culture (see section 3.4.2) The fatty acid concentration in the pellets in mg/ml is lower than that of the supernatant and therefore are not directly comparable. Results from the GLC analysis show that CLA was incorporated in the cell membranes, whereas the control culture contained negligible CLA. The cis-9, trans-11 isomer was the most abundant CLA isomer and accounted for 0.012 mg/ml, which represents 70 % of the total CLA isomers (assuming that the unidentified fatty acid peak was other CLA isomers). The content of the cis-9-C 18:1 (oleic acid) was increased (by 271 % compared with controls) in the membranes of B. breve 2258 cells incubated in LA (0.5 mg/ml), indicating the presence of a CLA reductase, which was capable of reducing the unsaturated trans-11 bond in CLA in B. breve 2258. The trans-11 C 18:1 (vaccenic acid) content of the cell membranes wes reduced (over 4-fold) in the LA treated cells compared with the control cells. As seen in the supernatant, a small increase of 28 % in C 18:0 (stearic acid) was detected in the membranes of the LA treated cells compared with the controls (Fig. 9). In order to evaluate if B. breve 2258 possesses enzymes other than the putative linoleic acid isomerase, involved in the biohydrogenation of linoleic acid, studies were undertaken using cis-9, trans-11 CLA as the substrate. Strain B. breve 2258 was inoculated in MRS containing 0.5 mg/ml of the pure cis-9, trans-11 CLA isomer (Matreya Inc. PA, USA) and incubated for 24 h at 37°C. Following incubation in the presence CLA (0.5 mg/ml), only 0.32 mg/ml (65 %) remained in the supernatant (Fig. 9) with the remaining 35 % converted to other fatty acids. The most predominantly formed fatty acid formed corresponded to the unidentified peak, observed following incubation with LA and eluted at 43 mins (Figs. 6 and 10). We propose this is another CLA isomer which was present at 0.12 mg/ml (71 % of the cis-9, trans-11 CLA peak). Oleic acid was also formed in the supernatant by B. breve 2258 following incubation with cis-9, trans-11 CLA with an increase of 85.5 % compared with the control supernatant. Smaller increases were also observed in the concentration of trans-11-C 18:1 (vaccenic acid) (74.5 % compared to control supernatant) and C 18:0 (stearic acid) (23.9 % compared to control supernatant). The lipid composition of the membrane following incubation of B. breve 2258 in cis-9, trans-11 CLA was also compared with control cells incubated in the absence of CLA. The fatty acid composition of the membranes from cultures inoculated in MRS containing 0.5 mg/ml of the pure cis-9, trans-11 CLA isomer shows that the membrane composition changed compared with the control. Cis-9, trans-11 CLA was incorporated into the membrane of the culture grown in the presence of CLA (0.03 mg/ml) compared with the culture, grown in the absence of CLA, which contained no CLA (Fig. 12.). The same unidentified fatty acid as observed in the supernatant was also present in the membrane following incubation with cis-9, trans-11 CLA (0.012 mg/ml). Verification of the identity of this fatty acid will be important, as clearly the bacterial cell has the capacity to convert cis-9, trans-11 CLA to this compound. the possibility is that it is another CLA isomer, although this was not verified in this study, nor was its exact identity confirmed. This could be accomplished using co-eluting fatty acid standards analyzed by GLC or by GCMS. An increase of 54.8 % in the cis-9-C 18:1 oleic acid membrane content was obtained following incubation of B. breve 2258 in CLA (0.5 mg/ml). This amount of oleic acid in the membranes, formed relative to the control, was greater in the LA treated cells (2.7-fold increase) than the CLA treated cells. As observed in the cell membranes obtained following incubation of B. breve 2258 in LA (0.5 mg/ml), the trans-11-C 18:1 vaccenic acid content of membranes was lower in the cells incubated with cis-9, trans-11 CLA than the control pellets (5.8-fold greater in the control). Only a very small increase was obtained in the content of C 18:0 stearic acid in the membranes of CLA treated cells compared with controls. The GLC analysis confirmed that B. breve 2258 converted LA to CLA and that a significant amount of another, as yet, unidentified isomer was also formed and the data also, it also indicates that a further biohydrogenation of cis-9, trans-11 CLA to C 18:1 isomers, preferably the cis-9-C 18:1 isomer occurs as a result of incubation with B. breve 2258 strain. Since also a small increase of C 18:0 was detected in the chromatogram, it is possible that additional enzymes are involved, but whether this activity is significant is unclear. The increase in saturation was obtained in both the supernatant and the bacterial pellets. Also when the pure CLA isomer was incubated with B. breve 2258 it was further hydrogenated to more saturated fatty acids, primarily cis-9-C 18:1. This may support the theory that incorporation of a trans fatty acids instead of cis, and saturation or trans conversion of cis double bonds is a strategy for the bacterial cell to counteract for the increased fluidity that occurs when LA and the cis-9, trans-11 CLA isomer (which has a cis bond) is interfering with the membranes which leads to expansion of membrane, elevation of membrane permeability and impairment of membrane functions (Junker and Ramos, 1999; Weber et al., 1994). Interestingly in this study, the differences in fatty acid composition when adding LA and CLA respectively, to the supernatant and pellets, is not very significant. When adding the pure cis-9, trans-11 CLA isomer to the supernatant it is converted to a great extent to other CLA isomer, which is not the case in the LA added supernatant. 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