Conjugated linoleic and linolenic acid production ...

International Journal of Food Microbiology 155 (2012) 234–240

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International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro

Conjugated linoleic and linolenic acid production kinetics by bifidobacteria differ among strains Lara Gorissen a, b, Luc De Vuyst b, Katleen Raes c, Stefaan De Smet a, Frédéric Leroy b,⁎ a

Laboratory for Animal Nutrition and Animal Product Quality, Department of Animal Production, Ghent University, Proefhoevestraat 10, B-9090 Melle, Belgium Research Group of Industrial Microbiology and Food Biotechnology (IMDO), Faculty of Sciences and Bioengineering Sciences, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium c Research Group EnBiChem, University College West-Flanders, Graaf Karel de Goedelaan 5, B-8500 Kortrijk, Belgium b

a r t i c l e

i n f o

Article history: Received 18 September 2011 Received in revised form 4 January 2012 Accepted 18 February 2012 Available online 23 February 2012 Keywords: Bifidobacteria Conjugated linoleic acid Conjugated linolenic acid Fermentation Kinetics

a b s t r a c t There is great interest in conjugated linoleic acid (CLA) and conjugated linolenic acid (CLNA) isomers because of their supposed health-promoting properties. Therefore, the differences in production kinetics of CLA and CLNA isomers from linoleic acid (LA) and α-linolenic acid (α-LNA), respectively, by bifidobacteria were investigated. Laboratory fermentations, supplemented with LA or α-LNA in the fermentation medium, were performed with Bifidobacterium bifidum LMG 10645, Bifidobacterium breve LMG 11040, B. breve LMG 11084, B. breve LMG 11613, B. breve LMG 13194, and Bifidobacterium pseudolongum subsp. pseudolongum LMG 11595. Conversion of LA and α-LNA to CLA and CLNA isomers, respectively, started immediately upon addition of the substrate fatty acids. During the active growth phase, the c9, t11-CLA isomer and the putative c9, t11, c15-CLNA isomer were formed. Further fermentation resulted in a reduction in the concentration of c9, t11-CLA and c9, t11, c15-CLNA and the subsequent production of the t9, t11-CLA isomer and the putative t9, t11, c15-CLNA isomer, respectively. Modelling of the growth and metabolite data indicated differences in production kinetics among the strains. Some strains displayed a high specific conversion of LA and α-LNA despite poor growth, whereas other strains grew well but displayed lower conversion. Production of specific CLA and CLNA isomers by bifidobacteria holds potential for the production of functional foods and could contribute to their probiotic properties. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Conjugated linoleic acid (CLA) and conjugated linolenic acid (CLNA) refer to a mixture of positional and geometrical isomers of linoleic acid (LA; c9, c12-C18:2) and α-linolenic acid (α-LNA; c9, c12, c15-C18:3), respectively, containing one or more conjugated double bonds. Dietary CLA and CLNA isomers are of interest because of their health effects (Benjamin and Spener, 2009; Bhattacharya et al., 2006; de Carvalho et al., 2010; Park, 2009; Tricon et al., 2005; Wahle et al., 2004). Research has focused on two CLA isomers, namely c9, t11- and t10, c12-CLA. However, these isomers show distinct biological activities and it cannot be excluded that some have adverse health effects as well, necessitating the identification of isomerspecific effects (Risérus et al., 2002; Tholstrup et al., 2008; Tricon et al., 2004). Furthermore, research has demonstrated that certain CLNA isomers, such as c9, t11, t13- and c9, t11, c13-CLNA, exhibit certain health-promoting properties in vitro, mainly anti-carcinogenic properties (Hennessy et al., 2011; Kohno et al., 2004; Suzuki et al., ⁎ Corresponding author at: Pleinlaan 2, B-1050 Brussels, Belgium. Tel.: + 32 2 629 3612; fax: + 32 2 629 2720. E-mail address: fl[email protected] (F. Leroy). 0168-1605/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2012.02.012

2001; Tsuzuki and Kawakami, 2008; Yasui et al., 2006) and effects on body fat mass (Hennessy et al., 2011; Yuan et al., 2009). CLA and CLNA isomers are primarily found in ruminant-derived foods, such as meat and dairy products. These isomers are formed as intermediates by rumen microorganisms during ruminal biohydrogenation of dietary unsaturated fatty acids. In this process, the latter undergo a series of isomerisation and hydrogenation reactions, eventually yielding saturated fatty acids, such as stearic acid. The isomerisation reaction, from LA and α-LNA to CLA and CLNA isomers, respectively, is catalysed by the linoleate isomerase (LAI) enzyme (Kepler and Tove, 1967). All intermediates, including CLA and CLNA isomers, are absorbed to a certain extent into the blood stream and integrated in milk and body fat (Jenkins et al., 2008). Additionally, c9, t11-CLA is produced endogenously in mammary and adipose tissues of both ruminants and humans from vaccenic acid (t11-C18:1) through Δ9-desaturase activity (Griinari et al., 2000; Turpeinen et al., 2002). The CLA content of ruminant meat and milk products varies from around 4–6 mg/g fat (Dhiman et al., 2005; Jiang et al., 1997; Schmid et al., 2006) of which approximately 80% to 90% is the c9, t11-CLA isomer (Chin et al., 1992; Parodi, 1977). Comparatively, the c9, t11, c15-CLNA content of milk is much lower (around 0.3 mg/g fat). The average daily intake of CLA by adults varies from 120 to 400 mg per day

L. Gorissen et al. / International Journal of Food Microbiology 155 (2012) 234–240

(Dhiman et al., 2005), however, including the endogenous conversion of dietary t11-C18:1 by Δ9-desaturase, a median daily CLA intake in humans of 650 mg might be achieved (van Wijlen and Colombani, 2010). On the other hand, a meta-analysis revealed that a dose of 3.2 g CLA per day is optimal for reduction of body fat mass (Whigham et al., 2007), but no distinction has been made between the different isomers. Therefore, enrichment of CLA and CLNA concentrations in dairy products is desirable, either through modification of the diet of ruminants or through the use of an appropriate starter culture in dairy processing. Indeed, several studies have indicated the ability of different genera of bacteria to form mainly c9, t11-CLA from LA. These bacteria, which could be used as starter cultures, include food-grade species of lactic acid bacteria (Alonso et al., 2003; Lin et al., 1999; Van Nieuwenhove et al., 2007), propionibacteria (Jiang et al., 1998), and bifidobacteria (Barrett et al., 2007; Coakley et al., 2003; Hennessy et al., 2009; Oh et al., 2003; Rosberg-Cody et al., 2004; Van Nieuwenhove et al., 2007). Furthermore, bifidobacterial cultures incubated with the c9, t11-CLA isomer are able to generate the t9, t11-CLA isomer, which suggests a conversion mechanism from c9, t11- to 9, t11-CLA (Coakley et al., 2006). Production of CLNA isomers from α-LNA by Bifidobacterium strains has been demonstrated as well (Coakley et al., 2009). However, detailed information on production kinetics of CLA and especially CLNA has not been provided yet. Previously, an intensive screening has revealed that six out of 36 Bifidobacterium strains, i.e. four Bifidobacterium breve strains, a Bifidobacterium bifidum strain, and a Bifidobacterium pseudolongum subsp. pseudolongum strain, are able to convert LA and α-LNA to different CLA and CLNA isomers, respectively (Gorissen et al., 2010). The main CLA and CLNA isomers formed have been identified as c9, t11- and t9, t11-CLA, and the putative c9, t11, c15- and t9, t11, c15-CLNA isomers, respectively. The present study aimed to examine the kinetics of the formation of these CLA and CLNA isomers by bifidobacteria through laboratory fermentations. Such information is required as a first step to assess the potential impact of bifidobacteria on CLA and CLNA formation during food fermentations and, possibly, in the (human) gut. 2. Materials and methods 2.1. Bacterial strains and culture conditions B. bifidum LMG 10645, B. breve LMG 11040, B. breve LMG 11084, B. breve LMG 11613, B. breve LMG 13194, and B. pseudolongum subsp. pseudolongum LMG 11595 were purchased from the BCCM™/LMG Bacteria Collection (Ghent, Belgium). All strains were stored at −80 °C in de Man–Rogosa–Sharpe (MRS) medium (Oxoid, Basingstoke, United Kingdom) supplemented with 0.4 g/l cysteine-HCl (Merck, Darmstadt, Germany), referred to as Cys-MRS, and 25% (v/v) glycerol as a cryoprotectant. Solid Cys-MRS was prepared by adding 1.5% (w/v) agar (Oxoid) to Cys-MRS broth. 2.2. Fermentation experiments Fermentations were carried out in 1.5-l Biostat B-DCU fermentors (Sartorius AG/B. Braun Biotech International, Melsungen, Germany). The inocula were prepared as follows: strains were transferred from stock cultures at −80 °C and subcultured twice in Cys-MRS medium while incubated anaerobically at 37 °C in a modular atmospherecontrolled system (MG anaerobic work station; Don Whitley Scientific, Shipley, United Kingdom) that was continuously sparged with a mixture of 80% nitrogen, 10% carbon dioxide, and 10% hydrogen (Air Liquide, Paris, France). The fermentation experiments were performed in Cys-MRS medium (containing 20 g/l glucose as energy source) under anaerobic conditions by continuously sparging the medium with a mixture of 90% nitrogen and 10% carbon dioxide (Air Liquide). The fermentation temperature was kept constant at 37 °C. The pH was maintained at 6.2 by automatic control using a 3 M solution of NaOH

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and a 1.5 M solution of H3PO4. To keep the medium homogeneous, gentle stirring (100 rpm) was applied. Temperature, pH, and stirring were controlled online (MFCS/win 2.1; Sartorius AG/B. Braun Biotech International). To minimise the inhibitory effect of the fatty acids on the growth of the cultures, 0.50 mg/ml LA or α-LNA (99% pure; Sigma-Aldrich, St. Louis, MO, USA) was added to the fermentation when the bacterial culture reached an optical density value between 0.1 and 0.2 at 600 nm (OD600), rather than at the start of the fermentation. Stock solutions of fatty acids were prepared as described before (Gorissen et al. 2010). The fermentations were monitored over 72 h and samples for further analysis were taken at regular time intervals, i.e. every hour during the first 4 h after addition of LA or α-LNA, then every 2 h until 12 h after the addition of LA or α-LNA, and at 24, 28, 32, 48, 52, 56, and 72 h of fermentation. Three fermentations, i.e. fermentations with B. breve LMG 11084 and B. breve LMG 11613, supplemented with LA, and B. breve LMG 11040, supplemented with α-LNA, were performed in triplicate to confirm reproducibility. 2.3. Analysis of growth, substrate consumption, and metabolite production Growth was monitored throughout the fermentations by measurements of OD600 and cell concentrations (expressed as colony forming units, CFU, per ml). Samples were plated on Cys-MRS agar and incubated under anaerobic conditions (MG anaerobic work station). Concentrations of glucose, lactate, acetate, formate, and ethanol were determined by high-performance liquid chromatography (HPLC). Therefore, the fermentation sample (0.7 ml) was centrifuged (3464 ×g for 20 min) and an equal volume of 20% (w/v) trichloroacetic acid was added to the supernatant for protein precipitation. After centrifugation (21,036 ×g for 20 min), the supernatant was filtered (pore size, 0.2 μm; Minisart RC4 filters; Sartorius AG, Göttingen, Germany) before injection into the column. All samples were analysed in triplicate. The HPLC analyses were performed on a chromatograph (Waters Corp., Milford, MA, USA) equipped with a 2414 differential refractometer, a 600S controller, a column oven, and a 717 plus autosampler. An ICSep ICE ORH-801 column (Interchim, Montluçon, France) was used with 10 mM of H2SO4 as a mobile phase at a flow rate of 0.4 ml/min. The column temperature was kept constant at 35 °C. External standards were used for quantification. 2.4. Lipid analysis Following centrifugation (3464 ×g for 20 min) of the fermentation sample (10 ml), lipids were extracted from cell pellets and cell-free culture supernatants, methylated, and analysed with gas chromatography (GC), as described previously (Gorissen et al., 2010). The combination of a basic methylation (at 50 °C for 30 min) and an acid methylation (at 50 °C for 10 min), did not lead to noticeable chemical isomerisation of the c9, t11- into the t9, t11-isomer (Raes et al., 2001). CLA and CLNA isomers were identified based on the retention times in the GC chromatograms, corresponding to the retention times of their respective compounds, as established previously (Gorissen et al., 2010). Fatty acid concentrations were calculated as the sum of fatty acids in the cell pellets and cell-free culture supernatants. 2.5. Kinetic analysis of growth and metabolite data The cell concentration X (in CFU/ml) was modelled with the following equations: dX=dt ¼ μ max X

when t b t d

ð1Þ

dX=dt ¼ −kd X

when t > t d :

ð2Þ

In these equations, t is time (in h), μmax is the maximal specific growth rate (in 1/h), kd is the maximal specific dying rate of the

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cells (in 1/h), and td is the moment at which the cell concentration started to decrease (in h). For those fermentations where a decrease in cell concentration was observed after addition of LA or α-LNA, an additional dying phase was introduced. The evolution of the concentration of LA (S, in mg/ml) was described with the following equations:

bifidobacteria tested (i.e. B. pseudolongum subsp. pseudolongum LMG 11595) was able to consume all glucose (Table 1). The metabolites produced were acetate, lactate, formate, and ethanol in fermentations supplemented with either LA or α-LNA (Table 1). In the fermentations with α-LNA, however, a lower production of metabolites was found due to a slower glucose breakdown.

dS=dt ¼ −kLA X

when t < t d

ð3Þ

3.2. Kinetics of LA conversion

dS=dt ¼ kLAþ

when t > t d

ð4Þ

with kLA the conversion rate of LA to CLA isomers during the active growth phase (in mg/CFU h) and kLA+ the rate of increase of LA in the medium, possibly due to dynamic changes in solubility of the initially added LA (in mg/ml h). The evolution of the concentration of the c9, t11-CLA isomer I1 (in mg/ml) was modelled with the following equations: dI1 =dt ¼ −dS=dt–kc X I1

when t b t d

ð5Þ

dI1 =dt ¼ −kc X I1

when t > t d

ð6Þ

with kc the conversion rate of the c9, t11-CLA isomer to the t9, t11CLA isomer (in ml/CFU h). The evolution of the concentration of the t9, t11-CLA isomer I2 (in mg/ml) was described with the following equation: dI2 =dt ¼ kc X I1 :

ð7Þ

Similar equations were used for the fermentations with α-LNA added to the medium leading to the production of c9, t11, c15- and t9, t11, c15-CLNA isomers. The corresponding parameters involved are kLNA (in mg/CFU h), kLNA+ (in mg/ml h), and kc (in ml/CFU h). The Eqs. (1) to (7) of the model were integrated with the Euler integration technique using Microsoft Excel (version 2007). The lag phase was modelled based on a Heaviside function, which forces the specific growth rate to zero during the duration of this phase. 3. Results 3.1. Growth and glucose metabolism All bifidobacterial cultures, to which LA was added, finished glucose uptake after 48 h of fermentation (Table 1), except for B. bifidum LMG 10645 and B. breve LMG 11084. When α-LNA was added, glucose consumption was much slower. Even after 72 h, only one strain of the six

In all fermentations with LA, conversion of LA started immediately after its addition to the medium. LA was first converted to the c9, t11CLA isomer (Fig. 1). While not all glucose was consumed by some strains after 48 h of fermentation (Table 1), conversion of LA to the c9, t11-CLA isomer already stabilised after 24 h of fermentation. Prolonged fermentation up to 72 h did not result in further appearance of c9, t11-CLA (Fig. 1). The maximal concentrations of c9, t11-CLA varied between 0.12 and 0.16 mg/ml. The formation of the c9, t11-CLA isomers was linked to the active growth phase of the cells. When cells entered the stationary phase or death phase, a decrease in c9, t11-CLA isomers together with a corresponding increase in the t9, t11-CLA isomers was seen. This simultaneous reduction and increase of c9, t11- and t9, t11isomers was more pronounced in certain strains, e.g. B. pseudolongum subsp. pseudolongum LMG 11595 (Fig. 1c) and B. breve LMG 13194 (data not shown), compared to the other strains. The maximal concentrations of t9, t11-CLA detected in the fermentations ranged from 0.02 to 0.09 mg/ml. The concentrations of t9, t11-CLA were much lower than the concentrations of the c9, t11-CLA isomer for all strains, except for B. pseudolongum subsp. pseudolongum LMG 11595 (Fig. 1c) and B. breve LMG 13194 (data not shown). For the latter two strains, concentrations of c9, t11-CLA and t9, t11-CLA were almost equal after 72 h of fermentation. A rapid increase in cell concentration does not seem to result in high conversion of LA to CLA isomers, as was the case for B. breve LMG 11084 (Fig. 1b). Similar results were observed for B. breve LMG 11040 and B. breve LMG 11613 (data not shown). On the contrary, growth of B. bifidum LMG 10645 (Fig. 1a) was inhibited by LA but good conversion of LA to c9, t11-CLA was obtained. Similar results were obtained for B. pseudolongum subsp. pseudolongum LMG 11595 (Fig. 1c) and B. breve LMG 13194 (data not shown). This was reflected in the biokinetic parameters of these fermentations as well (Table 2). B. bifidum LMG 10645 showed the highest kLA and kc values (10.1 · 10 − 10 mg/CFU h and 60.0 · 10 − 11 ml/CFU h), whereas B. breve LMG 11613 and B. breve LMG 11040 had the lowest kLA and kc values, respectively (1.7 · 10− 10 mg/CFU h and 1.1 · 10− 11 ml/CFU h). Reproducibility of the estimation of the biokinetic parameters was confirmed

Table 1 Metabolite production (in mM) by Bifidobacterium strains after fermentation in Cys-MRS medium supplemented with 0.50 mg/ml LA for 48 h and with 0.50 mg/ml α-LNA for 72 h from an initial substrate concentration of 111 ± 8.9 mM of glucose at 37 °C and constant pH 6.2. Bifidobacterium strain

Residual glucose

Lactate

Acetate

Formate

Ethanol

Fermentations with LA B. bifidum LMG 10645 B. breve LMG 11040 B. breve LMG 11084a B. breve LMG 11613a B. breve LMG 13194 B. pseudolongum subsp. pseudolongum LMG 11595

3.7 ± 0.1 0 1.8 ± 0.5 0 0 0

58.1 ± 0.5 49.8 ± 1.4 25.0 ± 4.1 20.1 ± 3.5 38.4 ± 1.5 45.0 ± 4.9

178.1 ± 1.0 196.7 ± 2.0 177.3 ± 22.0 202.1 ± 9.9 194.8 ± 3.7 187.9 ± 18.5

41.1 ± 0.2 68.2 ± 0.3 53.4 ± 5.9 81.9 ± 2.3 67.3 ± 0.7 59.1 ± 4.4

22.2 ± 0.1 19.5 ± 0.4 26.7 ± 4.4 33.3 ± 5.5 40.4 ± 0.4 36.9 ± 3.5

Fermentations with LNA B. bifidum LMG 10645 B. breve LMG 11040a B. breve LMG 11084 B. breve LMG 11613 B. breve LMG 13194 B. pseudolongum subsp. pseudolongum LMG 11595

91.2 ± 5.3 31.8 ± 2.3 84.7 ± 2.2 55.3 ± 6.4 75.7 ± 3.3 0

17.8 ± 0.9 32.0 ± 5.9 14.2 ± 0.5 7.9 ± 0.8 17.9 ± 0.5 53.9 ± 1.6

56.8 ± 6.2 165.1 ± 12.3 61.1 ± 3.0 117.7 ± 3.5 84.1 ± 1.2 208.4 ± 5.4

4.7 ± 1.1 56.0 ± 3.3 9.5 ± 0.1 50.5 ± 0.7 29.1 ± 0.2 65.3 ± 1.6

2.3 ± 0.1 19.8 ± 6.2 5.7 ± 1.3 23.3 ± 1.1 18.4 ± 0.4 22.6 ± 1.8

The results are presented as means ± standard deviations. Samples of single fermentation were analysed in triplicate (n = 3). a Average values and standard deviations of three replicates of the same fermentation analysed in triplicate (n = 9).


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0.5 · 10 − 11 ml/CFU h for the kc values for B. breve LMG 11084 and B. breve LMG 11613, respectively. Unexpectedly, towards the end of the fermentations an increase in the concentration in LA was found (Fig. 1). Additionally, a concentration of merely 0.15–0.20 mg/ml LA was found immediately after addition of this fatty acid to the fermentation and not the expected amount of 0.50 mg/ml LA. Nevertheless, the recovery of LA was similar for all fermentations and these initial levels were quantitatively converted into CLA isomers, enabling kinetic analysis of LA conversion. The fatty acid analysis revealed that LA and the CLA isomers were mainly found in the cell-free culture supernatants compared to the cell pellets. On average, 25% of LA and 22% of CLA were recovered in the cell pellets. 3.3. Kinetics of α-LNA conversion

Fig. 1. Cell concentration (×; log CFU/ml) and conversion of LA (□) into c9, t11-CLA (♦) and t9, t11-CLA (●) (in mg/ml) by Bifidobacterium bifidum LMG 10645 (a), Bifidobacterium breve LMG 11084 (b), and Bifidobacterium pseudolongum subsp. pseudolongum LMG 11595 (c) in Cys-MRS medium supplemented with 0.50 mg/ml LA at 37 °C and constant pH 6.2 (↓ indicates the time point when LA was added). Full lines are according to the model; (b) depicts a representative graph of three replicates.

in fermentations with B. breve LMG 11084 or B. breve LMG 11613 and LA supplemented to the Cys-MRS medium since the standard deviations, based on three replicates of the fermentations, were 0.4 · 10− 10 and 0.5 · 10 − 10 mg/CFU h for the kLA values for B. breve LMG 11084 and B. breve LMG 11613, respectively, and 1.2 · 10 − 11 and

In accordance with LA conversion, α-LNA conversion started immediately after its addition to the medium, leading to the formation of a first CLNA isomer, putatively the c9, t11, c15-CLNA isomer (Fig. 2). Conversion of α-LNA to CLNA isomers finished in less than 48 h of fermentation, with only one exception, i.e. B. bifidum LMG 10645 (Fig. 2a). Again, conversion of α-LNA by certain strains stopped before all glucose was consumed in these fermentations, namely for B. bifidum LMG 10645, B. breve LMG 11040, B. breve LMG 11084, and B. breve LMG 13194 (Table 1). In the case of B. bifidum LMG 10645 (Fig. 2a), a small initial dying phase was found after which cells grew very slowly. This resulted in a very slow conversion of α-LNA to c9, t11, c15-CLNA. In fermentations with the five other bifidobacterial strains, this initial dying phase was much less pronounced (B. pseudolongum subsp. pseudolongum LMG 11595, Fig. 2c) or even absent (B. breve LMG 13194, Fig. 2b). The maximal concentrations of c9, t11, c15-CLNA ranged from 0.17 to 0.34 mg/ml. Appearance of a second CLNA isomer, putatively the t9, t11, c15-CLNA isomer, was found between 24 h and 72 h of fermentation, especially in fermentations with B. breve LMG 13194 (Fig. 2b) and B. breve LMG 11613 (data not shown). Concentrations of this t9, t11, c15-CLNA isomer were lower than for the c9, t11, c15-CLNA. The maximal concentration of t9, t11, c15-CLNA ranged from 0.01 to 0.05 mg/ml. Of all strains, B. breve LMG 13194 showed the highest production of c9, t11, c15-CLNA and t9, t11, c15-CLNA, despite a poor growth (maximum cell concentration of 8.1 log CFU/ml; Fig. 2b). In contrast, B. pseudolongum subsp. pseudolongum LMG 11595 displayed a better growth resulting in lower conversion of fatty acids (Fig. 2c), which was also reflected in its lower kc value (Table 2). The standard deviations for the kLNA and kc values for fermentations with B. breve LMG 11040 and α-LNA supplemented to the Cys-MRS, performed in triplicate, were 5.8 · 10 − 9 mg/CFU h and − 11 0.3 · 10 ml/CFU h, respectively. For all strains, except B. bifidum LMG 10645, the kLNA values were higher than the kLA values (Table 2), indicating a more efficient production of c9, t11, c15CLNA compared to c9, t11-CLA. Similarly as the fermentations with LA, the highest amounts of LNA and CLNA isomers were found in the cell-free culture supernatants compared to the cell pellets. On average, 17% of LNA and 15% of CLNA were recovered in the cell pellets. 4. Discussion Considering the biological activity of CLA and CLNA isomers, laboratory fermentations were carried out in a complex medium with six LA- and α-LNA-converting Bifidobacterium strains (Gorissen et al. 2010) to assess their conversion kinetics. In agreement with results of earlier studies, formation of c9, t11-CLA from LA occurred during the logarithmic to early stationary growth phase of bifidobacteria (Coakley et al., 2003; Hennessy et al., 2009; Oh et al., 2003; Park

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Table 2 Biokinetic parameters for the modelling of CLA and CLNA production by Bifidobacterium (B.) strains in Cys-MRS medium supplemented with 0.50 mg/ml LA or α-LNA at 37 °C and constant pH 6.2.

Fermentations with LA kLA (mg/CFU h) kc (ml/CFU h)

B. bifidum LMG 10645

B. breve LMG 11040

B. breve LMG 11084

B. breve LMG 11613

B. breve LMG 13194

B. pseudolongum subsp. pseudolongum LMG 11595

10.1 · 10− 10 60.0 · 10− 11

3.1 · 10− 10 1.1 · 10− 11

3.6 · 10− 10a 5.0 · 10− 11a

1.7 · 10− 10a 2.5 · 10− 11a

4.0 · 10− 10 9.0 · 10− 11

2.4 · 10− 10 7.5 · 10− 11

23.3 · 10− 9a 1.8 · 10− 11a

14.0 · 10− 9 1.5 · 10− 11

11.0 · 10− 9 3.0 · 10− 11

2.0 · 10− 9 1.3 · 10− 11

7.0 · 10− 9 0.2 · 10− 11

Fermentations with α-LNA 0.6 · 10− 9 kLNA (mg/CFU h) kc (ml/CFU h) 2.0 · 10− 11 a

Average values of three replicates.

et al., 2009). In the stationary phase or death phase, the simultaneous decrease and increase of c9, t11- and t9, t11-isomers, respectively, seems to meet the hypothesis of further conversion of c9, t11-CLA to t9, t11-CLA after the active growth phase, as suggested previously for B. breve strains (Coakley et al., 2006; Hennessy et al., 2009), as well as further conversion of the putative c9, t11, c15CLNA to t9, t11, c15-CLNA (Gorissen et al., 2010). The LAI enzyme of Butyrivibrio fibrisolvens is known to convert LA and α-LNA to c9, t11-CLA and c9, t11, c15-CLNA, respectively (Kepler et al., 1966; Kepler and Tove, 1967). However, conversion of c9, t11-isomers into t9, t11-isomers seems not to be performed by this LAI. Yet, in the present study, B. breve LMG 13194 and B. pseudolongum subsp. pseudolongum LMG 11595 produced more t9, t11-CLA after 72 h of fermentation than the other four bifidobacterial strains tested, indicating that this conversion is strain-dependent, thereby suggesting an enzymatic rather than chemical conversion reaction of c9, t11isomers to t9, t11-isomers. The differences in fatty acid conversion among bifidobacterial strains were not as apparent in the fermentations with α-LNA compared to the fermentations with LA. However, all strains produced higher amounts of putative c9, t11, c15-CLNA than c9, t11-CLA and less of this putative c9, t11, c15-CLNA was further converted into the putative t9, t11, c15-CLNA isomer compared to the further conversion of c9, t11-CLA into t9, t11-CLA. These observations may be related to the toxicity of α-LNA. Since α-LNA is more toxic to the growing cells (Gorissen et al., 2010), detoxification through conversion to c9, t11, c15-CLNA could offer a benefit to the bifidobacterial strains capable to do so (Coakley et al., 2003; Jiang et al., 1998; Kim et al., 2000). Production of CLA and CLNA isomers was dependent on the growth kinetics of the tested bifidobacteria. The importance of cell density on CLA production by bifidobacterial strains has also been demonstrated in the case of B. breve NCIMB 702258, a strain for which increased cell numbers leads to increased isomerisation capacity (Hennessy et al., 2009). Such observations are most probably to be ascribed to increased levels of available LAI needed for conversion of LA to c9, t11-CLA. However, fast growth to high cell densities is no guarantee for a good conversion of LA and α-LNA to CLA and CLNA isomers, respectively. Strains displaying a high specific conversion (e.g. B. breve LMG 13194 and B. pseudolongum subsp. pseudolongum LMG 11595), i.e. a good conversion of LA to c9, t11-CLA and of c9, t11-CLA to t9, t11-CLA, showed rather poor growth whereas other strains (e.g. B. breve LMG 11613 and B. breve LMG 11084) grew well but performed poorly with respect to LA conversion. The dynamic increase in LA and α-LNA concentrations towards the end of the fermentations and the low concentrations of LA and α-LNA found at the beginning of the fermentations (0.15– 0.20 mg/ml instead of 0.50 mg/ml) were possibly due to their limited solubility, conversion of the fatty acids over time, or other experimental artefacts. Following the addition of LA and α-LNA

at 0.50 mg/ml, the excess amount might appear slowly in the medium once the soluble amount was converted, resulting in a small increase in the LA or α-LNA concentration. However, this did not result in supplementary CLA or CLNA formation. Similarly, washed cells of B. fibrisolvens A38 rapidly convert a dose of LA (350 μM) to CLA but are not capable of converting a second dose of LA, administered to these cells after 8 min (Kim et al., 2000). Furthermore, prolonged incubation of B. fibrisolvens cells with higher dosages of LA does not result in higher CLA production (Kepler and Tove, 1967; Kim et al., 2000). Kim et al. (2000) suggested that CLA production is likely to depend more on the cell density than on the concentrations of LA. Higher LA concentrations will lead to higher CLA production as long as these LA concentrations do not become too toxic to the cells. On the contrary, too low concentrations might not trigger isomerisation. Bifidobacteria seem to perform a cis-trans conversion of c9, t11isomers to t9, t11-isomers in the stationary growth phase, when LAI would be no longer available, as a reaction to the increasing concentrations of toxic substances (LA and α-LNA). A cis to trans conversion mechanism of unsaturated fatty acids exists in strains of the genera Vibrio and Pseudomonas, which enables them to change their membrane fluidity in response to an increase in temperature or as adaptation to toxic compounds, respectively (Heipieper et al., 2003). This conversion is not dependent on cell growth, as it occurs in non-growing cells as well (Heipieper et al., 1992). By converting cis fatty acids, which are bended fatty acids, to the corresponding trans fatty acids, whose linear shape resembles that of saturated fatty acids, the fluidity of the cell membrane is reduced and the permeability of the membrane may also be reduced (Heipieper et al., 2003). In conclusion, changes in bacterial growth and subsequent fatty acid metabolism, through modifications of the fermentation time, the initial amount of cells for fermentation, and the prevailing concentrations of LA and α-LNA, could be applied to steer the production of a desired isomer by selected bifidobacterial strains. For example, production of t9, t11-CLA by B. breve LMG 13194 or B. pseudolongum subsp. pseudolongum LMG 11595 seems interesting. The c9, t11-CLA isomer, the main isomer in food products from ruminants, has been extensively studied and is best known for its anti-carcinogenic and anti-inflammatory effects (Wahle et al., 2004). Recently, the t9, t11CLA isomer has been under investigation because of its potential anti-carcinogenic effects on human colon cancer cell lines, which is more effective than the c9, t11-CLA isomer (Beppu et al., 2006; Coakley et al., 2006), and its ability to express genes involved in lipid metabolism (Ecker et al., 2009). Furthermore, using one of these bifidobacteria as, or in combination with, a conventional starter culture could be a way of producing functional foods with elevated levels of certain specific CLA and CLNA isomers and holds an advantage over dietary supplements. The latter contain several CLA isomers, obtained through alkaline isomerisation of LA, with unknown biological activities (House et al., 2005). Bifidobacteria are also


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of CLA and CLNA isomer production in both functional foods and the human gut. Acknowledgements This work was financially supported by the Research FoundationFlanders (FWO-Vlaanderen) and the Research Council of the Vrije Universiteit Brussel. References

Fig. 2. Cell concentration (×; log CFU/ml) and conversion of α-LNA (□) into c9, t11, c15-CLNA (♦) and t9, t11, c15-CLNA (●) (mg/ml) by Bifidobacterium bifidum LMG 10645 (a), Bifidobacterium breve LMG 13194 (b), and Bifidobacterium pseudolongum subsp. pseudolongum LMG 11595 (c) in Cys-MRS medium supplemented with 0.50 mg/ml α-LNA at 37 °C and constant pH 6.2 (↓ indicates the time point when α-LNA was added). Full lines are according to the model.

common gut inhabitants (Trebichavsky et al., 2009) and some strains are applied as probiotics (Collins and Gibson, 1999; Ewaschuk et al., 2006; Gomes and Malcata, 1999; Ross et al., 2010). Considering the potential health advantages of CLA and CLNA isomers, the ability of bifidobacteria to produce these compounds could contribute to the probiotic properties of selected strains of these intestinal bacteria. Dedicated studies will be needed to further investigate the relevance

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