the production of conjugated α-linolenic, γ-linolenic and stearidonic acids by strains of...

ORIGINAL ARTICLE

The Production of Conjugated a-Linolenic, c-Linolenicand Stearidonic Acids by Strains of Bifidobacteriaand Propionibacteria

Alan A. Hennessy • Eoin Barrett • R. Paul Ross •

Gerald F. Fitzgerald • Rosaleen Devery •

Catherine Stanton

Received: 22 June 2011 / Accepted: 15 November 2011 / Published online: 10 December 2011

� AOCS 2011

Abstract Conjugated fatty acids are regularly found in

nature and have a history of biogenic activity in animals and

humans. A number of these conjugated fatty acids are mi-

crobially produced and have been associated with potent

anti-carcinogenic, anti-adipogenic, anti-atherosclerotic and

anti-diabetogenic activities. Therefore, the identification of

novel conjugated fatty acids is highly desirable. In this study,

strains of bifidobacteria and propionibacteria previously

shown by us and others to display linoleic acid isomerase

activity were assessed for their ability to conjugate a range of

other unsaturated fatty acids during fermentation. Only four,

linoleic, a-linolenic, c-linolenic and stearidonic acids, were

converted to their respective conjugated isomers, conjugated

linoleic acid (CLA), conjugated a-linolenic acid (CLNA),

conjugated c-linolenic acid (CGLA) and conjugated steari-

donic acid (CSA), each of which contained a conjugated

double bond at the 9,11 position. Of the strains assayed,

Bifidobacterium breve DPC6330 proved the most effective

conjugated fatty acid producer, bio-converting 70% of the

linoleic acid to CLA, 90% of the a-linolenic acid to CLNA,

17% of the c-linolenic acid to CGLA, and 28% of the

stearidonic acid to CSA at a substrate concentration of

0.3 mg mL-1. In conclusion, strains of bifidobacteria

and propionibacteria can bio-convert linoleic, a-linolenic,

c-linolenic and stearidonic acids to their conjugated isomers

via the activity of the enzyme linoleic acid isomerase. These

conjugated fatty acids may offer the combined health pro-

moting properties of conjugated fatty acids such as CLA and

CLNA, along with those of the unsaturated fatty acids from

which they are formed.

Keywords Bifidobacteria � Propionibacteria � CLA �a-Linolenic acid � c-Linolenic acid � Stearidonic acid

Abbreviations

CGLA Conjugated c-linolenic acid

CLA Conjugated linoleic acid

CLNA Conjugated a-linolenic acid

CSA Conjugated stearidonic acid

DAD Diode array detector

DMOX 4,4-Dimethyloxazoline

EPA Eicosapentaenoic acid

FAME Fatty acid methyl esters

FID Flame ionization detector

MRS DeMan-Rogosa-Sharpe

GLC Gas liquid chromatography

GLC-MS Gas liquid chromatography mass

spectrometry

GIT Gastrointestinal tract

MTAD 4-Methyl-1,2,4-triazoline-3,5-dione

PUFA Polyunsaturated fatty acids

RP-HPLC Reverse phase high performance liquid

chromatography

Introduction

Conjugated fatty acids are the positional and geometric

isomers of several polyunsaturated fatty acids (PUFA) with

A. A. Hennessy � E. Barrett � R. Paul Ross � C. Stanton (&)

Teagasc Food Research Centre, Moorepark, Fermoy, Co.,

Cork, Ireland

e-mail: [email protected]

A. A. Hennessy � R. Devery

National Institute for Cellular Biotechnology,

Dublin City University, Dublin, Ireland

E. Barrett � R. Paul Ross � G. F. Fitzgerald � C. Stanton

Alimentary Pharmabiotic Centre, BSI, University College Cork,

Cork, Ireland

123

Lipids (2012) 47:313–327

DOI 10.1007/s11745-011-3636-z

one or more conjugated double bonds. These conjugated

isomers are commonly found in nature, being detected in

the milkfat and tallow of ruminant animals [1, 2], plant

seed oils [3–6] and marine algae [7–10]. In addition, con-

jugated fatty acids may be formed chemically via the

alkaline isomerisation of unsaturated fatty acids [11, 12].

Of these natural and synthetic fatty acids, the conjugated

linoleic acid (CLA) isomers are the best characterised.

These fatty acids have been demonstrated to exhibit a range

of potentially health-promoting properties, including anti-

atherosclerotic, anti-obesogenic and anti-diabetogenic

activities; however, it is the anti-carcinogenic properties

which are most extensively reported [13, 14]. In addition to

CLA isomers, other conjugated fatty acids have been

shown to exhibit biological activities with relevance to

human health. Indeed, naturally occurring CLNA isomers

traditionally found in plant seed oils and in ruminant fats

have been reported to exhibit anti-carcinogenic and anti-

obese activities [15–18]. Other synthetically produced

conjugated isomers, such as conjugated eicosadienoic acids

(C20:2) and conjugated eicosatrienoic acids (C20:3) have

been associated with body fat reduction and increased lean

mass [12]. Conjugated eicosapentaenoic acids (C20:5)

have been reported to exhibit anti-carcinogenic activity,

and conjugated docosahexaenoic acids (C22:6) to exert

anti-carcinogenic and anti-adipogenic activity [11, 19–22].

The mechanisms of action of conjugated fatty acids are

reported to be either via competitive inhibition during the

metabolism of proinflammatory n-6 fatty acids to eicosa-

noids, via increased cellular lipid peroxidation, or at the

genetic level via their impact on the expression of cell

cycle arrest regulators, transcription factors, and key met-

abolic enzymes (for reviews see [13, 14, 17]). Given the

extent and significance of the health promoting biological

activities associated with conjugated fatty acids, economic

strategies for their production are highly desirable.

The potency of conjugated fatty acids has prompted

increased research into their identification and economic

production [23, 24]. These investigations have highlighted

in particular the ability of a range of microbes to produce

CLA [25, 26]. Indeed, it has been reported that dairy pro-

pionibacteria and intestinally isolated bifidobacteria pos-

sess the ability to conjugate the c9,c12 double bond of

linoleic acid (C18:2) yielding the c9,t11-C18:2 and t9,t11-

C18:2 CLA isomers, via the action of the enzyme linoleic

acid isomerase [27–29]. Furthermore, recent in vitro evi-

dence has highlighted the ability of strains of bifidobacteria

to produce the c9,t11,c15-C18:3 CLNA isomer from free

a-linolenic acid, and of Lactobacillus plantarum AKU

1009a to produce conjugated c-linolenic acid (CGLA) from

free c-linolenic acid [16, 30]. These observations demon-

strate the potential of CLA producing bacteria to conjugate

a range of PUFA. Similar to CLA, the ability of

propionibacteria and bifidobacteria to produce novel con-

jugated fatty acids may be used to enrich milk and yoghurt

with conjugated fatty acids through fermentation, or the

establishment of microbiota in the human gastrointestinal

tract (GIT) capable of producing these fatty acids in situ.

Indeed, further credence is given to this theory in light of

the recent evidence pertaining to the ex vivo production of

CLA by intestinally isolated strains of lactobacilli and

bifidobacteria reported by Ewaschuk et al. [31] and our

studies on in vivo production of CLA by bifidobacteria

from dietary linoleic acid in mice [32].

The aims of this study were (1) to assess the ability of

strains of bifidobacteria and propionibacteria previously

shown by us to conjugate linoleic acid to CLA to bio-

convert a range of PUFA during fermentation [27–29, 33],

and (2) to identify and characterise any novel microbially

produced conjugated fatty acids.

Materials and Methods

Bacterial Strains and Fatty Acid Substrates

The strains of Bifidobacterium and Propionibacterium used

in this study are listed in Table 1, and were selected based

on their ability to bioconvert linoleic acid (c9,c12-C18:2)

to the c9,t11 CLA isomer [27–29]. All strains were grown

in DeMan-Rogosa-Sharpe (MRS) medium (Difco, Detroit,

MI, USA) containing 0.05 mg mL-1L-cysteine hydro-

chloride (Sigma Aldrich., St Louis, MO, USA) (cys-MRS)

under anaerobic conditions (Anaerocult A Gas Packs (78%

N2, 18% CO2, 4% Other), Merck, Darmstadt, Germany)

at 37 �C for 18 h. Where solid media was required, 1.5%

(wt/vol) bacteriological agar (Oxoid, Basingstoke, Hampshire,

UK) was added to the cys-MRS medium. Cell counts were

determined by serial dilutions and plating using cys-MRS

agar.

All fatty acid substrates were of the highest purity

(Table 2) and were delivered to the growth medium in the

form of a 30 mg mL-1 stock solution containing 2% (wt/vol)

Tween 80 (Merck-Schuchardt, Hohenbrunn, Germany).

To ensure sterility, all substrates were filter sterilized through

a 0.45 lm Ministart filter (Sartorius AG, Goettingen,

Germany).

Microbial Conjugated Fatty Acid Production

A 4% (vol/vol) inoculum of the activated culture was added

to 10 mL cys-MRS medium containing 0.45 mg mL-1

[exceptions being 0.15 mg mL-1 for eicosapentaenoic acid

(EPA), and stearidonic acid] of the respective unsaturated

fatty acid substrates and incubated anaerobically (Anaero-

cult A, Merck) at 37 �C (bifidobacteria) or 30 �C

314 Lipids (2012) 47:313–327

123

(propionibacteria) for 72 h with regular spectrophotometric

monitoring of conjugate production (6, 12, 24, 48, and 72 h)

[29]. Fatty acids were extracted from 4 mL of the fermen-

tation medium using 2 mL of 2-propanol (Fisher Scientific

Ltd, Dublin, Ireland) and 4 mL of n-hexane (Fisher Scien-

tific Ltd), as described by Coakley et al. [27]. Following

extraction, the n-hexane was removed by heating under N2,

and the extracted fatty acids converted to fatty acid methyl

esters (FAME) by acid catalyzed methylation with 12 mL

of 4% (vol/vol) methanolic-HCl for 10 min under nitrogen

at ambient temperature, followed by extraction with 4 mL

n-hexane. Gas liquid chromatography (GLC) was used to

separate FAME using a CP-SELECT CB column

(100 m 9 0.25 mm id 9 0.25 lm film thickness, Varian

BV, Middelburgh, The Netherlands) and Varian 3400

Capillary GC (Varian, Walnut Creek, CA, USA) fitted with

a flame ionization detector (FID) as described by Coakley

et al. [27]. The presence of a conjugated double bond was

confirmed spectrophotometrically, using a modification of

the method of Barrett et al. [29] using FAME rather than

free fatty acids. Briefly, 200 ll of the methylated sample,

prepared as described above, was dispensed into a UV

transparent 96 well plate (Costar, Corning NY) and the

absorbance of UV light at a wavelength of 234 nm mea-

sured using a 96 well plate spectrophotometer (GENios

Plus, Medford, MA). All experiments were performed in

triplicate. Conjugated fatty acid production was expressed

as mg mL-1 and percentage bioconversion, the latter cal-

culated by:

Cc/Sc�100 ¼ % bioconverion

Sc ¼ Substrate concentration prior to fermentation:

Cc ¼ Conjugated fatty acid concentration

following fermentation

:

Table 1 Strains of

Bifidobacterium and

Propionibacterium

Strain Source References

Bifidobacterium

Bifidobacterium breve NCIMB 702258 Infant intestine [27]

Bifidobacterium breve NCIMB 8807 Nursling stools [27]

Bifidobacterium breve DPC6330 C. difficile (?) subject [29]

Bifidobacterium breve DPC6331 C. difficile (?) subject [29]

Bifidobacterium longum DPC6315 Healthy adult [29]

Bifidobacterium longum DPC6320 C. difficile (?) subject [29]

Propionibacterium

Propionibacterium freudenreichii subsp. shermanii JS Dairy starter [28]

Propionibacterium freudenreichii subsp. shermanii 9093 Dairy starter [28]

Propionibacterium freudenreichii subsp. freudenreichii Propioni-6 Dairy starter [28]

Propionibacterium freudenreichii subsp. freudenreichii ATCC 6207 Dairy starter [28]

Table 2 Fatty acid substratesName Chemical formula Source Purity (%)

Linoleic acid (C18:2-c9,c12) Sigma Aldrich, St Louis, MO 99

a-Linolenic acid (C18:3-c9,c12, c15) Sigma Aldrich, St Louis, MO 99

c-Linolenic acid (C18:3-c6,c9, c12) Nu-Chek Prep, Elysian, MN 99

Stearidonic acid (C18:4-c6,c9, c12,c15) Cayman Europe, Tallinn, Estonia 98

Nonadecadienoic acid (C19:2-c10,c13) Nu-Chek Prep, Elysian, MN 99

Eicosadienoic acid (C20:2-c11,c14) Nu-Chek Prep, Elysian, MN 99

Eicosatrienoic acid (C20:3-c11,c14, c17) Nu-Chek Prep, Elysian, MN 99

Homo-gamma linolenic (C20:3-c8,c11,c14) Nu-Chek Prep, Elysian, MN 99

Arachidonic acid (C20:4-c5,c8,c11,c14) Nu-Chek Prep, Elysian, MN 99

Eicosapentaenoic acid (C20:5-c5,c8,c11,c14, c17) Nu-Chek Prep, Elysian, MN 99

Heneicosadienoic acid (C21:2-c12,c15) Nu-Chek Prep, Elysian, MN 99

Docosadienoic acid (C22:2-c13,c16) Nu-Chek Prep, Elysian, MN 99

Docosatrienoic acid (C22:3-c13,c16,c19) Nu-Chek Prep, Elysian, MN 99

Docosatetraenoic acid (C22:4-c7,c10,c13,c16) Nu-Chek Prep, Elysian, MN 99

Docosapentaenoic acid (C22:5-c7,c10,c13,c16,c19) Nu-Chek Prep, Elysian, MN 99

Docosahexaenoic acid (C22:6-c4, c7,c10,c13,c16,c19) Nu-Chek Prep, Elysian, MN 99

Lipids (2012) 47:313–327 315

123

Purification and Identification of Microbially Produced

Conjugated Fatty Acids

Total fatty acids extracted using the method of Coakley

et al. [27] were partially concentrated using a rotary

evaporator (\50 �C) (Buchi Rotavapor R-210) and washed

once with 0.88% (wt/vol) KCl and twice with water/

methanol (1:1, vol/vol). The hexane was then evaporated

by heating at 45 �C under a steady flow of nitrogen. The

remaining lipid was resuspended at a concentration of

100 mg mL-1 in acetonitrile/acetate (100:0.14, vol/vol),

and stored at -20 �C under nitrogen until use. The con-

jugated fatty acids were isolated using RP-HPLC on a Luna

5l C18 (2) 100A preparative column (250 mm 9

21.20 mm) (Phenomenex, Macclesfield, Cheshire, UK) and

the Varian Prostar HPLC system (Varian). The mobile

phase used to obtain optimal separation was acetonitrile/

water/acetate (70:30:0.12, vol/vol) at a flow rate of

10 mL min-1. Conjugated fatty acids were detected using

a diode array detector (DAD) at an absorbance of 234 nm.

Fractions containing the conjugated fatty acids were col-

lected and pooled. Following removal of the acetonitrile

from the pooled fractions by rotary evaporation (\50 �C),

the conjugated fatty acids were re-extracted as described

above. Fatty acid compositions of the pure oils were con-

firmed by GLC as previously described [27]. Conjugated

fatty acids were identified by Mylnefield Lipid Analysis

(Dundee, Scotland) by the preparation and analysis of

FAME, 4,4-dimethyloxazoline (DMOX), 4-methyl-1,2,4-

triazoline-3,5-dione (MTAD) and/or pyrrolidide deriva-

tives by GLC-mass spectrometry (GLC-MS) [34].

Following, their identification by GC–MS, samples of

the purified oils produced by B. breve DPC6330 were used

as GLC standards to identify similar conjugated fatty acids

produced by the additional strains of bifidobacteria and

propionibacteria assayed. The methylation was conducted

as described above, while the GLC conditions were as

described by Coakley et al. [27].

CLNA, CGLA and CSA Production by B. breve

DPC6330

During the initial assessment of the strains for conjugated

fatty acid production B. breve DPC6330 proved most

efficient in terms of the bio-production of conjugated fatty

acids from linoleic, a-linolenic, c-linolenic and stearidonic

acid. Subsequently, to determine the impact of substrate

fatty acid concentration on conjugated fatty acid produc-

tion, B. breve DPC6330 was incubated anaerobically for

72 h at 37 �C in the presence of linoleic acid, a-linolenic

acid (Sigma Aldrich), c-linolenic acid (Nu-Chek Prep,

Elysian, MN) or stearidonic acid (Cayman Europe,

Akadeemia tee, Tallinn, Estonia) at concentrations of 0.15,

0.3 and 0.45 mg mL-1 (n = 3). To determine the time

course of CLNA, CGLA and conjugated stearidonic acid

(CSA) production by B. breve DPC6330, 16 individual

fermentations (10 mL volumes) were set up in triplicate for

each fatty acid at a substrate fatty acid concentration of

0.3 mg mL-1. At 16 different time points, over the 80-h

fermentation period, triplicate aliquots were removed and

assessed for bacterial cell numbers by serial dilution and

plating using cys-MRS agar and for fatty acid composition

by GLC following extraction and methylation of lipids.

Results

Conjugated Fatty Acid Production by Bifidobacteria

and Propionibacteria

The performance of the intestinally isolated bifidobacteria

and dairy propionibacteria when exposed to 0.45 mg mL-1

of the respective PUFA (0.15 mg mL-1 of EPA or steari-

donic acid) was visually observed to be strong (Table 3).

Using GLC and UV spectrophotometric analysis, it was

determined that four of the PUFA substrates were micro-

bially bioconverted to their respective conjugated fatty acid

isomers (Table 4). These fatty acid substrates were lino-

leic, a-linolenic, c-linolenic and stearidonic acids, all of

which contained the c9,c12 double bond. The production of

the c9,t11 and t9,t11 CLA isomers from linoleic acid by the

strains of bifidobacteria and propionibacteria was con-

firmed using GLC and a range of commercial CLA stan-

dards (Matreya LLC, PA), while the conjugated fatty acids

derived from a-linolenic, c-linolenic and stearidonic acids

were identified by GLC-MS.

Identification of Microbially Produced Conjugated

Fatty Acids

The predominant conjugated fatty acid isomer produced by

the strains of bifidobacteria and propionibacteria from free

a-linolenic acid was identified as 9,11,15-C18:3 (CLNA1).

The mass spectrum derived from the FAME confirmed the

molecular weight as 278 (Fig. 1a), while the mass spec-

trum of its DMOX derivative identified the molecule as the

9,11,15-C18:3 isomer (Fig. 1b). Further confirmation of

the structure was achieved by the comparison of the

DMOX spectra of this fatty acid with that of CLNA1

identified by both Destaillats et al. [18] and Winkler and

Steinhart [35].

The predominant conjugated fatty acid isomer produced

by the strains of bifidobacteria and propionibacteria from

free c-linolenic acid was identified as 6,9,11-C18:3

(CGLA1). The mass spectrum of the FAME confirmed the

molecular weight as 278 (Fig. 2a). However, due to the

316 Lipids (2012) 47:313–327

123

migration of the 9,11-double bond system to the 8,10-

position during the DMOX derivatization process (Fig. 2b)

MTAD and pyrrolidide derivatizations were also employed

in the identification of the molecule. The mass spectrum of

the MTAD derivative, with the ion at m/z = 250, con-

firmed that the fatty acid possessed a conjugated double

bond in the 9,11-position, with the third double bond

located on the carboxyl side of the conjugated double bond

(Fig. 2c) [36]. Pyrrolidide derivatization with the finger-

print of ions at m/z = 140, m/z = 154 and m/z = 166

characterised the position of a double bond in position-6 of

the carbon chain (Fig. 2d) [37].

The predominant conjugated fatty acid isomer produced

by strains of bifidobacteria and propionibacteria from

stearidonic acid was identified as 6,9,11,15-C18:4 (CSA1).

The mass spectrum of the FAME confirmed the molecular

weight as 276 (Fig. 3a). Once again, DMOX derivatization

of the fatty acid resulted in the migration of the 9,11-double

bond system to the 8,10-position (Fig. 3b). The spectrum

produced for the MTAD derivative, with the base ion now at

m/z = 248, confirmed the presence of a conjugated double

bond in the 9,11-position, along with the presence of a

double bond on either side of this (Fig. 3c). Although the

use of DMOX derivatives did not allow the identification of

the position of the first three double bonds, a double bond at

position-15 of the carbon backbone was confirmed by the

presence of a gap of 12 amu between m/z = 274 and

m/z = 286 (Fig. 3b). The mass spectrum observed with the

pyrrolidide derivative of this fatty acid confirmed the

presence of a double bond at position-6 (Fig. 3d).

Using GLC-MS analysis, it was not possible to deter-

mine the double bond conformation of CLNA1, CGLA1

and CSA1. However, given the specificity of the enzyme

linoleic acid isomerase for the conjugation of c9,c12 dou-

ble bond to the c9,t11 bond conformation, the most prob-

able bond conformation for CLNA1 is c9,t11,c15-C18:3,

while that of CGLA1 and CSA1 are likely to be c6,c9,t11-

C18:3 and c6,c9,t11,c15-C18:4, respectively [27, 28]. This

prediction is supported by the studies of Ogawa et al. [24]

and Kishino et al. [38] who successfully identified the

production of c9,t11,c15-C18:3 (CLNA1) and c6,c9,t11-

C18:3 (CGLA1) from free a-linolenic acid and c-linolenic

acid, respectively, by strains of lactobacilli exhibiting lin-

oleic acid isomerase activity.

In addition to the production of CLNA1, CGLA1 and

CSA1 by the strains of bifidobacteria and propionibacteria,

a second conjugated isomer was also produced from

a-linolenic acid, c-linolenic acid and stearidonic acid,

Table 3 Production of conjugated fatty acids by growing cultures of Bifidobacterium and Propionibacterium

Substrate Growth Species

B. brevea B. longumb Prop. freudenreichiisubsp. shermaniic

Prop. freudenreichiisubsp. freudenreichiid

Linoleic acid C18:2 (c9,c12) (?) (?) (?) (?) (?)

a-Linolenic acid C18:3 (c9,c12,c15) (?) (?) (?) (?) (?)

c-Linolenic acid C18:3 (c6,c9,c12) (?) (?) (?) (-) (?)e

Stearidonic acidf C18:4 (c6,c9,c12,c15) (?) (?) (?) (?) (?)

Nonadecanoic acid C19:2 (c10,c13) (?) (-) (-) (-) (-)

Eicosadienoic acid C20:2 (c11,c14) (?) (-) (-) (-) (-)

Homo-gamma linolenic C20:3 (c8,c11,c14) (?) (-) (-) (-) (-)

Eicosatrienoic acid C20:3 (c11,c14, c17) (?) (-) (-) (-) (-)

Arachidonic acid C20:4 (c5,c8,c11,c14) (?) (-) (-) (-) (-)

Eicosapentaenoic acidf C20:5 (c5,c8,c11,c14, c17) (?) (-) (-) (-) (-)

Heneicosadienoic acid C21:2 (c12,c15) (?) (-) (-) (-) (-)

Docosadienoic acid C22:2 (c13,c16) (?) (-) (-) (-) (-)

Docosatrienoic acid C22:3 (c13,c16,c19) (?) (-) (-) (-) (-)

Docosatetraenoic acid C22:4 (c7,c10,c13,c16) (?) (-) (-) (-) (-)

Docosapentaenoic acid C22:5 (c7,c10,c13,c16,c19) (?) (-) (-) (-) (-)

Docosahexaenoic acid C22:6 (c4,c7,c10,c13,c16,c19) (?) (-) (-) (-) (-)

a B. breve NCIMB 702258, NCIMB 8807, DPC6330, DPC6331b B. longum DPC6315, DPC6320c Prop. freudenreichii subsp. shermanii JS, 9093d Prop. freudenreichii subsp. freudenreichii Propioni-6, ATCC 6207e Only by Prop. freudenreichii subsp. freudenreichii Propioni-6f Strains assayed at a fatty acid concentration of 0.15 mg mL-1

Lipids (2012) 47:313–327 317

123

Table 4 Bioconversion of 0.45 mg mL-1 of selected PUFA to CLA, CLNA, CGLA and CSA by strains of Bifidobacterium and

Propionibacterium

Linoleic acid Concentration mg mL-1 Percentage bioconversion

Strain c9 t11 CLA t9 t11 CLA c9 t11 CLA t9 t11 CLA

Bifidobacterium

B. breve NCIMB 702258 0.254 ± 0.012 0.005 ± 0.001 58.94 1.08

B. breve NCIMB 8807 0.249 ± 0.019 0.028 ± 0.002 57.92 6.39

B. breve DPC6330 0.291 ± 0.004 0.009 ± 0.000 64.66 2.00

B. breve DPC6331 0.092 ± 0.001 0.004 ± 0.000 20.44 0.89

B. longum DPC6315 0.049 ± 0.002 0.003 ± 0.000 11.02 0.79

B. longum DPC6320 0.198 ± 0.004 0.007 ± 0.000 43.89 1.59

Propionibacterium

Prop. freudenreichii subsp. shermanii JSa 0.044 ± 0.001 0.005 ± 0.000 8.56 1.05

Prop. freudenreichii subsp. shermanii 9093a 0.243 ± 0.000 0.016 ± 0.000 47.37 3.17

Prop. freudenreichii subsp. freudenreichii Propioni-6a 0.214 ± 0.007 0.016 ± 0.003 41.63 3.02

Prop. freudenreichii subsp. freudenreichii ATCC 6207a 0.006 ± 0.000 0.002 ± 0.001 1.07 0.31

a-linolenic acid Concentration mg mL-1 Percentage bioconversion

Strain CLNA1 CLNA2 CLNA1 CLNA2

Bifidobacterium

B. breve NCIMB 702258 0.180 ± 0.013 0.019 ± 0.014 45.09 4.65

B. breve NCIMB 8807 0.234 ± 0.002 0.038 ± 0.000 58.85 9.44

B. breve DPC6330 0.282 ± 0.013 0.049 ± 0.002 70.79 12.18

B. breve DPC6331 0.028 ± 0.000 0.003 ± 0.000 6.91 0.79

B. longum DPC6315 0.000 ± 0.000 0.000 ± 0.000 0.10 0.00

B. longum DPC6320 0.000 ± 0.000 0.000 ± 0.000 0.00 0.00

Propionibacterium

Prop. freudenreichii subsp. shermanii JS 0.018 ± 0.001 0.001 ± 0.000 3.52 0.19

Prop. freudenreichii subsp. shermanii 9093 0.258 ± 0.017 0.017 ± 0.002 50.34 3.23

Prop. freudenreichii subsp. freudenreichii Propioni-6 0.043 ± 0.004 0.003 ± 0.000 8.41 0.53

Prop. freudenreichii subsp. freudenreichii ATCC 6207 0.008 ± 0.000 0.000 ± 0.000 1.65 0.00

c-linolenic acid Concentration mg mL-1 Percentage bioconversion

Strain CGLA1 CGLA2 CGLA1 CGLA2

Bifidobacterium

B. breve NCIMB 702258 0.100 ± 0.010 0.049 ± 0.012 25.13 12.34

B. breve NCIMB 8807 0.004 ± 0.000 0.001 ± 0.001 1.05 0.16

B. breve DPC6330 0.058 ± 0.003 0.023 ± 0.001 14.64 5.71

B. breve DPC6331 0.005 ± 0.001 0.002 ± 0.000 1.24 0.51

B. longum DPC6315 0.004 ± 0.005 0.004 ± 0.004 1.08 0.95

B. longum DPC6320 0.002 ± 0.003 0.000 ± 0.000 0.55 0.00

Propionibacterium





318 Lipids (2012) 47:313–327

123

60 80 100 120 140 160 180 200 220 240 260 280

10

20

30

40

50

60

70

80

90

m/z

Abu

nd

ance

%

55

6781

95

107

121 135

149

163

173

191

213223

243

261263

292

M+

91

CH3OOC

149163

223191

69

60 80 100 120 140 160 180 200 220 240 260 280 300 320

10

20

30

40

50

60

70

80

90

m/z

Abu

nd

ance

%

5567

7998

113

126

152 168 182196

222

208 234248

262

288302 316

331

M+

N

O

262

196

208

222

234 288

a

b

Fig. 1 a Mass spectrum of the

FAME, b Mass spectrum of the

DMOX derivative, of 9,11,15-

C18:3 (CLNA1)

Table 4 continued

Stearidonic acidb Concentration mg mL-1 Percentage bioconversion

Strain CSA1 CSA2 CSA1 CSA2

Bifidobacterium

B. breve NCIMB 702258 0.030 ± 0.001 0.008 ± 0.000 19.85 5.15

B. breve NCIMB 8807 0.014 ± 0.001 0.002 ± 0.000 9.49 1.46

B. breve DPC6330 0.039 ± 0.001 0.002 ± 0.000 25.81 1.52

B. breve DPC6331 0.022 ± 0.000 0.006 ± 0.000 14.80 3.74

B. longum DPC6315 0.004 ± 0.000 0.001 ± 0.000 3.04 0.87

B. longum DPC6320 0.029 ± 0.000 0.003 ± 0.000 19.25 2.11

Propionibacterium





a 0.5 mg mL-1

b 0.15 mg mL-1

Lipids (2012) 47:313–327 319

123

respectively. Comparisons of the GLC retention time of

these isomers and RP-HPLC analysis would suggest that

these isomers share equivalence with the t9,t11 CLA iso-

mer, which is found in small quantities along with the

predominant c9,t11 CLA isomer following conjugation of

linoleic acid by strains of bifidobacteria and propionibac-

teria (this isomer may also be produced as a methylation

artefact from c9,t11 CLA) [23, 27] (Fig. 4).

60 80 100 120 140 160 180 200 220 240 260 280

10

20

30

40

50

60

70

80

90

m/zA

bun

dan

ce %

Abu

nd

ance

%A

bun

dan

ce %

Abu

nd

ance

%

55

67

79

9193

105

121 135 147 161175

191 218 243

292M+

60 80 100 120 140 160 180 200 220 240 260 280 300 320

10

20

30

40

50

60

70

80

90

m/z

5572

91

98

126

113

152166 192

206

218

246

232

260

274 288302 316

331

M +

N

O

2 0 6

2 1 81 9 2

1 8 0

1 6 6

2 3 2

2 4 6

50 100 150 200 250 300 350

10

20

30

40

50

60

70

80

90

m/z

5582 109 135

166193

250

374 405

C H 3 O O C

N N

N

( C H 2 )5 C H 3

C H 3

OO

2 5 0

M +

60 80 100 120 140 160 180 200 220 240 260 280 300 320

10

20

30

40

50

60

70

80

90

m/z

55 7285 98

113

126

154140 166 194

206

220 232 246

331

M +

N C

O

19 4

206

22 0

232

2 46

a

b

c

d



DMOX derivative, c Mass

spectrum of the MTAD adduct,

d Mass spectrum of the

pyrrolidide derivative, of

6,9,11-C18:3 (CGLA1)

320 Lipids (2012) 47:313–327

123

Production of Conjugated Fatty Acids by B. breve

DPC6330

Of the strains assayed in the present study, B. breve

DPC6330 was the most effective in terms of ability to

convert linoleic, a-linolenic, c-linolenic and stearidonic

acids to their respective conjugated isomers (Table 4).

Consequently, this strain was selected for further studies to

assess the impact of incrementally increasing the concen-

tration of substrate fatty acids on the production of the

60 80 100 120 140 160 180 200 220 240 260 280

10

2030

4050

6070

8090

m/z

Abu

nd

ance

%A

bun

dan

ce %

Abu

nd

ance

%A

bun

dan

ce %

55

67

79

91

105

119

133

147

161

189

171

221

261

290

M+

60 80 100 120 140 160 180 200 220 240 260 280 300 320

10

20

30

40

50

60

70

80

90

m/z

5567 79

91

113

126

152166

180192

218 232

246

260

286300 314

329

274

M+

206

2 86

2 74N

O

50 100 150 200 250 300 350

10

20

30

40

50

60

70

80

90

m/z

5580 116

133166

191

248

288372 403

M +

24 8

C H 3O O C

N N

N

C H 3

OO

60 80 100 120 140 160 180 200 220 240 260 280 300 320

10203040

50607080

90

m/z

55

72

8598

113

126140 194

206

232 246 260286 300

329

M +

1 9 4

2 0 6

2 2 0

2 3 2

2 4 6

N C

O

2 6 0

2 8 6

a

b

c

d



DMOX derivative, c Mass

spectrum of the MTAD adduct

d Mass spectrum of the

pyrrolidide derivative, of

6,9,11,15-C18:4 (CSA1)

Lipids (2012) 47:313–327 321

123

conjugated fatty acid isomers CLNA, CGLA and CSA, as

well as to assess the time-scale of conjugated fatty acid

production.

Increasing the concentration of the substrate a-linolenic

acid from 0.15 to 0.45 mg mL-1 resulted in an increase in

the concentration of CLNA1 and the putative CLNA2 iso-

mers following 72 h incubation (Table 5). However, the

effect of increasing substrate concentration on the per-

centage bioconversion of a-linolenic acid to CLNA was less

profound (Table 5). In an attempt to elucidate the time-

scale for the production of both CLNA1 and the putative

CLNA2 isomers by B. breve DPC6330, growth and CLNA

production by the strain was monitored over 80 h in cys-

MRS containing 0.3 mg mL-1 a-linolenic acid (n = 3)

(Fig. 5). Following the initial inoculation at 8.0 log

cfu mL-1, production of both the CLNA1 and putative

CLNA2 isomers corresponded with a 16-h period of loga-

rithmic growth by the strain (0–0.292 mg mL-1 CLNA 1,

and 0–0.016 mg mL-1 CLNA 2, respectively) (Fig. 5).

Increasing the concentration of the substrate c-linolenic

acid from 0.15 to 0.45 mg mL-1 resulted in subsequent

increases in the total concentration of the CGLA1 isomer

produced by B. breve DPC6330, but a decrease in the

percentage bioconversion of c-linolenic acid to CGLA1

0

10

20

30

40

50

30 35 40 45 50

Retention time (min)In

tens

ity (

mV

)

1

3

2 4

5

6

5

15

25

30 35 40 45 50

Retention time (min)

1

8

7

3

2

9

5

10

15

20

25

30

30 35 40 45 50

Retention time (min)

3

2

1

12

11

10

b

c

a

Inte

nsity

(m

V)

Inte

nsity

(m

V)

Fig. 4 GLC profiles of B. breveDPC6330 grown in cys-MRS

containing 0.3 mg mL-1 of the

PUFA a a-linolenic acid,

b c-linolenic acid and

c stearidonic acid, following

80 h anaerobic incubation.

1 Stearic acid, 2 Vaccenic acid,

3 Oleic acid, 4 a-linolenic acid,

5 CLNA1, 6 CLNA2,

7 c-linolenic acid, 8 CGLA1,

9 CGLA2, 10 Stearidonic acid,

11 CSA1, 12 CSA2

322 Lipids (2012) 47:313–327

123

was obtained (Table 5). Increasing the concentration of the

substrate c-linolenic acid from 0.15 to 0.45 mg mL-1 did

not increase the concentration of c-linolenic acid biocon-

verted to the putative CGLA2 isomer (Table 5). Growth

and CGLA production by B. breve DPC6330 was moni-

tored over 80 h in cys-MRS containing 0.3 mg mL-1

c-linolenic acid (n = 3) (Fig. 5). Conjugated fatty acid

production occurred during a 16-h period of logarithmic

growth by B. breve DPC6330 (Fig. 5). This resulted in

CGLA1 concentrations increasing from 0 to 0.035 mg mL-1

during this period.

When the concentration of substrate stearidonic acid

was increased from 0.15 to 0.3 mg mL-1, a subsequent

increase in the concentration of CSA1 produced by

B. breve DPC6330 was observed along with a small

increase in the concentration of the putative CSA2 isomer

(Table 5). Increasing the substrate concentration from 0.15

to 0.3 mg mL-1 resulted in a 2.64% increase in the per-

centage bioconversion of stearidonic acid to CSA1, and a

0.55% decrease in the percentage bioconversion of steari-

donic acid to the putative CSA2 isomer (Table 5). At a

stearidonic acid concentration of 0.45 mg mL-1, growth of

B. breve DPC6330 was completely inhibited and neither

CSA isomer was produced (Table 5). CSA production by

B. breve DPC6330 from stearidonic acid was characterised

over an 80 h-period in cys-MRS containing 0.3 mg mL-1

of stearidonic acid (n = 3) (Fig. 5), and it was observed

that following 3 h exposure to stearidonic acid the viability

of the strain declined steadily (by 3.83 log cfu mL-1

following 20 h) (Fig. 5). Subsequently, following this time

an increase in cell numbers was observed from 4.30 to 6.70

log cfu mL-1 during which time the production of both

CSA1 and the putative CSA2 isomer was observed

(Fig. 5).

We assessed the substrate preference of B. breve

DPC6330 to isomerise the C18 unsaturated fatty acids

linoleic acid, a-linolenic acid, c-linolenic acid and steari-

donic acid (0.3 mg mL-1) to CLA, CLNA1, CGLA1 and

CSA1 over an 80-h fermentation period, relative to the

percentage bioconversion of linoleic acid to the c9,t11

CLA isomer which was set at 100% (Table 5). We found

that B. breve DPC6330 has a preference for the conjugation

of C18 fatty acid substrates in the order: a-linolenic acid

(130%) [linoleic acid (100%) [stearidonic acid (27%)

[c-linolenic acid (14%).

Discussion

In this study, we report that certain strains of bifidobacteria

and propionibacteria possess the ability to bioconvert

substrate fatty acids containing a c9,c12 double bond to

their conjugated isomers. Two conjugated fatty acids

products were produced by most strains from each

unconjugated fatty acid substrate, which were believed to

share equivalence with the c9,t11 CLA and t9,t11 CLA

isomers produced by certain bifidobacteria and propioni-

bacteria from linoleic acid, although the latter isomer has

Table 5 Effect of substrate concentration on the production of conjugated fatty acids CLNA, CGLA and CSA and the concentration of residual

substrate following 72 h anaerobic fermentation

Strain Substrate

concentration

(mg mL-1)

Concentration

mg mL-1Percentage

bioconversion

c9,t11 CLA t9,t11 CLA c9,t11 t9,t11

B. breve DPC6330 0.15 0.101 ± 0.000 0.003 ± 0.000 67.73 2.00

B. breve DPC6330 0.3 0.209 ± 0.002 0.003 ± 0.000 69.67 1.00

B. breve DPC6330 0.45 0.252 ± 0.004 0.010 ± 0.002 56.00 2.22

CLNA1 CLNA2 CLNA1 CLNA2

B. breve DPC6330 0.15 0.113 ± 0.001 0.002 ± 0.000 87.80 1.71

B. breve DPC6330 0.3 0.272 ± 0.007 0.004 ± 0.000 89.93 1.34

B. breve DPC6330 0.45 0.282 ± 0.013 0.049 ± 0.002 70.79 12.18

CGLA1 CGLA2 CGLA1 CGLA2

B. breve DPC6330 0.15 0.026 ± 0.001 0.009 ± 0.000 20.81 6.99

B. breve DPC6330 0.3 0.047 ± 0.011 0.009 ± 0.002 16.63 3.09

B. breve DPC6330 0.45 0.058 ± 0.003 0.023 ± 0.001 13.60 5.30

CSA1 CSA2 CSA1 CSA2

B. breve DPC6330 0.15 0.039 ± 0.001 0.002 ± 0.000 25.81 1.52

B. breve DPC6330 0.3 0.064 ± 0.005 0.002 ± 0.000 28.45 0.97

B. breve DPC6330 0.45 0.000 ± 0.000 0.000 ± 0.000 0.00 0.00

Lipids (2012) 47:313–327 323

123

also been known to be produced from c9,t11 CLA under

methylation conditions [23, 27, 28]. GLC-MS confirmed

that the strains used in the present study catalysed the

bioconversion of the c9,c12 double bond of the substrate

fatty acids linoleic, a-linolenic, c-linolenic and stearidonic

acids, to the conjugated 9,11-double bond form via linoleic

acid isomerase activity. These observations correspond

with the findings of others who identified the production of

CLNA1 and CGLA1 by Lactobacillus strains via the

activity of the enzyme linoleic acid isomerase [24, 38]. In

these studies, the bond conformation of CLNA1 was

identified as c9,t11,c15-C18:3, with that of CGLA1 deter-

mined to be c6,c9,t11-C18:3 [18, 24, 38].

The extent to which a-linolenic, c-linolenic and steari-

donic acids were bioconverted to their respective conjugated

fatty acid isomers differed substantially between the strains

and among the fatty acid substrates used in the present study.

Differences in the extent to which bacterial strains conju-

gated linoleic acid to CLA have been reported with some

regularity and attributed to variations in the toxicity of the

substrate fatty acids to the strains [26–29], but may also be a

result of the bond position of the substrate fatty acids [39].

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0 10 20 30 40 50 60 70 80

Time(h)Fa

tty a

cid

conc

entr

atio

n (m

g/m

l)Fa

tty a

cid

conc

entr

atio

n (m

g/m

l)Fa

tty a

cid

conc

entr

atio

n (m

g/m

l)

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

Cel

l cou

nt (

logc

fu/m

l)C

ell c

ount

(lo

gcfu

/ml)

Cel

l cou

nt (

logc

fu/m

l)

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0 10 20 30 40 50 60 70 80

Time(h)

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0 10 20 30 40 50 60 70 80

Time(h)

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

CLNA

CGLA

CSA

Fig. 5 Growth and CLNA/

CGLA/CSA production by

B. breve DPC6330 over 80 h in

the presence of 0.3 mg mL-1

a-linolenic acid/c-linolenic acid/

stearidonic acid. CLNA1/

CGLA1/CSA1 (squares),

CLNA2/CGLA/CSA2 (filledtriangles), a-linolenic acid/

c-linolenic acid/stearidonic acid

(diamonds) and log cfu/ml

(filled circles)

324 Lipids (2012) 47:313–327

123

Increasing the concentration of the substrate fatty acid

resulted in increased conjugated fatty acid production by

B. breve DPC6330, but also to increased residual substrate

concentrations. These observations may be, respectively,

attributed to the increased availability of substrate fatty acid,

and to the substrate concentration exceeding the isomerising

capacity of the growing culture [28, 40, 41].

Of the strains assayed, B. breve DPC6330 was most

efficient in terms of the bio-production of conjugated fatty

acids from linoleic, a-linolenic, c-linolenic and stearidonic

acid with results suggesting a higher affinity of the strain to

conjugate a-linolenic acid than c-linolenic acid. Although

stearidonic acid substantially inhibited the growth of

B. breve DPC6330, CSA production from stearidonic acid

exceeded CGLA production from c-linolenic acid at a

substrate fatty acid concentration of 0.3 mg mL-1. The

inhibition of bacterial growth by stearidonic acid is likely

related to the ability of certain fatty acids to disrupt cell

membrane composition and cell energetics [42–44].

However, when the conjugation of linoleic, a-linolenic,

c-linolenic, and stearidonic acids by the strain were viewed

together, it was apparent that the presence of an additional

double bond towards the distal end of the fatty acid mol-

ecule (e.g. a-linolenic acid) encouraged conjugation by the

strain, while the addition of a double bond between the

carboxyl group and the c9,c12 double bond (e.g. c-linolenic

acid) discouraged conjugation of the double bond.

In this study, we found that in particular, when c-lino-

lenic acid and stearidonic acid were provided as fatty acid

substrate to B. breve DPC6330 the sum of the concentra-

tion of conjugated fatty acids produced and residual sub-

strate following fermentation was not equivalent to the

quantity of substrate initially provided. Although it would

require the use of labelled fatty acids to discern the exact

fate of this missing substrate, it is likely that this difference

is attributable to bifidobacterial metabolic activity [45–47].

Overall, this study has demonstrated the ability of

strains of certain commensal bifidobacteria and dairy pro-

pionibacteria to produce a range of novel conjugated fatty

acids from PUFA containing the c9,c12 double bond sys-

tem. If bioactive in vivo, the production of such conjugated

fatty acids by commensals and potential probiotics offers a

valuable strategy for the production and delivery of these

novel conjugated fatty acids to the GIT [25, 48–50]. Fur-

thermore, the production of conjugated fatty acids such as

CLNA1, CGLA1 and CSA1 by potentially probiotic bifi-

dobacteria may present a valuable strategy for the in situ

production of bioactive molecules in the human GIT. As

the substrate fatty acids can be found in the human diet, it

is not unreasonable to assume their in vivo production

occurs [51–53]. Indeed, further credence is given to this

theory in light of the evidence pertaining to the ex vivo and

in vivo production of CLA from linoleic acid [31, 32].

Acknowledgments We would like to thank Seamus Aherne and

Lina Cordeddu for their technical assistance. The assistance with

GLC-MS by Mylnefield Lipid Analysis is gratefully acknowledged.

A. A. Hennessy is in receipt of a Teagasc Walsh Fellowship. This

research was funded by EU project QLK1-2001-02362 and by Ali-

mentary Pharmabiotic Centre (APC).

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