Development of a Rapid Method for the Screening of Conjugated Linoleic Acid (CLA)-Producing Strains of Bifidobacterium breve

Abstract

This study was performed to isolate some strains of Bifidobacteriumbreve from fecal materials of neonates and to screen them for the biotransformation activity of converting linoleic acid into conjugated linoleic acid (CLA). Fecal samples were collected from twenty healthy neonates between 14 and 100 days old, and four hundred colonies were randomly selected from a Bifidobacterium selective transoligosaccharide medium. A duplex polymerase chain reaction technique was developed for the rapid and accurate molecular characterization of the B. breve strains that have been reported to show the species-specific characteristic of CLA production. They are identified by 16S ribosomal DNA, fructose-6-phosphate phosphoketolase encoding genes (xfp), and rapid pulsed field gel electrophoresis. Thirty-six isolates were identified as B. breve, and just two of the 12 neonates were harboring B. breve strains. Each isolate showed different CLA-producing ability in the spectrophotometric assay. All of the positive strains from the primary spectrophotometric assay were confirmed for their CLA-producing activities using gas-chromatographic analysis, and their conversion rates were different, depending on the strain isolated in this study. Some strains of B. breve were successfully isolated and characterized based on the CLA-producing activity, and further studies are necessary to characterize the enzyme and the gene responsible for the enzyme activity.

Full text

Korean Journal for Food Science of Animal Resources
Korean J. Food Sci. An. 2018 August 38(4):806~815 pISSN : 1225-8563 eISSN : 2234-246X
DOI https://doi.org/10.5851/kosfa.2018.e34 www.kosfa.or.kr
© Korean Society for Food Science of Animal Resources. This is an open access article distributed under the terms of the Creative Commons Attribution Non-Commercial License
(http://creativecommons.org/licences/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and r eproduction in any medium, provided the original work is properly cited.
Received July 30, 2018
Revised August 7, 2018
Accepted August 8, 2018
*Corresponding author : Geun-Bae Kim
Department of Animal Science and
Technology, Chung-Ang University,
Anseong 17546, Korea
Tel: +82-31-670-3027
Fax: +82-31-676-5986
E-mail: kimgeun@cau.ac.kr
† These authors contributed equally to this
work.
Development of a Rapid Method for the Screening of
Conjugated Linoleic Acid (CLA)-Producing Strains
of Bifidobacterium breve
Sun-Hae Choi†, Kyoung-Min Lee†, Kwan-Hu Kim, and Geun-Bae Kim*
Department of Animal Science and Technology, Chung-Ang University, Anseong
17546, Korea
Abstract This study was performed to isolate some strains of Bifidobacterium breve
from fecal materials of neonates and to screen them for the biotransformation activity of
converting linoleic acid into conjugated linoleic acid (CLA). Fecal samples were
collected from twenty healthy neonates between 14 and 100 days old, and four hundred
colonies were randomly selected from a Bifidobacterium selective transoligosaccharide
medium. A duplex polymerase chain reaction technique was developed for the rapid and
accurate molecular characterization of the B. breve strains that have been reported to
show the species-specific characteristic of CLA production. They are identified by 16S
ribosomal DNA, fructose-6-phosphate phosphoketolase encoding genes (xfp), and rapid
pulsed field gel electrophoresis. Thirty-six isolates were identified as B. breve, and just
two of the 12 neonates were harboring B. breve strains. Each isolate showed different
CLA-producing ability in the spectrophotometric assay. All of the positive strains from
the primary spectrophotometric assay were confirmed for their CLA-producing activities
using gas-chromatographic analysis, and their conversion rates were different, depending
on the strain isolated in this study. Some strains of B. breve were successfully isolated
and characterized based on the CLA-producing activity, and further studies are necessary
to characterize the enzyme and the gene responsible for the enzyme activity.
Keywords probiotics, Bifidobacterium breve, duplex PCR, conjugated linoleic acid, rapid
screening
Introduction
The intestinal flora of an individual is composed of 100 trillion viable bacteria,
representing 100 or more different bacterial species. These organisms live together in
symbiotic or antagonistic relationships, growing on the food components ingested and
biocomponents secreted into the alimentary tract by the host (Mitsuoka, 2000).
Bifidobacteria are normal components of the intestinal flora throughout the life cycle.
They are ubiquitous, endosymbiotic inhabitants of the gastrointestinal tract, vagina, and
mouth. Bifidobacteria are the dominant species with Eubacterium, Clostridium, and
ARTICLE

Rapid Screening of CLA-producing Bifidobacterium breve Strains
807
Bacteroides in the colon flora, existing from 108 to 1011 per 1 g of colon contents (Mitsuoka, 1978).
Members of the genus Bifidobacterium are believed to exert various positive health benefits on their host. These beneficial
effects include anticarcinogenic activity (Biffi et al., 1997; Rafter et al., 2007; Reddy and Rivenson, 1993; Rowland et al.,
1998), modulation of immune response (Yasui and Ohwaki, 1991), improvement of the digestive system (Saavedra et al.,
1994), and lowering incidences of necrotizing enterocolitis in preterm neonates (Patole et al., 2016). According to a recent
report, there are some Bifidobacterium strains that can convert linoleic acid into bioactive conjugated linoleic acids (CLAs)
(Coakley et al., 2003; O’Shea et al., 2012).
The term “CLA” refers to a group of positional and geometric isomers of essential fatty-acid linoleic acid (LA, cis9, cis12-
C18:2) with conjugated double bonds, among which, the cis-9-,trans-11-ontadecadienoic acid (c9, t11 isomer) is approximately
80% of all possible CLA isomers (Pariza et al., 2001).
The conversion of linoleic acid to CLA takes place naturally in the rumen as part of the biohydrogenation process by the
action of rumen bacteria such as Butyrivibrio fibrisolvens (Griinari and Bauman, 1999). Therefore, CLA is present as a
natural component of ruminant meat as well as dairy products. There is increasing evidence of the potential health-promoting
properties of CLA isomers, including antiatherosclerosis (Lee et al., 1994), anticarcinogenesis (Tian et al., 2011),
enhancement of immunological function (Hayek et al., 1999), and reduction of body fat (Chaplin et al., 2015; Park et al.,
1997; West et al., 1998).
Many probiotic bacteria, especially some bifidobacteria, have been reported to exhibit bioconversion activity from LA
(linoleic acid) into CLA; however, this activity could be a strain-specific characteristic. From the screening of CLA
production with the major species of bifidobacteria, it has been demonstrated that the most-efficient CLA producers belong to
the species of B. breve (Barrett et al., 2007; Gorissen et al., 2010; Raimondi et al., 2016). In this study, a species-specific
duplex polymerase chain reaction (PCR) technique for the selection of many strains belonging to the species of B. breve was
developed, and this technique was successfully combined with a spectrophotometric assay for the rapid screening of probiotic
candidates harboring CLA-producing activity.
Materials and Methods
Bifidobacterium isolation from fecal materials of neonates
First, traditional colonies from neonatal feces were propagated under anaerobic conditions in transoligosaccharide (TOS)
agar (Yakult Honsha, Japan) at 37℃ for 48 h (Thitaram et al., 2005).
Bifidobacterium isolates were activated by successive subculturing into a Man-Rogosa-Sharpe (MRS) (Difco, USA) broth
supplemented with 0.05% L-cysteine·HCl and stored in 10% skim milk supplemented with glycerol in a –80℃ deep freezer.
As control strains, B. breve BB5 (Korea Yakult R & D Center) and B. breve LMC520 (Choi et al., 2008) were used for the
CLA production activity.
Identification of the isolates
Duplex PCR
Colonies grown on TOS agar were selected, and their genomic DNA was extracted with Lyse-N-Go (Pierce, USA) reagent
according to the manufacturer’s protocol. The primers used in the duplex PCR were a genus-specific primer based on xfp genes,
which encode fructose-6-phosphate phosphoketolase, and species-specific primers based on 16S rDNA from B. breve (Table 1).

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16S rDNA partial sequencing
Colonies grown on TOS agar were selected, and the genomic DNA was extracted with Lyse-N-Go reagent, as
recommended. Then, DNA was added in a mixture of 25 µL HotSTARTaq Master mix (Qiagen, USA), and a 1 µL forward
and reverse primer set (GP116 & GP120) was used. The PCR product was purified using a PCR purification kit (Qiagen,
USA) and identified by a Basic Local Alignment Search Tool (BLAST) search.
F6PPK encoding gene sequencing
Primers used in the duplex PCR were genus-specific primers (GP109 & GP111) based on the xfp gene. The PCR product
was ligated into the pGEM-T easy vector (Promega, USA). The ligation product was transformed E. coli JM109 (Takara,
Japan) and grown in Luria-Bertani (LB) agar supplemented with 50 µg/mL of ampicillin and X-gal (LAX agar) plate at 37℃
overnight. White colonies selected were inoculated in LB broth supplemented with 50 µg/mL of ampicillin and grown under
150 rpm at 37℃ for 8 h. Plasmids from recombinant colonies were prepared by a miniprep kit (Qiagen, USA) and sequenced
with a SP6 and T7 primer pair (Macrogen, Korea). The sequence was identified by BLAST search.
Rapid pulsed field gel electrophoresis (PFGE)
According to the method of Briczinski and Roberts (2006), 2 mL of the overnight cultured cell was centrifuged at 14,000
rpm for 10 min and resuspended with 600 µL of 100 mM Tris EDTA buffer (pH 7.6). 160 µL of sample was added to 40 µL
of lysozyme (100 mg/mL) and 10 µL of proteinase K (20 mg/mL) and mixed with an equal volume of 1.6% InCert agarose
(Bio-Rad Laboratories, USA) prepared in 0.1% SDS (International Biotechnologies, USA). Blended samples were dispended
into disposable plug molds. The plugs were incubated in 0.5 M EDTA, 1% sarkosyl buffer (pH 9.0) supplemented by 2 mL of
lysozyme (4 mg/mL) and mutanolysin (200 unit/mL) at 55℃ for 90 min. After lysis, samples were cultured in fresh sarkosyl
buffer supplemented with proteinase K (0.5 mg/mL) at 55℃ for 1 h and washed in preheated distilled water at 50℃ for 15
min. The plugs were washed three times with 10 mM EDTA buffer (pH 7.6) at 50℃ for 15 min under 75 rpm in a shaking
water bath. After lysis, the plugs were sliced at comb size and then transferred to 200 µL of a fresh restriction digest mixture
containing 50 units of Xba I and incubated at 37℃ for 2 h.
Electrophoresis was performed on 1% InCert agarose gel using a 0.5× TBE buffer (Bio-Rad Laboratories, USA). A lambda
Table 1. Primers used in this study
Primers Description Sequence (5’ to 3’) Product
size (bp) References
GP-107 Forward; 16S rDNA for B. breve CCGGATGCTCCATCACAC 250 Matsuki et al. (2002)
GP-108 Reverse; 16S rDNA for B. breve ACAAAGTGCCTTGCTCCCT Matsuki et al. (2002)
GP-109 Forward 1; xfp for Bifidobacterium TGGCAGTCCAACAAGCTC 923 This study
GP-111 Reverse ; xfp for Bifidobacterium TAGGAGCTCCAGATGCCGTG This study
GP-110 Forward 2; xfp for Bifidobacterium CATCGACGGCAAGAAGACCG 582 This study
GP-111 Reverse ; xfp for Bifidobacterium TAGGAGCTCCAGATGCCGTG This study
GP-116 Forward; 27F, universal for bacteria AGAGTTTGATCCTGGCTCAG 1,450
GP-120 Reverse; 1492R, universal for bacteria GGTTACCTTGTTACGACTT
GP-145 Forward; BSH gene for B. breve ATGTGCACTGGTGTCCGTTTC 954 This study
GP-146 Reverse; BSH gene for B. breve TCATCGGGCGACGCTGCTG This study

Rapid Screening of CLA-producing Bifidobacterium breve Strains
809
ladder (Bio-Rad Laboratories, USA) was included as a molecular size marker. An electrophoresis machine was operated
using a CHEF DR-Ⅲ (Bio-Rad Laboratories, USA). Operation times were increased linearly at 6 V/cm and 14℃. After
electrophoresis, the gel was stained with ethidium bromide (0.4 mg/L; Promega, USA) for 1 h and then destained for 2 h with
distilled water. The band pattern was confirmed using a UV transilluminator.
Screening of CLA-producing lactic-acid bacteria
Spectrophotometric assay
According to the method of Barrett et al. (2007), strains were incubated anaerobically in an mMRS broth containing
linoleic acid (0.5 mg/mL) and 2% (wt/vol) Tween 80 at 37℃ for 24 h. Following incubation, 1 mL of culture was centrifuged
at 20,800×g for 1 min, the pellet was discarded, and the supernatant (0.5 mL) was mixed with 0.5 mL of hexane by vortexing
for 2 min. 0.2 mL of the fatty acids extracted by vortexing the solution was mixed with 0.8 mL of methanol. The presence of
CLA in the culture supernatant was assayed using a spectrophotometer (smart plus SP-1900PC, Youngwoo, Korea) at 233 nm.
Gas chromatography (GC)
According to the method of Jung et al. (2006), strains were incubated anaerobically in a mMRS broth containing linoleic
acid (0.5 mg/mL) and 2% (wt/vol) Tween 80 at 37℃ for 24 h. 0.5 mL of the sample was mixed with 20 μL of stock (8 mL of
hexane, 100 mg of internal standard, C17:0) and 0.5 mL of hexane. After tilting for 10 min, the hexane layer was extracted by
centrifuge. The hexane layer was dried under nitrogen gas at 60℃ for 30 min.
Fatty acid was methylated in 1% HCl at 60℃ for 30 min. Fatty-acid methyl esters were produced by adding 2 mL of
saturated NaCl and 1 mL of hexane. The solvent was dried under nitrogen gas and concentrated with 100 µL of hexane.
Methylated fatty acids were analyzed with a gas chromatographer (Varian STAR 3400, USA) equipped with an SP-2560
column and a flame ionization detector.
Results and Discussion
Selection of bifidobacteria
Twelve neonatal feces were plated on TOS agar and incubated under anaerobic conditions at 37℃ for 48 h. Four hundred
typical milky-white colonies were selected from agar plate and were subcultured in MRS broth. Isolated strains were stored in
10% skim-milk-supplemented glycerol in a –80℃ deep freezer.
Identification of the genus Bifidobacterium
Duplex PCR
Conventional biochemical tests have some limitations in identifying specific species and discriminating a large number of
isolates. Thus, in this study, duplex PCR that can simultaneously accomplish the rapid identification and the correct
differentiation of B. breve was developed.
The PCR assay was conducted with genus-specific primers (Table 1; GP109 & GP111 and GP111 & GP110) for the genus
Bifidobacterium, producing products of 923 bp and 582 bp, respectively. As a result of PCR with species-specific primers
(GP107 & GP108), it was confirmed that 250 bp PCR product was obtained from only B. breve strains (Fig. 1).

Korean Journal for Food Science of Animal Resources Vol. 38, No. 4, 2018
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To increase the specificity of the PCR, a duplex PCR was conducted with multiplex primer sets, and two pairs of genus-
specific primer (GP109 & GP111, GP145 & GP146) and a pair of species-specific primer (GP107 & GP108) were used. After
the evaluation process, primer pairs (GP109 & GP111 and GP107 & GP108) producing products of 923 bp and 250 bp were
selected because this set was better than the other sets producing products of 582 bp and 250 bp in terms of the band
separation (Fig. 1A).
Through the duplex PCR assay developed in this study, we were able to confirm 36 strains B. breve from total 400 strains
of Bifidobacterium isolated from new born infants. Some of the typical PCR band pattern for B. breve strains, which produce
the PCR products of 923 bp and 250 bp are presented in Fig. 1B.
It is generally accepted that the predominant microorganisms in the colon of breast-fed babies are bifidobacteria, while the
colons of bottle-fed babies contain various other bacteria. Mitsou et al. (2007) reported that Bifidobacterium longum and
Bifidobacterium breve were the most frequently detected Bifidobacterium species in breast-fed infants.
In this study, B. breve was isolated from two infants among 12 infants. Only one strain among 40 strains from one person was
identified as a B. breve, but 31 strains among 40 strains from another person were identified as B. breve, proving significant
differences depending on the host.
16S rDNA partial sequencing
As a result of sequencing the 16S rDNA of B. breve by duplex PCR, all strains had high homology with B. breve,
indicating that all strains were B. breve. If the sequence of the 16S rRNA gene has more than 97% species sequence identity,
they are considered to be the same species (Stackebarandt and Goebel, 1994). Bifidobacterium breve ATCC 15700T and
commercial strain BB5 have 98% sequence identity, and B. breve ATCC 15700T and IB84 strain have 99% sequence identity.
As a result of comparing the nucleotide sequence by clustal W to confirm the difference between the strains of isolated B.
breve, all strains have the same 16S rDNA except BB5. Although it is difficult to identify the strain because of the high
homology of the DNA sequence between genus Bifidobacterium, studies, such as those on multiple PCR (Kwon et al., 2005;
Ventura et al., 2004) and DGGE (Satokari et al., 2001), have been conducted to identify Bifidobacterium, and it was found to
be a convenient and accurate method for the identification of strain level.
Fig. 1. Agarose gel electrophoresis of PCR products. (A) establishment of duplex PCR condition M: 1 kb ladder; lane 1: Bifidobacterium-
specific PCR product (923 kb) & B. breve-specific PCR product (250 bp); lane 2: Bifidobacterium-specific PCR product (582 bp) & B. breve-
specific PCR product (250 bp); lane 3: B. breve-specific PCR product (250 bp); lane 4: Bifidobacterium-specific PCR product (923 kb); lane 5
Bifidobacterium-specific PCR product (582 kb). (B) Identification of Bifidobactetrium breve. M: 1 kb ladder; lane 1–10: Bifidobacterium
strains isolated from infant feces. Dual bands of PCR products (lanes 1, 2, 4, 6, 7, 8, 9) represent B. breve strains and the others (lanes 3, 5,
and 10) are belonging to the other species of Bifidobacterium. PCR, polymerase chain reaction.

Rapid Screening of CLA-producing Bifidobacterium breve Strains
811
F6PPK encoding gene sequencing
PCRs were conducted with genus-specific primers, and the PCR products were cloned into a pGEM-T easy vector. As a
result of comparing the sequence by clustal W, the IB sample corresponded with B. breve ATCC 15700T, and the BB5 sample
and B. breve ATCC 15700T showed difference in six nucleotides.
Rosselló-Mora and Amman (2001) reported that the 16S rRNA gene sequence cannot distinguish clearly between
difference species belonging to the same genus of bacteria. Berthoud et al. (2005) identified Bifidobacterium by the xfp gene
and reported that the xfp gene sequencing is more accurate than the 16S rRNA gene sequencing, except for B. thermophilum,
B. thermacidophilum, and B. boum. In this study, it was also confirmed that in addition to 16S rRNA gene sequencing, xfp
gene sequencing could be used as a tool of identification for the isolated B. breve strains (data not shown).
Rapid PFGE
For the genotyping and genetic fingerprinting of the bacterial isolates, PFGE was applied using Xba I as restriction
enzymes. As shown in Fig. 2, PFGE analysis indicated that there was high correlation between new isolated strains (lane 2 to
lane 6) and different band pattern was observed from KCTC strains (land 8 to lane 11). Briczinski and Roberts (2006)
reported that there is no difference between a PFGE protocol consuming 5–7 days and a rapid protocol that takes 24 h. In this
study, PFGE was carried out using a rapid protocol, and it was confirmed that the band of the sample isolated from the same
infants also showed the same patterns (Fig. 2).
Screening of CLA producing lactic-acid bacteria
Spectrophotometric assay
The standard gas liquid chromatography-based screening process is laborious and time-consuming and can be a limiting
factor when a large number of strains are being tested. In this study, a simple and straightforward spectrophotometric method
was used for screening a large number of culture supernatants for the CLA production. Through the spectrophotometric assay,
the present study confirmed that B. breve isolated from neonatal feces produces CLA, but three species of B. breve received
Fig. 2. PFGE pattern of genomic DNA of Bifidobacterium strains after digestion by Xba I. Lane M, lambda ladder; lane 1, BB5; lane 2, IB26
lane 3, IB51; lane 4, IB55; lane 5, IB 62; lane 6, LMC520; lane 7, B. breve 2003; lane 8, B. breve KCTC 3220; lane 9, B. breve KCTC 3419; lane
10, B. breve KCTC 5081; lane 11, B. breve KCTC 3441; lane 12, B. longum BBL. PFGE, rapid pulsed field gel electrophoresis.

Korean Journal for Food Science of Animal Resources Vol. 38, No. 4, 2018
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from Korean Collection of Type Cultures (B. breve KCTC 3220, B. breve KCTC 3419, B. breve KCTC 5018) and B. breve
2003 do not produce CLA (Fig. 3).
As a result of the spectrophotometric assay, CLA-producing ability was different depending on the strains tested, and it was
observed that some of those isolates had higher CLA production activity than that of LMC520, which is one of the best CLA-
producing strains (Choi et al., 2008).
Pariza and Yang (1999) reported that CLA detection is possible through the spectrophotometric assay, and Barrett et al.
(2007) isolated and identified CLA-producing strains. In this study, the prescreening process was successful through the
spectrophotometric assay, and the fatty acid analysis was carried out by GC for a more accurate quantitative analysis (Fig. 3).
Gas chromatography (GC)
Strains that had a high optical density value in the spectrophotometer assay were analyzed by GC to find excellent strains
for the CLA production. First, the peak pattern of fatty-acid methyl ester (FAME) was analyzed using linoleic acid and
conjugated methyl ester (Sigma, USA).
It was observed that linoleic acid was converted to CLA by B. breve isolated from neonatal feces through chromatograms
(Fig. 4) with c9, t11-form as a major form of isomers of conjugated CLA. As shown in Fig. 5, conversion rates from LA into
CLA was different depending on the strains. Among the isolated B. breve strains, IB52 and IB84 strains showed more than 65%
conversion rate, which is comparable with LMC 520 strain (Choi et al., 2008). Many studies reported that free linoleic acid
inhibits the growth of CLA-producing strains (Jiang et al., 1998; Kim et al., 2000; Rainio et al., 2001), and Tween 80
neutralizes the antibacterial activity and increases the solubility of the fatty acid in the liquid medium (Jiang et al., 1998;
Rainio et al., 2001).
The study of the effect of the content of Tween 80 confirmed that the conversion rate of the medium containing 5% Tween
80 was higher than that of 2% Tween 80 (data not shown). The conversion rates of the isolates IB52, IB84, and LMC 520
were almost the same as that of LMC 520, and the conversion rate of IB84 was slightly higher than that of LMC 520.
Coakley et al. (2003) reported that B. breve and B. dentium have the highest productivity, comparing the conversation rate of
CLA by Bifidobacterium isolated from neonatal feces, and Oh et al. (2003) confirmed that B. breve and B. pseudocatenulatum
Fig. 3. Screening of B. breve isolates for the production of CLA using spectrophotometric assay. Negative control: B. longum ATCC
15700T; Positive control: LMC520; Commercial strain: BB5; Isolated strains from infant feces: IB7, IB13, IB29, IB30, IB31, IB32, IB52, IB54,
IB55, IB75, IB80, IB84. CLA, conjugated linoleic acid.

Rapid Screening of CLA-producing Bifidobacterium breve Strains
813
produce CLA. In this study, only B. breve had CLA-producing ability among 400 Bifidobacterium strains from neonatal feces.
Conclusion
Among the 400 strains identified out of 12 fecal isolates, 36 strains of B. breve were selected through duplex PCR. The
screening study showed that IB52, IB80, and IB84 had CLA-producing ability converted from linoleic acid. In this study,
using a duplex PCR for the selection of B. breve strains was a useful and precise method of selecting CLA-producing strains
from human feces. Selected strains are expected to be widely used in materials of fermented milk products and health food.
There are many studies that reported the efficacy of CLA-producing strains, including B. breve, through clinical trials. Further
studies are necessary to clarify CLA-producing ability from linoleic acid by B. breve in the body.
Acknowledgement
This research was supported by the Chung-Ang University Research Scholarship Grants in 2012.
Fig. 4. Chromatogram of conjugated linoleic acid (CLA) analysis with gas chromatograph. Heptadecanoic acid (C17:0) was used as an internal
standard. After 24 hr incubation, most of linoleic acid was converted into c9, t11-CLA and minor portion of t10, c12-CLA was also detected.
Fig. 5. Conversion rates of conjugated linoleic acids (cis-9, trans-11 isomer) from 24 h growth culture of Bifidobacterium breve strains in
MRS broth. The linoleic acid was added as a 50 mg/mL stock soluon containing 2% (v⁄v) Tween 80 and was previously filter sterilized
through a 0.45 μm filter. MRS, Man-Rogosa-Sharpe.

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