Conjugated Linoleic Acid Synthesis in Milk Fermented with Lactobacillus casei Strain AG

MATERIALS AND METHODS

Starter culture and growth conditions: The L. casei strain AG, isolated from a human intestine, was used in this study¹⁸. Bacterial cells were sub-cultured by plating on de Man-Rogosa-Sharpe agar (MRS, Merck, Jakarta, Indonesia) supplemented with 0.5 g L^–1 L-cysteine (Sigma-Aldrich Ltd., Singapore) and incubated at 37°C for 24 h under micro-aerobic conditions.

Milk fermentation and sample preparation: Fresh milk was pasteurised at 80°C for 30 min. The pasteurised milk was inoculated with a bulk starter L. casei strain AG (5% v/v) and incubated at 37°C overnight (18 h). Samples were then harvested by centrifugation at 5000 rpm/5°C for 30 min. The supernatants were collected for CLA analysis by gas chromatography-mass spectrometry (GC-MS), according to Alonso et al.¹

Fat extraction: Fat extraction was carried out according to Alonso et al.¹. Fermentation supernatants (6 mL) were mixed with 12 mL isopropanol and homogenised. The solution was then mixed with 9 mL hexane and centrifuged at 23,000 rpm/5°C for 5 min. The upper layer was collected, filtered using sodium sulphate and washed with 7 mL hexane. Evaporation of the hexane at room temperature (30-40°C) resulted in condensed fat. The esterification of the fat residue was carried out with 300 μL of 14% boron trifluoride in methanol at 50-60°C for 2 h. After esterification, 800 μL hexane was added and the sample was analyzed by GC-MS.

GC-MS analysis: GC-MS analysis was performed using a DB-1 Agilent J and W column (30 m×0.25 mm i.d.) with helium as the carrier gas and electron ionisation at 70 eV. The following conditions were used: a column heater temperature of 80°C, injection temperature of 310°C, injection model split, 16.5 kPa, 40 mL min^–1 total flow and 0.50 mL min^–1 column flow, with a split ratio of 73.2 mL injection volume. The CLA peak was identified by retention time comparisons.

Polymerase chain reaction (PCR) amplification: Genomic DNA was isolated using the Geneaid gSYNC™ DNA Extraction Kit (Geneaid Biotech Ltd., New Taipei City, Taiwan).

Table 1:	Primers used to amplify the partial homologue genes in L. casei strain AG

PCR amplification (Applied Biosystems thermal cycler 2720, Foster City, CA, USA) was performed using 2x KAPA Taq Readymix (Kapa Biosystems, Cape Town, South Africa), according to the manufacturer’s instructions. The primers used to detect CLA-synthesis genes were designed to recognise conserved regions of cla-hy, cla-dh and cla-dc genes, based on published L. casei genomes. The primer sequences were designed using primer 3 and are presented in Table 1. The following amplification programme was used: 1 min at 95°C, followed by 30 cycles of 30 sec at 94°C, 30 sec at 55°C, 2 min at 72°C and finally, 1 cycle of 5 min at 72°C. Amplified bands were resolved by electrophoresis on 2% (w/v) agarose gel (Sigma-Aldrich Ltd) and visualised by ethidium bromide staining.

The purified DNA was sequenced using the Applied Biosystem 3730-XL Analyser (1st Base, Singapore). The resulting sequences were compared using the National Centre for Biotechnology Information (NCBI) database. Phylogenetic trees were constructed in Mega 6.0.

Quantitative real-time PCR (qRT-PCR) analysis: Bacterial cultures were grown in a medium with or without the addition of 0.4 mg mL^–1 LA (Sigma-Aldrich Ltd.). LA was added as an aqueous solution at 1% (v/v) in Tween 80 (Merck). The broth medium was inoculated with 2% (v/v) of a bacterial starter culture and incubated at 37°C for 4 h. After incubation, cell cultures were harvested for total RNA isolation.

For qRT-PCR analysis, total RNA was extracted using the Geneaid Presto^TM Mini RNA Bacteria Kit (Geneaid Biotech Ltd., New Taipei City, Taiwan) with a DNAse treatment, according to the manufacturer’s instructions. RNA concentration was measured using spectrophotometry. RT-PCR (Real-Time PCR Bio-Rad CFX-96 thermal cycler, Singapore) was carried out using a KAPA SYBR^® FAST Universal One-Step qRT-PCR Kit (Kapa Biosystems) in duplicate. Relative quantification of the results was calculated using the equation 2^-ΔΔCT ¹⁹. Differences between the treatments and control were analyzed by independent t-tests using SPSS 16.0 (SPSS Inc., Chicago).

RESULTS AND DISCUSSION

CLA detection in fermented milk: CLA in milk fermented by the L. casei strain AG and corresponding non-fermented milk standard was detected in the form of fatty acid methyl esters (FAME). FAME forms of CLA and LA can be distinguished by GC-MS, due to their different retention times and same molecular weights. Fresh milk (non-fermented milk standard) used in this study contains LA but does not include CLA (data not shown). GC-MS results showed that milk fermented by the L. casei strain AG contained CLA (Fig. 1).

This study showed that the L. casei strain AG produced CLA isomers when it was used as a starter culture for milk fermentation (Fig. 1). Based on retention time, the chromatogram peak number 23 in milk fermented with the L. casei strain AG (Fig. 1) was close to the peak representing the CLA standard trans-10, cis-12-octadecadienoic acid. Meanwhile, the chromatogram peak number 19 was LA with a molecular weight (m/z) of 294. The presence of CLA in milk fermented by the L. casei strain AG was in agreement with the previous study by Alonso et al.¹. Using L. casei and L. acidophilus as starters to ferment skimmed milk, Alonso et al.¹ observed CLA production in milk supplemented with 0.02% LA. CLA isomers produced by L. casei were cis-9, trans-11-, trans-10, cis-12- and trans-9, trans-11-octadecadienoic acid. However, CLA isomers produced by the L. casei strain AG have not been previously identified.

Detection CLA-synthesising genes: The nucleotide sequences of cla-hy, cla-dh and cla-dc genes from L. plantarum AKU 1009a were aligned with the genome of L. casei strain Zhang and this approach revealed homologous of 1710, 861 and 876 bp, respectively. These homologue sequences were used to design primers cla-hy, cla-dh and cla-dc, with amplification products of 143, 206 and 161 bp, respectively. The length of the amplification products generated by these three primers was confirmed to be about 100-200 bp (Fig. 2).

The size of the amplification products obtained from L. casei strain AG, using the three selected primer pairs, correlated with the predicted target lengths.

Fig. 1:	Gas chromatography-mass spectrometry chromatogram, indicating the peaks corresponding to linolenic acid (LA) and conjugated linolenic acid (CLA)

Fig. 2:	Amplified bands obtained using the primers cla-hy (1), cla-dh (2) and cla-dc (3)

The amplified bands were then sequenced, to obtain nucleotide sequences. The nucleotide sequence of the cla-hy amplification product showed a 76% homology with the genes putatively encoding linoleate isomerase from Lactobacillus sakei strain LMG 13558 and 73% homology with the cla-hy gene from L. plantarum (Fig. 3). The nucleotide sequence of the cla-dh amplification product had 100% homology to the putative short-chain dehydrogenase/oxidoreductase gene from L. paracasei strain 1195 and 74% homology with the cla-dh gene from L. plantarum (Fig. 4). The nucleotide sequence of the cla-dc amplification product was 148 bp, instead of the expected 161 bp. This sequence was 77% homologous to the cla-dc gene for isomerase from L. plantarum (Fig. 5).

Some LAB, especially those belonging to the genus Lactobacillus, produce CLA from LA and ricinoleic acid. CLA production is a response to LA toxicity, as converting LA into CLA can reduce its toxicity²⁰. Previously, it was thought that CLA was generated as the product of LA catalysis by a single enzyme, linoleate isomerase. However, Kishino et al.¹⁶ showed that catabolism of LA into CLA involves multiple enzymes and reactions. The pathway for CLA synthesis has been described for L. plantarum AKU 1009a, which produces CLA isomers trans-10, cis-12-octadecadienoic acid and cis-9, trans-11-octadecadienoic acid. Synthesis of the CLA isomer trans-10, cis-12-octadecadienoic acid requires only a single enzyme that is expressed by the cla-hy gene. The other isomer, cis-9, trans-11-octadecadienoic acid is synthesized by two enzymes that are expressed by the cla-dh and cla-dc genes. The majority of species within the Lactobacillus genus possess one or more of these genes. The synthesis of CLA isomers depends on the enzyme cofactors FAD and NADH or NADPH.

Transcription of CLA-synthesising genes: The addition of LA in the growth medium was used to induce CLA synthesis.

Fig. 3:	Phylogenetic tree of cla-hy based on the sequence amplified from Lactobacillus casei AG

Fig. 4:	Phylogenetic tree of cla-dh based on the sequence amplified from Lactobacillus casei AG

Fig. 5:	Phylogenetic tree of cla-dc based on the sequence amplified from Lactobacillus casei AG

In this study, we measured the transcription levels of the cla-hy, cla-dh and cla-dc genes. The gene expression of cla-hy, cla-dh and cla-dc, when grown in m e dia with the addition of 0.4 mg mL^–1 LA, was normalised to an expression without the addition of the control.

The expression level of the cla-hy, cla-dh and cla-dc genes upon the addition of 0.4 mg mL^–1 LA was 0.74, 0.69 and 1.43, respectively. The addition of 0.4 mg mL^–1 LA in the growth medium did not change the expression of these genes (p>0.05). According to Alonso et al.¹, the addition of 0.4 mg mL^–1 LA in the growth medium of L. casei E4 and E10 increased CLA production. In the current study, the gene expression of cla-hy, cla-dh and cla-dc was not upregulated by the addition of 0.4 mg mL^–1 LA.

Milk fermented by the L. casei strain AG produced CLA and it possesses CLA-synthesising genes. However, the transcription activity of the cla-hy, cla-dh and cla-dc genes in the L. casei strain AG was not induced by the addition of 0.4 mg mL^–1 LA.

SIGNIFICANCE STATEMENT

This study discovers the synthesis of conjugated linoleic acid (CLA) in milk fermented with L. casei strain AG. The ability of L. casei AG to synthesise CLA in fermented milk can be beneficial for human health. This study will support the utilization of L. casei AG as starter culture for dairy fermentation and thus develop probiotic-based food industries.

ACKNOWLEDGMENT

This study was fully supported by Hibah Kompetensi, provided by the Indonesian Ministry for Research Technology and Higher Education.