Elsevier

Food Chemistry

Volume 125, Issue 4, 15 April 2011, Pages 1373-1378
Food Chemistry

Analytical Methods
Quantitative and qualitative determination of CLA produced by Bifidobacterium and lactic acid bacteria by combining spectrophotometric and Ag+-HPLC techniques

https://doi.org/10.1016/j.foodchem.2010.10.008Get rights and content

Abstract

Bifidobacterium and lactic acid bacteria (LAB), especially from the genera Lactobacillus and Lactococcus, are commonly used in the production of fermented dairy products due to their potential probiotic characteristics. Moreover, some strains of these microorganisms also have the ability to produce conjugated linoleic acid (CLA) from linoleic acid (LA), which has attracted much attention as a novel type of beneficial functional fermented milk. In the present work 22 probiotic bacteria were tested for the production of CLA, using a UV screening method and HPLC techniques. Five microorganisms, two strains of the genera Bifidobacterium, two Lactobacillus and one Lactococcus were selected for their ability to produce CLA after incubation in skim milk with free LA as a substrate. It was possible to quantify the production of CLA (in the range of 40–50 μg CLA/ml) and identify the CLA isomers produced as C18:2 cis 9, trans 11 (60–65%), C18:2 trans 10, cis 12 (30–32%), C18:2 trans 9, trans 11 and C18:2 trans 10, trans 12 (2–5%).

Introduction

Conjugated linoleic acid (CLA) is a mixture of positional and geometric conjugated isomers of the essential fatty acid linoleic acid (LA) with conjugated double bonds at carbon positions from 6–8 to 13–15. CLA isomers (C18:2 cis 9, trans 11 and C18:2 trans 10, cis 12) confer a number of beneficial biological effects that have been identified in a range of animal models and include anti-carcinogenesis, immuno-modulation, anti-atherosclerosis and reduction of whole body fat (Hur et al., 2007, Lin et al., 1999, Park and Pariza, 2007, Tanaka, 2005).

These compounds occur naturally in a variety of foods, including ruminant products such as milk-fat and meat which have been found to contain relatively large amounts of CLA. Dairy products from ruminants are very rich sources of CLA, among which 18:2 cis 9, trans 11, is the main isomer (Chin, Liu, Storkson, Ha, & Pariza, 1992). The presence of these compounds in dairy products is partly due to the isomerization and biohydrogenation of linoleic and linolenic acids that take place in the rumen; these processes are performed by ruminal bacteria, such as Butyrivibrio fibrisolvens and Megasphaera elsdenni (Bauman and Griinari, 2003, Jouany et al., 2007, Sieber et al., 2004). Such observation has raised the hypothesis that other microorganisms may also be able to produce CLA. This hypothesis and the fact that several fermented dairy products contain higher levels of CLA than non-fermented counterparts, created the possibility of producing fermented dairy products with high levels of CLA. Lactic acid bacteria (LAB), especially from the genera Lactobacillus, Bifidobacterium and Lactococcus, are commonly used, due to their potential probiotic characteristics, to produce fermented dairy products (Almeida et al., 2008, Antunes et al., 2009, Parvez et al., 2006). The identification of LAB able to produce CLA from a source of LA is of great importance since their use in the production of fermented dairy products will be of interest for human consumption as a probiotic dairy product with high CLA content.

Gas chromatography systems fitted with polar capillary columns and FID detectors are widely used in the fatty acid routine analysis (Jensen, 2002), as well as in the identification and quantification of minor compounds. However, when several isomers are presented, a combination of methodologies is needed. In analysis of CLA, GC has to be combined with Ag+-HPLC, in order to obtain a full resolution of all the CLA isomers in the sample (Bondia-Pons et al., 2007, Luna et al., 2005, Sehat et al., 1998). Furthermore HPLC and GC are time consuming but due to conjugated double bonds can be detected using a 233 nm wavelength; in this case, UV spectrophotometers are able to perform a simple and rapid measurement in the high CLA producer LAB screening assays (Barrett, Ross, Fitzgerald, & Stanton, 2007).

The aim of the present research work is to select CLA-producing bacteria in skim milk from a pool of potential probiotic LAB, by using a UV screening method to measure the CLA concentration followed by HPLC analytical techniques that are able to detect and identify the CLA isomers, toward their future application in the manufacture of fermented products.

Section snippets

Analytical reagents

All reagents used in the lab procedures were HPLC grade: hexane and isopropanol were obtained from Labscan (Dublin, Ireland), CLA isomers (C18:2 cis 9, trans 11 (rumenic acid) and C18:2 trans 10, cis 12) and linoleic acid (C18:2 cis 9, cis 12 from Sigma–Aldrich (St. Louis, MO, USA) and high CLA content oil (Tonalin®) from Cognis (Illertissen, Germany).

Bacterial strains

Twenty-two potentially probiotic strains were selected for this study; these included 16 strains of Lactobacillus, five strains of Bifidobacterium

Results

According to the spectrophotometric determination of CLA, the graph obtained from the standard curve demonstrated that an increase in the CLA concentration (from 0 to 0.30 μg/ml) coincided with a linear increase in absorbance (R2 = 0.993; y = 0.073x) for the C18:2 cis-9, trans 11 CLA isomer up to an absorbance of 2.2.

Nevertheless, in order to confirm the results, a second calibration curve was arranged using Tonalin® (CLA-TG80 oil; 80% CLA) in hexane solution applying the RA regression curve

Discussion

In addition to the increased interest in the physiological effects conferred upon humans following CLA consumption, there has been concomitant interest in the isolation of bacterial strains (Bifidobacterium and LAB, especially from the genera Lactobacillus and Lactococcus) with the ability to produce CLA in milk or dairy products (Alonso et al., 2003, Bisig et al., 2007, Ogawa et al., 2005, Sieber et al., 2004). Furthermore, the combination of UV spectrophotometric and chromatographic

Conclusion

This study has concluded that it is possible to use LAB in milk as CLA producing microorganisms using different sources of Linoleic acid (free acid or oil). The working conditions and the substrate used to perform these assays is critical, hence due to the isomer profile similar to that of the synthetic preparations (constituting a good alternative) and the high interest of dairy products containing CLA, corroborated by previous reports where LAB have shown high potential to isomerize the

Acknowledgements

This study was carried out with funds from the projects: CYTED-110AC0386, CM S-2009/AGR/1469, and CONSOLIDER-INGENIO CDS-2007-00063.

Financial support for author T.M. Braga was provided by a Ph.D. fellowship – BD/18667/2004 – issued by PRAXIS XXI (FCT, Portugal). The authors acknowledge Prof. Paula Teixeira and co-workers for the provision of probiotic isolates.

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