Analytical MethodsQuantitative and qualitative determination of CLA produced by Bifidobacterium and lactic acid bacteria by combining spectrophotometric and Ag+-HPLC techniques
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.
References (34)
- et al.
Properties and biotechnological methods to produce lipids containing conjugated linoleic acid
European Journal of Lipid Science and Technology
(2008) - et al.
Derivatization of fatty acids and its application for conjugated linoleic acid studies in ruminant meat lipids
Journal of the Science of Food and Agriculture
(2005) - et al.
Potentially probiotic açai yogurt
International Journal of Dairy Technology
(2008) - et al.
Production of free conjugated linoleic acid by lactobacillus acidophilus and lactobacillus casei of human intestinal origin
Journal of Dairy Science
(2003) - et al.
Probiotic buttermilk-like fermented milk product development in a semiindustrial scale: Physicochemical, microbiological and sensory acceptability
International Journal of Dairy Technology
(2009) - et al.
Detection of conjugated diene isomers of linoleic acid in liver lipids of rats fed a choline-devoid diet indicates that the diet does not cause lipoperoxidation
The Journal of Nutritional Biochemistry
(1995) - et al.
Rapid screening method for analyzing the conjugated linoleic acid production capabilities of bacterial cultures
Applied Environmental Microbiology
(2007) - et al.
Nutritional regulation of milk fat synthesis
Annual Review of Nutrition
(2003) - et al.
Influence of processing on the fatty acid composition and the content of conjugated linoleic acid in organic and conventional dairy products – A review
Lait
(2007) - et al.
Determination of conjugated linoleic acid in human plasma by fast gas chromatography
Journal of Chromatography A
(2007)
Dietary sources of conjugated dienoic isomers of linoleic acid, a newly recognized class of anticarcinogens
Journal of Food Composition and Analysis
Conjugated linoleic acid biosynthesis by human-derived Bifidobacterium species
Journal of Applied Microbiology
Optimization of a reconstituted skim milk based medium for enhanced CLA production by bifidobacteria
Journal of Applied Microbiology
Esterases of lactic acid bacteria and cheese flavour: Milk fat hydrolysis, alcoholysis and esterification
International Dairy Journal
Biological activities of conjugated linoleic acid (CLA) and effects of CLA on animal products
Livestock Science
The composition of bovine milk lipids: January 1995 to December 2000
Journal of Dairy Science
Dynamic features of the rumen metabolism of linoleic acid, linolenic acid and linseed oil measured in vitro
Lipids
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These authors contributed equally.