Effect of Pufa Substrates on Fatty Acid Profile of Bifidobacterium breve Ncimb 702258 and CLA/CLNA Production in Commercial Semi-Skimmed Milk

Abstract

Current research on lipids is highlighting their relevant role in metabolic/signaling pathways. Conjugated fatty acids (CFA), namely isomers of linoleic and linolenic acid (i.e. CLA and CLNA, respectively) can positively modulate inflammation processes and energy metabolism, promoting anti-carcinogenic and antioxidant effects, improved lipid profiles and insulin resistance, among others. Bioactive doses have been indicated to be above 1 g/d, yet these cannot be achieved through a moderate intake (i.e. 1-2 servings) of natural sources, and certain CLA-containing products have limited commercial availability. Such handicaps have fueled research interest in finding alternative fortification strategies. In recent years, screening of dairy products for CFA-producing bacteria has attracted much attention and has led to the identification of some promising strains, including Bifidobacterium breve NCIMB 702258. This strain has shown interesting producing capabilities in model systems as well as positive modulation of lipid metabolism activities in animal studies. Accordingly, the aim of this research work was to assay B. breve NCIMB 702258 in semi-skimmed milk to produce a probiotic fermented dairy product enriched in bioactive CLA and CLNA. The effect of substrates (LA, α-LNA and γ-LNA) on growth performance and membrane fatty acids profile was also studied, as these potential modifications have been associated to stress response. When tested in cys-MRS culture medium, LA, α-LNA and γ-LNA impaired the fatty acid synthesis by B. breve since membrane concentrations for stearic and oleic acids decreased. Variations in the C18:1 c11 and lactobacillic acid concentrations, may suggest that these substrates are also affecting the membrane fluidity. Bifidobacterium breve CFA production capacity was first assessed in cys-MRS with LA, α-LNA, γ-LNA or all substrates together at 0.5 mg/mL each. This strain did not produce CFA from γ-LNA, but converted 31.12% of LA and 68.20% of α-LNA into CLA and CLNA, respectively, after incubation for 24 h at 37 °C. In a second phase, B. breve was inoculated in a commercial semi-skimmed milk with LA, α-LNA or both at 0.5 mg/mL each. Bifidobacterium breve revealed a limited capacity to synthesize CLA isomers, but was able to produce 0.062-0.115 mg/mL CLNA after 24 h at 37 °C. However, organoleptic problems were reported which need to be addressed in future studies. These results show that although CFA were produced at too low concentrations to be able to achieve solely the bioactive dose in one daily portion size, fermented dairy products are a suitable vector to deliver B. breve NCIMB 702258.

Figure 1

Substrate tolerance of B. breve NCIMB 702258 in cys-MRS agar plates containing 0 (A), 1 (B), 2 (C) and 5 mg/mL (D) of LA and growth curves in the presence of 0.5 (E), 1 (F) and 2 mg LA/mL (G) after 48 h incubation at 37 °C, under anaerobic conditions. Optical density was recorded at 600 nm. Results are expressed as mean and standard deviation (n = 3).

Figure 2

Viable cell numbers of B. breve NCIMB 702258 [log (CFU/mL)] after 24 h incubation in cys-MRS culture medium or in commercial semi-skimmed UHT milk added with different unsaturated fatty acids. LA, α-LNA, γ-LNA or the three FA mixture (MIX) were in a final concentration of 0.5 mg/mL for each unsaturated fatty acid. γ-LNA was not assayed either alone or in MIX in semi-skimmed milk since previous assays showed no CFA production.

Figure 3

Substrate percentage reduction of different polyunsaturated fatty acids and conversion into corresponding conjugated fatty acids (%) by B. breve NCIMB 702258 in cys-MRS culture medium after 24 h incubation at 37 °C under anaerobic conditions.

Figure 4

Fatty acid composition of cys-MRS culture medium (A) and commercial semi-skimmed UHT milk (B) assayed for conjugated fatty acid production by B. breve NCIMB 702258 using linoleic (LA) and linolenic (α-LNA) acids as precursor substrates. A mixture of LA/α-LNA/γ-LNA in cys-MRS culture medium and LA/α-LNA in semi-skimmed UHT milk were assayed at a concentration of 0.5 mL/mg for each fatty acid.