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- Development of a new functional dairy product enriched in microbial bioactive conjugated fatty acids through an industrial-based approachPublication . Fontes, Ana Luiza Rodrigues; Gomes, Ana Maria Pereira; Alcalá, Luís Miguel Rodríguez; Domingues, Maria do Rosário Gonçalves dos Reis MarquesThe search for innovative food products with added-value properties has been an increasing tendency in the last years. Accordingly, different food compounds with potential bioactive properties have been identified, such as, conjugated linoleic acid (CLA) and conjugated linolenic acid (CLNA), which have been described with anti-carcinogenic, anti-obesity and anti-inflammatory activities, among others. These compounds are naturally found in meat and milk of ruminants or vegetables oils, but, due to availability and concentration concerns, it is not feasible to attain beneficial effects from CLA/CLNA through these sources. Alternatively, in situ microbial enrichment of food matrices has been studied, since several probiotic strains have been described with the capacity to isomerize linoleic acid (LA) and α-linolenic acid (α-LNA) into their corresponding conjugated forms. Thus, the objective of this work was to study the development of a new added-value dairy product through in situ microbial production of CLA and CLNA using previously hydrolyzed commercial vegetable oils as substrate sources, so as to evaluate possible side fatty acid (FA) metabolites released during the process, study the biochemical and organoleptic properties, and assess the shelf-life stability of the developed dairy product. First the selection of CLA/CLNA-producing strains was performed from among 85 probiotic strains through molecular detection of genes encoding enzymes involved in CLA/CLNA formation, namely, linoleate isomerase (LAI), myosin-cross-reactive antigen (MCRA) and fatty acid-hydratase (FA-HY), using reported primers and primers designed based on conserved motifs. Meanwhile, it was determined the strains’ maximum tolerance to LA after exposure to increasing concentrations thereof, namely 1, 2 and 5 mg/mL. About 34 strains revealed the presence of at least one of the screened genes, where the designed primers were more effective, but no association was found between their LA-tolerance and the CLA production potential. Moreover, only 4 genotypically-positive strains revealed the capacity to convert 0.5 mg/mL LA into CLA isomers. These were further tested for CLNA production from 0.5 mg/mL α-LNA. The strain Bifidobacterium breve DSM 20091 demonstrated the best yields of CLA and CLNA isomers (>50% of substrate conversion), being selected for the following assays. In a second phase, the utilization of commercial vegetables oils as substrate sources for the development of a CLA/CLNA-enriched fermented milk was studied. To increase the bioavailability of LA and α-LNA present in the selected oils, i.e., soybean (43.7 g LA/100 g oil) and flaxseed (41.3 g α-LNA/100 g oil) oils, different lipases were tested. The Candida rugosa lipase showed the best yields for all of the tested oils (>90% of hydrolysis). Bifidobacterium breve DSM 20091 was thereafter assayed in milk containing 0.5 mg/mL of LA and/or α-LNA obtained from the hydrolyzed oils. Results showed that this strain was not able to produce CLA simultaneously with CLNA to the same extent. Therefore, higher substrate concentrations (i.e., α-LNA) were further tested with only hydrolyzed flaxseed oil. The best yields were attained at 2 mg/mL α-LNA, registering ca. 1 mg/g of mainly CLNA isomers. To evaluate possible FA metabolites, a gas chromatography-mass spectrometry (GC-MS) analysis of milk fermented with pure LA and/or α-LNA or hydrolyzed flaxseed oil was performed. No further FA compounds were found that could result from LA or α-LNA metabolization; however, two additional CLNA isomers never reported before for bifidobacteria strains were discovered. The biochemical and organoleptic properties of the developed CLNA-enriched fermented milk were thereafter evaluated through analysis of sugars and organic acids content, titratable acidity, pH, nutritional composition, volatile compounds profile and sensory properties. The developed product showed comparable compositional properties; however, it lacked important flavor contributors, and bitterness and astringency prevailed. Finally, the stability of the CLNA-enriched fermented milk was assessed throughout 28 days of refrigerated (4 ºC) storage. To obtain an alternative delivery system of CLNA isomers, the enriched fermented milk was further lyophilized, and its stability during storage (12 weeks at room temperature) was also evaluated. For both products a free saturated FAs loss and an increment of conjugated isomers content was observed. In conclusion, the selection of potential CLA/CLNA-producing strains cannot rely solely on genotypic and/or substrate tolerance screening techniques. Even though, it was possible to obtain a fermented milk enriched in conjugated FAs with B. breve DSM 20091 using hydrolyzed flaxseed oil, and this strain showed a preference to produce CLNA isomers. Moreover, two additional CLNA isomers never reported before were discovered. The biochemical and nutritional characteristics of the CLNA-enriched fermented milk were acceptable, but there are organoleptic features that remain to be tackled. Although conjugated isomers in the developed dairy products increased throughout storage, the alterations in free saturated FA suggested the occurrence of oxidation processes.
- Effect of pufa substrates on fatty acid profile of bifidobacterium breve Ncimb 702258 and CLA/CLNA production in commercial semi-skimmed milkPublication . Fontes, Ana Luiza; Pimentel, Lígia; Rodríguez-Alcalá, Luis Miguel; Gomes, Ana MariaCurrent 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.