The 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.
| Date of Award | 5 Jul 2024 |
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| Original language | English |
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| Awarding Institution | - Universidade Católica Portuguesa
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| Supervisor | Ana Maria Gomes (Supervisor), Luís Miguel Rodríguez-Alcalá (Co-Supervisor) & Maria do Rosário Gonçalves dos Reis Marques Domingues (Co-Supervisor) |
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