Conjugated linolenic acid (CLnA) isomers are promising compounds due to their chemical similarities with the healthy omega 3 and Conjugated linoleic acid (CLA) and bioactivity at lower doses than this former compound[1, 2]. Moreover, some bacteria from foods transform linolenic (ALA) into CLnA and improved the in vivo fatty acid (FA) composition of liver, adipose tissue and brain in animals[3, 4]. It opens promising possibilities in the elaboration of new functional products. However, according to the existing bibliography and the experience of the research team of this proposal, some questions need to be addressed:

A. The number of identified CLnA producing bacteria is too low: Previous investigations focused on a “brute-force” approach: setting a general time and substrate concentration and assessing production by gas chromatography. This limits the number of the strains tested and the optimization of production. From the current bibliography, the assayed time varies from 24-78h and ALA concentration from 0.5-4 mg/mL wide transformation yield range (20-98%). In a previous work by members of the research team (TR1)[5], a novel approach was applied on basis that CLA producers can also transform ALA into CLnA. Only CLA producers were further assayed for CLnA. This was the first study describing the production of CLA and CLNA by strains isolated from human milk in reconstituted skim milk leading to publication of a patent[6]. These strains have membrane linoleate isomerases (LAI); thus molecular screening to detect the presence of the LAI genes is as a reliable useful tool[7]. Furthermore, members of the research team at CBQF (Professor Gomes, Dr. Pimentel and PI) are supervisors of a master degree student to identify CLnA producing bacteria. Molecular screening is being assayed together with FAME analysis, allowing a faster identification strains, avoiding rejection of possible candidates and a better optimization of production parameters.

B. Lack of knowledge about pathways involved in the microbial production of CLA/CLnA: Although the involved enzymes have been identified, mechanisms behind the bioconversion of ALA into CLnA are unknown. This knowledge would allow a better optimization of production conditions, increasing CLnA yields, improvement of the molecular screening and a better comprehension about how these microorganisms change the metabolism of the host.

C. Are CLnA isomers only produced as FFA?: CLA/CLnA producing bacteria mainly use free FA (FFA) as substrate. However, from the previous research works it is unclear if these conjugated FA (CFA) are in the free form or esterified (EFA) as the analytical methods assayed can derivatize FA from both moieties[8]. Moreover, all the previous studies assumed that all CLA/CLnA remains in the supernatants without transportation the bacteria. As pathways to transform FFA into CFA are not fully understood, all possibilities must be studied. Thus, in this investigation both supernatants and pellets will be analysed and specific preparation methods for EFA and FFA will be assayed to distinguish the origin of the D. Can microbial CLnA isomers exert positive health effects?: The bioactivity of CLnA isomers was showed using synthetic mixtures prepared lab scale via alkali isomerization of hydrolysed oils[9]. Despite the strong evidences pointing out to modulation of lipid metabolism[10] and anticancer effects[2], bioactivity of microbial CLnA has not been tested. However some studies reported that these bacteria altered the lipid metabolism of the host, remodelling the FA composition of liver, adipose tissue and brain in animal models[3, 4]. Therefore more research needed to deepen into this question.

E. Stability of High CLnA products: A bioactive compound must be stable after elaboration and during storage of the product. Otherwise it result in ingestion of hazardous compounds (e.g peroxides from lipid oxidation) and/or an insufficient intake. There is an utter lack of data describing the stability of high CLnA products (e.g dairy products) during storage and after elaboration. Previous studies conducted by members this research team, focused in the stability of naturally enriched CLA dairy products, reported that HTST pasteurization increased the content RA through isomerization of LA while sterilization of milk changed the distribution of isomers in the CLA fraction. All these reactions were oxidative in nature[11]. In previous studies it was found similar results after elaboration of powder milk using skim milk enriched with a commercial CLA oil while during storage of cheese, content of minor isomers decreased as result of microbiological growth (TR5)[12]. Elsewhere it was reported that CLnA has a lower thermal stability than CLA[13].

The PRO-TECh-CLNA project aims to cover these topics to bring cutting-edge knowledge and result into social and economic benefits.