Impact of microbial produced bioactive CLA and CLnA on acquired and genetic obesity and on gut microbiota in a murine model

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Effect of pufa substrates on fatty acid profile of bifidobacterium breve Ncimb 702258 and CLA/CLNA production in commercial semi-skimmed milk

Publication . Fontes, Ana Luiza; Pimentel, Lígia; Rodríguez-Alcalá, Luis Miguel; Gomes, Ana Maria

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.

2018Journal article

Open access

Microbiological in vivo production of CLNA as a tool in the regulation of host microbiota in obesity control

Publication . Pimentel, Lígia Leão; Fontes, Ana Luiza; Salsinha, Ana Sofia; Cardoso, Beatriz Batista; Gomes, Ana Maria; Rodríguez-Alcalá, Luís Miguel

Metabolic disorders associated with dietary patterns have become a social and economic problem. Obesity is indeed considered a central feature that increases the risks associated with a vast array of diseases (insulin resistance, type 2 diabetes, fatty liver disease, atherosclerosis, hypertension, stroke, cancer, and asthma), with significant morbidity and mortality. Although lipids may be involved in the development of these illnesses, recent studies have stated their role as cellular mediators and they have been assayed as bioactive compounds in possible treatments. Clear examples are the conjugated isomers of linoleic acid (CLA), which have been associated with antiatherogenic, antioxidative, immunostimulation, and body fat reduction activities. Recently, increased interest in other conjugated PUFAs has emerged, linked to the health-promoting properties of conjugated linolenic acid isomers (CLnA) like C18:3 c9t11c15 (rumelenic acid, RLA). These fatty acids combine in the same molecule a double conjugated bond system with the n3 structure of linolenic acid (ALA; C18:3 c9c12c15) resulting in a high bioactive potential. Animal studies have revealed that CLnA regulates leptin production and increases β-oxidation in the liver thus reducing perirenal and epididymal adipose tissue. Elsewhere it was reported that RLA increased PPARα levels in adipocytes, which suggests that it is a candidate functional ingredient for use in the prevention of obesity, diabetes, and dyslipidemia. CLA and CLnA are natural fatty acids (FAs) mainly found in dairy products and beef, because they are produced as intermediates of the biohydrogenation pathway of PUFAs by ruminal bacteria. Therefore it is hypothesized that other microorganisms may also be able to produce CLA and conjugated FAs. Indeed, recent investigations demonstrated that it is possible to elaborate fermented dairy products containing CLA and CLnA produced by these bacteria. Other authors have identified Bifidobacterium strains with a high rate of substrate conversion to yield CLA, CLnA, and stearidonic acids producing these FAs in vivo, increasing the RA concentration in murine and pigs livers as well as DHA and EPA in mice adipose tissue. An important issue arises from these results: host fatty acid composition can be manipulated by oral administration of CLA-producing microorganisms. In the last 5 years an increasing number of studies have found strong evidence of the role not only of diet but also of human gut microbiota in the development of diabetes and obesity. Indeed, host microbiota regulates the content of triglycerides, cholesterol esters, sphingomyelin, and phosphatidylcholine in the plasma, liver, and adipose tissue as well as energy metabolites. This chapter will review the current state-of-the-art microbiological in vivo production of CLA and CLnA and its effect on host microbiota and health.

2018Book part

Restricted access

Suitable simple and fast methods for selective isolation of phospholipids as a tool for their analysis

Publication . Pimentel, Lígia; Fontes, Ana Luiza; Salsinha, Sofia; Machado, Manuela; Correia, Inês; Gomes, Ana Maria; Pintado, Manuela; Rodríguez-Alcalá, Luis Miguel

Lipids are gaining relevance over the last 20 years, as our knowledge about their role has changed from merely energy/structural molecules to compounds also involved in several biological processes. This led to the creation in 2003 of a new emerging research field: lipidomics. In particular the phospholipids have pharmacological/food applications, participate in cell signalling/homeostatic pathways while their analysis faces some challenges. Their fractionation/purification is, in fact, especially difficult, as they are amphiphilic compounds. Moreover, it usually involves SPE or TLC procedures requiring specific materials hampering their suitableness for routine analysis. Finally, they can interfere with the ionization of other molecules during mass spectrometry analysis. Thus, simple high‐throughput reliable methods to selectively isolate these compounds based on the difference between chemical characteristics of lipids would represent valuable tools for their study besides that of other compounds. The current review work aims to describe the state‐of‐the‐art related to the extraction of phospholipids using liquid‐liquid methods for their targeted isolation. The technological and biological importance of these compounds and ion suppression phenomena are also reviewed. Methods by precipitation with acetone or isolation using methanol seem to be suitable for selective isolation of phospholipids in both biological and food samples.

2018Journal article

Restricted access

Lactobacillus mulieris sp. nov., a new species of Lactobacillus delbrueckii group

Publication . Rocha, Joana; Botelho, João; Ksiezarek, Magdalena; Ugarcina Perovic, Svetlana; Machado, Miguel; Carrico, João André; Pimentel, Lígia L.; Salsinha, Sofia; Rodríguez-Alcalá, Luis M.; Pintado, Manuela; Ribeiro, Teresa G.; Peixe, Luísa

One Gram-stain-positive, non-motile, non-spore-forming, catalase-negative, and coccobacilli-shaped strain, designated c10Ua161MT, was isolated from a urine sample from a reproductive-age healthy woman. Comparative 16S rRNA gene sequence analysis indicated that strain c10Ua161MT belonged to the genus Lactobacillus . Phylogenetic analysis based on pheS and rpoA gene sequences strongly supported a clade encompassing strains c10Ua161MT and eight other strains from public databases, distinct from currently recognized species of the genus Lactobacillus. In silico Average Nucleotide Identity (ANI) and Genome-to-Genome Distance Calculator (GGDC), showed 87.9 and 34.3 % identity to the closest relative Lactobacillus jensenii , respectively. The major fatty acids of strain c10Ua161MT were C18 : 1ω9c (65.0%), C16 : 0 (17.8%), and summed feature 8 (10.2 %; comprising C18 : 1ω7c, and/or C18 : 1ω6c). The DNA G+C content of the strains is 34.2 mol%. On the basis of data presented here, strain c10Ua161MT represents a novel species of the genus Lactobacillus , for which the name Lactobacillus mulieris sp. nov. is proposed. The type strain is c10Ua161MT (=CECT 9755T=DSM 108704T).

2020Journal article

Restricted access

Microbial production of conjugated linoleic acid and conjugated linolenic acid relies on a multienzymatic system

Publication . Salsinha, Ana S.; Pimentel, Lígia L.; Fontes, Ana L.; Gomes, Ana M.; Rodríguez-Alcalá, Luis M.

Conjugated linoleic acids (CLAs) and conjugated linolenic acids (CLNAs) have gained significant attention due to their anticarcinogenic and lipid/energy metabolism-modulatory effects. However, their concentration in foodstuffs is insufficient for any therapeutic application to be implemented. From a biotechnological standpoint, microbial production of these conjugated fatty acids (CFAs) has been explored as an alternative, and strains of the genera Propionibacterium, Lactobacillus, and Bifidobacterium have shown promising producing capacities. Current screening research works are generally based on direct analytical determination of production capacity (e.g., trial and error), representing an important bottleneck in these studies. This review aims to summarize the available information regarding identified genes and proteins involved in CLA/CLNA production by these groups of bacteria and, consequently, the possible enzymatic reactions behind such metabolic processes. Linoleate isomerase (LAI) was the first enzyme to be described to be involved in the microbiological transformation of linoleic acids (LAs) and linolenic acids (LNAs) into CFA isomers. Thus, the availability of lai gene sequences has allowed the development of genetic screening tools. Nevertheless, several studies have reported that LAIs have significant homology with myosin-cross-reactive antigen (MCRA) proteins, which are involved in the synthesis of hydroxy fatty acids, as shown by hydratase activity. Furthermore, it has been suggested that CLA and/or CLNA production results from a stress response performed by the activation of more than one gene in a multiple-step reaction. Studies on CFA biochemical pathways are essential to understand and characterize the metabolic mechanism behind this process, unraveling all the gene products that may be involved. As some of these bacteria have shown modulation of lipid metabolism in vivo, further research to be focused on this topic may help us to understand the role of the gut microbiota in human health.

2018Journal article

Restricted access