Identification and probiotic properties of lactobacilli isolated from two different fermented beverages

Purpose

Scientific information regarding the microbial content and functional aspects of fermented beverages traditionally produced in certain parts of Europe are scarce. However, such products are believed to have some health benefits and might contain functional bacterial strains, such as probiotics. The aim of the study was to identify such lactic acid bacteria strains isolated from water kefir and, for the first time, from braga, a Romanian fermented beverage made of cereals.

Methods

Lactic acid bacteria (LAB) were identified to species level based on (GTG)₅-PCR fingerprinting and 16S rRNA gene sequencing. Selected strains were screened for their antibacterial activity and probiotic potential.

Results

Eight isolates belonging to seven Lactobacillus species were recovered from the two drinks. The identification of LAB involved in the fermentation of braga (Lactobacillus plantarum, Lactobacillus fermentum, and Lactobacillus delbrueckii) is firstly reported here. Five of the Lactobacillus isolates showed antibacterial activity against pathogenic bacteria, including Listeria monocytogenes, Escherichia coli, Staphylococcus aureus, and Salmonella enterica. Moreover, most of them showed a good resistance to pH 2.5 and some survived at high concentrations of bile salts (up to 2%). Two L. plantarum isolates were able to inhibit all the indicator strains, and showed the best viability (about 70%) after a sequential treatment simulating the passage through the gastrointestinal tract.

Conclusion

Based on the results, the most promising candidates for designing new probiotic products are: L. plantarum BR9 from braga and L. plantarum CR1 from water kefir.

     

The hyperlipemic hamster - a model for testing the anti-atherogenic effect of amlodipine

Male Golden Syrian hamsters were subjected to a hyperlipemic diet. At intervals ranging from 2 to 14 weeks, the animals were examined for changes in serum constituents and structural modifications of lesion-prone areas: the cardiac valves, coronary arteries and aortic arch. Serum was characterized by a gradual increase in cholesterol, triglycerides and a decrease in total peroxyl-radical trapping potential. The sequence of modifications of the endothelial cells, smooth muscle cells, and migrating plasma monocytes as well as of the extracellular matrix were established. Amlodipine treatment of hyperlipemic hamster was assessed. Amlodipine exhibited an athero-protective effect, acting as antioxidant, reducing the LDL uptake by the vessel wall and consequently, limiting the size and extent of lesioned areas. The hyperlipemic hamster is a reliable model to unravel the cellular alterations leading to atheroma formation, and for testing the effect of drugs in this process.     

Vascular endothelium in atherosclerosis

Their strategic location between blood and tissue and their constitutive properties allow endothelial cells (EC) to monitor the transport of plasma molecules, by employing bidirectional receptor-mediated and receptor-independent transcytosis and endocytosis, and to regulate vascular tone, cellular cholesterol and lipid homeostasis. These cells are also involved in signal transduction, immunity, inflammation and haemostasis. Cardiovascular risk factors, such as hyperlipaemia/dyslipidaemia trigger the molecular machinery of EC to respond to insults by modulation of their constitutive functions followed by dysfunction and ultimately by injury and apoptosis. The gradual activation of EC consists initially in the modulation of two constitutive functions: (1) permeability, i.e. increased transcytosis of lipoproteins, and (2) biosynthetic activity, i.e. enhanced synthesis of the basement membrane and extracellular matrix. The increased transcytosis and the reduced efflux of β-lipoproteins (βLp) lead to their retention within the endothelial hyperplasic basal lamina as modified lipoproteins (MLp) and to their subsequent alteration (oxidation, glycation, enzymatic modifications). MLp generate chemoattractant and inflammatory molecules, triggering EC dysfunction (appearance of new adhesion molecules, secretion of chemokines, cytokines), characterised by monocyte recruitment, adhesion, diapedesis and residence within the subendothelium. In time, EC in the athero-prone areas alter their net negative surface charge, losing their non-thrombogenic ability, become loaded with lipid droplets and turn into foam cells. Prolonged and/or repeated exposure to cardiovascular risk factors can ultimately exhaust the protective effect of the endogenous anti-inflammatory system within EC. As a consequence, EC may progress to senescence, lose their integrity and detach into the circulation.     

The total peroxyl radical trapping potential in serum - an assay to define the stage of atherosclerosis

Lipid peroxides were identified among the factors that contribute to the atherosclerotic plaque formation in the arterial wall. We hypothesised that a correlation may exist between the content of antioxidant constituents in the serum and the gravity of atherosclerosis. To this purpose, we have determined the serum total peroxyl radical- trapping potential (TRAP), which is the combined capacity of all antioxidants to neutralize free radicals in serum and followed its variation in hyperlipemic animals in correlation with the stage of atherosclerosis. In addition, we compared TRAP values in the sera of coronary heart disease (CHD) patients, with or without type II diabetes mellitus. Results showed that after 18 weeks of hyperlipemic diet, the mean TRAP values measured in sera isolated from hyperlipemic hamsters exhibited an about 44% decrease, in good agreement with the increase of serum cholesterol and triglycerides. In the 3 groups of CHD patients, TRAP values decreased with about 10% in sera of stable angina patients, 20% in unstable patients, as compared with normal subjects. The lowest TRAP values were detected in the sera of patients with acute myocardial infarction. The results obtained for different experimental animals and for CHD patients sera indicate that the TRAP method, as adapted in our laboratory, is a reliable and reproducible assay, fit to be used in clinical studies as an ex vivo measurable parameter that correlates with the stage of the atherosclerosis.     

Ceruloplasmin and oxidized LDL colocalize in atherosclerotic lesions of hamster

Epidemiological studies show that the risk for cardiovascular diseases increases with increasing levels of free-copper in plasma. It is known that intact ceruloplasmin (CP), the major protein transporter of copper in human plasma, oxidizes low density lipoproteins (LDL) in vitro. Our aim was to study the interaction between LDL and CP in vitro and in vivo, in an animal model of diet-induced atherosclerosis. In order to visualize the pathway of LDL into the arterial wall, human native LDL was labeled with fluorescent DiI and injected into male, Golden Syrian hyperlipemic hamsters. In vitro results demonstrated that slightly degraded CP has a significant oxidation potential against LDL at neutral pH. In vivo, after 24 hours circulation, LDL-DiI was taken up by the enlarged intima and fatty streaks of the arterial wall. Immunohistochemical localization of oxidized LDL and CP revealed their presence in the same areas of the arteries that take up LDL-DiI. Co-localization of LDL and CP in the enlarged intima of pro-atherosclerotic areas might explain the possible copper-induced oxidation process that might occur after native LDL is taken-up from the blood, transcytosed through the endothelium and accumulated in focalized deposits.     

Anti-oxidant and anti-inflammatory mechanisms of amlodipine action to improve endothelial cell dysfunction induced by irreversibly glycated LDL

Amlodipine, alone or in combination with other drugs, was successfully used to treat hypertension. Our aim was to evaluate the potential of amlodipine (Am) to restore endothelial dysfunction induced by irreversibly glycated low density lipoproteins (AGE-LDL), an in vitro model mimicking the diabetic condition. Human endothelial cells (HEC) from EA.hy926 line were incubated with AGE-LDL in the presence/absence of Am and the oxidative and inflammatory status of the cells was evaluated along with the p38 MAPK and NF-κB signalling pathways. The cellular NADPH activity, 4-hydroxynonenal (4-HNE) and 3-nitrotyrosine levels in the culture medium and the adhesion of human monocytes to HEC were measured by chemiluminescence, UHPLC, Western Blot and spectrofluorimetric techniques. The gene expression of NADPH subunits (p22^phox, NOX4), eNOS and inflammatory molecules (MCP-1, VCAM-1) were determined by Real Time PCR, while the protein expression of p22^phox, MCP-1, iNOS, phospho-p38 MAPK and phospho-p65 NF-κB subunit were measured by Western Blot. Results showed that in HEC incubated with AGE-LDL, Am led to: (i) decrease of the oxidative stress: by reducing p22^phox, NOX4, iNOS expression, NADPH oxidase activity, 4-HNE and 3-nitrotyrosine levels; (ii) decrease of the inflammatory stress: by the reduction of MCP-1 and VCAM-1 expression, as well as of the number of monocytes adhered to HEC; (iii) inhibition of ROS-sensitive signalling pathways: by decreasing phosphorylation of p38 MAPK and p65 NF-κB subunits. In conclusion, the reported data demonstrate that amlodipine may improve endothelial dysfunction in diabetes through anti-oxidant and anti-inflammatory mechanisms.     

Characterization of some bacteriocins produced by lactic acid bacteria isolated from fermented foods

Lactic acid bacteria (LAB) isolated from different sources (dairy products, fruits, fresh and fermented vegetables, fermented cereals) were screened for antimicrobial activity against other bacteria, including potential pathogens and food spoiling bacteria. Six strains have been shown to produce bacteriocins: Lactococcus lactis 19.3, Lactobacillus plantarum 26.1, Enterococcus durans 41.2, isolated from dairy products and Lactobacillus amylolyticus P40 and P50, and Lactobacillus oris P49, isolated from bors. Among the six bacteriocins, there were both heat stable, low molecular mass polypeptides, with a broad inhibitory spectrum, probably belonging to class II bacteriocins, and heat labile, high molecular mass proteins, with a very narrow inhibitory spectrum, most probably belonging to class III bacteriocins. A synergistic effect of some bacteriocins mixtures was observed. We can conclude that fermented foods are still important sources of new functional LAB. Among the six characterized bacteriocins, there might be some novel compounds with interesting features. Moreover, the bacteriocin-producing strains isolated in our study may find applications as protective cultures.     

Statins: mechanism of action and effects

The beneficial effects of statins are the result of their capacity to reduce cholesterol biosyntesis, mainly in the liver, where they are selectively distributed, as well as to the modulation of lipid metabolism, derived from their effect of inhibition upon HMG-CoA reductase. Statins have antiatherosclerotic effects, that positively correlate with the percent decrease in LDL cholesterol. In addition, they can exert antiatherosclerotic effects independently of their hypolipidemic action. Because the mevalonate metabolism generates a series of isoprenoids vital for different cellular functions, from cholesterol synthesis to the control of cell growth and differentiation, HMG-CoA reductase inhibition has beneficial pleiotropic effects. Consequently, statins reduce significantly the incidence of coronary events, both in primary and secondary prevention, being the most efficient hypolipidemic compounds that have reduced the rate of mortality in coronary patients. Independent of their hypolipidemic properties, statins interfere with events involved in bone formation and impede tumor cell growth.