Prebiotic Effect of Lycopene and Dark Chocolate on Gut Microbiome with Systemic Changes in Liver Metabolism, Skeletal Muscles and Skin in Moderately Obese Persons.

Lycopene rich food and dark chocolate are among the best-documented products with a broad health benefit. This study explored the systemic effect of lycopene and dark chocolate (DC) on gut microbiota, blood, liver metabolism, skeletal muscle tissue oxygenation and skin. 30 volunteers were recruited for this trial, 15 women and 15 men with a mean age of 55 ± 5.7 years and with moderate obesity, 30 < BMI < 35 kg/m2. They were randomized and divided into five equal interventional groups: three received different formulations of lycopene, one of them with a 7 mg daily dose and two with 30 mg; another group was given 10 g of DC with 7 mg lycopene embedded into its matrix, and the last group received 10 g DC. The trial was double-blinded for the three lycopene groups and separately for the 2 DC groups; the trial lasted for 1 month. By the end of the trial there were dose-dependent changes in the gut microbiota profile in all three lycopene groups with an increase of relative abundance of, e.g., Bifidobacterium adolescentis and Bifidobacterium longum. This was also accompanied by dose-dependent changes in the blood, liver metabolism, skeletal muscle and skin parameters. Consumption of DC resulted in increased relative abundance of, e.g., Lactobacillus and a reduction of corneocyte exfoliation. This is the first study which reports the prebiotic potential of lycopene and DC.

Ingestion of Lycosome L-tug Formulation of Dark Chocolate Ameliorates Postprandial Hyperlipidemia and Hyperglycemia in Healthy Volunteers.

Twenty-eight healthy middle-aged volunteers (40-60 years old) with equal gender representation were randomized into 3 study groups to investigate the changes in postprandial glucose and lipids after ingestion of different formulations of dark chocolate (DC). The volunteers from the first group were requested to ingest 100 g of regular DC whereas the individuals from the third group were given 100 g of highly bioavailable lycosome formulated L-tug formulation of DC containing 23.3 mg of lycopene. A second group received a 23.3 mg lycopene capsule, a tomato-derived antioxidant carotenoid as a matching control. Serum specimens were obtained following 30 minutes as well as 1, 2, and 3 hours after study products intake. Ingestion of L-tug DC was accompanied by the reduced postprandial hyperglycemia with maximum difference seen at 3rd hour of the study and reduction of average AUCGluc values by 20% (P<0.05) as compared to regular DC. Moreover, ingestion of L-tug DC was accompanied by a statistically significantly reduced median concentration for postprandial triglycerides (to 390.7 mg⁎hr/dL; 5/95%% CIs: 363.2/405.7 versus regular DC value of 439.5mg⁎hr/dL and a lower range of confidence intervals - 5/95%CIs: 394.0/475.1). A similar tendency was observed in changes of total cholesterol concentration. Ingestion of L-tug DC completely abolished total cholesterol increase seen in volunteers at 3rd hour of postprandial period following intake of the control DC. Ingestion of lycopene alone did not cause any changes in postprandial changes of glucose or serum lipids. The observed postprandial changes can be related to the 56.2 % increase in serum lycopene level which was observed after ingestion of L-tug DC only. Higher serum lycopene levels following the ingestion of L-tug DC resulted in a corresponding increase in serum antioxidant capacity and reduction of oxidized LDL as well as a decline in malonic dialdehyde concentration in the serum of volunteers.

Lycopene presence in facial skin corneocytes and sebum and its association with circulating lycopene isomer profile: Effects of age and dietary supplementation.

Lycopene is a dietary antioxidant known to prevent skin photodamage. This study aimed to examine age-dependent presence of this carotenoid on the surface of the facial skin and in the serum as well as to measure the same parameters during supplementation with lycopene. Serum samples and samples from facial skin surface were obtained from 60 young (under 25 years old) and 60 middle-aged (over 50 years old) volunteers. Similar samples were taken from 15 middle-aged subjects during 4-week supplementation with lycopene (7 mg/day). Serum lycopene levels and isomer profiles were analyzed by HPLC. Lycopene in desquamated corneocytes and the sebum from facial skin surface was determined using lycopene-specific fluorescent monoclonal antibodies. The results demonstrated that there was no age-related difference in serum lycopene levels, but a higher proportion of (all-E)-lycopene was detected in the "young" group (37.5% vs 26.2% in the "middle-aged" group; p < 0.0001). "Young" volunteers also had a higher lycopene level in both corneocytes (p = 0.0071) and the sebum (p = 0.0139) from the skin surface. Supplementation with lycopene resulted in a sharp increase of lycopene concentrations in both serum and skin surface samples. There was also a clear change in the pattern of lycopene isomers in the serum manifested by a significant increase in the proportion of (all-E)-lycopene (from 22.1% to 44.0% after supplementation, p < 0.0001). It can be concluded that dietary supplementation with lycopene results in its accumulation in the serum and skin. This process is accompanied by significant changes in the circulating lycopene isomer profile which becomes similar to that typical for young individuals.

Systemic and skin-targeting beneficial effects of lycopene-enriched ice cream: A pilot study.

The health-promoting dietary antioxidant lycopene has limited natural bioavailability, but lycopene-rich functional foods can improve its bioavailability. We assessed a new lycopene-enriched ice cream for systemic antioxidant effects and influence on morphological characteristics of facial skin surface in healthy volunteers. In a randomized crossover study, we used 4-wk dietary interventions with either control or lycopene-enriched ice cream. Samples of serum and residual skin surface components (RSSC) from facial skin were taken before interventions, at 2 wk, and at intervention end. Lycopene concentration, conventional blood biochemistry, and oxidative stress biomarkers comprising inflammatory oxidative damage and low-density lipoprotein peroxidase proteins were assessed in the serum. Lycopene-associated immunofluorescence, lipid droplet size, corneocyte desquamation, and microbial presence were measured in the RSSC. The results show that lycopene concentrations in the serum and skin steadily increased during lycopene-enriched ice cream consumption. Whereas we found no intervention-dependent changes in conventional biochemical parameters, both inflammatory oxidative damage and low-density lipoprotein peroxidase protein values significantly decreased by the end of intervention with lycopene-enriched ice cream, but remained unchanged during control ice cream consumption. Control ice cream significantly increased corneocyte desquamation and bacterial presence in the RSSC. These adverse effects, which could potentially predispose consumers to acne development, were absent when volunteers consumed lycopene-enriched ice cream. We concluded that lycopene-enriched ice cream is a new functional food with clear antioxidant properties. In addition, enrichment with lycopene may alleviate proinflammatory action of ice cream at the level of facial skin, thus decreasing diet-associated acne development risk in young consumers.

Effect of lycopene supplementation on cardiovascular parameters and markers of inflammation and oxidation in patients with coronary vascular disease.

Oxidative stress and antioxidant deficiency play a pivotal role in initiation, development, and outcomes of cardiovascular disease. Pharmacokinetic parameters as well as the impact of highly bioavailable lycopene on cardiovascular variables, markers of inflammation and oxidation were investigated during a 30-day clinical trial in patients with coronary vascular disease. The patients were randomized into two major groups and were supplemented with a single 7 mg daily dose of lycopene ingested either in the form of lactolycopene (68 patients) or in the form of lycosome-formulated GA lycopene (74 patients). The endpoints included cardiovascular function parameters, serum lipids, and four markers of oxidative stress and inflammation. Ingestion of lycosome-formulated lycopene increased serum lycopene levels by 2.9- and 4.3-fold, respectively, after 2 and 4 weeks of the trial, whereas supplementation with lactolycopene upregulated serum lycopene by half-fold only after 4 weeks of ingestion. Lycosome formulation of lycopene resulted by the end of the trial in a threefold reduction in Chlamydia pneumoniae IgG and reduction to the same degree of the inflammatory oxidative damage marker. The decrease in oxidized LDL caused by lycosome-formulated lycopene was fivefold. Moreover, supplementation with lycosome-formulated lycopene was accompanied by a significant increase in tissue oxygenation and flow-mediated dilation by the end of the observational period. In contrast, lactolycopene did not cause any significant changes in the parameters studied. Therefore, enhanced bioavailability of lycopene promotes its antioxidant and anti-inflammatory functions and endorses a positive effect of lycopene on cardiovascular system.

Pharmacokinetics and Oxidation Parameters in Volunteers Supplemented with Microencapsulated Docosahexaenoic Acid.

CONTEXT: Docosahexaenoic acid (DHA) is an omega-3 fatty acid essential for cardiovascular health, brain development, and reproductive function. Due to hydrophobicity and low DHA bioavailability, new microencapsulated DHA formulations are under development. AIM: This study aims to evaluate DHA pharmacokinetics (PKs) and biological oxidation parameters in volunteers ingesting a newly developed lutein-containing lycosomal formulation of DHA (LF-DHA). MATERIALS AND METHODS: A total of 32 healthy volunteers (40-65 years old) with signs of oxidative stress (OS) and subclinical hypoxia were orally supplemented for a month with 250 mg of regular DHA (1st group) or a combination of lutein (7.0 mg) and zeaxanthin (1.4 mg) (2nd group). The third group received regular DHA (250 mg) co-ingested with lutein/zeaxanthin (7.0/1.4 mg), whereas the 4th group was given LF-DHA containing lutein/zeaxanthin (7.0/1.4 mg). PK, OS, and oxygenation parameters were analyzed. RESULTS: LF-DHA improved the PKs of DHA enhancing its serum concentrations time dependently by 34.6% and 94.1% after 2nd and 4th weeks, respectively. DHA and lutein ingested either alone or simultaneously as two separate formulations reduced the levels of OS markers. However, LF-DHA inhibited the malonicdialdehyde (MDA) and oxidized low-density lipoprotein values were better than other formulations. LF-DHA also enhanced the plasma oxygen and tissue oxygen saturation. This effect was significantly higher than in other groups. CONCLUSION: LF-DHA eliminates the need in high-dose DHA supplementation protocols and confers a higher DHA bioavailability, thereby improving the parameters of biological oxidation and tissue respiration in affected individuals.

Effect of Lycosome-Formulated Phosphatidylcholine on Parameters of Biological Oxidation after Single Intake of Moderate Amount of Alcohol.

Ingestion of a single dose of alcohol, ranging from the intake of a moderate amount alcohol to binge drinking, is the most frequent form of alcohol consumption with poorly understood medical consequences and obscure prophylactics. The study was aimed to determine whether lycosome formulated phosphatidylcholine (PC-Lyc) containing two highly bioavailable antioxidants (PC and lycopene) ingested shortly before the alcohol-containing beverage may alleviate the biochemical markers of liver damage and parameters of biological oxidation associated with the intake of a moderate amount of alcohol. Healthy middle-aged volunteers were requested to consume a moderate amount of alcohol - 0.5 ml/kg or 1.0 ml/kg shortly after ingestion of a capsule containing 450 mg of regular phosphatidylcholine (PC, n=10), PC-Lyc (n=10), or placebo pill (PP, n=10). Serum levels of ethanol (EtOH), acetaldehyde (AA), liver-specific enzymes, total antioxidant capacity of serum (TAC), oxidized LDL (LDL-Px), and malonic dialdehyde (MDA) were measured at 1, 2.5, and 5 hours after dosing with alcohol. Ingestion of PC regardless of the formulation used had no effect on serum EtOH concentration dynamics. However, volunteers supplemented with PC-Lyc showed a better clearance of AA in serum as compared to other groups. There was a reduction in serum TAC values by 18.5% and 16.1% in both placebo groups ingesting 0.5 and 1.0 ml/kg of alcohol, respectively, at the end of observational period. This decline was preventable by supplementation of volunteers with PC and especially with PC-Lyc. Moreover, PC-Lyc promoted a reduction of serum MDA and reversed an increase in serum LDL-Px. In addition, ingestion of alcohol at 1.0 ml/kg dose caused a transient increase in serum alanine-aminotransferase activity which was abolished by both formulations of PC. Therefore, combinatory lycosomal formulation of PC and lycopene may prevent some metabolic abnormalities associated with single intake of moderate amount of alcohol. This trial is registered with ACTRN12617001335381.

Non-Invasive Immunofluorescence Assessment of Lycopene Supplementation Status in Skin Smears.

Circulating lycopene level is negatively associated with the prevalence of cardiovascular disease, cancers (prostate and breast), type 2 diabetes mellitus, and aging. Traditionally, lycopene is measured in biological specimens by a combination of high-performance liquid chromatography (HPLC) and mass spectrometry methods. Moreover, as we recently reported, tissue/cell lycopene depositions can be observed by the immunohistochemistry method with a newly developed monoclonal antibody (mAb) against lycopene. A main objective of this study is to evaluate the performance of a new noninvasive immunofluorescence (IF) lycopene quantification skin test with mAbs against lycopene versus HPLC lycopene assay of serum lycopene in volunteers subjected to lycopene supplementation which represents a novel approach to lycopene measurement methodology. For this purpose, 32 healthy volunteers, 30-40 years old, were supplemented with lycopene (n = 15) or placebo (n = 17) for a period of 4 weeks. It was found that lycopene supplementation leads to a significant increase in serum lycopene concentration after 2 and 4 weeks by 2.6- and 3.4-fold over control, respectively. This was accompanied by a concordant step-wise rise in IF staining of skin corneocytes and sebum, quantifiable by arbitrary IF scores. Placebo supplementation did not affect serum lycopene values or intensity of IF staining of the skin samples. There was 86.6% agreement in paired HPLC/IF variants for the intermediate time point and 80.0% agreement at the end of the study in the lycopene group. Intraclass correlation between paired values in this group was +0.49 for the 2-week time point and +0.63 for the end point. These results indicate that the new antibody-based skin assay can be used for rapid detection of lycopene deficiencies. Moreover, the noninvasive nature of the skin swab test would allow using it to monitor, optimize, and personalize lycopene supplementation protocol of risk groups in the general population.

The degradation of three-ringed polycyclic aromatic hydrocarbons by wood-inhabiting fungus Pleurotus ostreatus and soil-inhabiting fungus Agaricus bisporus.

The degradation of two isomeric three-ringed polycyclic aromatic hydrocarbons by the white rot fungus Pleurotus ostreatus D1 and the litter-decomposing fungus Agaricus bisporus F-8 was studied. Despite some differences, the degradation of phenanthrene and anthracene followed the same scheme, forming quinone metabolites at the first stage. The further fate of these metabolites was determined by the composition of the ligninolytic enzyme complexes of the fungi. The quinone metabolites of phenanthrene and anthracene produced in the presence of only laccase were observed to accumulate, whereas those formed in presence of laccase and versatile peroxidase were metabolized further to form products that were further included in basal metabolism (e.g. phthalic acid). Laccase can catalyze the initial attack on the PAH molecule, which leads to the formation of quinones, and that peroxidase ensures their further oxidation, which eventually leads to PAH mineralization. A. bisporus, which produced only laccase, metabolized phenanthrene and anthracene to give the corresponding quinones as the dominant metabolites. No products of further utilization of these compounds were detected. Thus, the fungi's affiliation with different ecophysiological groups and their cultivation conditions affect the composition and dynamics of production of the ligninolytic enzyme complex and the completeness of PAH utilization.

Plasmid AZOBR_p1-borne fabG gene for putative 3-oxoacyl-[acyl-carrier protein] reductase is essential for proper assembly and work of the dual flagellar system in the alphaproteobacterium Azospirillum brasilense Sp245.

Azospirillum brasilense can swim and swarm owing to the activity of a constitutive polar flagellum (Fla) and inducible lateral flagella (Laf), respectively. Experimental data on the regulation of the Fla and Laf assembly in azospirilla are scarce. Here, the coding sequence (CDS) AZOBR_p1160043 (fabG1) for a putative 3-oxoacyl-[acyl-carrier protein (ACP)] reductase was found essential for the construction of both types of flagella. In an immotile leaky Fla- Laf- fabG1::Omegon-Km mutant, Sp245.1610, defects in flagellation and motility were fully complemented by expressing the CDS AZOBR_p1160043 from plasmid pRK415. When pRK415 with the cloned CDS AZOBR_p1160045 (fliC) for a putative 65.2 kDa Sp245 Fla flagellin was transferred into the Sp245.1610 cells, the bacteria also became able to assemble a motile single flagellum. Some cells, however, had unusual swimming behavior, probably because of the side location of the organelle. Although the assembly of Laf was not restored in Sp245.1610 (pRK415-p1160045), this strain was somewhat capable of swarming motility. We propose that the putative 3-oxoacyl-[ACP] reductase encoded by the CDS AZOBR_p1160043 plays a role in correct flagellar location in the cell envelope and (or) in flagellar modification(s), which are also required for the inducible construction of Laf and for proper swimming and swarming motility of A. brasilense Sp245.

Generation and Application of Monoclonal Antibody Against Lycopene.

A monoclonal antibody (Mab) against lycopene was developed from hybridoma clones obtained from BALB/c mice immunized with trans-isomer of lycopene (t-lycopene, t-LC) conjugated with colloidal gold particles. An alternating immunization schedule which included injection of both formulations of immunogen (without and with Freund's adjuvant) was most effective in the elucidation of a measurable immune response to the t-Lycopene conjugate. Selected hybridoma clones were able to produce an Mab positive in competition assay. In particular, preincubation of 6B9 Mabs with t-LC abolished the ability of 6B9 Mabs to bind LC in the competition assay. Mabs produced by other clones (4F10, 4A3, and 3B12) worked similarly. Analysis of antigen specificity showed that 6B9 Mab raised against t-LC did not recognize other carotenoids such as lutein and carotene. Mab 6B9 was shown to recognize lycopene on a glass surface and in the settings of indirect immunofluorescence experiments performed in cultured hepatocytes and alveolar macrophages incubated with and without lycopene, as well as in sebum and corneocyte specimens from the skin of volunteers supplemented with nutraceutical formulation of lycopene. Newly generated Mabs against lycopene may provide a valuable tool for different analytical assays of lycopene content in various biological, agricultural, and food products.

The coupling of the plant and microbial catabolisms of phenanthrene in the rhizosphere of Medicago sativa.

We studied the catabolism of the polycyclic aromatic hydrocarbon phenanthrene by four rhizobacterial strains and the possibility of enzymatic oxidation of this compound and its microbial metabolites by the root exudates of alfalfa (Medicago sativa L.) in order to detect the possible coupling of the plant and microbial metabolisms under the rhizospheric degradation of the organic pollutant. A comparative study of phenanthrene degradation pathways in the PAH-degrading rhizobacteria Ensifer meliloti, Pseudomonas kunmingensis, Rhizobium petrolearium, and Stenotrophomonas sp. allowed us to identify the key metabolites from the microbial transformation of phenanthrene, including 9,10-phenanthrenequinone, 2-carboxybenzaldehyde, and 1-hydroxy-2-naphthoic, salicylic, and o-phthalic acids. Sterile alfalfa plants were grown in the presence and absence of phenanthrene (0.03 g kg(-1)) in quartz sand under controlled environmental conditions to obtain plant root exudates. The root exudates were collected, concentrated by ultrafiltration, and the activity of oxidoreductases was detected spectrophotometrically by the oxidation rate for various substrates. The most marked activity was that of peroxidase, whereas the presence of oxidase and tyrosinase was detected on the verge of the assay sensitivity. Using alfalfa root exudates as a crude enzyme preparation, we found that in the presence of the synthetic mediator, the plant peroxidase could oxidize phenanthrene and its microbial metabolites. The results indicate the possibility of active participation of plants in the rhizospheric degradation of polycyclic aromatic hydrocarbons and their microbial metabolites, which makes it possible to speak about the coupling of the plant and microbial catabolisms of these contaminants in the rhizosphere.

Isolation and characterization of a glyphosate-degrading rhizosphere strain, Enterobacter cloacae K7.

Plant-growth-promoting rhizobacteria exert beneficial effects on plants through their capacity for nitrogen fixation, phytohormone production, phosphate solubilization, and improvement of the water and mineral status of plants. We suggested that these bacteria may also have the potential to express degradative activity toward glyphosate, a commonly used organophosphorus herbicide. In this study, 10 strains resistant to a 10 mM concentration of glyphosate were isolated from the rhizoplane of various plants. Five of these strains--Alcaligenes sp. K1, Comamonas sp. K4, Azomonas sp. K5, Pseudomonas sp. K3, and Enterobacter cloacae K7--possessed a number of associative traits, including fixation of atmospheric nitrogen, solubilization of phosphates, and synthesis of the phytohormone indole-3-acetic acid. One strain, E. cloacae K7, could utilize glyphosate as a source of P. Gas-liquid chromatography showed that E. cloacae growth correlated with a decline in herbicide content in the culture medium (40% of the initial 5mM content), with no glyphosate accumulating inside the cells. Thin-layer chromatography analysis of the intermediate metabolites of glyphosate degradation found that E. cloacae K7 had a C-P lyase activity and degraded glyphosate to give sarcosine, which was then oxidized to glycine. In addition, strain K7 colonized the roots of common sunflower (Helianthus annuus L.) and sugar sorghum (Sorghum saccharatum Pers.), promoting the growth and development of sunflower seedlings. Our findings extend current knowledge of glyphosate-degrading rhizosphere bacteria and may be useful for developing a biotechnology for the cleanup and restoration of glyphosate-polluted soils.

Versatile peroxidase of Bjerkandera fumosa: substrate and inhibitor specificity.

The inhibitor and substrate specificities of versatile peroxidase from Bjerkandera fumosa (VPBF) were studied. Two different effects were found: NaN(3), Tween-80, anthracene, and fluorene decreased the activity of VPBF, but p-aminobenzoic acid increased it. A mixed mechanism of effector influence on the activity of this enzyme was shown. The catalytic properties of VPBF in the oxidation of mono- and polycyclic aromatic compounds were studied also. 2,7-Diaminofluorene, ABTS, veratryl alcohol, and syringaldazine can be oxidized by VPBF in two ways: either directly by the enzyme or by diffusible chelated Mn(3+) as an oxidizing agent. During VPBF oxidation of 2,7-diaminofluorene, both with and without Mn(2+), biphasic kinetics with apparent saturation in both micromolar and millimolar ranges were obtained. In the case of ABTS, inhibition of VPBF activity by an excess of substrate was observed. Direct oxidation of p-aminobenzoic acid by versatile peroxidase was found for the first time. The oxidation of three- and four-ring PAHs by VPBF was investigated, and the oxidation of anthracene, phenanthrene, fluorene, pyrene, chrysene, and fluoranthene was shown. The products of PAH oxidation (9,10-anthraquinone, 9,10-phenanthrenequinone, and 9-fluorenone) catalyzed by VPBF were identified.