Tendentious effects of automated and manual metagenomic DNA purification protocols on broiler gut microbiome taxonomic profiling.

Here, we developed protocols to improve sensitivity, rigor and comparability of 16S rRNA gene amplification-based next-generation sequencing (NGS) results. A thorough study was performed by evaluating extraction efficiency with respect to the yield, purity, fragmentation of the purified DNA, and sequencing metrics considering the number of quality reads, amplicon sequence variants (ASVs), community structure and biodiversity. We identified batch-effects that significantly bias broiler gastrointestinal tract (GIT) community compositions and made recommendations to improve sensitivity, consistency, and cross-study comparability. We found that the purity of the extracted nucleic acid had a strong effect on the success rate of downstream library preparations. The preparation of stool bacterial suspensions from feces showed a significant positive influence on community biodiversity by enriching Gram-negative bacteria and cataloguing low abundant taxa with greater success than direct processing of fecal material. Applications relying on the automated Roche MagNa Pure 24 magnetic-bead based method provided results with high consistency therefore it seems to be the optimal choice in large-scale studies for investigating broiler GIT microbiota.

Effect of Anthocyanin-Rich Tart Cherry Extract on Inflammatory Mediators and Adipokines Involved in Type 2 Diabetes in a High Fat Diet Induced Obesity Mouse Model.

Male C57BL/6J mice were used to determine the possible therapeutic effects of our previously described tart cherry extract in a chronic obesity mouse model on metabolic parameters, glucose tolerance, inflammatory mediators, and antioxidant capacity. The control group received standard mouse chow, and the high fat control group was switched to a high fat diet and tap water supplemented with 5% sucrose. The high fat + anthocyanin group received the high fat and sucrose diet, but received the anthocyanin-rich tart cherry extract dissolved in their drinking water. After six weeks, an oral glucose tolerance test was performed, and the water-soluble antioxidant capacity (ACW), superoxide dismutase (SOD) activity, and the plasma levels of insulin, C-peptide, leptin, IL-6, MCP-1, adiponectin and resistin were measured. The high fat diet increased body weight, reduced glucose tolerance, and caused an elevation in leptin, IL-6, MCP-1, and resistin levels. Furthermore, antioxidant capacity was decreased with a significant elevation of SOD activity. Anthocyanin treatment failed to reverse the effects of the high fat diet on body weight and glucose tolerance, but significantly reduced the leptin and IL-6 levels. The tart cherry extract also made a significant enhancement in antioxidant capacity and SOD activity. Our results show that chronic anthocyanin intake has a potential to enhance redox status and alleviate inflammation associated with obesity.

Isolation of allithiamine from Hungarian red sweet pepper seed (Capsicum annuum L.).

A natural fat-soluble thiamine derivative, namely N-[(4-amino-2-methylpyrimidin-5-yl)methyl]-N-[(2E)-5-hydroxy-3-(prop-2-en-1-yldisulfanyl)pent-2-en-2-yl]formamide (allithiamine) has been identified only in garlic (Allium sativum) until now. Hungarian red sweet pepper (Capsicum annuum) was found as a new source of allithiamine. Extraction procedure and analytical method were developed for the isolation of allithiamine and a chemical synthesis of the compound was also developed. First solid-liquid extraction was performed with 96 % ethanol to isolate allithiamine from pepper seeds. Thereafter, solid phase extraction was applied from ethanolic extract using C18 cartridge to concentrate and purify samples for further analysis. The structure of the synthesized and the isolated compounds was verified by reverse phase HPLC, HPLC-MS, MALD-TOF MS and NMR. Furthermore, effect of allithiamine was investigated on streptozotocin-induced diabetic mice with neuropathy. The results show that neuropathic pain sensation is improved by allithiamine treatment similarly to benfothiamine.

Anthocyanin composition, antioxidant efficiency, and α-amylase inhibitor activity of different Hungarian sour cherry varieties (Prunus cerasus L.).

Five Hungarian sour cherry cultivars were studied to determine their anthocyanin contents and their possible inhibitory properties. The water and methanol soluble antioxidant capacities were separately assessed by photoluminescence showing values ranged from 3.4µgmg(-1) to 15.4µgmg(-1), respectively. The "VN1" variety (selected from "Csengodi csokros") showed the highest antioxidant capacity. The anthocyanin content, measured by pH differential method or isolated by solid phase extraction, was the highest also in "VN1". Correlation was found between the anthocyanin content and the high antioxidant capacity. The main anthocyanin components were cyanidin-3-O-rutinoside and cyanidin-3-O-glucoside. The presence of malvidin-3,5-O-diglycoside was verified by MALDI-TOF MS. Sour cherry extracts and selected anthocyanins inhibited the human salivary alpha-amylase catalyzed hydrolysis competitively. The lowest IC50 value, 55µgmL(-1) or 80µM, was measured for malvidin-3,5-O-diglycoside, for which possible binding modes within the alpha-amylase active site could be investigated in silico using molecular docking and molecular dynamics.