[Isolation, characterization and real-time RT-PCR for relative quantification of gene expression in H+-ATPase-defective mutants from Lactobacillus plantarum].

Objective: The aim of this study was to isolate Lactobacillus plantarum acid-sensitive mutants with lower H+-ATPase activity, and to study the mechanism of H+-ATPase regulation in Lactobacillus plantarum. Methods: We used neomycin to isolate acid-sensitive mutants of L. plantarum, and measured H+-ATPase activity and lactic acid production of wild-type and mutants. Genomic DNA was extracted from the wild-type ZUST and two mutants (ZUST-1, ZUST-2), and used as PCR templates. H+-ATPase genes of the strain were amplified, and the PCR products were sequenced. Sequence similarity of H+-ATPase was analyzed. Real-time RT-PCR was used to evaluate the relative quantification of the H+-ATPase genes expression. Results: The growth of the mutants was characterized in MRS broth, which revealed that their cell biomass and acid production were lower than that of the wild-type. H+-ATPase activity of the mutants ZUST-1 and ZUST-2 was 10.1% and 28.8% lower than that of the wild-type. Results showed that atpA gene of the mutants ZUST-1 and ZUST-2 existed 22 mutations by alignment of the wild-type sequence, and atpC gene of ZUST-2 existed 6 mutations. Mutants ZUST-1 and ZUST-2 atpA gene expression were 41.1% and 35.7% lower than that of the wild-type in exponential phase, 43.6% and 14.2% in stationary phase, respectively. The atpC gene expression of ZUST-1 was similar to that of the wild-type in exponential phase, and was 30% higher than that of the wild-type in stationary phase, and ZUST-2 atpC gene was not expressed. Conclusion: The mutants with lower H+-ATPase activity were found to up-regulate the expression of H+-ATPase genes in stationary phase, except ZUST-2 atpC gene was not expressed. H+-ATPase activity has an important connection with the difference in gene expression of atpA and atpC. The results of this study will pave the way for gaining further insights into the mechanism of the H+-ATPase-defective mutants.

Antioxidant Capacities of Fractions of Bamboo Shaving Extract and Their Antioxidant Components.

This research was conducted for evaluation of antioxidant activities of four fractions from bamboo shavings extract (BSE) and their antioxidant components. The antioxidant capacities of BSE and four fractions on ABTS, DPPH, FRAP and total antioxidant capacity assays exhibited the following descending order: DF > n-butanol fraction (BF) > BSE ≈ ethyl acetate fraction (AF) > water fraction (WF). Among the identified phenolic compounds, caffeic acid exhibited the highest antioxidant capacities on DPPH, FRAP and total antioxidant capacity assays. An extremely significant positive correlation between the antioxidant activities with the contents of total flavonoids, total phenolic acids, or total phenolics was observed in this study. The result indicated that the bamboo shaving extract and its solvent fractions could act as natural antioxidants in light of their potent antioxidant activities.

Salt gradient enhanced sensitivity in nanopores for intracellular calcium ion detection.

Intracellular calcium ion detection is of great significance for understanding the cell metabolism and signaling pathways. Most of the current ionic sensors either face the size issue or sensitivity limit for the intracellular solution with high background ion concentrations. In this paper, we proposed a calmodulin (CaM) functionalized nanopore for sensitive and selective Ca2+ detection inside living cells. A salt gradient was created when the nanopore sensor filled with a low concentration electrolyte was in contact with a high background concentration solution, which enhanced the surface charge-based detection sensitivity. The nanopore sensor showed a 10 × sensitivity enhancement by application of a 100-fold salt gradient, and a detection limit of sub nM. The sensor had a wide detection range from 1 nM to 1 mM, and allowed for quick calcium ion quantification in a few seconds. The sensor was demonstrated for intracellular Ca2+ detection in A549 cells in response to ionomycin.

Hydrolysis before Stir-Frying Increases the Isothiocyanate Content of Broccoli.

Broccoli is found to be a good source of glucosinolates, which can be hydrolyzed by endogenous myrosinase to obtain chemopreventive isothiocyanates (ITCs); among them, sulforaphane (SF) is the most important agent. Studies have shown that cooking greatly affects the levels of SF and total ITCs in broccoli. However, the stability of these compounds during cooking has been infrequently examined. In this study, we proved that the half-lives of SF and total ITCs during stir-frying were 7.7 and 5.9 min, respectively, while the myrosinase activity decreased by 80% after stir-frying for 3 min; SF and total ITCs were more stable than myrosinase. Thus, the contents of SF and total ITCs decreased during stir-frying largely because myrosinase was destroyed. Subsequently, it was confirmed that compared to direct stir-frying, hydrolysis of glucosinolates in broccoli for 90 min followed by stir-frying increased the SF and total ITC concentration by 2.8 and 2.6 times, respectively. This method provides large quantities of beneficial ITCs even after cooking.

Study on degradation kinetics of sulforaphane in broccoli extract.

The objective of this study was to investigate the thermal degradation kinetics of sulforaphane (SF) in broccoli extract at selected temperatures (60, 75, 82 and 100°C) and pH values (2.2, 3.0, 4.0, 5.0 and 6.0). The results indicated that SF is unstable at high temperatures, but is more heat-stable when present in acidic food products. The degradation rate constants of SF in broccoli extract were lower than those obtained in purified SF. The thermal degradation of SF followed first-order reaction kinetics, and the rate constant increased with increase of temperature and pH values. The rate constant vs temperature relationships, which yield linear Arrhenius plots, were described by a simpler exponential equation, and a mathematical model was developed, using the steady-state kinetic parameters obtained to predict the retentions of SF at various pH values, heating times and temperatures.

Analysis of phenolic composition of Noble muscadine (Vitis rotundifolia) by HPLC-MS and the relationship to its antioxidant capacity.

UNLABELLED: Phenolic compounds and anthocyanins in muscadines have attracted much attention due to their diverse biological activities. With bioassays of antioxidant activities in terms of total phenolic content (TPC), total anthocyanin content (TAC), total procyanidin content (TPA), oxygen radical absorbance capacity (ORAC), and ferric reducing antioxidant power (FRAP) of different parts of the Noble muscadine, the butanol (BuOH) extract of the muscadine skin showed the highest TPC (317.91 ± 1.83 mg GAE/100 g FW), which might be ascribed to its high TAC of 227.06 ± 1.29 mg/100 g fresh weight (FW). The ethyl acetate (EtOAc) extract of the muscadine seed contained the highest TPA (55.30 ± 0.63 mg CE/100 g FW). Correlation analyses demonstrated a significant linear relationship of TPC and TAC compared to their ORAC and FRAP values within the range of R(2) from 0.9283 to 0.9936, which suggested that phenolics and anthocyanins in the extracts contributed significantly to their antioxidant potential. Nineteen individual phenolics and 5 anthocyanins were identified by HPLC-MS, which indicated different chemical profiles of anthocyanins and other phenolics in the muscadine extracts. PRACTICAL APPLICATION: The paper has provided rich information of bioactive phytochemical profiles in different solvent extracts and their correlation with the antioxidant activity in the muscadine that is a very special regional fruit in U.S. Its high content of phenolic compounds demonstrates that muscadine could be beneficial to human health.

High-performance liquid chromatography-mass spectrometry and evaporative light-scattering detector to compare phenolic profiles of muscadine grapes.

Evaporative light scattering detector (ELSD) is considered as a universal detector able to detect almost any compound. This study successfully established a HPLC-ELSD method for the simultaneous determination of phenolic acids, flavonoids and anthocyanins in muscadine grape. Twenty-four phenolics, including 5 anthocyanins, were identified by HPLC-UV-MS, ten of them were selected to calibrate the ELSD. Results from the ELSD quantification suggested that gallic acid, proanthocyanidins, and ellagic acid were the main phenolics in muscadines. In addition, antioxidant tests suggested that the extracts of muscadine grapes possessed strong antioxidant activities. All extracts had a high total phenolic content (TPC). High total anthocyanins contents (TAC) were found in Noble muscadine, and high proanthocyanidins content (TPA) in the seed portion.