Biological Activities of Citrus aurantium Leaf Extract by Optimized Ultrasound-Assisted Extraction.

Several studies have explored the biological activities of Citrus aurantium flowers, fruits, and seeds, but the bioactivity of C. aurantium leaves, which are treated as waste, remains unclear. Thus, this study developed a pilot-scale ultrasonic-assisted extraction process using the Box-Behnken design (BBD) for the optimized extraction of active compounds from C. aurantium leaves, and their antityrosinase, antioxidant, antiaging, and antimicrobial activities were evaluated. Under optimal conditions in a 150× scaleup configuration (a 30 L ultrasonic machine) of a pilot plant, the total phenolic content was 69.09 mg gallic acid equivalent/g dry weight, which was slightly lower (3.17%) than the theoretical value. The half maximal inhibitory concentration of C. aurantium leaf extract (CALE) for 2,2-diphenyl-1-picrylhydrazyl-scavenging, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)-scavenging, antityrosinase, anticollagenase, antielastase and anti-matrix metalloprotein-1 activities were 123.5, 58.5, 181.3, 196.4, 216.3, and 326.4 mg/L, respectively. Moreover, the minimal inhibitory concentrations for bacteria and fungi were 150-350 and 500 mg/L, respectively. In total, 17 active compounds were detected in CALE-with linalool, linalyl acetate, limonene, and α-terpineol having the highest concentrations. Finally, the overall transdermal absorption and permeation efficiency of CALE was 95.9%. In conclusion, our CALE demonstrated potential whitening, antioxidant, antiaging, and antimicrobial activities; it was also nontoxic and easily absorbed into the skin as well as inexpensive to produce. Therefore, it has potential applications in various industries.

Improving the practicality of recombinant Escherichia coli biosensor in detecting trace Cr(VI) by modifying the cryogenic storage conditions of biosensors and applying simple pretreatment.

Hexavalent chromium (Cr(VI)) is a global environmental pollutant. To reduce the risk caused by Cr(VI), a simple, accurate, reproducible, and inexpensive method for quantifying Cr(VI) in water and soil should be developed. In this study, three types of recombinant Escherichia coli biosensors (namely T7-lux-E. coli, T3-lux-E. coli, and SP6-lux-E. coli biosensor) containing promoters (T7, T3, and SP6), chromate-sensing regulator chrB, and the reporter gene luxAB were constructed. This study investigated the effects of cryogenic freezing temperature and time on trace Cr(VI) measurement by using recombinant E. coli biosensors. The results indicated that the activity of thawed frozen SP6-lux-E. coli cells stored at -20 °C for 270 days did not differ from that of freshly prepared cells. Turbidity and conductivity in water samples and organic matter in soil interfered with Cr(VI) measurement using the biosensor. The SP6-lux-E. coli biosensor exhibited a wide measurement range and a low deviation of <5% for measuring Cr(VI) in various Cr(VI)-contaminated water and soil samples and required only a simple pretreatment or extraction process even after 270-day storage at -20 °C. To the best of our knowledge, this is the first study to report the use of recombinant biosensors for accurately measuring Cr(VI) in both water and soil.

Characterization of Tibetan kefir grain-fermented milk whey and its suppression of melanin synthesis.

Tibetan kefir grain as the starter of milk fermentation has been applied as functional food with many bioactive characteristics. In this study, the milk whey product (TKG-MW) was obtained through the milk fermentation of Tibetan kefir grain containing the dominant Lactobacillus, Acetobacter, and Bacillus after 3 and 6 days of cultivation. Antioxidant, anti-inflammatory, and melanogenesis inhibition capacities under TKG-MW treatment were analyzed. Results revealed that the antioxidation of TKG-MW at 6 days of fermentation was higher than that at 3 days of fermentation according to the DPPH and ABTS+ radical scavenging analysis. However, the anti-inflammation of TKG-MW was only observed at 6 days of fermentation by using lipopolysaccharide-stimulated RAW 264.7 macrophages. The inhibition of mushroom tyrosinase activity by TKG-MW was demonstrated. The decrease of melanin content was verified using α-melanocyte-stimulating hormone-stimulated B16-F10 cell. The real-time quantitative reverse transcription polymerase chain reaction result indicated that the mRNA levels of Tyr, Trp-1, and Trp-2 of the B16 cell involved in melanin synthesis were down-regulated over a two-fold change by the TKG-MW treatment. Additionally, the protein expressions of Tyr, Trp-1, Trp-2, and Mitf of the B16 cell were reduced with the TKG-MW treatment. Organic acids, such as lactic acid, succinic acid, 3-phenyllactic acid, l-pyroglutamic acid, and malic acid, were identified by liquid chromatography-mass spectrometry in TKG-MW and were found to significantly inhibit tyrosinase activity. To the best of our knowledge, this work is the first to report melanogenesis suppression by TKG-MW. Results suggested that the fermentation product of TKG could be applied as a depigmenting agent in food and cosmetics.

Highly Sensitive Luminescent Bioassay Using Recombinant Escherichia coli Biosensor for Rapid Detection of Low Cr(VI) Concentration in Environmental Water.

In this study, we constructed a recombinant Escherichia coli strain with different promoters inserted between the chromate-sensing regulator chrB and the reporter gene luxAB to sense low hexavalent chromium (Cr(VI)) concentrations (<0.05 mg/L); subsequently, its biosensor characteristics (sensitivity, selectivity, and specificity) for measuring Cr(VI) in various water bodies were evaluated. The luminescence intensity of each biosensor depended on pH, temperature, detection time, coexisting carbon source, coexisting ion, Cr(VI) oxyanion form, Cr(VI) concentration, cell type, and type of medium. Recombinant lux-expressing E. coli with the T7 promoter (T7-lux-E. coli, limit of detection (LOD) = 0.0005 mg/L) had the highest luminescence intensity or was the most sensitive for Cr(VI) detection, followed by E. coli with the T3 promoter (T3-lux-E. coli, LOD = 0.001 mg/L) and that with the SP6 promoter (SP6-lux-E. coli, LOD = 0.005 mg/L). All biosensors could be used to determine whether the Cr(VI) standard was met in terms of water quality, even when using thawing frozen cells as biosensors after 90-day cryogenic storage. The SP6-lux-E. coli biosensor had the shortest detection time (0.5 h) and the highest adaptability to environmental interference. The T7-lux-E. coli biosensor-with the optimal LOD, a wide measurement range (0.0005-0.5 mg/L), and low deviation (-5.0-7.9%) in detecting Cr(VI) from industrial effluents, domestic effluents, and surface water-is an efficient Cr(VI) biosensor. This unprecedented study is to evaluate recombinant lux E. coli with dissimilar promoters for their possible practice in Cr(VI) measurement in water bodies, and the biosensor performance is clearly superior to that of past systems in terms of detection time, LOD, and detection deviation for real water samples.

Analysis of bioavailable toluene by using recombinant luminescent bacterial biosensors with different promoters.

In this study, we constructed recombinant luminescent Escherichia coli with T7, T3, and SP6 promoters inserted between tol and lux genes as toluene biosensors and evaluated their sensitivity, selectivity, and specificity for measuring bioavailable toluene in groundwater and river water. The luminescence intensity of each biosensor depended on temperature, incubation time, ionic strength, and concentrations of toluene and coexisting organic compounds. Toluene induced the highest luminescence intensity in recombinant lux-expressing E. coli with the T7 promoter [T7-lux-E. coli, limit of detection (LOD) = 0.05 µM], followed by that in E. coli with the T3 promoter (T3-lux-E. coli, LOD = 0.2 µM) and SP6 promoter (SP6-lux-E. coli, LOD = 0.5 µM). Luminescence may have been synergistically or antagonistically affected by coexisting organic compounds other than toluene; nevertheless, low concentrations of benzoate and toluene analogs had no such effect. In reproducibility experiments, the biosensors had low relative standard deviation (4.3-5.8%). SP6-lux-E. coli demonstrated high adaptability to environmental interference. T7-lux-E. coli biosensor-with low LOD, wide measurement range (0.05-500 µM), and acceptable deviation (- 14.3 to 9.1%)-is an efficient toluene biosensor. This is the first study evaluating recombinant lux E. coli with different promoters for their potential application in toluene measurement in actual water bodies.

Removal of toluene vapor in the absence and presence of a quorum-sensing molecule in a biotrickling filter and microbial composition shift.

Toluene is highly toxic and mutagenic, and it is generally used as an industrial solvent. Thus, toluene removal from air is necessary. To solve the problem of reducing high toluene concentrations with a short gas retention time (GRT), a quorum-sensing molecule [N-(3-oxododecanoyl)-L-homoserine lactone] (OHL) was added to a biotrickling filter (BTF). In this study, a BTF was used to treat synthetic and natural waste gases containing toluene. An extensive analysis was performed to understand the removal efficiency, removal characteristics, and bacterial community of the BTF. The addition of 20 µM OHL to the BTF significantly improved toluene removal, and more than 99.2% toluene removal was achieved at a GRT of 0.5 min when natural waste gas containing toluene (590-1020 ppm or 2.21-3.83 g m-3) was introduced. The maximum inlet load for toluene was 337.9 g m-3 h-1. Moreover, the BTF exhibited satisfactory adaptability to shock loading and shutdown operations. Pseudomonadaceae (33.0%) and Comamonadaceae (26.3%) were predominant bacteria in the system after a 98-day operation. These bacteria were responsible for toluene degradation. The optimal moisture content and low pressure drop for system operations demonstrated that the BTF was energy and cost efficient. Therefore, processing through a BTF with OHL is a favorable technique for toluene treatment.

Anticandidal Potential of Stem Bark Extract from Schima superba and the Identification of Its Major Anticandidal Compound.

Plant-derived extracts are a promising source of new drugs. Schima superba is traditionally used in China for heat clearing, detoxification, and treatment of furuncles. In this study, the anticandidal properties and mechanism of action of S. superba (SSE) were explored using a stem bark extract. SSE possessed high polyphenol and saponin contents of 256.6 ± 5.1 and 357.8 ± 31.5 µg/mg, respectively. A clear inhibition zone was observed for C. albicans growth through the disc diffusion method and the 50% inhibition of C. albicans by SSE was 415.2 µg/mL. Transcriptomic analysis in C. albicans treated with different doses of SSE was conducted through RNA-seq. Average values of 6068 genes and 20,842,500 clean reads were identified from each sample. Among these samples, 1680 and 1956 genes were differentially expressed genes (DEGs) from the SSE treatments of 0.2 and 0.4 mg/mL, respectively. C. albicans growth was inhibited by the changes in gene expression associated with the cell wall and membrane composition including the regulation of chitin degradation and ergosterol biosynthesis. This result could be reflected in the irregularly wrinkled morphology of the ruptured cell as revealed through SEM analysis. ESI-MS and NMR analyses revealed that the major compound purified from SSE was sasanquasaponin III and the 50% inhibition of C. albicans was 93.1 µg/mL. In summary, the traditional Chinese medicine S. superba can be applied as an anticandidal agent in complementary and alternative medicine.

Evaluation of synthetic gene encoding α-galactosidase through metagenomic sequencing of paddy soil.

Many genes of industrial relevance can be found in soil. In this study, metagenome sequencing of paddy soil was performed with 55.68 Gb sequences and 1,787,113 putative open reading frames (ORFs). The functional profiles and metabolic pathway of soil metagenomes were examined using Gene Ontology, Metagenomics RAST, and Kyoto Encyclopedia of Genes and Genomes. To verify the protein function and assembly of ORFs, a putative gene encoding α-galactosidase, namely GalR, which shares 65% identity with an unpublished glycoside hydrolase (GH) 27 family protein, was synthesized using its optimal codon for overexpression in Escherichia coli. GalR was successfully obtained and characterized. The optimal temperature and pH for GalR activity were 30°C and pH 9, respectively. Enzymatic activity indicated that GalR was alkaliphilic and different from acidophilic α-galactosidase in the GH 27 family. Furthermore, 50% of the relative activity of GalR can be attained for 1.7 and 0.7 h preincubation at 40°C and 50°C, respectively. Significant inhibition of GalR was observed in the presence of ethylenediaminetetraacetic acid (EDTA), MgCl2, sodium dodecyl sulfate (SDS), and H2O2; however, it was resistant to 0.1% methanol and ethanol and was slightly activated with NaCl and KCl. The specific activity of GalR was achieved at 11.6 and 0.59 µmol/min/mg of protein using p-nitrophenyl-α-d-galactopyranoside and raffinose as substrates, respectively. Consequently, the metagenomic sequencing-based strategy can provide information for mining novel genes.

Three-Stage Single-Chambered Microbial Fuel Cell Biosensor Inoculated with Exiguobacterium aestuarii YC211 for Continuous Chromium (VI) Measurement.

Chromium (VI) [Cr(VI)] compounds display high toxic, mutagenic, and carcinogenic potential. Biological analysis techniques (e.g., such as enzyme-based or cell-based sensors) have been developed to measure Cr(VI); however, these biological elements are sensitive to the environment, limited to measuring trace Cr(VI), and require deployment offsite. In this study, a three-stage single-chambered microbial fuel cell (SCMFC) biosensor inoculated with Exiguobacterium aestuarii YC211 was developed for in situ, real-time, and continuous Cr(VI) measurement. A negative linear relationship was observed between the Cr(VI) concentration (5⁻30 mg/L) and the voltage output using an SCMFC at 2-min liquid retention time. The theoretical Cr(VI) measurement range of the system could be extended to 5⁻90 mg/L by connecting three separate SCMFCs in series. The three-stage SCMFC biosensor could accurately measure Cr(VI) concentrations in actual tannery wastewater with low deviations (<7%). After treating the wastewater with the SCMFC, the original inoculated E. aestuarii remained dominant (>92.5%), according to the next-generation sequencing analysis. The stable bacterial community present in the SCMFC favored the reliable performance of the SCMFC biosensor. Thus, the three-stage SCMFC biosensor has potential as an early warning device with wide dynamic range for in situ, real-time, and continuous Cr(VI) measurement of tannery wastewater.

Inspecting the genome sequence and agarases of Microbulbifer pacificus LD25 from a saltwater hot spring.

Neoagaro-oligosaccharides prepared by agar hydrolysis have various application fields, including the pharmaceutical, cosmetic, and food industries. In this study, an agarolytic strain was isolated from a saltwater hot spring and identified as Microbulbifer pacificus LD25 by 16S rRNA. The whole genome sequence of M. pacificus LD25 was obtained. It had a size of 4.27 Mb and comprised 3062 predicted genes in 37 contigs with a G+C content of 58.0%. Six agarases were annotated and classified into three families, namely, GH16 (AgaL1), GH86 (AgaL2, AgaL3), and GH50 (AgaL4, AgaL5, AgaL6), which shared 75-96% identities with unpublished hypothetical proteins and agarases. AgaL1, AgaL4, and AgaL6 can be successfully expressed and purified in Escherichia coli. AgaL1 and AgaL4 displayed a significantly agarolytic capability, whereas AgaL6 exhibited a rarely detectable enzymatic activity. The optimal temperature and pH required for the activity of AgaL1 and AgaL4 was 50°C and 60°C, respectively, at pH 7. The specific activities of AgaL1 and AgaL4 were achieved at 16.8 and 9.6 U per mg of protein. Both agarases were significantly inhibited in the presence of EDTA, MgO, ZnCl2, and H2O2. However, AgaL1 was resistant to 0.1% SDS and AgaL4 was slightly activated by CaCl2. Substrate hydrolysis detected by LC-MS/MS analysis indicated that neoagarobiose was the main product during AgaL1 and AgaL4 catalysis. Furthermore, AgaL4 was thermostable and retained over 93% of its relative activity after pre-incubation at 70°C for 180 min. Consequently, M. pacificus LD25 has a potential for agarase production in E. coli and industrial applications.