[Expression of ß-xylosidase An-xyl from Aspergillus niger and characterization of its xylose tolerance].

The hydrolysis of xylo-oligosaccharides catalyzed by ß-xylosidase plays an important role in the degradation of lignocellulose. However, the enzyme is easily inhibited by its catalytic product xylose, which severely limits its application. Based on molecular docking, this paper studied the xylose affinity of Aspergillus niger ß-xylosidase An-xyl, which was significantly differentially expressed in the fermentation medium of tea stalks, through cloning, expression and characterization. The synergistic degradation effect of this enzyme and cellulase on lignocellulose in tea stems was investigated. Molecular docking showed that the affinity of An-xyl to xylose was lower than that of Aspergillus oryzae ß-xylosidase with poor xylose tolerance. The Ki value of xylose inhibition constant of recombinant-expressed An-xyl was 433.2 mmol/L, higher than that of most ß-xylosidases of the GH3 family. The Km and Vmax towards pNPX were 3.6 mmol/L and 10 000 µmol/(min·mL), respectively. The optimum temperature of An-xyl was 65 ℃, the optimum pH was 4.0, 61% of the An-xyl activity could be retained upon treatment at 65 ℃ for 300 min, and 80% of the An-xyl activity could be retained upon treatment at pH 2.0-8.0 for 24 h. The hydrolysis of tea stem by An-xyl and cellulase produced 19.3% and 38.6% higher reducing sugar content at 2 h and 4 h, respectively, than that of using cellulase alone. This study showed that the An-xyl mined from differential expression exhibited high xylose tolerance and higher catalytic activity and stability, and could hydrolyze tea stem lignocellulose synergistically, which enriched the resource of ß-xylosidase with high xylose tolerance, thus may facilitate the advanced experimental research and its application.

Effects of management intensities on soil aggregate stability and carbon, nitrogen, phosphorus distribution in Phyllostachys edulis forests.

Soil aggregates are the main sites for the decomposition of soil organic matter and the formation of humus. The composition characteristics of aggregates with different particle sizes are one of the indicators for soil fertility. We explored the effects of management intensity (frequency of fertilization and reclamation) on soil aggregates in moso bamboo forests, including mid-intensity management (T1, fertilization and reclamation every 4 years), high-intensity management (T2, fertilization and reclamation every 2 years), and extensive management (CK). The water-stable soil aggregates (0-10, 10-20, and 20-30 cm layers) from moso bamboo forest were separated by a combination of dry and wet sieving method and the distribution of soil organic carbon (SOC), total nitrogen (TN) and available phosphorus (AP) across different soil layers were determined. The results showed that management intensities had significant effects on soil aggregate composition and stability, and SOC, TN, AP distribution of moso bamboo forests. Compared with CK, T1 and T2 decreased the proportion and stability of macroaggregates in 0-10 cm soil layer, but increased that in 20-30 cm soil layer, while reduced the content of organic carbon in macroaggregates, the contents of organic carbon, TN and AP in microaggregates. Such results indicated that the intensified management was not conducive to formation of macroaggregates in 0-10 cm soil layer and carbon sequestration in macroaggregates. It was beneficial to the accumulation of organic carbon in soil aggregates and nitrogen and phosphorus in microaggregates with lower human disturbance. Mass fraction of macroaggregates and organic carbon content of macroaggregates was significantly positively correlated with aggregate stability, which best explained the variations of aggregate stability. Therefore, macroaggregates and organic carbon content of macroaggregates were the most important factors affecting the formation and stability of aggregates. Appropriate reduction of disturbance was beneficial to the accumulation of macroaggregates in the topsoil, the sequestration of organic carbon by macro-aggregates, and the sequestration of TN and AP by microaggregates, and improving soil quality and sustainable management in moso bamboo forest from the point of view of soil aggregate stability.

Dissecting chemical markers for controlling "Paozhi" method of traditional Chinese medicine with untargeted metabolomics: Vinegar-baked Euphorbia kansui as a case study.

The processing of Traditional Chinese Medicine requires the appropriate parameters, while the specific chemical markers are still absent to obtain the optimized processing. In this study, we used vinegar-baked Euphorbia kansui as a case to dissect the chemical markers for the baking process using untargeted metabolomics. The robust chemical markers were selected based on the three rules, correlation, significant difference, and controllability. All the differential features were categorized based on their mass defects. After the differential analysis, 310 differential compounds were screened out and could be mainly divided into six categories: diacylglycerols and triacylglycerols demonstrated increasing trends with the baking time in the discriminant model, while ingenane-type diterpenes, jatrophane-type diterpenes, fatty acid esters, and fatty acids had decreasing trends. It was unexpected to find that the diterpenes did not correlate with the baking time. Only very few compounds meet the three rules. They were validated with a high-performance liquid chromatography method. Finally, only 13-Hydroxy-9,11-octadecadienoic acid and its isomer 9-Hydroxy-10,12-octadecadienoic acid could be used further to differentiate the commercial vinegar-baked Euphorbia kansui. It would be of interest to evaluate whether these two compounds could be utilized as markers to control more processing methods in future studies.

An improved strategy for identification and annotation of easily in-sourced dissociation diterpene lactones from plant natural products: Taking Andrographis paniculata (Burm. f.) as an example.

RATIONALE: Diterpene lactones (DL) in Andrographis paniculata (AP) are known as "natural antibiotics" for their excellent antibacterial activity. During mass spectrometry (MS) analysis, the hydroxyl groups in the AP DL skeleton are prone to neutral loss of H2 O, producing high in-source fragment peaks and affecting the characterization of these components. METHODS: Mass tags were applied during the MS data acquisition step, and special adduct ion form was used to guide the data processing and characterization steps. Besides, the total number of characterized AP DLs significantly increased when combining the number of neutrally lost H2 O from AP DLs, incorporating information on the diagnostic ions, and adopting molecular networks generated with the Global Natural Products Social Molecular Networking database. RESULTS: Ninety-nine DLs, comprising 6 monohydroxyl groups, 20 dihydroxyl groups, 27 trihydroxy groups, and 46 DLs with more than 3 hydroxyl groups, were characterized from AP. In addition, based on the characteristic fragments in the product ions (C3 H4 , Δm/z = 40.03 Da), it could be assumed that 90 DLs had the C19-OH structure among the identified DLs. The current study provides a new approach for collecting, processing, and characterizing MS analysis of natural DLs prone to in-source fragmentation. CONCLUSIONS: MS characterization of AP DLs was significantly improved, and many potential new compounds were identified in AP. This characterization provides new methods for the purification and identification of AP DLs.

In vitro fermentation of seaweed polysaccharides and tea polyphenol blends by human intestinal flora and their effects on intestinal inflammation.

A combination of polysaccharides and tea polyphenols can enhance immune activity synergistically, depending on the type and structure of polysaccharides, but the mechanism remains unknown. This study is aimed to investigate the regulating effects of different seaweed polysaccharide (ι-carrageenan, agarose) and tea polyphenol blends on intestinal flora and intestinal inflammation using an in vitro ascending-transverse-descending colon fermentation system and RAW264.7 cell model. The results showed that seaweed polysaccharides in the presence of tea polyphenol were almost completely degraded at transverse colon fermentation for 36 h. Agarose significantly enhanced the butyric acid production content by increasing the abundance of Lachnospiraceae, whereas agarose and tea polyphenol blends did not have a synergistic effect. On the contrary, ι-carrageenan and tea polyphenol blends synergistically increased the abundance of beneficial bacteria (e.g., Bacteroidetes and Bifidobacterium) and promoted the production of short-chain fatty acids (SCFAs), such as isobutyric acid. Such changes tended to alter the impacts of different seaweed polysaccharides and tea polyphenol blends on intestinal inflammation. Among them, ι-carrageenan and tea polyphenol blends were the most effective in inhibiting lipopolysaccharide-induced NO, ROS, IL-6, and TNF-α production in RAW264.7 cells, indicating the alleviated intestinal inflammation. The results suggest that the seaweed polysaccharide and tea polyphenol blends have prebiotic potential and can benefit intestinal health.

Simultaneous Inhibitory Effects of All-Trans Astaxanthin on Acetylcholinesterase and Oxidative Stress.

Alzheimer´s disease is a global neurodegenerative health concern. To prevent the disease, the simultaneous inhibition of acetylcholinesterase and oxidative stress is an efficient approach. In this study, the inhibition effect of all-trans astaxanthin mainly from marine organisms on acetylcholinesterase and oxidative stress was evaluated by a chemical-based method in vitro and cell assay model. The results show that all-trans astaxanthin was a reversible competitive inhibitor and exhibited a strong inhibition effect with half inhibitory concentration (IC50 value) of 8.64 µmol/L. Furthermore, all-trans astaxanthin inhibited oxidative stress through reducing malondialdehyde content and increasing the activity of superoxide dismutase as well as catalase. All-trans astaxanthin could induce the changes of the secondary structure to reduce acetylcholinesterase activity. Molecular-docking analysis reveals that all-trans astaxanthin prevented substrate from binding to acetylcholinesterase by occupying the space of the active pocket to cause the inhibition. Our finding suggests that all-trans astaxanthin might be a nutraceutical supplement for Alzheimer´s disease prevention.

In-depth exploration and comparison of chemical constituents from two Lilium species through offline two-dimensional liquid chromatography combined with multimode acquisition of high-resolution mass spectrometry.

Lilium lancifolium and Lilium brownii viridulum were two common cultivars of Lilium in China, which have been used as a source of food in ancient China, and as a traditional herbal medicine in most northern hemispheres countries continues today. However, only a few secondary metabolites in Lilium closely related to human health have been reported. In this research, an offline two-dimensional (HILIC and RP C18) separation system combined with multimode high-resolution mass spectrometry data acquisition was established for in-depth exploration and comparison of the chemical components in Lilium. In total, 331 components were identified, among which phenylpropanoid derivatives and steroidal saponins were the most abundant components. Furthermore, sulfur derivatives and steroidal alkaloids were systematically characterized in Lilium for the first time. These results provided valuable information for in-depth differentiating types of components characterization, which may be applied to assess and improve the edible and medicinal values of Lilium.

MGP Regulates Perivascular Adipose-Derived Stem Cells Differentiation Toward Smooth Muscle Cells Via BMP2/SMAD Pathway Enhancing Neointimal Formation.

Perivascular adipose-derived stem cells (PV-ADSCs) could differentiate into smooth muscle cells (SMCs), participating in vascular remodeling. However, its underlying mechanism is not well explored. Our previous single-cell RNA-sequencing dataset identified a unique expression of matrix Gla protein (MGP) in PV-ADSCs compared with subcutaneous ADSCs. MGP involves in regulating SMC behaviors in vascular calcification and atherosclerosis. In this study, we investigated MGP's role in PV-ADSCs differentiation toward SMCs in vitro and in vascular remodeling in vivo. PV-ADSCs were isolated from perivascular regions of mouse aortas. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR), Western blot, and immunofluorescence confirmed higher MGP expression in PV-ADSCs. The MGP secretion increased along PV-ADSCs differentiation toward SMCs in response to transforming growth factor-beta 1 (TGF-ß1). Lentivirus knockdown of MGP markedly promoted the bone morphogenetic protein 2 (BMP2) expression and phosphorylation of SMAD1/5/8 in PV-ADSCs, subsequently inhibiting its differentiation toward SMCs. Such inhibition could be partially reversed by further application of BMP2 inhibitors. On the contrary, exogenous MGP inhibited BMP2 expression and SMAD1/5/8 phosphorylation in PV-ADSCs, thereby promoting its differentiation toward SMCs. Transplantation of cultured PV-ADSCs, which was pretreated by MGP knockdown, in mouse femoral artery guide-wire injury model significantly alleviated neointimal hyperplasia. In conclusion, MGP promoted the differentiation of PV-ADSCs toward SMCs through BMP2/SMAD-mediated signaling pathway. This study offers a supplement to the society of perivascular tissues and PV-ADSCs.

Effects of Psychiatric Issues and Early Enteral Nutrition Therapy on Anxiety and Quality-of-Life of Patients with Gastric Cancer.

Objectives: In this study, we assessed the effects of psychiatric issues and early enteral nutrition therapy on anxiety and quality-of-life of patients with gastric cancer. Methods: We enrolled 60 patients with gastric cancer treated from January 2018 to November 2020 and divided them into 2 groups using a random number table (N=30 per group). All patients received early enteral nutrition therapy. The control group was given routine nursing; treatment of persons in the observation group was supplemented with additional psychiatric nursing. We then compared anxiety-depression scores, treatment compliance, nutritional status indices, and quality-of-life score. Results: The Self-rating Anxiety Scale (SAS) score and the Self-rating Depression Scale (SDS) score significantly declined in both groups after nursing compared with those before nursing (p<.05). After nursing, the SAS score and SDS score were lower in the observation group versus the control group (p<.05). The total rate of treatment compliance in the observation group (93.33%) was higher than that in the control group (73.33%) (p<.05). After nursing, the levels of hemoglobin, albumin, prealbumin, and transferrin were significantly higher in both groups than before nursing (p<.05), but higher in the observation group (p<.05). After nursing, the quality-of-life score was significantly higher in both groups than before nursing (p<.05), but higher in the observation group (p<.05). Conclusion: During enteral nutrition therapy for patients with gastric cancer, psychiatric treatment can effectively alleviate patient anxiety and depression, enhance treatment compliance, improve nutritional status, and enhance quality-of-life.

Untargeted metabolomics coupled with chemometric analysis deducing robust markers for discrimination of processing procedures: Wine-processed Angelica sinensis as a case study.

Wine-processed Angelica Sinensis is a widely used Chinese medicinal decoction piece in China. However, there are hardly any robust markers indicating the processing procedure of wine-processed Angelica Sinensis, including the amount of rice wine and processing degree. A strategy integrating untargeted metabolomics and chemometric analysis for deducing robust markers was provided and applied to the discrimination of processing procedure. First, 86 compounds were tentatively identified in wine-processed Angelica Sinensis by ultra-high-performance liquid chromatography coupled with quadrupole-time of flight mass spectrometry. Second, 93 potential chemical markers were selected using multivariate analysis, among which nine robust chemical markers were selected by verification with commercial samples. Finally, the effects of processing temperature, time, and amount of rice wine on the three selected chemical markers were investigated through a rapid analytical method. It was demonstrated that both m/z 258.1097 and 238.1189 were positively correlated with the amount of rice wine and processing degree. In summary, this study introduced two candidate processing markers as robust markers for discriminating the processing procedures of wine-processed Angelica sinensis. It also proposed a strategy to provide the reference for the research of other decoction pieces.

Sinisan Protects Primary Hippocampal Neurons Against Corticosterone by Inhibiting Autophagy via the PI3K/Akt/mTOR Pathway.

Objective: Corticosterone causes significant neurotoxicity in primary hippocampal neurons which is associated with depression. Dysfunctional autophagy is implicated in cognitive impairment and depressive-like behavior. The traditional Chinese medicine Sinisan (SNS) is highly effective in clinical treatment of depression. However, the molecular mechanisms underlying therapeutic effects of SNS are unknown. Purpose: The aim of this study was to elucidate the protective effect of SNS and the underlying mechanisms against corticosterone-induced neuronal damage. Study Design: The effects of serum derived from rats containing SNS (or untreated controls) on the expression of autophagy-related molecules in primary rat hippocampal neurons exposed to different concentrations of corticosterone for different intervals were explored. Methods: CCK-8 assay, LDH assay were used to analyze cell viability and LDH activity. Western blot, qRT-PCR, and immunofluorescence assays were used to determine protein and mRNA expression levels of molecules such as LC3, p62, Beclin1, ULK1, PI3K, p-PI3K, Akt p-Akt, mTOR, p-mTOR, p70S6, p-p70S6, 4ebp1 and p-4ebp1. Results: Corticosterone induced a dose- and time-dependent reduction in cellular viability. Moreover, corticosterone (100-400 µM) treatment for 24 h increased LC3-II/LC3-I protein ratio, increased Beclin1 and ULK1 protein expression levels, and decreased p62, PI3K, p-PI3K, p-Akt, p-mTOR, p-p70S6, and p-4ebp1 protein expression levels. Notably, SNS-containing serum reversed corticosterone-induced reduction of neuronal viability, and increased p62, PI3K, p-Akt, p-mTOR, p-p70S6, and p-4ebp1 protein and mRNA expression levels. In addition, SNS-containing serum decreased LC3-II/LC3-I protein ratio, and downregulated Beclin1, and ULK1 protein and mRNA expression in primary hippocampal neurons. Conclusion: SNS protects primary hippocampal neurons against corticosterone-induced neurotoxicity by preventing excessive autophagy through activation of PI3K/AKT/mTOR pathway.

Enzymatic hydrolysis and auto-isomerization during ß-glucosidase treatment improve the aroma of instant white tea infusion.

The aroma changes in instant white tea resulting from ß-glucosidase treatment was investigated by quantitative descriptive analysis (QDA), gas chromatography-mass spectrometry (GC-MS), odour activity value analysis (OAV), aroma reconstruction and omission tests. The grassy, floral and sweet notes increased significantly (P < 0.05), and the roasted note decreased significantly (P < 0.05) upon ß-glucosidase treatment. Quantitative analysis showed that the concentrations of benzaldehyde, benzeneacetaldehyde, (Z)-3-hexen-1-ol, linalool, phenylethyl alcohol, cis-linalool oxide, trans-linalool oxide, hexanol, hotrienol and (E)-2-hexen-1-ol increased significantly (P < 0.05) after treatment; however, (Z)-3-hexen-1-ol isomerized to (E)-2-hexen-1-ol. OAV analysis, aroma reconstruction and the omission test showed that the grassy, floral and sweet notes increased as the (Z)-3-hexen-1-ol, cis/trans-linalool oxide and benzeneacetaldehyde increased, whereas the roasted note declined under the same conditions. The enzymatic hydrolysis of glycosidic precursors and the auto-isomerization of volatile compounds provide new information for understanding how ß-glucosidase treatment improves the aroma of tea products.

Exolytic products of alginate by the immobilized alginate lyase confer antioxidant and antiapoptotic bioactivities in human umbilical vein endothelial cells.

Alginate is a natural polysaccharide resource abundant in brown algae and it can be cleaved into alginate oligosaccharides by alginate lyase. Alginate lyases and the bioactive alginate oligosaccharides have been applied in diverse fields such as pharmaceutical therapy and nutraceutical supplementation. Immobilized enzymes greatly facilitate their industrial application owing to their reusability, stability, and tunability. In this study, magnetic Fe3O4 nanoparticles were synthesized and used to immobilize an exolytic alginate lyase AlgL17 that was characterized previously. The immobilized AlgL17 demonstrated enhanced thermal and pH tolerance, extended storage stability, and moderate reusability. The mass spectrum indicated the specific activity of the immobilized AlgL17 to release alginate oligosaccharides (AOS) from alginate polysaccharide. The produced AOS exhibited their antioxidant and antiapoptotic activities in H2O2-stressed human umbilical vein endothelial cells by upregulation of reactive oxygen species scavenging activities and attenuation of the caspase-mediated apoptosis pathway.

Bioactive sesquiterpene coumarins from the resin of Ferula sinkiangensis targeted on over-activation of microglia.

Nine undescribed (1-4, 6-10) sesquiterpene coumarins, together with a new natural one (5) and ten known ones (11-20), were isolated from the low polarity fraction of the 95% ethanol extract of the resin of Ferula sinkiangensis. Their structures were elucidated based on the comprehensive analysis of HRESIMS, 1D and 2D NMR data. The absolute configurations were determined by comparison of experimental and calculated ECD spectra. All the identified SCs were evaluated for their anti-neuroinflammatory activities in LPS-induced BV-2 cells. Ferusingensine G (8) displayed a significant inhibitory effect on nitric oxide (NO) production with an IC50 value of 1.2 µM. The results suggested that natural SCs might be served as potential neuroinflammatory inhibitors.

Characterization of the Aroma of an Instant White Tea Dried by Freeze Drying.

The aroma of an instant white tea (IWT) was extracted through simultaneous distillation-extraction (SDE) and analyzed by sensory evaluation, gas chromatography-mass spectrometry-olfactometry (GC-MS-O), aroma reconstruction, omission test and synergistic interaction analysis. Sensory evaluation showed the IWT was dominated with floral and sweet notes. The SDE extract had the aroma similar to the IWT. The main volatile components in the SDE extract were benzyl alcohol, linalool, hotrienol, geraniol, α-terpineol, coumarin, camphene, benzeneacetaldehyde, 2-hexanone, cis-jasmin lactone and phenylethyl alcohol. GC-MS-O and aroma reconstruction experiments showed 16 aroma-active compounds. Linalool, trans-ß-damascenone and camphene were the major contributors to floral, sweet and green notes based on flavor dilution analysis and omission test. Linalool and trans-ß-damascenone had synergistic effect to promote floral and sweet notes. Camphene and trans-ß-damascenone had synergistic effect to reduce green and sweet notes. The study helps to understand the aroma of IWT and antagonism interactions among aroma-active volatiles.

Two-dimensional liquid chromatography analysis of all-trans-, 9-cis-, and 13-cis-astaxanthin in raw extracts from Phaffia rhodozyma.

An effective two-dimensional liquid chromatography method has been established for the analysis of all-trans-astaxanthin and its geometric isomers from Phaffia rhodozyma employing a C18 column at the first dimension and a C30 column in the second dimension, connected by a 10-port valve using the photo-diode array detector. The regression equation of astaxanthin calibration curve was established, and the precision and accuracy values were found to be in the range of 0.32-1.14% and 98.21-106.13%, respectively. By using two-dimensional liquid chromatography, it was found that day light, ultrasonic treatment, and heat treatment have significant influence on the content of all-trans-astaxanthin in the extract from P. rhodozyma due to the transformation of all-trans-astaxanthin to cis-astaxanthin. The day light and ultrasonic treatments more likely transform all-trans-astaxanthin to 9-cis-astaxanthin, and the thermal treatment transforms all-trans-astaxanthin to 13-cis-astaxanthin. These results indicate that the two-dimensional liquid chromatography method can facilitate monitoring astaxanthin isomerization in the raw extract from P. rhodozyma. In addition, the study will provide a general reference for monitoring other medicals and bioactive chemicals with geometric isomers.

Kellerin alleviates cognitive impairment in mice after ischemic stroke by multiple mechanisms.

Ischemic stroke is a global disease with high disability and mortality rates. Cognitive impairment is one of the major clinical features of ischemic stroke, and microglia-mediated inflammation has been shown to be an important contributor to the pathogenesis of ischemic stroke. Kellerin, extracted from Ferula sinkiangensis, was previously shown to inhibit microglial activation and exert a strong anti-neuroinflammatory effect. However, there is no report of the potential therapeutic effect of kellerin on ischemic stroke by targeting microglial cells. In this study, we wanted to examine the effects of kellerin on ischemic stroke in the bilateral common carotid artery occlusion (BCCAO) model and the lipopolysaccharide (LPS)-activated microglia model. We found that kellerin alleviated cognitive impairment, decreased neuronal loss, suppressed microglial activation, and transformed microglia from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype in BCCAO mice. Moreover, in in vitro studies, we found that kellerin regulated microglial polarization and inhibited the NLRP3 and MAPK signaling pathways after LPS treatment. These findings provide a new understanding of the function of kellerin in ischemic stroke, and suggest that kellerin could be a potential therapeutic agent for the treatment of ischemic stroke.

A Quantitative Analysis Model Established to Determine the Concentration of Each Source in Mixed Astaxanthin from Different Sources.

Astaxanthin from different sources possesses different biological activities and optical isomers. The ingredients of astaxanthin mixtures from different sources on the market have often been mislabeled. Therefore, it is important to determine the sources of astaxanthin and their respective concentrations in a mixture. To solve this problem, a quantitative analysis model was established and further verified. The results showed that the deviation between the calculated concentration and the actual concentration ranged from 0 to 7 µg/mL, and the recovery rate was between 88.90% and 103.56%. This indicates that the quantitative analysis model of astaxanthin was feasible and reliable. This study not only has important applications in the astaxanthin mixture component determination but may also shed light on the quantitative analysis of other sample mixtures with stereoisomers from different sources.

Aroma enhancement of instant green tea infusion using ß-glucosidase and ß-xylosidase.

ß-Glucosidase and ß-xylosidase were investigated for their ability to improve the aroma of instant green tea. The aroma and corresponding contributors were analyzed by sensory evaluation, gas chromatography-mass spectrometry, and odor activity value. Their specific contributions to aroma attributes were further examined by aroma reconstruction and omission experiments. The ß-glucosidase treatment significantly enhanced floral and grassy notes, on account of the increases of geraniol, nonanal, and cis-3-hexen-1-ol, and weakened the caramel note, attributable to the increases of nonanal, cis-3-hexen-1-ol, geraniol, methyl salicylate, and decanal. The co-treatment with ß-glucosidase and ß-xylosidase further enhanced the grassy note, with further increase in nonanal and cis-3-hexen-1-ol, and further weakened the caramel note, with additional increase in nonanal, cis-3-hexen-1-ol, methyl salicylate, and decanal. The synergistic action of ß-glucosidase and ß-xylosidase provides new clues to the production of instant green tea infusions with high aroma quality.

Two-component system AfrQ1Q2 involved in oxytetracycline biosynthesis of Streptomyces rimosus M4018 in a medium-dependent manner.

Regulation of secondary metabolism involves complex interactions of both pathway-specific regulators and global regulators, which may trigger or repress the expression of genes involved in antibiotic biosynthesis. Similarly, many of these global regulatory proteins belong to two-component systems. In this study, a new two-component system (TCS) AfrQ1Q2 homologous to AfsQ1Q2 of Streptomyces coelicolor was acquired from the genome sequence of Streptomyces rimosus M4018 by using bioinformatics analysis. RT-PCR results showed co-transcription of afrQ1 (RR) and afrQ2 (HK) in S. rimosus. Consequently, the significant enhancement in oxytetracycline (OTC) yield in afrQ1-disrupted mutant was observed when cultivated in the defined minimal medium (MM) with glycine as the sole nitrogen source. In order to further investigate the regulation mechanism of AfrQ1Q2 in OTC production, the transcriptional levels of five biosynthesis and regulation related genes such as oxyB, otrB, otcG, otcR and otrC were tested by qRT-PCR, which indicated a significantly up-regulatory trend in the afrQ1-disrupted mutant. Meanwhile, a down-regulatory trend of each gene was tested in the complementary mutant as compared to wild type M4018. Moreover, these selected five genes were positively correlated with OTC production. Conclusively, these findings suggested that the TCS AfrQ1Q2 could be one of the global regulators, which negatively regulates OTC production via activating pathway specific regulators in S. rimosus M4018.