Citrus flavonoids are attracting great interest due to their well-known beneficial effects, but many of them have not been characterized. In this work, ultra-high liquid chromatography coupled to high resolution mass spectrometry (UHPLCHRMS) was used for profiling flavonoids in citrus fruit. We proposed a strategy combining mass defect filtering (MDF) and MS/MS-based molecular networking (MMN) to handle complex UHPLC-HRMS data. The proposed strategy was explained and validated in the fruit of Citrus sinensis (L.) Osbeck, and when specific mass and mass defect windows were pre-defined, MDF enable removal of considerable un-related and/or interference MS1 peaks. In citrus fruit, the number of MS1 peaks in positive and negative modes were reduced by 70.80% (from 15,113 to 4413) and 55.30% (from 5617 to 2511), respectively, and thus the potential MS features of flavonoids were retained and exposed. After MDF, an MS/MS similarity-based MMN map was constructed to cluster flavonoids with similar chemical structures. MMN facilitated the annotation of 65 unknown citrus flavonoids by using only 21 pre-identified flavonoids as references. The compounds comprised 42 polymethoxylated flavonoids, 17 flavones, 24 flavanones, and 3 flavonols. Eleven of them had not been previously reported in Citrus sinensis (L.) Osbeck to our knowledge. Results of the current work indicated that the combination of MDF and MMN is a useful strategy for removing interference MS1 peaks and performing the structural annotation of unkonwn compounds in complex samples.

Biological approaches play an important role in lignin valorization, whereas many issues in this area remain unclear. Herein, ligninolytic enzymes in Pseudomonas putida NX-1 were systematically unraveled based on genome sequence technology. Particularly, a dye-decolorizing peroxidase was systematically studied by heterologous expression, enzyme purification, and enzymatic characterization, which suggested it possessed activities on both synthetic dyes and lignin-derived aromatics. Moreover, a complete pathway for polyhydroxyalkanoate biosynthesis was annotated, and the polyhydroxyalkanoate biosynthesis capability of P. putida NX-1 was experimentally confirmed with lignin as the sole carbon source. Furthermore, the monomer compositions, molecular weights, and thermal properties of polyhydroxyalkanoate from glucose and lignin-derived aromatics were comprehensively determined by gas chromatography-mass spectrometry, gel permeation chromatography, differential scanning calorimetry, and thermogravimetric analysis. The results indicated that physical properties of polyhydroxyalkanoate prepared from glucose and lignin-derived aromatics were similar, which suggested lignin could be an alternative feedstock for polyhydroxyalkanoate production without compromising its quality.

利用离子液体作为提取溶剂,结合超声辅助提取以及双水相萃取技术对柑橘果皮中的黄酮类化合物进行提取、分离及纯化.通过比较9种离子液体的提取率,发现三丁基己基溴化膦([P4446]Br)对柑橘果皮中的黄酮类化合物具有最高的提取率(56.15 mg/g).采用单因素试验结合响应面分析法优化超声辅助提取条件,获得最佳提取条件为:[P4446]Br质量分数54％、料液比1:10 (g/mL)、提取温度50℃、超声时间10 min.最佳条件下,柑橘果皮中的黄酮类化合物提取率为69.13 mg/g.超声辅助提取结束后,添加柠檬酸钠构建[P4446]Br-柠檬酸钠双水相体系,通过双水相萃取进一步分离纯化提取液中的黄酮类化合物.考察双水相体系的系线长度和相比对萃取率的影响,结果表明系线长度为71.95且相比为1.13时,黄酮类化合物的萃取效率最高,可达98.20％.双水相萃取结束后,采用正丁醇对黄酮类化合物进行反萃取,回收率可达80.11％.

As multiple biochemical pathways exist in nature for lignin synthesis and degradation, lignin valorization through biological pathways has attracted much attention. cis,cis-Muconate, a common metabolic intermediate in lignin biodegradation, is an important platform chemical for the synthesis of a variety of polymers. In this work, Rhodococcus opacus PD630, which is efficient in the assimilation of aromatic compounds, was modified for the first time to accumulate cis,cis-muconate from lignin and lignin-derived aromatics. To achieve efficient genome modification for R. opacus PD630, a markerless gene deletion/insertion system was constructed based on a mutant phenylalanyl-tRNA synthase gene as a counter-selection marker. With the developed gene deletion/insertion system, several R. opacus PD630 mutants were successfully constructed for cis,cis-muconate production from lignin-derived aromatics using catechol and protocatechuate as two key funnel nodes. Results indicated the constructed strains could assimilate at least 15 types of lignin-derived aromatics and real lignin for cis,cis-muconate production. Moreover, both lactase addition and H2SO4 + NaOH combinatorial pretreatment enhanced real lignin valorization to cis,cis-muconate. Ultimately, 1.63 g/L cis,cis-muconate was produced using lignin from corn stover as the substrate by a fed-batch fermentation process. Overall, these results highlight the great potential of using R. opacus as a cell factory for valorizing lignin to cis,cis-muconate and other valuable products.

Black tea (BT) is rich in dietary antioxidants, but its antioxidant composition has not been fully understood. To identify the true antioxidants occurring in BT, we established an approach based on 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay coupled with ultra-high performance liquid chromatography-high resolution mass spectrometry (DPPH-UHPLC-HRMS). The employment of HRMS enable us to detect trace antioxidants, resolve co-eluted antioxidants, and characterize chemical structures of unknown antioxidants. In total, 56 phenolic compounds were screened as potential antioxidants from 106 compounds identified in BT. Catechol and pyrogallol were revealed as the key substructures in enhancing the antioxidant abilities of phenolic compounds. During BT brewing, high temperature with extended time promote antioxidant leaching but may induce the degradation of esterified and glycosylated compounds such as theaflavin-3-gallate and rutin. In conclusion, this work identified the true antioxidant constituents of BT, their structural characteristics, and their dynamic changes under various brewing conditions.

An efficient in situ ionic liquid dispersive liquid-liquid microextraction followed by ultra-performance liquid chromatography was developed to determine four neonicotinoid insecticides in wild and commercial honey samples. In this method, a hydrophobic ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate, formed by in situ reaction between potassium hexafluorophosphate and 1-butyl-3-methylimidazolium bromide in sample solution, was used as the extraction solvent. In comparison with the traditional dispersive liquid-liquid microextraction method, the developed method required no dispersive solvent. To achieve high extraction efficiency and enrichment factor, the effects of various experimental parameters were studied in detail. Under the optimized conditions, the limits of detection and quantification were in the ranges of 0.30-0.62 and 1.20-2.50 μg/L, respectively. The method showed high enrichment factors (74-115) with the recoveries between 81.0 and 103.4%. The proposed method was finally applied to different wild and commercial honey samples.

Grape seeds are rich in proanthocyanidins, which are well-known natural antioxidants. In this work, an aqueous two-phase system (ATPS) based on ethanol/(NH4)(2)SO4 was proposed to extract and recover proanthocyanidins from grape seeds. Ionic liquids (IL) were used as adjuvants of ATPS to improve the extraction performance. The extraction efficiencies (EE) of proanthocyanidins were dramatically raised from 47.89 to 52.42% to 97.23 - 97.79% by adding 5 wt% [C(6)mirn]BF4 into ATPS. Extraction conditions were optimized as follows: 29 wt% ethanol, 19 wt% (NH4)(2)SO4, 4 wt% [C(6)mim]BF4, solid-liquid ratio = 1:20 g/mL, temperature (T) = 40 degrees C, pH = 5.0, and extraction time (t) = 20 min. Under optimum conditions, the yields of catechin, epicatechin, and procyanidin B2 reached 1.21, 1.22, and 0.14 mg/g dry weight, respectively, with EE of over 99.01%. Compared with conventional organic solvents such as methanol, ethanol, and isopropanol, the ATPS using ILs as adjuvants provided higher extraction yields. Results suggested that ethanol-based ATPS can extract value-added compounds from waste residues in the fruit-processing industry. In addition, low amount of IL addition can significantly enhance the extraction performance of ATPS.

Choline chloride-based deep eutectic solvents (DESs) were used as green alternatives to conventional organic solvents for extracting valuable flavonoids from citrus peel waste. The effects of viscosity, pH, and polarity of DESs on the extraction efficiency were investigated. Results revealed linear dependence of extraction yield of citrus flavonoids on the polarity of the hydrogen bond donor (HBD). To simultaneously and effectively extract polymethoxylated flavonoids (PMFs) and glycosides of flavonoids (GoFs) with different polarities, five ternary DESs were designed and prepared by combining the effective HBD components. Among the tailor-made DESs studied, choline chloride-levulinic acid-N-methyl urea showed the highest extraction yield of total flavonoids. Under optimized extraction conditions, 18.75 mg/g PMFs and 47.07 mg/g GoFs were extracted from citrus peel with recoveries of 95.87% and 86.32%, respectively. This study presents a useful strategy based on tailor-made DESs to selectively extracted value-added flavonoids from waste peels in the citrus-processing industry.

Ultra-performance liquid chromatography coupled to high-resolution mass spectrometry (UPLC-HRMS) has been used as a powerful tool to profile chemicals in traditional Chinese medicines. However, identification of potentially bioactive compounds is still a challenging work because of the large amount of information contained in the raw UPLC-HRMS data. Especially the ubiquitous matrix interference makes it more difficult to characterize the minor components. Therefore, rapid recognition and efficient extraction of the corresponding parent ions is critically important for identifying the attractive compounds in complex samples. Herein, we propose an integrated filtering strategy to remove un-related or interference MS1 ions from the raw UPLC-HRMS data, which helps to retain the MS features of the target components and expose the compounds of interest as effective as possible. The proposed strategy is based on the use of a combination of different filtering methods, including nitrogen rule, mass defect, and neutral loss/diagnostic fragment ions filtering. The strategy was validated by rapid screening and identification of 16 methoxylated flavonoids and 55 chlorogenic acids analogues from the raw UPLC-HRMS dataset of Folium Artemisiae Argyi. Particularly, successful detection of several minor components indicated that the integrated strategy has obvious advantages over individual filtering methods, and it can be used as a promising method for screening and identifying compounds from complex samples, such as herbal medicines.