Purification and characterization of limonin D-ring lactone hydrolase from sweet orange (Citrus sinensis (L.) Osbeck) seeds.

BACKGROUND: Citrus products often suffer from delayed bitterness, which is generated from the conversion of non-bitter precursors (limonoate A-ring lactone, LARL) to limonin under the catalysis of limonin D-ring lactone hydrolase (LDLH). In this study, LDLH was isolated and purified from sweet orange seeds, and a rapid and accurate high-performance liquid chromatography method to quantify LARL was developed and applied to analyze the activity and enzymatic properties of purified LDLH. RESULTS: Purified LDLH (25.22 U mg-1) showed bands of 245 kDa and 17.5 kDa molecular weights in native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate PAGE analysis respectively. After a 24 h incubation under strongly acidic (pH 3) or strongly alkaline (pH 9) conditions, LDLH still retained approximately 100% activity. Moreover, LDLH activity was not impaired by thermal treatment at 50 °C for 120 min. Enzyme inhibition assays showed that LDLH was inactivated only after ethylenediaminetetraacetic acid treatment, and other enzyme inhibitors showed no significant effect on its activity. In addition, the LDLH activity of calcium ion (Ca2+) intervention was 108% of that in the blank group, and that of zinc ion (Zn2+) intervention was 71%. CONCLUSION: LDLH purified in this study was a multimer containing 17.5 kDa monomer with a wide pH tolerance range (pH 3-9) and excellent thermal stability. Moreover, LDLH might be a metallopeptidase, and its activity was stimulated by Ca2+ and significantly inhibited by Zn2+. These findings improve our understanding of LDLH and provide some important implications for reducing the bitterness in citrus products in the future. © 2024 Society of Chemical Industry.

Effects of Separation and Purification Methods on Antioxidation, Hypoglycemic and DNA Protection Activity of Fenugreek Polysaccharide.

Six low molecular weight fenugreek polysaccharides (FP) were isolated and purified by ethanol stepwise precipitation (EFP-20, EFP-40, and EFP-60) and DEAE-52 cellulose column method (DFP-0, DFP-0.15, and DFP-0.3), respectively. The effects of different separation and purification techniques on the preliminary properties and biological activities of fenugreek polysaccharides were compared. The results showed that the DEAE-52 cellulose-eluted fractions had a higher total sugar content and displayed a looser structure. The molecular weights of all six fractions were in the range of 4-19 kDa, with significant changes in the ratio of galactose to mannose. All six fractions contained α-D-galactopyranose and ß-D-mannopyranose structures. Activity tests showed that all six fractions possessed antioxidant, hypoglycemic and DNA-protective activities. Among them, the DFP-0 fraction showed the highest activity. Overall, different isolation and purification methods lead to changes in the properties and bioactivities of FP, which provides a theoretical basis for the development and application of FP in functional foods and drugs.

Physical and oxidative stability of flaxseed oil-in-water emulsions prepared by natural lignin-carbohydrate complex.

Flaxseed oil, rich in α-linolenic acid, plays a crucial role in various physiological processes. However, its stability presents certain challenges. In this study, the natural lignin-carbohydrate complex (LCC) was used to prepare the physical and oxidative stability of flaxseed oil-in-water emulsions. The LCC was characterized by HPLC, GPC, and FT-IR. The stability of emulsions was evaluated by viscosity, modulus, and micro-morphology changes. Then, the oxidation products were monitored by UV-vis spectrophotometer and HPLC. The results revealed that the high internal phase emulsion (HIPE) was successfully prepared with 2.5 wt% LCC at an oil/water ratio of 75/25 (v/v). Small droplet size (13.361 µm) and high viscosity (36,500 mPa·s) were found even after 30-day storage. Steric interactions of the LCC play a crucial role in ensuring stability, intricately linked to the interfacial properties of the emulsion. Meanwhile, the oxidative stability of α-linolenic acid in the encapsulated flaxseed oil was significantly higher than that in the bulk flaxseed oil. The results revealed that the LCC as a suitable emulsifier opens a new window for the storage of functional lipids rich in polyunsaturated fatty acids.

Improvement of antibacterial activity of polysaccharides via chemical modification: A review.

Antibiotic residue and bacterial resistance induced by antibiotic abuse have seriously threatened food safety and human healthiness. Thus, the development and application of safe, high-efficiency, and environmentally friendly antibiotic alternatives are urgently necessary. Apart from antitumor, antivirus, anti-inflammatory, gut microbiota regulation, immunity improvement, and growth promotion activities, polysaccharides also have antibacterial activity, but such activity is relatively low, which cannot satisfy the requirements of food preservation, clinical sterilization, livestock feeding, and agricultural cultivation. Chemical modification not only provides polysaccharides with better antibacterial activity, but also promotes easy operation and large-scale production. Herein, the enhancement of the antibacterial activity of polysaccharides via acetylation, sulfation, phosphorylation, carboxymethylation, selenation, amination, acid graft, and other chemical modifications is reviewed. Meanwhile, a new trend on the application of loading chemically modified polysaccharides into nanostructures is discussed. Furthermore, possible limitations and future recommendations for the development and application of chemically modified polysaccharides with better antibacterial activity are suggested.

Molecular data and morphological characters clarify the taxonomic status of Pintara heringi pieridoides (Liu & Gu, 1994) (Hesperiidae, Pyrginae).

We found Albiphasma heringi (Mell, 1922) and A. pieridoides (Liu & Gu, 1994) to be conspecific by the 658 bp COI gene sequences and male genitalia characters. Considering the distinguishable wing patterns and allopatric distribution of the two taxa, we treat pieridoides as a subspecies of heringi. Therefore, the genus Albiphasma Huang, Chiba, Wang and Fan, 2016, which was established for heringi and pieridoides, becomes monotypic, and in light of morphological similarities and close genetic distance between heringi and Pintara bowringi (Joicey & Talbot, 1921), we propose its synonymy with Pintara Evans, 1932. The adults and male genitalia of both P. heringi heringi (Mell, 1922) and P. heringi pieridoides (Liu & Gu, 1994) are illustrated.

Notes on Halpe paupera Devyatkin, 2002 (Lepidoptera: Hesperiidae).

A series of specimens with some differences in wing patterns and male genitalia from different localities is recognized as Halpe paupera Devyatkin, 2002 by comparing the COI gene sequences. The morphological variability in male is discussed and illustrated. The intraspecific variation of the female of H. paupera walthewi Devyatkin, 2002 from the type locality is elucidated, and hence this subspecies is considered a synonym of H. paupera paupera. A distribution map and some bionomic information of the species are provided.

The transcription factor NtERF13a enhances abiotic stress tolerance and phenylpropanoid compounds biosynthesis in tobacco.

The AP2/ERF (APETALA2/ETHYLENE RESPONSE FACTOR) transcription factors play multiple roles in modulating the biosynthesis of diverse specialized metabolites in response to various environmental stresses. ERF13 has been shown to participate in plant resistance to biotic stress as well as in repressing the synthesis of fatty acid. However, its full roles in regulating plant metabolism and stress resistance still remains to be further studied. In this study, we identified two NtERF genes from N. tabacum genome that belong to Ⅸa subgroup of ERF family. Over-expression and knock-out of NtERF13a showed that NtERF13a could enhance plant resistance to salt and drought stresses, as well as promoted the biosynthesis of chlorogenic acid (CGA), flavonoids, and lignin in tobacco. Transcriptome analysis between WT and NtERF13a-OE plants revealed 6 differentially expressed genes (DEGs) that encode enzymes catalyzing the key steps of phenylpropanoid pathway. Chromatin immunoprecipitation, Y1H, and Dual-Luc assays further clarified that NtERF13a could directly bind to the fragments containing GCC box or DRE element in the promoters of NtHCT, NtF3'H, and NtANS genes to induce the transcription of these genes. Knock-out of NtHCT, NtF3'H, or NtANS in the NtERF13a-OE background significantly repressed the increase of phenylpropanoid compound contents caused by over-expression of NtERF13a, indicating that the promotion of NtERF13a on the phenylpropanoid compound contents depends on the activity of NtHCT, NtF3'H, and NtANS. Our study demonstrated new roles of NtERF13a in promoting plant resistance to abiotic stresses, and provided a promising target for modulating the biosynthesis of phenylpropanoid compounds in tobacco.

De novo Synthesis of 2-phenylethanol from Glucose by Metabolically Engineered Escherichia coli.

2-Phenylethanol (2- PE) is an aromatic alcohol with wide applications, but there is still no efficient microbial cell factory for 2-PE based on Escherichia coli. In this study, we constructed a metabolically engineered E. coli capable of de novo synthesis of 2-PE from glucose. Firstly, the heterologous styrene-derived and Ehrlich pathways were individually constructed in an L-Phe producer. The results showed that the Ehrlich pathway was better suited to the host than the styrene-derived pathway, resulting in a higher 2-PE titer of ∼0.76 ± 0.02 g/L after 72 h of shake flask fermentation. Furthermore, the phenylacetic acid synthase encoded by feaB was deleted to decrease the consumption of 2-phenylacetaldehyde, and the 2-PE titer increased to 1.75 ± 0.08 g/L. As phosphoenolpyruvate (PEP) is an important precursor for L-Phe synthesis, both the crr and pykF genes were knocked out, leading to ∼35% increase of the 2-PE titer, which reached 2.36 ± 0.06 g/L. Finally, a plasmid-free engineered strain was constructed based on the Ehrlich pathway by integrating multiple ARO10 cassettes (encoding phenylpyruvate decarboxylases) and overexpressing the yjgB gene. The engineered strain produced 2.28 ± 0.20 g/L of 2-PE with a yield of 0.076 g/g glucose and productivity of 0.048 g/L/h. To our best knowledge, this is the highest titer and productivity ever reported for the de novo synthesis of 2-PE in E. coli. In a 5-L fermenter, the 2-PE titer reached 2.15 g/L after 32 h of fermentation, suggesting that the strain has the potential to efficiently produce higher 2-PE titers following further fermentation optimization.

Tannic acid enhanced the physical and oxidative stability of chitin particles stabilized oil in water emulsion.

In this work, the stability of CP-TA complex stabilized emulsion was first characterized. It was found that the peak thickness, Turbiscan Stability Index (TSI) and droplet size of CP-TA complex stabilized emulsion gradually decreased with increasing content of TA, indicating the gradually enhanced physical stability of emulsion, which was attributed to the gradually decreased interfacial tension, zeta potential and increased viscosity of CP-TA complex. Moreover, the oxidative stability of CP-TA complex stabilized emulsion gradually enhanced with increasing of TA content due to the antioxidant activity of TA. XRD and FTIR results suggested that the interaction between CP and TA gradually enhanced with increasing content of TA in CP-TA complex, leading to the formation of larger CP-TA clusters shown in AFM results. In conclusion, the presence of tannic acid (TA) enhanced the physical and oxidative stability of chitin particles-tannic acid (CP-TA) complex stabilized oil in water emulsion.

Synergistic stabilization of oil in water emulsion with chitin particles and tannic acid.

The aim of the present study was to explore the effect of CP and TA on stability of oil in water emulsion stabilized by the two components, so as to fabricate the most efficient chitin based emulsifying agents. It was found that there was synergistic effect for CP and TA in stabilizing emulsion, specifically, the complex of chitin particles (CP) (3 g/L) with tannic acid (TA) (2 g/L) produced the most physically and oxidatively stable oil-in-water emulsion compared with other groups in this study. This is because CP-TA (3/5) complex had the lowest zeta potential, the lowest the oil water interfacial tension, the highest viscosity and the highest content of TA with excellent antioxidant activity. Furthermore, this is because there was intense interaction between CP and TA in CP-TA complex from results of FTIR, XRD and ITC, which then result in the formation of large CP-TA particles.

Improving emulsion stability based on ovalbumin-carboxymethyl cellulose complexes with thermal treatment near ovalbumin isoelectric point.

Ovalbumin (OVA) is an important protein emulsifier. However, it is unstable near the isoelectric point pH, which limits its applications in the food industry. Polysaccharides may be explored to tackle this challenge by improving its pH-dependent instability. In this work, carboxymethyl cellulose (CMC) was used as a model polysaccharide to mix with OVA near its isoelectric point (pH 4.7) with subsequent mild heating at 60 °C for 30 min. The molecular interactions between OVA and CMC were comprehensively studied via a series of characterizations, including turbidity, zeta potential, intrinsic fluorescence, surface hydrophobicity, circular dichroism (CD) spectra and Fourier transform infrared spectroscopy (FTIR). The droplet sizes of the emulsions prepared by OVA-CMC were measured to analyze emulsifying property and stability. The results indicated that free OVA was easily aggregated due to loss of surface charges, while complexing with CMC significantly inhibited OVA aggregation before and after heating owing to the strong electrostatic repulsion. In addition, OVA exposed more hydrophobic clusters after heating, which resulted in the growth of surface hydrophobicity. Altogether, the heated OVA-CMC complexes presented the best emulsifying property and stability. Our study demonstrated that complexing OVA with CMC not only greatly improved its physicochemical properties but also significantly enhanced its functionality as a food-grade emulsifying agent, expanding its applications in the food industry, as development of emulsion-based acidic food products.

Carbon nanoparticles enhance potassium uptake via upregulating potassium channel expression and imitating biological ion channels in BY-2 cells.

BACKGROUND: Carbon nanoparticles (CNPs) have been reported to boost plant growth, while the mechanism that CNPs enhanced potassium uptake for plant growth has not been reported so far. RESULTS: In this study, the function that CNPs promoted potassium uptake in BY-2 cells was established and the potassium accumulated in cells had a significant correlation with the fresh biomass of BY-2 cells. The K+ accumulation in cells increased with the increasing concentration of CNPs. The K+ influx reached high level after treatment with CNPs and was significantly higher than that of the control group and the negative group treated with K+ channels blocker, tetraethylammonium chloride (TEA+). The K+ accumulation was not reduced in the presence of CNPs inhibitors. In the presence of potassium channel blocker TEA+ or CNPs inhibitors, the NKT1 gene expression was changed compared with the control group. The CNPs were found to preferentially transport K+ than other cations determined by rectification of ion current assay (RIC) in a conical nanocapillary. CONCLUSIONS: These results indicated that CNPs upregulated potassium gene expression to enhance K+ accumulation in BY-2 cells. Moreover, it was speculated that the CNPs simulated protein of ion channels via bulk of carboxyl for K+ permeating. These findings will provide support for improving plant growth by carbon nanoparticles.

Foams Stabilized by ß-Lactoglobulin Amyloid Fibrils: Effect of pH.

ß-Lactoglobulin fibrils could serve as a surface-active component and form adsorption layers at the air/water interface. In this study, the physical parameters related to the surface adsorption, foaming, and surface properties of ß-lactoglobulin fibrils as a function of pH (2-8) were investigated. Results showed that an increase of pH from 2 to 5 led to a rise of the viscoelastic modulus of the surface adsorption layer and half-life time (t1/2) of foams, but it decreased foamability. When the pH was close to its isoelectric point (5.2), fibrils had the lowest electrostatic repulsion and entangled at the air/water interface resulting in a tightly packaged adsorption layer around bubbles to prevent coalescence and coarsening. When the pH (7-8) was higher than the pI of fibrils, the negatively charged ß-lactoglobulin fibrils possessed good foamability (∼80%) and high foam stability (t1/2 ≈ 8 h) simultaneously even at low concentration (1 mg/mL). It demonstrated that ß-lactoglobulin fibrils with negative charges presented a good foaming behavior and could be a potential new foaming agent in the food industry.