Characterization of aroma-active compounds in Dongli by quantitative descriptive analysis, gas chromatography-triple quadrupole tandem mass spectrometry, and gas chromatography-olfactometry.

Dongli, or frozen pear, is a traditional Chinese snack with a unique flavor. This study identified the aroma-active volatile compounds (VOCs) in Dongli using quantitative descriptive analysis (QDA), gas chromatography-triple quadrupole tandem mass spectrometry (GC-MS/MS), and gas chromatography-olfactometry (GC-O). QDA indicated that Dongli of all cultivars presented increased sweet and wine aromas. A total of 21 VOCs were identified by GC-MS/MS. Bidirectional orthogonal partial least square (O2PLS) analysis, GC-O analysis, detection frequency analysis (DFA), and relative odor activity values (ROAV) showed that: estragole and anethole contributing "anise, green" aromas were the key aromatic VOCs of fresh pears, while ethyl butanoate, butyl acetate, heptyl acetate, benzaldehyde, and geranyl acetone contributing "sweet, fruity, green" aromas were the key aromatic VOCs of Dongli. The results revealed that the repeated freezing treatment promoted a unique aroma in pears. This study would contribute to developing new pear products. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-022-05463-8.

Multivariate analyses of the volatile components in fresh and dried turnip (Brassica rapa L.) chips via HS-SPME-GC-MS.

Turnip is a vegetable that has many health promoting effects. To diversify the usage and increase the consumption of turnip, the effects of hot air drying, infrared drying, explosion puff drying and freeze drying (FD) on the volatiles of turnip chips were studied. The volatiles of fresh turnip and dried turnip chips were isolated by HS-SPME-GC-MS and a total of 67 volatiles were identified. However, the volatiles in turnip chips dried by different methods are quite different. Based on principal component analysis and hierarchical cluster analysis, the volatiles of fresh turnip were distinguished from those of the dried chips and FD was separated from the other drying methods. As the result of orthogonal projection on latent structure-discriminant analysis (OPLS-DA), isothiocyanato-cyclopropane and (2-isothiocyanatoethyl)-benzene were identified as the characteristic volatiles of fresh turnip. While, 2-azido-2,3,3-trimethyl-butane and hexanal were identified as the characteristic volatiles for FD dried chips.

Solubility and emulsifying activity of yam soluble protein.

Yam soluble protein (YSP) has been reported to have many physiological activities, such as scavenging free radicals, immune activation, and anti-hypertensive activities. Protein solubility and emulsifying activity are important protein-associated functional properties for the application of proteins in food systems. During this study, the factors of protein concentration, pH, temperature and salt concentration that influenced the solubility of YSP were investigated. As a result, the solubility was minimal near its isoelectric point (pH 3.5) and was highest at 45 °C in a temperature range of 40-60 °C. With an increase of protein concentration, the solubility decreased. According to the results of response surface methodology analysis, the interaction between pH and temperature on the solubility of YSP was significant, and the maximum solubility (87.5%) was obtained when the temperature was close to 40 °C, the pH was approximately 7 and the NaCl concentration approached 0.5 mol/L. As the protein concentration increased, the average particle size of the YSP emulsion decreased, and the particle size distribution gradually became balanced. Additionally, the microphotograph of the YSP emulsion reflected its distribution. The results of this study will provide data and a theoretical basis for the understanding of YSP's physicochemical properties and its application in the food industry.

Using steered molecular dynamics to study the interaction between ADP and the nucleotide-binding domain of yeast Hsp70 protein Ssa1.

Genetics experiments have identified six mutations located in the subdomain IA (A17V, R23H, G32D, G32S, R34K, V372I) of Ssa1 that influence propagation of the yeast [PSI+] prion. However, the underlining molecular mechanisms of these mutations are still unclear. The six mutation sites are present in the IA subdomain of the nucleotide-binding domain (NBD). The ATPase subdomain IA is a critical mediator of inter-domain allostery in Hsp70 molecular chaperones, so the mutation and changes in this subdomain may influence the function of the substrate-binding domain. In addition, ADP release is a rate-limiting step of the ATPase cycle and dysregulation of the ATPase cycle influences the propagation of the yeast [PSI+] prion. In this work, steered molecular dynamics (SMD) simulations were performed to explore the interaction between ADP and NBD. Results suggest that during the SMD simulations, hydrophobic interactions are predominant and variations in the binding state of ADP within the mutants is a potential reason for in vivo effects on yeast [PSI+] prion propagation. Additionally, we identify the primary residues in the ATPase domain that directly constitute the main hydrophobic interaction network and directly influence the ADP interaction state with the NBD of Ssa1. Furthermore, this in silico analysis reaffirms the importance of previously experimentally-determined residues in the Hsp70 ATPase domain involved in ADP binding and also identifies new residues potentially involved in this process.

Functional properties of Chinese yam (Dioscorea opposita Thunb. cv. Baiyu) soluble protein.

In this study, yam soluble protein (YSP) was extracted from Chinese yam (Dioscorea opposita Thunb. cv. Baiyu) and the functional properties were investigated under the influence of pH and ionic strength. As results, YSP was highly soluble and had better emulsifying activity over a wide range of pH. The solubility of YSP decreased in 0.5 and 1.0 M NaCl solution. An increment in NaCl concentration reduced the emulsion activity index and emulsion stability index of YSP. The oil absorption capacity of YSP was 3.2 ml/g protein. With the increase of pH, the foam capacity (FC) and stability (FS) decreased and then increased. FS of YSP increased as the salt concentration increased from 0 to 0.5 M, and then decreased. The minimal gelation concentration of YSP was 8% (w/v) and 10% YSP gel (w/v) had maximum gel strength in 0.1 M NaCl. These results suggested that YSP produced by acid precipitation may be used as a protein source with remarkable functional properties.

Investigating the impact of ultrasound on the structural, physicochemical, and emulsifying characteristics of Dioscorin: Insights from experimental data and molecular dynamics simulation.

This study investigated the impact of ultrasound treatment on dioscorin, the primary storage protein found in yam tubers. Three key factors, namely ultrasound power, duration, and frequency, were focused on. The research revealed that ultrasound-induced cavitation effects disrupted non-covalent bonds, resulting in a reduction in α-helix and ß-sheet contents, decreased thermal stability, and a decrease in the apparent hydrodynamic diameter (Dh) of dioscorin. Additionally, previously hidden amino acid groups within the molecule became exposed on its surface, resulting in increased surface hydrophobicity (Ho) and zeta-potential. Under specific ultrasound conditions (200 W, 25 kHz, 30 min), Dh decreased while Ho increased, facilitating the adsorption of dioscorin molecules onto the oil-water interface. Molecular dynamics (MD) simulations showed that at lower frequencies and pressures, the structural flexibility of dioscorin's main chain atoms increased, leading to more significant fluctuations between amino acid residues. This transformation improved dioscorin's emulsifying properties and its oil-water interface affinity.

Effects of spray drying and freeze drying on the structure and emulsifying properties of yam soluble protein: A study by experiment and molecular dynamics simulation.

This study focused on the effects of freeze drying (FD) and sprays drying (SD) on the structure and emulsifying properties of yam soluble protein (YSP). The results showed that the surface hydrophobicity (Ho) value, free sulfhydryl group (SH) content, turns content, denaturation temperature and enthalpy value of spray-dried YSP (SD-YSP) were higher than freeze-dried YSP (FD-YSP), but the apparent hydrodynamic diameter (Dh) value of SD-YSP was smaller. The smaller Dh, higher Ho and free SH led to higher percentage of adsorbed proteins and stronger binding between protein and oil droplet in emulsions. Thus, the emulsifying properties of SD-YSP were better, and the SD-YSP-stabilized emulsion had better dynamical rheological properties. Molecular dynamics (MD) simulations suggested that some intramolecular disulfide bonds and hydrogen bonds of dioscorin were broken, and some helices transformed into turns during the SD process. These structural changes resulted in better thermal stability and emulsification properties of SD-YSP.

Revealing the mechanism underlying the effects of γ-aminobutyric acid-dioscorin interactions on dioscorin structure and emulsifying properties by molecular dynamic simulations.

Many studies have shown that γ-aminobutyric acid (GABA) exhibits various beneficial biological activities, including gut-modulating, neuro-stimulating, and cardio-protecting activities. Naturally, GABA exists in small amounts in yam, which is primarily synthesized by the decarboxylation of L-glutamic acid in the presence of glutamate decarboxylase. Dioscorin, the major tuber storage protein of yam, has been shown to have good solubility and emulsifying activity. However, how GABA interacts with dioscorin and affects their properties has yet to be clarified. In this research, the physicochemical and emulsifying properties of GABA-fortified dioscorin, which was dried by spray drying and freeze drying, were studied. As results, the freeze-dried (FD) dioscorin produced more stable emulsions, while the spray-dried (SD) dioscorin adsorbed more rapidly to oil/water (O/W) interface. The fluorescence spectroscopy, ultraviolet spectroscopy and circular dichroism spectroscopy showed that GABA changed the structure of dioscorin, by exposing its hydrophobic groups. The addition of GABA significantly promoted the adsorption of dioscorin to the O/W interface and prevented droplets coalescence. The results of molecular dynamics simulation (MD) showed that GABA destroyed the H-bond network between dioscorin and water, increased surface hydrophobicity and finally improved the emulsifying properties of dioscorin.

Crystallization and preliminary X-ray crystallographic analysis of dioscorin from Dioscorea japonica.

Dioscorin, the major tuber storage protein in yam, has been reported to possess carbonic anhydrase, trypsin inhibitor, angiotensin-converting enzyme (ACE) inhibitor, free-radical scavenger, dehydroascorbate reductase and monodehydroascorbate reductase activities. Recent research has also found that dioscorin can enhance immune modulation via the toll-like receptor 4 (TLR-4) signal transduction pathway in RAW 264.7 cells, murine bone-marrow cells and human monocytes ex vivo. Resolving the structure of dioscorin would help in better understanding its activities and would provide clues to understanding the mechanism of its multiple functions. The full-length protein (residues 1-246) with an additional His(6) tag at the N-terminus was expressed in Escherichia coli Rosetta (DE3) cells. After His-tag cleavage and purification, the protein was crystallized by the sitting-drop vapour-diffusion method at 278 K. An X-ray diffraction data set was collected to a resolution of 2.11 Å using a synchrotron X-ray source. The crystal belonged to space group C222(1), with unit-cell parameters a = 83.5, b = 156.8, c = 83.6 Å, and was estimated to contain two protein molecules per asymmetric unit.

Expression, purification, crystallization and preliminary X-ray analysis of a novel N-substituted branched-chain L-amino-acid dioxygenase from Burkholderia ambifaria AMMD.

Ferrous ion- and α-ketoglutarate-dependent dioxygenase from Burkholderia ambifaria AMMD (SadA) catalyzes the C3-hydroxylation of N-substituted branched-chain L-amino acids, especially N-succinyl-L-leucine, coupled to the conversion of α-ketoglutarate to succinate and CO(2). SadA was expressed in Escherichia coli, purified and crystallized using the sitting-drop vapour-diffusion method at 293 K. Crystals of selenomethionine-substituted SadA were obtained using a reservoir solution containing PEG 3000 as the precipitant at pH 9.5 and diffracted X-rays to 2.4 Å resolution. The crystal belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 49.3, b = 70.9, c = 148.2 Å. The calculated Matthews coefficient (V(M) = 2.1 Å(3) Da(-1), 41% solvent content) suggested that the crystal contains two molecules per asymmetric unit.

Effect of different ionic liquids and organic solvents on the structural and physicochemical properties of cellulose-protein complexes extracted from Se-enriched peanut leaves.

Abundant cellulose and insoluble protein were contained in the Se-enriched peanut leaf residue, a by-product from leaf protein extraction. Ionic liquids (ILs) were used to extract the cellulose-protein complexes (CPCs) from Se-enriched peanut leaf residue. The effects of various ILs as extractants and organic solvents as regenerant on the physicochemical properties of CPCs were compared. The results showed that the yield of CPCs and recovery yield of [AMIM]Cl (1-allyl-3-methylimidazole chloride) were better than those of [BMIM]Cl (1-butyl-3-methylimidazolium chloride). Simultaneously, it could be seen from the infrared absorption peaks and secondary structure fitting results that [BMIM]Cl seemed stronger than [AMIM]Cl in destroying the secondary structure of CPCs. Scanning electron microscope (SEM) showed that the CPCs extracted by [BMIM]Cl were lamellate with holes on the surface, and the CPCs extracted by [AMIM]Cl were rough, almost without holes on the surface. Furthermore, the transmittance and tensile strength of the film which contained BA-CPC ([BMIM]Cl as extractant and acetonitrile as regenerant) film were better than those contained AA-CPC ([AMIM]Cl as extractant and acetonitrile as regenerant) film, which might be mainly because the types of ILs and regenerants affect the particle size of CPCs, thereby influencing the mechanical properties of the film.

Hydrogen peroxide modification affects the structure and physicochemical properties of dietary fibers from white turnip (Brassica Rapa L.).

Turnip (Brassica rapa L.) is widely consumed as a vegetable and traditional Chinese medicine with high dietary fiber content. Soluble dietary fiber (SDF) and insoluble dietary fiber (IDF) were obtained from white turnips, and the IDF was modified with alkaline hydrogen peroxide to obtain modified IDF (MIDF) and modified SDF (MSDF). The compositional, structural, and functional properties of the four samples were investigated. After modification, the modified dietary fibers (MDFs) showed smaller particle sizes and lower contents of pectin and polyphenol than those of unmodified dietary fibers (DFs) The results of scanning electron microscopy (SEM), Fourier transformed infrared (FT-IR) spectroscopy, X-ray diffraction (XRD) and differential scanning calorimetry (DSC) showed that compared to the DFs, the MDFs were smaller and had more exposed hydroxyl groups. Analysis of the microrheological behaviors showed that the MDFs had higher viscosity than that of the DFs, with a looser structure for the MSDF and a stable structure for the MIDF. Therefore, due to structural changes, the physical and functional properties of the MDFs were improved compared to those of the unmodified DFs. Pearson correlation analysis showed that the particle size was positively correlated with the pectin content. The water holding capacity (WHC), oil adsorption capacity (OAC) and water swelling capacity (WSC) showed positive correlations with each other. This work indicated that white turnip could be a potential new source of DFs, which presented desirable functional properties after modification.

Se-O Bond Is Unique to High Se Enriched Sweet Potato Stem Protein with Better Antioxidant Ability.

Sweet potato plants were treated with selenium (Se). Spraying Se on the sweet potato leaves was an effective Se enrichment method and proteins were extracted from the sweet potato stem. The structural characteristics of the protein were investigated. Fourier transform infrared spectroscopy (FT-IR) detected more signals from the Se-enriched sweet potato stem protein (SSP), and the number of forms of Se chemical bonds gradually increased with increasing Se content, such as the Se-O bond in high Se-enriched SSP, indicating altered secondary structures.Scanning electron microscopy-energy dispersive spectrometry (SEM-EDS) indicated more Se atoms in the Se-enriched SSPs (SSSPs). The DSC results revealed that Se enrichment enhanced the thermal stability of the samples. Moreover, selenomethionine (SeMet), selenocystine (SeCys2), and methylselenocysteine (MeSeCys) were determined to be the main Se forms in the SSSPs. Furthermore, the SSSPs showed relatively higher superoxide anion radical and DPPH radical scavenging activities than the blank, which indicates that SSSPs can be used as antioxidants. By recovering the proteins, the agricultural by-product-sweet potato stem can be further utilized, and the obtained Se-enriched proteins may contribute to human health.

Recovery of Yam Soluble Protein from Yam Starch Processing Wastewater.

Over the past two decades, many studies have shown that the yam storage protein dioscorin, which is abundant in the wastewater of starch processing, exhibits many biological activities both in vitro and in vivo. In the present study, the acid-precipitation method was optimized using Box-Behnken design (BBD) combined with response surface methodology (RSM) for the recovery of yam soluble protein (YSP) from wastewater. The experimental yield of YSP reached 57.7%. According to relative quantitative proteomics (LC-MS/MS), the crude YSP was mainly composed of 15 dioscorin isoforms, which was further verified by anion-exchange and size-exclusion chromatography. YSP was found to be rich in glutamic acid and aspartic acid, and the eight essential acids made up approximately 33.7% of the YSP. Moreover, the YSP demonstrated antioxidant activity, including scavenging DPPH, hydroxyl and superoxide anion radicals, and the possible structure-activity relationships were discussed. These results indicated that YSP produced by acid precipitation may be used as a protein source with antioxidant properties.

Foliar application is an effective method for incorporating selenium into peanut leaf proteins with antioxidant activities.

Proteins were extracted from Se-enriched peanut leaves, an agro-byproduct, and the foliar application of sodium selenite was indicated to be an effective method to incorporate Se into leaf selenoproteins with 75-80% incorporation rates. After trypsin digestion, the most abundant proteins from Se-enriched peanut leaf (PSPL) were identified as pathogenesis-related class 10 proteins, Ara h 8 allergen and its isoforms, using LC-MS/MS. The Se species in both the low Se PSPL and high Se PSPL were determined to be selenomethionine (SeMet), methylselenocysteine (MeSeCys) and selenocystine (SeCys2) with SeMet (15.6 mg/g) dominated the high Se PSPL. Their antioxidant activities were also evaluated using free radical scavenging assay, reducing power assay and ferric thiocyanate (FTC) test. As results, the PSPL exhibited potent DPPH radical (96.2%) and superoxide anion radical (98.4%) scavenging activities and showed strong reducing power in a Se-concentration-dependent manner, indicating that PSPL can be used as antioxidants and Se sources to improve health.

Optimization of the ultrafiltration-assisted extraction of Chinese yam polysaccharide using response surface methodology and its biological activity.

Ultrafiltration is a separation process for purifying and concentrating macromolecular solutions. Using Baiyu yam (Dioscorea opposita Thunb) as the raw material, single-factor experiments, Box-Behnken design (BBD) and response surface methodology (RSM) were employed to investigate the effects of the ultrafiltration pH, temperature and pressure on the extraction rate of Chinese yam polysaccharide (CYP). The constructed regression model is highly significant, and the optimal ultrafiltration-assisted extraction conditions were determined to be the following: pH 6.5, 20 °C and 0.03 MPa. Under these optimal conditions, a CYP extraction rate of 88.7% was achieved. After purification with anion exchange (DE-52) and size-exclusion (Sephadex G-100) columns, the monosaccharide composition of CYP was determined to be 50.8% glucose, 24.2% mannose and 11.8% galactose. Fourier transform infrared (FT-IR) spectroscopy characterization of CYP confirmed the characteristic absorption peaks of the polysaccharides. The microstructure of CYP exhibited characteristics typical of amorphous powders. CYP also exhibited antioxidant activities, including the scavenging of DPPH radicals, hydroxyl radicals and superoxide anion radicals. Moreover, CYP exhibited a relatively strong inhibitory effect on BGC-823 cell growth.

Molecular dynamics simulation and steered molecular dynamics simulation on irisin dimers.

Irisin is found closely associated with promoting the browning of beige fat cells in white adipose tissue. The crystal structure reveals that irisin forms a continuous inter-subunit ß-sheet dimer. Here, molecular dynamics (MD) simulation and steered molecular dynamics (SMD) simulation were performed to investigate the dissociation process and the intricate interactions between the two irisin monomers. In the process of MD, the interactions between the monomers were roughly analyzed through the average numbers of both hydrophobic contacts and H-bonds. Then, SMD was performed to investigate the accurate interaction energy between the monomers. By the analysis of dissociation energy, the van der Waals (vdW) force was identified as the major energy to maintain the dimer structure, which also verified the results of MD simulation. Meanwhile, 11 essential residues were discovered by the magnitude of rupture force during dissociation. Among them, residues Arg75, Glu79, Ile77, Ala88, and Trp90 were reported in a previous study using the method of mutagenesis and size exclusion chromatography, and several new important residues (Arg72, Leu74, Phe76, Gln78, Val80, and Asp91) were also identified. Interestingly, the new important residues that we discovered and the important residues that were reported are located in the opposite side of the ß-sheet of the dimer.

Molecular dynamics simulations of Hsp40 J-domain mutants identifies disruption of the critical HPD-motif as the key factor for impaired curing in vivo of the yeast prion [URE3].

Genetic screens using Saccharomyces cerevisiae have identified an array of Hsp40 (Ydj1p) J-domain mutants that are impaired in the ability to cure the yeast [URE3] prion through disrupting functional interactions with Hsp70. However, biochemical analysis of some of these Hsp40 J-domain mutants has so far failed to provide major insight into the specific functional changes in Hsp40-Hsp70 interactions. To explore the detailed structural and dynamic properties of the Hsp40 J-domain, 20 ns molecular dynamic simulations of 4 mutants (D9A, D36A, A30T, and F45S) and wild-type J-domain were performed, followed by Hsp70 docking simulations. Results demonstrated that although the Hsp70 interaction mechanism of the mutants may vary, the major structural change was targeted to the critical HPD motif of the J-domain. Our computational analysis fits well with previous yeast genetics studies regarding highlighting the importance of J-domain function in prion propagation. During the molecular dynamics simulations several important residues were identified and predicted to play an essential role in J-domain structure. Among these residues, Y26 and F45 were confirmed, using both in silico and in vivo methods, as being critical for Ydj1p function.

Steered molecular dynamics simulation of the binding of the bovine auxilin J domain to the Hsc70 nucleotide-binding domain.

The Hsp70 and Hsp40 chaperone machine plays critical roles in protein folding, membrane translocation, and protein degradation by binding and releasing protein substrates in a process that utilizes ATP. The activities of the Hsp70 family of chaperones are recruited and stimulated by the J domains of Hsp40 chaperones. However, structural information on the Hsp40-Hsp70 complex is lacking, and the molecular details of this interaction are yet to be elucidated. Here we used steered molecular dynamics (SMD) simulations to investigate the molecular interactions that occur during the dissociation of the auxilin J domain from the Hsc70 nucleotide-binding domain (NBD). The changes in energy observed during the SMD simulation suggest that electrostatic interactions are the dominant type of interaction. Additionally, we found that Hsp70 mainly interacts with auxilin through the surface residues Tyr866, Arg867, and Lys868 of helix II, His874, Asp876, Lys877, Thr879, and Gln881 of the HPD loop, and Phe891, Asn895, Asp896, and Asn903 of helix III. The conservative residues Tyr866, Arg867, Lys868, His874, Asp876, Lys877, and Phe891 were also found in a previous study to be indispensable to the catalytic activity of the DnaJ J domain and the binding of it with the NBD of DnaK. The in silico identification of the importance of auxilin residues Asn895, Asp896, and Asn903 agrees with previous mutagenesis and NMR data suggesting that helix III of the J domain of the T antigen interacts with Hsp70. Furthermore, our data indicate that Thr879 and Gln881 from the HPD loop are also important as they mediate the interaction between the bovine auxilin J domain and Hsc70.

Crystal structure of a novel N-substituted L-amino acid dioxygenase from Burkholderia ambifaria AMMD.

A novel dioxygenase from Burkholderia ambifaria AMMD (SadA) stereoselectively catalyzes the C3-hydroxylation of N-substituted branched-chain or aromatic L-amino acids, especially N-succinyl-L-leucine, coupled with the conversion of α-ketoglutarate to succinate and CO2. To elucidate the structural basis of the substrate specificity and stereoselective hydroxylation, we determined the crystal structures of the SadA.Zn(II) and SadA.Zn(II).α-KG complexes at 1.77 Å and 1.98 Å resolutions, respectively. SadA adopted a double-stranded ß-helix fold at the core of the structure. In addition, an HXD/EXnH motif in the active site coordinated a Zn(II) as a substitute for Fe(II). The α-KG molecule also coordinated Zn(II) in a bidentate manner via its 1-carboxylate and 2-oxo groups. Based on the SadA.Zn(II).α-KG structure and mutation analyses, we constructed substrate-binding models with N-succinyl-L-leucine and N-succinyl-L-phenylalanine, which provided new insight into the substrate specificity. The results will be useful for the rational design of SadA variants aimed at the recognition of various N-succinyl L-amino acids.