Low Isoelectric Point Research Articles

Plant-based marbled salami analogs: Emulsion-loaded microgels embedded within protein-polysaccharide hydrogel matrices

The inherent molecular differences between plant and animal proteins make it challenging to accurately simulate the optical and mechanical properties of real meat products using plant-derived ingredients. Soft matter physics approaches are therefore being employed to direct the assembly of plant proteins, polysaccharides, and lipids into meat-like structures. The main focus of the current study was to use soft matter physics approaches to create plant-based analogs of marbled meat products, such as salami, chorizo, or pepperoni. These products have whitish fatty regions embedded within reddish/brown lean regions. In this study, we mimicked the fatty regions in salami using emulsion-loaded alginate beads and the lean regions using composite hydrogels assembled from potato protein and gellan gum. Initially, the textural attributes of the plant-based lean regions were optimized by varying potato protein type (low or high isoelectric point) and concentration. As expected, the mechanical strength of the lean region analogs increased with increasing protein content and depended on protein type. Introducing alginate beads into the salami analogs had little impact on their textural attributes when measured under compression conditions but decreased their stiffness and fracture stress when measured under tensile conditions. In particular, introducing either small or large beads reduced the stiffness and breaking stress of the salami analogs, which was attributed to the introduction of weak points in the composite hydrogel. Finally, we showed that the appearance and textural attributes of real salami could be mimicked in plant-based salami by optimizing its composition and structure. This research provides valuable insights into methods of better simulating the properties of marbled meat products, like sausages and cold-cut meat products, thereby creating a more sustainable food supply.

Influence of life-history traits on mitochondrial DNA substitution rates exceeds that of metabolic rates in teleost fishes

Abstract Understanding the molecular relevance of metabolic rate (MR) is crucial for unveiling the mechanisms driving the evolution of animals. In this study, we investigated the association between mitochondrial DNA characteristics and both resting and maximal MRs in conjunction with life-history traits among 139 species of teleost fish. We gathered fish MR data from various sources and procured sequences of 13 mitochondrial protein-encoding genes. We calculated the absolute substitution rate for entire nucleotide sequences and 4-fold degenerate sites of each gene, along with encoding amino acid sequences. Using the phylogenetic comparative method, we then explored the associations between MR and mitochondrial DNA absolute substitution rate. Additionally, we screened MR-associated single nucleotide variants in mitochondrial DNA. The findings indicate no positive correlation between MRs and any substitution rate values of both combined sequences and individual mitochondrial protein-coding genes, refuting the MR hypothesis. Instead, both maximum body size and longevity correlated negatively with molecular substitution rates, suggesting their influences on both mutation and fixation within mitochondrial genes in fish. Results also revealed significant correlations between base variation at ATP6_169 and both resting MR and maximum MR, identifying the unique ATP6_169G in Scombridae fish, which results in an extremely low isoelectric point (pI) value of the ATP6 protein. Considering its functional significance, the ATP6_169G in Scombridae fish might link to their lifestyle characterized by fast locomotion and high metabolic demands alongside a slower molecular evolutionary rate.

Green Fluorescent Carbon Dots with Critically Controlled Surface States: Make Silk Shine via Feeding Silkworms.

Feeding silkworms with functional materials as additives to produce naturally modified silk is a facile, diverse, controllable, and environmentally friendly method with a low cost of time and investment. Among various additives, carbon dots (CDs) show unique advantages due to their excellent biocompatibility and fluorescence stability. Here, a new type of green fluorescent carbon dots (G-CDs) is synthesized with a high oil-water partition ratio of 147, a low isoelectric point of 5.16, an absolute quantum yield of 71%, and critically controlled surface states. After feeding with G-CDs, the silkworms weave light yellow cocoons whose green fluorescence is visible to the naked eye under UV light. The luminous silk is sewn onto the cloth to create striking patterns with beautiful fluorescence. Such G-CDs have no adverse effect on the survival rate and the life cycle of silkworms and enable their whole bodies to glow under UV light. Based on the strong fluorescence, chemical stability, and biological safety, G-CDs are found in the digestive tracts, silk glands, feces, cocoons, and even moth bodies. G-CDs accumulate in the posterior silk glands where fibroin protein is secreted, indicating its stronger combination with fibroin than sericin, which meets the requirements for practical applications.

Study on the Pb(II) adsorption mechanism on acid‐modified ceramsite made from sewage sludge and industrial solid wastes

AbstractCeramsite was made from sludge, phosphogypsum, and red mud and then modified with different concentrations of hydrochloric, nitric, or citric acid. The resulting composite ceramsite was subsequently utilized for the adsorption of Pb(II). The maximum adsorption capacities of hydrochloric acid– and nitric acid–modified ceramsites were 90.38 and 88.57 mg/g, respectively, which were much higher than those of unmodified ceramsite (66.86 mg/g) and citric acid–modified ceramsite (57.85 mg/g). The variation of isoelectric point, acidic and basic groups, and Fourier transform infrared spectra of ceramsite were compared before and after adsorption. A decrease of isoelectric point implied weakening of electrostatic repulsion between Pb(II) and the modified ceramsite. Functional groups, such as O─H, C═O (or C═C), and Si─O─Si (or Al─O─Al), were involved in the adsorption. In addition, phenol hydroxyl, which exhibited a significant increase in modified ceramsite and a noticeable decrease after adsorption, was the most important group. Besides, the modified ceramsite had larger specific surface areas and pores (30.66 g/m2, .11256 cm3/g), more functional groups, and lower isoelectric points (8.57), which were favorable for adsorption. The adsorption processes described by pseudo‐second‐order kinetics were controlled by chemical adsorptions. Isothermal studies indicated that monolayer adsorption occurred on hydrochloric acid–modified ceramsite and multiple processes occurred on nitric acid– and citric acid–modified ceramsites. Thermodynamic analysis confirmed Pb(II) adsorption on modified ceramsite was spontaneous and endothermic.

A novel wholly-Tröger's Base-based polyamide nanofiltration membrane constructed for the separation application in strong acidic condition via acid-induced segregation

In this study, a new kind of wholly-Tröger's base-based polyamide (wTB-PA) thin film composite (TFC) nanofiltration (NF) membrane was successfully fabricated for the separation application in strong acidic condition via the acid-induced segregation (AIS) effect. Firstly, the self-synthesized Tröger's base diamine monomer — 2,8-diamino-4,10-dimethyl-6H,12H-5,11-methanodibenzo [1,5]-diazocine (TBDA) was added in the polyethersulfone (PES) casting solution to prepare the TBDA-based PES substrate by synchronously coupling the non-solvent-induced phase transformation (NIPs) and AIS, and then the TBDA molecules on the PES substrate surface further reacted with trimesoyl chloride (TMC) via interfacial polymerization (IP) to obtain the wTB-PA TFC NF membranes. Meanwhile, the acid-induced segregation effect of on the microstructure and separation performance of the wTB-PA TFC NF membrane were systematically investigated. As compared with the conventional poly(piperazine amide) NF membrane (MPIP-TMC), the wTB-PA NF membranes exhibited thinner, smoother but more hydrophobic surface as well as lower isoelectric point, and especially showed much higher tolerance to 20 wt% H2SO4 after soaking 63 days and favorable dye rejection (～99.9 %). Thereinto, the acid segregation agent of citric acid monohydrate (CA) endowed the wTB-PA TFC NF membrane with relatively optimal acid resistance and SO42−/Cl− separation selectivity (about 11.8) than glacial acetic acid (AC), nitric acid (HNO3) and hydrochloric acid (HCl). Moreover, this wTB-PA NF membrane based on CA-induced segregation had higher recovery of the positively charged dye of methyl orange (MO) (＞98.0 %) in acidic condition. Therefore, the wTB-PA TFC NF membranes have great potential for the separation application in the strong acidic condition in the future.

Proteome encoded determinants of protein sorting into extracellular vesicles

AbstractExtracellular vesicles (EVs) are membranous structures released by cells into the extracellular space and are thought to be involved in cell‐to‐cell communication. While EVs and their cargo are promising biomarker candidates, sorting mechanisms of proteins to EVs remain unclear. In this study, we ask if it is possible to determine EV association based on the protein sequence. Additionally, we ask what the most important determinants are for EV association. We answer these questions with explainable AI models, using human proteome data from EV databases to train and validate the model. It is essential to correct the datasets for contaminants introduced by coarse EV isolation workflows and for experimental bias caused by mass spectrometry. In this study, we show that it is indeed possible to predict EV association from the protein sequence: a simple sequence‐based model for predicting EV proteins achieved an area under the curve of 0.77 ± 0.01, which increased further to 0.84 ± 0.00 when incorporating curated post‐translational modification (PTM) annotations. Feature analysis shows that EV‐associated proteins are stable, polar, and structured with low isoelectric point compared to non‐EV proteins. PTM annotations emerged as the most important features for correct classification; specifically, palmitoylation is one of the most prevalent EV sorting mechanisms for unique proteins. Palmitoylation and nitrosylation sites are especially prevalent in EV proteins that are determined by very strict isolation protocols, indicating they could potentially serve as quality control criteria for future studies. This computational study offers an effective sequence‐based predictor of EV associated proteins with extensive characterisation of the human EV proteome that can explain for individual proteins which factors contribute to their EV association.

Portable and Rapid Fluorescence Turn-On Detection of Total Pepsin in Saliva Based on Strong Electrostatic Interactions.

Salivary pepsin has been proposed as a promising diagnostic marker for gastroesophageal reflux disease (GERD). However, the activity of human pepsin is strongly influenced by pH, and the acidic condition (pH ∼ 2) is optimal, which is a contradiction for the pepsin detection kit based on its catalytic activity. Thus, its accurate quantification in saliva (neutral pH) by readily rapid tools with simplicity and low cost is still challenging. Herein, a convenient fluorescence assay has been developed for the rapid detection of pepsin at neutral pH based on its electrostatic interaction with SYBR Green (SG) rather than the bioactivity. At neutral pH, the positively charged SG fluorophore can be effectively adsorbed by the negatively charged pepsin due to the low isoelectric point (pI) and large molecular size of pepsin. Thus, the molecular rotation of SG is limited, and its fluorescence intensity is significantly increased. The strategy was further confirmed to have the same fluorescence response as that of normally active and inactivated pepsin. Due to the unique pI of pepsin, the fluorescence strategy is highly selective for pepsin compared to other proteins. On the basis of this strategy, a smartphone-based fluorescence capture device integrated with a programmed Python program was fabricated for both color recognition and the accurate detection of pepsin within 3 min. Under the optimal conditions, this turn-on sensor allowed for the on-site analysis of pepsin with a detection limit of 0.2 μg/mL. Moreover, this strategy was successfully used to assess salivary pepsin to aid in the noninvasive diagnosis of GERD.

Diversity and salinity adaptations of ammonia oxidizing archaea in three estuaries of China.

Ammonia-oxidizing archaea (AOA) are ubiquitously found in diverse habitats and play pivotal roles in the nitrogen and carbon cycle, especially in estuarine and coastal environments. Despite the fact that the diversity and distribution of AOA are thought to be tightly linked to habitats, little is known about the relationship that underpins their genomic traits, adaptive potentials, and ecological niches. Here, we have characterized and compared the AOA community in three estuaries of China using metagenomics. AOA were the dominant ammonia oxidizers in the three estuaries. Through phylogenetic analyses, five major AOA groups were identified, including the Nitrosomarinus-like, Nitrosopumilus-like, Aestuariumsis-like, Nitrosarchaeum-like, and Nitrosopelagicus-like groups. Statistical analyses showed that the aquatic and sedimentary AOA communities were mainly influenced by spatial factors (latitude and water depth) and environmental factors (salinity, pH, and dissolved oxygen) in estuaries, respectively. Compared to AOA dwelling in terrestrial and marine habitats, estuarine AOA encoded more genes involved in glucose and amino acid metabolism, transport systems, osmotic control, and cell motility. The low proteome isoelectric points (pI), high content of acidic amino acids, and the presence of potassium ion and mechanosensitive channels suggest a "salt-in" strategy for estuarine AOA to counteract high osmolarity in their surroundings. Our findings have indicated potential adaptation strategies and highlighted their importance in the estuarine nitrogen and carbon cycles. KEY POINTS: • Spatial and environmental factors influence water and sediment AOA respectively. • Estuarine AOA share low proteome isoelectric value and high acid amino acids content. • AOA adaptation to estuaries is likely resulted from their unique genomic features.

Molecular evolution steered structural adaptations in the DNA polymerase III α subunit of halophilic bacterium Salinibacter ruber.

A significant portion of the earth has a salty environment, and the literature on bacterial survival mechanisms in salty environments is limited. During molecular evolution, halophiles increase acidic amino acid residues on their protein surfaces which leads to a negatively charged surface potential that helps them to maintain the protein integrity and protect them from denaturation by competing with salt ions. Through protein family analysis, we have investigated the molecular-level adaptive features of DNA polymerase III's catalytic subunit (alpha) and its structure-function relationship. This study throws light on the novel understanding of halophilic bacterial replication and the molecular basis of salt adaptation. Comparisons of the amino acid contents and electronegativity of halophilic and mesophilic bacterial proteins revealed adaptations that allow halophilic bacteria to thrive in high salt concentrations. A significantly lower isoelectric point of halophilic bacterial proteins indicates the acidic nature. Also, an abundance of disordered regions in halophiles suggests the requirement of the salt ions that play a crucial role in their stable protein folding. Despite having similar topology, mesophilic and halophilic proteins, a set of very prominent molecular modifications was observed in the alpha subunit of halophiles.

Flocculation efficiency of phosphorylated hemoglobin on kaolin and hematite systems

AbstractHemoglobin recovered from slaughterhouse blood has proven to be an effective flocculant of negatively charged particulate contaminants, but it is ineffective against positively charged contaminants. Phosphorylation is useful for modifying proteins to increase the number of negatively charged sites. Dry‐heat phosphorylation and chemical phosphorylation were applied to hemoglobin. The kaolin/hematite clarification efficiencies of modified hemoglobin at different dosages and pH values were determined. ζ‐potential and total phosphorus content were also measured. Chemical phosphorylation was generally more effective than dry heat phosphorylation at achieving the desired modification of properties; it produced modified hemoglobin (CPhosHb) with higher phosphorus content and lower isoelectric point, and it had greater efficiency in flocculating hematite suspensions. At the optimal dosage of 5 mg/g hematite, treatment with CPhosHb resulted in a 99% turbidity reduction in the hematite suspension after 24 h. When 500 mM calcium chloride was applied to CPhosHb, it exhibited a positive charge in the overall pH range and was capable of clarifying suspensions of negatively charged kaolin particles at low doses. From these results, the effectiveness of chemical phosphorylation on hemoglobin is demonstrated and CPhosHb can be used as a flocculant of either negatively charged or positively charged contaminants in wastewater.

Modeling of lactic acid rejection from lactose in acidified cheese whey by nanofiltration

The continuously increasing demand of lactic acid opens a window for the integration of membrane technology in the dairy industry, improving the sustainability by avoiding the use of large amounts of chemicals and waste generation. Lactic acid recovery from fermentation broth without precipitation has been studied by numerous processes. In this work, a commercial membrane with high lactose rejection and a moderate lactic acid rejection, enabling a permselectivity up to 40%, is sought to perform the simultaneous removal of lactic acid and lactose separation from the acidified sweet whey from mozzarella cheese production in a single stage. The AFC30 membrane of the thin film composite nanofiltration (NF) type was selected because of its high negative charge, low isoelectric point, and divalent ion rejection, as well as a lactose rejection higher than 98% and a lactic acid rejection lower than 37%, at pH 3.5, to minimize the need of additional separation steps. The experimental lactic acid rejection was evaluated at varying feed concentration, pressure, temperature, and flow rate. As the dissociation degree of lactic acid is negligible in industrially simulated conditions, the performance of this NF membrane was validated by the irreversible thermodynamic Kedem-Katchalsky and Spiegler-Kedem models, with the best prediction in the latter case, with the parameter values: Lp = 3.24 ± 0.87 L × m-2 × h-1 × bar-1 and = 15.06 ± 3.17 L × m-2 × h-1, and σ = 0.45 ± 0.03. The results obtained in this work open the way for the up-scaling of membrane technology on the valorization of dairy effluents by simplifying the operation process and the model prediction and the choice of the membrane.

Kinetic Analysis of WO3-Modified Cr- and Sb-Codoped TiO2 for the Photocatalytic O2 Evolution Reaction

Photocatalysis–electrolysis hybrid systems are attractive candidates for cost-effective green hydrogen production. For these systems, it is crucial to improve the O2 evolution activity of the aqueous solution containing the redox mediators. In this study, a kinetic analysis was conducted, for the first time, to determine the role of WO3 modified at various temperatures onto Cr- and Sb-codoped TiO2. The kinetic analysis successfully elucidated the factor that contributed to the higher activity. Based on this implication, it was revealed that the WO3 species increased the amount of Fe3+ ions at the sliding surface owing to the low isoelectric point of WO3, resulting in improved O2 evolution activity. Thus, this study proposes a strategy based on kinetic analysis for the design and development of photocatalysts with improved activity in the O2 evolution reaction.

A “wrench-like” green amphoteric organic chrome-free tanning agent provides long-term and effective antibacterial protection for leather

Organic chrome-free tanning agents are an important part of the sustainable development in leather manufacturing industry. However, organic chrome-free tanning agents tanned leather have a low isoelectric point (pI < 5), which results in a low absorptivity of traditional anionic wet finishing materials; as a result, leather quality is poor, and tannery wastewater is difficult to treat. In addition, leather is easily eroded by bacteria and mold during its production, storage, and use, causing serious economic loss to enterprises and users. In this study, hexahydro-1,3,5-tris(hydroxyethyl)-s-triazine (HHTT), ethylene glycol glycidyl ether (EGDE), and collagen polypeptide (CP) were used as raw materials to prepare a wrench-like, antimicrobial, amphoteric, epoxy-terminated, organic chrome-free tanning agent (HHTT-EGDE-CP) with a pI of 5.21, and an epoxy value of 0.267 mol/100 g. HHTT-EHDE-CP can establish a stable covalent cross-linking with collagen fibers, thereby enhancing the physicochemical properties of leather, the absorptivity of anionic wet finishing materials, giving leather long-term and efficient antibacterial properties. Experimental results show, that the shrinkage temperature (78.8 °C) and the absorptivity of anionic fatliquors (93.1%) and dyes (97.5%) of HHTT-EGDE-CP-tanned leather surpassed those of leather tanned using commercial organic chrome-free tanning agents. Notably, the inhibition rates of HHTT-EGDE-CP-tanned leather against E. coli and S. aureus reached 99.93% and 99.99%, respectively. In addition, HHTT-EGDE-CP-tanned leather exhibited excellent inhibition against A. flavus and Asp. niger, with long-term and efficient antibacterial properties. This innovative technique offers new perspectives for designing and developing multifunctional green materials toward the sustainable development of industrial manufacturing.

Large-scale analysis of the genome of the rare alkaline-halophilic Stachybotrys microspora reveals 46 cellulase genes.

Fungi are of great importance in biotechnology, for example in the production of enzymes and metabolites. The main goal of this study was to obtain a high-coverage draft of the Stachybotrys microspora genome and to annotate and analyze the genome sequence data. The rare fungus S.microspora N1 strain is distinguished by its ability to grow in an alkaline halophilic environment and to efficiently secrete cellulolytic enzymes. Here we report the draft genome sequence composed of 3715 contigs, a genome size of 35 343 854 bp, with a GC content of 53.31% and a coverage around 20.5×. The identification of cellulolytic genes and of their corresponding functions was carried out through analysis and annotation of the whole genome sequence. Forty-six cellulases were identified using the fungicompanion bioinformatic tool. Interestingly, an S.microspora endoglucanase selected from those with a low isoelectric point was predicted to have a halophilic profile and share significant homology with a well-known bacterial halophilic cellulase. These results confirm previous biochemical studies revealing a halophilic character, which is a very rare feature among fungal cellulases. All these properties suggest that cellulases of S.microspora may have potential for use in the biofuel, textile, and detergent industries.

Characteristics and adaptability of Flavobacterium panici BSSL-CR3 in tidal flat revealed by comparative genomic and enzymatic analysis

Tidal flat microbes play an important ecological role by removing organic pollutants and providing an energy source. However, bacteria isolated from tidal flatsand their genomes have been scarcely reported, making it difficult to elucidate which genes and pathways are potentially involved in the above roles. In this study, strain BSSL-CR3, the third reported species among the tidal flat Flavobacterium was analyzed using whole-genome sequencing to investigate its adaptability and functionality in tidal flats. BSSL-CR3 is comprised of a circular chromosome of 5,972,859bp with a GC content of 33.84%. Genome annotation and API ZYM results showed that BSSL-CR3 has a variety of secondary metabolic gene clusters and enzyme activities including α-galactosidase. BSSL-CR3 had more proteins with a low isoelectric point (pI) than terrestrial Flavobacterium strains, and several genes related to osmotic regulation were found in thegenomic island(GI). Comparative genomic analysis with other tidal flat bacteria also revealed that BSSL-CR3 had the largest number of genes encoding Carbohydrate Active EnZymes (CAZymes) which are related to algae degradation. This study will provide insight into the adaptability of BSSL-CR3 to the tidal flats and contribute to facilitating future comparative analysis of bacteria in tidal flats.

Immunoreactivity profiling of Anti-Chinese hamster ovarian host cell protein antibodies by isobaric labeled affinity purification-mass spectrometry reveals low-recovery proteins

We evaluated the immunoreactivity profiles of eight commercial anti-host cell protein (anti-HCP) antibodies from different host animals and their antigens used for immunization by an isobaric labeled affinity purification-mass spectrometry (AP-MS) method. As a result, 34 proteins with high abundance but low recovery from harvest cell culture fluid were identified. Since they are likely to be underestimated in biopharmaceutical quality assessment, the features common to these proteins were investigated. Compared to other immunoprecipitated HCP proteins, proteins exhibiting lower molecular weight (ΔMW = -14600), lower isoelectric point (ΔpI = -0.86), and lower hydrophobicity (ΔGRAVY = -0.13) were enriched. This AP-MS method provides important information for HCP control strategies using immunological methods and is expected to contribute to the development of safe biopharmaceutics.

Construction of titanium-aluminum xerogel composite coagulant for removal of tetracycline in water: synergy effects and improvement mechanisms insight.

Titanium xerogel coagulant (TXC) is a new type of coagulant that has attracted much attention in recent years. However, the tetracycline removal performance of TXC was not satisfactory because low isoelectric point (pHiep) inhibited the electrical neutralization efficiency of TXC in an alkaline environment. To overcome this shortcoming, a composite xerogel coagulant (titanium-aluminum xerogel composite coagulant) was prepared. The removal of tetracycline and turbidity was used as evaluation indexes. It was proved that the combination of aluminum (III) and titanium (IV) enhanced the resistance of TXC to pH. The synthesized titanium-aluminum xerogel composite coagulant (TXAC) has an excellent removal ability of tetracycline in a wide pH range (pH = 5-10). At pH 8.8, the dosage required to remove 80% tetracycline from water decreased from 93 (TXC) to 35mg/L (TXAC). The reason for this improvement could be attributed to (i) aluminum (III) enhanced the electric neutralization of TXC to negatively charged pollutants in an alkaline environment; (ii) the complexing ability of organic matter and aluminum (III) was enhanced. This work provides a feasible scheme for the pretreatment of tetracycline in water to meet the pretreatment requirements of special water.

Role of Molecular Modification and Protein Folding in the Nucleation and Growth of Protein-Metal-Organic Frameworks.

Metal–organic frameworks (MOFs) are a class of porous nanomaterials that have been extensively studied as enzyme immobilization substrates. During in situ immobilization, MOF nucleation is driven by biomolecules with low isoelectric points. Investigation of how biomolecules control MOF self-assembly mechanisms on the molecular level is key to designing nanomaterials with desired physical and chemical properties. Here, we demonstrate how molecular modifications of bovine serum albumin (BSA) with fluorescein isothiocyanate (FITC) can affect MOF crystal size, morphology, and encapsulation efficiency. Final crystal properties are characterized using scanning electron microscopy (SEM), powder X-ray diffraction (PXRD), fluorescent microscopy, and fluorescence spectroscopy. To probe MOF self-assembly, in situ experiments were performed using cryogenic transmission electron microscopy (cryo-TEM) and X-ray diffraction (XRD). Biophysical characterization of BSA and FITC-BSA was performed using ζ potential, mass spectrometry, circular dichroism studies, fluorescence spectroscopy, and Fourier transform infrared (FTIR) spectroscopy. The combined data reveal that protein folding and stability within amorphous precursors are contributing factors in the rate, extent, and mechanism of crystallization. Thus, our results suggest molecular modifications as promising methods for fine-tuning protein@MOFs’ nucleation and growth.

Separation and purification of highly infectious enterovirus A71 particles using a strong anion-exchange column

Virions produced from cell culture is the primary source for production of formalin-inactivated whole virus vaccines for enteroviruses. EV-A71 particles produced from culture system comprise two major types, the immature/empty (E)-particle and the mature/full (F)-particle, which both exhibit low isoelectric point (pI) values but have distinct differences in infectivity and immunogenicity. Although EV-A71 particles can conventionally be separated into E-particle and F-particle using sucrose gradient ultracentrifugation, this procedure is cumbersome and difficult to put into practice for vaccine production. Methods based on ion-exchange chromatography have been exploited to improve the purification efficacy; however, none of them are capable of separating the E- and F-particles efficiently. In this study, we aimed to develop an approach to isolate and purify the highly immunogenic mature EV-A71 particles. By applying a step gradient elution procedure, we successfully isolated the viral structure protein VP0-cleaved particles of EV-A71 from a mixture of cultured viral solution using the Q-membrane anion-exchange chromatography. The elution started with 0.1x phosphate buffered saline (PBS) solution while increasing the percentage of 1x PBS containing 1M NaCl in sequential steps. By this procedure, the VP0-cleaved mature particles and VP0-uncleaved immature particles of EV-A71 could be separated into different fractions in Q-membrane with gradually increased NaCl concentration in elution buffer. The purified VP0-cleaved particles were shown to have characteristics equivalent to those of the highly infectious F-particles of EV-A71. The overall recovery rate for the mature EV-A71 particles by Q-membrane is 56% and its purity was shown to be equivalent to those isolated by the sucrose gradient ultracentrifugation. Our approach provides a simple and efficient purification method for recovering mature, highly infectious virus particles from the EV-A71 culture bulk.

Optimization of protein hydrolysates production from defatted peanut meal based on physicochemical characteristics and sensory analysis

Three enzymes, GA (Genencor Alkaline Protease), GN (Genencor Neutral Protease) and Protamex were used to prepare protein hydrolysates from defatted peanut meal (DPM). The physicochemical properties of defatted peanut meal hydrolysate (DPMH) and the changes of flavor were determined. DPMHs had lower isoelectric points, zeta potential values and better solubility. The DPMH of GN showed the best emulsifying activity and the emulsion stability. Volatile organic compounds (VOCs) changed significantly with the most drastic changes at 4–6 h. About 50 VOCs were labeled using GC-IMS. The fingerprint of smell change was established, and the enzymolysis reaction could be divided into different stages according to the change of flavor. DPMHs of GA and Protamex showed stronger astringency and bitterness, while stronger umami was shown in DPMH of GN. This research provides basic data support for enzyme selection, sensory and reaction stage control to obtain enzymatic polypeptides with improved properties from DPM.