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Ovomucin-Complex extracted from egg white is expected to have a barrier function similar to gastric mucin. In this study, the dynamic changes in structure, rheological properties and binding ability of Ovomucin-Complex during in vitro simulated gastric digestion were investigated. The results from HPLC and CLSM showed that extremely acidic pH (pH = 2.0) promoted Ovomucin-Complex to form aggregation. Acid-induced aggregation may hinder its binding to pepsin, thus rendering Ovomucin-Complex resistant to pepsin. Consequently, most of the polymer structure and weak gel properties of Ovomucin-Complex retained after simulated gastric digestion as verified by HPLC, CLSM and rheological measurement, although there was a small breakdown of the glycosidic bond as confirmed by the increased content of reducing sugar. The significantly reduced hydrophobic interactions of Ovomucin-Complex were observed under extremely acidic conditions and simulated gastric digestion compared with the native. Noticeably, the undigested Ovomucin-Complex after simulated gastric digestion showed a higher affinity (K D  = 5.0 ± 3.2 nm) for urease - the key surface antigen of Helicobacter pylori. The interaction mechanism between Ovomucin-Complex and urease during gastric digestion deserves further studies. This finding provides a new insight to develop an artificial physical mucus barrier to reduce Helicobacter pylori infection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Immune checkpoint inhibitors targeting PD-L1 lead to challenging patterns of efficacy and toxicity. Herein, by focusing on tracing the molecular biomarker of response to efficacy, we formulated a central hypothesis for the construction of theranostic functional monoclonal antibody incorporation with tracing ability based on fluorescence turn-on and controllable release strategies. Functional atezolizumab was constructed by in situ assembly of both biorthogonal group and controllable release group. The theranostic monoclonal antibodies achieved quantitative monitoring of PD-L1 on cells with different expression levels through biorthogonal light-up fluorescence, followed by the release of atezolizumab in combination with high tumor reduction conditions to promote immune activation. The combination of bio-orthogonal reaction-driven fluorescence turn-on and tumor microenvironment-responsive controllable release afforded theranostic bifunctional monoclonal antibodies for the detection of PD-L1 and combination therapy. Remarkably, these novel theranostics might be used as probes for fluorescent imaging and simultaneously achieving potent antitumor efficacy., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Ovomucin (OVM) is a natural glycoprotein with various biological activities but poor solubility. This study aimed to enhance the solubility of OVM by using an ultrasonic-assisted method. The effect of ultrasound (US) on the structure, thermal stability and biological functions of OVM aggregates was evaluated. It was found that insoluble OVM aggregates were dissociated and the solubility increased significantly to 90.0 % after US under 400 W for 45 min. US also improved the onset temperature (To) and denaturation temperature (Td) of OVM. More importantly, the cholesterol binding capacity of both OVM and its digestion products were significantly improved after US (p < 0.05). The gastrointestinal digestion products of US-OVM also showed higher α-amylase and α-glucosidase inhibition than native OVM aggregates. US-induced dissociation of OVM aggregates and the conversion of β-sheet and β-turn to random coil, resulting in the exposure of hydrophobic binding sites may be an important reason for the enhanced stability and adsorption capacity. These findings suggested that US was an effective method for preparing soluble OVM and improved its adsorption capacity, which can further facilitate the application of OVM in the food industry., Competing Interests: Declaration of competing interest The authors have declared that there is no conflict of interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Based on the scaffold hybridization strategy, twenty-four indole-guanidines were designed and synthesized. Subsequently, anti-proliferative activity against various cancer cells indicated that most of these hybrids exhibited moderate to high anti-proliferative activity, especially for human hepatoma cell lines. Selectivity investigation showed that these hybrids showed the best selectivity for SMMC-7721 subtype in human hepatoma cells. Particularly, (E)-3-((2-(N-pentylcarbamimidoyl)hydrazono)methyl)-1H-indol-5-yl 4-methylbenzoate (19) and (E)-3-((2-(N-pentylcarbamimidoyl)hydrazono)methyl)-1H-indol-5-yl 4-methoxybenzoate (22) exhibited potent inhibition against SMMC-7721 cells with IC 50 values of 0.057 μM and 0.042 μM, respectively, far outperforming that of Sorafenib. Meanwhile, hybrids 19 and 22 exhibited no significant cytotoxicity against normal cells such as HEK293 cells and HEK293T cells. Moreover, further investigations indicated that hybrid 22 effectively induced apoptosis, arrested the cell cycle at S phase, and selectively down regulated expression of p-STAT3, JAK2 and BRAF in SMMC-7721 cells in a dose-dependent manner. Molecular docking indicated that hybrid 22 exhibited high affinity with STAT3 and BRAF. In summary, hybrid 22 was developed as a potential and effective anti-hepatoma candidate, which was worthy of further investigation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

This study developed a closed-circuit biorefinery process for full conversion of lignocellulose into ethanol, biogas and organic fertilizer with zero waste on a pilot scale. In the process, subcritical water pretreatment could effectively break the structure of wheat straw (WS), and ethanol was obtained from pretreated wheat straw (PWS) using two batches of simultaneous saccharification and fermentation (SSF). The pretreatment and ethanol fermentation wastes were reused for biogas and organic fertilizer production by anaerobic digestion (AD), whereas the pretreatment and ethanol conversion efficiency were reduced when supernatant after AD was recovered for next batch pretreatment. The yields of ethanol (0.08-0.09 g/g), biogas (0.05-0.10 L/g) and organic fertilizer (0.55-0.79 g/g) were demonstrated through mass balance. Furthermore, the hidden problems were exposed on pilot-scale conversion process, and several strategies were provided for optimizing the biorefinery process in the future., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The conversion of lignocellulosic biomass to bioethanol is a potential approach to alleviate the energy crisis and environmental deterioration. To improve the conversion efficiency of bioethanol from wheat straw (WS), the optimization of subcritical water pretreatment and high solid hydrolysis were investigated in this study. Response surface methodology (RSM) accompanied with glucose concentration after enzymatic hydrolysis as a more reasonable response value was applied for the pretreatment optimization, and the optimum conditions were obtained as 220.51 °C of extraction temperature, 22.01 min of extraction time and 2.50% (w/v) of substrate loading. After pretreatment, the hemicellulose decreased by 18.37%, and the cellulose and lignin increased by 25.92% and 8.81%, respectively, which were consistent with the destroyed microstructure and raised crystallinity. The high efficiency of separate hydrolysis and fermentation (SHF) was verified by five commercial cellulases, and yields of hydrolysis and fermentation were 77.85-89.59% and 93.34-96.18%, respectively. Based on the high solid (15%) hydrolysis and fermentation, the ethanol concentration was significantly improved to 37.00 g/L. Interestingly, 64.47% of lignin was accumulated in the solid residue after enzymatic hydrolysis and it did not affect the efficiency of SHF, which further suggested that subcritical water mainly affected the structure of WS rather than the removal of lignin. Therefore, subcritical water pretreatment combined with high solid hydrolysis is a more effective solution for bioethanol conversion, which is also a promising strategy to utilize all components of lignocellulosic biomass., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

We previously developed two candidates with potency of inducing vascular normalization, BD7 and B14. However, the definite intracellular molecular target(s) responsible for their activity remains unknown. Herein, we report the discovery and functional assessment of several multifunctional photoaffinity probes for determining the potential biological targets of active compounds. The probes bear a photoaffinity moiety and a bioorthogonal unit attached to B7 or B14 and maintained the bioactivity of the parent active molecules. Using in vitro biological assays, we preliminarily identified VEGFR-2 as a potential intracellular target for the active candidates. Our results demonstrate the utility of these multifunctional photoaffinity probes for analyzing the biological activity and subcellular localization of the intracellular target proteins of active candidates., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Multidrug-resistant (MDR) bacterial infection still poses a serious threat to public health, therefore, effective and safe antimicrobial agents are urgently needed. In this study, recombinant bacteriocin BMP32 (BMP32r) prepared by the Escherichia coli expression system had a broad-spectrum antibacterial activity even against some MDR bacteria and its minimum inhibitory concentration ranged from 9.2 to 36.8 mg/L. Furthermore, BMP32r showed good stable performance in heat, pH and storage. Moreover, the scanning electron microscope and transmission electron microscope revealed that BMP32r killed indicator strains through cell wall destruction, pore formation, and the membrane permeability increasing which was proved by propidium iodide uptake investigation. The wound healing of an animal MDR S. aureus infected model was promoted by BMP32r, and the safety was verified by the cytotoxicity assay that the viability of HFF cells remained 87.3% in even when the concentration of BMP32r was as high as 147.2 mg/L. In addition, no abnormalities or damages to major organs was found in vivo assessments after treatment with BMP32r. In conclusion, BMP32r has great potential to be developed as a safe antimicrobial agent to treat MDR bacterial infections., Competing Interests: Declaration of competing interest The authors have declared no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Bacteriocins are a subclass of antibacterial peptides considered to be the most promising alternative to antibiotics. A large number of unknown bacteriocins are hidden in lactic acid bacteria. In this study, by combining the genome with LC-MS/MS, 14 novel bacteriocins produced by Lactobacillus rhamnosus LS-8 were detected. Moreover, these bacteriocins were successfully cloned via plasmid pET-28a(+) and pET-30a(+) and heterologously expressed in Escherichia coli BL21. Escherichia coli ATCC25922 and Staphylococcus aureus ATCC25923 were used to confirm their antibacterial activity. Subsequently, the four bacteriocins (pH 25, S68, S81, and S137) with the strongest antibacterial ability were selected, and their expression conditions were optimized. Purification was performed by cation exchange chromatography and high performance liquid chromatography, and the active parts were collected and analyzed by mass spectrometry. The mass spectrometry analysis revealed that peptide coverage was >71.39%. The MICs of the four bacteriocins against four pathogenic bacteria ranged from 5.38 to 19.84 μg/mL. In addition, these bacteriocins significantly inhibited the growth of four standard pathogenic bacteria. They also exhibited broad-spectrum bacteriostasis on Gram-positive and Gram-negative bacteria. Therefore, these new bacteriocins have great potential in the study of alternative antibiotics., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Intestinal dysfunction, which may cause a series of metabolic diseases, has become a worldwide health problem. In the past few years, studies have shown that consumption of poultry eggs has the potential to prevent a variety of metabolic diseases, and increasing attention has been directed to the bioactive proteins and their peptides in poultry eggs. This review mainly focused on the biological activities of an important egg-derived protein named ovomucin. Ovomucin and its derivatives have good anti-inflammatory, antioxidant, immunity-regulating and other biological functions. These activities may affect the physical, biological and immune barriers associated with intestinal health. This paper reviewed the structure and the structure-activity relationship of ovomucin，the potential role of ovomucin and its derivatives in modulation of intestinal health are also summarized. Finally, the potential applications of ovomucin and its peptides as functional food components to prevent and assist in the pretreatment of intestinal health problems are prospected., Competing Interests: Declaration of competing interest The authors have declared that there is no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Aconitine might have reproductive toxicity and the effects of aconitine on androgen synthesis in Leydig cells remain unclear. Here, we explore how aconitine affects androgen synthesis and metabolism in rat immature Leydig cells in vitro. Immature Leydig cells were isolated from 35-day-old male Sprague Dawley rats and cultured with 0-50 μM aconitine for 3 h in combination with LH, 8Br-cAMP, 22R-hydroxycholesterol, pregnenolone, progesterone, androstenedione, testosterone, and dihydrotestosterone, respectively. Medium androgens were measured. The levels of Leydig cell mRNAs, Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Srd5a1, and Akr1c14, were measured by qPCR. ROS and apoptosis were determined after 24-h aconitine treatment. Aconitine inhibited basal androgen production in Leydig cells at 0.05 μM and the higher concentrations. Aconitine blocked pregnenolone, progesterone, and androstenedione mediated androgen outputs without affecting 22R-hydroxycholesterol-mediated androgen production at 5 μM. Aconitine also inhibited LH and 8Br-cAMP stimulated androgen outputs at 5 μM. Further investigation showed that aconitine blocked androgen synthesis via down-regulating the expression of Scarb1, Hsd3b1, Cyp17a1, and Hsd17b3. At 50 μM, aconitine also induced ROS generation and increased apoptotic rate of Leydig cells. Aconitine lowered serum testosterone levels at 1.5 mg/kg after 7 days of oral exposure from postnatal day 35. In conclusion, aconitine inhibits androgen synthesis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A novel crude exopolysaccharide (EPS) produced by Lactobacillus plantarum KX041 possessed prominent antioxidant activity which was proved in our previous study. In our present study, the further purifications were conducted to obtain EPS fractions, which were called as EPS-1-1, EPS-2-1 and EPS-3. The structures and conformational characterizations were determined through FT-IR, UV, GC, HPLC, NMR, SEM and Congo red test analysis. The Mw of EPS-1-1, EPS-2-1 and EPS-3 were estimated to be 57,201, 70,734, and 26,387 Da, respectively. EPS-1-1 and EPS-2-1 had the similar structure, composed of arabinose, mannose, glucose and galactose with a ratio of 1.09:88.53:3.99:6.39 and 0.58:94.11:3.55:1.76, and both were α-type configurations. Whereas EPS-3 contained rhamnose, fucose, arabinose, xylose, mannose, glucose, galactose and galacturonic acid in a molar ratio of 2.01:2.65:10.95:4.62:4.07:27.81:44.16:3.73 and possessed a triple helical structure. SEM results indicated EPS-1-1 and EPS-2-1 appeared flake shapes piling up into compact structures with a rough surface. In addition, these purified EPS fractions all possessed the immune activity, DPPH/ABTS radicals scavenging activities and DNA damage protective effect., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Ovomucin is known to be critical for keeping the high quality and freshness of thick albumen, but there is lack of understanding on the dynamics changes of this important protein during storage. This study aimed to investigate the relationship between ovomucin content and egg freshness during storage. Firstly, the viscoelasticity of albumen was shown to be much higher than that of ovomucin-depleted albumen from rheological analysis results, indicating that ovomucin is an important component in maintaining the natural viscoelasticity of albumen. Then, the ovomucin content determined by ELISA method was compared to albumen pH, Haugh unit (HU), and yolk index in terms of egg white quality and to the time of storage in terms of egg freshness at 4°C, 25°C, and 37°C, respectively. Results of the transformation kinetic showed a decrease in ovomucin content with prolonged storage time (P ≤ 0.01). Correlation analysis showed a high positive correlation between ovomucin content and HU (P ≤ 0.01) and a high negative correlation between ovomucin content and the albumen pH (P ≤ 0.01) at the test temperatures. We therefore conclude that ovomucin content in albumen can be used as an index for egg freshness. At last, predictive models of the equivalent egg age (4°C and 25°C) for evaluating the egg freshness were established by means of exponential regression model with ovomucin content as the variable. These results can provide a theoretical and technical basis for the storage and fresh evaluation of shell eggs.

Ovomucin (OVM) plays an important role in inhibiting infection of various pathogens. However, this bioactivity mechanism is not much known. Here, the role of sialic acid in OVM anti-virus activity has been studied by ELISA with lectin or ligand. Structural changes of OVM after removing sialic acid were analyzed by circular dichroism and fluorescence spectroscopy. OVM could be binding to the hemagglutinin (HA) of avian influenza viruses H 5 N 1 and H 1 N 1 , this binding was specific and required the involvement of sialic acid. When sialic acid was removed, the binding was significantly reduced 71.5% and 64.35%, respectively. Therefore, sialic acid was proved as a recognition site which avian influenza virus bound to. Meanwhile, the endogenous fluorescence and surface hydrophobicity of OVM removing sialic acid were increased and the secondary structure tended to shift to random coil. This indicated that OVM molecules were in an unfolded state and spatial conformation disorder raising weakly. Remarkably, free sialic acid strongly promoted OVM binding to HA and thereby enhanced the interaction. It may contribute to the inhibition of host cell infection, agglutinate viruses. This study can be extended to the deepening of passive immunization field., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Ovomucin has a great potential because of its numerous bioactivities, which makes it an attractive molecule for industrials. However, to isolate soluble ovomucin without degradation and contamination remains a challenge. In this study, ovomucin of high purity (99.13%) was obtained in good yield (3.02 g kg -1 fresh egg white) via an improved two-step precipitation followed by gel filtration chromatography. The IC50 of the preparation for three representative new disease virus strains named LaSota, Mukteswar and V-4 is 1.99, 4.95 and 5.78 × 10 -3  g L -1 , respectively. Produced ovomucin showed limited reduction in the hemagglutination inhibition activity against all of the virus strains during the purification. Infrared spectroscopy of the ovomucin preparation indicated extensive glycosylation and sulfation. Amino acid analysis found that it was rich in alanine, glutamic acid, threonine and valine residues, which is typical in mucins. The improved process developed in this study is an alternative approach to obtain pure ovomucin with elevated antiviral activity and purity, which may significantly push forward the further research on bioactivities of ovomucin., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Many flavonoids and isoflavonoids have anti-diabetic effects in animal models. However, the mechanisms that are involved are generally unclear. Since 11β-hydroxysteroid dehydrogenases (HSD11Bs) play important roles in diabetes, we hypothesize that flavonoids and isoflavonoids may affect diabetes by targeting two isoforms of HSD11B differently. The inhibitory effects of flavonoids (apigenin and quercetin) and isoflavonoids [genistein and (±) equol] on rat and human HSD11B1 and HSD11B2 were analyzed. The potencies of inhibition on human HSD11B1 reductase was in the order of apigenin > quercetin > genistein > (±) equol, with IC 50 values of 2.19, 5.36, 11.00, and over 100 μM, respectively. Genistein also inhibited rat HSD11B1 reductase with IC 50 value of 24.58 μM, while other three chemicals showed no effects on the enzyme activity with IC 50 values over 100 μM. However, apigenin and (±) equol did not inhibit human HSD11B2 at concentrations as high as 100 μM, while genistein and quercetin inhibited human HSD11B2 by 60% and 50% at 100 μM, respectively. The effective flavonoids and isoflavonoids are noncompetitive inhibitors of HSD11B1 when steroid substrates were used. Docking analysis showed that they bound to the steroid-binding site of the human HSD11B1. These data indicate that apigenin is a selective inhibitor of human HSD11B1 of two HSD11B isoforms, which may be useful in managing symptoms of the metabolic syndrome., (Copyright © 2018. Published by Elsevier Inc.)

Phthalate diesters, including di-(2-ethylhexyl) phthalate (DEHP) and diethyl phthalate (DEP), are chemicals to which humans are ubiquitously exposed. Humans are exposed simultaneously to multiple environmental chemicals, including DEHP and DEP. There is little information available about how each chemical may interact to each other if they were exposed at same time. The present study investigated effects of the combinational exposure of rats to DEP and DEHP on fetal Leydig cell development. The results showed that the gestational (GD12-20) exposure of DEP + DEHP resulted in synergistic and/or dose-additive effects on the development of fetal Leydig cell. The lowest observed adverse-effect levels (LOAEL) for fetal Leydig cell (aggregation and cell size), and StAR expressions were of 10 mg/kg and, lower than when these chemicals were exposed alone. Also, mathematical modeling the response curves supports the dose-addition model over integrated-addition model. Overall, these data demonstrate that individual phthalate with a similar mechanism of action can elicit cumulative, dose additive, and sometimes synergistic, effects on the development of male reproductive system when administered as a mixture., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A rapid, sensitive and selective ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was developed and validated for the determination and pharmacokinetic investigation of acacetin in rat plasma. Sample preparation was accomplished through a simple one-step deproteinization procedure with 0.2 mL of acetonitrile to a 0.1 mL plasma sample. Plasma samples were separated by UPLC on an Acquity UPLC BEH C18 column using a mobile phase consisting of acetonitrile-0.1% formic acid in water with gradient elution. The total run time was 2.0 min and the elution of acacetin was at 0.83 min. The detection was performed on a triple quadrupole tandem mass spectrometer equipped with positive-ion electrospray ionization (ESI) by multiple reaction monitoring (MRM) of the transitions at m/z 285.3→242.2 for acacetin and m/z 237.2→194.3 for carbamazepine (internal standard). The calibration curve was linear over the range of 1-1600 ng/mL with a lower limit of quantitation (LLOQ) of 1.0 ng/mL. Mean recovery of acacetin in plasma was in the range of 78.4-85.2%. Intra-day and inter-day precision were both <10.5%. This method was successfully applied in pharmacokinetic study after intravenous administration of 5.0mg/kg acacetin in rats., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Diisononyl phthalate (DINP) is a synthetic material that has been widely used as a substitute for other plasticizers prohibited due to reproductive toxicity in consumer products. Some phthalates have been associated with testicular dysgenesis syndrome in male fetus when female pregnant dams were exposed to them. The present study investigated effects of DINP on fetal Leydig cell function and testis development. Female pregnant Sprague Dawley rats received control vehicle (corn oil) or DINP (10, 100, 500, and 1000 mg/kg) by oral gavage from gestational day (GD) 12 to 21. At GD 21.5, testicular testosterone production, fetal Leydig cell numbers and distribution, testicular gene and protein expression levels were examined. DINP showed dose-dependent increase of fetal Leydig cell aggregation with the low observed adverse-effect level (LOAEL) of 10 mg/kg and multinucleated gonocyte with LOAEL of 100 mg/kg. At 10 mg/kg, DINP also significantly increased fetal Leydig cell size, but inhibited insulin-like 3 and 3β-hydroxysteroid dehydrogenase gene expression and protein levels. DINP inhibited testicular testosterone levels at 1000 mg/kg. The results indicate that in utero exposure to DINP affects the expression levels of some fetal Leydig cell steroidogenic genes, gonocyte multinucleation and Leydig cell aggregation., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Metal-protein interaction for regulating the function and structure of proteins is of major interest. We demonstrate here that the addition of 9 μmol Mg2+ per mg ovomucin resulted in a 2.0-fold increase in the adhesion capacity of ovomucin to New Disease Virus (NDV) using ELISA method. The hemagglutinin inhibition rate of 100 μg/mL ovomucin-Mg2+ mixture (9 μmol Mg2+ per mg ovomucin) to NDV increased to 55.61%, which is significantly higher than that of the pure ovomucin (P<0.01). Binding characteristics of ovomucin to Mg2+ were then evident by fluorescence and FT-IR spectroscopy as well as dynamic light scattering (DLS) technology. Fluorescence and FT-IR spectroscopy results suggest that Mg2+ prefers to interact with the carbohydrate moiety of ovomucin without significant changes in the secondary structure when Mg2+ concentration is less than 9 μmol/mg ovomucin. When Mg2+ increases to 16 μmol/mg ovomucin, it gives rise to changes in secondary structure, and forms a more compact configuration. This is supported by the hydrodynamic radius of ovomucin with and without Mg2+ binding as examined by DLS. The Mg2+ bound in regulating the structure and activity of ovomucin provided important insight into the effect of metal ions on ovomucin binding to pathogens., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Purification of ovomucin is still an empirical technique and sometimes insufficient quantities of ovomucin are purified to allow characterization. Here we aimed to investigate the effect of MgCl(2) on the purity and bioactivity of ovomucin during isoelectric precipitation process and to develop an effective protocol to prepare pure ovomucin with high bioactivity. It was found that addition of MgCl(2) is an alternative approach to remove lysozyme from ovomucin, and that the hemagglutination inhibition (HI) activity of ovomucin with MgCl(2) against New Disease Virus (NDV) was about two times higher than the protein without salts. Thus, an improved procedure comprises a precipitation with 0.05 mol/L CaCl(2) followed by precipitation with 0.05 mol/L MgCl(2) was developed for the isolation of ovomucin. Better adhesion property of ovomucin was observed when low concentration of MgCl(2) was added in the designed ELASA test, whereas the adhesion property of the pure ovomucin without salts to NDV was lower. Thus, magnesium (II) plays an important role in the activity of ovomucin, and the alternative method developed in this study may significantly facilitate the further research on the mechanism of ovomucin activity., (Copyright © 2011 Elsevier B.V. All rights reserved.)