Endogenous reactive oxygen species (ROS)-driven protein oxidation regulates emulsifying and foaming properties of liquid egg white.

Highly oxidative reactive oxygen species (ROS) attack protein structure and regulate its functional properties. The molecular structures and functional characteristics of egg white (EW) protein (EWP) during 28 d of aerobic or anaerobic storage were explored to investigate the "self-driven" oxidation mechanism of liquid EW mediated by endogenous ROS signaling. Results revealed a significant increase in turbidity during the storage process, accompanied by protein crosslinking aggregation. The ROS yield initially increased and then decreased, leading to a substantial increase in carbonyl groups and tyrosine content. The free sulfhydryl groups and molecular flexibility in EWP exhibited synchronicity with ROS production, reflecting the self-repairing ability of cysteine residues in EWP. Fourier-transform infrared spectroscopy indicated stable crosslinking between EWP molecules in the early oxidation stage. However, continuous ROS attacks accelerated EWP degradation. Compared with the control group, the aerobic-stimulated EWP showed a significant decrease in foaming capacity from 30.5 % to 9.6 %, whereas the anaerobic-stimulated EWP maintained normal levels. The emulsification performance exhibited an increasing-then-decreasing trend. In conclusion, ROS acted as the predominant factor causing deterioration of liquid EW, triggering moderate oxidation that enhanced the superior foaming and emulsifying properties of EWP, and excessive oxidation diminished the functional characteristics by affecting the molecular structure.

Polysaccharide-addition order regulates sonicated egg white peptide stabilized nanoemulsions and ß-carotene digestion in vitro.

In this paper, the effects of the polysaccharide-addition order (before and after homogenisation) on the stability of nanoemulsion stabilised by sonicated egg white peptides and the in vitro digestive behaviour of loaded ß-carotene were investigated. The pyrene fluorescence results showed that the concentration of micelles formed by flaxseed gum (FG) in complex with peptides was significantly higher than that of peach gum (PG). The order of polysaccharide-addition affected the emulsion properties and stability; adding polysaccharides before homogenisation led to protein bridging flocculation, low polysaccharide coverage and a higher interfacial adsorbed protein content of the emulsion. PG enhanced potential spatial resistance and electrostatic repulsion, effectively prevented emulsion flocculation and improved electrostatic stability. After homogenisation, FG was added to emulsions to improve environmental stability, including ionic, temperature and storage stability. Due to the viscosity of polysaccharides and the formed polysaccharide-protein-lipid aggregates, the increasing degree of bridging flocculation promoted the prominent of apparent viscosity, and the G' and G'' exhibited a frequency-dependent increase. The polysaccharide type and mode changed the surface loading charge and droplet interface thickness, delayed the destruction of the droplet structure by protease, and slowed the release of ß-carotene to form micelles. In this study, a stable emulsion system and an efficient emulsion transport system for bioactive substances were obtained by regulating polysaccharides adding order, which is significant for constructing an efficient food emulsion delivery system.

The relationship between acylation degree and gelling property of NaOH-induced egg white gel: Efficient is better?

In this study, succinic anhydride (SA) and octenyl succinic anhydride (OSA) were used to modify the egg white protein (EWP), and acylated EWP was further prepared as a gel by adding NaOH and heat modification. The results showed that the acylation degree for the SA- and OSA-added groups reached 57.68% and 26.95%, respectively, and the average size, the absolute value of ζ-potential, increased significantly with SA and OSA addition, indicating that acylation improved the stability of EWP solution. Furthermore, the surface hydrophobicity increased for the SA-added group while decreasing for the OSA-added group, and the acylation process exposed the flexibility part of the protein molecule. Acylated EWP prepared gel (EWG) showed a translucent and slightly yellow appearance (except for the sol state of the OSA-added 1:50 group), the gel strength, water-holding capacity and rheological properties for SA-added EWG were remarkably reduced while OSA-added EWG improved apparently. Intermolecular forces analysis indicated that the addition of SA promoted the formation of disulfide bonds and strengthened proteins interactions while adding OSA weakened the interactions. Microstructural observations revealed a rougher gel network structure and weaker protein cross-linking in the gel prepared after the acylation of proteins. However, the high efficiency of SA and promotion of protein-molecule interactions were unable to counteract the negative effect of SA on the extension of the gel network structure, and the interaction between OSA expanded the formation of the gel network structure.

Characteristics of intermolecular forces, physicochemical, textural and microstructural properties of preserved egg white with Ca(OH)2 addition.

The effects of Ca(OH)2 on the physicochemical, mechanical and microstructural characteristics, intermolecular forces and protein patterns of preserved egg white (PEW) were investigated. Results suggested that Ca(OH)2 (0.1%) reduced the free alkalinity content and turbidity and increased the brightness of PEW. The surface hydrophobicity of PEW protein with added Ca(OH)2 decreased during the pickling period owing to the hydrophobic residues being hidden in the interior of the protein. Total content of sulfhydryl and disulfide bonds in PEW decreased. Non-specific cross-linking, hydrogen bonds and hydrophobic interactions were the primary intermolecular forces. For textural properties, hardness and springiness had obvious prominence. A loose porous and regular network-like microstructure formed as the Ca(OH)2 increased and Ca(OH)2 delayed denaturation of the PEW protein. The physical properties of PEW correlated with molecular interactions and the microenvironment. Ca(OH)2 improved the contribution of surface hydrophobicity, disulfide bonds and hydrophobic interactions to the gelation process.

The recombinant Lactococcus lactis oral vaccine induces protection against C. difficile spore challenge in a mouse model.

Clostridium difficile infection (CDI) causes nosocomial antibiotic-associated diarrhea and colitis in the developed world. Two potent cytotoxins, toxin A (TcdA) and toxin B (TcdB) are the virulence factors of this disease and can be a good vaccine candidate against CDI. In the present study, we genetically engineered Lactococcus lactis to express the nontoxic, recombinant fragments derived from TcdA and TcdB C-terminal receptor binding domains (Tcd-AC and Tcd-BC) as an oral vaccine candidate. The immunogenicity of the genetically engineered L. lactis oral vaccine delivery system (animal groups LAC and LBC or the combination of both, LACBC) was compared with the recombinant TcdA and TcdB C-terminal receptor binding domain proteins (animal groups PAC and PBC or the combination of both, PACBC), which were expressed and purified from E. coli. After the C. difficile challenge, the control groups received PBS or engineered L. lactis with empty vector, showed severe diarrhea symptoms and died within 2-3 days. However, both the oral vaccine and recombinant protein vaccine groups had significantly lower mortalities, body weight decreases and histopathologic lesions than the control sham-vaccine groups (p<0.05) except group LBC which only had a 31% survival rate after the challenge. The data of post infection survival showed that an average of 86% of animals survived in groups PAC and PACBC, 75% of animals survived in group LACBC, and 65% of animals survived in group LAC. All of the vaccinated animals produced higher titers of both IgG and IgA than the control groups (p<0.05), and the antibodies were able to neutralize the cytopathic effect of toxins in vitro. The results of this study indicate that there is a potential to use L. lactis as a delivery system to develop a cost effective oral vaccine against CDI.

Evaluation of a Salmonella vectored vaccine expressing Mycobacterium avium subsp. paratuberculosis antigens against challenge in a goat model.

Johnes disease (JD), caused by Mycobacterium avium subsp paratuberculosis (MAP), occurs worldwide as chronic granulomatous enteritis of domestic and wild ruminants. To develop a cost effective vaccine, in a previous study we constructed an attenuated Salmonella strain that expressed a fusion product made up of partial fragments of MAP antigens (Ag85A, Ag85B and SOD) that imparted protection against challenge in a mouse model. In the current study we evaluated the differential immune response and protective efficacy of the Sal-Ag vaccine against challenge in a goat model as compared to the live attenuated vaccine MAP316F. PBMCs from goats vaccinated with Sal-Ag and challenged with MAP generated significantly lower levels of IFN-γ, following in vitro stimulation with either Antigen-mix or PPD jhonin, than PBMC from MAP316F vaccinated animals. Flow cytometric analysis showed the increase in IFN-γ correlated with a significantly higher level of proliferation of CD4, CD8 and γδT cells and an increased expression of CD25 and CD45R0 in MAP316F vaccinated animals as compared to control animals. Evaluation of a range of cytokines involved in Th1, Th2, Treg, and Th17 immune responses by quantitative PCR showed low levels of expression of Th1 (IFN-γ, IL-2, IL-12) and proinflammatory cytokines (IL-6, IL-8, IL-18, TNF-α) in the Sal-Ag immunized group. Significant levels of Th2 and anti-inflammatory cytokines transcripts (IL-4, IL-10, IL-13, TGF-ß) were expressed but their level was low and with a pattern similar to the control group. Over all, Sal-Ag vaccine imparted partial protection that limited colonization in tissues of some animals upon challenge with wild type MAP but not to the level achieved with MAP316F. In conclusion, the data indicates that Sal-Ag vaccine induced only a low level of protective immunity that failed to limit the colonization of MAP in infected animals. Hence the Sal-Ag vaccine needs further refinement to increase its efficacy.

Evaluation of a Mycobacterium avium subsp. paratuberculosis leuD mutant as a vaccine candidate against challenge in a caprine model.

Johne's disease (JD) is prevalent worldwide and has a significant impact on the global agricultural economy. In the present study, we evaluated the protective efficacy of a leuD (Δleud) mutant and gained insight into differential immune responses after challenge with virulent M. avium subsp. paratuberculosis in a caprine colonization model. The immune response and protective efficacy were compared with those of the killed vaccine Mycopar. In vitro stimulation of peripheral blood mononuclear cells with johnin purified protein derivative showed that Mycopar and ΔleuD generated similar levels of gamma interferon (IFN-γ) but significantly higher levels than unvaccinated and challenged phosphate-buffered saline controls. However, only with ΔleuD was the IFN-γ response maintained. Flow cytometric analysis showed that the increase in IFN-γ correlated with proliferation and activation (increased expression of CD25) of CD4, CD8, and γδT cells, but this response was significantly higher in ΔleuD-vaccinated animals at some time points after challenge. Both Mycopar and ΔleuD vaccines upregulated Th1/proinflammatory and Th17 cytokines and downregulated Th2/anti-inflammatory and regulatory cytokines at similar levels at almost all time points. However, significantly higher levels of IFN-γ (at weeks 26 and 30), interleukin-2 (IL-2; week 18), IL-1b (weeks 14 and 22), IL-17 (weeks 18 and 22), and IL-23 (week 18) and a significantly lower level of IL-10 (weeks 14 and 18) and transforming growth factor ß (week 18) were detected in the ΔleuD-vaccinated group. Most importantly, ΔleuD elicited an immune response that significantly limited colonization of tissues compared to Mycopar upon challenge with wild-type M. avium subsp. paratuberculosis. In conclusion, the ΔleuD mutant is a promising vaccine candidate for development of a live attenuated vaccine for JD in ruminants.