Conjugation of chitopentaose with ß-lactoglobulin using Maillard reaction, and its effect on the allergic desensitization in vivo.

The conjugation of chitopentaose (CHP) on ß-lactoglobulin (ßLg) via Maillard reaction was used to desensitize ßLg. The stable ßLg-CHP conjugate (ßC-4) was formed at 4 h incubation, which contains 5 CHP attached molecules and a conjugated degree of 42 %. The conjugation promoted the thermal stability and emulsifying properties of ßLg, and inhibited the immunoglobulin E (IgE) combining capacity by decreasing the content of ß-sheet in ßLg. Moreover, ßLg-CHP conjugates were imparted with anti-oxidant properties and anti-inflammatory activities. Further, the combined action of inhibited IgE combining capacity and anti-inflammatory activities improved the allergy desensitization in ßLg sensitized mice. The results showed that overexpressed IgE and inflammatory factors, unbalanced Th1-/Th2- immune cytokines were significantly attenuated after ßLg was conjugated with CHP, avoiding the inflammatory lesions in spleen and colon. Additionally, the adverse changes in gut microbiota were alleviated in ßC-4 group with a decrease of Bacteroidetes and increase of Firmicutes at phylum level and the probiotic bacteria of Lactobacillaceae was significantly improved at the family level. Thus, the conjugation of CHP can desensitize allergic reaction caused by ßLg.

Granular architecture of lotus seed starch and its impact on physicochemical properties.

Lotus seed starch has high apparent amylose content (AAM). A representative definition of its granular architecture (e.g., lamellar structure) remained absent. This study defined the granular shape, crystalline and lamellar structures, and digestibility of twenty-two samples of lotus seed starch (LS) by comparing with those of potato and maize starches. LS granules had more elongated shape and longer repeat distance of lamellae than potato and maize starch granules. The enzymatic susceptibility of LS granules was more affected by AAM than granular architecture. Using these LSs as a model system, the relationships between lamellar structure of starch granules and properties of their gelatinized counterparts were investigated. In LSs, thinner amorphous lamella and thicker crystalline lamella were associated with higher swelling power and yield stress. The relationships were found to be connected via certain structural characteristics of amylopectin.

Gastrointestinal digestibility of micellar casein dispersions: Effects of caprine vs bovine origin, and partial colloidal calcium depletion using in vitro digestion models for the adults and elderly.

In vitro coagulation and digestion of caprine and bovine micellar casein concentrate (MCC) with or without partial colloidal calcium depletion (deCa) were studied under simulated adult and elderly conditions. Gastric clots were smaller and looser for caprine than bovine MCC, and were further looser with deCa and under elderly condition for both caprine and bovine MCC. Casein hydrolysis and concomitant formation of large peptides was faster for caprine than bovine MCC, and with deCa and under adult condition for caprine and bovine MCC. Formation of free amino groups and small peptides were faster for caprine MCC, and with deCa and under adult condition. Upon intestinal digestion, proteolysis occurred rapidly, and was faster under adult condition, but showed less differences with increasing digestion between caprine and bovine MCC, and with and without deCa. These results suggested weakened coagulation and greater digestibility for caprine MCC and MCC with deCa under both conditions.

Fabrication, characterization, and potential applications of re-assembled casein micelles.

Re-assembled casein micelles (rCMs), were formulated in the 1970s as a model system to understand native casein micelles (nCMs) in milk. These early works allowed an understanding of the critical factors involved in the formation of rCMs, such as minerals (citrate, phosphate, and calcium), casein type (αs-, ß-, and κ-casein) and the extent of their phosphorylation. rCMs were also used to understand the effect of treatments such as ethanol, high hydrostatic pressure and heating on the stability and integrity of the micelles. More recently, the applications of rCMs have been investigated, these include their use as a nanocarrier of bioactive molecules and as electrode-bound substrates to monitor chymosin activity by electrochemistry, to cite a few. Moreover, the potential to use rCMs in both food and non-food applications remains to be fully exploited. The advantage of choosing rCMs over nCMs as an encapsulant and a lucrative food ingredient is due to their more efficient preparation and being free from impurities. In this review, we report on the formulation of rCMs, their physico-chemical properties and their behavior under different physico-chemical treatments, along with the applications and challenges of rCMs in food systems and their industrial production as a dairy ingredient.

Physicochemical properties and molecular structure of lotus seed starch.

Current understanding of physicochemical properties of lotus seed starch (LS) is scarce partly due to its largely unknown molecular structure. This study compared the physicochemical and molecular characteristics of LSs of a wide collection to those of conventional starches (potato (PS) and maize starches (MS)). Variations were found in the chemical composition, physicochemical properties, and molecular structure of LSs. Amylose content and weight-based ratio of short to long chains of amylopectin (APS:APL) were principal factors affecting the physicochemical properties of LSs from different origins. Compared with PS and MS, LSs had higher gelatinization temperatures, lower amylose leaching, and faster retrogradation. These unique properties of LSs were related to their molecular structure and chemical composition. LSs had higher amylose contents than PS and MS as evaluated by various methods. A majority of amylose chains in LS were longer than those in MS but were shorter than those in PS. The APS:APL of LSs were higher than that of MS but lower than that of PS. The results provided a structural basis for understanding the properties of LS and suggested that this unconventional starch may be complementary to conventional starches for industrial applications.

Study of the in vitro properties of oligopeptides from whey protein isolate with high Fisher's ratio and their ability to prevent allergic response to ß-lactoglobulin in vivo.

High Fisher ratio oligopeptides (HFOPs) with molecular weight range from 100 to 800 Da derived from whey protein isolate (WPI) were used to prevent the allergic response induced by ß-lactoglobulin (ßLg) in vivo due to their anti-inflammatory activities to lipopolysaccharide (LPS) treated RAW 264.7 cells and anti-allergicproperties to anti-DNP mouse IgE sensitized RBL-2H3 cells in vitro. The results showed theoverexpressed immunoglobulin E (IgE), unbalanced Th1-/Th2-type immune cytokines and inflammatory factors in ßLg-allergic mice were significantly attenuated by oral administration of HFOPs, resulting in the prevention of inflammatory lesions in spleen and colonic histopathology. Moreover, HFOPs increased ratio of Bacteroidetes/Firmicutes at phylum level in sensitive mice, and improved the abundance of Lactobacillaceae at family level to maintain oral tolerance against ßLg and prevented allergic response. The use of HFOPs may provide a potential alternative for preventing the milk allergy induced by WPI.

Impacts of sonication and high hydrostatic pressure on the structural and physicochemical properties of quinoa protein isolate dispersions at acidic, neutral and alkaline pHs.

Herein, 1 wt% quinoa protein isolate (QPI) was exposed to sonication using a 20 kHz ultrasonicator equipped with a 6 mm horn (14.4 W, 10 mL, up to 15 min) or high hydrostatic pressure (HHP, up to 600 MPa, 15 min) treatments at pH 5, pH 7, and pH 9. The changes to physicochemical properties were probed by SDS-PAGE, FTIR, free sulfhydryl group (SH), surface hydrophobicity (H0), particle size and solubility. As revealed by SDS-PAGE, substantial amounts of 11S globulin participated in the formations of aggregates via SS bond under HHP, particularly at pH 7 and pH 9. However, protein profiles of QPI were not significantly affected by the sonication. Free SH groups and surface hydrophobicity were increased after the sonication treatment indicating protein unfolding and exposure of the embedded SH and/or hydrophobic groups. An opposite trend was observed in HHP treated samples, implying aggregation and reassociation of structures under HHP. HHP and sonication treatments induced a decrease in ordered secondary structures (random coil and ß-turn) accompanied with an increase in disordered secondary structures (α-helix and ß-sheet) as probed by FTIR. Finally, the sonication treatment induced a significant improvement in the solubility (up to ∼3 folds at pH 7 and ∼2.6 folds at pH 9) and a reduction in particle sizes (up to ∼3 folds at pH 7 and ∼4.4 folds at pH 9). However, HHP treatment (600 MPa) only slightly increased the solubility (∼1.6 folds at pH 7 and ∼1.2 folds at pH 9) and decreased the particle size (∼1.3 folds at pH 7 and ∼1.2 folds at pH 9). This study provides a direct comparison of the impacts of sonication and HHP treatment on QPI, which will enable to choose the appropriate processing methods to achieve tailored properties of QPI.

Supramolecular structure of quinoa starch affected by nonenyl succinic anhydride (NSA) substitution.

Quinoa starch granular structure as affected by nonenyl succinic anhydride (NSA) substitution was investigated by multiple approaches, including scattering, spectroscopic, and microscopic techniques. The modification had little impact on the morphology of starch granules. The NSA substitution was found mainly in the amorphous lamellae and amorphous growth rings. The NSA modification increased the thickness of the amorphous lamellae. The homogeneity of the ordered structure in the granules was improved, probably because the NSA modification reduced the amount of defects in the semi-crystalline growth ring. Compared to other chemical modifications such as acylation, succinylation was more effective in modifying the starch lamellar structure. A possible reaction pattern of NSA modification on quinoa starch is proposed, in which the NSA modification may follow the sequence of amorphous growth rings, the amorphous matrices among blocklets, amorphous and crystalline lamellae in semi-crystalline growth rings. This study provides new insights on the structural changes of starch granules induced by succinylation on the supramolecular level.

Modification of the interfacial structure of droplet-stabilised emulsions during in vitro dynamic gastric digestion: Impact on in vitro intestinal lipid digestion.

The in-vitro gastrointestinal digestion behaviour of an oil-in-water emulsion with an interface consisting of nano-sized droplets coated with caseinate particles, referred to as a droplet-stabilised emulsion (DSE), was explored using the human gastric simulator and pH-stat models. A caseinate-particle-stabilised emulsion (PSE) was used as a control, with a similar droplet size distribution and the same composition as the DSE. The nanodroplet-stabilised interface of the DSE was preserved during the first 180 min of gastric digestion. During 240 min, the droplet sizes of the DSE and the PSE increased from 22.71 ± 1.14 to 63.34 ± 6.57 µm and from 17.98 ± 1.16 to 85.11 ± 9.35 µm respectively. The small droplet size of the DSE that was released from the gastric phase contributed to slightly higher total free fatty acid (FFA) release (56.18 ± 3.55%) than that from the PSE (49.4 ± 2.67%). The FFA release rate of the DSE (1.21 % min-1) was greater than that of the PSE (1.06 % min-1) during the first 30 min of small intestinal digestion; similar FFA release rates (0.5 µmol s-1 m-2 × 10-4) were obtained for both emulsions beyond 30 min of digestion. This study provides new information on lipid digestion using a novel interfacial layer that was stabilised with nanodroplets.

Formation by high power ultrasound of aggregated emulsions stabilised with milk protein concentrate (MPC70).

In this work, oil-in-water emulsions stabilised by milk protein concentrate (MPC70) were investigated. The MPC70 concentration was kept constant at 5% (close to the protein content found in skim milk) and the oil volume fraction was varied from 20 to 65%. Sonication was performed at 20 kHz and at a constant power of 14.4 W for a total emulsion volume of 10 mL. Under certain oil concentration (≥35%) and sonication times (≥3s) the emulsion aggregated and formed high-viscosity pseudo plastic materials. However, the viscosity behaviour of the emulsion made with 35% oil reverted to that of a liquid if sonicated for longer times (≥15 s). Confocal laser scanning microscopy showed clearly that the oil droplets are aggregated under the sonication conditions and oil concentrations indicated above. An attempt to explain this behaviour through a simple model based on the bridging of oil droplets by the MPC70 particles and, taking into account the oil droplet and MPC70 particle sizes as well as the oil volume fraction, was made. The model fails to describe in details the aggregation behaviour of these emulsions, likely due to the inhomogeneous protein layer, where both free caseins and casein micelles are adsorbed, and to the packing of the oil droplets at concentrations ≤55%. Nonetheless, this work demonstrates the potential of ultrasound processing for the formation of dairy emulsions with tailored textures.

Unveiling the structure of the primary caseinate particle using small-angle X-ray scattering and simulation methodologies.

The low-resolution structure of casein (CN) clusters in sodium caseinate (NaCas) solution and its conformational dynamics were obtained by size-exclusion chromatography (SEC), analytical ultracentrifugation (AUC), small-angle X-ray scattering (SAXS), and molecular dynamics (MD) simulations. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and native PAGE revealed that the casein clusters consisted predominantly of α- and ß-CN complexes, and a trace amount of κ-CN. The AUC analysis indicated that the casein clusters were composed of 34.6% of casein monomers, 19.2%, 20.4%, and 25.8% of complexes with molar weight (Mw) of ~50.3, ~70.6, and ~133 kDa, respectively. The volume fractions of components in casein clusters were quantified as 64.3% of αs1-ß-αs2-CN, 22.3% of αs1-CN, 8.5% of αs2-CN, and 4.4% of αs1-αs2-CN, respectively. The ensemble optimization method (EOM) gave a fitting result where αs1-ß-αs2-CN species coexisted in ~35.3% under compact conformation and ~64.7% in elongated conformation in solution. The three-dimensional structures of αs1-ß-αs2-CN from EOM showed a good overlay on the casein clusters ab initio model obtained from DAMMIN and DAMMIX program. MD simulations revealed that αs1-ß-αs2-CN underwent a conformational change from the elongated state into the compact state within the initial 200 ns of simulations. The addition of nonionic surfactants affected little the backbone-to-backbone interactions in the formation of the casein clusters. We propose that αs1-CN, ß-CN, αs2-CN, and κ-CN associated in consecutive steps into casein clusters, and a trace of κ-CN may be located at the surface of the assemblies limiting the growth of casein aggregates.

Probing the conjugation of epigallocatechin gallate with ß-lactoglobulin and its in vivo desensitization efficiency.

Epigallocatechin gallate (EGCG) and ß-lactoglobulin (ßLg) were conjugated by covalent bonds to form EGCG-ßLg conjugates. This conjugation causes structural and bioactivity changes in ßLg, which in turn can be used as a possible approach for desensitization to allergens. In this study, the desensitization mechanism was investigated by monitoring ßLg secondary structure and immunoglobulin E (IgE) combining capacity changes on the basis of the conjugation mechanism. Furthermore, the desensitization efficiency in vivo was evaluated through animal experiments. The results show that temperature influenced the conjugation by decreasing the binding affinities (Ka) and binding numbers (n) of EGCG. The conjugation of EGCG decreased ßLg's IgE combining capacity by decreasing the ß-sheet component and imparted antioxidant properties by the introduction of hydroxyl groups. In addition, animal experiment results indicated that ßLg induced significant changes in the levels of IgE and inflammatory cytokines, and the relative abundance of small intestinal flora, linked to the inflammatory lesions and anaphylaxis symptoms. EGCG-ßLg conjugates can suppress the allergic response, attenuating serum IgE and relieving the anaphylaxis symptoms.

Covalently Immobilized Battacin Lipopeptide Gels with Activity against Bacterial Biofilms.

Novel antibiotic treatments are in increasing demand to tackle life-threatening infections from bacterial pathogens. In this study, we report the use of a potent battacin lipopeptide as an antimicrobial gel to inhibit planktonic and mature biofilms of S. aureus and P. aeruginosa. The antimicrobial gels were made by covalently linking the N-terminal cysteine containing lipopeptide (GZ3.163) onto the polyethylene glycol polymer matrix and initiating gelation using thiol-ene click chemistry. The gels were prepared both in methanol and in water and were characterised using rheology, Fourier transform infrared (FT-IR) spectroscopy and scanning electron microscopy (SEM). Antibacterial and antibiofilm analyses revealed that the gels prepared in methanol have better antibacterial and antibiofilm activity. Additionally, a minimum peptide content of 0.5 wt% (relative to polymer content) is required to successfully inhibit the planktonic bacterial growth and disperse mature biofilms of P. aeruginosa and S. aureus. The antibacterial activity of these lipopeptide gels is mediated by a contact kill mechanism of action. The gels are non-haemolytic against mouse red blood cells and are non-cytotoxic against human dermal fibroblasts. Findings from this study show that battacin lipopeptide gels have the potential to be developed as novel topical antibacterial agents to combat skin infections, particularly caused by S. aureus.

Extraction of tetracycline in food samples using biochar microspheres prepared by a Pickering emulsion method.

We report for the first time the use of biochar as a stabilizer for oil-in-water (o/w) Pickering emulsion. The emulsion is subsequently used to prepare tetracycline-imprinted biochar composite microspheres (MIPMs) with tailored sizes and good uniformity. The adsorption properties of tetracycline to the MIPMs were investigated using different adsorption experiments including adsorption kinetic experiment, equilibrium binding experiment, selectivity evaluation and competitive adsorption tests. The MIPMs were used as adsorbent for solid phase extraction (SPE) for the extraction of tetracycline present in drinking water, fish, and chicken samples. Under optimal conditions, the results showed good recovery yield ranging from ~73% to ~95% with a relative standard deviation (RSD) ranging from ~0.3% to ~8.4%, respectively.

Effect of sonication, microwaves and high-pressure processing on ACE-inhibitory activity and antioxidant potential of Cheddar cheese during ripening.

Dairy processing provides acceptable safety and shelf-life to final products, and improves their bioactivity. The present study evaluated the potential of different milk processing techniques to improve the antioxidant and angiotensin-I converting enzyme (ACE)-inhibitory activity of Cheddar cheese, during ripening. Cheese was made from milk subjected to different pre-treatments (C = untreated control, US-1 = ultrasonication, specific energy = 23 J/g, 20 kHz frequency; US-2 = Ultrasonication specific energy = 41 J/g, 20 kHz; HPP = high-pressure processing, 400 MPa for 15 min, at temperature < 40 °C; MW = microwave, temperature<40 °C, specific energy = 86.5 J/g) and analysed after ripening for 0, 3, 6 and 9 months. The results showed that the rate of proteolysis during both cheese making and subsequent ripening was significantly affected by the pre-treatment. Antioxidant activity and ACE-inhibitory potential of cheeses made from pre-treated milk significantly increased (p < 0.05) in the following order: US-2 > HPP > US-1 > MW > C. These findings demonstrate the possibility of using ultrasound, microwaves or high-pressure processing as pre-treatments to improve the nutritional attributes of cheese.

Solubilisation of micellar casein powders by high-power ultrasound.

High protein milk ingredients, such as micellar casein powder (MCP), exhibit poor solubility upon reconstitution in water, particularly after long-time storage. In this study, ultrasonication (20 kHz, power density of 0.75 W/ml) was used to improve the solubility of aged MCP powders. For all the MCP powders (concentration varying from 0.5 to 5%, and storage of MCP at 50 °C for up to 10 days) it was found that short time ultrasonication (2.5 min) reduced the size of the protein particles from >30 µm to ∼0.1 µm, as measured by light scattering. This resulted in an improvement of solubility (>95%) for all the MCP powders. Cryo-electron microscopy and small x-ray angle scattering showed that the MCP powders dissolved into particles with morphologies and internal structure similar to native casein micelles in bovine milk. SDS-PAGE and RP-HLPC showed that ultrasonication did not affect the molecular weight of the individual casein molecules. Compared to overhead stirring using a 4-blade stirrer, ultrasonication required less than 10 times the drawn electrical energy density to achieve a particle size 10 times smaller.

Size reduction of "reformed casein micelles" by high-power ultrasound and high hydrostatic pressure.

We investigated the effect of ultrasound (US) and high hydrostatic pressure (HHP) on the size of reformed casein micelles (RMCs) obtained by titrating calcium and phosphorous solution into sodium caseinate solutions. Both US and HHP reduced the size of the RMCs. A decrease in size from ~200 nm to ~170 nm when US (20 kHz, 0.46 W/mL) was applied for 30 min; and down to ~85 nm when HHP was applied 500 MPa for 15 min. Electron microscopic analysis showed that the RMCs before and after US are similar to milk native casein micelles, and that HHP extensively disintegrated the RMCs. Small angle X-ray scattering and SDS-PAGE showed that the internal structure of the RMCs as well as the casein molecules are not affected by the US and HHP treatments.

Improvement of the rheological and textural properties of calcium sulfate-induced soy protein isolate gels by the incorporation of different polysaccharides.

In this study, the effects of the addition of various polysaccharides (konjac gum, gellan gum, and curdlan gum) on the rheological and textural properties of calcium sulfate-induced soy protein isolate gels were investigated. The incorporation of konjac gum and curdlan gum at 0.3 and 0.5% (w/v) concentrations and gellan gum at 0.5% concentration significantly enhanced (P < 0.05) the hardness and water-holding capacity of the resultant gels. The increased elastic moduli during and after gelation, reinforced fracture stress, and lowered onset gelling temperature indicated that the addition of the abovementioned polysaccharides strengthened gel structures and accelerated gelation. Confocal laser scanning microscopy analysis revealed that the polysaccharides also improved gel microstructures, with the gels containing konjac gum displaying the highest homogeneity. The findings of this study may provide important information for the development of innovative soy protein isolate-based gel products with improved texture.

Glycerol induced stability enhancement and conformational changes of ß-lactoglobulin.

The protective mechanism of glycerol on ß-lactoglobulin were studied in 0-60% glycerol solutions by experimental and molecular simulation approaches. Results showed that the stability of ß-lactoglobulin increased with glycerol concentration, with little secondary structure changes induced by glycerol. The tertiary structure altered slightly with glycerol concentration, resulting in a stronger near UV circular dichroism signal and intrinsic tryptophan fluorescence quenching, indicating aromatic side chains closer to hydrophobic microenvironment. The Rg of ß-lactoglobulin increased with glycerol concentration without dimer dissociation, due to expansion of the quaternary structures. Moreover, the flexibility (RMSF) of ß-lactoglobulin decreased by glycerol. Distance between areas enclosing Asp33 and Arg40 from separate chains did not increase, suggesting possibly reinforced electrostatic attractions. In conclusion, the stabilization of ß-lactoglobulin in glycerol solution is probably the comprehensive results of the decreased molecular flexibility, the strengthened hydrophobic interaction in individual chain, and the possibly reinforced electrostatic attractions between two chains of ß-lactoglobulin.

Molecular characterization of the ß-lactoglobulin conjugated with fluorescein isothiocyanate: Binding sites and structure changes as function of pH.

Protein conjugated with dyes is a method which can be used for analyzing food components. For example ß-lactoglobulin (ßlg) can be conjugated with amine-reactive dyes to form ßlg-dye conjugates. In this study, the effect of pH on the conjugation of ßlg with fluorescein isothiocyanate (FITC) was investigated using MALDI-TOF MS, LC-MS, dynamic light scattering (DLS) and fourier transform infrared spectroscopy (FTIR). The results showed that the binding numbers increased with the increase in pH, which leading to a greater change in the zeta-potential and the secondary structure of ßlg after dye conjugation. In particular, the degree of labelling (DOL) was 94.9 ±â€¯7.9%, and the conjugation was mono-labelled at pH 8, indicating no significant changes in the physicochemical properties of ßlg. Furthermore, LC-MS revealed that the most probable conjugated lysine is located at position 100, 47 and 77 of ßlg.