Complexation of bovine lactoferrin with selected phenolic acids via non-covalent interactions: binding mechanism and altered functionality.

Non-covalent interactions of 4 selected phenolic acids, including gallic acid (GA), caffeic acid (CA), chlorogenic acid (CGA), and rosmarinic acid (RA) with lactoferrin (LF) were investigated. Compound combined with LF in the binding constant of CA > GA > RA > CGA, driven by van der Waals (vdW) and hydrogen bonding (H-bonds) for GA, and hydrophobic forces for others. Conformation of LF was impacted at secondary and ternary structure levels. Molecular docking indicated that GA and CA located in the same site near the iron of the C-lobe, while RA and CGA bound to the C2 and N-lobe, respectively. Significantly enhanced antioxidant activity of complexes was found compared with pure LF (P < 0.05), as demonstrated by DPPH, ABTS, FRAP models. CA, CGA, and RA significantly decreased the ESI and improved foam ability of LF (P < 0.05), and the effect of CA and RA was the most remarkable, respectively.

Structure and functionality of whey protein, pea protein, and mixed whey and pea proteins treated by pH shift or high-intensity ultrasound.

Three modifications (pH shift, ultrasound, combined pH shift and ultrasound) induced alterations in pure whey protein isolate (WPI), pea protein isolate (PPI), and mixed whey and pea protein (WPI-PPI) were investigated. The processing effect was related to the protein type and technique used. Solubility of WPI remained unchanged by various treatments. Particle size was enlarged by pH shift while reduced by ultrasound and combined approach. All methods exposed more surface hydrophobic groups on WPI, while pH shift and joint processing was detrimental to its emulsifying activity. The PPI and mixture exhibited similar responses toward the modifications. Solubility of PPI and the blend enhanced in the sequence of pH shift and ultrasound > ultrasound > pH shift. Individual approach expanded while co-handling diminished the particle diameter. Treatments also caused more disclosure of hydrophobic regions in PPI and WPI-PPI and emulsifying activity was ameliorated in the order of pH shift and ultrasound > ultrasound > pH shift.

Mixed whey and pea protein based cold-set emulsion gels induced by calcium chloride: Fabrication and characterization.

The cold-set gels of oil-in-water emulsions stabilized by mixtures of whey protein isolate (WPI) and pea protein isolate (PPI) with mass ratios of 10:0, 7:3, 5:5, 3:7, and 0:10 were investigated to evaluate the possibility of pea protein to replace milk protein. Particle size and surface charge of emulsions increased and decreased with raised PPI content, respectively. The redness and yellowness of emulsion gels were strengthened with elevated pea protein percentage and independent of calcium concentration applied. Considerable differences in water holding capacity were observed between samples with different mixed proteins and high percentage of pea protein gave better water retaining ability. Gradual decreases in hardness and chewiness of emulsion gels were observed at three calcium levels with the increased PPI proportion. FT-IR spectra indicated no new covalent bonds were generated between samples with different whey and pea protein mass ratios. As PPI concentration elevated, the network structure of emulsion gels gradually became loose and disordered. The established cold-set calcium-induced whey/pea protein composite gels may have the potential to be utilized as a new material to encapsulate and deliver environment sensitive bio-active substances.

Insights from 4D Label-Free Proteomic Analysis into Variation of Milk Fat Globule Membrane Proteins of Human Milk Associated with Infant's Gender.

Milk fat globule membrane (MFGM) protein profiles of breast milk collected from women in northeast China with male or female babies were investigated using a four-dimensional (4D) label-free proteomic technique. Altogether, 2538 proteins were detected and quantified and 249 were differentially expressed, with 198 decreased proteins compared to the samples of mothers with female babies. Different proteins associated with infant's gender were principally located in nuclear. The differentially expressed proteins were mainly involved in gene ontology (GO) functions of the cellular process, binding, and cell and found to be distributed in lipid-related biological processes and molecular functions to a large extent. The pathway of neurodegeneration-multiple disease ranked top for the altered proteins. The screened proteins were observed to contain some proteins related to typical functions of immunity, lipid metabolism, digestion, and growth and development. 114 proteins formed a relatively compact network (269 interactions) and dolichyl-diphospho-oligosaccharide-protein glycosyltransferase subunit 2 interacted the most with other proteins as the hub protein. MFGM proteins of breast milk were affected by the sex of offspring, and these findings may provide useful information for reasonable adjustments of infant formula powder specifically for boys or girls in the market.

Deciphering the difference of casein fraction in human milk associated with infant gender using quantitative proteomics.

Human milk is an ideal natural food for infants, and the infant's gender may have impact on protein composition of breast milk. In this study, we used 4D label-free quantitative proteomics techniques to identify and quantitatively analyze casein fraction in breast milk secreted for male and female infants. The results showed that a total of 2064 proteins were identified in human milk, and 95 of them were differentially abundant proteins. Compared to breast milk secreted by mothers of female infants, 21 proteins were up-regulated, and 59 proteins were down-regulated in breast milk secreted by mothers of male infants. The most abundant domain among the differentially abundant proteins was the immunoglobulin V-set domain, which may be involved in immune regulation. Gene Ontology functional analysis revealed that, the main biological processes, molecular functions, and cellular components corresponded to cellular process, binding, and cell part, respectively. The Kyoto Encyclopedia of Genes and Genomes pathways were mainly associated with human diseases and metabolism, with biosynthesis of cofactors being the most involved pathway. The results contribute to our understanding of the composition of casein in breast milk, and may provide information about the nutritional differences in breast milk from mothers of newborns of different genders.

Understanding molecular interaction between thermally modified ß-lactoglobulin and curcumin by multi-spectroscopic techniques and molecular dynamics simulation.

This study elucidated the binding of curcumin (CUR) onto preliminary thermally modified ß-lactoglobulin (ß-LG). ß-LG at pH 8.1 was heated at 75 °C, 80 °C and 85 °C for 10 min to construct denatured proteins (ß-LG75, ß-LG80, ß-LG85). Steady and time-resolved fluorescence studies uncovered that CUR quenched proteins in simultaneous static and dynamic mode. Pre-heating ß-LG improved its binding with CUR and the strongest affinity occurred in ß-LG80. Fluorescence resonance energy transfer (FRET) analysis indicated that binding distance between CUR and ß-LG80 was the smallest and energy transfer was the most efficient. ß-LG80 had the highest surface hydrophobicity. Fourier-transform infrared (FT-IR) spectroscopy and differential scanning calorimeter (DSC) confirmed that CUR transferred from crystal to amorphous state after association with protein and revealed the contribution of hydrogen bonds. Combination of ß-LG80 with CUR retained the antioxidant capacity of each component. Molecular dynamics simulation demonstrated enhanced hydrophobic solvent accessible surface area of ß-LG80 compared with native protein. Data obtained from this study may provide useful information for comprehensively understanding the ability of ß-lactoglobulin to bind hydrophobic substances under different environmental conditions like high temperature and alkaline medium.

Proteomic characterization and comparison of milk fat globule membrane proteins of Saanen goat milk from 3 habitats in China using SWATH-MS technique.

Saanen goats are among the major dairy goats in China. In present study, variation of milk fat globule membrane proteins profile of Saanen goat milk caused by geographic location was investigated using sequential window acquisition of all theoretical fragment ions data-independent acquisition mass spectrometry based proteomic approach. A total of 1,001 proteins were quantified in goat milk collected from 3 habitats of China [Guangdong (GD); Inner Mongolia (IM); Shannxi (SX)]. Most of the proteins were found to act cellular process of biological process, cell of cellular component, binding of molecular function after Gene Ontology annotation and metabolic of pathway indicated by Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Differentially expressed proteins (DEP) for GD versus IM, GD versus SX, IM versus SX were identified to be 81, 91, and 44, respectively. Gene Ontology enrichment analysis showed that the greatest DEP for 3 groups (GD vs. IM, GD vs. SX, IM vs. SX) were cellular process, cellular process and organonitrogen compound biosynthetic process/immune system process for biological process. For cellular component, the largest number of DEP for 3 comparison groups were organelle, organelle and organelle/intracellular. For molecular function, DEP of the 3 comparison groups were expressed most in structural molecule activity, binding and anion binding, respectively. Pathways with the majority of DEP were ribosome, systemic lupus erythematosus and primary immunodeficiency/systemic lupus erythematosus/amoebiasis/PI3K-Akt signaling pathway for GD versus IM, GD versus SX and IM versus SX, severally. Protein-protein interaction network analysis showed that DEP interacted most were 40S ribosomal protein S5, fibronectin and Cytochrome b-c1 complex subunit 2, mitochondrial for GD versus IM, GD versus SX and IM versus SX, separately. Data may give useful information for goat milk selection and milk authenticity in China.

Lycopene-loaded emulsions stabilized by whey protein covalently modified with pectin or/and chlorogenic acid: Enhanced physicochemical stability and reduced bio-accessibility.

Lycopene-loaded emulsions were formulated with whey protein isolate (WPI) covalently modified with high methoxylated pectin (HMP) or/and chlorogenic acid (CA) prepared by dry heating or/and alkali grafting. Covalent WPI products were confirmed by SDS-PAGE and degree of graft/CA binding equivalent values. The α-helix and ß-sheet percentage, surface hydrophobicity and fluorescence intensity of WPI decreased significantly (p < 0.05) upon binding. Both binary and ternary complexes enhanced the stability of the emulsions, and lycopene retained more after UV irradiation, thermal treatment, storage, compared with emulsions stabilized by WPI, with the best protection by both ternary complexes. In vitro simulated digestion results showed that free fatty acids were released in the order of WPI > WPI-HMP > WPI-CA > WPI-HMP-CA ≈ WPI-CA-HMP. Bio-accessibility analysis showed the same trend as the fatty acid release rate. These results may provide a theoretical basis for applications of conjugating protein with polysaccharide or/and polyphenol emulsions.

Comparative proteomics of human milk casein fraction collected from women of Korean and Han ethnic groups in China.

Introduction: Human breast milk provides neonates with indispensable nutrition and function. Milk protein is one of the main constituents of breast milk. Human milk profiles can be influenced by many factors. Methods: The present study aimed to investigate the difference in casein isolated from mature milk of healthy mothers of Korean and Han ethnic groups in China using data-independent acquisition (DIA) proteomics. Results: A total of 535 proteins were identified and quantified in casein fraction samples from both groups. A total of 528 proteins were annotated to 52 Gene Ontology (GO) terms, the majority (94.13%) of which were distributed in the cell and cell parts of the cellular component. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that 106 proteins were involved in 23 pathways, the greatest (36.79%) in carbohydrate metabolism. There were 39 differentially expressed proteins (DEPs)-10 upregulated and 29 downregulated-between Korean and Han milk. The GO function of blood microparticles and KEGG pathway of Staphylococcus aureus infection for DEPs were the most significantly enriched (p < 0.05). Protein-protein interaction analysis revealed a network with 23 DEPs in 47 interactions, and the fibrinogen alpha chain ranked first as the hub protein. Discussion: These data may provide useful technical guidance for the development of specific infant foods for certain populations.

Comparative proteomics analyses of whey proteins from breastmilk collected from two ethnic groups in northeast China.

The current study aims to investigate differences in whey protein of breastmilk of volunteered mother collected from two ethnic groups (Korean and Han) in China using data-independent acquisition (DIA) based proteomics technique. The total detected 624 proteins were principally allocated to cellular process of biological process (BP), cell and cell part of cell component (CC) and binding of molecular function (MF) according to Gene Ontology (GO) annotation; and carbohydrate metabolism of Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Among the 54 differently expressed proteins, 8 were related with immunity. Enrichment data showed that intracellular of GO functions and viral myocarditis of KEGG pathways were most significantly enriched (p < 0.05). Protein-protein interaction (PPI) network suggested that 40S ribosomal protein S27a and 60S ribosomal protein L10a which interacted most with other proteins ranked the top two hub proteins by MCC (Maximal Clique Centrality) method. This study may have guiding role for development of infant formula powder for specific infants of Han or Korean groups according to responding breastmilk composition.

Formation, stability and in vitro digestion of curcumin loaded whey protein/ hyaluronic acid nanoparticles: Ethanol desolvation vs. pH-shifting method.

Curcumin (CUR) was encapsulated in whey protein isolate/hyaluronic acid (WPI/HA) electrostatic nanoparticles at pH 5.4, 4.4, 3.4 and 2.4 using ethanol desolvation (DNP) or pH-shifting (PSNP) method. The prepared nanoparticles were characterized and compared for physiochemical properties, structure, stability, and in vitro digestion. PSNPs had smaller particle size, more uniform distribution, and higher encapsulation efficiency than DNPs. Main driving forces involved for fabricating the nanoparticles were electrostatic forces, hydrophobic forces, and hydrogen bonds. PSNP exhibited better resistance towards salt, thermal treatment, and long-term storage while DNPs showed stronger protection for CUR against thermal degradation and photodegradation. Stability of nanoparticles increased with decreasing pH values. In vitro simulated digestion exhibited that DNPs had lower release rate of CUR in SGF and higher antioxidant activity of its digestion products. Data may provide a comprehensive reference for selection of loading approach when constructing nanoparticles based on proteins/polysaccharides electrostatic complexes.

Digestion behavior, in vitro and in vivo bioavailability of cannabidiol in emulsions stabilized by whey protein-maltodextrin conjugate: Impact of carrier oil.

An emulsion delivery system may be affected significantly by oil phase composition in terms of digestion behavior and bioavailability of the delivered substance. In this study, emulsions loaded with cannabidiol (CBD) were prepared with medium chain triglyceride (MCT), long chain triglyceride (LCT) or MCT/LCT(1:1) as carrier oil and whey protein-maltodextrin conjugate as emulsifier, and the digestion behavior of emulsion and bioavailability of CBD were assessed in vitro and in vivo. The particle size of emulsions throughout the in vitro digestion process was in the order of MCT < MCT/LCT < LCT, and three emulsions showed consistent particle size changes: stable in oral phase, sharply increased in gastric phase, and decreased in small intestine. After intestinal digestion, about 90% of free fatty acids (FFA) was released in MCT emulsion, followed by MCT/LCT (76%) and then LCT (45%). CBD was degraded during gastrointestinal digestion and the transformation stability of CBD in oil phase was in the order of LCT > MCT/LCT > MCT. Although CBD had higher bioaccessibility in MCT and MCT/LCT emulsions, the bioavailability of CBD in LCT was the highest (43%), followed by MCT/LCT (39%), MCT (33%). In vivo pharmacokinetic study showed that MCT/LCT and LCT were more favorable for CBD transport and absorption. The results may provide useful information for the construction of delivery systems, protecting CBD molecules, and improving their bioavailability.

Towards understanding the interaction between ultrasound-pretreated ß-lactoglobulin monomer with resveratrol.

Increasingly, studies are using ultrasound to elevate the functional properties of proteins, so the interaction between phenolic compounds and proteins induced by ultrasound needs to be further understood. ß-Lactoglobulin (ß-LG) at pH 8.1, which exists mainly as monomers, was ultrasound treated at 20 kHz ultrasonic intensity and 30% amplitude for 0-5 min and subsequently interacted with resveratrol. Fluorescence data showed that ultrasound pretreatment improved binding constant (Ka ) from (1.62 ± 0.45) × 105 to (9.43 ± 0.55) × 105 M-1 and binding number from 1.13 ± 0.09 to 1.28 ± 0.11 in a static quenching mode. Fluorescence resonance energy transfer (FRET) analysis indicated that resveratrol bound to the surface hydrophobic pocket of native and treated proteins with no obvious changes in energy transfer efficiency (E) and Föster's distance (r). Thermodynamic parameters indicated that ultrasonication shifted the main driving force from the hydrophobic force for native and 1-min treated ß-LG to van der Waals forces and hydrogen bonding for both 3-min and 5-min treated proteins. Ultrasonication and resveratrol addition generated significant differences in surface hydrophobicity and the surface charge of the protein (P < 0.05), whereas they had little influence on the secondary structure of ß-LG. Compared with the native ß-LG/resveratrol complex, ultrasound-treated protein complexes showed significantly stronger 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) scavenging capacity (P < 0.05), and kept relatively stable after 180-min irradiation. Data provided by this study can lead to a better comprehension of the structure and molecular events occurring during the complexing process between an ultrasound-pretreated protein with polyphenol.

Pea protein based nanocarriers for lipophilic polyphenols: Spectroscopic analysis, characterization, chemical stability, antioxidant and molecular docking.

The current research aims to construct and assess pea protein isolate (PPI) nanocarriers for lipophilic polyphenols of curcumin (CUR), quercetin (QUE) and resveratrol (RES), respectively. Fluorescence analysis demonstrated that the binding affinity declined in sequence of QUE > CUR > RES and about one polyphenol compound was bound to protein. Thermodynamic parameters revealed that hydrophobic interaction was mainly responsible for complexation between CUR/RES and PPI, while hydrogen bonding for QUE with PPI. All nanoparticles showed particle size of 154-159 nm. Three lipophilic polyphenols were successfully encapsulated into PPI, with loading capacity of RES > QUE > CUR. Complexation of three polyphenols did not change the secondary structure of PPI. Results of FTIR, DSC and XRD confirmed that polyphenols changed from crystalline to amorphous state after combination with PPI. SEM pictures exhibited regular spherical microstructure of nanocomplexes. PPI shielded polyphenols from sensitive environment of ultraviolet light and thermal treatment. ABTS and DPPH radical scavenging activity of polyphenols were considerably improved through complexation with PPI. Molecular docking studies showed binding energy with 11S legumin in sequence of QUE > RES > CUR, and stronger hydrogen bonds were built between QUE and the protein than the other two polyphenols. Data in the present work may provide helpful information for encapsulation of lipophilic polyphenols with pea protein and the potential application in food science, pharmaceutical and cosmetics industries in the future.

Fabrication and characterization of a cannabidiol-loaded emulsion stabilized by a whey protein-maltodextrin conjugate and rosmarinic acid complex.

A cannabidiol (CBD)-loaded oil-in-water emulsion stabilized by a whey protein (WP)-maltodextrin (MD) conjugate and rosmarinic acid (RA) complex was fabricated, and its stability characteristics were investigated under various environmental conditions. The WP-MD conjugates were formed via dry-heating. The interaction between WP and MD was assessed by browning intensity, reduced amount of free amino groups, the formation of high molecular weight components in sodium dodecyl sulfate-PAGE, and changes in secondary structure of whey proteins. The WP-MD-RA noncovalent complex was prepared and confirmed by fluorescence quenching and Fourier-transform infrared spectroscopy spectra. Emulsions stabilized by WP, WP-MD, and WP-RA were used as references to evaluate the effect of WP-MD-RA as a novel emulsifier. Results showed that WP-MD-RA was an effective emulsifier to produce fine droplets for a CBD-loaded emulsion and remarkably improved the pH and salt stabilities of emulsions in comparison with WP. An emulsion prepared with WP-MD-RA showed the highest protection of CBD against UV and heat-induced degradation among all emulsions. The ternary complex kept emulsions in small particle size during storage at 4°C. Data from the current study may offer useful information for designing emulsion-based delivery systems which can protect active substance against environmental stresses.

Comparative analysis of caseins in Saanen goat milk from 3 different regions of China using quantitative proteomics.

A quantitative proteomic technique based on data-independent acquisition (DIA) was used to analyze differentially expressed caseins of Saanen goat milk samples collected from 3 regions in China (Guangdong, GD; Inner Mongolia, IM; Shaanxi, SX). A total of 345 proteins were quantified in each sample. Gene Ontology (GO) analysis showed that proteins were mainly involved in cellular process and cell and binding functions. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that proteins were mainly involved in metabolic pathways. Differentially expressed proteins (DEP) between goat milk from 3 comparison groups composed of paired regions were compared and analyzed. The number of DEP was 114, 69, and 79 for GD versus IM, GD versus SX, and IM versus SX, respectively. The GO enrichment analysis of the 3 comparison groups showed that differences were mainly related to the regulation of biological quality, biological regulation, and response to stimulus in terms of biological process; extracellular region for cellular component; and binding function for molecular function. Pathways in which DEP of GD versus IM, GD versus SX, and IM versus SX were mostly protein processing in endoplasmic reticulum for the first 2 groups and metabolic pathways for the last. Protein-protein interaction network analysis demonstrated that the most prominent DEP was heat shock protein 90 ß family member 1 for both the GD versus IM and the GD versus SX groups, and haptoglobin for the IM versus SX group. Data from this study may offer useful information for further investigation of the protein composition of Saanen goat milk and its application in the dairy industry.

Enhanced Stability and Oral Bioavailability of Cannabidiol in Zein and Whey Protein Composite Nanoparticles by a Modified Anti-Solvent Approach.

Wide applications of cannabidiol (CBD) in the food and pharmaceutical industries are limited due to its low bioavailability, sensitivity to environmental pressures and low water solubility. Zein nanoparticles were stabilized by whey protein (WP) for the delivery of cannabidiol (CBD) using a modified anti-solvent approach. Particle size, surface charge, encapsulation efficiency, and re-dispersibility of nanoparticles were influenced by the zein to WP ratio. Under optimized conditions at 1:4, zein-WP nanoparticles were fabricated with CBD (200 µg/mL) and further characterized. WP absorbed on zein surface via hydrogen bond, hydrophobic forces, and electrostatic attraction. The zein-WP nanoparticles showed excellent storage stability (4 °C, dark) and effectively protected CBD degradation against heat and UV light. In vivo pharmacokinetic study demonstrated that CBD in zein-WP nanoparticles displayed 2-times and 1.75-fold enhancement in maximum concentration (C max) and the area under curve (AUC) as compared to free-form CBD. The data indicated the feasibility of developing zein-WP based nanoparticles for the encapsulation, protection, and delivery of CBD.

Formation and characterization of noncovalent ternary complexes based on whey protein concentrate, high methoxyl pectin, and phenolic acid.

Protein-polysaccharide-polyphenol noncovalent ternary complexes possess unique physicochemical, structural, and functional properties. In the present study, ternary complexes based on whey protein concentrate (WPC; 2%, wt/vol) and high methoxyl pectin (HMP; 0.5%, wt/vol) complexes and 0.2 to 0.6% (wt/vol) chlorogenic acid (CA) or rosmarinic acid (RA) were formed and characterized at 3 pH values (4, 4.5, and 5). The pH conditions were decided according to phase diagram of WPC and HMP during acidification. Fluorescence quenching experiments indicated that WPC-HMP complexes bound RA stronger than CA and the binding constant increased with increasing pH for both phenolic acids. Particle size of ternary complexes decreased and absolute ζ-potential increased with pH values changing from 4 to 5, and RA influenced the particle size of WPC-HMP complexes greater than CA. The CA and RA in ternary complexes showed good stability against UV light with pH order of pH 5 > pH 4.5 > pH 4. Fourier-transform infrared spectroscopy spectra indicated the involvement of hydrogen bonding between WPC-HMP and CA or RA. Antibacterial tests showed that ternary complexes had good antibacterial activity against Staphylococcus aureus and Escherichia coli at concentrations of 6.2 mg/mL and the ability increased with decreasing pH values. All ternary complexes possessed strong scavenging radical capacities with median inhibitory concentration (IC50) values ranging from 2.71 ± 0.05 to 6.20 ± 0.41 µg/mL. Antioxidative ability of ternary complexes increased as pH went up and WPC-HMP-RA showed significantly higher antioxidative property compared with WPC-HMP-CA. Data may provide useful information for rational design of ternary complexes and applications of the formed complexes in food matrices such as beverages and emulsions.

Proteomic analysis of differentially expressed whey proteins in Saanen goat milk from different provinces in China using a data-independent acquisition technique.

Whey proteins of Saanen goat milk samples from 3 provinces in China (Guangdong, GD; Inner Mongolia, IM; Shaanxi, SX) were characterized and compared using data-independent acquisition quantitative proteomics technique. A total of 550 proteins were quantified in all 3 samples. There were 44, 44, and 33 differentially expressed proteins (DEP) for GD versus IM, GD versus SX, and IM versus SX, respectively. Gene ontology annotation analysis showed that the largest number of DEP for the 3 comparisons were as follows: for biological processes: response to progesterone, glyceraldehyde-3-phosphate metabolic process, and negative regulation of megakaryocyte differentiation; for molecular functions: antioxidant activity, binding, and peroxiredoxin activity; and for cellular components: the same category of extracellular regions for the 3 comparisons, respectively. Pathways for the DEP of 3 comparisons were (1) disease; (2) synthesis and metabolism; and (3) synthesis, degradation, and metabolism. Protein-protein interaction network analysis showed that DEP for GD versus SX had the most interactions.

Characterization of interactions between whey protein isolate and hyaluronic acid in aqueous solution: Effects of pH and mixing ratio.

Interactions between whey protein isolate (WPI) and hyaluronic acid (HA) were characterized as functions of pH (6.0-1.0) and protein to polysaccharide ratio (R, 1:4-10:1). Intramolecular soluble complexes formed at pHc of 5.6-5.8, followed by intermolecular insoluble complexes formed at pHΦ1 of 4.4-4.6. Complexes at ratios below 4:1 reached maximum optical value at pH 2.4 while samples above 4:1 peaked at pH 3-3.4 then precipitated. WPI/HA coacervates completely dissociated into soluble complex at pH 1.6-1.8 (pHΦ2). WPI/HA mixtures showed shear thinning behavior and elastic property. Whey protein underwent significant α-helix structure change when interacting with HA in range of pHΦ1>pH > pHΦ2 and at low R values (1:4 and 1:2). Scanning electronic microscope (SEM) pictures showed pH and mixing ratio dependent microstructural changes corresponding with phase transition. Data may provide helpful information for further application of WPI/HA complexes in medical, food and cosmetic fields.