Interfacial protein adsorption behavior can be connected across a wide range of timescales using the microfluidic EDGE (Edge-based droplet GEneration) tensiometer.

HYPOTHESIS: Our hypothesis is that dynamic interfacial tension values as measured by the partitioned-Edge-based Droplet GEneration (EDGE) tensiometry can be connected to those obtained with classical techniques, such as the automated drop tensiometer (ADT), expanding the range of timescales towards very short ones. EXPERIMENTS: Oil-water and air-water interfaces are studied, with whey protein isolate solutions (WPI, 2.5 - 10 wt%) as the continuous phase. The dispersed phase consists of pure hexadecane or air. The EDGE tensiometer and ADT are used to measure the interfacial (surface) tension at various timescales. A comparative assessment is carried out to identify differences between protein concentrations as well as between oil-water and air-water interfaces. FINDINGS: The EDGE tensiometer can measure at timescales down to a few milliseconds and up to around 10 s, while the ADT provides dynamic interfacial tension values after at least one second from droplet injection and typically is used to also cover hours. The interfacial tension values measured with both techniques exhibit overlap, implying that the techniques provide consistent and complementary information. Unlike the ADT, the EDGE tensiometer distinguishes differences in protein adsorption dynamics at protein concentrations as high as 10 wt% (which is the highest concentration tested) at both oil-water and air-water interfaces.

Study on the interaction mechanism of whey protein isolate with phosphatidylcholine: By multispectral methods and molecular docking.

The elucidation of the interaction mechanism between phospholipids and milk proteins within emulsions is pivotal for comprehending the properties of infant formula fat globules. In this study, multispectral methods and molecular docking were employed to explore the relationship between phosphatidylcholine (PC) and whey protein isolate (WPI). Observations indicate that the binding constant, alongside thermodynamic parameters, diminishes as temperature ascends, hinting at a predominantly static quenching mechanism. Predominantly, van der Waals forces and hydrogen bonds constitute the core interactions between WPI and PC. This assertion is further substantiated by Fourier transform infrared spectroscopy, which verifies PC's influence on WPI's secondary structure. A detailed assessment of thermodynamic parameters coupled with molecular docking reveals that PC predominantly adheres to specific sites within α-lactalbumin, ß-lactoglobulin, and bovine serum albumin, propelled by a synergy of hydrophobic interactions, hydrogen bonding, and van der Waals forces, with binding energies noted at -5.59, -6.71, and -7.85 kcal/mol, respectively. An increment in PC concentration is observed to amplify the emulsification properties of WPI whilst concurrently diminishing the zeta potential. This study establishes a theoretical foundation for applying the PC-WPI interaction mechanism in food.

A Protocol for Preparing Mucoadhesive Emulsion Microgels and Assessing Their Mucoadhesion Properties In Vitro.

Intravesical instillation is an efficient therapeutic technique based on targeted administration of a drug directly into the lesion for the treatment of bladder diseases. This is an alternative to traditional systemic administration of drugs. However, this technique requires repeated procedures, which can lead to even greater inflammation and infection of the urethra. To date, novel systems that allow prolonged drug retention in the bladder cavity are actively being developed. We recently reported a targeted drug delivery system based on the mucoadhesive emulsion microgels consisting of the natural component whey protein isolate. Such micron-sized carriers possess high loading capacity, a prolonged drug release profile, and efficient mucoadhesive properties to the bladder urothelium. As a continuation of this work, we present a protocol for the synthesis of mucoadhesive emulsion microgels. Detailed procedures for preparing precursor solutions as well as studying the physico-chemical parameters of microgels (including loading capacity and drug release rate) and the mucoadhesive properties using the model of porcine bladder urothelium are discussed. Precautionary measures and nuances that are worth paying attention to during each experimental stage are given as well. Key features • The protocol for the synthesis of mucoadhesive emulsion microgels based on whey protein isolate is presented. The experimental conditions of emulsion microgels synthesis are discussed. • Methods for studying the physico-chemical properties of mucoadhesive emulsion microgels (size of emulsion microgels particles, loading capacity, release kinetics) are described. • The method for assessing mucoadhesive properties of emulsion microgels is demonstrated using the porcine bladder tissue model ex vivo.

Fabrication and Characterization of Docosahexaenoic Acid Algal Oil Pickering Emulsions Stabilized Using the Whey Protein Isolate-High-Methoxyl Pectin Complex.

In this study, the whey protein isolate-high-methoxyl pectin (WPI-HMP) complex prepared by electrostatic interaction was utilized as an emulsifier in the preparation of docosahexaenoic acid (DHA) algal oils in order to improve their physicochemical properties and oxidation stability. The results showed that the emulsions stabilized using the WPI-HMP complex across varying oil-phase volume fractions (30-70%) exhibited consistent particle size and enhanced stability compared to emulsions stabilized solely using WPI or HMP at different ionic concentrations and heating temperatures. Furthermore, DHA algal oil emulsions stabilized using the WPI-HMP complex also showed superior storage stability, as they exhibited no discernible emulsification or oil droplet overflow and the particle size variation remained relatively minor throughout the storage at 25 °C for 30 days. The accelerated oxidation of the emulsions was assessed by measuring the rate of DHA loss, lipid hydroperoxide levels, and malondialdehyde levels. Emulsions stabilized using the WPI-HMP complex exhibited a lower rate of DHA loss and reduced levels of lipid hydroperoxides and malondialdehyde. This indicated that WPI-HMP-stabilized Pickering emulsions exhibit a greater rate of DHA retention. The excellent stability of these emulsions could prove valuable in food processing for DHA nutritional enhancement.

The non-covalent and covalent interactions of whey proteins and saccharides: influencing factor and utilization in food.

During the application of Whey proteins (WPs), they often have complex interactions with saccharides (Ss), another important biopolymer in food substrate. The texture and sensory qualities of foods containing WPs and Ss are largely influenced by the interactions of WPs-Ss. Moreover, the combination of WPs and Ss is possible to produce many excellent functional properties including emulsifying properties and thermal stability. However, the interactions between WPs-Ss are complex and susceptible to some processing conditions. In addition, with different interaction ways, they can be applied in different fields. Therefore, the non-covalent interaction mechanisms between WPs-Ss are firstly summarized in detail, including electrostatic interaction, hydrogen bond, hydrophobic interaction, van der Waals force. Furthermore, the existence modes of WPs-Ss are introduced, including complex coacervates, soluble complexes, segregation, and co-solubility. The covalent interactions of WPs-Ss in food applications are often formed by Maillard reaction (dry or wet heat reaction) and occasionally through enzyme induction. Then, two common influencing factors, pH and temperature, on non-covalent/covalent bonds are introduced. Finally, the applications of WPs-Ss complexes and conjugations in improving WP stability, delivery system, and emulsification are described. This review can improve our understanding of the interactions between WPs-Ss and further promote their wider application.

Investigating the impact of ultrasound-assisted treatment on the crafting of mulberry leaf protein and whey isolate complex: A comprehensive analysis of structure and functionality.

Mulberry leaf protein (MLP) is a nutrient-rich protein, but its applicability is limited because of its poor solubility. To address this issue, this study combines MLP with whey protein isolates (WPI), known for the high nutritional value, and subsequently forms composite protein nanoparticles using the ultrasound-assisted pH shifting method. Microscopic observation and SDS-PAGE confirmed the binding between these two proteins. Fluorescence spectra and Fourier Transform infrared spectroscopy (FTIR) analysis supported the involvement of electrostatic interactions, hydrophobic attractions, and hydrogen bonding in the formation of stable complex nanoparticles. The interactions between the proteins became stronger after ultrasound-assisted pH-shifting treatment. Solubility, emulsification capacity, foaming, and antioxidant activity, among other indicators, demonstrate that the prepared composite nanoparticles exhibit favorable functional properties. The study successfully illustrates the creation of protein-based complex nanoparticles through the ultrasound-assisted pH shifting method, with potential applications in the delivery of bioactive compounds.

Dietary intake of a MFGM/EV-rich concentrate promotes accretion of very long odd-chain sphingolipids and increases lipid metabolic turnover at the whole-body level.

Lipids from cow milk fat globule membranes (MFGMs) and extracellular vesicles (EVs) are considered beneficial for neurodevelopment, cognitive maintenance and human health in general. Nevertheless, it is largely unknown whether intake of infant formulas and medical nutrition products rich in these particles promote accretion of specific lipids and whether this affects metabolic homeostasis. To address this, we carried out a 16-week dietary intervention study where mice were supplemented with a MFGM/EV-rich concentrate, a control diet supplemented with a whey protein concentrate and devoid of milk lipids, or regular chow. Assessment of commonly used markers of metabolic health, including body weight, glucose intolerance and liver microanatomy, demonstrated no differences across the dietary regimes. In contrast, in-depth lipidomic analysis revealed accretion of milk-derived very long odd-chain sphingomyelins and ceramides in blood plasma and multiple tissues of mice fed the MFGM/EV diet. Furthermore, lipidomic flux analysis uncovered that mice fed the MFGM/EV diet have increased lipid metabolic turnover at the whole-body level. These findings help fill a long-lasting knowledge gap between the intake of MFGM/EV-containing foods and the health-promoting effects of their lipid constituents. In addition, the findings suggest that dietary sphingomyelins or ceramide-breakdown products with very long-chains can be used as structural components of cellular membranes, lipoprotein particles and signaling molecules that modulate metabolic homeostasis and health.

Antibacterial mechanism of whey protein isolated-citral nanoparticles and stable synergistic antibacterial eugenol encapsulated Pickering emulsion for grapes preservation.

The purpose of this study was to prepare Pickering emulsion with synergistic antibacterial effect using whey protein isolated-citral (WPI-Cit) nanoparticles with eugenol for grape preservation. In this emulsion, eugenol was encapsulated in oil phase. The particle size, ζ-potential, and antibacterial mechanism of the nanoparticles were characterized. The rheological properties, antibacterial effects and preservation effects of WPI-Cit Pickering emulsion were measured. The results showed that the optimal preparation condition was performed at WPI/Cit mass ratio of 1:1, WPI-Cit nanoparticles were found to damage the cell wall and membrane of bacteria and showed more effective inhibition against S. aureus. Pickering emulsion prepared with WPI-Cit nanoparticles exhibited a better antibacterial effect after eugenol was encapsulated in it, which extended the shelf life of grapes when the Pickering emulsion was applied as a coating. It demonstrated that the Pickering emulsion prepared in this study provides a new way to extend the shelf life.

Composite-structure oleogels constructed by glycerol monolaurate and whey protein isolate: Preparation, characterization and in vitro digestion.

The Glycerol monolaurate (GML) oleogel was induced using Camellia oil by slowly raising the temp to the melting point (MP) of GML. Whey protein isolate (WPI) solution with different ratios was composited with GML oleogel by emulsion template methods, forming dense spines and honeycomb-like networks and impressed with an adjustable composite structure. Textural results showed that compared with single GML-based oleogels, the GML/WPI composite oleogels had the advantages of high hardness and molding, and structural stability. The composite oleogels had moderate thermal stability and maximal oil binding (96.36%). In particular, as up to 6 wt% GML/WPI, its modulus apparent viscosity was significantly increased in rheology and similar to commercial fats. Moreover, it achieved the highest release of FFA (64.07%) and the synergy provided a lipase substrate and reduced the body's burden. The resulting composite oleogel also showed intermolecular hydrogen bonding and van der Waals force interactions. These findings further enlarge the application in the plant and animal-based combined of fat substitutes, delivery of bioactive molecules, etc., with the desired physical and functional properties according to different proportions.

Performance of spray-dried Ziziphus jujuba extract using insoluble fraction of Persian gum-sodium alginate and whey protein: Microstructural and physicochemical attributes of micro- and nano-capsules.

The study focused on the impact of the insoluble fraction of Persian gum-sodium alginate and a blend of the insoluble fraction of Persian gum-sodium alginate (IFPG-Al) with whey protein isolate (WPI) on sprayed Ziziphus jujuba extract (JE) powder. The addition of whey protein led to powders with higher moisture (10%), higher solubility (99.19%), and lower powder yield (27.82%). The powders fabricated with WPI depicted the best protection of polyphenolic compounds (3933.4 mg/L) and the highest encapsulation efficiency activity (74.84%). Additionally, they had a higher T g (62.63°C), which indicates more stability of the powders during shelf life. The sphericity of the majority of the particles was noticeable in powders, but multi-sided concavities were visible in the protein-containing particles. Based on the particle size's results, IFPG-Al/WPI capsules fabricated relatively smaller particles (2.54 µm). It can be acknowledged that the presence of protein in particles can bring fruitful results by preserving valuable bioactive compounds.

Monitoring sodium caseinate and whey protein isolate interaction with mild preheat treatment using ultrasound spectroscopy and confocal laser scanning microscopy.

Ultrasound spectroscopy and confocal laser scanning microscopy (CLSM) methods were developed to visualize the interaction between sodium caseinate (SC) and whey protein isolate (WPI) with a mild preheat treatment (57°C, 10 min) followed by adding glucono-δ-lactone (GDL). Ultrasonic velocity changes during incubation at 25°C after adding GDL for four kinds of mixtures (no-treated SC plus no-treated WPI, preheated SC plus no-treated WPI, no-treated SC plus preheated WPI and preheated SC plus preheated WPI) were monitored. The results reveal that the mild preheating treatment of the proteins affected the timing of the increase in compressibility of each system. CLSM observation with individualized dyes which have different maxima of excitation and emission wavelengths, showed the preheated SC plus no-treated WPI mixture had a slightly coarse structure and the highest correlation coefficient, suggesting the highest colocalization of the SC and WPI among the four kinds of mixed-protein systems. Furthermore, the scanning electron microscopy (SEM) observation suggests that there are some differences among the gels, namely, preheated WPI leads to the formation of developed three-dimensional gel networks with filamentous structures, whereas SC promotes the formation of cluster-like crowded networks composed of more fine aggregated particles instead of developed filamentous structures. These results demonstrated that although SC is known as a heat-stable protein, pretreated SC could lead to an increase of the collaboration with WPI in the presence of GDL. This finding anticipated the possibility creating a food material with another texture using a milk-protein mixed system.

Influence of thermal denaturation on whey protein isolates in combination with chitosan for fabricating Pickering emulsions: a comparison study.

Composite natural emulsifiers such as whey protein isolate (WPI) and chitosan (CS) are commonly used in Pickering emulsions to address the effect of thermal deformation of proteins before complexation with CS and heating after complexation. In this study, the properties of WPI and CS composites were investigated by complexing CS with either unmodified WPI or thermally denatured WPI (DWPI). Three types of composite particles were prepared, WPI-CS, DWPI-CS, and D(WPI-CS). Atomic force microscopy revealed that the composite particles formed larger aggregates with increased contour size and surface roughness compared to CS and WPI, whereas the interfacial tension decreased, indicating improved emulsifying abilities. Fourier-transform infrared analysis revealed differences in the hydrogen bonds between CS and WPI/DWPI. All three composite particles formed stable emulsions with droplet sizes of 20.00 ± 0.15, 27.80 ± 0.35, and 16.77 ± 0.51 µm, respectively. Thermal stability experiments revealed that the curcumin emulsion stabilized with WPI-CS and DWPI-CS exhibited relatively better thermal stability than that stabilized with D(WPI-CS). In vitro experiments results indicated that the bioaccessibility of the curcumin emulsion stabilized with WPI-CS was 61.18 ± 0.16%, significantly higher than that of the emulsions prepared with the other two composite particles (p < 0.05). This study will enable the customized design of WPI composite-based Pickering emulsions for application in the food and nutrition industries.

Understanding regulating effects of protein-anionic octenyl succinic anhydride-modified starch interactions on the structural, rheological, digestibility and release properties of starch.

BACKGROUND: Proteins and anionic octenyl succinic anhydride (OSA)-modified starch (OSA-starch) are common ingredients in food systems. The interactions between OSA-starch and protein are found to alter the structural and functional properties of the protein-OSA-starch complexes. In this regard, the close understanding of the relationship among the molecular interactions between whey protein isolate (WPI) and OSA-high amylose corn starch (HAS), structure changes and rheological, digestibility and release properties of WPI-OSA-HAS was investigated. RESULTS: The molecular interactions of WPI-OSA-HAS were significant for increasing the surface rough, solubility, storage modulus and loss modulus, but decreasing the R1047/1022 values. For the nutritional evaluation, the anti-digestibility of WPI-OSA-HAS was enhanced with increased resistant starch + slowly digestible starch contents and decreased equilibrium hydrolysis percentage and kinetic constant. During the digestion, part of the starch granule, OSA groups and WPI were lost, but the loss was lower than for OSA-HAS. Furthermore, the results of curcumin-loaded WPI-OSA-HAS in simulated gastrointestinal fluids demonstrated that curcumin could be gradually released to simulate colonic fluid. Notably, the interaction between WPI and OSA-HAS depended on the WPI concentration with the stronger molecular interactions obtained at 35% concentration. CONCLUSION: These results provided important information concerning how to adjust the rheological, anti-digestibility and release properties of WPI-OSA-HAS through altering the electrostatic interactions and hydrophobic interactions of WPI-OSA-HAS. © 2024 Society of Chemical Industry.

Study on the fabrication and controlled release behavior of N-Acetylneuraminic acid-loaded hydrogels stabilized by gelatin/whey protein isolate.

Gelatin (GEL), pectin (PEC), carboxymethyl cellulose (CMC), and whey protein isolate (WPI) were employed to formulate hydrogels for stabilizing N-Acetylneuraminic Acid (NeuAc). GEL/WPI-NeuAc hydrogels, irrespective of the ratio, exhibited a flexible and smooth surface with a continuous three-dimensional network structure internally. Porosity of the three types of hydrogels increased from 3.69% to 86.92% (GEL/WPI), 41.67% (PEC/WPI), and 87.62% (CMC/WPI), rendering them suitable as carriers for NeuAc encapsulation. The dynamic swelling behavior of all hydrogels followed Schott's second-order kinetics model. The degradation performance of GEL, PEC, and CMC/WPI-NeuAc hydrogels was optimal at a 5: 5 ratio, with degradation rates of 80.39 ± 1.26%, 82.38 ± 1.96%, and 81.39 ± 1.57%, respectively. GEL, PEC, CMC/WPI-NeuAc hydrogels demonstrated decreased release rates of 44.56%, 31.04%, and 41.26%, respectively, compared to free NeuAc, post gastric digestion. The present investigation suggests the potential of GEL/WPI hydrogels as effective carriers for delivering NeuAc encapsulation.

Mitigation of malondialdehyde-induced protein lipoxidation by epicatechin in whey protein isolate.

Malondialdehyde (MDA) can induce lipoxidation in whey protein isolate (WPI). The physicochemical changes in this reaction with or without the presence of a phenolic compound epicatechin (EC) were characterized in this study. Results suggested the content of MDA was significantly reduced during co-incubation of MDA and EC. The addition of EC dose-dependently alleviated MDA-induced protein carbonylation, Schiff base formation and loss of tryptophan fluorescence. The interruption of MDA-binding to WPI was directly visualized by immunoblotting analysis. Observation of the surface microstructure of WPI showed that MDA-induced protein aggregation was partially restored by EC. Meanwhile, EC was found to promote loss of both protein sulfhydryls and surface hydrophobicity due to possible phenol-protein interactions. These observations suggested the potential of EC in the relief of MDA-mediated protein lipoxidation.

Non-covalent complexes of lutein/zeaxanthin and whey protein isolate formed at different pH levels: Binding interactions, storage stabilities, and bioaccessibilities.

Lutein (Lut) and zeaxanthin (Zx) are promising healthy food ingredients; however, the low solubilities, stabilities, and bioavailabilities limit their applications in the food and beverage industries. A protein-based complex represents an efficient protective carrier for hydrophobic ligands, and its ligand-binding properties are influenced by the formulation conditions, particularly the pH level. This study explored the effects of various pH values (2.5-9.5) on the characteristics of whey protein isolate (WPI)-Lut/Zx complexes using multiple spectroscopic techniques, including ultraviolet-visible (UV-Vis), fluorescence, and Fourier transform infrared (FTIR) spectroscopies and dynamic light scattering (DLS). UV-Vis and DLS spectra revealed that Lut/Zx were present as H-aggregates in aqueous solutions, whereas WPI occurred as nanoparticles. The produced WPI-Lut/Zx complexes exhibited binding constants of 104-105 M-1, which gradually increased with increasing pH from 2.5 to 9.5. FTIR spectra demonstrated that pH variations and Lut/Zx addition caused detectable changes in the secondary WPI structure. Moreover, the WPI-Lut/Zx complexes effectively improved the physicochemical stabilities and antioxidant activities of Lut/Zx aggregates during long-term storage and achieved bioaccessibilities above 70% in a simulated gastrointestinal digestion process. The comprehensive data obtained in this study offer a basis for formulating strategies that can be potentially used in developing commercially available WPI complex-based xanthophyll-rich foods.

The Effect of Whey Protein Isolate Hydrolysate on Digestive Properties of Phytosterol.

Phytosterol (PS) is a steroid, and its bioavailability can be enhanced by interacting with protein in the C-24 hydroxyl group. The interaction between sterols and amino acid residues in proteins can be enhanced by enzymatic hydrolysis. Phytosterol and whey insulation hydrolysates (WPH1-4) fabricated by the Alcalase enzyme at different enzymatic hydrolysis times were selected as delivery systems to simulate sterol C-24 hydroxyl group interaction with protein. Increasing hydrolysis time can promote the production of ß-Lg, which raises the ratio of ß-turn in the secondary structure and promotes the formation of interaction between WPH and PS. The correlation coefficient between hydrogen bonds and encapsulation efficiency (EE) and bioaccessibility is 0.91 and 0.88 (P < 0.05), respectively, indicating that hydrogen bonds of two components significantly influenced the combination by concealing the hydrophobic amino acids and some residues, which improved PS EE and bioavailability by 3.03 and 2.84 times after PS was combined with the WPI hydrolysate. These findings are expected to enhance the absorption of PS and other macromolecules by protein enzymatic hydrolysis to broaden their applications for food.

Effect of pH on the emulsifying performance of protein-polysaccharide complexes.

BACKGROUND: Protein-polysaccharide complexes have been successfully used for emulsion stabilization. However, it is unclear how the complex's surface charge influences aggregation stability and coalescence stability of emulsions, and whether a low charged interfacial film can still maintain the coalescence stability of oil droplets. In the present study, the effects of pH (around the pI of protein) on the aggregation and coalescence stability of emulsions were investigated. RESULTS: Whey protein isolate (WPI) and peach gum polysaccharides (PGP) complexes (WPI-PGP complexes) were synthesized at pH 3, 4 and 5. Their sizes were 598, 274 and 183 nm, respectively, and their ζ-potentials were +2.9, -8.6 and -22.8 mV, respectively. Interface rheological experiments showed that WPI-PGP complex at pH 3 had the lowest interfacial tension, and formed the softest film compared to the complexes at pH 4 and 5. Microfluidic experiments showed that all WPI-PGP complexes were able to stabilize droplets against coalescence within short timescales (milliseconds). At pH 3, no coalescence was observed even under conditions where the continuous phase flow influenced the shape of oil droplets (from spheres to ellipsoids). At pH 4 and 5, the model emulsions were stable over 16 days of storage, extensive aggregation and creaming occurred at pH 3 after 8 days. Importantly, no coalescence took place. CONCLUSION: The present study confirmed that the aggregation stability of the emulsions was mainly determined by the surface charge of the complex, whereas the coalescence stability of emulsions is expectedly determined by steric repulsion, providing new insights into how to prepare stable food emulsions. © 2024 Society of Chemical Industry.

Whey Protein Isolate as a Substrate to Design Calendula officinalis Flower Extract Controlled-Release Materials.

The use of natural active substances and the development of new formulations are promising directions in the cosmetic and pharmacy industries. The primary purpose of this research was the production of microparticles based on whey protein isolate (WPI) and calcium alginate (ALG) containing Calendula officinalis flower extract and their incorporation into films composed of gelatin, WPI, and glycerol. Both swollen and dry microparticles were studied by optical microscopy and their sizes were measured. Water absorption by the microparticles, their loading capacity, and the release profile of flower extract were also characterized. The films were analyzed by mechanical tests (Young's modulus, tensile strength, elongation at break), swelling capacity, contact angle, and moisture content measurements. The presented data showed that the active ingredient was successfully enclosed in spherical microparticles and completely released after 75 min of incubation at 37 °C. The incorporation of the microparticles into polymer films caused a decrease in stiffness and tensile strength, simultaneously increasing the ductility of the samples. Moreover, the films containing microparticles displayed higher swelling ability and moisture content compared to those without them. Hence, the materials prepared in this study with Calendula officinalis flower extract encapsulated into polymeric microspheres can be a starting point for the development of new products intended for skin application; advantages include protection of the extract against external factors and a controlled release profile.

Fucoidan alleviates the inhibition of protein digestion by chitosan and its oligosaccharides.

Chitosan (CTS) and chitosan oligosaccharides (COS) have been widely applied in food industry due to their bioactivities and functions. However, CTS and COS with positive charges could interact with proteins, such as whey protein isolate (WPI), influencing their digestion. Interaction among CTS/COS, FUC, and WPI/enzymes was studied by spectroscopy, chromatography, and chemical methods in order to reveal the role of FUC in relieving the inhibition of protein digestibility by CTS/COS and demonstrate the action mechanisms. As shown by the results, the addition of FUC increased degree of hydrolysis (DH) and free protein in the mixture of CTS and WPI to 3.1-fold and 1.8-fold, respectively, while raise DH value and free protein in the mixture of COS and WPI to 6.7-fold and 1.2-fold, respectively. The interaction between amino, carboxyl, sulfate, and hydroxyl groups from carbohydrates and protein could be observed, and notably, FUC could interact with CTS/COS preferentially to prevent CTS/COS from combining with WPI. In addition, the addition of FUC could also relieve the combination of CTS to trypsin, increasing the fluorescence intensity and concentration of trypsin by 83.3 % and 4.8 %, respectively. Thus, the present study demonstrated that FUC could alleviate the inhibitory effect of CTS/COS on protein digestion.