A protectant for Lactobacillus rhamnosus based on whey protein isolate and isomalt: Stress resistance and underlying mechanisms.

Probiotics are exposed to a variety of abiotic and biotic stresses during food fermentation and production, such as acidity, heat, osmolality, and oxidation, which affect their metabolic activity and efficiency. Therefore, it is essential to develop new protective agents to maintain the activity and stability of probiotics. This study introduces a new protectant, spray-dried whey protein isolate (WPI) and isomaltose (ISO). We evaluated the effects of four WPI-ISO ratios (1:0, 2:1, 1:1, 1:2) on the physical properties, including moisture content, water activity (aw), wettability, and glass transition temperature. In addition, we evaluated the environmental tolerance of Lactobacillus rhamnosus to different WPI-ISO ratios under thermal, storage, and simulated gastrointestinal conditions. The results showed that the moisture content (< 7 %) and water activity (< 0.3) of the protectant and probiotic powders met storage stability requirements. The moisture content, water activity, wettability index (WI), and glass transition temperature decreased significantly with the addition of isomalt, thereby improving the pressure resistance of L. rhamnosus through the synergistic effect of WPI and ISO. The WPI-ISO protectant not only improved the environmental tolerance and wettability of probiotics by reducing the moisture content and water activity but also significantly improved the survival rate of L. rhamnosus under various stress conditions such as high temperature and gastrointestinal environment. L. rhamnosus maintains good activity with a viable bacterial count of over 9 lg CFU/g after 90 days of storage, demonstrating effective protection against the environment stress. This study provides a promising new strategy to improve the stability of probiotics in the food industry.

Development of Oleogel-in-Water High Internal Phase Emulsions with Improved Physicochemical Stability and Their Application in Mayonnaise.

Egg-free mayonnaise is receiving greater attention due to its potential health benefits. This study used whey protein isolate (WPI) as an emulsifier to develop high internal phase emulsions (HIPEs) based on beeswax (BW) oleogels through a simple one-step method. The effects of WPI, NaCl and sucrose on the physicochemical properties of HIPEs were investigated. A novel simulated mayonnaise was then prepared and characterized. Microstructural observation revealed that WPI enveloped oil droplets at the interface, forming a typical O/W emulsion. Increase in WPI content led to significantly enhanced stability of HIPEs, and HIPEs with 5% WPI had the smallest particle size (11.9 ± 0.18 µm). With the increase in NaCl concentration, particle size was increased and ζ-potential was decreased. Higher sucrose content led to reduced particle size and ζ-potential, and slightly improved stability. Rheological tests indicated solid-like properties and shear-thinning behaviors in all HIPEs. The addition of WPI and sucrose improved the structures and viscosity of HIPEs. Simulated mayonnaises (WE-0.3%, WE-1% and YE) were then prepared based on the above HIPEs. Compared to commercial mayonnaises, the mayonnaises based on HIPEs exhibited higher viscoelastic modulus and similar tribological characteristics, indicating the potential application feasibility of oleogel-based HIPEs in mayonnaise. These findings provided insights into the development of novel and healthier mayonnaise alternatives.

Oral food-derived whey protein isolate-Tremella fuciformis polysaccharides pickering emulsions with adhesive ability to delivery magnolol for targeted treatment of ulcerative colitis.

Magnolol (Mag) is a promising natural compound with therapeutic potential for ulcerative colitis (UC). Here we designed and fabricated an oral food-grade whey protein isolate-Tremella fuciformis polysaccharides (WPI-TFPS) stabilized pickering emulsions to encapsulate Mag (Mag-WPI-TFPS) for targeted treatment of UC. With the assistance of the WPI-TFPS, pickering emulsions were well encapsulated and formed stable microparticles with a particle size of approximately 9.49 ± 0.047 µm, a 93.63 ± 0.21 % encapsulation efficiency and a loading efficiency of 21.53 ± 0.01 %. In vitro, the formulation exhibited sustained-release properties in simulated colon fluid with a cumulative release rate of 60.78 % at 48 h. In vivo, the Mag-WPI-TFPS specifically accumulated in the colon tissue for 24 h with stronger fluorescence intensity, which demonstrated that TFPS and WPI had a good adherence ability to inflamed mucosa by electrostatic attraction and ligand-receptor interactions. As expected, compared with Free-Mag, the oral administration of Mag-WPI-TFPS remarkably alleviated the symptoms of UC and protected the colon tissue in DSS-induced UC mice. More importantly, WPI-TFPS enhanced gut microbiota balance by increasing the diversity and relative abundances of Lactobacillaceae and Firmicutes. Overall, this study presents a convenient, eco-friendly, food-derived oral formulation with potential as a dietary supplement for targeted UC treatment.

Mechanistic insights into the interaction of Lycium barbarum polysaccharide with whey protein isolate: Functional and structural characterization.

Protein-polysaccharide interactions are crucial for food system structure and stability. This study investigates the interaction of Lycium barbarum polysaccharide (LBP) at 0-2.00 % concentrations with whey protein isolate (WPI), focusing on functionality and structural changes. LBP covalently grafted onto WPI, as confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), forming WPI-LBP complexes with a maximum degree of grafting (DG) of 44.58 % at 2.00 % LBP. This grafting reduced WPI's surface hydrophobicity (H0) and improved solubility, emulsifying properties, and digestibility under certain conditions, with optimal antioxidant activity at 1.00 % LBP. Multispectral analysis and microscopy showed LBP grafting alters WPI's secondary, tertiary, crystalline, and micro/nanostructures. The comprehensive analysis indicates that the interaction between LBP and WPI involves covalent bonding, hydrogen bonding, hydrophobic interactions, and electrostatic forces, as supported by zeta potential and chemical forces results. These findings suggest LBP-protein complexes as promising food materials for enhancing functionality and stability in the food industry.

Dynamic high-pressure microfluidization assisted with galactooligosaccharide-modified whey protein isolate: Investigating its effect on relieving intestinal barrier damage.

The study aimed to investigating the mechanisms of relieved intestinal barrier damage by dynamic high-pressure microfluidization assisted with galactooligosaccharide- glycated whey protein isolate. The modifications changed the multi-structure, and the modified whey protein isolate could promote the proliferation of IEC-6 cells and contributed to the restoration of LPS-induced occludin damage in IEC-6 cells. Also, it could repair cyclophosphamide-induced ileal villus rupture and crypt destruction in BALB/c mice, significantly altered the abundance of dominant bacteria, which were associated with propionic acid, butyric acid, isovaleric acid, and valeric acid. Ileum transcriptomics revealed that the modified whey protein isolate significantly regulate of the levels of Cstad, Cyp11a1, and Hs6st2 genes, relating to the increase of propionic acid, isovaleric acid, and valeric acid. In conclusion, galactooligosaccharide- modified whey protein isolate could regulate the level of Cstad, Cyp11a1 and Hs6st2 genes by altering the gut microbial structure and the level of SCFAs, thereby repairing the intestinal barrier.

Co-cold extrusion synergized with cysteine for enhancing physicochemical, rheological characteristics and in vitro digestibility of whey protein isolate.

Impacts of co-cold extrusion (≤50 °C) of whey protein isolate (WPI) and cysteine (Cys, 0, 20, 40, 60, 80 and 100 mmol/L) on its physicochemical, in vitro digestion and rheological properties were investigated. As Cys concentration increased, the emulsifying properties and in vitro digestibility of co-extruded WPI-Cys products showed an increasing trend. Specifically, when Cys reached 100 mmol/L, surface hydrophobicity, emulsification activity index (EAI), emulsification stability index (ESI) and in vitro stomach digestibility of the co-extruded WPI-Cys products increased by 205.07%, 77.51%, 193.95% and 71.81% compared with WPI, respectively. Principal component analysis (PCA) results further indicated that co-extruded WPI-Cys at a concentration of 100 mmol/L had the best functional properties. In addition, co-extruded WPI-Cys exhibited the strongest Péclet number (Pe) value and apparent viscosity at a Cys concentration of 100 mmol/L among all samples. Therefore, co-extrusion would be an effective method for modifying WPI, providing whey protein-based ingredients with excellent functional properties for food processing.

Characterization of Peptide Profiles and the Hypoallergenic and High Antioxidant Activity of Whey Protein Hydrolysate Prepared Using Different Hydrolysis Modes.

Food proteins and peptides are generally considered a source of dietary antioxidants. The aim of this study was to investigate the antioxidant activity, allergenicity, and peptide profiles of whey protein hydrolysates (WPHs) using different hydrolysis methods. The results demonstrated that the degrees of hydrolysis of the hydrolysates with one-step (O-AD) and two-step (T-AD) methods reached 16.25% and 17.64%, respectively. The size exclusion chromatography results showed that the O-AD had a higher content of >5 and <0.3 kDa, and the distribution of peptide profiles for the two hydrolysates was significantly different. Furthermore, 5 bioactive peptides and 15 allergenic peptides were identified using peptidomics. The peptide profiles and the composition of the master proteins of the O-AD and T-AD were different. The DPPH and ABTS radical scavenging abilities of WPHs were measured, and hydrolysates were found to exhibit a strong radical scavenging ability after being treated using different hydrolysis methods. An enzyme-linked immunosorbent assay showed that the sensitization of WPHs was significantly reduced. This study may provide useful information regarding the antioxidant properties and allergenicity of WPHs.

Improvement in probiotic intestinal survival by electrospun milk fat globule membrane-pullulan nanofibers: Fabrication and structural characterization.

Studies have demonstrated the protective effect of milk fat globule membrane (MFGM) on probiotics in harsh environments. However, currently, there are no reports on the encapsulation of probiotics using MFGM. In this study, MFGM and pullulan (PUL) polysaccharide fibers were prepared by electrostatic spinning and used to encapsulate probiotics, with whey protein isolates (WPI)/PUL as the control. The morphology, physical properties, mechanical properties, survival, and stability of the encapsulated Lacticaseibacillus rhamnosus GG (LGG) were studied. The results showed that the MFGM/PUL solution had significant effects on pH, viscosity, conductivity, and stability. Electrostatic spinning improved the mechanical properties and encapsulation ability of the polymer formed by MFGM/PUL. LGG encapsulated in MFGM/PUL nanofibers survived rate was higher than WPI/PUL nanofibers in mimic intestinal juice, which could be attributed to the phospholipid content contained in MFGM. These results demonstrate that MFGM is a promising material for probiotic encapsulation, providing an important basis for the potential use of MFGM/PUL nanofibers as a robust encapsulation matrix.

Unveiling the transformative influence of sonochemistry on formation of whey protein isolate and green tea extract (WPI-GTE) conjugates.

This study investigated the formation of conjugates between whey protein isolate (WPI) and green tea extract (GTE) using three methods: redox-pair (R), ultrasound-assisted redox-pair (RU), and ultrasonication (UL). Ultrasonication significantly reduced the reaction time for synthesizing WPI-GTE conjugates compared to the standard R method (p < 0.05). The UL methods had the highest conjugate yield determined by polyphenol binding (p < 0.05). Fourier-transform infrared spectroscopy (FTIR) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) confirmed the conjugate formation, indicating an increased molecular weight due to protein binding with polyphenols through covalent and non-covalent bonds. Conjugates produced via ultrasonication exhibited enhanced solubility, smaller particle size, better emulsifying capacity, and improved foaming ability compared to those formed using the traditional R method (p < 0.05). However, conjugates from the R method showed higher antioxidant activity, as evidenced by DPPH•and ABTS•+ scavenging activities (p < 0.05). In conclusion, WPI-GTE conjugates created through ultrasonic treatment demonstrate potential as dual-functional ingredients, serving as both antioxidant and emulsifier.

Galacto-oligosaccharides modified whey protein isolate ameliorates cyclophosphamide-induced immunosuppression.

The effect of whey protein isolate (WPI)- galacto-oligosaccharides (GOS)/fructo-oligosaccharides (FOS) conjugates on RAW264.7 cells, and further the effect of WPI-GOS conjugates on CTX-induced immunosuppressed mice were investigated. Compared to WPI-FOS conjugates, WPI-GOS conjugates exhibited deeper glycation extent, more pronounced structural unfolding and helix-destabilizing, and obviously improved functional indicators of RAW264.7 macrophages. In addition, WPI-GOS conjugates also repaired immune organ and intestinal barrier and increased IL-1ß and IFN-γ levels in immunosuppressed mice. The alteration of gut microbiota induced by WPI-GOS conjugates changed the serum metabolites, causing the activation of NFκB pathway, which strengthens the immune system. The activation of NFκB pathway maybe associated with the mTOR signal pathway and ABC transporters. However, the precise mechanisms by which NFκB pathway interacts with mTOR signal pathway and ABC transporters to modulate the immune response need for further research.

Biointerfacial supramolecular self-assembly of whey protein isolate nanofibrils on probiotic surface to enhance survival and application to 3D printing dysphagia foods.

Personalized three-dimensional (3D) printed foods rich in probiotics were investigated. Lactiplantibacillus plantarum (Lp), as a representative of probiotics, was used to investigate the 3D printing of probiotic-rich dysphagia foods. Here, whey protein isolate nanofibrils (WPNFs) were coated and anchored on bacterial surfaces via biointerfacial supramolecular self-assembly, providing protection against environmental stress and the 3D printing process. The optimized composite gels consisting of High acyl gellan gum (0.25 g), whey protein isolate (1.25 g), fructooligosaccharides (0.75 g), Lp-WPNFs-Glyceryl tributyrate emulsion (Φ = 40%, 3.75 mL) can realize 3D printing, and exhibit high resolution, and stable shape. The viable cell count is higher than 8.0 log CFU/g. They are particularly suitable for people with dysphagia and are classified as level 5-minced & moist in the international dysphagia diet standardization initiative framework. The results provide new insights into the development of WPNFs-coating on bacterial surfaces to deliver probiotics and 3D printed food rich in probiotics.

Effect of curdlan on the gel properties and interactions of whey protein isolate gels.

This study investigated the influence of curdlan on the gel properties of whey protein isolate (WPI). Results demonstrated that curdlan significantly improved the water-holding capacity, gel strength and rheological properties of the WPI gels. Moreover, it promoted the unfolding of the molecular structures of WPI, which was manifested by the transition from α-helix to ß-sheet, an increase in free sulfhydryl content and a decrease in surface hydrophobicity. Furthermore, 4 % curdlan promoted the formation of WPI with uniform and compact elastic gel network structures, primarily attributed to disulphide bonds, hydrogen bonds and hydrophobic interactions. However, when the addition of curdlan exceeds 4 %, excessive entanglement of curdlan chains and steric hindrance effects hinder the unfolding and folding of protein structures, weaken their interaction, result in a loose network structure and affect the gel properties. In conclusion, this study demonstrates that curdlan can effectively improve the gelling properties of WPI, suggesting its potential application in low-calorie gel-based dairy products.

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.

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.

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.

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.

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.

3D Printing Properties of Heat-Induced Sodium Alginate-Whey Protein Isolate Edible Gel.

The objective of this study was to develop a food 3D printing gel and investigate the effects of whey protein isolate (WPI), sodium alginate (SA), and water-bath heating time on the 3D printing performance of the gel. Initially, the influence of these three factors on the rheological properties of the gel was examined to determine the suitable formulation ranges for 3D printing. Subsequently, the formulation was optimized using response surface methodology, and texture analysis, scanning electron microscopy (SEM), and Fourier-transform infrared (FTIR) spectroscopy were conducted. The rheological results indicated that gels with WPI concentrations of 6-7 g, SA concentrations of 0.8-1.2 g, and water-bath heating times of 10-12 min exhibited lower yield stress and better self-supporting properties. The optimized formulation, determined through response surface methodology, consisted of 1.2 g SA, 6.5 g WPI, and a heating time of 12 min. This optimized formulation demonstrated enhanced extrusion capability and superior printing performance. SEM analysis revealed that the optimized gel possessed good mechanical strength, and FTIR spectroscopy confirmed the successful composite formation of the gel. Overall, the results indicate that the optimized gel formulation can be successfully printed and exhibits excellent 3D printing performance.

Development and Characterization of Hybrid Meat Analogs from Whey Protein-Mushroom Composite Hydrogels.

There is a need to reduce the proportion of animal-derived food products in the human diet for sustainability and environmental reasons. However, it is also important that a transition away from animal-derived foods does not lead to any adverse nutritional effects. In this study, the potential of blending whey protein isolate (WPI) with either shiitake mushroom (SM) or oyster mushroom (OM) to create hybrid foods with enhanced nutritional and physicochemical properties was investigated. The impact of OM or SM addition on the formation, microstructure, and physicochemical attributes of heat-set whey protein gels was therefore examined. The mushroom powders were used because they have relatively high levels of vitamins, minerals, phytochemicals, and dietary fibers, which may provide nutritional benefits, whereas the WPI was used to provide protein and good thermal gelation properties. A variety of analytical methods were used to characterize the structural and physicochemical properties of the WPI-mushroom hybrids, including confocal microscopy, particle electrophoresis, light scattering, proximate analysis, differential scanning calorimetry, thermogravimetric analysis, dynamic shear rheology, textural profile analysis, and colorimetry. The charge on whey proteins and mushroom particles went from positive to negative when the pH was raised from 3 to 9, but whey protein had a higher isoelectric point and charge magnitude. OM slightly increased the thermal stability of WPI, but SM had little effect. Both mushroom types decreased the lightness and increased the brownness of the whey protein gels. The addition of the mushroom powders also decreased the hardness and Young's modulus of the whey protein gels, which may be because the mushroom particles acted as soft fillers. This study provides valuable insights into the formation of hybrid whey protein-mushroom products that have desirable physiochemical and nutritional attributes.