Regulatory Mechanism of Humulinone on Protein Foam: applications in whey and pea protein

Regulatory Mechanism of Humulinone on Protein Foam: applications in whey and pea protein | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Regulatory Mechanism of Humulinone on Protein Foam: applications in whey and pea protein Yang Gao, Chen Xu, Hanhan Liu, Junyu Lin, Chenyan Lv This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6811741/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Foam is one of the most common and important systems in food, contributing to both the visual appeal and sensory experience. This work investigated the impact of humulinone on the foam properties and physicochemical characteristics of two widely used proteins in food systems: whey protein (WP), a traditional animal-derived protein, and pea protein (PP), a novel plant-based protein alternative. The results showed that humulinone significantly enhanced the foamability of both proteins, increasing the foaming capacity of WP by 2.34 times and PP by 1.66 times at a mass ratio of protein to humulinone of 1:0.1, while having limited effect on foam stability. Spectroscopic analysis and molecular docking revealed that humulinone interacted mainly through hydrogen bonding, leading to conformational changes in secondary structure-promoting. Surface tension measurements indicated that humulinone reduced the surface tension of WP and PP, which accelerated the diffusion stage during interfacial adsorption. Additionally, in food matrix models such as milk and commercial pea protein beverages, humulinone showed potential in improving foamability. These findings provide both mechanistic insights and theoretical support for the application of humulinone in foam-based food products. Humulinone Protein Foamability Interactions Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Foams play a vital role in food products, enhancing both their visual appeal and taste experience. Despite their importance, foams are inherently thermodynamically unstable, prone to breaking and collapsing over time. To enhance the stability of foams, surfactants are essential, as they effectively reduce the surface tension at the air-water interface [ 1 ]. Proteins are particularly valuable in this context due to their high surface activity [ 2 ]. Dairy-derived proteins, in particular, are widely utilized as foaming agents in food processing. They are abundant, cost-effective, and possess excellent foaming properties along with high nutritional value [ 3 ]. However, as the food industry increasingly shifts towards sustainable practices, there is a growing interest in plant-based proteins. Pea proteins, for instance, have gained popularity in various food products due to their high nutritional profile, extensive cultivation, affordability, and hypoallergenic nature. This trend highlights the potential of plant proteins to complement or even replace traditional dairy proteins in foamed applications, aligning with the demand for healthier and more sustainable food options [ 4 ]. Physical modification, chemical modification or addition of small molecules have been widely used to improve the foam properties of proteins for better application in food processing and production [ 5 , 6 ]. Interaction between multicomponent food matrices plays a key role in regulating foam properties, and some studies have been conducted to improve the foam properties of proteins by adding small molecule surfactants or mixing multi-components. Nooshkam et al. devised mixtures of licorice extract/whey protein/sodium alginate, which have high foaming properties and produce highly stable foam [ 7 ]. Improvement of protein foam characteristics by adding small molecule actives, such as flavonoids (quercetin, rutin) interacting with plant proteins through non-covalent bonds, which on the one hand improves the foamability and emulsification of soy and pea proteins [ 8 ], and at the same time foamed proteins act as delivery carriers to protect the activity of flavonoids as well as to improve the bioavailability. Hops are one of the indispensable ingredients in beer brewing that brings a distinctive bitter flavor and aroma. Substances in hops such as iso-α-acid (an isomerization product of α-acids) also have a significant effect on beer foam. It has been found that the addition of iso-α-acid or its hydrogenated reduced derivatives to a beer liquid without hops increases the foam stability of that beer and enhances the adhesion of the foam to the walls of the glass [ 9 ]. Similar to iso-α-acids, humulinone are oxidation products of α-acids (Fig. 1 ), and in a previous study by our group, we found that the “bubble cap” formed during beer fermentation contained significant amounts of iso-α-acids and humulinone [ 10 , 11 ]. In addition, dry-hopped beers with fine foam have much higher levels of humulinone than regular ales [ 12 ]. These results imply an inextricable relationship between humulinone and beer foam. However, the mechanism of action between humulinone and protein foaming has been less studied. It is worth mentioning that in our previous study, we found that humulinone enriched in aged hops greatly improved the foaming and foam stability of protein Z in beer as well as the beer's frothiness [ 13 ]. Therefore, it is hypothesized that humulinone can interact with other proteins and be applied in foamed food systems to improve the foaming properties of proteins, thereby improving the structure and texture of food products. Therefore, we chose one of the most commonly used animal proteins in food systems, whey protein (WP), and a novel alternative protein product, pea protein (PP) of plant origin, to investigate the effects of humulinone on the foaming properties, physicochemical properties, and mechanisms of both. The effects of humulinone on the foam properties of WP and PP were characterized, and then the interactions between humulinone and PP were investigated by multispectroscopy and computer simulation, and the mechanism of humulinone affecting the molecular level of WP/PP was examined in terms of the interfacial properties and the protein structure, respectively. Finally, the effect of humulinone on the foam properties of WP and PP-based real food systems was investigated, providing theoretical basis for the application of humulinone in foam food. Materials and Methods Chemicals and materials Whey protein and pea protein were purchased from commercial maltsters in China. CO 2 hops extract was purchased from BarthHaas (Beijing China). 8-anilino-1-naphthalenesulfonic acid (ANS), and potassium bromide (KBr) were purchased from Bio Dee Biotechnology Co. Ltd (Beijing, China). SDS electrophoresis marker and reagents used for the gel electrophoresis were purchased from Solarbio Co. Ltd (Beijing, China). The pea milk and cow's milk are from Chipmunks (Shandong, China) and Mongoose (Beijing, China), respectively. All other reagents used were of analytical grade, and ultrapure water was used throughout the research. Preparation of proteins and humulinone WP and PP were dissolved in 50 mM phosphate buffer (pH 6.5) at a concentration of 100.00 mg/mL, stirred at room temperature for 1 hour, and subsequently stored at 4°C overnight to ensure complete hydration. The resulting solutions were centrifuged at 10,000 rpm for 10 mins to remove insoluble materials. The supernatants were then dialyzed against 50 mM phosphate buffer (pH 6.5) to eliminate residual metal ions and low-molecular-weight impurities. The purified protein solutions were diluted to the desired concentrations with phosphate buffer for use in subsequent experiments. Protein composition was assessed using SDS-PAGE according to Laemmli’s method [ 14 ]. Protein concentrations were determined by the Lowry method, using bovine serum albumin (BSA) as the standard. Humulone was prepared following the method described by Taniguchi [ 10 ], with slight modifications. Briefly, hop extract was dissolved in anhydrous ethanol at a 1:1 ratio (m:v), followed by the addition of a base to remove insoluble oily substances through heating at 40°C, yielding a clear filtrate. Dilute hydrochloric acid was then slowly added to the filtrate to adjust the pH to 1–2. The solution was refrigerated at 4°C overnight to facilitate the precipitation of α-acid paste. Subsequently, 10.00 g of the α-acid paste was dissolved in 50 mL of diethyl ether along with 5 mL of cumene hydroperoxide, and the mixture was added to 350 mL of saturated sodium bicarbonate solution. The resulting solution was stored in a sealed container at room temperature, protected from light, for 4 days. After this period, the precipitated sodium salt of humulone was collected by filtration and washed three times with cold ether and deionized water, respectively. Finally, 1 g of the washed sodium salt was dissolved in 100 mL of methanol containing 1% phosphoric acid, and 1 L dilute hydrochloric acid (0.5 M) was added to induce precipitation. The resulting humulone powder was obtained by vacuum filtration and its purity was determined by high-performance liquid chromatography (HPLC). Fluorescence titration The fluorescence quenching titration experiments were run on a Cary Eclipse spectrophotometer (Cary, Varian, USA). 1 mL of 0.4 mg/mL WP or PP solution was placed in a quartz cuvette with a path length of 1 cm, to which humulinone solution was added, and the fluorescence intensity was recorded after blowing uniformly. The experimental temperature was 25 ℃, the excitation wavelength was 280 nm, and the absorption wavelength was 290–500 nm. The excitation slit width was 5 nm, and the emission slit width was 5 nm for WP and 10 nm for PP [ 15 ]. Fourier transform infrared spectroscopy (FTIR) spectra The FTIR spectra were obtained using a Thermo Nicolet iS50 FTIR spectrometer (Thermo Nicolet Analytical Instruments, MA, USA). The WP/PP solutions were mixed with humulinone solutions in mass ratios of 1:0, 1:0.01, 1:0.05, and 1:0.1. The mixtures were freeze-dried to obtain solid powders. In addition, the mixed solutions were whipped to form foams, which were immediately collected and rapidly frozen using liquid nitrogen. The frozen foams were then freeze-dried to obtain solid powders for FTIR analysis. The freeze-dried powders were ground with dried KBr under infrared light. The mixture was then pressed into transparent, uniform thin films using a pellet press and placed into the FTIR instrument. Scans were conducted in the range of 4000 ~ 400 cm − 1 , with 32 scans performed at a resolution of 4 cm − 1 . Data in the range of 1600 ~ 1700 cm − 1 were fitted to analyze the secondary structure of the proteins. Protein-ligand docking The binding interactions between humulinone and WP and PP were investigated using Autodock 4.2.6 software. β-Lactoglobulin (β-LG), the predominant protein in WP, was selected for molecular docking analysis. For PP, 7S vicilin and 11S legumin, the major storage proteins, were chosen as representative docking targets. The X-ray crystallography data for β-LG (PDB ID: 1BEB) at 1.80 Å resolution [ 16 ] was obtained from the RCSB Protein Data Bank ( http://www.rcsb.org ). Natural pea globulin does not have a crystal structure, and molecular docking was performed by predicting the structures of 7S and 11S using AlphaFold 3 based on the amino acid sequences in the UniProt database ( http://www.uniprot.org ), and the amino acid sequences are shown in Fig. S1 . The three-dimensional structure of humulinone was retrieved from PubChem ( https://pubchem.ncbi.nlm.nih.gov/ ). Molecular Graphics Laboratory (MGL) tools 1.5.7 were employed for the docking studies between proteins and humulinone. Before initiating the docking process, water molecules were removed from the protein macromolecules, and polar hydrogen atoms were added. Subsequently, AutoGrid4 was utilized to organize all atoms in β-LG, 7S and 11S into grids. For β-LG, a grid box with dimensions of 72×82×124 Å and a grid spacing of 0.531 Å was set up. For 7S and 11S, a grid box size of 126×126×126 Å and 112×100×114 Å, then a grid spacing of 0.806 Å and 0.703 Å were used separately. AutoDock4 was then applied for docking calculations. The program was run 50 times using a Genetic Algorithm, and the Lamarckian GA module ranked the results. The optimal complex conformation was determined by calculating the minimal energy score for each docking result. The obtained optimal result was further visualized in a two-dimensional graph displaying the molecular model using Pymol 2.4.1 [ 17 ]. Dynamic surface tension The humulinone solution and WP/PP (1 mg/mL) mixed with different proportions (mass ratio = 1:0, 1:0.01, 1:0.05, 1:0.1). The dynamic surface tension (γ) of the solutions was measured using a tensiometer (DCAT21, Dataphysics, Germany) by Wilhelmy method, with continuous monitoring for 1800 seconds [ 18 ]. Surface hydrophobicity analysis The surface hydrophobicity was determined using the ANS fluorescence probe, as described by Tang et al. [ 19 ]. Solutions of WP/PP and humulinone were adjusted to protein concentrations of 0.1, 0.2, 0.3, 0.4, and 0.5 mg/mL. ANS (8 mM, 5 µL) was then added to 1 mL of the sample solutions. Fluorescence intensity was measured at an excitation wavelength of 390 nm and an emission wavelength of 470 nm. The initial slope of the fluorescence intensity curve as a function of protein concentration was used to determine the relative surface hydrophobicity. Foaming performance The method for foaming performance measurement was adapted from [ 20 ]. WP and PP (1 mg/mL) were mixed with humulinone in mass ratios of 1:0, 1:0.01, 1:0.05, and 1:0.1 to prepare the sample solutions. A 15 mL aliquot of the sample solution was added to a 50 mL graduated cylinder and subjected to high-speed shear mixing at 9000 rpm for 90 seconds to generate foam. The initial foam volume V 0 (mL) was recorded over time. The foamability was calculated according to the Eq. ( 1 ). After the initial foam formation, some liquid remains within the foam, causing it to settle and the liquid surface to rise rapidly, which compresses the foam volume and does not accurately reflect foam stability. Therefore, in the calculation of foam stability, measurements were recorded starting from the point when the liquid surface remained relatively constant (after 5 mins). V 5 and V 30 represent the foam volumes at 5 mins and 30 mins, respectively. Foam stability was calculated according to Eq. ( 2 ). Furthmore, to characterize the fineness of the foam, place the newly formed foam on a slide and observe the foam size distribution under an optical microscope. The foam size was counted and analyzed by Image J. To evaluate the application of humulinone in real samples, humulinone was added to cow's milk and pea milk to achieve mass concentrations of 0.1% and 0.2%. A 15 mL sample was placed into a 50 mL graduated cylinder and whipped using a high-speed shear mixer at 9000 rpm for 90 seconds to generate foam. The formation and changes in the foam were photographed and recorded. Statistical analysis All experiments were performed in triplicate, and all data are presented as mean ± standard deviation (n = 3). Statistical analysis was conducted using one-way analysis of variance (ANOVA), with significance determined at p < 0.05. Results and discussion Preparation and identification of WP/PP and humulinone The protein concentrations were determined using the Lowry method, revealing that the soluble protein contents obtained from 100 mg/mL solutions of WP and PP were 44.45 mg/mL and 3.50 mg/mL, respectively. As shown in Fig. S2, WP primarily consists of β-lactoglobulin (18.4 kDa), α-lactalbumin (14 kDa), and a small amount of bovine serum albumin (66.6 kDa) [ 21 ]. In contrast, the composition and classification of PP are more complex, making it difficult to distinguish specific bands and their corresponding proteins on the gel. Based on sedimentation coefficients, pea protein can be classified into 11S legumin, 7S vicilin, and albumin [ 22 ]. The 11S legumin is a hexameric protein with a molecular weight of approximately 360 kDa, the 7S vicilin and albuminis a trimeric protein with a molecular weight of approximately 150 kDa and 210 kDa, respectively [ 23 ]. Humulinone consists of cohumulinone, n-humulinone, and adhumulinone [ 24 ]. The purification method for humulinone was carried out based on protocols previously developed by our research group, resulting in a purity of 84.60% [ 13 ]. The purified humulinone sample was then used for subsequent experiments. Effects of humulinone on foamability of WP and PP To eliminate the potential interference from humulinone itself in the protein foaming experiments, a control test was conducted in which humulinone alone was subjected to stirring. As shown in Fig. S3, humulinone did not generate any foam under these conditions, indicating that it does not possess intrinsic surfactant properties. Therefore, the observed enhancement in protein foaming can be attributed to the interaction between humulinone and the proteins, rather than to any surface-active behavior of humulinone itself. Foaming is one of the important properties of proteins, the foam volume was recorded as shown in Fig. S4. As shown in Fig. 2 A-B, humulinone significantly enhanced the foamability of WP. In the absence of humulinone, the foamability of a 1.00 mg/mL WP solution was 78.89 ± 8.39%. As the concentration of humulinone increased, the foamability of WP progressively improved. When the mass ratio of WP to humulinone reached 1:0.1, the foamability of WP increased to 184.44 ± 3.85%, which is 2.34 times higher than that of WP alone. Furthermore, humulinone did not significantly affect the foam stability of WP. Regardless of the presence of humulinone, the foam stability of WP remained within the range of 68%~75%. Although foam stability did not improve, the substantial increase in foamability led to a higher foam volume for the WP solution with a 1:0.1 (w/w) humulinone ratio after 60 mins of foam formation (13.17 ± 0.76 mL), compared to the WP solution alone (11.83 ± 1.26 mL). As shown in Fig. 2 C-D, humulinone also significantly enhanced the foamability of PP. In the absence of humulinone, the foamability of a 1.00 mg/mL PP solution was 116.67 ± 3.33%. As the concentration of humulinone increased, the foamability of PP progressively improved. When the mass ratio of PP to humulinone reached 1:0.1, the foamability of PP increased to 193.33 ± 6.67%, which is 1.66 times higher than that of PP alone. Similarly, humulinone did not significantly affect the foam stability of PP. Regardless of the presence of humulinone, the foam stability of PP remained within the range of 67%~73%. At a protein concentration of 1.00 mg/mL, the foamability of PP (116.67 ± 3.33%) was higher than that of WP (78.89 ± 8.39%). The effect of humulinone on the foamability of WP was stronger than that on PP. When the mass ratio of humulinone reached 1:0.1 (w/w), the foamability of WP increased by approximately 2.34 times, while the foamability of PP increased by approximately 1.66 times, with a difference of about onefold between the two. The foam stability of both WP and PP was similar, remaining around 70%, regardless of the addition of humulinone. Moreover, humulinone did not have a significant effect on the foam stability of either WP or PP. This may be attributed to the fact that WP and PP are mixtures of various proteins, some of which inherently exhibit poor foam stability or consist of low molecular weight peptides, rendering humulinone's impact on these components minimal. Furthermore, as shown in Fig. 3 A-B, optical microscopy revealed that the bubbles formed in PP and WP solutions containing humulone (protein: humulone = 1:0.1, w/w) were more uniform and exhibited a more spherical morphology compared to those formed by the proteins alone. Bubble size distribution was analyzed using ImageJ software, and the results (Fig. S5) indicated that the average bubble diameter was smaller in the presence of humulone. This suggests that humulone contributes to the formation of more homogeneous and finer foam structures. Interactions between humulinone and WP/PP Due to differences in the content of tryptophan, tyrosine, and phenylalanine between WP and PP, the fluorescence spectra exhibited distinct characteristics [ 25 ]. Figure 4 A-B show the effect of humulinone on the fluorescence spectra of WP (A) and PP (B). The maximum absorption peak for WP was observed at 335 nm, while PP showed its maximum absorption peak at 352 nm, with an additional peak at 307 nm. Upon the addition of humulinone, the fluorescence intensity of both WP and PP decreased, indicating that humulinone interacted with both proteins. During the fluorescence quenching process, no red shift or blue shift in the maximum absorption peak was observed, suggesting that humulinone did not cause significant changes in the polarity of the microenvironment around the tryptophan, tyrosine, and phenylalanine residues in WP and PP [ 26 ]. As shown in Fig. 4 C, a characteristic peak corresponding to the stretching vibration of hydrogen bonds and the -NH group was observed at 3364 cm⁻¹ for WP. After the addition of humulinone, this peak shifted to a lower wavenumber, indicating that the interaction between WP and humulinone is associated with hydrogen bonding [ 27 , 28 ]. The characteristic peak at 2933 cm⁻¹ corresponds to the -CH vibration on saturated carbon. It is evident that this peak also underwent a noticeable shift under the influence of humulinone. Furthermore, in the amide I band (1600 ~ 1700 cm⁻¹), the characteristic peak of WP also changed, suggesting that the secondary structure of WP was affected. Similar to WP, as shown in Fig. 4 D, the absorption peak of PP at 3339 cm⁻¹ corresponds to the -NH stretching vibration and hydrogen bonding. Upon interaction with humulinone, this characteristic peak shifted, indicating that the binding between PP and humulinone is associated with hydrogen bonding. Additionally, in the amide I band, the characteristic peak of PP shifted from 1617 cm⁻¹ to 1672 cm⁻¹, and the peak shape also underwent significant changes, suggesting that humulinone affected the secondary structure of PP. Furthermore, the characteristic peak of PP at 1029 cm⁻¹ shifted to around 1060 cm⁻¹, further confirming the interaction between PP and humulinone. Protein-ligand docking studies were conducted to identify potential binding sites for humulinone and to investigate the interaction mechanisms between humulinone and proteins. A total of 50 molecular docking runs were performed between β-LG and humulinone. The lowest-energy binding conformation, with a binding energy of -23.12 kJ/mol, was selected for further analysis, as shown in Fig. 5 A. Based on the 3D molecular docking model, the humulinone molecule was found to be embedded between the two subunits of β-LG, forming hydrogen bonds with Asp137, Leu143, and Met145, with respective bond distances of 1.9 Å, 2.0 Å, and 2.2 Å [ 29 ]. For PP, 7S vicilin and 11S legumin were selected as representative proteins for molecular docking with humulinone. Due to the large molecular weight and hexameric structure of 11S legumin, which makes full docking computation challenging, a single subunit of 11S was used for docking analysis. The results are presented in Fig. 5 B and Fig. S6, respectively. The lowest binding energies were − 24.20 kJ/mol for the 7S-humulinone complex and − 21.03 kJ/mol for the 11S-humulinone complex. In the 7S model, humulinone is embedded within the hydrophobic cavities formed by two adjacent β-barrel domains, establishing hydrogen bonds with several β-sheet-associated residues, including Gln16, Lys31, Gln34, Arg46, Gln130, and Lys307. For 11S, humulinone interacts with regions comprising β-folds and irregular coils, forming hydrogen bonds with Tyr356, Pro501, and Lys503. In summary, molecular docking analysis confirmed the presence of hydrogen bonding interactions between humulinone and both WP and PP, supporting the findings from FTIR spectroscopy, and providing a theoretical basis for elucidating the mechanism by which humulinone modulates the foaming behavior of WP/PP systems. Effect of humulinone on the structure of WP/PP By analyzing the infrared spectra of the protein in the range of 1600–1700 cm − 1 , the secondary structure content of WP/PP and WP/PP-humulinone complexes was calculated. The secondary structure of proteins reflects the stability of protein foam to some extent. The stronger the rigid structure, i.e., the protein that maintains a certain folding structure, the better the foam stability [ 30 , 31 ]. As shown in Fig. 6 A, WP is primarily composed of β-turns, α-helices, and random coils, with a low content of β-sheets, indicating that WP predominantly adopts a more flexible, loose structure. Similar to the effect of humulinone on protein Z, the interaction with humulinone also reduces the content of β-turns in WP and increases the content of β-sheets [ 13 ]. The difference is that the α-helix content of WP also increases slightly. Overall, under the influence of humulinone, the rigidity of the secondary structure of WP increases. Additionally, after foaming, there was no significant change in the secondary structure of WP, indicating that during protein adsorption, the folded structure of WP did not undergo significant extension, and only a small portion of the flexible structure was altered. As shown in Fig. 6 B, PP is a protein predominantly composed of α-helix, followed by β-turn and random coil. Unlike WP, the addition of humulinone did not decrease the β-turn content in PP, but rather slightly increased it. Additionally, the content of random coils also increased, while the content of α-helices and β-sheets decreased. This suggests that humulinone enhances the flexibility of PP, which could be one of the reasons for the increased foaming ability [ 32 ]. Similar to WP, the secondary structure of PP did not show significant changes after foaming. Overall, humulinone altered the secondary structure of WP/PP to some extent, increasing the rigidity of WP and enhancing the flexibility of PP. However, these changes did not follow a consistent pattern. This is because protein structure is complex, and different proteins possess distinct structures, requiring specific analysis based on the situation [ 33 ]. Several studies have shown that protein surface hydrophobicity is related to foaming properties [ 34 ]. As shown in Fig. 6 C-D, humulinone did not significantly alter the surface hydrophobicity of WP and PP, suggesting that the binding with humulinone did not expose the hydrophobic regions that are typically buried within the protein structure. Hydrophobic interactions are essential for maintaining protein conformation, allowing proteins to remain stable in aqueous solutions. The interaction between humulinone and the proteins primarily involves hydrogen bonding, without causing significant disruption to the proteins’ native structures. In addition, the lack of significant change in surface hydrophobicity also coincides with the fact that the maximum absorption wavelength was not red-shifted or blue-shifted in the fluorescence quenching results. Based on these results, it can be speculated that humulinone binds extensively to the surfaces of WP and PP through hydrogen bonds, thereby increasing the overall hydrophobicity of the complexes. This in turn accelerates the protein adsorption process and enhances foamability. Effect of humulinone on the interfacial properties of WP/PP To elucidate the role of humulinone in the foaming process of proteins, the effects of humulinone on the surface tension and adsorption kinetics of WP/PP were investigated. To explore the impact of humulinone on the interfacial properties of WP/PP, the dynamic surface tension of the sample solutions was measured using the Wilhelmy method. Surface tension can reflect the rate of adsorption and swelling of proteins at the air-water interface, thus determining the foaming ability of proteins [ 35 ]. As shown in Fig. S7A-B, during the measurement, the surface tension of all samples decreased as the adsorption experiment progressed. Specifically, the addition of humulinone resulted in a noticeable reduction in surface tension: for WP, it decreased from 53.9 mN/m to 49.3 mN/m, corresponding to an 8.7% reduction; for PP, the surface tension decreased from 46.8 mN/m to 41.5 mN/m, reflecting an 11.3% reduction. Within the first 200 seconds, the surface tension dropped rapidly, and then the rate of decrease slowed down, indicating that WP/PP underwent adsorption at the interface. With the addition of humulinone, the surface tension of WP/PP continued to decrease, suggesting that humulinone enhanced the surface activity of the proteins. This result also explains the observed increase in foaming properties with the addition of humulinone. Additionally, when the ratio of humulinone to protein increased to 1:0.1 (w/w), a noticeable inflection point in the surface tension curve was observed between 150 ~ 210 seconds, indicating that humulinone accelerated the interfacial adsorption process of the proteins [ 36 ]. These findings suggest that humulinone facilitates the initial diffusion and interfacial adsorption of protein molecules, thereby enhancing their foaming capacity. Generally, protein adsorption at the air/water or oil/water interface involves diffusion, unfolding, and rearrangement [ 37 ]. Diffusion refers to the process by which proteins gradually move from the bulk solution to the air/water interface. The variation of dynamic surface pressure (π) with adsorption time (t) can be analyzed using the modified form of the Ward-Tordai equation Eq. ( 3 ): $$\:\text{π=2}{\text{C}}_{\text{0}}{\text{K}}_{\text{B}}\text{T}\sqrt{\text{D}\text{t}\text{/3.14}}$$ 3 where C₀, K B , T, D, and t represent the sample concentration, Boltzmann constant, absolute temperature, diffusion coefficient, and adsorption time, respectively. After a brief period of diffusion-dominated adsorption, the adsorption rate gradually slows down, and the process becomes increasingly influenced by molecular unfolding and rearrangement [ 38 ]. The rate of protein unfolding and molecular rearrangement at the interface can be calculated using Eq. ( 4 ): $$\:\text{ln[(}{\pi}_{\text{f}}\text{-}{\pi}_{\text{t}}\text{)/(}{\pi}_{\text{f}}\text{-}{\pi}_{\text{0}}\text{)]=-}{\text{k}}_{\text{i}}\text{t}$$ 4 Where π f , π t , and π 0 represent the surface pressure at final adsorption time (3600 s), any time and initial time, respectively, and k i represents the first-order rate constant. The slope of the first linear stage corresponds to the penetration rate (K P ), while the slope of the second slope corresponds to the molecular rearrangement rate (K R ). As shown in Fig. S7C-D, the surface pressure during the initial stage of diffusion is expected to be close to zero. However, the surface pressure observed in the experimental results started from a relatively high value, meaning that the entire diffusion process was not monitored in this experiment. Nonetheless, the surface pressure at 0 seconds clearly shows that the samples with added humulinone exhibited higher surface pressures, indicating that humulinone facilitated the diffusion process of WP/PP [ 18 ]. Furthermore, the calculations in Table 1 shows an increase in the diffusion coefficient (K diff ), suggesting that during the monitored diffusion phase, humulinone enhanced the diffusion rate of WP, while no similar effect was observed for PP. As the protein adsorption time increases, the concentration of protein at the interface gradually increases, leading to a corresponding rise in surface pressure. When the relationship between the surface pressure (π) and the time (t 1/2 ) is no longer linear, the adsorption process is no longer diffusion-controlled but is instead governed by the capacity for interface expansion (K P ) and molecular rearrangement (K R ) (Fig. S8A-B) [ 39 ]. Interface expansion refers to the structural changes of proteins at the air/water interface, which result in the exposure of hydrophobic groups to air and hydrophilic groups to the aqueous phase. Molecular rearrangement is a slow and complex process in which protein molecules at the interface undergo further rearrangement to form a stable interfacial film. As shown in Table 1 , under the influence of humulinone, K P for WP increased, suggesting that humulinone not only enhanced the diffusion process of WP but also accelerated the expansion process. However, with the addition of humulinone, K R exhibited an initial decrease followed by an increase. For PP, humulinone reduced the K P , but no significant pattern was observed for K R . Table 1 Characteristic parameters, including the apparent diffusion rate (K diff ), constants of penetration and structural rearrangement at the interface (K P and K R ) for WP/PP and WP/PP-humulinone. Protein Protein: Humulinone K diff (mN/m/s 1/2 ) K P (×10 − 3 ) K R (×10 − 2 ) Whey protein 1:0 0.2156 ± 0.0017 1.2557 ± 0.0398 9.3187 ± 0.1383 1:0.01 0.2286 ± 0.0020 1.2747 ± 0.0278 5.8094 ± 0.1114 1:0.05 0.2353 ± 0.0025 1.4246 ± 0.0260 14.3989 ± 0.2183 1:0.10 0.2718 ± 0.0037 1.3433 ± 0.0167 13.6639 ± 0.2775 Pea protein 1:0 0.1413 ± 0.0016 1.5716 ± 0.016 3.9392 ± 0.1794 1:0.01 0.1019 ± 0.0036 1.4519 ± 0.0028 4.1087 ± 0.5681 1:0.05 0.07591 ± 0.0009 1.5153 ± 0.0038 3.5237 ± 0.2163 1:0.10 0.1074 ± 0.0017 1.3452 ± 0.0039 3.9398 ± 0.1794 Effect of humulinone on the foam characteristics of milk and pea protein milks Figure 7 shows the changes in foam produced by milk and pea protein milk under the influence of humulinone over time. The milk used in the experiment was labeled as containing 3.2 g/100 mL of protein. As shown in Fig. 7 A-B, the milk exhibited poor foamability, which could be attributed to the complex composition of milk. Components like lipids may interfere with foam formation and stability, and the foam formation method used could also be a contributing factor. Since both the milk and the foam are opaque and white, it is difficult to accurately measure foam volume. However, from the overall volume of foam and liquid, it can be observed that the addition of humulinone slightly improved the foamability of the milk, although it did not significantly contribute to foam stability. The pea protein milk used in the experiment is labeled with a protein content of 3.3 g/100 g. The ingredient list includes: water, pea protein, fructose syrup, coconut milk, vegetable oil, microcrystalline cellulose, dipotassium phosphate, and salt. The foamability of pea protein milk is similar to that of milk, producing relatively little foam (Fig. 7 C-D). However, the addition of humulinone has a more pronounced effect on enhancing the foamability of pea protein milk and helps to better maintain the foam. Milk and pea protein milk can be frothed into foam for use in coffee, desserts, and other applications. The experimental results indicate that humulinone can enhance the foam properties of whey protein and pea protein-based food products. However, the enhancement of foaming properties by humulinone in real food systems is less pronounced compared to pure protein systems, likely due to the complexity of actual food matrices, which contain various non-protein components such as lipids, carbohydrates, and mineral ions (e.g., sodium and calcium) that may interfere with protein interfacial behavior and foam formation. Conclusion This study explored the effects and mechanisms of humulinone on the foaming properties of two representative food proteins: whey protein (WP) and pea protein (PP). The addition of humulinone significantly improved the foamability of both proteins, with maximum increases of 2.34 times for WP and 1.66 times for PP at a mass ratio of 1:0.1 (protein: humulinone). However, no notable enhancement in foam stability was observed. Mechanistic analyses indicated that humulinone interacted with WP and PP mainly through hydrogen bonding, leading to reduced surface tension and accelerated interfacial diffusion. These interfacial changes are closely associated with the observed improvements in foam formation. Furthermore, humulinone induced structural modulation in both proteins—enhancing the rigidity of WP and increasing the flexibility of PP—suggesting that different structural adaptations can both contribute to better foamability via improved interfacial behavior. Although the results in real food matrices were not as good as those of single protein systems due to the complexity of the system, the results of the study provide a theoretical basis for the application of putrescine in protein-based foamed products. Future work may focus on optimizing formulation conditions and further exploring its role in multi-component food systems. Declarations Competing Interests The authors have no competing interests to declare that are relevant to the content of this article. Funding No funding was received to assist with the preparation of this manuscript. Author Contribution Yang Gao: Formal analysis, Investigation, Data curation, Writing – original draft. Chen Xu: Data curation, Resources, Validation. Hanhan Liu: Visualization, Methodology. Junyu Lin: Investigation, Resources. Chenyan Lv: Conceptualization, Methodology, Writing – review & editing. Data Availability No datasets were generated or analysed during the current study. References Bureiko A, Trybala A, Kovalchuk N, Starov V (2015) Current applications of foams formed from mixed surfactant–polymer solutions. Advances in Colloid and Interface Science 222:670–677. https://doi.org/10.1016/j.cis.2014.10.001 Shen P, Peng J, Sagis LMC, Landman J (2024) Air-water interface properties and foam stabilization by mildly extracted lentil protein. 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Langmuir 19:2349–2356. https://doi.org/10.1021/la020720e Jarpa-Parra M, Bamdad F, Tian Z, et al (2015) Impact of pH on molecular structure and surface properties of lentil legumin-like protein and its application as foam stabilizer. Colloids and Surfaces B: Biointerfaces 132:45–53. https://doi.org/10.1016/j.colsurfb.2015.04.065 Sheng L, Wang J, Huang M, et al (2016) The changes of secondary structures and properties of lysozyme along with the egg storage. International Journal of Biological Macromolecules 92:600–606. https://doi.org/10.1016/j.ijbiomac.2016.07.068 Zhan F, Li J, Wang Y, et al (2018) Bulk, foam, and interfacial properties of tannic acid/sodium caseinate nanocomplexes. Journal of Agricultural and Food Chemistry 66:6832–6839. https://doi.org/10.1021/acs.jafc.8b00503 Foegeding EA, Luck PJ, Davis JP (2006) Factors determining the physical properties of protein foams. Food Hydrocolloids 20:284–292. https://doi.org/10.1016/j.foodhyd.2005.03.014 Ding L, Lu L, Sheng L, et al (2020) Mechanism of enhancing foaming properties of egg white by super critical carbon dioxide treatment. Food Chemistry 317:126349. https://doi.org/10.1016/j.foodchem.2020.126349 Dickinson E (2011) Mixed biopolymers at interfaces: Competitive adsorption and multilayer structures. Food Hydrocolloids 25:1966–1983. https://doi.org/10.1016/j.foodhyd.2010.12.001 Graham DE, Phillips MC (1979) Proteins at liquid interfaces: III. Molecular structures of adsorbed films, Journal of Colloid and Interface Science 70 (1979) 70:427–439. https://doi.org/10.1016/0021-9797(79)90050-X Tang C-H, Shen L (2015) Dynamic adsorption and dilatational properties of BSA at oil/water interface: Role of conformational flexibility. Food Hydrocolloids 43:388–399. https://doi.org/10.1016/j.foodhyd.2014.06.014 Additional Declarations No competing interests reported. Supplementary Files SupplementalMaterials.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 14 Jul, 2025 Reviews received at journal 02 Jul, 2025 Reviewers agreed at journal 24 Jun, 2025 Reviews received at journal 19 Jun, 2025 Reviewers agreed at journal 16 Jun, 2025 Reviewers agreed at journal 16 Jun, 2025 Reviewers invited by journal 15 Jun, 2025 Editor assigned by journal 06 Jun, 2025 Submission checks completed at journal 06 Jun, 2025 First submitted to journal 03 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6811741","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":472194611,"identity":"6cbf7cb5-3545-443d-a2ac-c1a4fbbbf9e4","order_by":0,"name":"Yang Gao","email":"","orcid":"","institution":"China Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Gao","suffix":""},{"id":472194612,"identity":"296381ae-9462-481f-baff-af8b81066985","order_by":1,"name":"Chen Xu","email":"","orcid":"","institution":"China Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Chen","middleName":"","lastName":"Xu","suffix":""},{"id":472194613,"identity":"6c64298b-72af-46ec-a518-4e6a21b01fd0","order_by":2,"name":"Hanhan Liu","email":"","orcid":"","institution":"China Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Hanhan","middleName":"","lastName":"Liu","suffix":""},{"id":472194614,"identity":"8dabdaab-48e2-424e-93a6-bcdbce6dcfac","order_by":3,"name":"Junyu Lin","email":"","orcid":"","institution":"China Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Junyu","middleName":"","lastName":"Lin","suffix":""},{"id":472194615,"identity":"1e9791ae-bc75-49ae-84ba-ab16373f9f24","order_by":4,"name":"Chenyan Lv","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA30lEQVRIiWNgGAWjYBACfmbGxgcJFTVyEC4bEVok25ubDT6cOWZMvBaDM8fbJGe2MSc2EK2F4UZimzQPG1t6f/sZA4YPZYcZ+Gc34NfBOCOx2ZqHRyZ3xpkcA8YZ5w4zSNw5gF8Ls0Ri420eCbbcDQw5Bsy8bYcZDCQS8Gthk0hskOYxYE434H9jwPyXGC08PAebJGckMCcYSABtYSRGiwR7IzCQDxwznHHjWcHBnnPpPBI3CGixP8z+8EHivxp5/v7kjQ9+lFnL8c8goAUFHAC5lAT1o2AUjIJRMApwAQCZWkLcLPBz0QAAAABJRU5ErkJggg==","orcid":"","institution":"China Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Chenyan","middleName":"","lastName":"Lv","suffix":""}],"badges":[],"createdAt":"2025-06-03 13:23:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6811741/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6811741/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84826174,"identity":"a90d6610-47ad-4ea3-9952-6fb28b74b8f4","added_by":"auto","created_at":"2025-06-17 17:13:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":49472,"visible":true,"origin":"","legend":"\u003cp\u003eChemical structures α-acid and humulinone.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6811741/v1/ef1aff263a886c1af762c9f4.png"},{"id":84825462,"identity":"ffbbbabb-dddb-4c9a-9d11-0d1fbfcbe35b","added_by":"auto","created_at":"2025-06-17 17:05:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":477200,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of humulinone on the foam properties of WP/PP. (A) The visual images of foam after whipping of WP-humulinone solutions with different mass ratios; (B) Foamability and foam stability of WP-humulinone. (C) The visual images of foam after whipping of PP-humulinone solutions with different mass ratios; (D) Foamability and foam stability of PP-humulinone.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6811741/v1/0c444696528f8cb536da8adc.png"},{"id":84825464,"identity":"b61f5cc1-4ae5-4509-88ff-40f02892ea87","added_by":"auto","created_at":"2025-06-17 17:05:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":439140,"visible":true,"origin":"","legend":"\u003cp\u003eOptical microscope images of the effect of humulinone on the foam size of WP (A) and PP (B) (protein: humulone = 1:0.1, w/w). Scale bar is 1 cm.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6811741/v1/235b09b7e1a8688529865889.png"},{"id":84825467,"identity":"8794b8b5-3c3d-47f9-b040-4f5911d00f6b","added_by":"auto","created_at":"2025-06-17 17:05:21","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":239510,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of humulinone on the fluorescence spectra of WP (A) and PP (B). FTIR spectra of WP-humulinone complex (C) and PP-humulinone complex (D) with different mass ratios (1:0.05 and 1:0.1).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6811741/v1/4378ffce7408e19bc7b0f007.png"},{"id":84826175,"identity":"fa18ed71-8a70-4af6-b182-dd287fbf6dce","added_by":"auto","created_at":"2025-06-17 17:13:21","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":425741,"visible":true,"origin":"","legend":"\u003cp\u003eThree-dimensional view of the possible presence of binding sites for β-LG (A) and 7S vicilin (B) interacting with humulinone by hydrogen bonding.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6811741/v1/0f5e493be778f287512e0d27.png"},{"id":84827316,"identity":"f5bc1c37-2378-4975-aa25-1c46268e2ed5","added_by":"auto","created_at":"2025-06-17 17:29:21","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":135052,"visible":true,"origin":"","legend":"\u003cp\u003eSecondary structural components of WP-humulinone (A) and PP-humulinone (B) with different molar ratios in liquid and foam. Effects of humulinone on surface hydrophobicity of WP (C) and PP (D).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6811741/v1/8b1bdb1e5914a77dd38eea17.png"},{"id":84825481,"identity":"c28a36d2-8d80-4a79-b52f-402a4f44bc90","added_by":"auto","created_at":"2025-06-17 17:05:22","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":521723,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of humulinone on milk foam and pea protein milk foam. (A) Visual image of foam change: (A) milk, (C) pea protein milk; Line diagram of foam volume change over time: (B) milk, (D) pea protein milk.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6811741/v1/08d3e0ceb6d4508787877cb7.png"},{"id":84827673,"identity":"a043a429-9235-4b64-a72c-7b4ffca30b95","added_by":"auto","created_at":"2025-06-17 17:37:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2986074,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6811741/v1/e121f03a-8ee8-4314-9850-f2de9409f26b.pdf"},{"id":84826183,"identity":"b3503d1a-3118-4790-9e0f-2037fdf6912d","added_by":"auto","created_at":"2025-06-17 17:13:22","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":2503354,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalMaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-6811741/v1/7bcb46203688f0ec9b74088d.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Regulatory Mechanism of Humulinone on Protein Foam: applications in whey and pea protein","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFoams play a vital role in food products, enhancing both their visual appeal and taste experience. Despite their importance, foams are inherently thermodynamically unstable, prone to breaking and collapsing over time. To enhance the stability of foams, surfactants are essential, as they effectively reduce the surface tension at the air-water interface [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Proteins are particularly valuable in this context due to their high surface activity [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Dairy-derived proteins, in particular, are widely utilized as foaming agents in food processing. They are abundant, cost-effective, and possess excellent foaming properties along with high nutritional value [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, as the food industry increasingly shifts towards sustainable practices, there is a growing interest in plant-based proteins. Pea proteins, for instance, have gained popularity in various food products due to their high nutritional profile, extensive cultivation, affordability, and hypoallergenic nature. This trend highlights the potential of plant proteins to complement or even replace traditional dairy proteins in foamed applications, aligning with the demand for healthier and more sustainable food options [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePhysical modification, chemical modification or addition of small molecules have been widely used to improve the foam properties of proteins for better application in food processing and production [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Interaction between multicomponent food matrices plays a key role in regulating foam properties, and some studies have been conducted to improve the foam properties of proteins by adding small molecule surfactants or mixing multi-components. Nooshkam et al. devised mixtures of licorice extract/whey protein/sodium alginate, which have high foaming properties and produce highly stable foam [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Improvement of protein foam characteristics by adding small molecule actives, such as flavonoids (quercetin, rutin) interacting with plant proteins through non-covalent bonds, which on the one hand improves the foamability and emulsification of soy and pea proteins [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], and at the same time foamed proteins act as delivery carriers to protect the activity of flavonoids as well as to improve the bioavailability.\u003c/p\u003e \u003cp\u003eHops are one of the indispensable ingredients in beer brewing that brings a distinctive bitter flavor and aroma. Substances in hops such as iso-α-acid (an isomerization product of α-acids) also have a significant effect on beer foam. It has been found that the addition of iso-α-acid or its hydrogenated reduced derivatives to a beer liquid without hops increases the foam stability of that beer and enhances the adhesion of the foam to the walls of the glass [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Similar to iso-α-acids, humulinone are oxidation products of α-acids (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), and in a previous study by our group, we found that the \u0026ldquo;bubble cap\u0026rdquo; formed during beer fermentation contained significant amounts of iso-α-acids and humulinone [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In addition, dry-hopped beers with fine foam have much higher levels of humulinone than regular ales [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. These results imply an inextricable relationship between humulinone and beer foam.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHowever, the mechanism of action between humulinone and protein foaming has been less studied. It is worth mentioning that in our previous study, we found that humulinone enriched in aged hops greatly improved the foaming and foam stability of protein Z in beer as well as the beer's frothiness [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Therefore, it is hypothesized that humulinone can interact with other proteins and be applied in foamed food systems to improve the foaming properties of proteins, thereby improving the structure and texture of food products.\u003c/p\u003e \u003cp\u003eTherefore, we chose one of the most commonly used animal proteins in food systems, whey protein (WP), and a novel alternative protein product, pea protein (PP) of plant origin, to investigate the effects of humulinone on the foaming properties, physicochemical properties, and mechanisms of both. The effects of humulinone on the foam properties of WP and PP were characterized, and then the interactions between humulinone and PP were investigated by multispectroscopy and computer simulation, and the mechanism of humulinone affecting the molecular level of WP/PP was examined in terms of the interfacial properties and the protein structure, respectively. Finally, the effect of humulinone on the foam properties of WP and PP-based real food systems was investigated, providing theoretical basis for the application of humulinone in foam food.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eChemicals and materials\u003c/h2\u003e\n \u003cp\u003eWhey protein and pea protein were purchased from commercial maltsters in China. CO\u003csub\u003e2\u003c/sub\u003e hops extract was purchased from BarthHaas (Beijing China). 8-anilino-1-naphthalenesulfonic acid (ANS), and potassium bromide (KBr) were purchased from Bio Dee Biotechnology Co. Ltd (Beijing, China). SDS electrophoresis marker and reagents used for the gel electrophoresis were purchased from Solarbio Co. Ltd (Beijing, China). The pea milk and cow's milk are from Chipmunks (Shandong, China) and Mongoose (Beijing, China), respectively. All other reagents used were of analytical grade, and ultrapure water was used throughout the research.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003ePreparation of proteins and humulinone\u003c/h3\u003e\n\u003cp\u003eWP and PP were dissolved in 50 mM phosphate buffer (pH 6.5) at a concentration of 100.00 mg/mL, stirred at room temperature for 1 hour, and subsequently stored at 4°C overnight to ensure complete hydration. The resulting solutions were centrifuged at 10,000 rpm for 10 mins to remove insoluble materials. The supernatants were then dialyzed against 50 mM phosphate buffer (pH 6.5) to eliminate residual metal ions and low-molecular-weight impurities. The purified protein solutions were diluted to the desired concentrations with phosphate buffer for use in subsequent experiments. Protein composition was assessed using SDS-PAGE according to Laemmli’s method [\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e]. Protein concentrations were determined by the Lowry method, using bovine serum albumin (BSA) as the standard.\u003c/p\u003e\n\u003cp\u003eHumulone was prepared following the method described by Taniguchi [\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e], with slight modifications. Briefly, hop extract was dissolved in anhydrous ethanol at a 1:1 ratio (m:v), followed by the addition of a base to remove insoluble oily substances through heating at 40°C, yielding a clear filtrate. Dilute hydrochloric acid was then slowly added to the filtrate to adjust the pH to 1–2. The solution was refrigerated at 4°C overnight to facilitate the precipitation of α-acid paste. Subsequently, 10.00 g of the α-acid paste was dissolved in 50 mL of diethyl ether along with 5 mL of cumene hydroperoxide, and the mixture was added to 350 mL of saturated sodium bicarbonate solution. The resulting solution was stored in a sealed container at room temperature, protected from light, for 4 days. After this period, the precipitated sodium salt of humulone was collected by filtration and washed three times with cold ether and deionized water, respectively. Finally, 1 g of the washed sodium salt was dissolved in 100 mL of methanol containing 1% phosphoric acid, and 1 L dilute hydrochloric acid (0.5 M) was added to induce precipitation. The resulting humulone powder was obtained by vacuum filtration and its purity was determined by high-performance liquid chromatography (HPLC).\u003c/p\u003e\n\u003ch3\u003eFluorescence titration\u003c/h3\u003e\n\u003cp\u003eThe fluorescence quenching titration experiments were run on a Cary Eclipse spectrophotometer (Cary, Varian, USA). 1 mL of 0.4 mg/mL WP or PP solution was placed in a quartz cuvette with a path length of 1 cm, to which humulinone solution was added, and the fluorescence intensity was recorded after blowing uniformly. The experimental temperature was 25 ℃, the excitation wavelength was 280 nm, and the absorption wavelength was 290–500 nm. The excitation slit width was 5 nm, and the emission slit width was 5 nm for WP and 10 nm for PP [\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eFourier transform infrared spectroscopy (FTIR) spectra\u003c/h3\u003e\n\u003cp\u003eThe FTIR spectra were obtained using a Thermo Nicolet iS50 FTIR spectrometer (Thermo Nicolet Analytical Instruments, MA, USA). The WP/PP solutions were mixed with humulinone solutions in mass ratios of 1:0, 1:0.01, 1:0.05, and 1:0.1. The mixtures were freeze-dried to obtain solid powders. In addition, the mixed solutions were whipped to form foams, which were immediately collected and rapidly frozen using liquid nitrogen. The frozen foams were then freeze-dried to obtain solid powders for FTIR analysis. The freeze-dried powders were ground with dried KBr under infrared light. The mixture was then pressed into transparent, uniform thin films using a pellet press and placed into the FTIR instrument. Scans were conducted in the range of 4000 ~ 400 cm\u003csup\u003e− 1\u003c/sup\u003e, with 32 scans performed at a resolution of 4 cm\u003csup\u003e− 1\u003c/sup\u003e. Data in the range of 1600 ~ 1700 cm\u003csup\u003e− 1\u003c/sup\u003e were fitted to analyze the secondary structure of the proteins.\u003c/p\u003e\n\u003ch3\u003eProtein-ligand docking\u003c/h3\u003e\n\u003cp\u003eThe binding interactions between humulinone and WP and PP were investigated using Autodock 4.2.6 software. β-Lactoglobulin (β-LG), the predominant protein in WP, was selected for molecular docking analysis. For PP, 7S vicilin and 11S legumin, the major storage proteins, were chosen as representative docking targets. The X-ray crystallography data for β-LG (PDB ID: 1BEB) at 1.80 Å resolution [\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e] was obtained from the RCSB Protein Data Bank (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.rcsb.org\u003c/span\u003e\u003c/span\u003e). Natural pea globulin does not have a crystal structure, and molecular docking was performed by predicting the structures of 7S and 11S using AlphaFold 3 based on the amino acid sequences in the UniProt database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.uniprot.org\u003c/span\u003e\u003c/span\u003e), and the amino acid sequences are shown in Fig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e. The three-dimensional structure of humulinone was retrieved from PubChem (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubchem.ncbi.nlm.nih.gov/\u003c/span\u003e\u003c/span\u003e). Molecular Graphics Laboratory (MGL) tools 1.5.7 were employed for the docking studies between proteins and humulinone.\u003c/p\u003e\n\u003cp\u003eBefore initiating the docking process, water molecules were removed from the protein macromolecules, and polar hydrogen atoms were added. Subsequently, AutoGrid4 was utilized to organize all atoms in β-LG, 7S and 11S into grids. For β-LG, a grid box with dimensions of 72×82×124 Å and a grid spacing of 0.531 Å was set up. For 7S and 11S, a grid box size of 126×126×126 Å and 112×100×114 Å, then a grid spacing of 0.806 Å and 0.703 Å were used separately. AutoDock4 was then applied for docking calculations. The program was run 50 times using a Genetic Algorithm, and the Lamarckian GA module ranked the results. The optimal complex conformation was determined by calculating the minimal energy score for each docking result. The obtained optimal result was further visualized in a two-dimensional graph displaying the molecular model using Pymol 2.4.1 [\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eDynamic surface tension\u003c/h2\u003e\n \u003cp\u003eThe humulinone solution and WP/PP (1 mg/mL) mixed with different proportions (mass ratio = 1:0, 1:0.01, 1:0.05, 1:0.1). The dynamic surface tension (γ) of the solutions was measured using a tensiometer (DCAT21, Dataphysics, Germany) by Wilhelmy method, with continuous monitoring for 1800 seconds [\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eSurface hydrophobicity analysis\u003c/h3\u003e\n\u003cp\u003eThe surface hydrophobicity was determined using the ANS fluorescence probe, as described by Tang et al. [\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e]. Solutions of WP/PP and humulinone were adjusted to protein concentrations of 0.1, 0.2, 0.3, 0.4, and 0.5 mg/mL. ANS (8 mM, 5 µL) was then added to 1 mL of the sample solutions. Fluorescence intensity was measured at an excitation wavelength of 390 nm and an emission wavelength of 470 nm. The initial slope of the fluorescence intensity curve as a function of protein concentration was used to determine the relative surface hydrophobicity.\u003c/p\u003e\n\u003ch3\u003eFoaming performance\u003c/h3\u003e\n\u003cp\u003eThe method for foaming performance measurement was adapted from [\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e]. WP and PP (1 mg/mL) were mixed with humulinone in mass ratios of 1:0, 1:0.01, 1:0.05, and 1:0.1 to prepare the sample solutions. A 15 mL aliquot of the sample solution was added to a 50 mL graduated cylinder and subjected to high-speed shear mixing at 9000 rpm for 90 seconds to generate foam. The initial foam volume V\u003csub\u003e0\u003c/sub\u003e (mL) was recorded over time. The foamability was calculated according to the Eq. (\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" width=\"531\" height=\"57\"\u003e\u003c/p\u003e\n\u003cp\u003eAfter the initial foam formation, some liquid remains within the foam, causing it to settle and the liquid surface to rise rapidly, which compresses the foam volume and does not accurately reflect foam stability. Therefore, in the calculation of foam stability, measurements were recorded starting from the point when the liquid surface remained relatively constant (after 5 mins). V\u003csub\u003e5\u003c/sub\u003e and V\u003csub\u003e30\u003c/sub\u003e represent the foam volumes at 5 mins and 30 mins, respectively. Foam stability was calculated according to Eq. (\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\"\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eFurthmore, to characterize the fineness of the foam, place the newly formed foam on a slide and observe the foam size distribution under an optical microscope. The foam size was counted and analyzed by Image J.\u003c/p\u003e\n\u003cp\u003eTo evaluate the application of humulinone in real samples, humulinone was added to cow's milk and pea milk to achieve mass concentrations of 0.1% and 0.2%. A 15 mL sample was placed into a 50 mL graduated cylinder and whipped using a high-speed shear mixer at 9000 rpm for 90 seconds to generate foam. The formation and changes in the foam were photographed and recorded.\u003c/p\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eStatistical analysis\u003c/h2\u003e\n \u003cp\u003eAll experiments were performed in triplicate, and all data are presented as mean ± standard deviation (n = 3). Statistical analysis was conducted using one-way analysis of variance (ANOVA), with significance determined at \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results and discussion","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ePreparation and identification of WP/PP and humulinone\u003c/h2\u003e \u003cp\u003eThe protein concentrations were determined using the Lowry method, revealing that the soluble protein contents obtained from 100 mg/mL solutions of WP and PP were 44.45 mg/mL and 3.50 mg/mL, respectively. As shown in Fig. S2, WP primarily consists of β-lactoglobulin (18.4 kDa), α-lactalbumin (14 kDa), and a small amount of bovine serum albumin (66.6 kDa) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In contrast, the composition and classification of PP are more complex, making it difficult to distinguish specific bands and their corresponding proteins on the gel. Based on sedimentation coefficients, pea protein can be classified into 11S legumin, 7S vicilin, and albumin [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The 11S legumin is a hexameric protein with a molecular weight of approximately 360 kDa, the 7S vicilin and albuminis a trimeric protein with a molecular weight of approximately 150 kDa and 210 kDa, respectively [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Humulinone consists of cohumulinone, n-humulinone, and adhumulinone [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The purification method for humulinone was carried out based on protocols previously developed by our research group, resulting in a purity of 84.60% [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The purified humulinone sample was then used for subsequent experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEffects of humulinone on foamability of WP and PP\u003c/h2\u003e \u003cp\u003eTo eliminate the potential interference from humulinone itself in the protein foaming experiments, a control test was conducted in which humulinone alone was subjected to stirring. As shown in Fig. S3, humulinone did not generate any foam under these conditions, indicating that it does not possess intrinsic surfactant properties. Therefore, the observed enhancement in protein foaming can be attributed to the interaction between humulinone and the proteins, rather than to any surface-active behavior of humulinone itself.\u003c/p\u003e \u003cp\u003eFoaming is one of the important properties of proteins, the foam volume was recorded as shown in Fig. S4. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B, humulinone significantly enhanced the foamability of WP. In the absence of humulinone, the foamability of a 1.00 mg/mL WP solution was 78.89\u0026thinsp;\u0026plusmn;\u0026thinsp;8.39%. As the concentration of humulinone increased, the foamability of WP progressively improved. When the mass ratio of WP to humulinone reached 1:0.1, the foamability of WP increased to 184.44\u0026thinsp;\u0026plusmn;\u0026thinsp;3.85%, which is 2.34 times higher than that of WP alone. Furthermore, humulinone did not significantly affect the foam stability of WP. Regardless of the presence of humulinone, the foam stability of WP remained within the range of 68%~75%. Although foam stability did not improve, the substantial increase in foamability led to a higher foam volume for the WP solution with a 1:0.1 (w/w) humulinone ratio after 60 mins of foam formation (13.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.76 mL), compared to the WP solution alone (11.83\u0026thinsp;\u0026plusmn;\u0026thinsp;1.26 mL).\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-D, humulinone also significantly enhanced the foamability of PP. In the absence of humulinone, the foamability of a 1.00 mg/mL PP solution was 116.67\u0026thinsp;\u0026plusmn;\u0026thinsp;3.33%. As the concentration of humulinone increased, the foamability of PP progressively improved. When the mass ratio of PP to humulinone reached 1:0.1, the foamability of PP increased to 193.33\u0026thinsp;\u0026plusmn;\u0026thinsp;6.67%, which is 1.66 times higher than that of PP alone. Similarly, humulinone did not significantly affect the foam stability of PP. Regardless of the presence of humulinone, the foam stability of PP remained within the range of 67%~73%.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAt a protein concentration of 1.00 mg/mL, the foamability of PP (116.67\u0026thinsp;\u0026plusmn;\u0026thinsp;3.33%) was higher than that of WP (78.89\u0026thinsp;\u0026plusmn;\u0026thinsp;8.39%). The effect of humulinone on the foamability of WP was stronger than that on PP. When the mass ratio of humulinone reached 1:0.1 (w/w), the foamability of WP increased by approximately 2.34 times, while the foamability of PP increased by approximately 1.66 times, with a difference of about onefold between the two. The foam stability of both WP and PP was similar, remaining around 70%, regardless of the addition of humulinone. Moreover, humulinone did not have a significant effect on the foam stability of either WP or PP. This may be attributed to the fact that WP and PP are mixtures of various proteins, some of which inherently exhibit poor foam stability or consist of low molecular weight peptides, rendering humulinone's impact on these components minimal.\u003c/p\u003e \u003cp\u003eFurthermore, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-B, optical microscopy revealed that the bubbles formed in PP and WP solutions containing humulone (protein: humulone\u0026thinsp;=\u0026thinsp;1:0.1, w/w) were more uniform and exhibited a more spherical morphology compared to those formed by the proteins alone. Bubble size distribution was analyzed using ImageJ software, and the results (Fig. S5) indicated that the average bubble diameter was smaller in the presence of humulone. This suggests that humulone contributes to the formation of more homogeneous and finer foam structures.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eInteractions between humulinone and WP/PP\u003c/h2\u003e \u003cp\u003eDue to differences in the content of tryptophan, tyrosine, and phenylalanine between WP and PP, the fluorescence spectra exhibited distinct characteristics [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B show the effect of humulinone on the fluorescence spectra of WP (A) and PP (B). The maximum absorption peak for WP was observed at 335 nm, while PP showed its maximum absorption peak at 352 nm, with an additional peak at 307 nm. Upon the addition of humulinone, the fluorescence intensity of both WP and PP decreased, indicating that humulinone interacted with both proteins. During the fluorescence quenching process, no red shift or blue shift in the maximum absorption peak was observed, suggesting that humulinone did not cause significant changes in the polarity of the microenvironment around the tryptophan, tyrosine, and phenylalanine residues in WP and PP [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, a characteristic peak corresponding to the stretching vibration of hydrogen bonds and the -NH group was observed at 3364 cm⁻\u0026sup1; for WP. After the addition of humulinone, this peak shifted to a lower wavenumber, indicating that the interaction between WP and humulinone is associated with hydrogen bonding [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The characteristic peak at 2933 cm⁻\u0026sup1; corresponds to the -CH vibration on saturated carbon. It is evident that this peak also underwent a noticeable shift under the influence of humulinone. Furthermore, in the amide I band (1600\u0026thinsp;~\u0026thinsp;1700 cm⁻\u0026sup1;), the characteristic peak of WP also changed, suggesting that the secondary structure of WP was affected.\u003c/p\u003e \u003cp\u003eSimilar to WP, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, the absorption peak of PP at 3339 cm⁻\u0026sup1; corresponds to the -NH stretching vibration and hydrogen bonding. Upon interaction with humulinone, this characteristic peak shifted, indicating that the binding between PP and humulinone is associated with hydrogen bonding. Additionally, in the amide I band, the characteristic peak of PP shifted from 1617 cm⁻\u0026sup1; to 1672 cm⁻\u0026sup1;, and the peak shape also underwent significant changes, suggesting that humulinone affected the secondary structure of PP. Furthermore, the characteristic peak of PP at 1029 cm⁻\u0026sup1; shifted to around 1060 cm⁻\u0026sup1;, further confirming the interaction between PP and humulinone.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eProtein-ligand docking studies were conducted to identify potential binding sites for humulinone and to investigate the interaction mechanisms between humulinone and proteins. A total of 50 molecular docking runs were performed between β-LG and humulinone. The lowest-energy binding conformation, with a binding energy of -23.12 kJ/mol, was selected for further analysis, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA. Based on the 3D molecular docking model, the humulinone molecule was found to be embedded between the two subunits of β-LG, forming hydrogen bonds with Asp137, Leu143, and Met145, with respective bond distances of 1.9 \u0026Aring;, 2.0 \u0026Aring;, and 2.2 \u0026Aring; [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFor PP, 7S vicilin and 11S legumin were selected as representative proteins for molecular docking with humulinone. Due to the large molecular weight and hexameric structure of 11S legumin, which makes full docking computation challenging, a single subunit of 11S was used for docking analysis. The results are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and Fig. S6, respectively. The lowest binding energies were \u0026minus;\u0026thinsp;24.20 kJ/mol for the 7S-humulinone complex and \u0026minus;\u0026thinsp;21.03 kJ/mol for the 11S-humulinone complex. In the 7S model, humulinone is embedded within the hydrophobic cavities formed by two adjacent β-barrel domains, establishing hydrogen bonds with several β-sheet-associated residues, including Gln16, Lys31, Gln34, Arg46, Gln130, and Lys307. For 11S, humulinone interacts with regions comprising β-folds and irregular coils, forming hydrogen bonds with Tyr356, Pro501, and Lys503. In summary, molecular docking analysis confirmed the presence of hydrogen bonding interactions between humulinone and both WP and PP, supporting the findings from FTIR spectroscopy, and providing a theoretical basis for elucidating the mechanism by which humulinone modulates the foaming behavior of WP/PP systems.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eEffect of humulinone on the structure of WP/PP\u003c/h2\u003e \u003cp\u003eBy analyzing the infrared spectra of the protein in the range of 1600\u0026ndash;1700 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, the secondary structure content of WP/PP and WP/PP-humulinone complexes was calculated. The secondary structure of proteins reflects the stability of protein foam to some extent. The stronger the rigid structure, i.e., the protein that maintains a certain folding structure, the better the foam stability [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, WP is primarily composed of β-turns, α-helices, and random coils, with a low content of β-sheets, indicating that WP predominantly adopts a more flexible, loose structure. Similar to the effect of humulinone on protein Z, the interaction with humulinone also reduces the content of β-turns in WP and increases the content of β-sheets [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The difference is that the α-helix content of WP also increases slightly. Overall, under the influence of humulinone, the rigidity of the secondary structure of WP increases. Additionally, after foaming, there was no significant change in the secondary structure of WP, indicating that during protein adsorption, the folded structure of WP did not undergo significant extension, and only a small portion of the flexible structure was altered.\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, PP is a protein predominantly composed of α-helix, followed by β-turn and random coil. Unlike WP, the addition of humulinone did not decrease the β-turn content in PP, but rather slightly increased it. Additionally, the content of random coils also increased, while the content of α-helices and β-sheets decreased. This suggests that humulinone enhances the flexibility of PP, which could be one of the reasons for the increased foaming ability [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Similar to WP, the secondary structure of PP did not show significant changes after foaming. Overall, humulinone altered the secondary structure of WP/PP to some extent, increasing the rigidity of WP and enhancing the flexibility of PP. However, these changes did not follow a consistent pattern. This is because protein structure is complex, and different proteins possess distinct structures, requiring specific analysis based on the situation [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSeveral studies have shown that protein surface hydrophobicity is related to foaming properties [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC-D, humulinone did not significantly alter the surface hydrophobicity of WP and PP, suggesting that the binding with humulinone did not expose the hydrophobic regions that are typically buried within the protein structure. Hydrophobic interactions are essential for maintaining protein conformation, allowing proteins to remain stable in aqueous solutions. The interaction between humulinone and the proteins primarily involves hydrogen bonding, without causing significant disruption to the proteins\u0026rsquo; native structures. In addition, the lack of significant change in surface hydrophobicity also coincides with the fact that the maximum absorption wavelength was not red-shifted or blue-shifted in the fluorescence quenching results. Based on these results, it can be speculated that humulinone binds extensively to the surfaces of WP and PP through hydrogen bonds, thereby increasing the overall hydrophobicity of the complexes. This in turn accelerates the protein adsorption process and enhances foamability.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eEffect of humulinone on the interfacial properties of WP/PP\u003c/h2\u003e \u003cp\u003eTo elucidate the role of humulinone in the foaming process of proteins, the effects of humulinone on the surface tension and adsorption kinetics of WP/PP were investigated. To explore the impact of humulinone on the interfacial properties of WP/PP, the dynamic surface tension of the sample solutions was measured using the Wilhelmy method.\u003c/p\u003e \u003cp\u003eSurface tension can reflect the rate of adsorption and swelling of proteins at the air-water interface, thus determining the foaming ability of proteins [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. As shown in Fig. S7A-B, during the measurement, the surface tension of all samples decreased as the adsorption experiment progressed. Specifically, the addition of humulinone resulted in a noticeable reduction in surface tension: for WP, it decreased from 53.9 mN/m to 49.3 mN/m, corresponding to an 8.7% reduction; for PP, the surface tension decreased from 46.8 mN/m to 41.5 mN/m, reflecting an 11.3% reduction. Within the first 200 seconds, the surface tension dropped rapidly, and then the rate of decrease slowed down, indicating that WP/PP underwent adsorption at the interface. With the addition of humulinone, the surface tension of WP/PP continued to decrease, suggesting that humulinone enhanced the surface activity of the proteins. This result also explains the observed increase in foaming properties with the addition of humulinone. Additionally, when the ratio of humulinone to protein increased to 1:0.1 (w/w), a noticeable inflection point in the surface tension curve was observed between 150\u0026thinsp;~\u0026thinsp;210 seconds, indicating that humulinone accelerated the interfacial adsorption process of the proteins [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. These findings suggest that humulinone facilitates the initial diffusion and interfacial adsorption of protein molecules, thereby enhancing their foaming capacity.\u003c/p\u003e \u003cp\u003eGenerally, protein adsorption at the air/water or oil/water interface involves diffusion, unfolding, and rearrangement [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Diffusion refers to the process by which proteins gradually move from the bulk solution to the air/water interface.\u003c/p\u003e \u003cp\u003eThe variation of dynamic surface pressure (π) with adsorption time (t) can be analyzed using the modified form of the Ward-Tordai equation Eq.\u0026nbsp;(\u003cspan refid=\"Equ3\" class=\"InternalRef\"\u003e3\u003c/span\u003e):\u003cdiv id=\"Equ3\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ3\" name=\"EquationSource\"\u003e\n$$\\:\\text{\u0026pi;=2}{\\text{C}}_{\\text{0}}{\\text{K}}_{\\text{B}}\\text{T}\\sqrt{\\text{D}\\text{t}\\text{/3.14}}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e3\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere C₀, K\u003csub\u003eB\u003c/sub\u003e, T, D, and t represent the sample concentration, Boltzmann constant, absolute temperature, diffusion coefficient, and adsorption time, respectively.\u003c/p\u003e \u003cp\u003eAfter a brief period of diffusion-dominated adsorption, the adsorption rate gradually slows down, and the process becomes increasingly influenced by molecular unfolding and rearrangement [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The rate of protein unfolding and molecular rearrangement at the interface can be calculated using Eq.\u0026nbsp;(\u003cspan refid=\"Equ4\" class=\"InternalRef\"\u003e4\u003c/span\u003e):\u003cdiv id=\"Equ4\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ4\" name=\"EquationSource\"\u003e\n$$\\:\\text{ln[(}{\\pi}_{\\text{f}}\\text{-}{\\pi}_{\\text{t}}\\text{)/(}{\\pi}_{\\text{f}}\\text{-}{\\pi}_{\\text{0}}\\text{)]=-}{\\text{k}}_{\\text{i}}\\text{t}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e4\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eWhere π\u003csub\u003ef\u003c/sub\u003e, π\u003csub\u003et\u003c/sub\u003e, and π\u003csub\u003e0\u003c/sub\u003e represent the surface pressure at final adsorption time (3600 s), any time and initial time, respectively, and k\u003csub\u003ei\u003c/sub\u003e represents the first-order rate constant. The slope of the first linear stage corresponds to the penetration rate (K\u003csub\u003eP\u003c/sub\u003e), while the slope of the second slope corresponds to the molecular rearrangement rate (K\u003csub\u003eR\u003c/sub\u003e).\u003c/p\u003e \u003cp\u003eAs shown in Fig. S7C-D, the surface pressure during the initial stage of diffusion is expected to be close to zero. However, the surface pressure observed in the experimental results started from a relatively high value, meaning that the entire diffusion process was not monitored in this experiment. Nonetheless, the surface pressure at 0 seconds clearly shows that the samples with added humulinone exhibited higher surface pressures, indicating that humulinone facilitated the diffusion process of WP/PP [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Furthermore, the calculations in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows an increase in the diffusion coefficient (K\u003csub\u003ediff\u003c/sub\u003e), suggesting that during the monitored diffusion phase, humulinone enhanced the diffusion rate of WP, while no similar effect was observed for PP.\u003c/p\u003e \u003cp\u003eAs the protein adsorption time increases, the concentration of protein at the interface gradually increases, leading to a corresponding rise in surface pressure. When the relationship between the surface pressure (π) and the time (t\u003csub\u003e1/2\u003c/sub\u003e) is no longer linear, the adsorption process is no longer diffusion-controlled but is instead governed by the capacity for interface expansion (K\u003csub\u003eP\u003c/sub\u003e) and molecular rearrangement (K\u003csub\u003eR\u003c/sub\u003e) (Fig. S8A-B) [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Interface expansion refers to the structural changes of proteins at the air/water interface, which result in the exposure of hydrophobic groups to air and hydrophilic groups to the aqueous phase. Molecular rearrangement is a slow and complex process in which protein molecules at the interface undergo further rearrangement to form a stable interfacial film. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, under the influence of humulinone, K\u003csub\u003eP\u003c/sub\u003e for WP increased, suggesting that humulinone not only enhanced the diffusion process of WP but also accelerated the expansion process. However, with the addition of humulinone, K\u003csub\u003eR\u003c/sub\u003e exhibited an initial decrease followed by an increase. For PP, humulinone reduced the K\u003csub\u003eP\u003c/sub\u003e, but no significant pattern was observed for K\u003csub\u003eR\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCharacteristic parameters, including the apparent diffusion rate (K\u003csub\u003ediff\u003c/sub\u003e), constants of penetration and structural rearrangement at the interface (K\u003csub\u003eP\u003c/sub\u003e and K\u003csub\u003eR\u003c/sub\u003e) for WP/PP and WP/PP-humulinone.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProtein\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProtein: Humulinone\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eK\u003csub\u003ediff\u003c/sub\u003e (mN/m/s\u003csup\u003e1/2\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eK\u003csub\u003eP\u003c/sub\u003e (\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eK\u003csub\u003eR\u003c/sub\u003e (\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eWhey protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1:0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.2156\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0017\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e1.2557\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0398\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e9.3187\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1383\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1:0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.2286\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0020\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e1.2747\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0278\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e5.8094\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1114\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1:0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.2353\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0025\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e1.4246\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0260\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e14.3989\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2183\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1:0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.2718\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0037\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e1.3433\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0167\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e13.6639\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2775\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003ePea protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1:0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.1413\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0016\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e1.5716\u0026thinsp;\u0026plusmn;\u0026thinsp;0.016\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e3.9392\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1794\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1:0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.1019\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0036\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e1.4519\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0028\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e4.1087\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5681\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1:0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.07591\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0009\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e1.5153\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0038\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e3.5237\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2163\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1:0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.1074\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0017\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e1.3452\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0039\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e3.9398\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1794\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eEffect of humulinone on the foam characteristics of milk and pea protein milks\u003c/h2\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e shows the changes in foam produced by milk and pea protein milk under the influence of humulinone over time. The milk used in the experiment was labeled as containing 3.2 g/100 mL of protein. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-B, the milk exhibited poor foamability, which could be attributed to the complex composition of milk. Components like lipids may interfere with foam formation and stability, and the foam formation method used could also be a contributing factor. Since both the milk and the foam are opaque and white, it is difficult to accurately measure foam volume. However, from the overall volume of foam and liquid, it can be observed that the addition of humulinone slightly improved the foamability of the milk, although it did not significantly contribute to foam stability.\u003c/p\u003e \u003cp\u003eThe pea protein milk used in the experiment is labeled with a protein content of 3.3 g/100 g. The ingredient list includes: water, pea protein, fructose syrup, coconut milk, vegetable oil, microcrystalline cellulose, dipotassium phosphate, and salt. The foamability of pea protein milk is similar to that of milk, producing relatively little foam (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC-D). However, the addition of humulinone has a more pronounced effect on enhancing the foamability of pea protein milk and helps to better maintain the foam.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMilk and pea protein milk can be frothed into foam for use in coffee, desserts, and other applications. The experimental results indicate that humulinone can enhance the foam properties of whey protein and pea protein-based food products. However, the enhancement of foaming properties by humulinone in real food systems is less pronounced compared to pure protein systems, likely due to the complexity of actual food matrices, which contain various non-protein components such as lipids, carbohydrates, and mineral ions (e.g., sodium and calcium) that may interfere with protein interfacial behavior and foam formation.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study explored the effects and mechanisms of humulinone on the foaming properties of two representative food proteins: whey protein (WP) and pea protein (PP). The addition of humulinone significantly improved the foamability of both proteins, with maximum increases of 2.34 times for WP and 1.66 times for PP at a mass ratio of 1:0.1 (protein: humulinone). However, no notable enhancement in foam stability was observed. Mechanistic analyses indicated that humulinone interacted with WP and PP mainly through hydrogen bonding, leading to reduced surface tension and accelerated interfacial diffusion. These interfacial changes are closely associated with the observed improvements in foam formation. Furthermore, humulinone induced structural modulation in both proteins\u0026mdash;enhancing the rigidity of WP and increasing the flexibility of PP\u0026mdash;suggesting that different structural adaptations can both contribute to better foamability via improved interfacial behavior. Although the results in real food matrices were not as good as those of single protein systems due to the complexity of the system, the results of the study provide a theoretical basis for the application of putrescine in protein-based foamed products. Future work may focus on optimizing formulation conditions and further exploring its role in multi-component food systems.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eCompeting Interests\u003c/strong\u003e \u003cp\u003eThe authors have no competing interests to declare that are relevant to the content of this article.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eNo funding was received to assist with the preparation of this manuscript.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eYang Gao: Formal analysis, Investigation, Data curation, Writing \u0026ndash; original draft. Chen Xu: Data curation, Resources, Validation. Hanhan Liu: Visualization, Methodology. Junyu Lin: Investigation, Resources. Chenyan Lv: Conceptualization, Methodology, Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e \u003cp\u003eNo datasets were generated or analysed during the current study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBureiko A, Trybala A, Kovalchuk N, Starov V (2015) Current applications of foams formed from mixed surfactant\u0026ndash;polymer solutions. Advances in Colloid and Interface Science 222:670\u0026ndash;677. https://doi.org/10.1016/j.cis.2014.10.001\u003c/li\u003e\n\u003cli\u003eShen P, Peng J, Sagis LMC, Landman J (2024) Air-water interface properties and foam stabilization by mildly extracted lentil protein. Food Hydrocolloids 147:109342. https://doi.org/10.1016/j.foodhyd.2023.109342\u003c/li\u003e\n\u003cli\u003eKornet R, Yang J, Venema P, et al (2022) Optimizing pea protein fractionation to yield protein fractions with a high foaming and emulsifying capacity. 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Food Hydrocolloids 43:388\u0026ndash;399. https://doi.org/10.1016/j.foodhyd.2014.06.014\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"european-food-research-and-technology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [European Food Research and Technology](https://link.springer.com/journal/217)","snPcode":"217","submissionUrl":"https://submission.springernature.com/new-submission/217/3","title":"European Food Research and Technology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Humulinone, Protein, Foamability, Interactions","lastPublishedDoi":"10.21203/rs.3.rs-6811741/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6811741/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFoam is one of the most common and important systems in food, contributing to both the visual appeal and sensory experience. This work investigated the impact of humulinone on the foam properties and physicochemical characteristics of two widely used proteins in food systems: whey protein (WP), a traditional animal-derived protein, and pea protein (PP), a novel plant-based protein alternative. The results showed that humulinone significantly enhanced the foamability of both proteins, increasing the foaming capacity of WP by 2.34 times and PP by 1.66 times at a mass ratio of protein to humulinone of 1:0.1, while having limited effect on foam stability. Spectroscopic analysis and molecular docking revealed that humulinone interacted mainly through hydrogen bonding, leading to conformational changes in secondary structure-promoting. Surface tension measurements indicated that humulinone reduced the surface tension of WP and PP, which accelerated the diffusion stage during interfacial adsorption. Additionally, in food matrix models such as milk and commercial pea protein beverages, humulinone showed potential in improving foamability. These findings provide both mechanistic insights and theoretical support for the application of humulinone in foam-based food products.\u003c/p\u003e","manuscriptTitle":"Regulatory Mechanism of Humulinone on Protein Foam: applications in whey and pea protein","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-17 17:05:17","doi":"10.21203/rs.3.rs-6811741/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-14T21:58:48+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-02T08:31:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"230214829958104989618129714108396937321","date":"2025-06-25T03:31:41+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-19T15:09:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"122748205837201427918603108424643289771","date":"2025-06-17T03:36:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"49902164142989161654140819151495177783","date":"2025-06-16T08:53:11+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-15T21:46:13+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-06T04:46:53+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-06T04:46:19+00:00","index":"","fulltext":""},{"type":"submitted","content":"European Food Research and Technology","date":"2025-06-03T13:14:05+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"european-food-research-and-technology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [European Food Research and Technology](https://link.springer.com/journal/217)","snPcode":"217","submissionUrl":"https://submission.springernature.com/new-submission/217/3","title":"European Food Research and Technology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"4db968b3-0c53-48ea-b797-aa56e5ad3a04","owner":[],"postedDate":"June 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-08-24T13:38:26+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-17 17:05:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6811741","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6811741","identity":"rs-6811741","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}