Preparation and characterization of plant protein-mushroom hybrids: Toward more healthy and sustainable foods

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This study focusses on the formation and properties of hybrid protein-rich foods consisting of potato protein and mushroom, specifically Oyster ( Pleurotus ostreatus ) and Shiitake ( Lentinula edodes ) mushrooms. Hybrid products with the same total solids content (20% w/w) were formed by combining potato protein (10% or 15% w/w) with powdered mushroom (10% or 5% w/w) in aqueous solutions (100 mM NaCl). Measurements of the z-potential versus pH profile showed that the electrical charge of both the proteins and mushrooms went from positive at pH 3 to negative at pH 8, but the point of zero charge was around pH 5.0, 4.0, and 3.5 for potato protein, Oyster mushroom, and Shitake mushroom, respectively. Consequently, there were intermediate pH conditions where there should be an electrostatic attraction between the proteins and mushrooms. Differential scanning calorimetry showed that the potato proteins were originally in their native state but underwent irreversible thermal denaturation around 66 o C, whereas the mushroom dispersions exhibited no thermal transitions. Thermal denaturation of the potato proteins was still observed in the presence of mushrooms. The potato protein was soluble at low and high pH values, but insoluble around its isoelectric point (pI 5). In contrast, the mushroom dispersions contained insoluble particles across the entire pH range. The protein-mushroom hybrids were heated at 90°C for 30 minutes to promote thermal denaturation and gelation of the proteins. Texture profile analysis showed that the hybrids were harder and chewier than protein alone, especially when shiitake mushrooms were added, making them more meat-like. Dynamic shear rheology showed that strong irreversible heat-set gels were formed when the proteins were thermally denatured. Tristimulus color analysis showed that the L*, a*, and b* values changed upon adding the mushrooms, leading to a browner appearance. Microscopy analysis showed that the hybrids had a heterogeneous microstructure, which was attributed to the dispersion of insoluble mushroom particles in a potato protein matrix. These results suggest that potato protein and mushroom hybrids could be healthy, eco-friendly, and tasty substitutes for meat, but further research is required on their nutritional and sensory attributes. Potato protein oyster mushroom shiitake mushroom textural analysis rheology plant-based food sustainability Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1 Introduction Recently, there has been an appreciable shift towards the development of protein-rich food products that can be used as alternatives to animal-derived foods, like meat, fish, egg, or dairy products, which has mainly been driven by concerns about reducing the negative environmental food print of the global food supply [ 1 , 2 ] . Furthermore, the "clean-label" movement is encouraging food producers to replace synthetic additives with natural alternatives [ 3 ] . In this study, we focused on the formulation of protein-rich hybrid food products from plant proteins and mushrooms because of their good sustainability and nutritional profiles. Potato protein is a non-allergenic food protein that has been classified as Generally Recognized as Safe (GRAS) for use as a food ingredient in the United States. Proteins derived from potatoes can be categorized into three main groups: patatins, protease inhibitors, and oxidative enzymes [ 4 – 6 ] . These proteins possess notable nutritional value, as they comprise all essential amino acids. In this study, we used patatin, a glycoprotein as an irreversible heat-set gelling agent. It has an isoelectric point between about pH 4.5 and 5.2 and a molecular weight between about 39 and 43 kDa. It represents about 40% of the total protein content of potatoes. Patatin has been reported to have a thermal denaturation temperature between about 50 to 60°C [ 7 , 8 ] . Ground mushroom powder was used as a filler to modify the texture and appearance of the potato protein gels, as well as to provide key nutrients, including protein, fiber, vitamins, and minerals [ 9 , 10 ] . Indeed, mushrooms are especially high in dietary fiber, proteo-glucans, B vitamins, selenium, and potassium, which are crucial for maintaining health and wellbeing [ 11 – 14 ] . Additionally, mushrooms naturally contain antioxidants that can fight oxidative stress and reduce the risk of chronic diseases [ 15 , 16 ] . Moreover, mushrooms can be produced sustainability, as they require minimal resources, space, and time to grow [ 17 ] . Forestry and agricultural by-products, such as wooden logs and sawdust, can be used during mushroom cultivation, which contributes to the establishment of a more circular economy where little is wasted [ 18 ] . Edible mushrooms also flourish in other kinds of lignocellulosic waste, such as wheat or rice straw, offering an efficient approach to recycle agricultural residues [ 19 – 21 ] . The cultivation of mushrooms also requires minimum use of synthetic pesticides and fertilizers, which reduces environmental pollution and promotes sustainable agricultural practices by minimizing chemical runoff and preserving soil health [ 17 ] . Another advantage of utilizing mushrooms to create alternatives to meat and seafood products is that they have an appealing umami flavor due to the presence of glutamic acid, aspartic acid, and nucleotides [ 22 ] . Finally, mushrooms may also be used to create meat analogs that have fibrous structures and textures that mimic those of real meat [ 23 , 24 ] . In summary, integrating more mushrooms into our diets could mitigate some of the negative environmental impacts of traditional animal agriculture, thus fostering a more sustainable food system. Food protein ingredients are typically marketed in powder form and necessitate dispersion and/or solubilization in water before they can be employed [ 25 ] . Mushroom ingredients can also be obtained in a dried form, which can be converted into a powder by simple grinding and sieving. However, it is important to control the grinding and sieving conditions to obtain uniform powder particles with the required dimensions. In our study, potato protein (patatin) was used as a model plant protein because of its good nutritional profile, high water solubility, and good thermal gelling properties [ 26 – 28 ] . Oyster and shiitake mushrooms were used as model mushroom samples because of their good nutritional value and commercial importance. Hybrid potato protein-mushroom products were then created by heating to induce irreversible thermal gelation of the potato proteins. The microstructure, physicochemical properties, and functionality of these hybrid products was then characterized using a variety of methods. The results of this study may provide fundamental knowledge about the interactions of plant proteins and mushrooms that can be used to create more nutritious and sustainable alternatives to animal-derived food products. 2 Materials and Methods 2.1 Materials Dried oyster and shiitake mushrooms were purchased from New Tiger International Company (Westbury, NY, USA). Sodium chloride (NaCl) was purchased from Sigma-Aldrich Co., Ltd. (St. Louis, MO, USA). Potato protein (“PP200”, Solanic®200) was kindly supplied by the Royal Avebe Company (Veendam, Netherlands). The manufacturer stated that this ingredient contained 90.5% protein, 7.0% water, 2.9% salt, < 0.2% carbohydrates, and < 0.1 grams fat (w/w). The Kjeldahl method was used to determine the protein quantity in accordance with the International Organization for Standardization protocol (ISO 3188), employing a nitrogen-to-protein conversion factor of 6.25. Double distilled water was used for the preparation of all solutions, which was produced using a laboratory-scale water purification unit (Nanopure Infinity, Barnstaeas International, Dubuque, IA, USA). 2.2 Preparation of oyster and shiitake mushroom powders The house-dried mushrooms were initially blended for 90 seconds at 6000 rpm to achieve a finer, consistent texture. The finely ground mushroom powders were then passed through a 20 mm sieve to remove large particles and ensure consistency in particle size for the subsequent experiments. In particular, creating a fine powder facilitated mixing and the preparation of uniform mushroom/potato protein hybrids. 2.3 Preparation of potato protein and mushroom solutions Potato protein solutions of varying concentration (20, 15, 10, and 0.1% w/w) were prepared by dispersing and dissolving powdered PP200 in double distilled water, based on a method described previously [ 29 ] . Briefly, small amounts of PP200 were gradually added to solution at regular intervals to reach the target protein concentration (20% w/w), and then the sample was covered with parafilm to avoid moisture loss and stirred overnight at 350 rpm and 4°C. Solutions with different protein concentrations were then prepared by dilution of the stock. Separate oyster and shiitake mushroom suspensions were prepared by dispersing them in double distilled water at specific concentrations (10 and 5% w/w) and then adding salt to reach 100 mM NaCl. These solutions were then combined with the potato protein stock solutions to achieve the desired final concentrations, aiming for a total solids content of 20 wt.% from both potato and mushroom components. To aid in dissolving the mushrooms, the potato protein solution was introduced during the mixing process in distilled water. The resulting solutions were mixed in various ratios to obtain the required final concentration. Then, pH adjustments were made using HCl and NaOH stock solutions. Pure potato protein (no mushroom) solutions were prepared with concentrations of 20, 15, and 10% w/w (20PP, 15PP, and 10PP, respectively). Mixed protein-mushroom solutions were prepared that contained 15% w/w PP200 and either 5% w/w oyster mushroom (15PP + 5OM) or 5% w/w shiitake mushroom (15PP + 5SM), and that contained 10% w/w PP200 and either 10% w/w oyster mushroom (10PP + 10OM) or 10% w/w shiitake mushroom (10PP + 10SM). Subsequently, these solutions were poured into 100 ml beakers, sealed with parafilm and aluminum wraps, and then heated at 90°C in a water bath for 30 minutes. This process promoted the unfolding and crosslinking of the potato proteins leading to the formation of irreversible heat-set gels. After heating, the beakers were transferred to an ice-filled tray for a 30-minute cooling period and then refrigerated for 2 hours before being analyzed. 2.4 Proximate composition analysis The proximate composition of the mushrooms was expressed as a percentage on a dry weight basis. Standard proximate analysis methods specified by the AOAC [ 30 ] were used to determine moisture, crude protein, crude fat, and total ash contents. Moisture content was determined by drying in a hot air oven at 100 ± 5°C until a constant weight was achieved. Crude protein content was determined using the Dumas method, employing a conversion factor of 4.38 (rather than the more common factor of 6.25), due to mushrooms containing significant non-protein nitrogen levels [ 13 , 31 , 32 ] . Crude fat content was determined using the cold extraction method with hexane. Ash content was measured by incineration at 550 ± 5°C using samples that had previously been dried in hot air. Total carbohydrate content was calculated by deducting the sum of the moisture, crude protein, crude fat, and total ash percentages from 100. 2.5 Zeta Potential and turbidity The zeta-potential values of the samples containing potato protein, oyster mushroom, and shiitake mushroom, either individually or combined (total concentration of 0.1%) were measured at pH values ranging from 3 to 8. These measurements were performed using a particle electrophoresis instrument (Zetasizer Nano ZS, Malvern Instruments Ltd., Malvern, UK). Samples were prepared by dispersing 0.1% w/v of the proteins or mushroom powders into aqueous solutions, and then adjusting the pH using HCl and NaOH solutions. Each data point was measured at least four times to ensure accuracy. Additionally, the transmittance of the protein and mushroom dispersions was measured at 600 nm as an indication of their turbidity using a UV–visible spectrophotometer. The samples were vortexed for 1–2 mins before loading into the measurement cell for analysis. Deionized water was used as a blank refence with the same adjusted pH value. A minimum of four independent measurements were performed for each sample. 2.6 Differential scanning calorimetry Differential scanning calorimetry (DSC) was used to identify and characterize any thermal transitions in the protein or mushroom dispersions when they were heated (DSC25, TA instruments, New Castle, DE, USA). Protein and mushroom dispersions with different compositions (pH 7) were analyzed. Around 50 to 100 mg of each sample was then precisely weighed into a high-volume aluminum pan. These pans were then sealed hermetically, and an additional sealed pan without any sample acted as a reference. Once the samples were equilibrated at 25°C, the samples were heated to 110°C at a rate of 3°C/min under a N 2 atmosphere. Throughout this phase, the instrument continuously recorded heat flow against temperature profiles. The instrument software was used to ascertain the temperatures and enthalpy changes associated with any thermal transitions. 2.7 Dynamic shear rheology The rheological properties of the samples were assessed using a dynamic shear rheometer (HR20, TA Instruments, DE, United States). Approximately 1.3–1.5 ml of unheated sample (pH 7.0) was placed onto a parallel plate (stainless steel, 40.00 mm diameter, crosshatched, Peltier plate) and the measurement gap was set to 1000 µm. Any excess sample was removed using a tissue, ensuring no contact with the sample on the plate. A solvent trap was used to reduce evaporation of the sample during the measurement process. A series of oscillation tests was then conducted: Temperature ramp : The samples were placed in the measurement cell and held at 25°C for 5 minutes. Rheological measurements were then conducted as the samples were heated from 25 to 90°C at 6°C/min. Upon reaching 90°C, the samples were held at this temperature for 20 minutes before being cooled down to 25°C at the same rate (6°C/min). Subsequently, they were incubated at this temperature for an additional 10 minutes. Throughout these measurements, a strain of 0.1% and a frequency of 1.0 Hz were used. Frequency sweep : The dynamic shear modulus was measured as a function of oscillation frequency (0.1–100.0 rad/s) at 25 ℃. To remain within the linear viscoelastic region, a 0.1% strain amplitude was used for these measurements. After reaching the measurement temperature, the samples were incubated for 5 min. Strain sweep : Following the completion of the temperature ramp, rheological measurements were conducted by increasing the strain from 0.01 to 1000% at a constant temperature of 25°C and frequency of 1 Hz. The complex shear modulus (G*), storage modulus (G’), loss modulus (G"), and axial force of the samples were recorded throughout the measurements. 2.8 Texture Profile Analysis Gels were prepared by heating 50 g of covered samples in 100 ml beakers at 90°C for 30 minutes using a water bath. Afterward, the samples were cooled in an ice bath for 30 minutes and then refrigerated for 2 hours before analysis. The samples were then cut into 1 ×1 × 1 cm³ cubes. A commercial texture analyzer (TA-XT2, Stable Micro System, Surrey, UK) with a cylindrical probe was then used to assess the textural and fracture properties of these cubes using different measurement programs: Double-compression test : The texture profile analysis (TPA) parameters of the samples were determined utilizing a double-compression test, which involved a two-cycle compression/decompression program. The testing conditions were based on prior studies [ 33 ] . Pre-test speed, test speed, and post-test speed values were set at 2 mm/s each and a final strain target of 50% was used. A 5 sec gap separated the cycles, and the trigger type was set as auto force with a trigger force of 15 g. A cylindrical probe (P/50, 50 mm stainless cylinder) was used. Parameters such as fracture, hardness, resilience, cohesion, springiness, gumminess, and chewiness were calculated from the resulting force-distance profiles [ 34 ] S ingle-compression test : The fracture properties of the samples were evaluated using a single-compression test utilizing a one-cycle compression program. Similar probe, pre-test speed, and trigger force parameters were used as for the double-compression test. However, in this case, the test speed was set to 1mm/s, the post-test speed was set to 10 mm/s, and the strain was set to 90%. The instrument software recorded the stress versus strain relationship. The Young’s Modulus of the samples was calculated from the initial slope of these curves. The fracture stress and fracture strain were determined from the first break in the stress-strain profiles. 2.9 Water-holding Capacity The water holding capacity (WHC) of the samples was determined using a method described previously [ 35 , 36 ] . Briefly, protein and mushroom samples with different compositions were prepared in double distilled water containing 100 mM NaCl and adjusted to pH 7.0. After allowing them to solubilize for two hours, approximately 1 ml of the samples were loaded into tared microcentrifuge tubes (1.5 ml MCT Graduated yellow microcentrifuge tubes, Fisherbrand) using a positive displacement pipette and their weight was noted as (T 1 ). Subsequently, the samples were heated to 90°C for 20 minutes in a water bath, cooled to room temperature, and weighed again (T 2 ). During these measurements, it was essential to remove any water drops appearing on the surfaces of the heated tubes to reduce errors. After centrifugation at 1000×g for 5 minutes, the tubes were inverted on a paper towel for 10 minutes to drain excess water from the formed gels. The centrifuged samples were then weighed (T 3 ). The WHC was calculated as the percentage of water retained within the gel matrix using the following equation: WHC = 100 × \(({T}_{3}-{T}_{1})/({T}_{2}-{T}_{1})\) Here, T 1 is the weight of samples before heating, T 2 is the weight of samples + microcentrifuge tubes after cooling, and T 3 is the weight of the samples + microcentrifuge tubes after centrifugation and excess water was drained (Boyle et al., 2018). 2.10 Colorimetry analysis. The tristimulus color coordinates, L* (lightness/ brightness), a* (redness/greenness) and b* (yellowness/blueness) of each sample were measured using an instrumental colorimeter (ColorFlex EZ 45/0 LAV, Hunter Colorimeter, Hunter, Virginia, US) following heating and gel formation. The instrument was calibrated using a standardized light source (D65) and detecting angle (10°) by measuring the reflectance of standardized white and black plates. Following this, the samples were placed in a transparent petri plate and their color was measured under similar conditions, while they were covered with a black cup. The black cup was used to reduce the external light interference to obtain more accurate and consistent color measurements. The general appearance of the gels was also recorded using a digital camera. 2.11 Confocal microscopy Confocal scanning microscopy equipped with a 10× eye lens and a 40× objective lens (Nikon DEclipse C1 80i, Nikon, NY) was used to determine the microstructure of the samples. To discriminate between different components, the samples were stained with selective fluorescent dyes: fluorescein isothiocyanate (FITC) (1 mg/ml in ethanol) for proteins, Nile red (1 mg/ml in ethanol) for oils, and calcofluor white (1 mg/mL in water) for polysaccharides. After being heated in the water bath, the samples were thinly sliced (0.1–0.2 mm) and then mounted on glass slides after mixing with the dyes. Incubation for approximately 1 minute followed, and cover slips were used to seal the samples before observation under the microscope. Any extra dye on the slide was carefully removed using a tissue without disturbing the samples. The acquired images were stored and analyzed using the microscope’s software package (NIS-Elements, Nikon, Melville, NY, USA). 2.12 Statistical analysis Experiments were conducted in triplicate for all analyses using separate sets of freshly prepared samples. However, for rheological analysis, experiments were performed in duplicate. The results were expressed as mean values and standard deviations calculated in Excel (Microsoft Corp., Redmond, WA, USA). The statistical difference was calculated at a confidence level of 95% using ANOVA with a Tukey test, which was carried out using R program. 3 Results and discussion 3.1 Proximate composition analysis The proximate compositions of the different mushrooms on a dry weight basis are shown in Table 1 . The oyster and shiitake mushrooms contained 11.6% and 9.77% moisture, respectively. These relatively low values are because the mushrooms were dried prior to packaging. Except for their ash contents, the proximate compositions of the two types of dried mushrooms were significantly different (p < 0.05). Both mushrooms had similar ash contents of about 5%, which can mainly be attributed to the presence of minerals within them. The protein content of the oyster and shiitake mushrooms were around 17.1% and 14.6%, respectively. The fat content of oyster and shiitake mushrooms were around 5% and 3%, respectively. Relatively high amounts of total carbohydrate were found in both mushroom varieties, with the shiitake mushrooms having higher values than the oyster mushrooms. Mushrooms contain digestible carbohydrates like sugars, glycogen, and starches [ 12 ] , as well as indigestible ones like beta glucans, chitin, hemicelluloses, and pectic substances [ 13 ] . 3.2 pH dependence of zeta potential Protein functionality is impacted by the pH of the surrounding aqueous solution, as it alters the charge distribution on the surface of the protein molecules, thereby altering their electrostatic interactions [ 37 ] . For this reason, the pH-dependence of the zeta potential of the potato protein, oyster mushroom, and shiitake mushroom and their hybrids were measured (Figs. 1 a and 1 b ) . For the potato protein solution, the zeta potential changed from highly positive (+ 31 mV) at pH 3 to highly negative (-33 mV) at pH 8, with a point of zero charge close to pH 5. This effect can be attributed to the change in protonation of the carboxyl and amino groups with pH. At low pH, these groups are protonated (–COOH and –NH 3 + ) leading to a positive charge, but at high pH they are non-protonated (-COO − and –NH 2 ) leading to a negative charge [ 7 ] . The point of zero charge can be defined as the isoelectric point of the proteins. Around this pH, the protein molecules have a tendency to aggregate with each other because of the weakening of the electrostatic repulsion between them [ 38 ] . The zeta-potential of both mushroom dispersions also changed from positive at low pH to negative at high pH, but they had isoelectric points around pH 4 (Fig. 1 a). This suggests that the mushroom particles also had proteins at their surfaces. The fact that the isoelectric point of the mushroom dispersions was below that of the potato protein may have been for several reasons. First, there mushroom proteins may have had a higher carboxyl-to-amino group ratio than the potato proteins. Second, the mushroom particles may have had some anionic polysaccharides or mineral ions associated with them. As expected, the hybrid potato protein/mushroom samples had a zeta-potential that was intermediate between the protein only and mushroom only samples. Interestingly, however, the isoelectric point of the hybrid samples was similar to that of the potato protein samples (pH 5). Moreover, the overall zeta-potential versus pH profile of the hybrid samples was closer to that of the protein only samples than the mushroom only samples. This suggests that the potato protein molecules may have adsorbed onto the surfaces of the particles in the mushroom dispersions, where they then dominated the overall charge. 3.3 pH-dependence of transmittance The pH-dependence of the transmittance of the potato protein, oyster mushroom, and shiitake mushroom dispersions were measured to provide insights into their tendency to aggregate (Figs. 2 a and 2 b). Typically, aggregation leads to a reduction in the transmittance because more light is scattered by colloidal particles than by individual molecules [ 8 ] . For the potato protein dispersions, the transmittance values were relatively small at low and high pH values, which can be attributed to strong electrostatic repulsion between the protein molecules. However, there was an appreciable decrease in the transmittance at pH 4 and 5, which can be attributed to protein aggregation close to the isoelectric point where there is only a weak electrostatic repulsion between the protein molecules. The transmittance measurements were supported by digital photographs of the samples, which showed that appreciable protein aggregation and sedimentation occurred between pH 4 to 6, with the pH 5 samples having the cloudiest appearance and thickest sediment layer. The transmittance of the individual oyster mushroom and shiitake mushroom dispersions remained relatively constant cross the whole pH range, being considerably lower than 100%, indicating that there were aggregates present that scattered light at all pH values. This was confirmed by the digital photographs of these samples, which showed that they contained whitish particles that in some cases were visible to the naked eye. In summary, these results indicate that the mushroom samples consisted of colloidal particles across the entire pH range, whereas the potato protein samples only consisted of colloidal particles near their isoelectric point. This knowledge is important for understanding the potential impact of the mushroom dispersions on the gelling behavior of the protein solutions. 3.4 Differential Scanning Calorimetry The heat flow versus temperature profiles of the potato protein and mushroom dispersions were quantified using differential scanning calorimetry at pH 7 (Fig. 3 ). Under these conditions, the pH is well above the isoelectric point of the potato proteins, which helps to reduce their aggregation. A single endothermic peak was observed for the potato protein solutions when they were heated, which was attributed to thermal denaturation of the globular proteins. The denaturation temperature of the potato protein depended slightly on its concentration, being 66.1, 66.1, and 65.3°C for 20%, 15%, and 10% protein solutions, respectively. These values are close to those reported in previous studies on potato proteins [ 3 , 8 ] . No thermal transitions were detected when the mushroom dispersions were heated (data not shown), which may have been because the proteins were already denatured during the preparation of the powdered ingredients. The protein-mushroom hybrids exhibited thermal transitions with peak temperatures that depended on their composition: 69.1°C (10% PP + 10% SM), 68.2°C (10% PP + 10% OM), 65.3°C (15% PP + 5% SM), and 67.2°C (15% PP + 5% OM), which are fairly similar to the denaturation temperature of the potato protein samples. In principle, any changes in the denaturation temperature of the potato proteins may have been due to their interactions with components in the mushroom dispersions that either increased or decreased their thermal stability. However, these interactions did not appear to have a major impact on the stability of the globular proteins because all the thermal denaturation temperatures were fairly similar. 3.5 Rheological properties Further insights into the interactions between the potato proteins and mushroom dispersions was obtained by measuring the rheological properties of the protein-mushroom hybrids. 3.5.1 Temperature-dependent properties Changes in the storage modulus (G') and loss modulus (G") of the protein-mushroom hybrids were measured when they were heated and cooled. As an example, Fig. 4 shows the results for the samples containing only 10% potato protein, only 10% oyster mushroom, and both 10% potato protein and 10% oyster mushroom. The data for the other samples followed a similar trend, but the absolute values were different. Initially, we considered the sample containing only potato protein. Prior to heating, the storage and loss modulus were relatively low, but G' was greater than G", which indicates that a weak gel was formed. When the samples were heated from about 40 to 55 o C, there was a steep decline in both the storage and loss modulus, and G' became less than G". This suggests that the weak gel formed at ambient temperature dissociated during mild heating, possibly because of weakening of the hydrogen bonding between the protein molecules at higher temperatures. However, when the potato protein solution was further heated from 55 to 90 o C, there was a steep increase in both the storage and loss modulus, and G' again exceeded G". This effect can be attributed to thermal denaturation and aggregation of the potato protein molecules, leading to the formation of a 3D protein network throughout the sample, which gave some elastic-like characteristics [ 7 ] . When the samples were held at 90°C for 20 minutes, the gel strength continued to increase, which can be attributed to more protein molecules unfolding and being incorporated into the gel network. When the samples were then cooled from 90 to 25 o C there was an increase in their storage and loss modulus, which can be attributed to an increase in gel strength at lower temperatures due to strengthening of hydrogen bonding. Notably the samples did not return back to their original gel strength after heating and cooling to ambient temperature, which indicated that irreversible heat-set gels were formed. The rheological properties of the mushroom samples were much less sensitive to temperature (Fig. 4 ). Prior to heating, the storage and loss modulus were considerably higher than observed for the potato protein solutions, and G' exceeded G", which indicated that they formed gels. However, the gel strength did not change appreciably during heating from 25 to 90 o C, which can be attributed to the fact that no thermal transitions occurred, as highlighted by the DSC results (Section 3.4). There was a slight increase in the G' and G" values when the samples were held at 90 o C for 20 min, which may have been due to a small amount of water evaporation. However, the storage and loss modulus values remained constant when the samples were cooled back to ambient temperature. The potato protein/mushroom-hybrids exhibited rheological behavior that combined that of the pure protein and the pure mushroom samples. Prior to heating, the rheology was dominated by the properties of the mushroom dispersion, with the hybrid samples having fairly similar G' and G" values as the oyster mushroom samples. However, during heating, a pronounced increase in the storage and loss modulus of the hybrid samples was observed between 55 and 90 o C, which can be attributed to unfolding and aggregation of the potato proteins. Moreover, there was a pronounced increase in the G' and G" values during cooling of the samples, which is probably due to strengthening of the hydrogen bonding in the system. Notably, the final values of G' and G" after heating and cooling were higher for the hybrid samples than for the potato protein only samples, which suggests that the presence of the mushroom dispersion reinforced the gel strength. The final gel strength (G' final and G" final ) and gelation temperature (T gel ), defined as the temperature when G' first exceeded 1000 Pa) of the different samples tested are shown in Table 2 . As expected, the final gel strength increased with increasing protein concentration in the samples containing only potato protein because there were more protein molecules to participate in gel network formation. Moreover, the gelation temperature of these samples decreased with increasing protein concentration because the number of unfolded protein molecules needed to form a 3D gel network occurred at lower temperatures. The final gel strengths of both pure mushroom dispersions were relatively low, and no gelation temperature was detected. However, the final gel strength of the hybrid samples was greater than the final gel strength of either the pure protein dispersions or the pure mushroom dispersions with the same protein and mushroom content, which suggests the presence of the mushroom particles reinforced the strength of the protein gels. Possible reasons for this effect are given later (Section 3.7.). Table 2 Final gel strengths (G' and G") and gelation temperatures of samples with different compositions. Sample G' final (Pa) G" final (Pa) T gel ( o C) 10% PP 11484.35 ± 13.36 2651.2 ± 10.69 80.77 15% PP 43174.45 ± 492.92 10283.325 ± 3.64 72.87 20% PP 148922 ± 1238.85 35923.15 ± 865.43 68.61 10%PP + 10%OM 25389.4 ± 718.28 5004.05 ± 173.91 68.59 10%PP + 10%SM 29311.35 ± 8846.024 6064 ± 979.39 68.58 15%PP + 5%OM 36972.95 ± 2382.03 7795.595 ± 594.03 73.46 15%PP + 5%SM 78075.05 ± 1451.38 17848.9 ± 625.93s 71.64 3.5.2 Strain sweep Additional information about the rheological characteristics of the hybrid gels was gained by studying the impact of the applied shear strain on their dynamic shear moduli. Prior to testing, the samples were heated from 25 to 90°C to promote protein unfolding and aggregation, held at 90 o C, and then cooled from 90 to 25°C to promote gel network strengthening, as described in Section 3.5.1. The viscoelastic properties of the pure protein and hybrid composite gels were then measured by determining their storage and loss moduli as the strain was increased from 0.01 to 1000% (Fig. 5 ). For the sake of clarity, the data is plotted as the complex shear modulus (G*) and tangent of the phase angle (tan delta) versus shear strain. At relatively low strains, the storage modulus was always higher than the loss modulus (G'>G′′) and tan delta was less than 1, which is indicates the samples exhibited mainly solid-like behavior under these conditions [ 39 ] . At higher strains, however, G′′ exceeded G' and tan delta exceeded 1, which can be attributed to some disruption and flow of the samples. The complex shear modulus remained relatively constant when the strain was increased from around 0.01 to 10%, which suggests that this was the linear viscoelastic region (LVR), where no permanent damage is done to the samples by the stresses applied. However, beyond this range, a notable decline in G* occurred, indicating the onset of irreversible deformation. The decrease in shear modulus observed at higher strains is likely due to the partial disruption and subsequent flow of the gel network. The general pattern of the G* versus frequency profiles was similar for all samples, however, the magnitude of the shear modulus dependent on sample composition. For the samples containing 10% potato protein, the addition of both types of mushroom powder increased G*, suggesting that they acted as active rigid fillers. In contrast, for the samples containing 15% potato protein, the addition of 5% shiitake mushroom increased the shear modulus, whereas the addition of 5% oyster mushroom decreased it. This effect may have been due to differences in the rigidity of the particles in the different mushroom dispersions. It is possible that the particles in the shiitake mushroom were more rigid than the 15% protein gel, whereas those in the oyster mushroom were not. In contrast, both types of mushroom particles may have been more rigid than the 10% protein gel, which meant that they both reinforced it. 3.5.3 Frequency-sweep Further insights into the properties of the hybrid gels were obtained by measuring the change in their dynamic shear rheology when the frequency of the applied shear stress was raised from 0.1 to 100 rad/s. All samples were heated from 25 to 90°C, held at 90 o C, and then cooled from 90 to 25°C to promote gelation prior to analysis (see Section 3.5.1). For all samples, G′ exceeded G′′ across the entire frequency range, which indicated that the gels were predominantly elastic-like. The G′ and Gʹʹ values increased slightly with increasing frequency, suggesting that they became more rigid and more viscous at higher frequencies. This effect likely occurred because the biopolymer molecules within the gel networks required some time to respond to the applied shear stress: at lower frequencies, the molecules had sufficient time to relax, but at higher frequencies, they did not, making the gels more rigid and more viscous [ 29 , 40 ] . The shear modulus versus frequency profiles of 10% potato protein, 10% mushroom, and their hybrid samples (10% potato protein + 10% mushroom) are compared in Fig. 6 a. The shear modulus of the 10% potato proteins was much higher than that of the 10% mushroom ones. However, the shear modulus of the hybrid samples was appreciably higher than that of the pure 10% protein ones. This suggests that the presence of the mushrooms increased the shear modulus of the gels. The fact that the pure mushroom gels were much softer than the pure protein gels suggests that the mushroom particles would not have increased the gel strength simply by acting as fillers. Consequently, the observed increase in gel strength may have been due to some phase separation effects. The potato protein molecules could not occupy the same volume as the mushroom particles. As a result, there was an increase in the effective protein concentration in the matrix surrounding the mushroom particles. This led to an increase in the gel strength of the overall system, as the rheology of composite dispersed materials is dominated by the rheology of the continuous phase. The shear modulus versus frequency profiles of 15% potato protein and 15% potato protein + 5% mushroom hybrids are compared in Fig. 6 b. In this case, it was not possible to measure the rheology of the 5% mushroom samples because the gels were too weak and quickly separated within the rheometer. In this case, the addition of 5% shiitake mushroom increased the overall gel strength, whereas the addition of 5% oyster mushroom decreased it. As mentioned earlier, this may have been because the particles in the oyster mushroom were softer than those in the shiitake mushroom. Analysis of dynamic frequency sweep data can provide valuable insights into the nature of the gels formed [ 41 ] . For instance, entangled gel networks exhibit G′~ω 2 and G″~ω 1 behavior at lower frequencies, with a crossover point between G′ and G″ at higher frequencies; covalently crosslinked gel networks have G' and G″ values independent of frequency; and physically crosslinked gel networks exhibit only a slight frequency dependence [ 41 ] . Typically, covalently crosslinked gels have the highest strength due to the presence of strong covalent bonds; physically crosslinked gels have an intermediate strength due to the presence of weaker hydrogen, electrostatic, and hydrophobic bonds; and entangled gels have the lowest strength because there are no bonds formed [ 41 , 42 ] . The degree of frequency dependence of a gel can be determined by fitting the following equation to the G' versus frequency data [ 43 ] : log G′ = z′ log ω + K Here, ω is the oscillation frequency, z′ is a constant (related to the frequency dependence), and K is a constant (related to the strength of the molecular interactions). The constant z′ represents the slope of a log-log plot of G′ versus ω, where z′ > 0 and z′ = 0 are characteristics of physical and covalent linkages within the gel structure, respectively [ 43 , 44 ] . The goodness of fit is evaluated from the coefficient of determination (R 2 ). In our study, R 2 was always greater than 0.99, which indicates a good fit between the theory and the experimental data. The z′ and K values of pure potato protein and hybrid gels are shown in Table 2 . The z′ value of all the samples was above 0 (0.125 to 0.143), indicating that they had the characteristics of physical gels. This is to be expected because the potato protein gels should mainly be held together by attractive hydrophobic and hydrogen bonding. The K values ranged from 9.03 to 11.90, which suggests that there were differences in the overall strength of the molecular interactions within the different gel matrices. The tan δ values of all the gels ranged from around 0.19 to 0.25 across all frequencies (data not shown), indicating that they remained predominantly elastic-like [ 45 ] . 3.6 Textural attributes The textural attributes of the different hybrid gels were also assessed using large deformation uniaxial compression testing, as this is more closely related to the behavior in practical applications, such as mastication. The properties of the hybrid gels were compared to those containing a similar protein concentration (either 10 or 15%). All pure protein and hybrid samples were prepared by heating the solutions at 90°C for 30 minutes (100 mM NaCl, pH 7). 3.6.1 Double compression testing Initially, the textural attributes of the hybrid gels were characterized using texture profile analysis (TPA), which involves compressing and decompressing each sample twice, using a maximum strain of 50%. This method is commonly used to mimic the repetitive and large-scale deformation that foods experience during chewing, thereby serving as an indirect means of assessing food perception [ 46 ] . The force versus distance profiles of each sample was measured and then the TPA parameters were calculated, including hardness, cohesion, springiness, and chewiness (Table 3 ). Table 3 Texture profile analysis parameters of pure potato protein and potato protein/mushroom hybrid gels with different compositions. Group Hardness (g) Adhesiveness Resilience Cohesion Springiness Gumminess Chewiness 20% PP 2770 ± 120 a -0.20 ± 0.1 a 9.66 ± 0.22 a 0.27 ± 0.04 abc 90.24 ± 0.94 a 7.44 ± 1.85 a 6.71 ± 1.62 a 15% PP 700 ± 101 b -0.19 ± 0.08 a 6.66 ± 1.25 b 0.23 ± 0.02 c 92.34 ± 3.43 a 1.61 ± 0.31 b 1.49 ± 0.32 c 15%PP + 5%OM 420 ± 124 c -0.12 ± 0.04 a 7.43 ± 0.26 b 0.24 ± 0.01 c 75.55 ± 7.42 b 1.0 ± 0.3 b 0.75 ± 0.16 c 15%PP + 5%SM 1630 ± 109 ab -0.29 ± 0.13 a 9.6 ± 0.6 a 0.35 ± 0.02 a 88.06 ± 3.18 a 5.56 ± 0.08 a 4.90 ± 0.30 b 10% PP 130 ± 20 d -0.32 ± 0.04 a 7.06 ± 0.22 b 0.34 ± 0.05 a 96.98 ± 0.15 a 0.44 ± 0.12 b 0.43 ± 0.12 c 10%PP + 10%OM 190 ± 19 d -0.34 ± 0.05 a 4.44 ± 0.21 c 0.26 ± 0.02 bc 64.14 ± 6.80 b 0.48 ± 0.05 b 0.31 ± 0.06 c 10%PP + 10%SM 350 ± 37 c -0.27 ± 0.11 a 7.63 ± 0.19 b 0.32 ± 0.01 ab 93.08 ± 3.76 a 1.10 ± 0.15 b 1.03 ± 0.15 c As expected, the hardness of the samples increased with increasing protein concentration, as there would have been more crosslinking within the protein gel network, which is consistent with previous studies on other kinds of proteins [ 47 , 48 ] . For the samples containing 10% potato protein, the hardness was greater in the presence of mushroom than in its absence. In contrast, for the samples containing 15% potato protein, the hardness increased after addition of the shiitake mushroom but decreased after the addition of the oyster mushroom. These results were therefore consistent with the measurements of the shear modulus discussed earlier. The addition of the mushrooms also altered the other TPA parameters of the gels, especially the springiness, chewiness, and gumminess. However, there were few clear trends in the data. Other researchers have also reported that incorporating mushrooms into meat analogs can enhance their texture profile analysis properties [ 24 ] . 3.6.2 Single compression testing Additional insights into the impact of the mushrooms on the mechanical strength and fracture properties of the heat-set potato protein gels were obtained using a single-compression test, where the stress versus strain curves were measured as the samples were compressed to a final strain of 90% (Fig. 7 ) . In all samples, there was an initial linear increase in stress with strain, followed by a break point in the curve, which corresponded to the first observed disruption of their structure. However, there were differences between the samples depending on the composition. These differences were determined by calculating the Young's modulus from the initial slope of the stress versus strain curves, and the breaking stress and breaking strain from the break point. As expected, the Young’s modulus increased with increasing protein concentration for both the pure protein gels and for the hybrid gels (Fig. 7 b), since there were more protein molecules available to participate in the formation of the 3D gel network. For the gels containing 10% potato protein, Young’s modulus was higher in the presence of the mushroom than in its absence. In contrast, for the gels containing 15% potato protein, the Young's Modulus was higher in the presence of shiitake mushroom but lower in the presence of oyster mushroom. These results are therefore consistent with the shear modulus and hardness measurements discussed earlier. The breaking stress and breaking strain of the gels also depended strongly on their composition (Fig. 7 c). Interestingly, the breaking stress and strain could be modulated by incorporating different types and amounts of mushroom into the potato protein/mushroom hybrid gels. For example, samples that were relatively tough and rubbery could be formed from the samples containing 10%PP + 10%SM or 15%PP + 5%OM. This kind of behavior may be useful for creating hybrid products that better match the properties of real meat. 3.7 Possible origin of mushroom effects on texture and rheology of potato protein gels The mushroom samples used in this study are multicomponent systems containing protein, carbohydrates, lipids, water, and minerals. Some of these components are water soluble, whereas others are water insoluble, and so tend to exist as particulate matter that can vary in its size, shape, rigidity, interactions, and aggregation state. It is therefore challenging to accurately elucidate the impact of the different mushroom samples on the rheological properties of the hybrid samples. Nevertheless, it is useful to examine mathematical models that have been developed to described the mechanical properties of composite hybrid materials consisting of particles (“fillers”) dispersed within a polymer network (“matrix”) [ 49 ] . The following equation has been derived to relate the elastic modulus of a composite hybrid material to the nature of the particles it contains [ 49 , 50 ] : $${E}_{C}={E}_{M}\left(\frac{1+\frac{2M}{3}}{\left(\frac{2}{3}-\frac{5\varphi }{3}\right)M+\left(1+\frac{5\varphi }{3}\right)}\right)$$ 1 Here, M = E F / E M , E C , E F , and E M are the elastic moduli of the composite materials, fillers, and matrix, respectively, and φ is the volume fraction of the filler. This equation shows that the elastic modulus of the hybrid material is proportional to that of the elastic modulus of the matrix surrounding the filler particles. In our study, it is anticipated that the hybrid materials consist of insoluble particles arising from the mushrooms (filler) embedded in a 3D gel network formed by the aggregated potato protein molecules (matrix). The above theoretical model provides some useful insights into factors that might potentially impact the mechanical properties of the hybrids: Any factor that increases or decreases the elastic modulus of the potato protein matrix ( E M ), would be expected to either increase or decrease the elastic modulus of the hybrid composite material, respectively. The presence of the mushroom particles may have promoted phase separation in the composite hybrid gels. The potato protein molecules could not occupy the same volume as the mushroom particles. As a result, there was an increase in the effective protein concentration in the matrix surrounding the mushroom particles. This led to an increase in the gel strength of the matrix ( E M ), and therefore of the overall composite system ( E C ). Alternatively, the mushroom may have contained free mineral ions (like calcium or magnesium) that could promote crosslinking of the protein molecules, therefore increasing the elastic modulus of the matrix. The particles in the mushroom may have acted as fillers that could increase or decrease the elastic modulus of the overall hybrid system depending on their rigidity relative to that of the surrounding matrix (E F /E M ). At 10% potato protein, both of the mushroom dispersions may have contained particles that were more rigid than the surrounding protein matrix, thereby increasing the overall gel strength. In contrast, at 15% potato protein, only the particles in the shiitake mushroom were more rigid than those in the protein matrix. 3.8 Water holding capacity (WHC) The water holding capacity (WHC) is used to quantify the amount of water that is held within a protein gel network structure [ 51 ] . The WHC plays an important role in determining the quality and sensory attributes of many foods [ 52 ] , such as the juiciness of plant-based meat products [ 36 ] . Nearly all the pure potato protein gels or hybrid potato protein/mushroom gels tested in this study had remarkably high WHC values: ≥ 99.3% (data not shown). This was probably because the potato protein (10 or 15%) formed a dense biopolymer network containing small uniform pores that held in the water strongly through capillary forces. This characteristic may be important for the creation of plant-based foods that have juicy mouthfeels. 3.9 Color coordinates and appearance The appearance of food products plays a vital role in determining their appeal to consumers. For this reason, we characterize the influence of mushroom addition on the overall appearance and color of the potato protein/mushroom hybrids. The color was quantified in terms of the tristimulus color coordinates (L*, a*, b*). The lightness (L*) is mainly determined by the total amount of light reflected from the surface of a material, which depends on light scattering and absorption phenomena. The greater the fraction of light reflected, the higher the lightness. The chroma values (a* and b*) are mainly determined by selective absorption of light waves by pigments over specific wavelength ranges [ 53 ] . The L* value typically ranges from 0 (pure black) to 100 (pure white). The a* value ranges from strongly positive (red) to strongly negative (green), while the b* value ranges from strongly positive (yellow) to strongly negative (blue) [ 53 , 54 ] . The appearance and color coordinates of the different composite hydrogels measured using an instrumental colorimeter are shown in Fig. 8 . There were appreciable changes in the appearance of the hybrid hydrogels depending on their composition. The L* values decreased and the b* values became more positive after the addition of the mushrooms to the potato protein gels, which indicated that they became less light and more yellow. This effect can be attributed to the inherent color of the mushroom powders. The shiitake mushrooms led to a bigger reduction in the lightness, whereas the oyster mushrooms led to a bigger increase in the yellowness, which can again be reflected to differences in the appearance of the different mushroom powders. 3.10 Microstructure Finally, the microstructure of the pure potato protein and hybrid gels was assessed using confocal fluorescence microscopy. The proteins were stained green, the lipids were stained red, and the polysaccharides were stained blue with appropriate fluorescent dyes (Fig. 9 ). In the absence of mushroom, there were relatively large irregular-shaped protein aggregates dispersed throughout the system. In addition, there appeared to be a uniform distribution of proteins dispersed throughout the sample (uniform green background). The microscopy images also showed that there were large irregular-shaped protein aggregates in the hybrid samples, with some polysaccharide regions around too. We did not observe any lipid-rich regions in the hybrid samples, which can be attributed to the relatively low fat contents of the original mushroom powders ( Table 1 ). The negative charges on the proteins and mushroom particles at pH 7 may have generated some electrostatic repulsive forces between them, which inhibited excessive aggregation, thereby promoting the formation of a more open uniform gel network. 4 Conclusion The study reveals the potential of blending plant proteins with mushrooms to create hybrid materials that may be useful for the formulation of plant-based foods. Protein/mushroom hybrids with different textures and appearances could be produced by varying the composition of the system, such as the type and amount of mushroom added. The electrical charge on the particles in the mushrooms went from positive at low pH to negative at high pH, suggesting that they contained proteins at their surfaces. The incorporation of mushroom into the potato protein gels did not alter the denaturation temperature of the proteins. The rheological properties of potato protein/mushroom hybrids were measured during heating and cooling. The presence of the mushroom led to a weak gel being formed at ambient temperatures before heating, whereas the presence of the potato proteins led to the formation of an irreversible heat set gel. The rheological and textural properties of the gels, such as their shear modulus, hardness, and Young’s modulus, could be manipulated by controlling the type and amount of mushroom blended with the potato proteins. The presence of the mushrooms could either increase or decrease the gel strength (relative to a sample with the same potato protein content), which was attributed to several mechanisms. First, there may have been components in the mushrooms that promote aggregation in the protein matrix (such as mineral ions). Second, there may have been components in the mushrooms that promoted phase separation in the system, thereby leading to an increase in the protein concentration in the protein matrix. Third, the colloidal particles in the mushroom samples may have acted as fillers that increased or decreased gel strength depending on their rigidity relative to that of the protein matrix. Further research is needed to elucidate the precise nature of these interactions. The incorporation of the mushroom powders also altered the appearance of the hybrid samples, reducing their lightness and increasing their yellowness, by an amount that depended on mushroom type. These color changes may influence consumer perception. All of the hybrid samples were shown to have a high water holding capacity, which may lead to a more juicy mouthfeel during mastication. Overall, this research highlights the potential of creating hybrid protein/mushroom products with a range of different physicochemical and sensory attributes. Moreover, the incorporation of the mushrooms may increase the nutritional profile of the hybrid products because they naturally contain vitamins, minerals, and dietary fibers. This would lead to products with a cleaner label, which would be desirable for many consumers. In conclusion, the results of this research could help to facilitate the transition to a healthier and more sustainable diet by creating a wider variety of high-quality plant-based foods for consumers. Declarations Acknowledgements This material was partly based upon work supported by the National Institute of Food and Agriculture, USDA, Massachusetts Agricultural Experiment Station (MAS00559) and USDA, AFRI (2020-03921 and 2022-09185) grants, as well as the Good Food Institute. 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McClements, Current opinion in colloid & interface science 7 (5-6), 451-455 (2002). Table 1 Table 1 is not available with this version Additional Declarations Competing interest reported. Professor DJ McClements serves on the scientific advisory board of a plant-based cheese and a tempeh company Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 31 Jul, 2024 Reviews received at journal 30 Jul, 2024 Reviews received at journal 28 Jul, 2024 Reviewers agreed at journal 08 Jul, 2024 Reviewers agreed at journal 07 Jul, 2024 Reviewers agreed at journal 06 Jul, 2024 Reviewers invited by journal 29 Jun, 2024 Editor assigned by journal 16 Jun, 2024 Submission checks completed at journal 16 Jun, 2024 First submitted to journal 10 Jun, 2024 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. 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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-4559769","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":321091570,"identity":"c768a0da-ef01-46b9-9b5c-e6542817701c","order_by":0,"name":"Disha Jayakumar","email":"","orcid":"","institution":"University of Massachusetts","correspondingAuthor":false,"prefix":"","firstName":"Disha","middleName":"","lastName":"Jayakumar","suffix":""},{"id":321091571,"identity":"7b360354-6d21-40da-99b2-cb55972d5a40","order_by":1,"name":"Ramdattu Santhapur","email":"","orcid":"","institution":"University of Massachusetts","correspondingAuthor":false,"prefix":"","firstName":"Ramdattu","middleName":"","lastName":"Santhapur","suffix":""},{"id":321091573,"identity":"a64a784c-f6fb-4308-bc6a-9930f0d0962f","order_by":2,"name":"David Julian McClements","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEElEQVRIiWNgGAWjYHACxgMQmvnAByABRjBggEsPWAsPA1viDFK18BjOQFGPS4tu++EHBz7m2OTZ85/52PChwtqev52BTepGTZ09A3vzNgksWszOpBkcnLktrZhHIndj44wz6YkzDjOwSeccO5zYwHOsDKuWGzwMh3m3HU7skeDd/pi37XCCATNIC9uBBAaJHDOcWv5u+5/Yw3/mYTNQiz1Eyz+gw+Tf4NbCuO1AYg9DDiNIC+MGkJbcNmbGBgke7FpAfundlpzYcyPNEOoXxmbr3L7DiW08acUW2LQcP/zwwc9tdont/YcfQkKs//DB2znf6uz52Q9vvIEtlLEAxgYwxUak8lEwCkbBKBgFmAAAHiBlBr57sIoAAAAASUVORK5CYII=","orcid":"","institution":"University of Massachusetts","correspondingAuthor":true,"prefix":"","firstName":"David","middleName":"Julian","lastName":"McClements","suffix":""}],"badges":[],"createdAt":"2024-06-10 18:55:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4559769/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4559769/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":59518956,"identity":"a105a9a2-bf0a-4852-97fe-0a4a42a7d827","added_by":"auto","created_at":"2024-07-02 18:38:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":129097,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e Impact of pH on the zeta potential of 0.1% (w/v) potato protein (PP), 0.1% (w/v) oyster mushroom (OM), 0.1% (w/v) 15%PP+5%OM and 0.1% (w/v) 10%PP+10%OM mixtures\u003cstrong\u003e. (b) \u003c/strong\u003eImpact of pH on the zeta potential of 0.1% (w/v) potato protein (PP), 0.1% (w/v) shiitake mushroom (SM), 0.1% (w/v) 0.1% (w/v) 15%PP+5%SM and 0.1% (w/v) 10%PP+10%SM mixtures.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4559769/v1/6dc31165d433e6cba171cf39.png"},{"id":59518955,"identity":"a9f1a8da-1efd-4499-a904-33f1bff28bb1","added_by":"auto","created_at":"2024-07-02 18:38:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":268860,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e Impact of pH on the transmittance of 0.1% (w/v) potato protein (PP), and its physical appearance.\u003cstrong\u003e (b)\u003c/strong\u003e Impact of pH on the transmittance of 0.1% (w/v) oyster mushroom (OM), 0.1% (w/v) shiitake mushroom (SM)and its physical appearance.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4559769/v1/7ebbdb2b1c1b907def0c1482.png"},{"id":59518954,"identity":"0202feea-ddc8-446f-93e6-3aea77b857ca","added_by":"auto","created_at":"2024-07-02 18:38:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":52504,"visible":true,"origin":"","legend":"\u003cp\u003eDifferential scanning calorimetry profile of 10 wt% potato protein solutions in the absence and presence of 10 wt% mushroom (oyster or shitake) during heating from 25 to 110° C with at 3° C/ min.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4559769/v1/f1572f927295cefd79e62edf.png"},{"id":59518957,"identity":"a828d7f7-f9aa-4896-a690-e9eb0bb2e93a","added_by":"auto","created_at":"2024-07-02 18:38:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":137955,"visible":true,"origin":"","legend":"\u003cp\u003eTemperature sweep results of pure potato protein, pure oyster mushroom, and potato protein-oyster mushroom hybrid gels.\u0026nbsp; The storage (G’) and loss (G”) moduli of the samples were measured as they were heated from 25 to 90\u003csup\u003eo\u003c/sup\u003eC, held at 90\u003csup\u003eo\u003c/sup\u003eC, and then cooled from 90 to 25\u003csup\u003eo\u003c/sup\u003eC (strain = 0.1, frequency = 1 Hz).\u0026nbsp;\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4559769/v1/41f3f845118616cd65572dc2.png"},{"id":59519396,"identity":"00ab632f-1dcb-49c5-ba3c-4f01cd8ad574","added_by":"auto","created_at":"2024-07-02 18:46:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":957150,"visible":true,"origin":"","legend":"\u003cp\u003eStrain sweep results of pure potato protein and potato protein-mushroom hybrid gels with different compositions. The complex shear modulus (G*) and phase angle (Tan delta) of the samples were measured as the strain was increased at 1 Hz and 25°C. Results are shown for samples containing 10% potato protein (a and c) or 15% potato protein (b and d).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4559769/v1/48abd82c874663035b8b105b.png"},{"id":59518960,"identity":"72664d11-258b-407e-a2c2-e592b20c3e82","added_by":"auto","created_at":"2024-07-02 18:38:44","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":844842,"visible":true,"origin":"","legend":"\u003cp\u003eFrequency sweep results of pure potato protein and potato protein-mushroom hybrid gels with different compositions. The storage (G’) and loss (G”) moduli of the samples were measured as the frequency was increased at a strain of 0.1% and 25°C. Results are shown for samples containing 10% potato protein (a and c) or 15% potato protein (b and d).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4559769/v1/6158cbf06c731a1db32f5537.png"},{"id":59518958,"identity":"a7ef89fb-e97d-4e68-a608-786ad89223d8","added_by":"auto","created_at":"2024-07-02 18:38:44","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":503258,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of sample composition on the stress-strain relationship of potato protein and potato protein-mushroom hybrids during single compression-decompression experiments (25°C): \u003cstrong\u003e(a)\u003c/strong\u003e 10% potato protein and \u003cstrong\u003e\u0026amp; \u003c/strong\u003e15% potato protein \u003cstrong\u003e(b) \u003c/strong\u003eYoung’s modulus\u003cstrong\u003e (c) \u003c/strong\u003eFracture stress-fracture stain map of potato protein-mushroom gels (pH 7)\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4559769/v1/4737fe1def1c8bc24fd120d1.png"},{"id":59518961,"identity":"0da2c62e-78fc-4b61-8f28-ee9411e663a6","added_by":"auto","created_at":"2024-07-02 18:38:44","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1225999,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a) \u003c/strong\u003eTristimulus coordinates (L*, a*, and b*) of hybrid gels with different potato protein and mushroom (OM/ SM) contents. Different letters (a-g) indicate significant differences (p \u0026lt; 0.05) between the samples’ color coordinates. \u003cstrong\u003e(b)\u003c/strong\u003eDigital photographs of the pure protein and hybrid gels.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-4559769/v1/4b77e0c20b7930dab2941252.png"},{"id":59519397,"identity":"f5b3b3e3-0576-481a-b8b3-8b318912105d","added_by":"auto","created_at":"2024-07-02 18:46:44","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1283573,"visible":true,"origin":"","legend":"\u003cp\u003eConfocal fluorescence microscopy images of composite hydrogels containing different potato protein and mushroom (OM/ SM) ratios.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-4559769/v1/3fa031f2e662266a115958ce.png"},{"id":59519770,"identity":"aa9621f9-ade5-45ee-ba1b-810c715911c3","added_by":"auto","created_at":"2024-07-02 18:54:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8577049,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4559769/v1/b4f43c31-3bf1-4c45-af66-26792ebeb501.pdf"}],"financialInterests":"Competing interest reported. Professor DJ McClements serves on the scientific advisory board of a plant-based cheese and a tempeh company","formattedTitle":"Preparation and characterization of plant protein-mushroom hybrids: Toward more healthy and sustainable foods","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eRecently, there has been an appreciable shift towards the development of protein-rich food products that can be used as alternatives to animal-derived foods, like meat, fish, egg, or dairy products, which has mainly been driven by concerns about reducing the negative environmental food print of the global food supply \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Furthermore, the \"clean-label\" movement is encouraging food producers to replace synthetic additives with natural alternatives \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. In this study, we focused on the formulation of protein-rich hybrid food products from plant proteins and mushrooms because of their good sustainability and nutritional profiles.\u003c/p\u003e \u003cp\u003ePotato protein is a non-allergenic food protein that has been classified as Generally Recognized as Safe (GRAS) for use as a food ingredient in the United States. Proteins derived from potatoes can be categorized into three main groups: patatins, protease inhibitors, and oxidative enzymes \u003csup\u003e[\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. These proteins possess notable nutritional value, as they comprise all essential amino acids. In this study, we used patatin, a glycoprotein as an irreversible heat-set gelling agent. It has an isoelectric point between about pH 4.5 and 5.2 and a molecular weight between about 39 and 43 kDa. It represents about 40% of the total protein content of potatoes. Patatin has been reported to have a thermal denaturation temperature between about 50 to 60\u0026deg;C \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Ground mushroom powder was used as a filler to modify the texture and appearance of the potato protein gels, as well as to provide key nutrients, including protein, fiber, vitamins, and minerals \u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. Indeed, mushrooms are especially high in dietary fiber, proteo-glucans, B vitamins, selenium, and potassium, which are crucial for maintaining health and wellbeing \u003csup\u003e[\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. Additionally, mushrooms naturally contain antioxidants that can fight oxidative stress and reduce the risk of chronic diseases \u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Moreover, mushrooms can be produced sustainability, as they require minimal resources, space, and time to grow \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Forestry and agricultural by-products, such as wooden logs and sawdust, can be used during mushroom cultivation, which contributes to the establishment of a more circular economy where little is wasted \u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. Edible mushrooms also flourish in other kinds of lignocellulosic waste, such as wheat or rice straw, offering an efficient approach to recycle agricultural residues \u003csup\u003e[\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. The cultivation of mushrooms also requires minimum use of synthetic pesticides and fertilizers, which reduces environmental pollution and promotes sustainable agricultural practices by minimizing chemical runoff and preserving soil health \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Another advantage of utilizing mushrooms to create alternatives to meat and seafood products is that they have an appealing umami flavor due to the presence of glutamic acid, aspartic acid, and nucleotides \u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Finally, mushrooms may also be used to create meat analogs that have fibrous structures and textures that mimic those of real meat \u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. In summary, integrating more mushrooms into our diets could mitigate some of the negative environmental impacts of traditional animal agriculture, thus fostering a more sustainable food system.\u003c/p\u003e \u003cp\u003eFood protein ingredients are typically marketed in powder form and necessitate dispersion and/or solubilization in water before they can be employed \u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. Mushroom ingredients can also be obtained in a dried form, which can be converted into a powder by simple grinding and sieving. However, it is important to control the grinding and sieving conditions to obtain uniform powder particles with the required dimensions. In our study, potato protein (patatin) was used as a model plant protein because of its good nutritional profile, high water solubility, and good thermal gelling properties \u003csup\u003e[\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. Oyster and shiitake mushrooms were used as model mushroom samples because of their good nutritional value and commercial importance. Hybrid potato protein-mushroom products were then created by heating to induce irreversible thermal gelation of the potato proteins. The microstructure, physicochemical properties, and functionality of these hybrid products was then characterized using a variety of methods. The results of this study may provide fundamental knowledge about the interactions of plant proteins and mushrooms that can be used to create more nutritious and sustainable alternatives to animal-derived food products.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials\u003c/h2\u003e \u003cp\u003eDried oyster and shiitake mushrooms were purchased from New Tiger International Company (Westbury, NY, USA). Sodium chloride (NaCl) was purchased from Sigma-Aldrich Co., Ltd. (St. Louis, MO, USA). Potato protein (\u0026ldquo;PP200\u0026rdquo;, Solanic\u0026reg;200) was kindly supplied by the Royal Avebe Company (Veendam, Netherlands). The manufacturer stated that this ingredient contained 90.5% protein, 7.0% water, 2.9% salt, \u0026lt; 0.2% carbohydrates, and \u0026lt;\u0026thinsp;0.1 grams fat (w/w). The Kjeldahl method was used to determine the protein quantity in accordance with the International Organization for Standardization protocol (ISO 3188), employing a nitrogen-to-protein conversion factor of 6.25. Double distilled water was used for the preparation of all solutions, which was produced using a laboratory-scale water purification unit (Nanopure Infinity, Barnstaeas International, Dubuque, IA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Preparation of oyster and shiitake mushroom powders\u003c/h2\u003e \u003cp\u003eThe house-dried mushrooms were initially blended for 90 seconds at 6000 rpm to achieve a finer, consistent texture. The finely ground mushroom powders were then passed through a 20 mm sieve to remove large particles and ensure consistency in particle size for the subsequent experiments. In particular, creating a fine powder facilitated mixing and the preparation of uniform mushroom/potato protein hybrids.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Preparation of potato protein and mushroom solutions\u003c/h2\u003e \u003cp\u003ePotato protein solutions of varying concentration (20, 15, 10, and 0.1% w/w) were prepared by dispersing and dissolving powdered PP200 in double distilled water, based on a method described previously \u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. Briefly, small amounts of PP200 were gradually added to solution at regular intervals to reach the target protein concentration (20% w/w), and then the sample was covered with parafilm to avoid moisture loss and stirred overnight at 350 rpm and 4\u0026deg;C. Solutions with different protein concentrations were then prepared by dilution of the stock.\u003c/p\u003e \u003cp\u003eSeparate oyster and shiitake mushroom suspensions were prepared by dispersing them in double distilled water at specific concentrations (10 and 5% w/w) and then adding salt to reach 100 mM NaCl. These solutions were then combined with the potato protein stock solutions to achieve the desired final concentrations, aiming for a total solids content of 20 wt.% from both potato and mushroom components. To aid in dissolving the mushrooms, the potato protein solution was introduced during the mixing process in distilled water. The resulting solutions were mixed in various ratios to obtain the required final concentration. Then, pH adjustments were made using HCl and NaOH stock solutions.\u003c/p\u003e \u003cp\u003ePure potato protein (no mushroom) solutions were prepared with concentrations of 20, 15, and 10% w/w (20PP, 15PP, and 10PP, respectively). Mixed protein-mushroom solutions were prepared that contained 15% w/w PP200 and either 5% w/w oyster mushroom (15PP\u0026thinsp;+\u0026thinsp;5OM) or 5% w/w shiitake mushroom (15PP\u0026thinsp;+\u0026thinsp;5SM), and that contained 10% w/w PP200 and either 10% w/w oyster mushroom (10PP\u0026thinsp;+\u0026thinsp;10OM) or 10% w/w shiitake mushroom (10PP\u0026thinsp;+\u0026thinsp;10SM). Subsequently, these solutions were poured into 100 ml beakers, sealed with parafilm and aluminum wraps, and then heated at 90\u0026deg;C in a water bath for 30 minutes. This process promoted the unfolding and crosslinking of the potato proteins leading to the formation of irreversible heat-set gels. After heating, the beakers were transferred to an ice-filled tray for a 30-minute cooling period and then refrigerated for 2 hours before being analyzed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Proximate composition analysis\u003c/h2\u003e \u003cp\u003eThe proximate composition of the mushrooms was expressed as a percentage on a dry weight basis. Standard proximate analysis methods specified by the AOAC \u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e were used to determine moisture, crude protein, crude fat, and total ash contents. Moisture content was determined by drying in a hot air oven at 100\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u0026deg;C until a constant weight was achieved. Crude protein content was determined using the Dumas method, employing a conversion factor of 4.38 (rather than the more common factor of 6.25), due to mushrooms containing significant non-protein nitrogen levels \u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. Crude fat content was determined using the cold extraction method with hexane. Ash content was measured by incineration at 550\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u0026deg;C using samples that had previously been dried in hot air. Total carbohydrate content was calculated by deducting the sum of the moisture, crude protein, crude fat, and total ash percentages from 100.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Zeta Potential and turbidity\u003c/h2\u003e \u003cp\u003eThe zeta-potential values of the samples containing potato protein, oyster mushroom, and shiitake mushroom, either individually or combined (total concentration of 0.1%) were measured at pH values ranging from 3 to 8. These measurements were performed using a particle electrophoresis instrument (Zetasizer Nano ZS, Malvern Instruments Ltd., Malvern, UK). Samples were prepared by dispersing 0.1% w/v of the proteins or mushroom powders into aqueous solutions, and then adjusting the pH using HCl and NaOH solutions. Each data point was measured at least four times to ensure accuracy. Additionally, the transmittance of the protein and mushroom dispersions was measured at 600 nm as an indication of their turbidity using a UV\u0026ndash;visible spectrophotometer. The samples were vortexed for 1\u0026ndash;2 mins before loading into the measurement cell for analysis. Deionized water was used as a blank refence with the same adjusted pH value. A minimum of four independent measurements were performed for each sample.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Differential scanning calorimetry\u003c/h2\u003e \u003cp\u003eDifferential scanning calorimetry (DSC) was used to identify and characterize any thermal transitions in the protein or mushroom dispersions when they were heated (DSC25, TA instruments, New Castle, DE, USA). Protein and mushroom dispersions with different compositions (pH 7) were analyzed. Around 50 to 100 mg of each sample was then precisely weighed into a high-volume aluminum pan. These pans were then sealed hermetically, and an additional sealed pan without any sample acted as a reference. Once the samples were equilibrated at 25\u0026deg;C, the samples were heated to 110\u0026deg;C at a rate of 3\u0026deg;C/min under a N\u003csub\u003e2\u003c/sub\u003e atmosphere. Throughout this phase, the instrument continuously recorded heat flow against temperature profiles. The instrument software was used to ascertain the temperatures and enthalpy changes associated with any thermal transitions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Dynamic shear rheology\u003c/h2\u003e \u003cp\u003eThe rheological properties of the samples were assessed using a dynamic shear rheometer (HR20, TA Instruments, DE, United States). Approximately 1.3\u0026ndash;1.5 ml of unheated sample (pH 7.0) was placed onto a parallel plate (stainless steel, 40.00 mm diameter, crosshatched, Peltier plate) and the measurement gap was set to 1000 \u0026micro;m. Any excess sample was removed using a tissue, ensuring no contact with the sample on the plate. A solvent trap was used to reduce evaporation of the sample during the measurement process. A series of oscillation tests was then conducted:\u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e \u003cem\u003eTemperature ramp\u003c/em\u003e: The samples were placed in the measurement cell and held at 25\u0026deg;C for 5 minutes. Rheological measurements were then conducted as the samples were heated from 25 to 90\u0026deg;C at 6\u0026deg;C/min. Upon reaching 90\u0026deg;C, the samples were held at this temperature for 20 minutes before being cooled down to 25\u0026deg;C at the same rate (6\u0026deg;C/min). Subsequently, they were incubated at this temperature for an additional 10 minutes. Throughout these measurements, a strain of 0.1% and a frequency of 1.0 Hz were used.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e \u003cem\u003eFrequency sweep\u003c/em\u003e: The dynamic shear modulus was measured as a function of oscillation frequency (0.1\u0026ndash;100.0 rad/s) at 25 ℃. To remain within the linear viscoelastic region, a 0.1% strain amplitude was used for these measurements. After reaching the measurement temperature, the samples were incubated for 5 min.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e \u003cem\u003eStrain sweep\u003c/em\u003e: Following the completion of the temperature ramp, rheological measurements were conducted by increasing the strain from 0.01 to 1000% at a constant temperature of 25\u0026deg;C and frequency of 1 Hz.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003eThe complex shear modulus (G*), storage modulus (G\u0026rsquo;), loss modulus (G\"), and axial force of the samples were recorded throughout the measurements.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Texture Profile Analysis\u003c/h2\u003e \u003cp\u003eGels were prepared by heating 50 g of covered samples in 100 ml beakers at 90\u0026deg;C for 30 minutes using a water bath. Afterward, the samples were cooled in an ice bath for 30 minutes and then refrigerated for 2 hours before analysis. The samples were then cut into 1 \u0026times;1 \u0026times; 1 cm\u0026sup3; cubes. A commercial texture analyzer (TA-XT2, Stable Micro System, Surrey, UK) with a cylindrical probe was then used to assess the textural and fracture properties of these cubes using different measurement programs:\u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e \u003cem\u003eDouble-compression test\u003c/em\u003e: The texture profile analysis (TPA) parameters of the samples were determined utilizing a double-compression test, which involved a two-cycle compression/decompression program. The testing conditions were based on prior studies \u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. Pre-test speed, test speed, and post-test speed values were set at 2 mm/s each and a final strain target of 50% was used. A 5 sec gap separated the cycles, and the trigger type was set as auto force with a trigger force of 15 g. A cylindrical probe (P/50, 50 mm stainless cylinder) was used. Parameters such as fracture, hardness, resilience, cohesion, springiness, gumminess, and chewiness were calculated from the resulting force-distance profiles \u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eS\u003cem\u003eingle-compression test\u003c/em\u003e: The fracture properties of the samples were evaluated using a single-compression test utilizing a one-cycle compression program. Similar probe, pre-test speed, and trigger force parameters were used as for the double-compression test. However, in this case, the test speed was set to 1mm/s, the post-test speed was set to 10 mm/s, and the strain was set to 90%. The instrument software recorded the stress \u003cem\u003eversus\u003c/em\u003e strain relationship. The Young\u0026rsquo;s Modulus of the samples was calculated from the initial slope of these curves. The fracture stress and fracture strain were determined from the first break in the stress-strain profiles.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Water-holding Capacity\u003c/h2\u003e \u003cp\u003eThe water holding capacity (WHC) of the samples was determined using a method described previously \u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. Briefly, protein and mushroom samples with different compositions were prepared in double distilled water containing 100 mM NaCl and adjusted to pH 7.0. After allowing them to solubilize for two hours, approximately 1 ml of the samples were loaded into tared microcentrifuge tubes (1.5 ml MCT Graduated yellow microcentrifuge tubes, Fisherbrand) using a positive displacement pipette and their weight was noted as (T\u003csub\u003e1\u003c/sub\u003e). Subsequently, the samples were heated to 90\u0026deg;C for 20 minutes in a water bath, cooled to room temperature, and weighed again (T\u003csub\u003e2\u003c/sub\u003e). During these measurements, it was essential to remove any water drops appearing on the surfaces of the heated tubes to reduce errors. After centrifugation at 1000\u0026times;g for 5 minutes, the tubes were inverted on a paper towel for 10 minutes to drain excess water from the formed gels. The centrifuged samples were then weighed (T\u003csub\u003e3\u003c/sub\u003e). The WHC was calculated as the percentage of water retained within the gel matrix using the following equation:\u003c/p\u003e \u003cp\u003eWHC\u0026thinsp;=\u0026thinsp;100 \u0026times;\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(({T}_{3}-{T}_{1})/({T}_{2}-{T}_{1})\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003cp\u003eHere, T\u003csub\u003e1\u003c/sub\u003e is the weight of samples before heating, T\u003csub\u003e2\u003c/sub\u003e is the weight of samples\u0026thinsp;+\u0026thinsp;microcentrifuge tubes after cooling, and T\u003csub\u003e3\u003c/sub\u003e is the weight of the samples\u0026thinsp;+\u0026thinsp;microcentrifuge tubes after centrifugation and excess water was drained (Boyle et al., 2018).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Colorimetry analysis.\u003c/h2\u003e \u003cp\u003eThe tristimulus color coordinates, L* (lightness/ brightness), a* (redness/greenness) and b* (yellowness/blueness) of each sample were measured using an instrumental colorimeter (ColorFlex EZ 45/0 LAV, Hunter Colorimeter, Hunter, Virginia, US) following heating and gel formation. The instrument was calibrated using a standardized light source (D65) and detecting angle (10\u0026deg;) by measuring the reflectance of standardized white and black plates. Following this, the samples were placed in a transparent petri plate and their color was measured under similar conditions, while they were covered with a black cup. The black cup was used to reduce the external light interference to obtain more accurate and consistent color measurements. The general appearance of the gels was also recorded using a digital camera.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Confocal microscopy\u003c/h2\u003e \u003cp\u003eConfocal scanning microscopy equipped with a 10\u0026times; eye lens and a 40\u0026times; objective lens (Nikon DEclipse C1 80i, Nikon, NY) was used to determine the microstructure of the samples. To discriminate between different components, the samples were stained with selective fluorescent dyes: fluorescein isothiocyanate (FITC) (1 mg/ml in ethanol) for proteins, Nile red (1 mg/ml in ethanol) for oils, and calcofluor white (1 mg/mL in water) for polysaccharides. After being heated in the water bath, the samples were thinly sliced (0.1\u0026ndash;0.2 mm) and then mounted on glass slides after mixing with the dyes. Incubation for approximately 1 minute followed, and cover slips were used to seal the samples before observation under the microscope. Any extra dye on the slide was carefully removed using a tissue without disturbing the samples. The acquired images were stored and analyzed using the microscope\u0026rsquo;s software package (NIS-Elements, Nikon, Melville, NY, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12 Statistical analysis\u003c/h2\u003e \u003cp\u003eExperiments were conducted in triplicate for all analyses using separate sets of freshly prepared samples. However, for rheological analysis, experiments were performed in duplicate. The results were expressed as mean values and standard deviations calculated in Excel (Microsoft Corp., Redmond, WA, USA). The statistical difference was calculated at a confidence level of 95% using ANOVA with a Tukey test, which was carried out using R program.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results and discussion","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Proximate composition analysis\u003c/h2\u003e \u003cp\u003eThe proximate compositions of the different mushrooms on a dry weight basis are shown in \u003cb\u003eTable\u0026nbsp;1\u003c/b\u003e. The oyster and shiitake mushrooms contained 11.6% and 9.77% moisture, respectively. These relatively low values are because the mushrooms were dried prior to packaging. Except for their ash contents, the proximate compositions of the two types of dried mushrooms were significantly different (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Both mushrooms had similar ash contents of about 5%, which can mainly be attributed to the presence of minerals within them. The protein content of the oyster and shiitake mushrooms were around 17.1% and 14.6%, respectively. The fat content of oyster and shiitake mushrooms were around 5% and 3%, respectively. Relatively high amounts of total carbohydrate were found in both mushroom varieties, with the shiitake mushrooms having higher values than the oyster mushrooms. Mushrooms contain digestible carbohydrates like sugars, glycogen, and starches \u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e, as well as indigestible ones like beta glucans, chitin, hemicelluloses, and pectic substances \u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.2 pH dependence of zeta potential\u003c/h2\u003e \u003cp\u003eProtein functionality is impacted by the pH of the surrounding aqueous solution, as it alters the charge distribution on the surface of the protein molecules, thereby altering their electrostatic interactions \u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. For this reason, the pH-dependence of the zeta potential of the potato protein, oyster mushroom, and shiitake mushroom and their hybrids were measured (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e. For the potato protein solution, the zeta potential changed from highly positive (+\u0026thinsp;31 mV) at pH 3 to highly negative (-33 mV) at pH 8, with a point of zero charge close to pH 5. This effect can be attributed to the change in protonation of the carboxyl and amino groups with pH. At low pH, these groups are protonated (\u0026ndash;COOH and \u0026ndash;NH\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e) leading to a positive charge, but at high pH they are non-protonated (-COO\u003csup\u003e\u0026minus;\u003c/sup\u003e and \u0026ndash;NH\u003csub\u003e2\u003c/sub\u003e) leading to a negative charge \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. The point of zero charge can be defined as the isoelectric point of the proteins. Around this pH, the protein molecules have a tendency to aggregate with each other because of the weakening of the electrostatic repulsion between them \u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe zeta-potential of both mushroom dispersions also changed from positive at low pH to negative at high pH, but they had isoelectric points around pH 4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). This suggests that the mushroom particles also had proteins at their surfaces. The fact that the isoelectric point of the mushroom dispersions was below that of the potato protein may have been for several reasons. First, there mushroom proteins may have had a higher carboxyl-to-amino group ratio than the potato proteins. Second, the mushroom particles may have had some anionic polysaccharides or mineral ions associated with them. As expected, the hybrid potato protein/mushroom samples had a zeta-potential that was intermediate between the protein only and mushroom only samples. Interestingly, however, the isoelectric point of the hybrid samples was similar to that of the potato protein samples (pH 5). Moreover, the overall zeta-potential versus pH profile of the hybrid samples was closer to that of the protein only samples than the mushroom only samples. This suggests that the potato protein molecules may have adsorbed onto the surfaces of the particles in the mushroom dispersions, where they then dominated the overall charge.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.3 pH-dependence of transmittance\u003c/h2\u003e \u003cp\u003eThe pH-dependence of the transmittance of the potato protein, oyster mushroom, and shiitake mushroom dispersions were measured to provide insights into their tendency to aggregate (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Typically, aggregation leads to a reduction in the transmittance because more light is scattered by colloidal particles than by individual molecules \u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. For the potato protein dispersions, the transmittance values were relatively small at low and high pH values, which can be attributed to strong electrostatic repulsion between the protein molecules. However, there was an appreciable decrease in the transmittance at pH 4 and 5, which can be attributed to protein aggregation close to the isoelectric point where there is only a weak electrostatic repulsion between the protein molecules. The transmittance measurements were supported by digital photographs of the samples, which showed that appreciable protein aggregation and sedimentation occurred between pH 4 to 6, with the pH 5 samples having the cloudiest appearance and thickest sediment layer. The transmittance of the individual oyster mushroom and shiitake mushroom dispersions remained relatively constant cross the whole pH range, being considerably lower than 100%, indicating that there were aggregates present that scattered light at all pH values. This was confirmed by the digital photographs of these samples, which showed that they contained whitish particles that in some cases were visible to the naked eye.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn summary, these results indicate that the mushroom samples consisted of colloidal particles across the entire pH range, whereas the potato protein samples only consisted of colloidal particles near their isoelectric point. This knowledge is important for understanding the potential impact of the mushroom dispersions on the gelling behavior of the protein solutions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Differential Scanning Calorimetry\u003c/h2\u003e \u003cp\u003eThe heat flow \u003cem\u003eversus\u003c/em\u003e temperature profiles of the potato protein and mushroom dispersions were quantified using differential scanning calorimetry at pH 7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Under these conditions, the pH is well above the isoelectric point of the potato proteins, which helps to reduce their aggregation. A single endothermic peak was observed for the potato protein solutions when they were heated, which was attributed to thermal denaturation of the globular proteins. The denaturation temperature of the potato protein depended slightly on its concentration, being 66.1, 66.1, and 65.3\u0026deg;C for 20%, 15%, and 10% protein solutions, respectively. These values are close to those reported in previous studies on potato proteins \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. No thermal transitions were detected when the mushroom dispersions were heated (data not shown), which may have been because the proteins were already denatured during the preparation of the powdered ingredients. The protein-mushroom hybrids exhibited thermal transitions with peak temperatures that depended on their composition: 69.1\u0026deg;C (10% PP\u0026thinsp;+\u0026thinsp;10% SM), 68.2\u0026deg;C (10% PP\u0026thinsp;+\u0026thinsp;10% OM), 65.3\u0026deg;C (15% PP\u0026thinsp;+\u0026thinsp;5% SM), and 67.2\u0026deg;C (15% PP\u0026thinsp;+\u0026thinsp;5% OM), which are fairly similar to the denaturation temperature of the potato protein samples. In principle, any changes in the denaturation temperature of the potato proteins may have been due to their interactions with components in the mushroom dispersions that either increased or decreased their thermal stability. However, these interactions did not appear to have a major impact on the stability of the globular proteins because all the thermal denaturation temperatures were fairly similar.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Rheological properties\u003c/h2\u003e \u003cp\u003eFurther insights into the interactions between the potato proteins and mushroom dispersions was obtained by measuring the rheological properties of the protein-mushroom hybrids.\u003c/p\u003e \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e \u003ch2\u003e3.5.1 Temperature-dependent properties\u003c/h2\u003e \u003cp\u003eChanges in the storage modulus (G') and loss modulus (G\") of the protein-mushroom hybrids were measured when they were heated and cooled. As an example, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e shows the results for the samples containing only 10% potato protein, only 10% oyster mushroom, and both 10% potato protein and 10% oyster mushroom. The data for the other samples followed a similar trend, but the absolute values were different. Initially, we considered the sample containing only potato protein. Prior to heating, the storage and loss modulus were relatively low, but G' was greater than G\", which indicates that a weak gel was formed. When the samples were heated from about 40 to 55\u003csup\u003eo\u003c/sup\u003eC, there was a steep decline in both the storage and loss modulus, and G' became less than G\". This suggests that the weak gel formed at ambient temperature dissociated during mild heating, possibly because of weakening of the hydrogen bonding between the protein molecules at higher temperatures. However, when the potato protein solution was further heated from 55 to 90\u003csup\u003eo\u003c/sup\u003eC, there was a steep increase in both the storage and loss modulus, and G' again exceeded G\". This effect can be attributed to thermal denaturation and aggregation of the potato protein molecules, leading to the formation of a 3D protein network throughout the sample, which gave some elastic-like characteristics \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. When the samples were held at 90\u0026deg;C for 20 minutes, the gel strength continued to increase, which can be attributed to more protein molecules unfolding and being incorporated into the gel network. When the samples were then cooled from 90 to 25\u003csup\u003eo\u003c/sup\u003eC there was an increase in their storage and loss modulus, which can be attributed to an increase in gel strength at lower temperatures due to strengthening of hydrogen bonding. Notably the samples did not return back to their original gel strength after heating and cooling to ambient temperature, which indicated that irreversible heat-set gels were formed.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe rheological properties of the mushroom samples were much less sensitive to temperature (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Prior to heating, the storage and loss modulus were considerably higher than observed for the potato protein solutions, and G' exceeded G\", which indicated that they formed gels. However, the gel strength did not change appreciably during heating from 25 to 90 \u003csup\u003eo\u003c/sup\u003eC, which can be attributed to the fact that no thermal transitions occurred, as highlighted by the DSC results (Section 3.4). There was a slight increase in the G' and G\" values when the samples were held at 90\u003csup\u003eo\u003c/sup\u003eC for 20 min, which may have been due to a small amount of water evaporation. However, the storage and loss modulus values remained constant when the samples were cooled back to ambient temperature.\u003c/p\u003e \u003cp\u003eThe potato protein/mushroom-hybrids exhibited rheological behavior that combined that of the pure protein and the pure mushroom samples. Prior to heating, the rheology was dominated by the properties of the mushroom dispersion, with the hybrid samples having fairly similar G' and G\" values as the oyster mushroom samples. However, during heating, a pronounced increase in the storage and loss modulus of the hybrid samples was observed between 55 and 90\u003csup\u003eo\u003c/sup\u003eC, which can be attributed to unfolding and aggregation of the potato proteins. Moreover, there was a pronounced increase in the G' and G\" values during cooling of the samples, which is probably due to strengthening of the hydrogen bonding in the system. Notably, the final values of G' and G\" after heating and cooling were higher for the hybrid samples than for the potato protein only samples, which suggests that the presence of the mushroom dispersion reinforced the gel strength.\u003c/p\u003e \u003cp\u003eThe final gel strength (G'\u003csub\u003efinal\u003c/sub\u003e and G\"\u003csub\u003efinal\u003c/sub\u003e) and gelation temperature (T\u003csub\u003egel\u003c/sub\u003e ), defined as the temperature when G' first exceeded 1000 Pa) of the different samples tested are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e2\u003c/span\u003e. As expected, the final gel strength increased with increasing protein concentration in the samples containing only potato protein because there were more protein molecules to participate in gel network formation. Moreover, the gelation temperature of these samples decreased with increasing protein concentration because the number of unfolded protein molecules needed to form a 3D gel network occurred at lower temperatures. The final gel strengths of both pure mushroom dispersions were relatively low, and no gelation temperature was detected. However, the final gel strength of the hybrid samples was greater than the final gel strength of either the pure protein dispersions or the pure mushroom dispersions with the same protein and mushroom content, which suggests the presence of the mushroom particles reinforced the strength of the protein gels. Possible reasons for this effect are given later (Section 3.7.).\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 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFinal gel strengths (G' and G\") and gelation temperatures of samples with different compositions.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG'\u003csub\u003efinal\u003c/sub\u003e (Pa)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eG\"\u003csub\u003efinal\u003c/sub\u003e (Pa)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eT\u003csub\u003egel\u003c/sub\u003e (\u003csup\u003eo\u003c/sup\u003eC)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10% PP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e11484.35\u0026thinsp;\u0026plusmn;\u0026thinsp;13.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2651.2\u0026thinsp;\u0026plusmn;\u0026thinsp;10.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e80.77\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e15% PP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e43174.45\u0026thinsp;\u0026plusmn;\u0026thinsp;492.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10283.325\u0026thinsp;\u0026plusmn;\u0026thinsp;3.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e72.87\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e20% PP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e148922\u0026thinsp;\u0026plusmn;\u0026thinsp;1238.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e35923.15\u0026thinsp;\u0026plusmn;\u0026thinsp;865.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e68.61\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10%PP\u0026thinsp;+\u0026thinsp;10%OM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e25389.4\u0026thinsp;\u0026plusmn;\u0026thinsp;718.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5004.05\u0026thinsp;\u0026plusmn;\u0026thinsp;173.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e68.59\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10%PP\u0026thinsp;+\u0026thinsp;10%SM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e29311.35\u0026thinsp;\u0026plusmn;\u0026thinsp;8846.024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6064\u0026thinsp;\u0026plusmn;\u0026thinsp;979.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e68.58\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e15%PP\u0026thinsp;+\u0026thinsp;5%OM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e36972.95\u0026thinsp;\u0026plusmn;\u0026thinsp;2382.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7795.595\u0026thinsp;\u0026plusmn;\u0026thinsp;594.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e73.46\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e15%PP\u0026thinsp;+\u0026thinsp;5%SM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e78075.05\u0026thinsp;\u0026plusmn;\u0026thinsp;1451.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17848.9\u0026thinsp;\u0026plusmn;\u0026thinsp;625.93s\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e71.64\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=\"Sec22\" class=\"Section3\"\u003e \u003ch2\u003e3.5.2 Strain sweep\u003c/h2\u003e \u003cp\u003eAdditional information about the rheological characteristics of the hybrid gels was gained by studying the impact of the applied shear strain on their dynamic shear moduli. Prior to testing, the samples were heated from 25 to 90\u0026deg;C to promote protein unfolding and aggregation, held at 90 \u003csup\u003eo\u003c/sup\u003eC, and then cooled from 90 to 25\u0026deg;C to promote gel network strengthening, as described in Section 3.5.1. The viscoelastic properties of the pure protein and hybrid composite gels were then measured by determining their storage and loss moduli as the strain was increased from 0.01 to 1000% (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). For the sake of clarity, the data is plotted as the complex shear modulus (G*) and tangent of the phase angle (tan delta) \u003cem\u003eversus\u003c/em\u003e shear strain. At relatively low strains, the storage modulus was always higher than the loss modulus (G'\u0026gt;G\u0026prime;\u0026prime;) and tan delta was less than 1, which is indicates the samples exhibited mainly solid-like behavior under these conditions \u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e. At higher strains, however, G\u0026prime;\u0026prime; exceeded G' and tan delta exceeded 1, which can be attributed to some disruption and flow of the samples.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe complex shear modulus remained relatively constant when the strain was increased from around 0.01 to 10%, which suggests that this was the linear viscoelastic region (LVR), where no permanent damage is done to the samples by the stresses applied. However, beyond this range, a notable decline in G* occurred, indicating the onset of irreversible deformation. The decrease in shear modulus observed at higher strains is likely due to the partial disruption and subsequent flow of the gel network. The general pattern of the G* versus frequency profiles was similar for all samples, however, the magnitude of the shear modulus dependent on sample composition. For the samples containing 10% potato protein, the addition of both types of mushroom powder increased G*, suggesting that they acted as active rigid fillers. In contrast, for the samples containing 15% potato protein, the addition of 5% shiitake mushroom increased the shear modulus, whereas the addition of 5% oyster mushroom decreased it. This effect may have been due to differences in the rigidity of the particles in the different mushroom dispersions. It is possible that the particles in the shiitake mushroom were more rigid than the 15% protein gel, whereas those in the oyster mushroom were not. In contrast, both types of mushroom particles may have been more rigid than the 10% protein gel, which meant that they both reinforced it.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003e3.5.3 Frequency-sweep\u003c/h2\u003e \u003cp\u003eFurther insights into the properties of the hybrid gels were obtained by measuring the change in their dynamic shear rheology when the frequency of the applied shear stress was raised from 0.1 to 100 rad/s. All samples were heated from 25 to 90\u0026deg;C, held at 90 \u003csup\u003eo\u003c/sup\u003eC, and then cooled from 90 to 25\u0026deg;C to promote gelation prior to analysis (see Section 3.5.1). For all samples, G\u0026prime; exceeded G\u0026prime;\u0026prime; across the entire frequency range, which indicated that the gels were predominantly elastic-like. The G\u0026prime; and Gʹʹ values increased slightly with increasing frequency, suggesting that they became more rigid and more viscous at higher frequencies. This effect likely occurred because the biopolymer molecules within the gel networks required some time to respond to the applied shear stress: at lower frequencies, the molecules had sufficient time to relax, but at higher frequencies, they did not, making the gels more rigid and more viscous \u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe shear modulus \u003cem\u003eversus\u003c/em\u003e frequency profiles of 10% potato protein, 10% mushroom, and their hybrid samples (10% potato protein\u0026thinsp;+\u0026thinsp;10% mushroom) are compared in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea. The shear modulus of the 10% potato proteins was much higher than that of the 10% mushroom ones. However, the shear modulus of the hybrid samples was appreciably higher than that of the pure 10% protein ones. This suggests that the presence of the mushrooms increased the shear modulus of the gels. The fact that the pure mushroom gels were much softer than the pure protein gels suggests that the mushroom particles would not have increased the gel strength simply by acting as fillers. Consequently, the observed increase in gel strength may have been due to some phase separation effects. The potato protein molecules could not occupy the same volume as the mushroom particles. As a result, there was an increase in the effective protein concentration in the matrix surrounding the mushroom particles. This led to an increase in the gel strength of the overall system, as the rheology of composite dispersed materials is dominated by the rheology of the continuous phase.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe shear modulus \u003cem\u003eversus\u003c/em\u003e frequency profiles of 15% potato protein and 15% potato protein\u0026thinsp;+\u0026thinsp;5% mushroom hybrids are compared in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb. In this case, it was not possible to measure the rheology of the 5% mushroom samples because the gels were too weak and quickly separated within the rheometer. In this case, the addition of 5% shiitake mushroom increased the overall gel strength, whereas the addition of 5% oyster mushroom decreased it. As mentioned earlier, this may have been because the particles in the oyster mushroom were softer than those in the shiitake mushroom.\u003c/p\u003e \u003cp\u003eAnalysis of dynamic frequency sweep data can provide valuable insights into the nature of the gels formed \u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. For instance, entangled gel networks exhibit G\u0026prime;~ω\u003csup\u003e2\u003c/sup\u003e and G\u0026Prime;~ω\u003csup\u003e1\u003c/sup\u003e behavior at lower frequencies, with a crossover point between G\u0026prime; and G\u0026Prime; at higher frequencies; covalently crosslinked gel networks have G' and G\u0026Prime; values independent of frequency; and physically crosslinked gel networks exhibit only a slight frequency dependence \u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. Typically, covalently crosslinked gels have the highest strength due to the presence of strong covalent bonds; physically crosslinked gels have an intermediate strength due to the presence of weaker hydrogen, electrostatic, and hydrophobic bonds; and entangled gels have the lowest strength because there are no bonds formed \u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e. The degree of frequency dependence of a gel can be determined by fitting the following equation to the G' \u003cem\u003eversus\u003c/em\u003e frequency data \u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e:\u003c/p\u003e \u003cp\u003elog G\u0026prime; = z\u0026prime; log ω\u0026thinsp;+\u0026thinsp;K\u003c/p\u003e \u003cp\u003eHere, ω is the oscillation frequency, z\u0026prime; is a constant (related to the frequency dependence), and K is a constant (related to the strength of the molecular interactions). The constant z\u0026prime; represents the slope of a log-log plot of G\u0026prime; versus ω, where z\u0026prime; \u0026gt; 0 and z\u0026prime; = 0 are characteristics of physical and covalent linkages within the gel structure, respectively \u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e. The goodness of fit is evaluated from the coefficient of determination (R\u003csup\u003e2\u003c/sup\u003e). In our study, R\u003csup\u003e2\u003c/sup\u003e was always greater than 0.99, which indicates a good fit between the theory and the experimental data. The z\u0026prime; and K values of pure potato protein and hybrid gels are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The z\u0026prime; value of all the samples was above 0 (0.125 to 0.143), indicating that they had the characteristics of physical gels. This is to be expected because the potato protein gels should mainly be held together by attractive hydrophobic and hydrogen bonding. The K values ranged from 9.03 to 11.90, which suggests that there were differences in the overall strength of the molecular interactions within the different gel matrices. The tan δ values of all the gels ranged from around 0.19 to 0.25 across all frequencies (data not shown), indicating that they remained predominantly elastic-like \u003csup\u003e[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Textural attributes\u003c/h2\u003e \u003cp\u003eThe textural attributes of the different hybrid gels were also assessed using large deformation uniaxial compression testing, as this is more closely related to the behavior in practical applications, such as mastication. The properties of the hybrid gels were compared to those containing a similar protein concentration (either 10 or 15%). All pure protein and hybrid samples were prepared by heating the solutions at 90\u0026deg;C for 30 minutes (100 mM NaCl, pH 7).\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003e3.6.1 Double compression testing\u003c/h2\u003e \u003cp\u003eInitially, the textural attributes of the hybrid gels were characterized using texture profile analysis (TPA), which involves compressing and decompressing each sample twice, using a maximum strain of 50%. This method is commonly used to mimic the repetitive and large-scale deformation that foods experience during chewing, thereby serving as an indirect means of assessing food perception \u003csup\u003e[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/sup\u003e. The force \u003cem\u003eversus\u003c/em\u003e distance profiles of each sample was measured and then the TPA parameters were calculated, including hardness, cohesion, springiness, and chewiness (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTexture profile analysis parameters of pure potato protein and potato protein/mushroom hybrid gels with different compositions.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\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=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHardness (g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAdhesiveness\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eResilience\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCohesion\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSpringiness\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eGumminess\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eChewiness\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e20% PP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2770\u0026thinsp;\u0026plusmn;\u0026thinsp;120 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-0.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 \u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e90.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.94 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7.44\u0026thinsp;\u0026plusmn;\u0026thinsp;1.85 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e6.71\u0026thinsp;\u0026plusmn;\u0026thinsp;1.62 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e15% PP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e700\u0026thinsp;\u0026plusmn;\u0026thinsp;101 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-0.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.66\u0026thinsp;\u0026plusmn;\u0026thinsp;1.25 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e92.34\u0026thinsp;\u0026plusmn;\u0026thinsp;3.43 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e15%PP\u0026thinsp;+\u0026thinsp;5%OM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e420\u0026thinsp;\u0026plusmn;\u0026thinsp;124 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-0.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e75.55\u0026thinsp;\u0026plusmn;\u0026thinsp;7.42 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e15%PP\u0026thinsp;+\u0026thinsp;5%SM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1630\u0026thinsp;\u0026plusmn;\u0026thinsp;109 \u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-0.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e88.06\u0026thinsp;\u0026plusmn;\u0026thinsp;3.18 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10% PP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e130\u0026thinsp;\u0026plusmn;\u0026thinsp;20 \u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-0.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e96.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10%PP\u0026thinsp;+\u0026thinsp;10%OM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e190\u0026thinsp;\u0026plusmn;\u0026thinsp;19 \u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-0.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 \u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e64.14\u0026thinsp;\u0026plusmn;\u0026thinsp;6.80 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10%PP\u0026thinsp;+\u0026thinsp;10%SM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e350\u0026thinsp;\u0026plusmn;\u0026thinsp;37 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-0.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e93.08\u0026thinsp;\u0026plusmn;\u0026thinsp;3.76 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAs expected, the hardness of the samples increased with increasing protein concentration, as there would have been more crosslinking within the protein gel network, which is consistent with previous studies on other kinds of proteins \u003csup\u003e[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/sup\u003e. For the samples containing 10% potato protein, the hardness was greater in the presence of mushroom than in its absence. In contrast, for the samples containing 15% potato protein, the hardness increased after addition of the shiitake mushroom but decreased after the addition of the oyster mushroom. These results were therefore consistent with the measurements of the shear modulus discussed earlier. The addition of the mushrooms also altered the other TPA parameters of the gels, especially the springiness, chewiness, and gumminess. However, there were few clear trends in the data. Other researchers have also reported that incorporating mushrooms into meat analogs can enhance their texture profile analysis properties \u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003e3.6.2 Single compression testing\u003c/h2\u003e \u003cp\u003eAdditional insights into the impact of the mushrooms on the mechanical strength and fracture properties of the heat-set potato protein gels were obtained using a single-compression test, where the stress \u003cem\u003eversus\u003c/em\u003e strain curves were measured as the samples were compressed to a final strain of 90% (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. In all samples, there was an initial linear increase in stress with strain, followed by a break point in the curve, which corresponded to the first observed disruption of their structure. However, there were differences between the samples depending on the composition. These differences were determined by calculating the Young's modulus from the initial slope of the stress \u003cem\u003eversus\u003c/em\u003e strain curves, and the breaking stress and breaking strain from the break point.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs expected, the Young\u0026rsquo;s modulus increased with increasing protein concentration for both the pure protein gels and for the hybrid gels (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb), since there were more protein molecules available to participate in the formation of the 3D gel network. For the gels containing 10% potato protein, Young\u0026rsquo;s modulus was higher in the presence of the mushroom than in its absence. In contrast, for the gels containing 15% potato protein, the Young's Modulus was higher in the presence of shiitake mushroom but lower in the presence of oyster mushroom. These results are therefore consistent with the shear modulus and hardness measurements discussed earlier.\u003c/p\u003e \u003cp\u003eThe breaking stress and breaking strain of the gels also depended strongly on their composition (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec). Interestingly, the breaking stress and strain could be modulated by incorporating different types and amounts of mushroom into the potato protein/mushroom hybrid gels. For example, samples that were relatively tough and rubbery could be formed from the samples containing 10%PP\u0026thinsp;+\u0026thinsp;10%SM or 15%PP\u0026thinsp;+\u0026thinsp;5%OM. This kind of behavior may be useful for creating hybrid products that better match the properties of real meat.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Possible origin of mushroom effects on texture and rheology of potato protein gels\u003c/h2\u003e \u003cp\u003eThe mushroom samples used in this study are multicomponent systems containing protein, carbohydrates, lipids, water, and minerals. Some of these components are water soluble, whereas others are water insoluble, and so tend to exist as particulate matter that can vary in its size, shape, rigidity, interactions, and aggregation state. It is therefore challenging to accurately elucidate the impact of the different mushroom samples on the rheological properties of the hybrid samples. Nevertheless, it is useful to examine mathematical models that have been developed to described the mechanical properties of composite hybrid materials consisting of particles (\u0026ldquo;fillers\u0026rdquo;) dispersed within a polymer network (\u0026ldquo;matrix\u0026rdquo;) \u003csup\u003e[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe following equation has been derived to relate the elastic modulus of a composite hybrid material to the nature of the particles it contains \u003csup\u003e[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]\u003c/sup\u003e:\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$${E}_{C}={E}_{M}\\left(\\frac{1+\\frac{2M}{3}}{\\left(\\frac{2}{3}-\\frac{5\\varphi }{3}\\right)M+\\left(1+\\frac{5\\varphi }{3}\\right)}\\right)$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eHere, \u003cem\u003eM\u003c/em\u003e\u0026thinsp;=\u0026thinsp;\u003cem\u003eE\u003c/em\u003e\u003csub\u003eF\u003c/sub\u003e/\u003cem\u003eE\u003c/em\u003e\u003csub\u003eM\u003c/sub\u003e, \u003cem\u003eE\u003c/em\u003e\u003csub\u003eC\u003c/sub\u003e, \u003cem\u003eE\u003c/em\u003e\u003csub\u003eF\u003c/sub\u003e, and \u003cem\u003eE\u003c/em\u003e\u003csub\u003eM\u003c/sub\u003e are the elastic moduli of the composite materials, fillers, and matrix, respectively, and φ is the volume fraction of the filler. This equation shows that the elastic modulus of the hybrid material is proportional to that of the elastic modulus of the matrix surrounding the filler particles.\u003c/p\u003e \u003cp\u003eIn our study, it is anticipated that the hybrid materials consist of insoluble particles arising from the mushrooms (filler) embedded in a 3D gel network formed by the aggregated potato protein molecules (matrix). The above theoretical model provides some useful insights into factors that might potentially impact the mechanical properties of the hybrids:\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eAny factor that increases or decreases the elastic modulus of the potato protein matrix (\u003cem\u003eE\u003c/em\u003e\u003csub\u003eM\u003c/sub\u003e), would be expected to either increase or decrease the elastic modulus of the hybrid composite material, respectively. The presence of the mushroom particles may have promoted phase separation in the composite hybrid gels. The potato protein molecules could not occupy the same volume as the mushroom particles. As a result, there was an increase in the effective protein concentration in the matrix surrounding the mushroom particles. This led to an increase in the gel strength of the matrix (\u003cem\u003eE\u003c/em\u003e\u003csub\u003eM\u003c/sub\u003e), and therefore of the overall composite system (\u003cem\u003eE\u003c/em\u003e\u003csub\u003eC\u003c/sub\u003e). Alternatively, the mushroom may have contained free mineral ions (like calcium or magnesium) that could promote crosslinking of the protein molecules, therefore increasing the elastic modulus of the matrix.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eThe particles in the mushroom may have acted as fillers that could increase or decrease the elastic modulus of the overall hybrid system depending on their rigidity relative to that of the surrounding matrix (E\u003csub\u003eF\u003c/sub\u003e/E\u003csub\u003eM\u003c/sub\u003e). At 10% potato protein, both of the mushroom dispersions may have contained particles that were more rigid than the surrounding protein matrix, thereby increasing the overall gel strength. In contrast, at 15% potato protein, only the particles in the shiitake mushroom were more rigid than those in the protein matrix.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003e3.8 Water holding capacity (WHC)\u003c/h2\u003e \u003cp\u003eThe water holding capacity (WHC) is used to quantify the amount of water that is held within a protein gel network structure \u003csup\u003e[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]\u003c/sup\u003e. The WHC plays an important role in determining the quality and sensory attributes of many foods \u003csup\u003e[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]\u003c/sup\u003e, such as the juiciness of plant-based meat products \u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. Nearly all the pure potato protein gels or hybrid potato protein/mushroom gels tested in this study had remarkably high WHC values: \u0026ge; 99.3% (data not shown). This was probably because the potato protein (10 or 15%) formed a dense biopolymer network containing small uniform pores that held in the water strongly through capillary forces. This characteristic may be important for the creation of plant-based foods that have juicy mouthfeels.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003e3.9 Color coordinates and appearance\u003c/h2\u003e \u003cp\u003eThe appearance of food products plays a vital role in determining their appeal to consumers. For this reason, we characterize the influence of mushroom addition on the overall appearance and color of the potato protein/mushroom hybrids. The color was quantified in terms of the tristimulus color coordinates (L*, a*, b*). The lightness (L*) is mainly determined by the total amount of light reflected from the surface of a material, which depends on light scattering and absorption phenomena. The greater the fraction of light reflected, the higher the lightness. The chroma values (a* and b*) are mainly determined by selective absorption of light waves by pigments over specific wavelength ranges \u003csup\u003e[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]\u003c/sup\u003e. The L* value typically ranges from 0 (pure black) to 100 (pure white). The a* value ranges from strongly positive (red) to strongly negative (green), while the b* value ranges from strongly positive (yellow) to strongly negative (blue) \u003csup\u003e[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe appearance and color coordinates of the different composite hydrogels measured using an instrumental colorimeter are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e. There were appreciable changes in the appearance of the hybrid hydrogels depending on their composition. The L* values decreased and the b* values became more positive after the addition of the mushrooms to the potato protein gels, which indicated that they became less light and more yellow. This effect can be attributed to the inherent color of the mushroom powders. The shiitake mushrooms led to a bigger reduction in the lightness, whereas the oyster mushrooms led to a bigger increase in the yellowness, which can again be reflected to differences in the appearance of the different mushroom powders.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec30\" class=\"Section2\"\u003e \u003ch2\u003e3.10 Microstructure\u003c/h2\u003e \u003cp\u003eFinally, the microstructure of the pure potato protein and hybrid gels was assessed using confocal fluorescence microscopy. The proteins were stained green, the lipids were stained red, and the polysaccharides were stained blue with appropriate fluorescent dyes (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). In the absence of mushroom, there were relatively large irregular-shaped protein aggregates dispersed throughout the system. In addition, there appeared to be a uniform distribution of proteins dispersed throughout the sample (uniform green background). The microscopy images also showed that there were large irregular-shaped protein aggregates in the hybrid samples, with some polysaccharide regions around too. We did not observe any lipid-rich regions in the hybrid samples, which can be attributed to the relatively low fat contents of the original mushroom powders (\u003cb\u003eTable\u0026nbsp;1\u003c/b\u003e). The negative charges on the proteins and mushroom particles at pH 7 may have generated some electrostatic repulsive forces between them, which inhibited excessive aggregation, thereby promoting the formation of a more open uniform gel network.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4 Conclusion","content":"\u003cp\u003eThe study reveals the potential of blending plant proteins with mushrooms to create hybrid materials that may be useful for the formulation of plant-based foods. Protein/mushroom hybrids with different textures and appearances could be produced by varying the composition of the system, such as the type and amount of mushroom added. The electrical charge on the particles in the mushrooms went from positive at low pH to negative at high pH, suggesting that they contained proteins at their surfaces. The incorporation of mushroom into the potato protein gels did not alter the denaturation temperature of the proteins. The rheological properties of potato protein/mushroom hybrids were measured during heating and cooling. The presence of the mushroom led to a weak gel being formed at ambient temperatures before heating, whereas the presence of the potato proteins led to the formation of an irreversible heat set gel. The rheological and textural properties of the gels, such as their shear modulus, hardness, and Young\u0026rsquo;s modulus, could be manipulated by controlling the type and amount of mushroom blended with the potato proteins. The presence of the mushrooms could either increase or decrease the gel strength (relative to a sample with the same potato protein content), which was attributed to several mechanisms. First, there may have been components in the mushrooms that promote aggregation in the protein matrix (such as mineral ions). Second, there may have been components in the mushrooms that promoted phase separation in the system, thereby leading to an increase in the protein concentration in the protein matrix. Third, the colloidal particles in the mushroom samples may have acted as fillers that increased or decreased gel strength depending on their rigidity relative to that of the protein matrix. Further research is needed to elucidate the precise nature of these interactions. The incorporation of the mushroom powders also altered the appearance of the hybrid samples, reducing their lightness and increasing their yellowness, by an amount that depended on mushroom type. These color changes may influence consumer perception. All of the hybrid samples were shown to have a high water holding capacity, which may lead to a more juicy mouthfeel during mastication.\u003c/p\u003e \u003cp\u003eOverall, this research highlights the potential of creating hybrid protein/mushroom products with a range of different physicochemical and sensory attributes. Moreover, the incorporation of the mushrooms may increase the nutritional profile of the hybrid products because they naturally contain vitamins, minerals, and dietary fibers. This would lead to products with a cleaner label, which would be desirable for many consumers. In conclusion, the results of this research could help to facilitate the transition to a healthier and more sustainable diet by creating a wider variety of high-quality plant-based foods for consumers.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; This material was partly based upon work supported by the National Institute of Food and Agriculture, USDA, Massachusetts Agricultural Experiment Station (MAS00559) and USDA, AFRI (2020-03921 and 2022-09185) grants, as well as the Good Food Institute. The authors thank Sisheng Li, Jae Kun Ryu, Xiaoyan Hu, and Zeynap Aksoylu for valuable advice. \u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eD. J. McClements and L. Grossmann, Comprehensive Reviews in Food Science and Food Safety \u003cstrong\u003e20\u003c/strong\u003e (4), 4049-4100 (2021).\u003c/li\u003e\n\u003cli\u003eD. Y. Lee, S. Y. 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Jin, L. Hu, J. Lyu and X. Wu, Food Chemistry \u003cstrong\u003e351\u003c/strong\u003e, 129230 (2021).\u003c/li\u003e\n\u003cli\u003eD. J. McClements, Advances in colloid and interface science \u003cstrong\u003e97\u003c/strong\u003e (1-3), 63-89 (2002).\u003c/li\u003e\n\u003cli\u003eD. J. McClements, Current opinion in colloid \u0026amp; interface science \u003cstrong\u003e7\u003c/strong\u003e (5-6), 451-455 (2002).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table 1","content":"\u003cp\u003eTable 1 is not available with this version\u003c/p\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":"food-biophysics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Food Biophysics](https://www.springer.com/journal/11483)","snPcode":"11483","submissionUrl":"https://submission.nature.com/new-submission/11483/3","title":"Food Biophysics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Potato protein, oyster mushroom, shiitake mushroom, textural analysis, rheology, plant-based food, sustainability","lastPublishedDoi":"10.21203/rs.3.rs-4559769/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4559769/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThere is growing interest in finding more sustainable alternatives to animal-derived foods, like meat, fish, egg, and dairy products. This study focusses on the formation and properties of hybrid protein-rich foods consisting of potato protein and mushroom, specifically Oyster (\u003cem\u003ePleurotus ostreatus\u003c/em\u003e) and Shiitake (\u003cem\u003eLentinula edodes\u003c/em\u003e) mushrooms. Hybrid products with the same total solids content (20% w/w) were formed by combining potato protein (10% or 15% w/w) with powdered mushroom (10% or 5% w/w) in aqueous solutions (100 mM NaCl). Measurements of the z-potential \u003cem\u003eversus\u003c/em\u003e pH profile showed that the electrical charge of both the proteins and mushrooms went from positive at pH 3 to negative at pH 8, but the point of zero charge was around pH 5.0, 4.0, and 3.5 for potato protein, Oyster mushroom, and Shitake mushroom, respectively. Consequently, there were intermediate pH conditions where there should be an electrostatic attraction between the proteins and mushrooms. Differential scanning calorimetry showed that the potato proteins were originally in their native state but underwent irreversible thermal denaturation around 66 \u003csup\u003eo\u003c/sup\u003eC, whereas the mushroom dispersions exhibited no thermal transitions. Thermal denaturation of the potato proteins was still observed in the presence of mushrooms. The potato protein was soluble at low and high pH values, but insoluble around its isoelectric point (pI 5). In contrast, the mushroom dispersions contained insoluble particles across the entire pH range. The protein-mushroom hybrids were heated at 90\u0026deg;C for 30 minutes to promote thermal denaturation and gelation of the proteins. Texture profile analysis showed that the hybrids were harder and chewier than protein alone, especially when shiitake mushrooms were added, making them more meat-like. Dynamic shear rheology showed that strong irreversible heat-set gels were formed when the proteins were thermally denatured. Tristimulus color analysis showed that the L*, a*, and b* values changed upon adding the mushrooms, leading to a browner appearance. Microscopy analysis showed that the hybrids had a heterogeneous microstructure, which was attributed to the dispersion of insoluble mushroom particles in a potato protein matrix. These results suggest that potato protein and mushroom hybrids could be healthy, eco-friendly, and tasty substitutes for meat, but further research is required on their nutritional and sensory attributes.\u003c/p\u003e","manuscriptTitle":"Preparation and characterization of plant protein-mushroom hybrids: Toward more healthy and sustainable foods","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-02 18:38:39","doi":"10.21203/rs.3.rs-4559769/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-31T11:53:07+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-31T03:52:39+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-29T02:58:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"132841959460859837545865770760068899196","date":"2024-07-09T01:33:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"296695280487709467186654070480733615489","date":"2024-07-07T16:43:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"238719588803051412198446145459357797372","date":"2024-07-07T01:26:15+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-30T01:47:46+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-17T01:49:13+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-17T01:48:26+00:00","index":"","fulltext":""},{"type":"submitted","content":"Food Biophysics","date":"2024-06-10T18:53:54+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"food-biophysics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Food Biophysics](https://www.springer.com/journal/11483)","snPcode":"11483","submissionUrl":"https://submission.nature.com/new-submission/11483/3","title":"Food Biophysics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a0f83460-f106-45cc-a767-5260fdd1f3fd","owner":[],"postedDate":"July 2nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-08-14T12:48:04+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-02 18:38:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4559769","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4559769","identity":"rs-4559769","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}