Effect of differences preheating temperatures on the functional and physical properties of soy protein concentrate

Soy protein is widely used in the food industry due to its nutritional value as well as its functional and physical properties, which form the sensory characteristics of food. With thermal process, the functional and physical properties of native soy protein can result the texture of food products becoming excessive. This has a negative impact on the sensory characteristics of food products with high concentrations of soy protein. The functional and physical properties of soy protein can be modified using the preheating method by controlling the protein aggregation behavior through the temperature setting used. Thus, this study aims to observe the effect of differences in preheating temperature on the functional and physical properties of soy protein concentrate. Soy protein was observed in the form of soy protein concentrate. Preheating was carried out at 70, 80 and 90°C with a protein concentration of 6% (w/v). The functional and physical properties observed were solubility, gel-forming capacity, voluminosity, microstructure, and protein digestibility. The results showed that the preheating of soy protein concentrate had a significant effect on solubility, gelling capacity, voluminosity, and protein digestibility. However, the difference in preheating temperature did not have a significant effect on the voluminosity and microstructure of the soy protein concentrate.


Introduction
Plant protein has become popular in recent years, supported by increasing research on plant protein which shows significant health benefits compared to animal protein [1][2][3].Plant protein is also considered to be an alternative to environmental issues caused by livestock agriculture emission [4].Soy protein is one of the most common plant proteins.Soy protein contains 35-40% protein and various types of essential amino acids such as lysine and leucine [5][6][7].Based on the percentage of protein, soy protein products are divided into soy flour (50-65%), soy protein concentrate (65-90%), and soy protein isolate (> 90%) [8].Soy protein products facilitate the utilization of soy protein in the food industry.
Protein is an important macronutrient that humans need.Besides its role as nutritional properties of foods, functional and physical properties of proteins are needed in the food industry [9].Functional and physical properties of proteins depend on the type and structure of amino acids which can be modified by various physical (heat, ultrasound, radiation), chemical (deamidation, cross-linking, acylation, glycosylation) and enzymatic (oxidase, transglutaminase, endopeptidase) methods [10][11][12][13].Protein modification controls the functional properties of protein in food such as solubility, water binding 1230 (2023) 012150 IOP Publishing doi:10.1088/1755-1315/1230/1/012150 2 capacity, fat binding capacity, emulsifier, foaming, gelling capacity, as well as physical properties in protein food such as viscosity, density, and particle size [14,15].Through controlling functional and physical properties of protein, protein food products with the desired properties can be obtained.
The application of protein modification is partly due to the relationship between the functional and physical properties of the native protein and the resulting food product textures.The addition of protein in its native form can cause the texture to become harder through the thermal process.Thus, the higher protein content added, the harder the resulting texture [16].This condition has a negative impact on the sensory properties of some food products [17].Therefore, protein modification is needed to add a large amount of protein content to food without changing the texture considerably [18].Thermal process/heating are extensively used modification method to improve the functional and physical properties of soy protein.Proper heating contributes to increased sensory properties and nutritional value [19].Different heating temperatures are extrinsic factors that can affect the functional and physical properties of proteins [14].Heating soy protein dispersion above 70°C causes protein denaturation and formation of new intra and intermolecular bonds that allow changes in protein properties [20].Preheating can control the denaturation process and protein aggregation behaviour which affects the functional and physical properties of proteins [21].
Various studies have shown changes and improvements in the functional and physical properties of proteins after heating [22][23][24][25].However, research regarding the relationship between preheating and the functional and physical properties of soy protein is still limited.Thus, in this study, preheating was applied to soy protein concentrate to prevent negative effects on texture and sensory properties of food products.Modification of soy protein concentrate through preheating was carried out to observe the effect of temperature differences on the functional and physical properties of soy protein concentrate to obtain the best sensory properties.

Materials and methods
The main ingredient used in this study was soy protein concentrate obtained from PT. Kaira Food Berkah Lestari with non-GMO soybeans imported from China.Another ingredient used is distilled water and chilled water.

Preheating treatment
Samples were dispersed distilled water with a protein concentration of 6% (w/v).The soy protein concentrate suspension was stirred until homogeneous, then heated for 30 minutes at a holding temperature of 70, 80 and 90°C.The soy protein concentrate suspension was then cooled with cold water until it reached room temperature, which was ±25°C.A vacuum oven at 55 °C was used to dry the soy protein concentrate suspension.The dried soy protein concentrate suspension was ground to a powder form and sifted with a size of 80 mesh to remove coarse particles.The modified soy protein concentrate is stored in a dry container [23].

Solubility
Samples were dispersed in water with a concentration of 5% (w/v) and homogenized using a vortex for 5 minutes.Then the suspension was centrifuged at 1800 rpm for 5 minutes.The precipitate obtained is dried in an oven at ±105°C until it reaches a constant mass, which is known as the mass of insoluble solid.Solubility is defined as the percentage of dissolved solid mass compared to the total solid mass [24].

Gelling capacity
Samples and skim milk were dispersed in water with a protein concentration of 5% (w/v).The suspension was stirred for 15 minutes at 50°C.At 35 °C, glucono-delta lactone was added at a concentration of 2.5% (w/v) and stirred for 2 minutes.After that, incubation was carried out at ±35°C until it acquired a pH of 4.3.Gel hardness was observed at pH 4.7; 4,6; 4.5; 4,4; and 4.3 to determine the gelling capacity.Gel hardness was tested using the Texture Analyzer [26].

Voluminosity
Samples are dispersed in water at different concentrations.The viscosity of each dilution was measured at 100 rpm using a viscometer.Relative viscosity (ηr) is calculated by the following equation: The volume fraction of each suspension, ϕv (ml/l) is calculated using Lee's equation as follows: Thus, the voluminosity calculated using: =    Where, C is the concentration of the sample (g/l) [26].

Microstructure (SEM)
The sample is inserted into the specimen stub.Then, the stub specimen is placed on the specimen stage.Observations were made in a vacuum by shooting electrons at the object.The results of the observations are in the form of images at a specified magnification along with particle sizes.

In vitro protein digestibility
A sample of 200 mg that had been analyzed for total N was dispersed into 9 ml of Walphole Buffer 0.2 N pH 2.Then, 1 ml of 2% pepsin enzyme was added to the suspension.The suspension was then incubated for 5 hours in a water bath shaker at 37°C.After incubation, the suspension was centrifuged at 3000 rpm for 20 minutes.The supernatant obtained was then added with 5 ml of 20% TCA and allowed to stand for 90 minutes.After that, the supernatant was filtered with Whatman No. 41.The resulting filtrate was analyzed using the Micro Kjeldahl method to determine the total N [27].Thus, protein digestibility is calculated using the following formula: Protein Digestibility (%) = filtrate total N Sample total N x dilution factor x 100%

Solubility
The solubility of soy protein concentrate, with control and preheating treatment to 70, 80, and 90°C respectively, were 68.83% ± 0.07; 32.7% ± 1.16; 35% ± 1.66; and 35.93% ± 2.14, respectively.Based on the T-test, the solubility of soy protein concentrate in the control treatment was significantly higher than that of the preheating treatment at all temperatures.Furthermore, the solubility increased significantly from the preheating treatment at 70°C to 90°C.The decrease in solubility in the preheating treatment indicated the occurrence of denaturation and the formation of insoluble aggregates.The formation of aggregates can increase protein stability, thereby preventing further aggregation [28].Therefore, proteins with low solubility can be utilized to minimize excessive aggregation when proteins are heated during the cooking process, which might result in hardness in foods.Heating during the preheating treatment promotes the mobility of protein molecules, breaks intra-and intermolecular bonds, and induces the opening of secondary and tertiary structures.This results in hydrophobic amino acids (eg free sulfur or thiol groups) that were originally buried in the protein structure becoming exposed and interacting with other protein molecules.Interactions that can occur include the formation of cross-links between protein molecules, including hydrophobic and electrostatic interactions, hydrogen bonds, and disulfide-sulfhydryl exchanges.This interaction encourages the formation of aggregates with higher molecular weights, resulting in decreased solubility, and precipitation occurs [29,30].

Gelling capacity
Based on the data, the gel hardness increased with decreasing pH in all treatments.The similar phenomenon happened in the previous study [31], where gels from skim milk of goats, cows, red deer, and sheep induced with GDL formed in the pH range of casein's isoelectric point (pH 4.6); and the storage modulus increased gradually with a decrease in pH [31].If the gel hardness in all treatments were compared with the T-test, the soy protein concentrates with the control treatment significantly formed the hardest gel hardness compared to the preheating treatment at all temperatures.This means that there is a decrease in the gelling capacity of soy protein concentrate after preheating.This was caused by a decrease in the solubility of soy protein concentrate with preheating treatment.Soy protein concentrate with lower solubility causes it to precipitate more easily during the incubation period, so that protein interactions in solution decrease.The decrease in gelling capacity was also influenced by differences in aggregation behavior during the preheating and gelling treatments.The formation of aggregates with smaller particle sizes can reduce the gelling capacity of soy protein and cause the gel hardness to become weaker [23].

Figure 2. Gel hardness of soy protein concentrate control and preheating treatment
By altering the concentration, preheating can affect protein aggregation behavior.Lower concentration preheating results in the development of aggregates with smaller particle sizes, which inhibits further aggregation when the protein is stimulated to form a gel [21].Thus, the control-treated soy protein concentrate in this study was able to form larger protein aggregates and produce a harder gel hardness due to the fact that soy protein had not undergone previous aggregation through preheating treatment.Reducing the gelling capacity can be beneficial if it is aimed at avoiding a food texture that is too hard when protein is added in large quantities.The temperature of preheating also influences particle size and gelling capacity.As shown in Figure 3, the preheating treatment at 80, 70, and 90 °C resulted in the soy protein concentrate with the highest hardness to the lowest.Based on the T-test, the gelling capacity increased significantly from 70°C to 80°C, then significantly decreased again at 90°C.Unlike in the similar research, the hardness of the gel increased at higher degrees of denaturation and preheating temperature [32].In this study, soy protein was denatured at preheating temperatures of 80, 90, and 100 °C with onset times of 180, 40, and 10 seconds, respectively.A higher heating intensity causes a higher level of denaturation and aggregation, so that the texture of the gel that is formed becomes harder [32,33].
The heating time can impact the difference in the trend of gel hardness and preheating temperature.In this study, the holding time used was 30 minutes, longer than the time used in previous studies [32].The decrease in the gelling capacity of the 90°C treatment (the treatment with the longest heating time) associated with the decrease in aggregate particle size at a longer initial heating time.Research showed that particle size of whey protein that had gone through heating at 85 °C tended to increase first, but after a long time the particle size decreased again [34].The particle size decreases as the temperature rises to a certain limit for protein polymerization, causing the aggregate to fail to stabilize the serum phase, precipitate, and the particle size to decrease [35].

Voluminosity
According to Lee's equation, the viscosity of the protein solution increases with increased protein concentration.The viscosity of protein solutions is affected by various types of interactions between complex protein molecules, so that an increase in protein concentration can increase the interactions between protein molecules and the viscosity of protein solutions.Electrostatic interactions are the dominant factor determining the viscosity of protein solutions at low concentrations through longdistance interactions.This interaction includes the attraction of opposite charges and the repulsion of like charges.Electrostatic repulsion between like charges suppresses the mobility of protein molecules, which causes an increase in the viscosity of the solution.In addition, the increase in viscosity is also caused by the attraction of hydrophobic interactions through close interactions [36].Hydrophobic interaction is an important phenomenon that drives interactions between nonpolar solutes in aqueous solutions [37].The intermolecular attraction interactions inhibit the mobility of protein molecules in solution, resulting in an increase in viscosity [36].

Figure 3. Voluminosity of soy protein concentrate control and preheating treatment
Based on the results obtained, the voluminosity of soy protein concentrates in the control and preheating treatments at 70, 80, and 90 °C were: 4.03 ± 0.18; 2.49 ± 0.04; 2.53 ± 0.11; and 2.47 ± 0.08 ml/g, respectively.These results indicate a decrease in the voluminosity of soy protein concentrate in the preheating treatment compared to the control treatment.The decrease in voluminosity is affected by the properties of the protein when it interacts with water, such as protein swelling [38].Protein swelling occurs through various intermolecular attractions that trap water in the protein matrix [39].Trapping water in the protein matrix increases the hydrodynamic volume of protein molecules, which affects the flow properties of the solution [40].Thus, protein swelling can be an indicator of the viscosity of a solution.Several factors that affect protein structure and intermolecular interactions also affect the swelling and viscosity of proteins in food [41].
Denaturation during the preheating process causes the opening of the protein structure, exposing previously buried hydrophobic bonds.Exposure to hydrophobic bonds increases the hydrophobicity of the protein surface, which results in decreased protein stability [33].Proteins with low stability will more easily interact, form bonds with other proteins, and form protein aggregates, which are generally irreversible and very stable [28].This results in reduced interactions between proteins in solution and a decrease in viscosity.Thus, the preheating treatment resulted in a lower volume of soy protein concentrate than the control.These are supported by the research of Nöbel et al. (2016), where the voluminosity value of casein micelles decreased with increasing temperature up to 70 °C.The voluminosity of casein micelles obtained at 70 °C was 3.5 mL/g [42].Soy protein concentrate with a lower voluminosity can be utilized to prevent further protein aggregation during the heating process in food processing, which results in excessive texture in food products.
Based on the T-test, the voluminosity of soy protein concentrates at preheating treatment temperatures of 70, 80, and 90 °C did not show a significant difference.This can be influenced by the drying process, which is carried out using a vacuum oven.Although drying in a vacuum oven is carried out at temperatures below the denaturation temperature of soy protein, which is 55 °C, it is possible that during heating, further changes in the protein structure will occur.Research by Liang et al. (2020) found that preheating soy protein at 55 °C for 30 minutes increased the degree of soy protein hydrolysis.The increase in the degree of hydrolysis at 55 °C indicates that at that temperature, the opening of the protein structure has begun to occur, which makes it easier for the protein to be hydrolyzed.Therefore, drying using a vacuum oven at 55 °C can be a factor affecting changes in protein structure in all preheated soy protein concentrate.Thus, the effect of temperature differences on preheated soy protein concentrate was not clearly seen.Another alternative drying method that can be used to prevent the effect of further heating on proteins is freeze drying.

Microstructure
Sample characteristics were observed microscopically using a Scanning Electron Microscope (SEM) to determine the shape and size of the particles.Based on the observation of SEM results (Table 1.), soy protein concentrate in the control treatment, some particles seemed to stick together, indicating a denaturation process and the formation of aggregates beforehand.The denaturation process is possible during the drying process using a spray dryer.Most of the control treatment particles are spherical.Meanwhile, the shape of the preheated soy protein concentrate at all temperatures looks similar; some are round or oval and have a rough surface.The difference is influenced by the preheating process and size reduction through milling.
The size of the particles was calculated to support particle observations via SEM.The particle sizes of the control soy protein concentrate sampling and the preheating treatment at 70, 80, and 90°C were 152.85 ± 10.73; 199.76 ± 12.37; 119.66 ± 12.54; and 122.75 ± 6.13 μm, respectively.Based on the Ttest, the particle size of all treatments did not show a significant difference.Particles of this size are categorized as fine flour, which is ≤200 μm in size [44].In the other study [45], particle size increases with increasing heating intensity, which is associated with aggregate formation.The heating treatment causes the protein structure to unfold, and the hydrophobic groups in the molecule become exposed to the protein surface, resulting in hydrophobic aggregation between protein molecules [45].In this study, the preheating treatment had a smaller particle size.Size reduction occurs due to the milling process in the preheating treatment.Because the aggregate formed in the preheating process is brittle, the grinding process results in a smaller particle size.This is marked by an increase in particle size with a size of <99 µm, which is categorized as dust size (≤100 µm) [44].

Protein digestibility
Based on the results, the digestibility of soy protein in the control treatment was 50.59% ± 0.26.Meanwhile, in the initial heating treatment at 90 °C, it was 48.11% ± 0.81, respectively.Based on the T-test, soy protein concentrate preheated at 90 °C experienced a significant decrease in protein digestibility when compared to the control treatment.Thermal processes can basically have a positive impact on protein digestibility [46].However, this depends on the treatment conditions and the degree of denaturation.Heating can inactivate trypsin inhibitors, which have a positive impact on protein digestibility.In addition, the opening of protein structures during the denaturation process can increase the accessibility of enzymes [47].However, the opening of the protein structure can eventually lead to the formation of aggregates, which causes a decrease in protein digestibility [48].The higher digestibility of soy protein in the control treatment compared to the preheating treatment at 90 °C can also be affected by the thermal process used in the manufacture of soy protein concentrate.The spray drying process in the manufacture of soy protein concentrate involves a thermal process, which allows for partial denaturation and increased protein digestibility.The decrease in protein digestibility in the preheating treatment compared to the control treatment can also be caused by the denaturation process and the formation of protein aggregates during the preheating.Protein aggregates are generally irreversible and very stable [28].Thus, the formation of aggregates inhibits the binding of enzymes to proteins, so that the digestibility of proteins decreases [25].Although the pre-heating process results in a decrease in digestibility, high-protein food products with a lower hardness can increase the amount of protein that can be consumed, thereby allowing a greater amount of protein to be digested.A decrease in protein digestibility with heat treatment also occurred in other study [49].In that study, protein digestibility was related to the degree of hydrolysis (DH), namely the ratio of the number of broken peptide bonds to the total bonds available for proteolytic hydrolysis [50].Some study conducted research on quinoa protein isolate at various pH by heating it to 90 °C for 30 minutes; the results obtained showed a decrease in DH in the heating treatment compared to controls [49].This decrease is thought to correlate with the level of protein aggregation, which causes reduced pepsin accessibility [49].

Conclusion
Based on the results, preheating affects the functional properties of soy protein concentrate.Preheating resulted in a significant decrease in solubility, gel-forming capacity, voluminosity, and protein digestibility, when compared to the control.The difference in preheating temperature significantly affected the solubility, gelling capacity, and protein digestibility.However, the difference in preheating temperature did not affect the voluminosity and microstructure significantly.Decreases in solubility, gel-forming capacity, voluminosity, and digestibility indicate the formation of protein aggregates through a denaturation process.The formation of irreversible protein aggregates increases protein stability, thereby preventing excessive aggregation during heating in the food production process, which results in a hard food texture.

Figure 1 .
Figure 1.Solubility of soy protein concentrate control and preheating treatment

Figure 4 .
Figure 4. Particle sizes of soy protein concentrate control and preheating treatment

Table 1 .
SEM image of soy protein concentrate control and preheating treatment