Effect of heating temperature on physical, functional, and digestibility properties of Whey Protein Concentrate (WPC)

Whey protein is a substance that is derived from the production of cheese. In its native form, whey protein has problems in its application in food products because it can cause a hard texture that affects sensory acceptance. It is necessary to modify the functional properties of whey protein so that it can be used more widely in the food industry. One of the modifications of whey protein is the heating treatment. The process was carried out by preheating at 70, 80, and 90°C. Then centrifuged for 15 minutes at 6000 rpm, then dried the solids using an oven vacuum at 50°C with a pressure of 25 inHg. The functional properties tested in this study were microstructure morphology, solubility, gel texture, voluminosity, and protein digestibility. The morphology of WPC powder produced significant differences between native and heating treatment. The microstructure results using TEM showed that the shape of native WPC was spherical and porous, while the heating treatment was flake-shaped. In terms of particle size distribution, native WPC and 80°C treatment have a bimodal pattern, while 70 and 90°C treatment have a unimodal pattern. As the heating temperature increases, the protein solubility will be getting lower. The lowest solubility was obtained at 90°C (16.04 ± 0.74%). Hardness produced in the gel with heating temperature significantly different than the native with the decreasing pH, the hardness produced will be higher. The fastest acidification rate produced until the final pH of 4.4 was at 80 and 90°C (270 minutes), which was faster than native (295 minutes). The heating treatment increase significantly differently in voluminosity than the native. Protein digestibility resulted in a significant difference between native and 80°C, where at 80°C (30.76 ± 0.07%), protein digestibility was higher than native (27.38 ± 0.09%).


Introduction
Milk is an emulsion of fat in water which contains several dissolved compounds [1].The main composition of milk is water, fat, milk sugar (lactose), and protein [2].Part of the milk protein is in solution and the rest is in colloidal suspension [3].Cow's milk contains 2.5-3.7%protein, the amount of protein contained in cow's milk is 30-35 g/L [4,5].The two largest types of protein in milk are casein which represents 80% milk protein, and whey protein which represents 20% milk protein [5].
Whey protein is a substance that is derived from the production of cheese.Often in the manufacturing process, whey protein is considered as waste.Even though whey protein can be applied well in the food industry because it is cheap, has good nutrition for health, and its functional properties can be regulated [6,7].An example of a dry whey product is whey protein concentrate (WPC) powder, which has a variety of functional properties that can enhance the nutritional value of food items such as cookies, 1230 (2023) 012155 IOP Publishing doi:10.1088/1755-1315/1230/1/012155 2 bread, cakes, crackers, pasta, confectionery products, baby food formulations, sports supplements, and special diet foods [8,9].WPC is abundant in calcium, phosphorus, essential amino acids, and watersoluble vitamins, which makes it a highly nutritious substance [10].
Whey protein as a protein source for high protein foods has problems in its original form because it has functional properties in increasing texture where this is not expected.Therefore, to minimize these properties it is necessary to carry out a denaturation process, one of which is the heating method [11].
Heating is a modification method that can affect the functional properties and physical characteristics of whey protein resulting in denatured protein.The functional properties of whey are generally associated with whey protein.The molecule that plays a major role in experiencing these changes is β-Lg.has been investigated that the temperature, protein concentration, ionic strength, pH, and the presence of whey protein or casein are all factors that can affect changes in β-Lactoglobulin.[12].
Whey protein has the ability to form a gel, which can enhance the texture of food products.This gelling property is an important functional characteristic that is needed for consumer acceptance, especially in the processing of meat, milk, bread, cakes, and to enhance the visual appeal of food products like yogurt.[13].
The solubility of protein is determined by the interaction between the hydrophobic surface of the protein molecules and the hydrophilic properties of the solvent, which in the case of food is water.As a result, the solubility of proteins is considered a hydrophilic characteristic [14].
The method for determining the nutritional value of protein for humans typically involves using the protein digestibility corrected amino acid score.This process has a significant impact on how amino acids and nitrogen are absorbed and utilized in the body's metabolism [15,16].Previous studies have demonstrated that not all proteins are created equal in terms of their digestion rates, with some being classified as slow or fast proteins based on their molecular structure.Additionally, the way that proteins denature or clump together in the digestive system can either speed up or slow down the process of gastric emptying [17,18].While the unique peptides found in different proteins may also play a role in digestion kinetics, the overall structure of the protein is a crucial factor [19].
According to this explanation, this study will focus on examining how changes in heating temperature affect the functional properties of WPC by looking at solubility, gel characteristics seen based on microstructure and texture observations, and voluminosity to determine the ability of whey protein as a component that can improve functional properties in food both as a structure builder and/or structure breaker.Then an in vitro protein digestibility test was carried out to find out how easily the WPC protein could be digested after being given a heating treatment.

Solubility.
The solubility of WPC powder was carried out at room temperature (25 -30 o C) using the method [20] with slight modifications.The WPC Powder (80 w/w) and skim milk sample was dissolved in 50 ml of distilled water using a vortex for 5 minutes.Then, the solution was transferred to a falcon tube and centrifuged for 10 minutes at 700 x g (2010 rpm).After centrifugation, the total solids content (TS) obtained was heated in an oven at 105°C for 24 hours.The solubility (S %) of the WPC powder was calculated using the following equation [21] : where, Wts is the total weight of solids (dissolved and insoluble) in solution (g), W is the weight of insoluble solids (g).
2.2.2.Microstructure.Particle analysis was performed using TM-EDS with 250x and 500x magnification.Taken with an electron beam acceleration of 5 kV.The particle diameter is calculated using the Image J application.
2.2.3.Gel texture.Gel texture was measured using a texture analyzer.Measurements were made using the puncture test method.The gel strength of a cylindrical gel sample (with a height of 24mm and a diameter of 25mm) was measured using texture analysis with a cylindrical aluminum probe P/0.5R and a TA.XT Plus machine.The measurement was carried out under specific parameters, including pre-test speed 2.0 mm/s, test speed 1.0 mm/s, test speed up 2.0 mm/s, test distance 5.0 mm, and trigger force 5.0 g, with the maximum strength obtained from continuous compression being recorded as the gel strength value (in grams) [22].

Voluminosity.
The voluminosity of whey protein was determined according to the method outlined by [23].The procedure involved diluting heat-induced casein micelles and whey protein aggregates to different concentrations and measuring the viscosity of each dilution at 20 o C using a viscometer.The volume fraction (ϕv) of each dilution was calculated based on the relative viscosity data (ƞr ).Lee's equation was then used to calculate the voluminosity of the whey protein aggregate [24].
Voluminosity, (mL g -1 ) is calculated using the formula: Where c is the protein concentration g L -1 2.2.5.Protein digestibility.In vitro protein digestibility test was based on the method of [25] with slight modifications.Protein digestibility was carried out in vitro by analyzing total N using the Micro-Kjeldahl method.Samples without the addition of enzyme and with the addition of enzyme are compared to the total N value so that the protein digestibility of the sample is known.Here's the calculation formula.

Solubility
The functional properties of a food product are influenced significantly by its solubility.The effect of protein solubility in food products greatly affects the level of consumer preference.The following are the solubility of heating treated WPC.

Figure 1. Solubility index between WPC native and WPC preheated
The solubility results in Figure 1 show that native produces the highest solubility compared to the heating temperature treatments of 70, 80, and 90 o C. WPC with heating treatment produces a very significant difference to the control.This happens because the pre-heat or pre-heating with a temperature of > 60 o C can denature proteins so that hydrophobic bonds are formed, where these bonds produce solids called aggregates.The treatment between temperatures also has significant differences, as in the treatment between temperatures of 70 with 80 o C, where the heating temperature treatment of 70 o C resulted in a higher solubility.However, the heating treatment between 80 o C and 90 o C did not show a significant difference in solubility.From the graph it can be seen that the higher the temperature, the resulting lower solubility.This is the same as what happened to [26] that the higher the heating temperature, the lower the protein solubility.Higher temperatures will cause protein solubility to decrease, but it is stated that solubility will increase in the temperature range of 40 -50 o C. Research conducted by [27] proved that whey protein looked very compact and produced a lot of insoluble aggregates compared to samples which were dominated by soy protein.
An increase in protein solubility and surface hydrophobicity can lead to a decrease in the Water Holding Capacity (WHC) of proteins.Research by [28], has shown that heat treatment of whey can lower WHC values, as the denaturation of whey proteins during heating makes it easier to remove water during centrifugation, resulting in a more open network structure of the gel.The susceptibility of each whey protein to denaturation and protein interactions is ultimately determined by the composition and sequence of the inherent amino acids that control the molecular structure.Hydrophobic interactions are entropy driven, therefore they often occur at higher temperatures.Hydrophobic interactions, which are driven by entropy, tend to occur at higher temperatures.These interactions arise from unfavorable interactions between water molecules and nonpolar residues on protein molecules, resulting in a decrease in entropy and a change in the water structure.To minimize this unfavorable entropy change, nonpolar amino acid residues are forced to interact and form a hydrophobic core, reducing their contact area with water [29].This leads to intramolecular conformational changes or intermolecular aggregations of protein molecules to protect the hydrophobic residues from the aqueous environment.Another theory suggests that the hydrophobic effect arises from the mutual affinity between nonpolar chains via van der Waals forces [30].
Solubility in the food industry is needed because solubility can affect the protein can form structures in food.In this study, it was observed that heating treatment caused a decrease in the solubility of WPC.In general, low protein solubility is not beneficial in terms of foaming agent, emulsion, gelation, and whipping properties [31].However, some applications such as high-protein beverages, yoghurt, juices, and the manufacture of high-protein biscuits low solubility is required for colloidal stability and avoiding the formation of hard structures in foods [32].Figure 2 shows that heat-treated and native WPC powder have significant differences in shape.It can be seen that native WPC powder has a spherical shape with pores scattered on its surface.The heating treatment at 70, 80, and 90 o C can be seen to produce fine flakes without pores.Assessing the morphological parameters of WPI samples have spherical particles [33].In general, native WPC powder and WPI produce smooth powder particle surfaces, while denatured whey protein produces microwrinkles on the powder surface [34].However, in this study, the results are opposite.
Based on the graph in Figure 3, it can be seen that the particle size distribution in native WPC produces a bimodal size distribution graph, which has 2 size peaks where this indicates that the particle size of native WPC has a size that tends to be heterogeneous with an average particle size in the range of 40-49 µm and 70-79 µm.The 70°C heating temperature treatment produces a unimodal size distribution graph, which has 1 peak which indicates that the 70°C temperature treatment has a particle size distribution that tends to be homogeneous in the range of 40-49 µm.The 80 o C heating temperature treatment produces a bimodal particle size distribution in the range of 20-29 µm and 40-49 µm, where these results are lower than native WPC.Then the 90 o C temperature treatment has a unimodal particle size distribution, but the particle size range is wider at 30-59 µm.When whey protein was heated, it causes the protein particles to become larger and less dense, resulting in powder particles that are more porous.This process also increases the cohesiveness of the powder, particularly when there are high levels of native whey protein present [35].However, the results showed that the native WPC had a larger particle size similar to that of the heating treatment.

Voluminosity
A common way to measure hydration degree is to measure a related quantity, voluminosity which is defined as the number of "ml" of solution occupied by one gram of dry micellar material [36].

Figure 4. Voluminosity of WPC preheated
According to Figure 4, native WPC has lower voluminosity compared to heated WPC at of 70, 80, and 90 o C treatment.The voluminosity between heating temperatures of 70 to 80 o C was not significantly increase, but between 70 and 90 o C was significantly increase.Within the heating temperatures of 80 and 90 o C, the voluminosity of 90 o C was lower than that of 80 o C treatment.The graph indicates that the heating temperature of 80 o C resulted in the highest voluminosity value of 5.77 ml/g.This is consistent with research conducted by [37] which showed that heating WPI (6% w/w) at 78-82 o C increased viscosity and voluminosity values compared to control temperature (24 o C).As expected, thermally denatured WPI or individual whey proteins increased voluminosity values [38].However, the graph shows that the voluminosity value decreased between heating temperatures of 80 and 90 o C.This is in agreement with research on casein micelles which showed that the voluminosity value at 20 o C (4.1 ml/g) was higher than at 70 o C (3.5 ml/g) [39,40].Increasing temperature can cause a decrease in voluminosity, which continues to slow down within the covered temperature range.
When whey protein was heated, it causes the β-lactoglobulin to unfold, leading to the formation of disulfide bonds which make the sulfhydryl groups reactive to other types of chemical bonds.This process results in a decrease in the ratio of whey protein, while increasing both the voluminosity and volume fraction of whey protein.The voluminosity of whey protein is affected by factors such as the total solids, the ratio of casein whey protein, and the extent of protein denaturation [41].

Gel texture characteristics
The way in which WPI forms a gel is impacted by various factors such as its concentration, the surrounding conditions (including temperature, pH, and ionic strength), and how it interacts with other components of the food.[42].The process of gelation or gel formation has a series of reactions produced, one of which is ionic bonding where the right balance is needed between protein-protein and proteinwater bonds [29].The bonds that occur in the gelation process are the disulfide bonds produced by heatinduced whey protein gels that produce rubbery characteristics, then brittleness which is responsible for non-covalent bonds, and hardness [43,44].Hardness refers to the maximum force during the first pressing.Figure 5 shows that the hardness of native WPC (C) tends to increase as pH decreases, and the heating treatment at 70, 80, and 90°C also tends to increase hardness as pH decreases at various measurements.The hardness WPC native results trend in a lower at temperature measurements between 70 o C and 80 o C, it can be seen that trend of increasing hardness has increased as the pH decreases with a temperature treatment of 80 o C which tends to be higher, although it can be seen from measuring pH 4.7 that there is no significant difference between the two.At temperature measurements between 70 o C and 90 o C, it can be seen that hardness increases as the pH decreases hardness.The resulting which tends to be higher.The hardness at the heating temperature between 80 o C and 90 o C produces trend an increasing.But between the two, there was no significant difference in the range of pH 4.7 -4.57and it was only seen that the difference hardness at pH 4.5 with a temperature treatment of 90 o C tended to be higher.The findings demonstrate that increasing the heating temperature and decreasing the pH result in increased gel hardness.This is due to the gradual breakdown of GDL into gluconic acid during incubation at 35-37°C, which leads to the release of protons.These protons then decrease the electrostatic repulsion between protein aggregates and induce their aggregation into a three-dimensional gel network by protonating the charged carboxyl groups.[45].These results are directly proportional to previous research conducted by [46], where hardness of the whey protein gel has a higher value at 70 o C than 60 and 65 o C. According to [47], the pH of the solution is a crucial factor affecting the appearance and strength of the WPC gel.

Figure 6. Graphic acidification rate of WPC preheated
The graph in Figure 6 shows that the time required for gel formation is affected by pH levels.Native WPC starts to form a gel at pH 4.7 within 90 minutes, while WPC treated at 70°C reaches pH 4.69 in ).The 80°C treatment takes about 270 minutes to reach pH 4.4, which is faster than native WPC.The 90°C treatment takes about 270 minutes to reach pH 4.4, which is slower than the 80°C treatment.At 80°C, the pH reduction rate is faster than at 70°C to reach pH 4.4, while the rate is the same between 80°C and 90°C.The pH reduction rate is faster at 90°C than at 70-80°C to reach pH 4.4.These results indicate that higher heating temperatures lead to faster acidification rates during the gelation process.This finding is consistent with a previous study by [48], which showed that WPI with a concentration of 4% (w/w) can reach pH 4.4 within 135 minutes.
In the results of this study, it can be seen that the higher the heating temperature, the faster the acidification rate and the higher the hardness of the resulting gel.This is because the acidification rate depends on the protein: GDL concentration ratio as well as the temperature that affects the gel formation process [48].Therefore, the gel that becomes hard as the pH decreases will harden depending on how much protein: acidulant ratio and temperature is used.
According to a study, gelation of WPI at a 7% concentration was examined after the addition of varying amounts of GDL.The acidification process was found to be faster at higher GDL levels, resulting in a lower final pH [49].The pH at which the elastic modulus of the aggregates increased was observed to decrease with decreasing pH, but the value was consistently lower when the acidification process was faster.The study concluded that the stiffness of the gel at a certain pH is influenced by the rate of acidification, with slower acidification allowing more time for the gel to develop and become more rigid at that particular pH [50].
The ability to form a gel is a valuable characteristic that can be employed in a variety of food products, including baked goods, processed meats, surimi, desserts, and sour cream.However, in most food formulations, multiple functional properties are required, making it challenging to determine the specific importance of each property in achieving successful product outcomes [51].

Protein digestibility
Digestive enzymes break down the proteins in whey into polypeptides, which are further broken down into shorter peptides such as tripeptides, dipeptides, and some free amino acids in the gut by pancreatic enzymes.These shorter peptides are mainly absorbed by the intestinal epithelial cells (IECs) present in the small intestinal mucosa [52].Protein digestibility is closely related to bio accessibility, which is a measurement of the proportion of compounds consumed in the diet that are released from the food matrix during digestion in the lumen content and are accessible for absorption in the small intestine or biologically transformed by intestinal micro-biota [53].Whey protein has a high main content of β-Lg, but β-Lg in its native form (without heating treatment) is resistant to the digestive enzymes pepsin and chymotrypsin due to its stable structure.Therefore, it is necessary to change the structure of whey protein, namely by heating [54].It can be seen in Figure 7 the results of protein digestibility of native WPC powder with 80 o C heating temperature treatment.The treatment was taken only at 80 o C heating temperature because at that temperature whey protein produces significant boundary size distribution and lower particle size than other samples (native and 70 o C treatment) [55].From these results, it can be seen that the heating process at 80°C treatment (30.76%) produces a significant difference with native (27.39%).These results show that under 80 o C heating temperature treatment can increase protein digestibility.This was proven by previous research which stated that the bio accessibility of essential amino acids of WPI in 80 o C heating treatment (35.5 ± 1.2%) resulted in higher bio accessibility than the control (34.1 ± 1.5%) (without heating treatment).This occurs due to an increase in susceptibility to pepsin hydrolysis [56].Heating time is also very influential, the longer the heating time, the smaller the particle size of the peptides in whey protein, so that it greatly affects the digestibility of proteins in the stomach and duodenum [55].Heating above 70°C causes dimers to break apart into individual monomers, which in turn exposes hydrophobic amino acids and free thiol/sulfhydryl groups.Heating at 80 to 90°C results in a change in the shape of the β-LG molecule which can increase exposure to the stomach thereby increasing the hydrolysis of β-LG by pepsin [57].
Breaking the native structure and arrangement of β-lactoglobulin through cleavage of disulfide bonds or heating leads to a notable rise in the vulnerability of the protein to be digested by pepsin and chymotrypsin.Heating process at 80 and 90 o C can reduce the resistance of β-Lg to the digestive enzymes pepsin and chymotrypsin, which in turn can increase the absorption of β-Lg in the digestive system.The susceptibility to proteolysis gradually increases with heating temperature; however, the effect is more obvious at 80 and 90 o C than at 50, 60, and 70 o C [54].

Conclusion
Protein denaturation by heating treatment affects the functional properties of WPC powder, including microstructure, solubility, texture, voluminosity, and protein digestibility.The microstructure of WPC powder seen through TEM showed a difference in structural shape between native and heating treatment, but not between treatments.The particle size of heating-treated does not differ much from that of native WPC.The solubility of WPC decreases with increasing heating temperature.The texture of the gel in the acid cold-set gelation process resulted in a highest the fastest acidification rate produced until the final pH of 4.4 was in the 80 and 90 o C (270 min).Voluminosity from various heating treatments have a significant difference with a trend that tends to increase with increasing temperature.However, when the heating treatment was 90 o C, there was a decrease in voluminosity value.Protein digestibility resulted significant difference between native WPC and 80 o C treatment, where in the 80 o C, protein digestibility was higher than native.

Figure 5 .
Figure 5. Gel hardness of WPC preheated graphic At 80°C, it takes 83 minutes to reach pH 4.70, and at 90°C, it takes only 60 minutes to reach pH 4.70.On the other hand, native WPC takes 295 minutes to reach pH 4.44, which is slower than the 70°C treatment (280 minutes with a final pH of 4.42

Figure 7 .
Figure 7. Protein digestibility diagram of native and WPC preheated