The effect of preheated whey protein concentrate addition on high protein biscuit

WPC (Whey Protein Concentrate) is a product that has a high biological value (BV) and nutritional composition with a protein content ranging from 34–80%. WPC can be applied in the development of high protein food products such as biscuits. However, the excessive use of WPC might result in a hard texture that will have an impact on decreasing the palatability of the product. Preheated treatment can be used to modify WPC so that it loses its functional properties as a structure builder. WPC will be denatured and cause the formation of whey protein aggregates. The denaturation that occurs makes WPC tend to lose its reactivity and become more stable. The aim of this study was to determine the effect of preheated WPC in a high protein biscuit. There were 7 treatments; control biscuit (without WPC), biscuits with the addition of non-preheated (NPH) and preheated (PH) WPC with 11%, 13%, and 15% protein content. The results showed that the addition of preheated WPC could produce better physical and sensory characteristics when compared to biscuits using non-preheated WPC. Biscuits PH 11% can produce the best characteristics with a hardness value of 1,171.543 g; crumb structure with small pores; porosity 18.944%; and a DF value of 2.482. This is also supported by the results of the triangle test, where the panellists could not distinguish the colour, taste, and texture of the biscuit when it was compared with the control.


Introduction
Protein is a macronutrient component that has an essential role in humans as the source of nitrogen and essential amino acids [1].Protein in the body has a variety of functions, including building or forming substances, regulating metabolic processes, means of transportation, playing a role in the body's defense mechanisms (antibodies), generating and conducting nerve impulses, regulating growth and differentiation, and being the main source of energy besides carbohydrates and fats [2].Every individual need adequate protein intake because a lack or excess of protein can have a bad impact on health.Lack of body protein can disrupt protein synthesis and ultimately affect body activities including growth, while excess can lead to obesity [3].Fulfillment of protein intake can be done by developing highprotein food products.
High protein food is a product that is formulated by adding certain food ingredients that contain high concentrations of protein [4].A high protein food claim can be used if 100 grams of food product in solid form has a protein content of 20% NRV (Nutrient Reference Values) [5].The NRV of protein that must be used for labeling purposes in international standardization is 50 grams [6].Based on these, claims for high protein foods can be fulfilled if 100 grams of product have a protein content of at least 10 grams.High protein food products can be developed from various types of products, one of which is 2 biscuits.Biscuit is a small baked product made from flour, sugar, and fat with a low moisture content (less than 5%) and a long shelf life [7,8].Biscuits were chosen because it has an easy production and distribution process and is also popular with various groups (from children to adults) [9].According to [1], commercial biscuits on the market generally have a low protein content of around 7-8%.The protein content in biscuits can be enriched by adding food sources of protein, one of which is whey protein.
Whey protein or cheese serum is a greenish-yellow solution obtained from separating coagulated milk curd using proteolytic enzymes or acids [10].Whey protein has a nutritional composition and a high biological value (BV), which is 100 [11,12].Biological value (BV) is a value that can be used to find out how much the body uses protein from a food ingredient [2].Whey protein also has good benefits for human health such as being able to control hypertension and cholesterol, reducing the risk of type 2 diabetes, improving cognitive function and memory, preventing malnutrition and obesity, and providing nutrition for children [13].Whey Protein Concentrate (WPC) is a whey protein derivative product that has a protein content (total solid) in the range of 34% to 80% [14].WPC has high nutrition because it is rich in calcium, phosphorus, essential amino acids, and water-soluble vitamins [15].WPC also contains several essential amino acids such as histidine, isoleucine, leucine, lysine, methionine/cysteine, phenylalanine/tyrosine, threonine, and tryptophan which are especially needed by children [14].
The use of WPC in a high-protein biscuit formulation will increase the nutrition of the resulting product, but the excessive use of protein will cause problems.The resulting biscuits will have a hard texture, which will impact on decreasing product palatability [4].Modification of WPC can be done by preheating treatment to produce biscuits with a texture that is not too hard [16].WPC when given by heat treatment, will change so that it loses its functional properties as a structure builder and can reduce its solubility [14].Research conducted by [17] shows that the use of WPC made through a denaturation process at 90˚C for 60 minutes using a batch pasteurizer produces biscuits with a texture that is not too hard when compared with the commercial or native WPC.Research by [4] showed that the optimal formula of biscuits made from denatured WPC can be obtained by using 12.49% protein and 3.28% minerals, however this formulation still has a high hardness value (5,432.18g) so it still has a hard texture.The aim of this study was to examine the effect of adding preheated (PH) WPC with certain protein contents (11%, 13%, and 15%) which were compared with the addition of non-preheated (NPH) WPC and the control (without WPC) to produce high protein biscuits with a good texture and sensory characteristics.This formulation is also made by adding yellow sweet potato flour and the minerals (calcium lactate, zinc gluconate, and Iron Polymaltose Complex (IPC)) to complement the nutritional content of the biscuits.

Materials
The main materials used in this research were water, baking powder, baking soda, powdered sugar, egg yolks, margarine, minerals (calcium lactate, zinc gluconate, and Iron Polymaltose Complex (IPC)), skim milk powder, wheat flour (low protein), yellow sweet potato flour, vanilla flavoring, and commercial WPC 80 with a protein content of 82.78%.

Preheating treatment
The preheating process of commercial WPC80 begins with weighing the WPC and water according to the formula that has been specified.The WPC and water were put into a beaker glass and heated using a hot plate magnetic stirrer to a temperature of 90˚C and held for 15 minutes.Cooling was carried out until the WPC temperature reached 25˚C (room temperature).The WPC that has cooled can be added to the biscuit dough for PH 11%, PH 13%, or PH 15%.

Preparation of biscuit
The formulation and procedure of high-protein biscuits refer to [4] with modifications.The formula was made using the principle of mass balance by adding non-preheated (NPH) WPC or preheated (PH) WPC to produce biscuits with a protein content of 11%, 13%, and 15%.The biscuit was made using the creaming method.Margarine and powdered sugar were mixed using a mixer at 860 rpm for 3 minutes.Wet ingredients including egg yolks and Iron Polymaltose Complex (IPC) were added and then mixed for 2 minutes.Dry ingredients including wheat flour, yellow sweet potato flour, skim milk powder, minerals (calcium lactate and zinc gluconate), vanilla flavoring, and leavening agents (baking powder and baking soda) were added, then mixed for 3 minutes until all the ingredients blended.Commercial WPC (preheated or non-preheated according to treatment) was added, then mixed 3 minutes.The dough is rested for 15 minutes.The biscuit dough is formed in circular molds with a diameter of 5 cm and a thickness of 0.3 cm and baked at 120 -135ºC for 20 -25 minutes.

Texture analysis
The texture property of biscuits was analyzed with the texture analyzer TA.XTExpress (Stable Micro Systems).Biscuit samples are placed on two plates with a same height.The analysis was carried out using project biscuit bending and P/6 probe with a pre-test speed of 1 mm/s, a test speed of 4 mm/s, a post-test speed of 10 mm/s, and a distance of 10 mm.The measurement results in the form of hardness values will be displayed automatically on the software Exponent Lite Express that has been connected to the texture analyzer.

Image analysis
Image analysis of biscuits is observed by capturing cross-sectional images of biscuits using a camera lens.The resulting images are then processed using the software ImageJ (http://rsb.info.nih.gov/ij/).The biscuit crumbs shown in the image are cut into a rectangular shape measuring 150x1500 pixels and 4 converted to grayscale image.The porosity of the biscuit crumb can be analyzed descriptively through that image.
Quantitative porosity and fractal dimension analysis is performed by further processing grayscale images.Image segmentation was carried out using Otsu thresholding [18].Quantitative porosity analysis is carried out using the menu available in the ImageJ software.Fractal dimension analysis is carried out using the box counting method in the ImageJ.The principle of the box counting method is to place several grids with various sizes in the image, and the number of boxes containing pixels from the image (N) is calculated for each grid size (L) used.The plot of a log-log graph between N as the ordinate (y) and L as the abscissa (x) is made.The slope value of the graph is the fractal dimension on a twodimensional space (D) because it is obtained from a two-dimensional image, so it is necessary to add an extra dimension to the D value to represent a three-dimensional as follows: DF = D + 1 [19].

Sensory analysis with triangle test
Triangle test was conducted to determine sensory differences between products that have been given certain treatments.There are six sets of triangles presented to the panelists.Each set contains three biscuit samples, consisting of two same samples and one different sample.Panelists were asked to identify different samples among the three samples presented sequentially.The sensory properties tested were color, taste, and texture parameters.The interpretation of the data from the test results is done by counting the number of correct answers and then comparing it to the minimum number of correct answers [20].The "no difference" assumption is rejected if the number of correct answers is greater than or equal to the minimum number of answers.The minimum number of correct responses with 15 panelists and α = 10% is 8.

Texture analysis
The texture of biscuits in this study was analyzed using a texture analyzer instrument to determine the hardness profile.Hardness is a force that is given as pressure on the object until it is deformed [21].A high hardness value in biscuits is not expected because it indicates a hard texture that is difficult to consume.The data from the biscuit hardness test results in force (g) and time (s) from each repetition were averaged and then plotted on a graph in Figure 1.The maximum peak in each sample occurs at different times.The maximum peak of control biscuits and PH 11% was achieved in the shortest time, namely 0.14 seconds; followed by NPH 13% at 0.15 seconds; NPH 11% at 0.16 seconds; PH 13% at 0.17 seconds; NPH 15% at 0.18 seconds; and PH 15% at 0.19 seconds.The difference in sample time required to reach the maximum peak can indicate the strength of the sample structure being tested.Products with a high hardness values require a longer time to reach their maximum peak [22].Based on this, the long time to reach the maximum peak indicates a strong sample structure because it can withstand the applied force longer.Research by [22] related to MOSEP (Modified Sweet Potato) and mung bean flour-based snackbars shows a hardness value of 1,861.03gF can be achieved in 0.57 seconds, while a hardness value of 1,961.08 gF can be achieved in 0.69 seconds.
The results showed, there are several samples of biscuits that have a relationship between time and hardness values that is not in suitable with the statement of [22], where the time for biscuits with NPH 13%, NPH 15%, PH 11%, PH 13%, dan PH 15% to reach the maximum peak does not represent the highest force or hardness values.This is because the resulting data is the average of each repetition performed for each treatment.It is possible that each repetition of each treatment has a maximum peak that is achieved at a different time, so the resulting force and time have lower or higher values.This data can also be affected by the level of accuracy and precision of the texture analyzer instrument used [22].Hardness is the maximum amount of force that a product can accept before deformation.The results of testing the biscuit hardness values obtained by calculating the average of maximum force from each repetition in various treatments are shown in Table 2.  Based on the results of the t-test, all treatments including PH 11%, NPH 13%, NPH 15%, PH 11%, PH 13%, and PH 15% had a hardness value that was significantly different from the control biscuits.The average of the test results in Table 2. shows that the control biscuit sample has the lowest hardness value, namely 923.336 g ± 42.023 g, followed by PH 11% (1,171.543g ± 105.593 g), PH 13% (1,253.769g ± 121.870 g), NPH 11% (1,334.710g ± 113.193 g), NPH 13% (1,561.769g ± 65.21 g), PH 15% (1,650.751g ± 131.355 g), dan NPH 15% (2,164.367g ± 207.517 g).The hardness of the PH biscuit group (added preheated WPC80) had a lower hardness value than the NPH biscuit group (added non-preheated WPC80) at the same protein content.However, both the NPH and PH biscuit groups showed an increase in hardness values along with an increase in protein content.
Control biscuits were made without the addition of WPC80 and had a protein content of 9%.The hardness value that appears in the control biscuit can be generated from protein interactions in ingredients used in the formulation such as yellow sweet potato flour, egg yolks, wheat flour, skim milk powder, and other ingredients.The lower hardness value in the PH biscuit group, when compared to the NPH biscuit group at the same protein content, indicates that the preheating treatment for commercial (native) WPC80 have an effect as a texture controller and its application to high protein biscuits can reduce the hardness value.The native protein could easily react with other components to form complex compounds.The high solubility due to the large hydrophilic surface on native whey protein causes the protein to be spread evenly on the food system and forms whey protein aggregates when heated so that it can produce an unfavorable texture [23].Modification of commercial WPC by heat treatment at the right temperature and time causes protein denaturation, the formation of aggregates, and changes in functional properties [24].Denatured WPC causes exposure to hydrophobic regions and the formation of aggregates due to thiol groups interacting with disulfide bonds through oxidation reactions or thiol-disulfide exchange reactions [25].The aggregates that are formed are irreversible and non-reactive because the available protein has been denatured before being processed with other ingredients [26].Research by [27] also stated that the preheating treatment could be applied to induce whey protein aggregation and improve heat stability in subsequent processing.Based on this, the addition of preheated WPC80 to the biscuit dough did not result in the formation of an excessively hard texture in the biscuits.This is suitable with research by [17] that showed the addition of WPC made by a heating treatment until it was denatured in a high protein biscuit formulation resulted in lower hardness (1,880.31± 647.70 -3,154.62 ± 55. 43) compared to biscuits with the addition of commercial (native) WPC (2,786.41± 800.20 -3,698.33 ± 136.95).
The results also showed that the NPH and PH biscuit groups had an increase in hardness as the protein content increased.The protein content of biscuits correlates positively with the value of biscuits.The hardness value of the biscuit will be higher as the protein content increases.The interaction of biscuit ingredients will determine the physical characteristics of the final product.The increasing addition of WPC to the biscuit dough causes the percentage of other ingredients to decrease and affects the texture of the biscuits.This is in line with several previous studies which also showed a trend toward increased hardness with the addition of whey protein.Substitution of wheat flour with 5-15% WPC increases the hardness from 4.3-6.5 (kg) [1]; 5-30% WPC increases the hardness from 4,110.2-7,504 [28]; and the substitution of sweet potato flour in gluten-free biscuits with 5-15% WPC increases the hardness from 18.16-22.26N [29].
The increase in hardness in the PH biscuit also occurred in line with the increase in protein content, although the value was lower than the NPH biscuit.This could be due to the preheating process carried out on the WPC solution, which did not completely denature the protein.To denature whey protein until it is completely denatured needs the right temperature and time.When the amount of WPC80 in the formulation is increased without an increase in temperature or preheating time, some proteins may not be denatured.The amount of undenatured protein will increase along with the increase of the protein content in the formulation, so it will react during the baking process and produce a harder texture in the biscuit.Research by [4] also shows that a biscuit with the addition a large amounts of denatured WPC has a harder texture, whereas biscuit with 10% protein has a hardness of 4,030.54gF while biscuits with 15% protein has a hardness of 6,266.63 gF.
The hardness of the NPH and PH biscuits in this study still have a good texture.The hardness of the tested NPH and PH biscuits had a value range of 1,171.543-2,164.367g.Research by [30] showed that the hardness values of several samples of commercial semi-sweet biscuits ranged from 2,039.43 to 3,059.15 gF.This shows that the texture of NPH and PH biscuits can be accepted by consumers because it still included in the hardness value range for commercial biscuits.As for in this study, PH 11% biscuits were the biscuits with the best treatment because it had the lowest hardness value of 1,171.543± 105.593 which was closest to the control biscuits (923.336± 42.023).

Image analysis
Food products have a distinctive structure that is obtained naturally or through processing [31].Image analysis is a method that can be used as an alternative for determining the structural properties of food products [32].Biscuit structure in this study was carried out on the crumb by analyzing the porosity and fractal dimensions using ImageJ software.

Porosity.
Porosity is a parameter that indicates the open structure of a material [33].According to [34], porosity in food products is the fraction of the volume of air space in a network.Porosity in biscuits is the porous part or air cells that are found on the inside of the biscuit as a result of the interaction of its constituent components and formed during the baking process.The porosity of the biscuit crumb can be observed in Figure 2. The results showed that the control biscuit has a structure 1230 (2023) 012167 IOP Publishing doi:10.1088/1755-1315/1230/1/0121677 with large-sized pores and is distributed evenly in large quantities.The NPH and PH biscuit groups showed a crumb structure with smaller pore sizes than the control biscuits and a trend of decreasing pore size as the protein content increased.The number of pores contained in NPH and PH biscuits has a different data trend.The number of pores in NPH biscuits increased as the protein content increased, while the number of pores in PH biscuits decreased as the protein content increased.The decrease in pore size in the NPH and PH biscuit groups along with an increase in protein content could indicate that the biscuits have a denser structure and a harder texture.According to [35], the denser cookie structure shows a greater level of hardness and less brittle cookies.An increase in the number of pores in NPH biscuits along with an increase in protein content indicates an increase in the volume and thickness of the biscuits as a result of the functional properties of native protein as a structure builder.Native protein in the dough has the ability to form structures related to emulsifying and swelling properties, which proteins are able to hold water and stabilize starch gels during the gelatinization process [36].According to [37], native whey protein has a similar properties with egg whites in terms of its ability to incorporate air in the formation of foam structures.Research by [38] also stated that the addition of native WPC to bakery products could increase its volume up to 18%.
Research by [39] stated that there is a relationship between the interaction of water in the dough and the physical properties of cookies.The use of non-preheated WPC80 which is hydrophilic in biscuits will increase the number of pores, while the addition of preheated WPC80 which changes to become hydrophobic will decrease the number of pores in the resulting biscuits.The higher water absorption in the biscuit dough results in the formation of more pores due to water evaporation during the baking process, which will leave a porous structure in the biscuits [40].Research by [41] also stated that the addition of more native whey protein flour to the biscuit formulation could cause greater water absorption and produce a higher crumb structure.
An increased number of pores in a biscuit can give it a crunchy texture, but if it is too numerous, it can increase the hardness value due to the increased thickness of the biscuit.This was confirmed based on the results of texture analysis using a texture analyzer which showed that at the same protein content, the NPH biscuit with a higher number of pores showed a higher hardness value than PH biscuits with a lower number of pores.The testing of biscuit texture with three-point bending can be affected by the thickness of the sample [42].The lower hardness value of soy protein-based biscuits is probably due to their lower thickness [28].Research by [29] also showed that the addition of 5-10% native WPC to a gluten-free biscuit formulation resulted in an increase in thickness (9.03 mm -10.03 mm) and hardness (18.16 N -22.26N).
Quantitative porosity analysis was also carried out in this study.Based on the t-test results, biscuits with PH 15% had a value that was not significantly different from the control biscuits, while biscuits with NPH 11%, NPH 13%, NPH 15%, PH 11%, and PH 13% were significantly different from the control biscuits.The average test results are shown in Table 2.It shows that the control biscuit sample has the highest porosity value of 22.765% ± 4.072%, followed by 15% PH (19.656% ± 6.277%), 11% NPH (19.621% ± 3.481%), PH 11% (18.944% ± 3.975%), NPH 13% (18.683% ± 4.713%), NPH 15% (18.634% ± 4.439%), and PH 13% (16.144% ± 4.290%).The porosity of the PH biscuit group tends to have a lower value than the NPH biscuit group at the same protein content.Both the NPH and PH biscuits have a porosity value that decreased as the protein content increased; however, the porosity value increased for 15% PH biscuits.Increasing the protein content in the biscuit resulted in a decrease in the porosity value, possibly due to a reduction in the pore size of the biscuit.The results of the descriptive analysis showed an increase in the number of pores in the NPH biscuit group; however, their small size resulted in a trend toward lower porosity values as the protein content increased.The biscuit porosity analysis in this study was carried out by calculating the percentage of porous area compared to the total area of each biscuit using ImageJ.The intensity of color in the image can also affect the porosity value.

Fractal Dimensions (DF).
Fractal dimension is a popular technique that is useful for digital image analysis to determine the roughness of an object and can be applied to measuring and classifying shapes, texture and segmentation analysis, and graphic fields [43].According to [44], the fractal dimension describes the dimensions between conventional dimensions (1, 2, and 3).Fractal dimensions from 1 to 2 can be used to describe the area filling capacity of a convoluted line, while fractal dimensions 2 to 3 describe the volume filling capacity of a highly rugged surface [43].Fractal dimension analysis in this study used the box-counting method carried out on the biscuit crumbs.The results of fractal dimension analysis on various biscuit treatments are shown in Table 2.
Based on the t-test results, biscuits with PH 11% and PH 15% had a DF value that was not significantly different from the control biscuits, while biscuits with NPH 11%, NPH 13%, NPH 15% and PH 15% were significantly different.DF values of biscuits vary within a very narrow range, namely between 2.440 -2.518.The DF value of the control biscuits showed the highest value among the other treatments (2.518 ± 0.046), followed by PH 11% (2.482 ± 0.051), NPH 11% (2.473 ± 0.056), NPH 13% (2.467 ± 0.063), NPH 15% (2.466 ± 0.066), PH 15% (2.464 ± 0.091), and PH 13% (2.440 ± 0.056).This narrow range indicates that products have similar specifications [45].The fractal dimension of the biscuit crumb structure does not show a big difference.The fractal dimension is a representation of the geometry and irregularity of the pores [45].A high fractal dimension value indicates that the pores have a more random pattern and a more irregular geometry [46].Research by [47] also stated that a high fractal dimension value describes a rougher crumb while a lower value describes a smoother or simpler surface.The range of biscuit fractal dimensions in this study is in accordance with [47] who stated that the fractal dimension value of porous products such as bread has a value between 2 and 3, which value describes a wrinkled surface.The fractal dimension values for several types of bread (yeasted sweet bread, danish, pound cake) analyzed showed a range of values from 2.75 to 2.77 [47].
The concept of fractal dimensions can also be used to describe and determine the structure of aggregate particles, where the DF value has a positive relationship with the number of particles and aggregate size [48].The higher the DF value, the denser the aggregate structure formed [49].However, in this study, the DF value was obtained from the segmentation of the biscuit's pore as the object and the cell wall as the background.The value of DF has important consequences for the mechanical properties of aggregate networks and is proven to be used to study two limiting regimes of fractal aggregation, namely diffusion-limited (DLCA) and reaction-limited cluster-cluster aggregation (RLCA) [49].Aggregates formed in the DLCA mechanism can be indicated by a DF value of 1.7-1.8while in the RLCA mechanism, the DF value is greater than 2 [49].The results of fractal dimension analysis showed that all treatments of biscuits had an RLCA aggregation mechanism because the DF value obtained was greater than 2. RLCA is an aggregation mechanism that occurs more slowly due to electrostatic repulsion between approaching particles [48].This RLCA aggregation will produce aggregates with denser and irreversible properties [50].

Sensory analysis with triangle test
The test results in Table 2. showed that the panelists could not distinguish the difference of NPH 11% (color, taste), PH 11% (color, taste, texture), and PH 13% (color) biscuits when compared to the control biscuits, whereases for biscuits NPH 11% (texture), NPH 13% (color, taste, texture), NPH 15% (color, taste, texture), PH 13% (taste, texture), and PH 15% (color, taste, texture) panelists can differentiate the sensory attributes when compared to control biscuits.This shows that the addition of preheated and non-preheated WPC80 can affect the sensory attributes of the biscuits.Research by [28] stated that the addition of 10-30% native whey protein resulted in a decrease in the values of all sensory parameters tested.Research by [51] also stated that biscuits using 10% native WPC had the best characteristics based on color and appearance, taste and flavor, body and texture, as well as overall acceptability compared to biscuits with higher addition of native WPC.Based on the results of the triangle test, the biscuits with PH 11% were the best treated biscuits because the biscuit had sensory attributes such as color, taste, and texture that were similar or not significantly different from the control biscuits.

Conclusion
Modification of commercial WPC80 through the preheating treatment on high protein biscuits with variations in protein content (11%, 13%, and 15%) has an effect as a texture controller and produces better physical and sensory characteristics when compared to biscuits using non-preheated WPC80.Biscuits with the addition of preheated WPC80 with a protein content of 11% (PH 11%) can produce biscuits with the best physical and sensory characteristics.PH 11% biscuits have physical characteristics with a crisper texture, a structure with small pores, and an RLCA (reaction-limited cluster-cluster aggregation) mechanism.These results were supported by sensory test results using the triangle test method that showed PH 11% biscuits had similar color, taste, and texture attributes to the control biscuits.

Figure 1 .
Figure 1.The graph of the biscuit texture analysis

Table 1 .
Formulation of high protein biscuit

Table 2 .
The result of hardness, fractal dimensions, and sensory analysis with triangle test