Effect of Mealworm Powder Substitution on the Properties of High-Gluten Wheat Dough and Bread Based on Different Baking Methods

Mealworms (Tenebrio molitor) are protein-rich edible insects that have been regarded as novel food ingredients. In this study, high-gluten wheat flour was formulated with dried mealworm powder at various levels (0%, 5%, 10%, 15%, and 20%) to study its influence on the pasting, farinograph, and extensograph properties and microstructure of the dough. A subsequent decrease in the pasting parameters was observed due to starch dilution. The water absorption, dough development time, and dough stability time decreased gradually from 71.9% to 68.67%, 13.6 min to 10.43 min, and 14.1 min to 5.33 min, respectively, with the increase in the substitution of mealworm powder from 0% to 20%. The farinograph characteristics corresponded to a weak gluten network formed through the dilution of gluten by the replacement of wheat flour with a non-gluten ingredient. The stretch ratio of the high-gluten dough increased gradually from 4.37 (M0) to 6.33 (M15). The increased stretching resistance and extensibility of the dough with 5% and 10% mealworm powder indicated that mealworm powder can act as a plasticizer in the gluten network, which might contribute to the decreased strength and increased elasticity and flexibility of the dough network. The bread made with three different baking methods showed similar increases in specific volume and decreased hardness up to the 10% substitution level, owing to the increased elasticity and flexibility of the dough. The GB/T 35869-2018 Rapid-baking method, GB/T 14611-2008 Straight dough method, and automatic bread maker method exhibited the highest specific volumes of 3.70 mL/g, 3.79 mL/g, and 4.14 mL/g when the wheat flour was substituted with 10% mealworm powder. However, 15% and 20% mealworm powder substitution markedly reduced the bread quality owing to the dilution effect and mealworm powder phase separation. These results provide a perspective on the relationship between the rheological properties of mealworm powder-substituted high-gluten dough and application suggestions for insect food development in the food industry.


Introduction
To deal with the global population increase, the loss of farmland, and global climate change, there is an urgent need for alternative protein sources to replace animal protein production [1]. Proteins of vegetable origin were identified as the first candidates, but they are not the best choices owing to the low protein and limited amino acid contents of plants [2]. Insects have traditionally been regarded as a part of the diet in most countries. In comparison to the proteins of plant and common livestock origin, those from insects have the advantages of being environmentally friendly, requiring less land and feed use, and having a high food conversion ratio. From a nutritional perspective, insects have a high content of protein (35-61%), lipids (13-33%), and dietary fiber. In addition to macronutrients, some insects are also known for their rich mineral and vitamin profiles [3]. The yellow mealworm (Tenebrio molitor), as a commonly farmed insect used for pet food, has attracted attention in the food industry. The dried yellow mealworm was regarded of Cereal Chemists (AACC) method 76-21 (AACC, 2000), a Rapid Visco Analyzer (RVA) (RVA-Eritm, Perkin Elmer, Waltham, MA, USA) was used to study the pasting properties of the control wheat flour and the blends [22].

Scanning Electron Microscopy (SEM)
The wheat dough prepared from the mixed flour was dried at −80 • C overnight, and the samples were then sputtered with a thin layer of gold under vacuum conditions. The freeze-dried dough was observed using a scanning electron microscope (TESCAN MIRA LMS, Brno-Kohoutovice, Czech Republic) at an acceleration voltage of 15 kV.
GB/T 35869-2018 Rapid-baking method [21]: All raw materials were mixed and kneaded for 20 min using a dough mixer (AM-CG108-1, ACA). The resultant dough was then subjected to pre-fermentation at 38 • C with 85% relative humidity for 20 min in a controlled fermentation cabinet (DHTHM-16-0-P-SD, Doaho Test Co., Ltd., Shanghai, China). The fermented dough was divided into several pieces with a weight of approximately 75 g, and each piece was then molded into a round shape. After fermentation (40 min, 38 • C, 85% relative humidity), the bread was baked in a steam oven (K6, Daewoo) at 175 • C for 20 min.
GB/T 14611-2008 Straight dough method [20,24]: All raw materials were mixed thoroughly using a dough mixer (AM-CG108-1, ACA) for 20 min. The dough was leavened for 70 min at 30 • C in a glass bowl sealed with food-grade cling wrap (DHTHM-16-0-P-SD, Doaho Test Co., Ltd., Shanghai, China). Thereafter, the fermented dough was divided into pieces weighing approximately 75 g, and each piece was formed into a round shape and sealed with food-grade cling wrap for secondary fermentation (20 min at 30 • C). The bread was baked at 175 • C with top and bottom heat for 20 min in a steam oven (K6, Daewoo).
Automatic bread maker method [25]: To prepare the bread, 250 g of mixed flour, 15 g of butter, 90 g of water, 18 g of sugar, 3 g of salt, 12 g of milk powder, and 3 g of yeast were put into a bread maker (MM-ESC1510, Midea). The wheat flour was replaced at ratios of 0%, 5%, 10%, 15%, and 20% with mealworm powder to make the bread. The operating conditions of the bread maker were set as basic bread, light color, and 500 g.
2.6. Physiochemical Analyses of Bread 2.6.1. Specific Volume, Porosity, and Colorimetric Analyses The specific volume of bread was determined using the millet displacement method. The specific volume (mL/g) was calculated as the volume (mL)/weight (g) of bread. Image analysis was employed for the evaluation of porosity and cell density (crumb porosity), according to the method of Kowalski et al. [5]. Images of the bread cross-sections were obtained, and the porosity of the bread was analyzed using ImageJ software. The images were converted into 8-bit gray images, and the corresponding regions were selected for analysis to obtain the cross-sectional porosity.
The crumb colors of the bread samples were detected using a colorimeter (CS-820N, Hangzhou CHNSpec Technology Co., Ltd., Hangzhou, China). The parameters L* (darkness/brightness), a* (shade of red/green), and b* (shade of blue/yellow) were analyzed. The determination was based on the L*, a*, and b* color systems.

Texture
Texture profile analysis (TPA) was performed using a TA-XT plus texture analyzer (Stable Micro Systems Ltd., Surrey, UK) with a 36 mm diameter cylinder probe P/36R, according to the method described by de Castro et al. [26]. The bread was cut into pieces with a thickness of 5 cm. The pre-test, test, and post-test speeds were 2 mm/s, and the sample compression was 25% of its original height with a 5.0 g trigger force.

Statistical Analysis
All experiments were performed at least in triplicate. OriginPro 2022b (OriginLab, Northampton, MA, USA) was used for data processing. Data were expressed as the mean ± standard deviation. Statistical differences in the correlation analysis were determined by performing a one-way analysis of variance (ANOVA). Tukey's test at p < 0.05 was used to determine significant differences between mean values.

Pasting Properties
The RVA curves and pasting parameters are shown in Figure 1 and Table 2, respectively. The pasting parameters are related to the extent of starch granule swelling [27]. The peak viscosity of the high-gluten wheat flour decreased nearly linearly with an increase in the mealworm powder content, from 1073.67 cp for wheat flour (M0) to 580.33 cp for that formulated with 20% mealworm powder (M20). The other parameters showed similar decreasing trends, which were ascribed to the starch dilution effect mediated by mealworm powder substitution [28]. Khuenpet et al. [29] also observed similar decreasing trends in peak viscosity and breakdown viscosity. The increased pasting temperature of the wheat flour with mealworm powder might be attributed to the slow heat transfer of the mixed flour due to the increase in fat and dietary contents [30]. worm powder substitution [28]. Khuenpet et al. [29] also observed similar decreas trends in peak viscosity and breakdown viscosity. The increased pasting temperature the wheat flour with mealworm powder might be attributed to the slow heat transfer the mixed flour due to the increase in fat and dietary contents [30].

Farinograph Properties
The farinograph curves and parameters are shown in Figure 2 and Table 3, resp tively. Water absorption is a parameter that is indicative of the ability of the mixed flo to absorb water and form dough of optimal consistency. The water absorption decreas from 71.9% for the control (M0) to 68.67% for the wheat flour formulated with a 20% me worm substitution (M20). This decrease in water absorption was ascribed to the glut dilution effect, which is consistent with the pasting results. A previous study has repor that the nutritional components of mealworm powder include 4.43% water, 3.5% a 1.17% carbohydrate, 43.5% protein, 25.3% fat, and 22.1% dietary fiber [8]. It can be inferr

Farinograph Properties
The farinograph curves and parameters are shown in Figure 2 and Table 3, respectively. Water absorption is a parameter that is indicative of the ability of the mixed flour to absorb water and form dough of optimal consistency. The water absorption decreased from 71.9% for the control (M0) to 68.67% for the wheat flour formulated with a 20% mealworm substitution (M20). This decrease in water absorption was ascribed to the gluten-dilution effect, which is consistent with the pasting results. A previous study has reported that the nutritional components of mealworm powder include 4.43% water, 3.5% ash, 1.17% carbohydrate, 43.5% protein, 25.3% fat, and 22.1% dietary fiber [8]. It can be inferred that the high content of fat and dietary fiber retarded the water absorption of the mixed flours [31]. Waseem et al. [32] also observed a similar decrease in water absorption when wheat flour was substituted with 20% spinach powder. Fang et al. [33] also attributed a 10% isomaltodextrin substitution resulting in decreased water absorption to the incorporation of high dietary fiber content.  [32] also observed a similar decrease in water absorption whe wheat flour was substituted with 20% spinach powder. Fang et al. [33] also attributed 10% isomaltodextrin substitution resulting in decreased water absorption to the incorp ration of high dietary fiber content.  The dough development time, stability time, and farinograph quality number a usually positively related to the dough strength [23]. The dough development time d creased significantly from 13.6 min for M0 to approximately 11 min for the dough wi various mealworm powder contents. Furthermore, the stability time showed a substit tion level-dependent decrease. This decreased tolerance to the mixing of high-glute dough can be partially explained by the interactions between mealworm powder and gl ten, which prevent the complete hydration of proteins, thereby impeding the proper fo mation of the high-gluten matrix during dough mixing. The mealworm powder might a  The dough development time, stability time, and farinograph quality number are usually positively related to the dough strength [23]. The dough development time decreased significantly from 13.6 min for M0 to approximately 11 min for the dough with various mealworm powder contents. Furthermore, the stability time showed a substitution level-dependent decrease. This decreased tolerance to the mixing of high-gluten dough can be partially explained by the interactions between mealworm powder and gluten, which prevent the complete hydration of proteins, thereby impeding the proper formation of the high-gluten matrix during dough mixing. The mealworm powder might act as a plasticizer of the gluten network, potentially weakening the interactions between glutenin chains. Similar tendencies, such as a decrease in dough stability following supplementation with dietary fiber-rich plant materials, have also been reported by other researchers. Tian et al. [27] observed an increasing trend in the dough development time and stability time upon adding a certain amount of Chlorella pyrenoidosa powder to high-gluten wheat flour (11.76 g/100 g protein). Further, Zarzycki et al. [34] found that the dough stability of highgluten wheat flour decreased from 12.7 min (control) to 10.5 min for wheat flour formulated with 12 g/100 g defatted Moldavian dragonhead seed residue flour. The farinograph quality number, which is a measure of the ability of the dough to retain its structure over time during mixing, showed a change similar to that observed in the stability time values. A low degree of softening is desirable and indicates a good tolerance of the dough to mixing. The degree of softening of high-gluten dough increased significantly after mealworm substitution, which corresponded to the weak gluten network resulting from the dilution of gluten by replacing wheat flour with non-gluten ingredients. Similar to the present study, a previous study reported an increase in the degree of high-gluten dough softening after dough supplementation with 0.5% Chlorella pyrenoidosa powder [27]. The color of the dough was influenced by the addition of mealworm powder. Owing to the brown color of the mealworm, the lightness was significantly decreased after this substitution. Moreover, the redness (a*) increased significantly, and the yellowness (b*) showed an increasing trend.

Extensograph and Texture Properties
The farinograph properties reflect the strength of the wheat dough, whereas the extensograph properties reflect the viscoelastic properties of the dough [35]. The extensograph curves are presented in Figure 3, with the extensograph parameters shown in Table 4. The total energy required to stretch the dough from the beginning of stretching to breaking is expressed as the stretching energy. The dough mixed with a 5% mealworm powder substitution gradient showed significantly increased stretching energy and stretching resistance compared to the dough without mealworm powder. This result could be attributed to the interactions between mealworm proteins, lipids, and dietary fiber and gluten proteins. With an increasing substitution gradient, the stretching resistance showed an increasing trend up to a 15% substitution; however, no significant difference was observed among the dough samples with different substitution gradients. The extensibility of the high-gluten dough formulated with or without mealworm powder showed no significant changes between samples. Zhang et al. [36] found that bamboo shoot dietary fiber increased the tensile resistance and improved the viscoelasticity, extensibility, and plasticity of wheat dough. The rearrangement of starch granules and mealworm powder within the dough structure during extensional deformation could interfere with the protein network [37]. The high-gluten dough showed decreased strength and resistance to mixing as well as high stretching resistance and extensibility. Based on these findings and observations, it could be inferred that mealworm powder can act as a plasticizer in the gluten network, which might contribute to the diminished strength but increased elasticity and flexibility of the dough network.

Dough Microstructure
SEM observations were used to investigate the continuity of the wheat dough with various levels of mealworm powder (Figure 4). The starch granules were embedded in the gluten network of the wheat dough without mealworm powder (M0). Continuous gluten networks containing starch granules and gluten films were observed in mealworm-free and 5% and 10% mealworm-substituted dough samples, probably because the small amount of mealworm powder components could properly fill the gap between the gluten network and act as a plasticizer. Upon substitution with up to 15% and 20% mealworm powder, some starch granules and mealworm powder particles were released from the gluten network, causing it to become discontinuous. Accordingly, it has been reported that insoluble fiber can destroy the network structure of gluten [38].

Physical Properties of Bread
The visual appearances of the bread and crumb and the binarized images of the crosssection are shown in Figure 5. The visual appearance gave the initial impression that the bread made using the high-gluten wheat flour formulated with 20% mealworm powder (M20) was unacceptable to consumers owing to the dark color and decreased volume. Table 4 shows the L* (lightness), a* (redness), b* (yellowness), specific volume, and porosity values calculated from the binarized images. As the mealworm powder substitution

Physical Properties of Bread
The visual appearances of the bread and crumb and the binarized images of the cross-section are shown in Figure 5. The visual appearance gave the initial impression that the bread made using the high-gluten wheat flour formulated with 20% mealworm powder (M20) was unacceptable to consumers owing to the dark color and decreased volume. Table 4 shows the L* (lightness), a* (redness), b* (yellowness), specific volume, and porosity values calculated from the binarized images. As the mealworm powder substitution level was increased, the L* value of bread decreased, whereas the redness and yellowness increased accordingly. The color change could be ascribed to the incorporation of brown mealworm powder and the occurrence of the Maillard reaction.
As can be seen from Table 5, with an increase in the mealworm powder content, the specific volume of bread increased by up to 10% and then decreased gradually. The bread produced using all three methods showed the highest specific volume with a 10% mealworm powder substitution. The GB/T 35869-2018 Rapid-baking method, GB/T 14611-2008 Straight dough method, and automatic bread maker method exhibited the highest specific volumes of 3.70, 3.79, and 4.14 mL/g, respectively. Even though a subsequent decrease in the pasting parameters was observed due to starch dilution. The farinograph properties corresponded to a weak gluten network formed through the dilution of gluten by the replacement of wheat flour with a non-gluten ingredient. The high-gluten dough showed increased stretching resistance and extensibility. It can be inferred that mealworm powder can act as a plasticizer in the gluten network, which might contribute to the decreased strength and increased elasticity and flexibility of the dough network, resulting in an increased gas retention ability at 5% and 10% mealworm substitution levels. When the substitution level exceeded 10%, the specific volume decreased, indicating that the high level of mealworm substitution decreased the bread dough strength and gas retention stability. At substitution levels of up to 20%, the continuity of the bread dough might have been affected due to the sedimentation of mealworm powder. The porosity of the bread based on cross-sections showed a trend similar to that of the specific volume. The specific volume and porosity commonly exhibited positive correlations with the bread quality. However, compared to the bread with a 15% substitution level, the bread with a 20% substitution level showed an increasing trend in porosity with all production methods.
Roncolini et al. [6] also observed a similar increase in the specific volume after substituting soft wheat flour with 5% and 10% mealworm powder; they found that the 5% substitution level achieved the highest specific volume, but they did not study a substitution level higher than 10%. It is known that mealworm powder is rich in not only protein but also lipids, which can be adsorbed at the gas cell-dough interface and then increase gas retention during leavening [39]. However, some previous studies have shown the opposite effect of mealworm powder on the bread-specific volume. Khuenpet et al. [29] found that the specific volume of bread decreased gradually with an increase in mealworm substitution of up to 15% and that the hardness of the bread increased by approximately four times compared with the control bread. González et al. [16] used mealworm powder to replace 5% of the wheat flour and observed a significantly decreased specific volume compared to the control. However, by examining the bread-making method used in previous research, it can be inferred that the opposite trend was attributed to an inappropriate dough mixing time; Khuenpet et al. [29] mixed ingredients using a food mixer machine for 30 min, whereas González et al. [16] mixed the dough for only 5 min. Another noteworthy point is whether adjusting the water content in bread making is suggestive of the farinograph property. In the present study, the water content was adjusted according to the farinograph property for the GB/T 35869-2018 Rapid-baking method and GB/T 14611-2008 Straight dough method, whereas the water content remained the same for the automatic bread maker method. The similar trend in the specific volume and porosity indicates that the change in bread quality is less susceptible to an adjustment of the water content. As can be seen from Table 5, with an increase in the mealworm powder content, the specific volume of bread increased by up to 10% and then decreased gradually. The bread produced using all three methods showed the highest specific volume with a 10% mealworm powder substitution. The GB/T 35869-2018 Rapid-baking method, GB/T 14611-2008  Values in the same column followed by different superscript letters are significantly different (p < 0.05). L*, lightness; a*, redness; b*, yellowness. The wheat flour was substituted by mealworm powder at weight ratios of 0% (M0), 5% (M5), 10% (M10), 15% (M15), and 20% (M20), respectively.

Texture Analysis
Texture parameters of the bread were shown in Table 6. Hardness is generally negatively related to the bread quality. With the three different baking methods, the hardness of the bread decreased significantly with up to 10% mealworm substitution and then increased significantly for the bread with a 15% mealworm substitution. For the straight dough method, the hardness of the bread decreased significantly from 415 g (M0) to 350 g (M5). The bread made by the automatic bread maker showed relatively low hardness compared to the other methods, which might be due to the increased portion of butter and sugar in the ingredients. The hardness significantly decreased to 262 g (M5) and 179 g (M10) compared to the control M0 (324 g), which become coccoid with the highest specific volume (4.41 mL/g) of the bread made by the bread maker with 10% mealworm powder. Interestingly, the hardness of the bread with 20% mealworm powder was decreased slightly compared to that with 15%, which might be ascribed to the phase separation of mealworm powder and bread dough. Roncolini et al. [6] also found that the addition of 5% and 10% mealworm powder yielded softer bread, as indicated by the significantly decreased hardness compared with the control bread. However, González et al. [16] observed no significant differences in the texture parameters between the bread with 5% mealworm powder and the control. Higher specific volume is usually related to the decreased hardness of bread. Khuenpet et al. [29] found that the hardness of bread increased gradually with an increase in mealworm substitution of up to 15%. The chewiness and gumminess of the bread with various methods showed a similar trend to that of hardness. The cohesiveness of the bread showed no significant change with up to a 15% mealworm substitution level, indicative of the continuity of the bread dough network, which was coccoid based on SEM observations. The cohesiveness of bread with 20% mealworm powder using the GB/T 35869-2018 method was decreased significantly to 0.564. The resilience showed no significant change with up to a 5% mealworm substitution for the GB/T 35869-2018 Rapid-baking method and GB/T 14611-2008 Straight dough method, whereas with the automatic bread maker method, the resilience was maintained at up to a 10% substitution level. Compared with the other two standard bread-making methods, the automatic bread