Impact of Extrusion Parameters on Soy Protein Microstructure

Soy protein fibers are a new type of soy protein produced using unmodified defatted soybean meal and soy protein isolate (SPI) through twin-screw extrusion technology, commonly used as a meat substitute. During the extrusion process, various complex processing operations can lead to changes in the structure and physicochemical properties of food polymers.

Professors Zhang Haojia, Zhu Xiuqing*, and Sun Ying from the School of Food Engineering at Harbin University of Commerce systematically studied the effects of extruder barrel temperature, screw speed, and material moisture content on the solubility, particle size, and secondary structure of soy protein, to explore the conformational changes of soy protein during the extrusion process, aiming to clarify the mechanism of extrudate formation and provide a theoretical basis for the quality optimization of soy protein fibers.

01

Results and Analysis

1.1 Impact of Extrusion System Parameters on SPI Nitrogen Solubility Index

1.1.1 Impact of Different Barrel Temperatures on SPI Nitrogen Solubility Index

As shown in Figure 1A, with the increase in extrusion temperature, the nitrogen solubility index (NSI) of the extrudate significantly decreases. Under the condition of an extrusion temperature of 170 °C, the NSI drops to its lowest value (14.45%), indicating that the extrusion treatment significantly reduces the solubility of soy protein, mainly due to the destruction of the original natural structure of SPI by high temperature and high shear force in the extruder. With the exposure of hydrophobic groups and the action of non-covalent bonds, larger molecular weight protein aggregates are formed; however, as the extrusion temperature continues to rise, the NSI does not continue to decrease but slightly increases at an extrusion temperature of 180 °C, possibly because excessive extrusion temperature destroys the structure of protein aggregates, increasing the proportion of low molecular weight peptides, leading to a slight recovery of SPI’s NSI.

1.1.2 Impact of Different Material Moisture Contents on SPI Nitrogen Solubility Index

As the material moisture content increases, the NSI of SPI first increases and then decreases. From Figure 1B, when the material moisture content increases from 16% to 22%, the NSI rises to its highest value (16.30%), indicating that extrusion treatment at a moisture content of 22% is beneficial for improving the solubility of SPI; when the material moisture content is below 22%, the low moisture content leads to severe expansion of the material at the die of the extruder; this indicates that the lack of moisture lubrication protection results in high temperature, high pressure, and high shear force in the extruder severely damaging the natural structure of the SPI sample; upon observing the extruded samples, when the material moisture content exceeds 22%, the extrudate is severely fractured, and material spouting occurs at the extruder die, possibly due to the instantaneous vaporization of water molecules under high temperature conditions leading to excessive die pressure, which is not conducive to the formation of SPI aggregates, causing a trend of decreased solubility and unfavorable conditions for the formation of stable extrudates.

1.1.3 Impact of Different Screw Speeds on SPI Nitrogen Solubility Index

With the increase in screw speed, the NSI of SPI first increases and then decreases. As shown in Figure 1C, when the screw speed increases from 100 r/min to 130 r/min, the NSI rises to its maximum value (16.37%). This is presumed to be due to the higher mechanical energy input at higher screw speeds, leading to protein depolymerization and increased solubility; however, lower screw speeds result in longer residence times, causing excessive thermal denaturation of the protein and lower solubility. When the screw speed exceeds 130 r/min, the NSI decreases, dropping to 12.93% at a screw speed of 160 r/min. This may be due to the high shear force at excessive screw speeds promoting the destruction of the original natural structure of SPI, exposing hydrophobic groups, and forming larger molecular weight protein aggregates under the action of non-covalent bonds, further decreasing the solubility of SPI.

1.2 Impact of Extrusion System Parameters on SPI Free Sulfhydryl and Disulfide Bond Content

1.2.1 Impact of Different Barrel Temperatures on SPI Free Sulfhydryl and Disulfide Bond Content

As shown in Figure 2A, with the increase in extrusion temperature, the content of disulfide bonds in SPI first increases and then decreases, while the content of free sulfhydryl groups shows a trend of first decreasing and then stabilizing. This is presumed to be because after SPI undergoes extrusion treatment, its natural structure is destroyed, molecular chains are opened, and intramolecular sulfhydryl groups are exposed. Additionally, some free sulfhydryl groups are converted into disulfide bonds, leading to insignificant changes in free sulfhydryl content. It can be inferred that disulfide bonds play an important role in the formation of SPI aggregates; when the temperature exceeds 160 °C, the content of disulfide bonds slightly decreases, indicating that excessively high temperatures are not conducive to the exposure of intramolecular sulfhydryl groups and the formation of disulfide bonds. Relevant studies also indicate that excessively high extrusion temperatures can destroy the molecular bonds formed directly by protein molecules.

1.2.2 Impact of Different Material Moisture Contents on SPI Free Sulfhydryl and Disulfide Bond Content

As shown in Figure 2B, as the material moisture content increases from 16% to 22%, the content of disulfide bonds gradually decreases, while the change in the content of free sulfhydryl groups is not significant. This indicates that under these moisture conditions, the material, due to the protective lubrication effect of water, is not conducive to the exposure of internal sulfhydryl groups and the formation of disulfide bonds. Both lower or higher material moisture contents exacerbate the damage to the natural conformation of SPI during extrusion treatment; at a material moisture content of 22%, the content of disulfide bonds is at its lowest, resulting in the highest solubility of extruded SPI.

1.2.3 Impact of Different Screw Speeds on SPI Free Sulfhydryl and Disulfide Bond Content

As shown in Figure 2C, with the increase in screw speed, the content of free sulfhydryl groups decreases while the content of disulfide bonds shows a trend of first increasing and then decreasing. This indicates that after SPI undergoes the shear force of the screw, the natural structure of the protein is damaged, and the internal groups are exposed, leading to an increase in disulfide bond content. The highest content of disulfide bonds is reached at a screw speed of 130 r/min, at which point SPI aggregates to a higher degree under the influence of disulfide bonds and has higher solubility. However, when the screw speed exceeds 130 r/min, the content of disulfide bonds slightly decreases, indicating that excessively high screw speeds are not conducive to the exposure of internal sulfhydryl groups and the formation of disulfide bonds, further reducing the solubility of SPI and decreasing the proportion of low molecular weight components in SPI subunits.

1.3 Impact of Extrusion System Parameters on SPI Particle Size Volume Distribution

1.3.1 Impact of Different Barrel Temperatures on SPI Particle Size Volume Distribution

In its natural state, SPI samples exhibit a unimodal distribution, and the particle size distribution is relatively uniform. After extrusion treatment, the SPI particle size volume distribution graph changes from unimodal to bimodal or trimodal. With the increase in extrusion temperature, the particle size distribution gradually shifts to the right, and the average particle size of SPI shows an increasing trend. Larger protein particle sizes will reduce their solubility. As shown in Table 2, the average particle size of SPI increases to its maximum value at 150 °C, and a larger absorption peak for particle size appears in the spectrum in Figure 3A, presumed to be due to the fact that as the temperature increases, the protein molecular chains depolymerize and re-aggregate into larger aggregates, leading to an increase in average particle size. However, when the temperature continues to increase beyond 160 °C, the larger absorption peaks gradually disappear, but the distribution remains broad, indicating that high-temperature extrusion conditions will destroy some of the larger SPI aggregates. This phenomenon may also be due to the fact that the loose structure formed by SPI after high-temperature treatment is easily damaged.

1.3.2 Impact of Different Material Moisture Contents on SPI Particle Size Volume Distribution

As shown in Figure 3B, with the increase in material moisture content, the particle size volume distribution graph remains unimodal, indicating that samples with different moisture contents can still achieve relatively uniform SPI aggregates. From Table 3, it can be seen that when the material moisture content is low, the average particle size of SPI is also low. This phenomenon may be due to the fact that lower moisture conditions severely damage the natural conformation of SPI, increasing the content of insoluble components in SPI. The components that can dissolve into the particle size testing solution are mostly small molecular peptides, leading to a trend of decreasing particle size; when the material moisture content increases to 22%, the average particle size increases to its maximum value, and further increasing the material moisture content leads to a decrease in average particle size. This indicates that higher material moisture content is beneficial for forming more stable SPI aggregates, but due to the lubricating protective effect of water, the degree of protein denaturation is lower under this moisture condition, and more proteins maintain their natural conformation, resulting in higher solubility. This may be because higher material moisture inhibits the exposure of internal hydrophobic groups of SPI, and the binding of water molecules to SPI somewhat inhibits the damage to SPI’s natural conformation and the formation of aggregates.

1.3.3 Impact of Different Screw Speeds on SPI Particle Size Volume Distribution

After extrusion treatment at different screw speeds, the SPI particle size volume distribution graph changes from the natural state of unimodal to multimodal. With the increase in screw speed, the peak positions in the particle size distribution graph gradually shift to the right, and the average particle size of SPI shows an increasing trend. As shown in Figure 3C, at a screw speed of 130 r/min, the particle size reaches its maximum value, and the distribution is relatively uniform, indicating that under these conditions, SPI has the highest solubility. This suggests that this extrusion condition is conducive to forming stable SPI aggregates. However, as the screw speed continues to increase, under conditions of 140-160 r/min, the PDI of SPI molecules is low, and the particle size volume distribution gradually returns to a uniform state with larger particle sizes.

1.4 Impact of Extrusion System Parameters on SPI Zeta Potential

1.4.1 Impact of Different Barrel Temperatures on SPI Zeta Potential

The absolute value of Zeta potential indicates the amount of positive or negative charges on the molecular surface. The higher the absolute value of SPI Zeta potential, the more charges on the protein molecular surface, and under greater electrostatic repulsion, SPI structure can maintain a relatively stable state. Compared to unextruded SPI, extrusion treatment significantly increases the absolute value of SPI Zeta potential; as shown in Figure 4A, with the increase in extrusion temperature, the absolute value of SPI Zeta potential first increases and then decreases. At an extrusion temperature of 160 °C, the absolute value of SPI Zeta potential reaches its highest point, indicating that the electrostatic repulsion between SPI molecules is maximized. This shows that after extrusion treatment, the number of protein aggregates formed through disulfide bonds, hydrophobic interactions, and electrostatic interactions increases. As the extrusion temperature further rises, the absolute value of Zeta potential no longer continues to increase, and the electrostatic charge density on the surface of SPI molecules decreases, indicating that excessively high extrusion temperatures are not conducive to the formation of protein aggregates. Extrusion treatment can destroy the natural structure of SPI while promoting the exposure of internal hydrophobic residues and charged groups, resulting in an increase in the absolute value of SPI Zeta potential.

1.4.2 Impact of Different Material Moisture Contents on SPI Zeta Potential

After extrusion treatment, the absolute value of the Zeta potential of the extrudate significantly increases. As shown in Figure 4B, with the increase in material moisture content, the trend shows first a decrease and then an increase. At a material moisture content of 22%, the absolute value of SPI Zeta potential reaches its lowest point, indicating that the electrostatic repulsion between SPI molecules is minimal. This shows that under these conditions, the protein aggregates formed after extrusion treatment are fewer; both higher and lower material moisture contents lead to an increase in the absolute value of Zeta potential, indicating that under conditions of higher or lower material moisture contents, it is beneficial for the formation of insoluble protein aggregates. Combining with the particle size analysis data, both higher and lower material moisture contents lead to lower average particle sizes of the extrudate and higher PDI, making it impossible to form a uniform SPI aggregate system. In summary, the impact of different moisture contents during extrusion treatment on SPI aggregation is significant; lower material moisture contents facilitate the formation of SPI aggregates but cannot form a uniform aggregation system, and higher moisture contents can also disrupt the formation of stable aggregates due to the vaporization of water molecules.

1.4.3 Impact of Different Screw Speeds on SPI Zeta Potential

With the increase in screw speed, the absolute value of SPI Zeta potential first increases and then decreases. As shown in Figure 4C, at a screw speed of 130 r/min, the absolute value of SPI Zeta potential reaches its highest point, indicating that at this point, the electrostatic repulsion between SPI molecules is maximized. Existing studies also indicate that after extrusion treatment, the number of protein aggregates formed through disulfide bonds, hydrophobic interactions, and electrostatic interactions increases. However, as the screw speed further increases, the absolute value of Zeta potential begins to decrease, indicating that excessively high screw speeds input higher mechanical energy, leading to the destruction of SPI aggregates or hindering the formation of more protein aggregates. However, higher screw speeds are beneficial for achieving a more uniform aggregate system of SPI.

1.5 Impact of Extrusion System Parameters on SPI Secondary Structure

1.5.1 Impact of Different Extrusion Temperatures on the Relative Content of SPI Secondary Structure

As shown in Figure 5A, after extrusion treatment, the relative content of α-helical and β-folding structures in the secondary structure of soy protein decreases, while the relative content of β-turn and random coil structures increases. With the increase in extrusion temperature, the relative content of α-helical, β-turn, and random coil structures shows a trend of first increasing and then decreasing, while the relative content of β-folding shows a trend of first decreasing and then increasing. At 160 °C, the relative content of α-helical and β-turn structures reaches their highest values at 25.20% and 17.98%, respectively, while the relative content of β-folding reaches its lowest value (42.45%); the relative content of random coil reaches its highest value (14.56%) at 170 °C.

1.5.2 Impact of Different Material Moisture Contents on the Relative Content of SPI Secondary Structure

As shown in Figure 5B, with the increase in material moisture content, the relative content of random coil structures shows slight fluctuations, while the relative content of α-helical and β-folding structures shows a trend opposite to that of β-turn. At a moisture content of 22%, the relative content of α-helical structures reaches its highest value, while at a moisture content of 16%, the relative content of β-turn structures is highest at 26.75%, and the relative content of β-folding reaches its lowest value (41.30%). Under the conditions of 22% material moisture content, due to the protective lubrication effect of water, the degree of protein denaturation is lowest, and the proportions of secondary structures are closest to the original stable state. Moisture contents above or below this level will increase the disorder of the protein; based on previous analysis of SPI average particle size and Zeta potential after extrusion treatment, the increase in the content of SPI aggregates is primarily influenced by the β-turn structure, while the formation of uniform stable aggregates of SPI after extrusion treatment requires the participation of α-helical structures. Studies have shown that protein aggregates with higher contents of α-helical and β-folding structures also exhibit higher stability.

1.5.3 Impact of Different Screw Speeds on the Relative Content of SPI Secondary Structure

After extrusion treatment of soy protein at different screw speeds, the relative content of α-helical and β-folding structures in the secondary structure decreases, while the relative content of β-turn and random coil structures slightly increases. As shown in Figure 5C, with the increase in screw speed, the relative content of α-helical and β-folding structures first increases and then decreases, while the relative content of β-turn shows a trend opposite to that of α-helical and β-folding contents. Based on existing research results, it can be inferred that after extrusion treatment, the degree of denaturation of SPI increases, and the secondary structures of α-helical and β-folding convert into β-turn and random coil structures. When the screw speed ranges from 120 to 130 r/min, the proportions of protein secondary structures are closer to the original state. Being lower or higher than this range will lead to an increase in protein disorder, possibly because excessively low screw speeds result in longer residence times, increasing the degree of protein denaturation, while excessively high screw speeds lead to increased mechanical energy input, causing damage to the natural structure of the protein.

02

Conclusion

This experiment used SPI as the raw material to study the effects of different extrusion temperatures (120-180 °C), screw speeds (100-160 r/min), and material moisture contents (22%-28%) on the conformation of soy protein in cold-pressed soybean meal. The results show that at 150 °C, the protein polymerization degree of the extrudate is higher, and the fiber formation effect is better. Both excessively high and low temperatures are not conducive to protein aggregation and the formation of a stable state; when the material moisture content is between 20% and 22%, the protective effect of water results in the best state of the extrudate, forming a uniform system of SPI aggregates. When the material moisture content is too low or too high, excessive die pressure makes it difficult to form a more complete extrudate; lower screw speeds result in insufficient mechanical energy input and excessive residence time of SPI, preventing complete denaturation and depolymerization of the protein, while higher screw speed conditions promote the destruction of SPI’s original natural structure through high shear forces, forming larger molecular weight protein aggregates. In summary, under appropriate system parameters, extrusion is conducive to obtaining the best quality extrudate; too low results in incomplete protein denaturation, lower polymerization degree, and poor fiber formation effect, while too high will cause excessive protein denaturation, damaging the already formed fiber structure and degrading the quality of the extrudate.

This article “The Impact of Extrusion Parameter Control on the Microstructure of Soy Protein” is sourced from “Food Science”, 2023, Volume 44, Issue 15, pages 103-112, authors: Zhang Haojia, Zhu Xiuqing, Sun Ying. DOI:10.7506/spkx1002-6630-20220721-246. Click below to read the original text for more information about the article.

Intern Editor: Mei Juan; Editor: Zhang Ruimei. Click below to read the original text to view the full text.

Recent Research Hotspots

“Food Science”: Academician Xie Mingyong from Nanchang University et al.: The Influence of Frying Technology on the Formation of Acrylamide and 5-Hydroxymethylfurfural in French Fries

“Food Science”: Associate Professor Chen Pei from Shaanxi Open University et al.: Analysis of Microbial Diversity in Liquor Fermentation

“Food Science”: Professor Sun Dongliang from Beijing University of Petroleum and Chemical Technology et al.: Integrated Mushroom Hot Air Drying Simulation Method and Application

“Food Science”: Researcher Bi Jinfeng from the Chinese Academy of Agricultural Sciences: Research Progress on the Impact of Drying on the Color of Fruit and Vegetable Processing

“Food Science”: Associate Professor Wang Wenxiu from Hebei Agricultural University et al.: Analysis of the Relationship between Protein and Quality Changes during Hot Air Drying of Litopenaeus vannamei

“Food Science”: Senior Engineer Sun Lei from Hebei Provincial Key Laboratory of Food Safety et al.: Determination of 13 Diuretics in Animal-Derived Foods by Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry