
==== Front
Food Chem X
Food Chem X
Food Chemistry: X
2590-1575
Elsevier

S2590-1575(24)00627-8
10.1016/j.fochx.2024.101739
101739
Research Article
Co-cold extrusion synergized with cysteine for enhancing physicochemical, rheological characteristics and in vitro digestibility of whey protein isolate
Mu Sinan a1
Mu Zhishen b1
Gantumur Munkh-Amgalan a
Yang Nan a
Sukhbaatar Narantuya c
Sun Yuxue yuxue20@neau.edu.cn
a⁎
Jiang Zhanmei zhanmeijiang@neau.edu.cn
a⁎
a Key Laboratory of Dairy Science (Northeast Agricultural University), Ministry of Education, College of Food Science, Northeast Agricultural University, Harbin 150030, PR China
b National Enterprise Technology Center, Inner Mongolia Mengniu Dairy (Group) Co., Ltd., Huhhot 011500, PR China
c School of Industrial Technology, Mongolian University of Science and Technology, 14191, Baga toiruu 34, Sukhbaatar district Ulaanbaatar, Mongolia
⁎ Corresponding authors. yuxue20@neau.edu.cnzhanmeijiang@neau.edu.cn
1 Sinan Mu and Zhishen Mu, both contribute equally to this article.

13 8 2024
30 10 2024
13 8 2024
23 10173914 1 2024
3 8 2024
12 8 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc/4.0/ This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-nc/4.0/).
Impacts of co-cold extrusion (≤50 °C) of whey protein isolate (WPI) and cysteine (Cys, 0, 20, 40, 60, 80 and 100 mmol/L) on its physicochemical, in vitro digestion and rheological properties were investigated. As Cys concentration increased, the emulsifying properties and in vitro digestibility of co-extruded WPI-Cys products showed an increasing trend. Specifically, when Cys reached 100 mmol/L, surface hydrophobicity, emulsification activity index (EAI), emulsification stability index (ESI) and in vitro stomach digestibility of the co-extruded WPI-Cys products increased by 205.07%, 77.51%, 193.95% and 71.81% compared with WPI, respectively. Principal component analysis (PCA) results further indicated that co-extruded WPI-Cys at a concentration of 100 mmol/L had the best functional properties. In addition, co-extruded WPI-Cys exhibited the strongest Péclet number (Pe) value and apparent viscosity at a Cys concentration of 100 mmol/L among all samples. Therefore, co-extrusion would be an effective method for modifying WPI, providing whey protein-based ingredients with excellent functional properties for food processing.

Highlights

• Co-extrusion of WPI with Cys is a fast method for modifying WPI.

• Co-extrusion improved rheological properties and in vitro digestibility of WPI.

• Co-extruded WPI-Cys had better emulsifying properties than WPI or EWPI.

Keywords

Whey protein isolate
Cysteine
Co-extrusion
==== Body
pmc1 Introduction

Whey protein isolate (WPI) is extensively used in food systems such as yogurt, juice and processed meat because of its high nutritional value and functionality (Li et al., 2024). However, functional characteristics including emulsification and gelation ability of WPI still need further improvement to satisfy the demands of the food industry (Uluko, Liu, Lv, & Zhang, 2016). The modification of WPI could be achieved by physical, chemical and enzymatic methods. Among these methods, the application of physical methods was more common, such as extrusion, ultrasonication, ultra-high pressure, microwave and other technologies (Hu et al., 2024).

Extrusion is an operation that uses shear force, heating and mechanical pressure to modify food raw materials. At present, extrusion technology has been used in the modification of soybean protein, pea protein and WPI to meet different requirements (Chen, Zhang, Zhang, Meng, & Wang, 2021). Extrusion includes cold extrusion and thermal extrusion. In general, thermal extrusion leads to aggregation and fragmentation of protein molecules, reducing their solubility (Afizah & Rizvi, 2014). Specifically, Li and Lee (1996) reported that solubility of wheat protein in many solvents has been significantly decreased after thermal extrusion at 160, 170, and 185 °C. Relatively, WPI retained its native protein value, functionality, and digestibility when extruded at 50 °C or below (Onwulata, Konstance, Cooke, & Farrell Jr, 2003). Cold-extrusion technology can change the overall orientation of the molecular chain and break the secondary bonds in the protein molecule at low temperatures (≤ 50 °C) by the driving force of the extruder screw. Yang et al. (2019) demonstrated that cold extrusion treatment improved the surface hydrophobicity, emulsification, and rheology of WPI. And Onwulata et al. (2003) found that gel strength of cold-extruded-WPI at 35 °C and 50 °C increased by 115% and 145%, respectively, but this trend disappeared at 100 °C. Therefore, cold-extrusion may provide technical support for improving the functional properties of proteins.

Cysteine (Cys) residues and disulfide bonds in proteins have a role in regulating the structure and function of proteins. With the addition of exogenous Cys, the free sulfhydryl groups of Cys could undergo SH/S-S exchange reactions with the disulfide bonds and free sulfhydryl groups of proteins, but free sulfhydryl groups of the protein were blocked by sulfhydryl oxidation reactions (Schmidt, Illingworth, Deng, & Cornell, 1979). These two types of reactions proceed simultaneously and functional properties of the corresponding protein products were affected. Based on this principle, Cys was often used for the modification of proteins. For instance, Peng et al. (2022) found that high moisture extrusion accompanied by Cys addition resulted in a weakened network structure of pea proteins, and its oil holding capacity reached a maximum with the addition of 0.09% Cys. Furthermore, the shelf-life of whey protein nutrition bar was significantly extended by 14 days when the molar ratio of WPI to Cys was 0.05 (Zhu & Labuza, 2010). In addition, Cys was added with D-xylose or d-glucose to wheat flour for extrusion, and the content of garlic-like and onion-like flavor substances in extrusion products was significantly increased (Bredie, Hassell, Guy, & Mottram, 1997). Therefore, Cys can enhance functional properties of natural protein, it can be used as a flavor precursor in food processing.

Generally, individual Cys or cold-extrusion for the modification of WPI was tedious and not suitable for industrial production, while co-extrusion of WPI and Cys was a faster and more direct modification approach (Floris, Bodnár, Weinbreck, & Alting, 2008). Although previous reports have demonstrated that individual Cys or cold-extrusion treatment could improve functional properties of WPI, it will not be expounded whether co-cold extrusion of WPI and Cys will be another better selection for obtaining WPI ingredients with excellent characteristics. Therefore, the effects of 0, 20, 40, 60, 80 and 100 mmol/L concentrations of Cys and WPI co-cold-extrusion (≤ 50 °C) on physicochemical properties, rheological properties and in vitro digestion of WPI would be investigated. This will potentially broaden the application of Cys in industrialized food production, and provide the food industry with whey protein-based functional food ingredients with excellent functional properties.

2 Materials and methods

2.1 Materials and chemicals

Whey protein isolate (WPI, 93.77% protein content) was sourced by Mullins (Whey Inc., Mosinee USA). L-cysteine (Cys), trifluoroacetic acid (TFA), trypsin (4000 U/g) and pepsin (3000 U/g) were obtained from Sigma-Aldrich (St. Louis, MO, USA). All reagents were analytical grade.

2.2 Preparation of co-extruded WPI with Cys

The co-extrusion of WPI and Cys was accomplished by a co-rotating twin-screw extruder (EV 25, Clextral, Fomeny, France). The extruder was equipped with a high-shear screw with a length-to-diameter ratio of 24:1 and a diameter of 25 mm. (Yang et al., 2019). Initially, WPI powders were added to the extruder at a rate of 3.5 kg/h through the feed screw. Cys solutions at concentrations of 0, 20, 40, 60, 80, and 100 mmol/L were also added to the extruder at a rate of 3.5 kg/h through an automatic valve. Then, the moisture content of the extrudate was controlled to 50% (w/w) by controlling the flow rate of Cys solution. The temperatures of extruder barrel feeding zone (T1), mixing zone (T2) and cooking zone (T3) were set to 25, 30 and 35 °C, respectively, to control that the temperature of the extrudate reaching the output zone (T4) was still below 50 °C. The screw speed was set at 300 r/min. Afterwards, the extruded products were freeze-dried. And the co-extruded WPI-Cys products corresponding to the Cys concentration at 0, 20, 40, 60, 80 and 100 mmol/L were named as EWPI, EWPI-Cys20, EWPI-Cys40, EWPI-Cys60, EWPI-Cys80 and EWPI-Cys100, respectively.

2.3 Particle size and zeta potential

The protein samples were diluted to 1 mg/mL using phosphate buffer (0.01 mol/L, pH 7.0), and the particle size distribution, median particle size (D50, μm), and zeta-potential of the protein samples were measured at 25 °C using a particle size analyzer (Zetasizer Nano-ZS 90, Malvern Ltd., UK) (Li et al., 2024).

2.4 Surface hydrophobicity (H0)

8-anilino-1-naphthalenesulfonic acid (ANS) was used as a fluorescent probe to obtain surface hydrophobicity of co-extruded WPI-Cys. The excitation and emission wavelengths of the fluorescence spectrophotometer (Hitachi, Science Systems, Ibaraki, Japan) were set to 390 and 470 nm. The fluorescence intensity was plotted as the vertical coordinate and the protein concentration as the horizontal coordinate. The slope of the obtained curve was the H0 of proteins (Jiang et al., 2023).

2.5 Solubility

Specifically, protein solutions (pH 7.0) stirred for >3 h were centrifuged at 8000g for 15 min at room temperature. The protein concentration of supernatant was measured by the biuret method (Fields & Chodosh, 1970). Solubility was defined as the ratio of protein content in the supernatant to the total protein content. The calculation formula is as follows:(1) Solubility%=C1C2×100

where C1 and C2 are the protein content of the supernatant and solution before centrifugation, respectively.

2.6 Intrinsic fluorescence spectra

Samples were prepared to 0.5 mg/mL with phosphate buffer solution (PBS) (0.01 mol/L, pH 7.0). The fixed excitation and emission wavelengths of the fluorescence photometer were set as 280 and 290–420 nm, respectively. The slit widths were all 5 nm, and the running speed was 240 nm/s.

2.7 Emulsifying properties

The co-extruded samples were diluted to 5 mg/mL using PBS buffer (0.01 mol/L, pH 7.0), and 3 mL of the sample was mixed with 1 mL of soybean oil, then the solution was dispersed for 2 min at 12,000 g using a high-speed disperser (T-18, IKA, Germany). 50 μL of the obtained emulsion was left to stand for 0 and 10 min, respectively, and then rapidly dispersed into 5 mL of SDS solution (1 mg/mL). The absorbance of the emulsion was measured at 500 nm. The emulsification activity index (EAI) (m2/g) and emulsification stability index (ESI) (%) were calculated as the following equation (Cui et al., 2024):(2) EAIm2g=2×2.303C×1−θ×104×A0×dilution

where A0 is the absorbance of emulsion, C is the concentration of protein solution (g/mL), θ is the volume fraction occupied by oil phase, and dilution is the dilution factor.(3) ESI%=A10A0×100

where A10 and A0 are the absorbance measured at 10 and 0 min, respectively.

2.8 Rheological properties

Rheological properties of protein samples (20%, W/W) were determined using a DHR-1 rotational rheometer. The rheometer was equipped with a titanium steel fixture with a diameter of 60 mm and a taper angle of 1°. The variation of apparent viscosity (η) with shear rate (γ ˙) in the protein solution was measured. After determining the linear viscoelastic region, the strain was set to 1%. Subsequently, change of viscoplastic modulus (G′ and G′′) in protein solution with the oscillation angular frequency (ω) was observed (Wei et al., 2024).

2.9 In vitro simulated digestion

The model simulating digestion in vitro was developed according to Zhou, Zhao, Cui, and Sun (2015). First, pepsin was prepared to 5 mg/mL using 0.01 mol/L HCl to obtain simulated gastric fluid (SGF). The protein sample solution (3 mg/mL) was adjusted to pH 1.5 using 1 mol/L HCl. The sample solution was mixed with the pepsin storage solution to give a pepsin concentration of 4% (w/w). After shaking for 30 min at 37 °C, the pH of the gastric digest was adjusted to 6 to interrupt the digestion process.

Subsequently, simulated intestinal fluid (SIF) digestion was performed. The pH of the gastric digest was regulated to 7.8. The trypsin (250 U/mg) stock solution (5 mg/mL PBS buffer) was added to the pepsin predigests and shaken at 40 °C for 60 min. Then, Na2CO3 solution (150 mmol/L) was added to terminate the reaction.

2.10 Digestibility

The content of protein in the digestive fluid was measured by the method of biuret (Fields & Chodosh, 1970), and standard curve was made with BSA. The calculation formula of the in vitro digestibility (%) is as follows:(4) Digestibility%=A−A1A×100

where A is the protein content of the sample before simulated digestion, A1 is the protein content in the digestive fluid.

2.11 Ferric reducing power

The ferric reducing power of the sample was determined by the approach of Benjakul, Lertittikul, and Bauer (2005). Digested protein samples, K3Fe (CN6) (1%) and PBS (0.2 mol/L, pH 6.6) were each taken 1 mL and mixed. The solution was reacted in a 50 °C water bath for 20 min and then mixed with 1 mL of trichloroacetic acid (10%). Then, centrifuged it with 750 g centrifugal force for 10 min at 25 °C, the obtained supernatant was treated with distilled water and 0.1% FeCl3. The OD700 was used as the standard to determine the reducing power.

2.12 Inhibitory rate of α-glucosidase activity

50 μL of sample was mixed with 100 μL of α-glucosidase (0.075 U/mL) and p-nitrophenyl-β-D-glucopyranoside (3 mmol/L), and the reaction was carried out at 37 °C for 30 min. 2 mL Na2CO3 (0.1 mol/L) was used as a terminating agent and finally the absorbance was obtained at 400 nm (Huang et al., 2023). The α-glucosidase inhibitory activity was obtained as follows:(5) Inhibitory rate ofα−glucosidase%=A−A1A×100

where A is the absorbance value without adding samples, A1 is the absorbance value of samples.

2.13 Inhibitory activity of xanthine oxidase (XOD)

Following the method of Li et al. (2019), 100 μL of XOD solution (0.2 U /mL 0.2 M PBS, pH 7.5) was mixed with 100 μL of the sample. Then 200 μL of xanthine solution (0.04 mmol/L) was used to initiate the reaction at 37 °C for 15 min. The reaction was terminated with 200 μL (1 mol/L) HCl and the absorbance was recorded at 290 nm. The XOD inhibitory activity was obtained using the formula below:(6) Inhibitory activity ofXOD%=A0A×A0×100

where A0 and A are the absorbance values of the blank and sample groups, respectively.

2.14 Statistical analysis

The results of this work were presented as mean ± standard deviation (triplicate experiments, n = 3). The SPSS software 17.0 (SPSS, Inc., Chicago, IL, USA) was used to analyze the significance of differences by one-way analysis of variance (ANOVA) and Duncan's multiple comparisons (P < 0.05). The Origin 8.5 software was used for drawing. The principal component analysis (PCA) and heatmap were obtained using surface hydrophobicity, solubility, EAI, ESI, digestibility, reducing power, α-glucosidase inhibitory activity and XOD inhibitory activity as indexes.

3 Results and discussion

3.1 Particle size and zeta potential

The particle size, D50 and zeta potential of co-extruded WPI-Cys samples are shown in Fig. 1A, B and C, respectively. As shown in Fig. 1A and B, all the particle sizes of WPI or EWPI were <10 μm, and their D50 was around 1.11 and 1.28 μm, respectively. This indicated that during the cold-extrusion process, no aggregation and polymers of macromolecular proteins were detected. However, as the Cys concentration gradually ranged from 20 to 100 mmol/L, the particle size distribution of co-extruded WPI-Cys samples was further widen and shifted to the right, and their D50 also increased significantly compared to that of WPI or EWPI. Moreover, D50 of EWPI-Cys100 showed the largest value and was about 29.41 times higher than that of WPI (P < 0.05). It was possible that extrusion unfolded the tertiary structure of WPI, while the addition of Cys reduced the intramolecular disulfide bonds of WPI. This led to the active-SH group being exposed and the occurrence of SH-SS exchange reactions (Yang, Qian, Jiang, & Hou, 2021), which caused aggregation and polymerization of protein molecules. In addition, hydrophobic interactions also caused extensive aggregation of protein molecules, forming larger particles.Fig. 1 Particle size (A), median particle size (B) and zeta potential (C) of co-extruded WPI-Cys conjugates. Error bars represent the standard deviation of the mean of triplicate experiments. Values with different letters are significantly different (p < 0.05).

Fig. 1

From Fig. 1C, the difference in absolute value of zeta potential between WPI or EWPI was not significant. However, as the addition of Cys increased, absolute value of zeta potential of co-extruded WPI-Cys samples increased gradually. After the addition of Cys reached 100 mmol/L, its absolute value of zeta potential was the maximum (P < 0.05). This result might be explained because of the SH/S-S exchange reaction, which caused disulfide bonds to break and free sulfhydryl groups to be exposed to the surface of proteins (Zhu & Labuza, 2010). This induced more negative charges on the surface of protein, resulting in an enhanced absolute value of the zeta potential.

3.2 Surface hydrophobicity (H0)

Surface hydrophobicity can characterize exposure of hydrophobic amino acids residues (Kao, Allara, & Tadigadapa, 2011). The H0 of co-extruded WPI-Cys is shown in Fig. 2A. It can be seen that the H0 of WPI or EWPI was both small, and since most of hydrophobic amino acids in globular proteins were buried inside the molecules and the single extrusion treatment would fully not expose them. However, as the concentration of added Cys reached from 20 to 100 mmol/L, H0 of co-extruded WPI-Cys samples showed a significant increasing trend, and their H0 increased by 157.88%, 186.75%, 193.05%, 198.71% and 205.07%, respectively, compared to WPI. The explanation was that Cys led to the further exposure of the hydrophobic region hidden in the spherical structure, and hydrophobic binding sites on the surface of the molecular also increased with the increase of Cys concentration (Wen et al., 2024).Fig. 2 Surface hydrophobicity (A), solubility (B) and endogenous fluorescence (C) of co-extruded WPI-Cys conjugates. Error bars represent the standard deviation of the mean of triplicate experiments. Values with different letters are significantly different (p < 0.05).

Fig. 2

3.3 Solubility

The solubility of protein represents its degree of denaturation. The solubility of co-extruded WPI-Cys samples is shown in Fig. 2B. It could be seen that both single extrusion and co-extrusion led to a decrease in solubility. This might be explained that tertiary structures of proteins unfolded after extrusion and the side chains of non-polar amino acid residues buried in the molecule were exposed, leading to a decrease in the solubility of protein samples. As the addition of Cys ranged from 0 to 100 mmol/L, the solubility of co-extruded WPI-Cys samples decreased by 15.39%, 19.22%, 26.68%, 25.38%, 29.86%, and 32.18%, respectively, compared to WPI (P < 0.05). During single extrusion, EWPI would renature as the pressure and shear force disappeared (Dahl & Villota, 1991), resulting in less reduction in solubility. Furthermore, co-extrusion caused the aggregation and polymerization of proteins and prevent the renaturation of WPI (Floris et al., 2008). And with the increasing Cys concentration, solubility of co-extruded WPI-Cys significantly decreased. In this test, surface hydrophobicity and solubility of co-extruded WPI-Cys samples were negatively correlated (correlation coefficient = −0.8977). Co-extrusion treatment exposed hydrophobic groups in WPI, which induced hydrophobic aggregation of protein molecules, resulting in the decrease of its solubility. The study by Yu et al. (2018) also demonstrated that surface hydrophobicity of the mussel protein was negatively associated with their solubility.

3.4 Intrinsic fluorescence spectroscopy

Amino acid residues of proteins, especially tryptophan (Trp), have fluorescence absorption properties. Intrinsic fluorescence spectroscopy is often used to reveal the interactions between small molecules and proteins (Wei et al., 2006). The intrinsic fluorescence spectroscopy of co-extruded WPI-Cys samples is shown in Fig. 2C. Fluorescence intensity of EWPI-Cys increased gradually with the addition of Cys from 0 to 100 mmol/L, and EWPI-Cys100 reached the maximum fluorescence intensity. Co-extrusion treatment could break the disulfide bond of protein molecules through SH/S-S exchange reactions, which further promoted the exposure of Trp inside the protein (Li et al., 2022), resulting in a greater increase in its fluorescence intensity.

3.5 Emulsifying properties

The variation of emulsifying properties of WPI-Cys samples is shown in Fig. 3A and B. EAI of EWPI was increased by 32.37% (P < 0.05), compared with WPI (Fig. 3A). This was because that cold extrusion expanded the total surface area of WPI, enhancing its mobility and adsorption capacity at the oil-water interface. While the concentration of Cys varied from 20 to 100 mmol/L, EAI of co-extruded WPI-Cys samples was increased by 37.39%, 46.13%, 49.48%, 60.19% and 77.51% (P < 0.05), respectively, compared to WPI. The reason might be that co-extrusion of Cys and WPI further promoted the change of tertiary structure of protein molecules and the increase of total surface area, while the flexibility of proteins was also increased. Another explanation was that the increase in surface hydrophobicity resulted in greater surface activity of protein molecules (Thongzai, Matan, Ganesan, & Aewsiri, 2022), thus increasing the EAI of protein molecules.Fig. 3 EAI (A), ESI (B), apparent viscosity (C), Pe (D) and viscoplastic modulus (E) of co-extruded WPI-Cys conjugates. Error bars represent the standard deviation of the mean of triplicate experiments. Values with different letters are significantly different (p < 0.05).

Fig. 3

As shown in Fig. 3B that EWPI had a larger ESI than the WPI. And with the addition of Cys increasing from 20 to 100 mmol/L, the ESI of the co-extruded WPI-Cys showed an increasing trend. Especially, the ESI of EWPI-Cys100 was 193.95% higher than that of WPI (P < 0.05). The result indicated that co-extrusion could further increase ESI of protein samples compared to single extrusion. The reason for this result was that Cys promoted the polymerization of protein molecules to form viscoelastic membranes through disulfide bonds and hydrophobic interactions after co-extrusion (Lavoisier, Vilgis, & Aguilera, 2019). And with the increasing amount of Cys, the content of intermolecular disulfide bonds increased, which could promote the increase of the viscoelasticity of interfacial film, thereby making the formed emulsion more stable. In addition, the addition of Cys promoted the increase of free sulfhydryl content, which increased the net negative charge on the surface and the absolute value of zeta potential. This also enhanced the electrostatic repulsion between protein molecules (Rodríguez Patino, Rodríguez Niño, & Sánchez, 2002), which inhibited the aggregation of oil droplets and facilitated the formation of stable emulsions. Thus EWPI-Cys100 had the highest ESI.

3.6 Steady state shear properties

The variation of apparent viscosity of samples with shear rate is shown in Fig. 3C. Furthermore, apparent viscosity of samples all decreased significantly with the increased shear rate. This demonstrated that WPI, EWPI and EWPI-Cys100 showed shear-thinning behavior and exhibited pseudoplastic fluid properties. The possible reason was that shear force broke the hydrogen bonds and electrostatic interactions between the protein and water molecules, prompting the rearrangement of peptide chains along the shear plane, thus reducing the flow resistance.

In addition, the apparent viscosity of EWPI-Cys100 was the highest among all samples in the shear rate range of 0.01–100 s−1. The reason might be that the absolute value of zeta potential of co-extruded WPI-Cys samples increased during co-extrusion treatment (Fig. 1C), which represented an increase in the surface charge of protein molecules. This led to the enhancement of electrostatic repulsion between protein molecules, which increased the resistance to flow. (Benoit, Afizah, Ruttarattanamongkol, & Rizvi, 2013).

3.7 Péclet number

The dependence of the Péclet number (Pe) of samples on shear rate is shown in Fig. 3D. The physical meaning of Pe in rheology is the ratio of the convection rate to diffusion rate (Chen, Ding, & Tan, 2007). As is seen from Fig. 3D, in the shear rate range of 0.01–100 s−1, the Pe of WPI was 0.0053–0.0099 (≪1). This indicated that apparent viscosity of the sample during its flow was dominated by the Brownian motion of protein molecules (Tan, Zhong, & Langrish, 2019). However, Pe of EWPI was 4.46 at 0.01 s−1 and 7.30 at 100 s−1 (>1) (Pitkowski, Durand, & Nicolai, 2008), indicating that intermolecular interactions of proteins influenced the flow behavior, and the shear-induced rearrangement of peptide chains affected the apparent viscosity of EWPI.

Moreover, Pe of EWPI-Cys100 in the shear rate range of 0.01–100 s−1 was 157,348.03–5079.06 (≫1), indicating that flow behavior of the solution was dominated by the protein intermolecular interaction. And even at high shear rate, no Newtonian plateau appeared, indicating the strong polymeric structure formed among protein molecules (Xia et al., 2022). This polymeric structure hindered the rearrangement of peptide chains along the shear plane during its flow, leading to the significant increase in Pe.

3.8 Dynamic frequency sweep

The frequency dependence of the viscoplastic modulus in protein samples is shown in Fig. 3E. Storage modulus (G′) was less than loss modulus (G′′) at all frequencies of WPI, which demonstrated that WPI solution exhibited the liquid-like flow behavior. And at the maximum angular frequency (100 rad·s−1), G ′ and G ′′ of WPI were 0.47 Pa and 0.44 Pa, respectively, which were the smallest among all samples. And the G′ of EWPI was larger than G′′ at all frequencies, indicating that EWPI exhibited the solid behavior. At the maximum angular frequency (100 rad·s−1), G′ and G′′ of EWPI were 71.94 Pa and 22.50 Pa, respectively, which were 163.50 and 47.87 times higher than those of WPI (P < 0.05). It may be due to the increase in hydrophobic interactions of cold extrusion-induced WPI molecules, resulting in a tighter arrangement of protein aggregates, which exhibited a solid-like flow behavior.

Furthermore, at an angular frequency of 100 rad·s−1, the G′ and G′′ of EWPI-Cys100 were 2089.12 and 7555.89 Pa, respectively, which were the highest among all protein samples. Potentially, co-extrusion led to the unfolding of tertiary structures of proteins and the enhancement of its hydrophobic and covalent interactions. This induced the formation of strongly aggregated structure, which remarkably increased the viscoplastic modulus of the EWPI-Cys100 solution.

3.9 In vitro digestibility

Gastrointestinal simulated digestibility of co-extruded WPI-Cys samples is shown in Fig. 4A. During simulated gastric and intestinal digestion, co-extruded WPI-Cys samples had significantly higher digestibility than WPI or EWPI. Specifically, when the added Cys changed from 0 to 40 mmol/L, the digestibility of co-extruded WPI-Cys in simulated stomach and intestine gradually increased (P < 0.05). Probably, with the addition of Cys, the molecular structure of WPI unfolded, exposing hydrophobic amino acid residues which could react with the digestive enzymes, thus improving the digestibility. Faris, Wang, and Wang (2008) treated soy protein with NADP-thioredoxin system (NTS), which effectively reduced intramolecular disulfide bonds in soy protein powder. The digestibility of NTS-treated soy protein increased compared to untreated soy protein. Furthermore, when the concentration of Cys exceeded the critical point (60 mmol/L), the digestibility of co-extruded WPI-Cys would not continue to increase. This was due to the formation of extensive protein aggregates, which masked some protease action sites and prevented the recognition of protease and peptide bond (Zhang et al., 2020).Fig. 4 In vitro digestibility (A) and ferric reducing power (B) of co-extruded WPI-Cys conjugates.

Fig. 4

3.10 Ferric reducing power

Antioxidant properties of simulated gastrointestinal digests of co-extruded WPI-Cys samples are expressed as ferric reducing power, and the results are shown in Fig. 4B. During simulated gastric and intestinal digestion, as the Cys concentration gradually changed from 0 to 100 mmol/L, the ability of co-extruded WPI-Cys digests to reduce Fe3+ gradually increased, and EWPI-Cys100 reached the maximum. In protein hydrolysates, amino acid residues such as cysteine, methionine, lysine, histidine, tyrosine and tryptophan had antioxidant capacity (Zhang et al., 2013). Cold extrusion treatment promoted the hydrolysis of proteins, exposing amino acid residues with antioxidant activity to reduce Fe3+ to Fe2+. This might be the main reason for the increase in ferric reducing power of the co-extruded samples compared to WPI. In addition, another reason was probably that the added exogenous Cys had antioxidant capacity (Elias, McClements, & Decker, 2005). The iron-reducing capacity of the co-extruded WPI-Cys digest gradually increased with increasing Cys concentration. Wen et al. (2020) treated lotus root slices with Cys and found that Cys-treated lotus root slices had significantly higher iron reducing power than the control.

3.11 α-glucosidase inhibitory activity

The inhibitory rate of α-glucosidase activity is often used to represent the antidiabetic ability of proteins. The α-glucosidase inhibitory rates in simulated gastrointestinal digests of co-extruded WPI-Cys samples are shown in Fig. 5A. Simulated gastric and intestinal digests of co-extruded EPWI-Cys samples showed significantly higher α-glucosidase inhibitory activity than those of WPI or EWPI (P < 0.05). Furthermore, as the concentration of Cys increased, the α-glucosidase inhibitory rate in gastric and intestinal digests of co-extruded WPI-Cys was increased. For instance, at the Cys concentration of 80 mmol/L, the α-glucosidase inhibitory rate of gastric and intestinal WPI-Cys digests increased by 29.92% and 25.25%, respectively (P < 0.05), compared to WPI digests. Lacroix and Li-Chan (2013) proposed that as competitive inhibitors, peptides can bind to active sites of α-glucosidase through hydrophobic interactions, thereby delaying the absorption of glucose. Furthermore, co-extruded WPI-Cys with high digestibility would produce more peptides that WPI or EWPI (shown in Fig. 4A), thus inhibiting α-glucosidase activity.Fig. 5 α-glucosidase inhibitory activity (A) and XOD inhibitory activity (B) of co-extruded WPI-Cys conjugates.

Fig. 5

3.12 XOD inhibitory activity

The inhibitory of XOD activity could block the synthesis and deposition of uric acid, thus preventing the occurrence of gout and hyperuricemia (Emmerson, 1996). The XOD inhibitory rates in simulated gastrointestinal digests of co-extruded WPI-Cys samples are shown in Fig. 5B. Furthermore, from Fig. 5B, with the addition of Cys changing from 0 to 100 mmol/L, the XOD inhibitory rates in stomach and intestine showed a gradual increase (P < 0.05). Su, He, Zhao, Waterhouse, and Sun-Waterhouse (2018) confirmed that Trp could bind to the specific domain of XOD and inhibit the activity of XOD. It is possible that the endogenous fluorescence intensity of co-extruded WPI-Cys increased substantially with increasing Cys concentration (shown in Fig. 2C), indicating that more Trp residues was exposed, thus increasing the XOD inhibitory rates.

3.13 PCA and heatmap analysis

PCA is one of the most widely used linear dimensionality reduction algorithms. According to the result of PCA (Fig. 6A), the cumulative contribution rate of principal component 1 (PC1) and principal component 2 (PC2) was 98%. Between them, the variance contribution rate of PC1 was 86.8%, which was related to solubility, EAI, ESI, surface hydrophobicity, ferric reducing power, α-glucosidase inhibitory activity and XOD inhibitory activity. And the variance contribution rate of PC2 was 11.2%, which was related to digestibility. Furthermore, the score of the EWPI-Cys100 sample was the highest, and the second was the EWPI-Cys80 sample. These results indicated that co-extruded WPI-Cys samples at a concentration of 100 MPa had better emulsifying properties and in vitro digestibility. It was further demonstrated that co-extrusion of Cys with WPI promoted the SH/S-S exchange reaction and exposed the surface sulfhydryl groups of WPI. Additionally, co-extrusion promoted aggregation of proteins and exposed protease cleavage sites, thus improving the emulsification, surface hydrophobicity, and in vitro digestive properties of WPI.Fig. 6 PCA (A) and heatmap (B) of co-extruded WPI-Cys conjugates.

Fig. 6

The heatmap of co-extruded WPI-Cys samples is shown in Fig. 6B. As shown in Fig. 6B, the Cys concentration was positively correlated with surface hydrophobicity, EAI, ESI of co-extruded WPI-Cys, and in vitro digestibility, ferric reducing power, α-glucosidase inhibitory activity and XOD inhibitory activity of its digests. It was illustrated that with the concentration of Cys increasing to 100 mM, the emulsification properties, in vitro digestibility and bioactivity of the digests of co-extruded WPI-Cys gradually increased compared to WPI. This indicated that co-extrusion was an effective method of modifying whey proteins.

4 Conclusion

It was the first time to demonstrate that the co-extrusion of WPI and Cys at low temperature could effectively improve the physicochemical, rheological properties and in vitro digestion of WPI. In general, the emulsifying characteristics and biological activities of co-extruded WPI-Cys were positively correlated with the concentration of Cys. The result of PCA indicated that co-extruded WPI-Cys at a concentration of 100 mmol/L had the highest score, and its surface hydrophobicity, EAI, ESI, ferric reducing power, α-glucosidase inhibitory rate and XOD inhibitory rate reached the maximum. In addition, compared to WPI, co-extruded WPI-Cys samples showed an increase in apparent viscosity increase and Pe. Moreover, the G′ and G′′ of EWPI-Cys100 were the highest among all the samples. Therefore, this investigation proposes that co-extrusion of WPI and Cys is a fast, environmentally friendly, and convenient method to modify WPI, which would provide food protein ingredients with outstanding functional properties for food industry.

CRediT authorship contribution statement

Sinan Mu: Writing – original draft, Methodology, Investigation, Formal analysis, Conceptualization. Zhishen Mu: Writing – original draft, Methodology, Investigation, Data curation. Munkh-Amgalan Gantumur: Visualization, Validation, Data curation. Nan Yang: Visualization, Validation, Data curation. Narantuya Sukhbaatar: Writing – original draft, Investigation, Data curation. Yuxue Sun: Writing – review & editing, Supervision, Resources, Funding acquisition, Conceptualization. Zhanmei Jiang: Writing – review & editing, Supervision, Resources, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

The authors are unable or have chosen not to specify which data has been used.

Acknowledgement

This study was supported by project for 10.13039/501100001809 Natural Science Foundation of China (No. 32172164 ), Natural Key Science Foundation of Heilongjiang Province of China (No. ZD2021C007 ) and Cooperative Innovation Project of Heilongjiang Province Education Department (LJGXCG2022-029 ).
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