Effect of Ionic Strength and Mixing Ratio on Complex Coacervation of Soy Protein Isolate/Flammulina Velutipes Polysaccharide

Introduction

Proteins and polysaccharides are essential components of the food nutrition system, and the complex coacervation of protein/polysaccharide plays a crucial role in the texture, structure, and flavor of food. Due to their excellent physicochemical properties such as emulsifying ability, gelation, and biodegradability, protein/polysaccharide coacervates can serve as emulsion stabilizers, fat replacers, and biodegradable packaging materials, playing an irreplaceable role in the development and application of functional foods.

Soy protein isolate (SPI) is a commercially available soybean protein with more than 90% protein content and a balanced amino acid composition, with lysine and other essential amino acids close to the recommended values by FAO/WHO, thus possessing high nutritional value. SPI is readily available in the market and has a wide range of applications, making it a highly valuable commercial economic protein in the food industry. In addition to its high nutritional value, SPI also exhibits certain biological activities such as lowering blood lipids, reducing cholesterol, and decreasing the risk of cardiovascular diseases. However, issues such as poor solubility and low gel mechanical strength limit the application of SPI in traditional food industries. To better utilize the excellent properties of SPI, modifications are made using the non-covalent interactions between proteins and polysaccharides. Flammulina velutipes polysaccharide (FVP) is an anionic mixed polysaccharide extracted from the fruiting body of Flammulina velutipes, primarily composed of glucan with multiple polysaccharide components. FVP is widely used in the food industry due to its excellent physicochemical properties. For instance, FVP nanoparticles can be used as stabilizers to prepare oil/water Pickering emulsions, and edible films made from FVP exhibit high mechanical strength, ductility, and antibacterial properties.

Professor Junmiao Zhang and Professor Qiuhui Hu from the College of Food Science and Engineering at Nanjing University of Finance and Economics explore the effects of pH, ionic strength, and mixing ratio on the interactions between soy protein isolate and Flammulina velutipes polysaccharide based on protein and polysaccharide complex coacervation, providing a theoretical basis and reference for the development of traditional functional foods using soy protein isolate and Flammulina velutipes polysaccharide.

Results and Discussion

Soy Protein Isolate/Flammulina Velutipes Polysaccharide Complex Coacervation

As shown in Figures 1A-C, the complex coacervation of Flammulina velutipes polysaccharide and soy protein occurs at pH 6.2, with maximum coacervation of the mixed solution at pH 4.5. Fourier-transform infrared spectroscopy shows a slight blue shift in the absorption peak of the amide I band of the Flammulina velutipes polysaccharide/soy protein complex, with a significant increase in the intensity of the absorption peak at 1043 cm^-1, indicating that there are electrostatic interactions in the formation of the Flammulina velutipes polysaccharide/soy protein complex coacervates (Figure 1D). Figure 2 shows the laser confocal images of Flammulina velutipes polysaccharide/soy protein under different pH conditions. The results indicate that as the pH decreases, the solution transitions from an initially homogeneous dispersed state to local aggregation, and when the pH reaches 4.5, the solution overall exhibits an aggregated state.

Effect of Ionic Strength and Mixing Ratio on Complex Coacervation of Soy Protein Isolate/Flammulina Velutipes Polysaccharide

Figure 1 Turbidity Curves of SPI, FVP, and FVP/SPI Mixtures

Effect of Ionic Strength and Mixing Ratio on Complex Coacervation of Soy Protein Isolate/Flammulina Velutipes Polysaccharide

Figure 2 Microstructure of FVP/SPI at Different pH Levels, Red Indicates Soy Protein under CLSM

Complex Coacervation of Soy Protein Isolate/Flammulina Velutipes Polysaccharide at Different Ionic Strengths and Mixing Ratios

When the salt ion concentration is 50 mol/L, the maximum turbidity value of the SPI/FVP mixed solution, the span from pH_φ1 to pH_φ2, and the surface charge of soy protein all reach their maximum values (Figures 3A, B). As the salt ion concentration increases, the surface charge of the Flammulina velutipes polysaccharide shows an overall increasing trend (Figures 3C, D). When the mixing ratio of protein and polysaccharide is adjusted from 20:1 to 1:1, the turbidity of the SPI/FVP solution shows a trend of first increasing and then decreasing, achieving a maximum near the isoelectric point of soy protein (Figures 4A, B). Moreover, the apparent charge of the SPI/FVP mixed solution is between that of Flammulina velutipes polysaccharide and soy protein isolate (Figure 4C).

Effect of Ionic Strength and Mixing Ratio on Complex Coacervation of Soy Protein Isolate/Flammulina Velutipes Polysaccharide

Figure 3 Turbidity Curves of FVP/SPI Mixed Solutions at Different Ionic Strengths

Effect of Ionic Strength and Mixing Ratio on Complex Coacervation of Soy Protein Isolate/Flammulina Velutipes Polysaccharide

Figure 4 Turbidity Curve of FVP/SPI Mixtures at Different Mixing Ratios

Soy Protein Isolate/Flammulina Velutipes Polysaccharide Coacervate Secondary Structure

Table 1 shows the spatial conformation changes of coacervates under different preparation conditions. Under constant pH and mixing ratio, the influence of ionic strength on the secondary structure of SPI/FVP complex primarily manifests as: ionic strength can increase the content of α-helix and decrease the content of β-sheet. Under constant pH and ionic strength, as the FVP content increases, the α-helix and random coil content of SPI/FVP complex coacervates remain constant, while the β-sheet content increases and the β-turn decreases. The results indicate that salt ions can enhance the α-helix and reduce the β-sheet; increasing the amount of Flammulina velutipes polysaccharide raises the β-sheet content and lowers the β-turn.

Table 1 Secondary Structure Composition of Flammulina Velutipes Polysaccharide/Soy Protein Coacervates under Different pH, Salt Ion Concentrations, and Mixing Ratios

Effect of Ionic Strength and Mixing Ratio on Complex Coacervation of Soy Protein Isolate/Flammulina Velutipes Polysaccharide

Soy Protein Isolate/Flammulina Velutipes Polysaccharide Coacervate Rheological Properties

Figure 5 shows that the G’ values of all samples are much higher than the G” values, indicating that the FVP/SPI complex coacervates exhibit elastic behavior and possess solid-like properties. When pH and mixing ratio are constant, lower NaCl concentrations (C_NaCl = 10 and 50 mmol/L) result in a higher storage modulus of the SPI/FVP complex coacervate layer compared to the layer without NaCl. At higher NaCl concentrations (C_NaCl ≥ 100 mmol/L), the storage modulus of the SPI/FVP complex coacervate layer is lower than that of the layer without NaCl. When pH and salt ion concentration are constant, as the amount of FVP increases, both the storage modulus and loss modulus show a decreasing trend, with lower storage modulus indicating a looser structure of the complex coacervate. Notably, when the SPI:FVP ratio is 10:1 and 15:1, they exhibit similar rheological behaviors, which may be related to the charge saturation of SPI or FVP.

Effect of Ionic Strength and Mixing Ratio on Complex Coacervation of Soy Protein Isolate/Flammulina Velutipes Polysaccharide

Figure 5 Storage Modulus (A, C) and Loss Modulus (B, D) of SPI/FVP Coacervates

Conclusion

This study investigates the phase behavior, secondary structure, and rheological properties of soy protein isolate/Flammulina velutipes polysaccharide complex coacervation under different pH, ionic strength, and mixing ratios. The results indicate that at a sodium chloride concentration of 50 mmol/L and a soy protein to Flammulina velutipes polysaccharide mixing ratio of 15:1, the complex coacervates exhibit stable structures and good rheological properties, indicating their application potential in the development of traditional foods.

Effect of Ionic Strength and Mixing Ratio on Complex Coacervation of Soy Protein Isolate/Flammulina Velutipes Polysaccharide

Figure 6 Schematic Diagram of SPI/FVP Complex or Copolymer Formation

First Author

Effect of Ionic Strength and Mixing Ratio on Complex Coacervation of Soy Protein Isolate/Flammulina Velutipes Polysaccharide

Junmiao Zhang, male. Master’s degree from the College of Food Science and Engineering, Nanjing University of Finance and Economics, graduated in June 2021 with a Master’s degree in Food Science and Engineering. Currently a PhD student in the Food Science and Technology College of Nanjing Agricultural University, class of 2021, under the supervision of Professor Qiuhui Hu. During his master’s studies, he participated in the National Key Research and Development Program on the interactions of proteins and active polysaccharides and their immunomodulatory effects, studying the preparation of Flammulina velutipes polysaccharide and soy protein complex coacervates and their immune-enhancing effects on macrophages. He has published two SCI papers as the first author, applied for two patents, and completed the Jiangsu Province Graduate Innovation Program. During his graduate studies, he received titles such as Excellent Graduate Student, Excellent Graduate Student Cadre, and Outstanding Graduate.

Corresponding Author

Effect of Ionic Strength and Mixing Ratio on Complex Coacervation of Soy Protein Isolate/Flammulina Velutipes Polysaccharide

Qiuhui Hu, Director of the Processing Research Room of the National Edible Mushroom Industry Technology System, Professor/Doctoral Supervisor at Nanjing Agricultural University/Nanjing University of Finance and Economics, enjoys special government allowances from the State Council. Selected as a “New Century Excellent Talent Support Program” by the Ministry of Education, leader of the Jiangsu Province College Blue and Green Project Technology Innovation Team, young and middle-aged scientific and technological leadership in Jiangsu Province’s “333 High-level Talent Training Project”, and outstanding young and middle-aged experts in Jiangsu Province. He has presided over several projects including the National Key Research and Development Program’s “Strategic International Science and Technology Innovation Cooperation” key project, National Natural Science Foundation, sub-project of the “13th Five-Year Plan” National Key Research and Development Program, Ministry of Agriculture 948 Project, and Jiangsu Province Modern Agriculture Key Project. His key technology innovations and applications in the deep processing of edible mushrooms won the first prize for scientific and technological progress in Jiangsu Province in 2019 (first contributor), the first prize for teaching achievements in Jiangsu Province in 2017 for the innovative and practical training model of food science and engineering professionals integrating “economics”, “management”, and “law” (first contributor), and the second prize for outstanding achievements in scientific and technological progress from the Ministry of Education in 2014 (first contributor). He serves on the editorial board of the journal Food Science and Human Wellness. Over 200 papers have been published, and he has been continuously listed in the Elsevier’s list of highly cited scholars in the field of agriculture and biological sciences in China from 2017 to 2021, with more than 30 authorized invention patents.

Effect of Ionic Strength and Mixing Ratio on Complex Coacervation of Soy Protein Isolate/Flammulina Velutipes Polysaccharide

Junmiao Zhang^a, Hengjun Du^b, Ning Ma^a, Lei Zhong^c, Gaoxing Ma^a, Fei Pei^a, Hui Chen^a,d, Qiuhui Hu^a,*

^a Key Laboratory of Edible Mushroom Processing, Ministry of Agriculture and Rural Affairs, College of Food Science and Engineering, Nanjing University of Finance and Economics, Nanjing 210023, China

^b Department of Food Science, University of Massachusetts, Amherst MA 01003, USA

^c College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China

^d Jiangsu Alphay Bio-technology Co., Ltd., Nantong 226009, China

*Corresponding author.

Abstract

Soy protein isolate (SPI) is a commercial protein with balanced amino acids, while the poor solubility impedes its use in traditional foods. To overcome the problem, the complex coacervation of SPI/Flammulina velutipes polysaccharide (FVP) were investigated. Initial results revealed that the suitable amounts of FVP contributed to reducing the turbidity of SPI solution. Under electrostatic interaction, the formation of SPI/FVP coacervates were spontaneous and went through a nucleation and growth process. Low salt concentration (C_NaCl = 10, 50 mmol/L) led to an increase in the critical pH values (pHc, pH_φ1) while the critical pH values decreased when C_NaCl ≥ 100 mmol/L. The concentration of NaCl ions increased the content of α-helix. With the increase of FVP, the critical pH values decreased and the content of β-sheet increased through electrostatic interaction. At SPI/FVP ratio of 10:1 and 15:1, the complex coacervation of SPI/FVP were saturated, and the coacervates had the same storage modulus value. SPI/FVP coacervates exhibited solid-like properties and presented the strongest storage modulus at C_NaCl = 50 mmol/L. The optimal pH, SPI/FVP ratio and NaCl concentration of complex coacervation were collected, and the coacervates demonstrated a valuable application potential to protect and deliver bioactives and food ingredients.

Reference:

ZHANG J M, DU H J, MA N, et al. Effect of ionic strength and mixing ratio on complex coacervation of soy protein isolate/Flammulina velutipes polysaccharide[J]. Food Science and Human Wellness, 2023, 12(1): 183-191. DOI:10.1016/j.fshw.2022.07.006.

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Effect of Ionic Strength and Mixing Ratio on Complex Coacervation of Soy Protein Isolate/Flammulina Velutipes Polysaccharide

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Editor: Liang Anqi; Responsible editor: Sun Yong

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Effect of Ionic Strength and Mixing Ratio on Complex Coacervation of Soy Protein Isolate/Flammulina Velutipes Polysaccharide

Effect of Ionic Strength and Mixing Ratio on Complex Coacervation of Soy Protein Isolate/Flammulina Velutipes Polysaccharide

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