Extraction of proteins from grass pea (Lathyrus sativus L.) and Persian everlasting pea (Lathyrus rotundifolius L.) seeds using aqueous two-phase systems

Document Type : Research Paper

Authors

1 Department of Food Science & Technology, College of Agriculture & Natural Resources of University of Tehran, Karaj, Iran

2 Department of Food Science & Technology, College of Agriculture & Natural Resources of University of Tehran, Karaj, Iran

3 Research Center for Biochemistry and Nutrition in Metabolic Diseases, Institute for Basic Sciences, Kashan University of Medical Sciences, Kashan, Iran

Abstract

This study investigated the extraction of proteins from grass pea (Lathyrus sativus L.) and Persian everlasting pea (Lathyrus rotundifolius L.) seeds under varying pH levels and sodium chloride (NaCl) concentrations, as well as using aqueous two-phase systems (ATPSs). The lowest protein extractibility was observed at pH 4, whereas the highest extractibility occurred at pH 9.5 in distilled water. The results indicate that the presence of salt under alkaline conditions did not significantly enhance extractibility compared to salt-free alkaline solutions. An ATPS comprising polyethylene glycol (PEG, 4000 g/mol) and arabic gum was developed and evaluated for protein extraction and separation. In the formed system, PEG and arabic gum preferentially migrated to the top and bottom phases, respectively. Optimal extraction conditions—20% PEG, 1% arabic gum, a solid-to-liquid ratio of 1:10, particle size of 50 mesh, and a temperature of 25 °C—yielded a maximum extraction efficiency of approximately 63%. SDS-PAGE electrophoresis profiles revealed a clear phase-dependent partitioning pattern, where low-molecular-weight subunits (≤25 kDa) preferentially migrated to the top phase, while higher-molecular-weight subunits (>50 kDa) predominantly partitioned into the bottom phase. This research demonstrates that aqueous two-phase systems (ATPS) are an effective, environmentally friendly, and cost-effective method for the extraction and fractionation proteins from grass pea and Persian everlasting pea seeds.

Keywords

Main Subjects

EXTENDED ABSTRACT

Introduction

In recent years, due to population growth and increasing economic and environmental concerns, plant-based proteins have gained significant attention as sustainable alternatives to animal-derived proteins. Lathyrus species are annual legumes from the Fabeae tribe that exhibit remarkable adaptability to harsh environmental conditions. Among these, grass pea (Lathyrus sativus L.) and Persian everlasting pea (Lathyrus rotundifolius L.) are underutilized yet promising protein sources. The efficiency of protein extraction depends on factors such as the target application, available resources, and extraction technique. Aqueous two-phase systems (ATPSs) have recently attracted attention as a mild and efficient approach for the extraction and purification of biomolecules. This study aimed to investigate protein extraction from grass pea (GP) and Persian everlasting pea (PEP) seeds using different extraction conditions, and to characterize the extracted proteins using SDS-PAGE.

Materials and Methods

Protein was extracted from GP and PEP seed flours using conventional alkaline extraction at different pH values and NaCl concentrations (0, 0.2, 0.6, and 1 M). An aqueous two-phase system was also applied for protein extraction. The binodal curve for PEG (polyethylene glycol, 4000 g/mol) and arabic gum was determined using the turbidity (cloud point) method. The optimal ATPS parameters were then selected for protein extraction. SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) was used to estimate the molecular weight distribution of the extracted polypeptides.

Results

The results showed that the highest and lowest protein extractability occurred at pH 9.5 and pH 4, respectively, for both GP and PEP in distilled water. Around the isoelectric point (pI), protein extractability decreased due to reduced electrostatic repulsion between protein molecules as the net charge approached zero. Although maximum extractability was also observed at pH 9.5 with NaCl concentrations of 0.2 and 0.6 M, it was lower than the extractability achieved with distilled water. At 1 M NaCl, the lowest and highest protein extractability occurred at pH 2.5 and pH 8.5, respectively. Overall, the use of NaCl solutions did not significantly improve protein extractability compared to distilled water.

In ATPS, increasing PEG concentration enhanced the volume of the top phase (PEG-rich phase), and extraction efficiency improved up to 20% PEG but declined at 25% due to increased viscosity. Similarly, raising gum arabic concentration from 0.5% to 1% improved extraction, while higher concentrations reduced yield. Smaller particle sizes (up to 50 mesh) increased extraction efficiency, likely due to more effective release of intracellular contents. The optimal solid-to-liquid ratio was 1:10, and extraction yield improved with increasing temperature up to 25 °C, but declined beyond 30 °C, possibly due to protein denaturation or reduced phase separation efficiency.

SDS-PAGE revealed polypeptide bands ranging from 19 to 120 kDa in PEP and 18 to 140 kDa in GP. In the top phase of ATPS, dominant subunits for PEP were observed at approximately 19, 24, 35, and 40 kDa, and for GP at 18, 26, 35, and 40 kDa. In the bottom phase, subunits around 35, 50, 75, and 120 kDa were detected in PEP, and 35, 60, 70, and 140 kDa in GP.

Conclusions

The results indicated that salt addition did not significantly improve protein extraction over distilled water under alkaline conditions. The ATPS composed of PEG 4000 and arabic gum effectively facilitated the extraction and separation of proteins from GP and PEP seeds. PEG and arabic gum preferentially migrated to the top and bottom phases, respectively. Maximum extraction efficiency (~63%) was achieved under optimal conditions: 20% PEG, 1% arabic gum, a solid–liquid ratio of 1:10, particle size of 50 mesh, and temperature of 25 °C. Electrophoretic analysis demonstrated that low-molecular-weight polypeptides (≤25 kDa) migrated primarily to the top phase, whereas higher-molecular-weight polypeptides (>50 kDa) accumulated in the bottom phase. Overall, this study confirms that aqueous two-phase systems are efficient, eco-friendly, and cost-effective tools for the extraction and fractionation of both hydrophilic and hydrophobic proteins from underutilized legumes such as grass pea and Persian everlasting pea.

Author Contributions

Yeganeh Azimi (YA), Hossein Kiani (HK), Seyed Mohammad Mousavi (SMM), Mahsa Mohseni (MM), Mina Homayoonfal (MH). Conceptualization was contributed by (YA) and (HK). Data Curation was involved by (YA) and (MM). Formal analysis was done by (YA) and (MH). Investigation was performed by (YA). Methodology did by (YA) and (HK). Project administration was carried out (HK) and (SMM). Supervision was responsible by (HK) and (SMM). Writing–original draft was done by (YA). Writing–review and editing did by (YA), (MK) and (MH). All authors have read and agreed to the final version of the manuscript.

Data Availability Statement

Research data are not shared.

Acknowledgment

This research was supported by University of Tehran, Iran. We thank our colleagues from Bioprocessing and Biodetection lab of Department of Food Science and Technology, University of Tehran.

Ethical considerations

The authors avoided data fabrication, falsification, plagiarism, and misconduct.

Conflict of interest

The author declares no conflict of interest.

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Iranian Journal of Biosystem Engineering
Volume 56, Issue 3
October 2025
Pages 21-42

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History

  • Receive Date: 22 July 2025
  • Revise Date: 26 September 2025
  • Accept Date: 01 October 2025

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