Food Analytical Methods

, Volume 10, Issue 6, pp 1669–1680 | Cite as

Establishment of an Aqueous PEG 200-Based Deep Eutectic Solvent Extraction and Enrichment Method for Pumpkin (Cucurbita moschata) Seed Protein

  • Rui-Lin Liu
  • Pei Yu
  • Xian-Li Ge
  • Xiu-Feng Bai
  • Xing-Qiang Li
  • Qiang Fu


A novel approach for ultrasound–microwave synergistic extraction (UMSE) of pumpkin seed protein was developed using aqueous poly (ethylene glycol) (PEG 200)-based deep eutectic solvent (DES) as a green extraction medium. Key factors controlling the extraction and optimal operating conditions were optimized by combining the one variable at a time and response surface methodology. Results showed that the PEG 200 as a hydrogen bond donor combined with choline chloride as a typical hydrogen bond acceptor had a highest extraction efficiency among different solvents. The optimal extraction parameters were optimized as follows: PEG 200-based DES concentration, 28% w/w; solid to liquid ratio, 28 g mL−1; microwave power, 140 W; and extraction temperature, 43 °C. Under the optimal parameters, the actual extraction yield was 93.95 ± 0.23% (n = 3). The precipitation rate of pumpkin seed protein was 97.97% with a precipitation time of only 4 min by using an isoelectric point-ethanol-PEG 200 DES ternary co-precipitation method. Overall, this integrated method of PEG 200-based DES and UMSE exhibits a powerful tool for the rapid and efficient extraction of pumpkin seed protein.


Deep eutectic solvent Polyethylene glycol Pumpkin seed protein Ultrasound–microwave synergistic extraction Response surface methodology 


Compliance with Ethical Standards

The manuscript has not been published previously (partly or in full). The manuscript has not been submitted to more than one journal for simultaneous consideration.

Consent to submit has been received explicitly from all co-authors, as well as from the institute/organization where the work has been carried out before the work is submitted.

Authors whose names appear on the submission have contributed sufficiently to the scientific work and, therefore, share collective responsibility and accountability for the results.


The project were supported by the China Postdoctoral Science Foundation (No. 2016M592774) and the Open Projects Program of the Key Laboratory of Shaanxi Province Craniofacial Precision Medicine Research, Xi’an Jiaotong University (No. 2016LHM-KFKT002).

Conflict of Interest

Rui-Lin Liu declares that he has no conflict of interest. Pei Yu declares that she has no conflict of interest. Xian-Li Ge declares that he has no conflict of interest. Xiu-Feng Bai declares that he has no conflict of interest. Xing-Qiang Li declares that he has no conflict of interest. Qiang Fu declares that he has no conflict of interest.

Ethical Approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Informed Consent

Informed consent is not applicable.


  1. Aremu CY (1990) Proximate and amino acid composition of cowpea (Vigna unguiculata, walp) protein concentrate prepared by isoelectric point precipitation. Food Chem 37:61–68CrossRefGoogle Scholar
  2. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefGoogle Scholar
  3. Bucko S, Katona J, Popovic L, Petrovic L, Jelena MJ (2016) Influence of enzymatic hydrolysis on solubility, interfacial and emulsifying properties of pumpkin (Cucurbita pepo) seed protein isolate. Food Hydrocoll 60:271–278CrossRefGoogle Scholar
  4. Buyel JF, Bautista JA, Fischer R, Yusibov VM (2012) Extraction, purification and characterization of the plant-produced HPV16 subunit vaccine candidate E7 GGG. J Chromatogr B 880:19–26CrossRefGoogle Scholar
  5. Capriotti AL, Cavaliere C, Piovesana S, Satampachiacchiere S, Ventura S, Chiozzi RZ, Lagana A (2015) Characterization of quinoa seed proteome combining different protein precipitation techniques: improvement of knowledge of nonmodel plant proteomics. J Sep Sci 38:1017–1025CrossRefGoogle Scholar
  6. Celli GB, Ghanem A, Brooks MS-L (2015) Optimization of ultrasound-assisted extraction of anthocyanins from haskap berries (Lonicera caerulea L.) using response surface methodology. Ultrason Sonochem 27:449–455CrossRefGoogle Scholar
  7. Chan C-H, Yusoff R, Ngoh G-C, Kung FW-L (2011) Microwave-assisted extractions of active ingredients from plants. J Chromatogr A 1218:6213–6225CrossRefGoogle Scholar
  8. Chiesa S, Gnansounou E (2011) Protein extraction from biomass in a bioethanol refinery-possible dietary applications: use as animal feed and potential extension to human consumption. Bioresour Technol 102:427–436CrossRefGoogle Scholar
  9. Ding X, Zhang H, Chen H, Wang L, Qian H, Qi X (2015) Extraction, purification and identification of antifreeze proteins from cold acclimated malting barley (Hordeum vulgare L.). Food Chem 175:74–85CrossRefGoogle Scholar
  10. Feng S, Luo Z, Zhong Z, Jiang L, Tang K (2014) Extraction optimization by response surface methodology: purification and characterization of phytosterol from sugarcane (Saccharum officinarum L.) rind. J Sep Sci 37:1308–1314CrossRefGoogle Scholar
  11. Francisco M, van den Bruinhorst A, Kroon MC (2013) Low-transition-temperature mixtures (LTTMs): a new generation of designer solvents. Angewandte Chemie-International Edition 52:3074–3085CrossRefGoogle Scholar
  12. Ge X-L, Shi T, Wang H, Zhang J, Zhang Z-Q (2016) Development of an aqueous polyethylene glycol-based extraction and recovery method for almond (Prunus armeniaca L.) protein. Food Anal Methods. doi: 10.1007/s12161-016-0525-3 Google Scholar
  13. Huang Y, Wang Y, Pan Q, Wang Y, Ding X, Xu K, Li N, Wen Q (2015) Magnetic graphene oxide modified with choline chloride-based deep eutectic solvent for the solid-phase extraction of protein. Anal Chim Acta 877:90–99CrossRefGoogle Scholar
  14. Kadam SU, Tiwari BK, Alvarez C, O'donnell CP (2015) Ultrasound applications for the extraction, identification and delivery of food proteins and bioactive peptides. Trends Food Sci Technol 46:60–67CrossRefGoogle Scholar
  15. Kim YJ, Lee HM, Wang Y, Wu J, Kim SG, Kang KY, Park KH, Kim YC, Choi IS, Agrawal GK, Rakwal R, Kim ST (2013) Depletion of abundant plant RuBisCO protein using the protamine sulfate precipitation method. Proteomics 13:2176–2179CrossRefGoogle Scholar
  16. Kumara V, Sharma VK, Kalonia DS (2009) Effect of polyols on polyethylene glycol (PEG)-induced precipitation of proteins: impact on solubility, stability and conformation. Int J Pharm 366:38–43CrossRefGoogle Scholar
  17. Li Q, Fu C (2005) Application of response surface methodology for extraction optimization of germinant pumpkin seeds protein. Food Chem 92:701–706CrossRefGoogle Scholar
  18. Li N, Wang Y, Xu K, Huang Y, Wen Q, Ding X (2016) Development of green betaine-based deep eutectic solvent aqueous two-phase system for the extraction of protein. Talanta 152:23–32CrossRefGoogle Scholar
  19. Liu R-L, Song S-H, Wu M, He T, Zhang Z-Q (2013) Rapid analysis of fatty acid profiles in raw nuts and seeds by microwave-ultrasonic synergistic in situ extraction-derivatisation and gas chromatography-mass spectrometry. Food Chem 141:4269–4277CrossRefGoogle Scholar
  20. Liu R-L, Ge X-L, Gao X-Y, Zhan H-Y, Shi T, Su N, Zhang Z-Q (2016) Two angiotensin-converting enzyme-inhibitory peptides from almond protein and the protective action on vascular endothelial function. Food & Function 7:3733–3739CrossRefGoogle Scholar
  21. Mondal D, Mahto A, Veerababu P, Bhatt J, Prasad K, Nataraj SK (2015) Deep eutectic solvents as a new class of draw agent to enrich low abundance DNA and proteins using forward osmosis. RSC Adv 5:89539–89544CrossRefGoogle Scholar
  22. Nam MW, Zhao J, Lee MS, Jeong JH, Lee J (2015) Enhanced extraction of bioactive natural products using tailor-made deep eutectic solvents: application to flavonoid extraction from Flos sophorae. Green Chem 17:1718–1727CrossRefGoogle Scholar
  23. Oliveira FS, Pereiro AB, Rebelo LPN, Marrucho IM (2013) Deep eutectic solvents as extraction media for azeotropic mixtures. Green Chem 15:1326–1330CrossRefGoogle Scholar
  24. Ordonez-santos LE, Pinzon-zarate LX, Gonzalez-salcedo LO (2015) Optimization of ultrasonic-assisted extraction of total carotenoids from peach palm fruit (Bactris gasipaes) by-products with sunflower oil using response surface methodology. Ultrasonic Sonochemistry 27:560–566CrossRefGoogle Scholar
  25. Paiva A, Craveiro R, Aroso I, Martins M, Reis RL, Duarte ARC (2014) Natural deep eutectic solvents-solvents for the 21st century. ACS Sustain Chem Eng 2:1063–1071CrossRefGoogle Scholar
  26. Purkayastha MD, Dutta G, Barthakur A, Mahanta CL (2015) Tackling correlated responses during process optimisation of rapeseed meal protein extraction. Food Chem 170:62–73CrossRefGoogle Scholar
  27. Salgado PR, Drago SR, Ortiz SEM, Petruccelli S, Andrich O, Gonzalez RJ, Mauri AN (2012) Production and characterization of sunflower (Helianthus annuus L.) protein-enriched products obtained at pilot plant scale. LWT-Food Science and Technology 45:65–72CrossRefGoogle Scholar
  28. Sharma VK, Kalonia DS (2004) Polyethylene glycol-induced precipitation of interferon alpha-2a followed by vacuum drying: development of a novel process for obtaining a dry stable powder. AAPS PharmSciTech 6:1–14Google Scholar
  29. Vergara-barberan M, Lerma-garcia MJ, Herrero-martinez JM, Simo-alfonso EF (2015) Use of an enzyme-assisted method to improve protein extraction from olive leaves. Food Chem 169:28–33CrossRefGoogle Scholar
  30. Wagle DV, Zhao H, Baker GA (2014) Deep eutectic solvents: sustainable media for nanoscale and functional materials. Acc Chem Res 47:2299–2308CrossRefGoogle Scholar
  31. Wessel D, Flugge UI (1984) A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal Biochem 138:141–143CrossRefGoogle Scholar
  32. Wu D, Gao T, Yang H, Du Y, Li C, Wei L, Zhou T, Lu J, Bi H (2015) Simultaneous microwave/ultrasonic-assisted enzymatic extraction of antioxidant ingredients from Nitraria tangutorun Bobr. juice by-products. Ind Crop Prod 66:229–238CrossRefGoogle Scholar
  33. Xu K, Wang Y, Huang Y, Li N, Wen Q (2015) A green deep eutectic solvent-based aqueous two-phase system for protein extracting. Anal Chim Acta 864:9–20CrossRefGoogle Scholar
  34. Xu K, Wang Y, Ding X, Huang Y, Li N, Wen Q (2016) Magnetic solid-phase extraction of protein with deep eutectic solvent immobilized magnetic graphene oxide nanoparticles. Talanta 148:153–162CrossRefGoogle Scholar
  35. Zhang QH, Vigier KD, Royer S, Jerome F (2012) Deep eutectic solvents: syntheses, properties and applications. Chem Soc Rev 41:7108–7146CrossRefGoogle Scholar
  36. Zhao X, Chen F, Chen J, Gai G, Xue W, Li L (2008) Effects of AOT reverse micelle on properties of soy globulins. Food Chem 111:599–605CrossRefGoogle Scholar
  37. Zhao B-Y, Xu P, Yang F-X, Wu H, Zong M-H, Lou W-Y (2015) Biocompatible deep eutectic solvents based on choline chloride: characterization and application to the extraction of rutin from Sophora japonica. ACS Sustain Chem Eng 3:2746–2755CrossRefGoogle Scholar
  38. Zheng Y, Tang Q, Wang T, Wang J (2015) Molecular size distribution in synthesis of polyoxymethylene dimethyl ethers and process optimization using response surface methodology. Chem Eng J 278:183–189CrossRefGoogle Scholar
  39. Zhu C-Z, Zhang W-G, Zhou G-H, Xu X-L, Kang Z-L, Yin Y (2013) Isolation and identification of antioxidant peptides from Jinhua ham. J Agric Food Chem 61:1265–1271CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.School of PharmacyXi’an Jiaotong UniversityXi’anPeople’s Republic of China
  2. 2.Key Laboratory of Shaanxi Province Craniofacial Precision Medicine Research, College of StomatologyXi’an Jiaotong UniversityXi’anPeople’s Republic of China

Personalised recommendations