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Licensed Unlicensed Requires Authentication Published by De Gruyter September 26, 2019

Optimization of Aqueous Two Phase Extraction of Proteins from Litopenaeus Vannamei Waste by Response Surface Methodology Coupled Multi-Objective Genetic Algorithm

  • P. Saravana Pandian , S. Sindhanai Selvan , A. Subathira and S. Saravanan EMAIL logo


Waste generated from industrial processing of seafood is an enormous source of commercially valuable proteins. One among the underutilized seafood waste is shrimp waste, which primarily consists of head and carapace. Litopenaeus vannamei (L. vannamei) is the widely cultivated shrimp in Asia and contributes to 90 % of aggregate shrimp production in the world. This work was focused on extraction as well as purification of value-added proteins from L. vannamei waste in a single step aqueous two phase system (ATPS). Polyethylene glycol (PEG) and trisodium citrate system were chosen for the ATPS owing to their adequate partitioning and less toxic nature. Response surface methodology (RSM) was implemented for the optimization of independent process variables such as PEG molecular weight (2000 to 6000), pH (6 to 8) and temperature (25 to 45 °C). The results obtained from RSM were further validated using a Multi-objective genetic algorithm (MGA). At the optimized condition of PEG molecular weight 2000, pH 8 and temperature 35 °C, maximum partition coefficient and protein yield were found to be 2.79 and 92.37 %, respectively. Thus, L. vannamei waste was proved to be rich in proteins, which could be processed industrially through cost-effective non-polluting ATPS extraction, and RSM coupled MGA could be a potential tool for such process optimization.


[1] Kandra P, Challa MM. Kalangi Padma Jyothi H. Efficient use of shrimp waste: present and future trends. Appl Microbiol Biotechnol. 2012;93:17–29.10.1007/s00253-011-3651-2Search in Google Scholar PubMed

[2] Christie AE. Expansion of the Litopenaeus vannamei and Penaeus monodon peptidomes using transcriptome shotgun assembly sequence data. Gen Comp Endocrinol. 2014;206:235–54.10.1016/j.ygcen.2014.04.015Search in Google Scholar PubMed

[3] Briggs M, Funge-Smith S, Subasinghe R, Phillips M. Introductions and movement of Penaeus vannamei and Penaeus stylirostris in Asia and the Pacific. RAP Publ. 2004;10:92.Search in Google Scholar

[4] Iqbal M, Tao Y, Xie S, Zhu Y, Chen D, Wang X, et al. Aqueous two-phase system (ATPS): an overview and advances in its applications. Biol Proced Online. 2016;18:18.10.1186/s12575-016-0048-8Search in Google Scholar PubMed PubMed Central

[5] Nagaraja VH, Iyyaswami R. Aqueous two phase partitioning of fish proteins: partitioning studies and ATPS evaluation. J Food Sci Technol. 2014;52:3539–48.10.1007/s13197-014-1425-4Search in Google Scholar PubMed PubMed Central

[6] Vernau J, Kula MR Extraction of proteins from biological raw material using aqueous poly(ethylene) glycol-citrate phase systems. Biotechnol Appl Biochem. 1990;12:397–404.Search in Google Scholar

[7] Chang C, Xu G, Yang J, Wang D. Optimization of cellulase production using agricultural wastes by artificial neural network and genetic algorithm. Chem Prod Process Model. 2011;6. DOI: 10.2202/1934-2659.1553.Search in Google Scholar

[8] Kazeem MA, Hossain SM, Hossain MM, Razzak SA. Application of central composite design to optimize culture conditions of chlorella vulgaris in a batch photobioreactor: an efficient modeling approach. Chem Prod Process Model. 2018;13: 2017008.10.1515/cppm-2017-0082Search in Google Scholar

[9] Konak A, Coit DW, Smith AE. Multi-objective optimization using genetic algorithms: a tutorial. Reliab Eng Syst Safe. 2006;91:992–1007.10.1016/j.ress.2005.11.018Search in Google Scholar

[10] Nabavi SR. Multi-objective optimization of preparation conditions of asymmetric polyetherimide membrane for prevaporation of isopropanol. Chem Prod Process Model. 2016;11:47–50.10.1515/cppm-2015-0062Search in Google Scholar

[11] Poonsin T, Simpson BK, Benjakul S, Visessanguan W, Klomklao S. Albacore tuna (Thunnus alalunga) spleen trypsin partitioning in an aqueous two-phase system and its hydrolytic pattern on Pacific white shrimp (Litopenaeus vannamei) shells. Int J Food Prop. 2017;20:2409–22.10.1080/10942912.2016.1240180Search in Google Scholar

[12] Leal AL, de Castro PF,de Lima JP, de Souza Correia E. de Souza Bezerra R. Use of shrimp protein hydrolysate in Nile tilapia (Oreochromis niloticus, L.). Feeds Aquac Int. 2010;18:635–46.10.1007/s10499-009-9284-0Search in Google Scholar

[13] Rosenberg IM. Protein analysis and purification: benchtop techniques. 2nd rev. ed. Boston: Springer Science and Business Media, 2013.Search in Google Scholar

[14] Hanaei H, Murugesan T. Phase diagram of an aqueous salt-polymer system composed of poly ethylene glycol 4000 + Na3C6H5O7 + H2O. Appl Mech Mater. 2014;625:574–7.10.4028/ in Google Scholar

[15] Zhang ZF, Wang R, Ye F, Wang H, Zhao W. Extraction of glycyrrhizic acid by aqueous two-phase system formed by PEG and two environmentally friendly organic acid salts - sodium citrate and sodium tartrate. Green Process Synth. 2019;8:551–6.10.1515/gps-2019-0024Search in Google Scholar

[16] Xavier L, Freire MS, Vidal-Tato I. Recovery of phenolic compounds from Eucalyptus wood wastes using ethanol-salt-based aqueous two-phase systems. Maderas Cienc y Tecnol. 2017;19:0–0.10.4067/S0718-221X2017005000001Search in Google Scholar

[17] Herbst J, Pott RW. The effect of temperature on different aqueous two-phase diagrams of polyethylene glycol (PEG 6000, PEG 8000, and PEG 10000) + potassium sodium tartrate + water. J Chem Eng Data. 2019;64:3036–43.10.1021/acs.jced.9b00133Search in Google Scholar

[18] Bommenahalli Shashidhara R, Iyyaswami R. Aqueous two phase partitioning of Pisum sativum lectin in PEG/citrate salt system. Prep Biochem Biotechnol. 2018;48:759–67.10.1080/10826068.2018.1504220Search in Google Scholar

[19] Settu S, Velmurugan P, Jonnalagadda RR, Nair BU. Extraction of bovine serum albumin using aqueous two-phase poly (ethylene glycol) – poly (acrylic acid) system. J Sci Ind Res. 2015;74:348–53.Search in Google Scholar

[20] Glyk A, Heinisch SL, Scheper T, Beutel S. Comparison of colorimetric methods for the quantification of model proteins in aqueous two-phase systems. Anal Biochem. 2015;477:35–7.10.1016/j.ab.2015.02.007Search in Google Scholar

[21] Engel S, Vyazmensky M, Barak Z, Chipman DM, Merchuk JC. Determination of the dissociation constant of valine from acetohydroxy acid synthase by equilibrium partition in an aqueous two-phase system. J Chromatogr B Biomed Sci Appl. 2000;743:225–9.10.1016/S0378-4347(00)00050-5Search in Google Scholar

[22] Xu K, Wang Y, Huang Y, Li N, Wen Q. A green deep eutectic solvent-based aqueous two-phase system for protein extracting. Anal Chim Acta. 2015;864:9–20.10.1016/j.aca.2015.01.026Search in Google Scholar PubMed

[23] Saravanan S, Rao JR, Nair BU, Ramasami T. Aqueous two-phase poly (ethylene glycol)-poly (acrylic acid) system for protein partitioning: influence of molecular weight, pH, and temperature. Process Biochem. 2008;43:905–11.10.1016/j.procbio.2008.04.011Search in Google Scholar

[24] Perumalsamy M, Murugesan T. Liquid-liquid equilibrium of aqueous two-phase system (PEG 2000-sodium citrate-water) using potential difference as a key tool. Phys Chem Liq. 2014;52:26–36.10.1080/00319104.2013.795857Search in Google Scholar

[25] De Oliveira RM, Dos Reis Coimbra JS, Minim LA, Da Silva LH, Fontes MP. Liquid-liquid equilibria of biphasic systems composed of sodium citrate + polyethylene(glycol) 1500 or 4000 at different temperatures. J Chem Eng Data. 2008;53:895–9.10.1021/je7004209Search in Google Scholar

[26] Shahriari S, Doozandeh SG, Pazuki G. Partitioning of cephalexin in aqueous two-phase systems containing poly(ethylene glycol) and sodium citrate salt at different temperatures. J Chem Eng Data. 2012;57:256–62.10.1021/je201033fSearch in Google Scholar

[27] Hu M, Zhai Q, Jiang Y, Jin L, Liu Z. Liquid-liquid and liquid-liquid-solid equilibrium in PEG + Cs2SO4 + H2O. J Chem Eng Data. 2004;49:1440–3.10.1021/je0498558Search in Google Scholar

[28] Ramyadevi D, Subathira A, Saravanan S. Use of response surface methodology to evaluate the extraction of protein from shrimp waste by aqueous two-phase system (polyethylene glycol and ammonium citrate). J Environ Res Develop. 2012;6:4.Search in Google Scholar

[29] Kammoun R, Chouayekh H, Abid H, Naili B, Bejar S. Purification of CBS 819.72 α-amylase by aqueous two-phase systems: modelling using response surface methodology. Biochem Eng J. 2009;46:306–12.10.1016/j.bej.2009.06.003Search in Google Scholar

[30] Ozer A, Gurbuz G, Calimli A, Korbahti BK. Biosorption of copper (II) ions on Enteromorpha prolifera: application of response surface methodology (RSM). Chem Eng J. 2009;146:377–87.10.1016/j.cej.2008.06.041Search in Google Scholar

[31] Sharma S, Malik A, Satya S. Application of response surface methodology (RSM) for optimization of nutrient supplementation for Cr (VI) removal by Aspergillus lentulus AML05. J Hazard Mater. 2009;164:1198–204.10.1016/j.jhazmat.2008.09.030Search in Google Scholar PubMed

[32] Abdelhafez AA, Husseiny SM, Abdel-Aziz Ali A, Sanad HM. Optimization of β-carotene production from agro-industrial by-products by Serratia marcescens ATCC 27117 using Plackett–Burman design and central composite design. Ann Agric Sci. 2016;61:87–96.10.1016/j.aoas.2016.01.005Search in Google Scholar

[33] Santos JH, Carretero G, Coutinho JA, Rangel-Yagui CO, Ventura SP. Multistep purification of cytochrome c PEGylated forms using polymer-based aqueous biphasic systems. Green Chem. 2017;19:5800–8.10.1039/C7GC02600ESearch in Google Scholar

[34] Patil G, Raghavarao KS. Aqueous two phase extraction for purification of C-phycocyanin. Biochem Eng J. 2007;34:156–64.10.1016/j.bej.2006.11.026Search in Google Scholar

Supplementary Material

The online version of this article offers supplementary material (DOI:

Received: 2019-02-25
Revised: 2019-07-26
Accepted: 2019-09-09
Published Online: 2019-09-26

© 2019 Walter de Gruyter GmbH, Berlin/Boston

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