Optimization of protein extraction from detoxified Jatropha seed cake using response surface methodology and amino acid analysis

  • S. AhluwaliaEmail author
  • R. Bidlan
  • A. K. Shrivastav
  • R. K. Goswami
  • P. Singh
  • J. G. Sharma
Original Paper


Jatropha seed cake is a by-product of biodiesel industry which is rich in proteins and carbohydrates and contains many bioactive compounds. Besides having a good protein quality, the essential amino acid content (except lysine) is also high in Jatropha seed cake, and therefore, Jatropha proteins can be exploited for many technical applications. The major hindrance for its use as a protein supplement is the presence of anti-nutritional factors and toxins, especially the phorbol esters. The detoxification of Jatropha seed cake for the removal of phorbol esters has been shown in our earlier studies. Proteins were extracted from the detoxified Jatropha seed cake and analyzed for amino acid content. Various parameters such as temperature, solubilization pH, precipitation pH and extraction time were studied for their effect on protein content, and the process was optimized using response surface methodology among 5 best samples selected based on protein content and protein yield from dry concentrates. The amount of dry matter, protein content and protein yield of 17.1%, 41.98% and 38.66%, respectively, was obtained under the optimized conditions of 60 °C temperature, solubilization pH 11.0, precipitation pH 4.41 and extraction time of 0.78 h. Amino acid contents revealed that the extracted samples contained a higher concentration of essential amino acids, especially leucine (4.40 ± 0.98 g/100 g). The results indicated that the protein extraction procedure from Jatropha seed cake affected the amino acid content, yet the values were close to FAO/WHO reference protein, and thus, the Jatropha proteins can be explored further for their use as feed supplement.


Jatropha proteins Isoelectric precipitation Amino acids Response surface methodology Optimization process 



The first two authors acknowledge DTU and UGC for providing research fellowships.


  1. AACC (2000) Approved methods of American Association of Cereal Chemists. American Association of Cereal Chemists Inc., St. PaulGoogle Scholar
  2. Achten WMJ, Almeida J, Fobelets V, Bolle E, Mathijs E, Singh VP, Tewari DN, Verchot LV, Muys B (2010) Life cycle assessment of Jatropha biodiesel as transportation fuel in rural India. Appl Energy 87(12):3652–3660CrossRefGoogle Scholar
  3. Ahluwalia S, Sharma JG, Singh P (2017) Degradation of phorbol esters In Jatropha seed cake by Pseudomonas aeruginosa DS1. Int J Pharm Bio Sci 8(2):542–546CrossRefGoogle Scholar
  4. Ahluwalia S, Sharma JG, Singh P (2018) Modeling and Analysis of critical factors for phorbol ester degradation in Jatropha seed cake using ISM and MICMAC. Int J Environ Waste Manag 21(4):287–305CrossRefGoogle Scholar
  5. Aleksandra G, Eduardo M, Angel C, Radosław G, Filip B, Avtar SM (2018) Enhanced protein extraction from oilseed cakes using glycerol-choline chloride deep eutectic solvents: a biorefinery approach. ACS Sustain Chem Eng 6(11):15791–15800. CrossRefGoogle Scholar
  6. Beuk JF, Chornock FW, Rice EE (1948) The effect of severe heat treatment upon the amino acids of fresh and cured pork. J Biol Chem 175:291–298Google Scholar
  7. Calull M, Fabregas J, Marce RM, Borrull F (1991) Determination of free amino acids by precolumn derivatization with phenylisothiocyanate. Application to wine samples. Chromatographia 31:272–276CrossRefGoogle Scholar
  8. Chen J, Huang PT, Zhang KY, Ding FR (2012) Isolation of biosurfactant producers, optimization and properties of biosurfactant produced by Acinetobacter sp. from petroleum-contaminated soil. J Appl Microbiol 112:660–671CrossRefGoogle Scholar
  9. Chenyan LV, Xiaoling J, Meiliang L, Jingyun Y, Guanghua Z (2011) Optimization of extraction process of crude protein from grape seeds by RSM. Food Sci Technol Res 17(5):437–445CrossRefGoogle Scholar
  10. Csapo J, Csapo ZK, Albert C, Loki K (2008) Hydrolysis of proteins performed at high temperatures and for short times with reduced racemization, in order to determine the enantiomers of D- and L-amino acids. Acta Univ Sapientiae Aliment 1:31–48Google Scholar
  11. Devappa RK, Swamylingappa B (2008) Biochemical and nutritional evaluation of Jatropha protein isolate prepared by steam injection heating for reduction of toxic and antinutritional factors. J Sci Food Agric 88:911–919CrossRefGoogle Scholar
  12. Gabriele G, Markus S, Andreas S (2015) Characterization of Jatropha curcas L. Protein cast films with respect to packaging relevant properties. Int J Political Sci 2015:1–9. Google Scholar
  13. Ghosh A, Chikara J, Chaudhary DR (2012) Value addition of Jatropha cake and its utilisation as manure in Jatropha and other crops. In: Carels N, Sujatha M, Bahadar B (eds) Jatropha, challenges for a new energy crop. Farming, economics and biofuel, vol 1. Springer, New YorkGoogle Scholar
  14. Gisele LAM, Roseli SL, Hulda NCM, Patricia AC, Ana MQBB, Catarina AG, Paulo JAS (2014) Effect of alkaline agent and pH on the composition of freeze-dried proteins extracted from castor bean (Ricinus communis L.) cake. Chem Eng Trans 37:697–702Google Scholar
  15. Gofferje G, Klingele S, Stabler A, Schweiggert-Weisz U, Floter E (2014) Comparison of two protein extraction techniques utilizing aqueous de-oiled residue from Jatropha curcas L. Waste Biomass Valor 5:33–41CrossRefGoogle Scholar
  16. Hamarneh AI, Heeres HJ, Broekhuis AA, Picchioni F (2010) Extraction of Jatropha curcas proteins and application in polyketone-based wood adhesives. Int J Adhes Adhes 30:615–625CrossRefGoogle Scholar
  17. Jakub E, Petr S, Magdalena DV, Lukas PA, Petr S, Jindrich K, Zdenek HD, Martin B, Eliska K (2016) Jatropha seed cake and organic waste compost: the potential for improvement of soil fertility. Ecol Chem Eng S 23(1):131–141Google Scholar
  18. Jimenez ME, Ruiz J, Perez Palacios T, Silva A, Antequera T (2012) Gas chromatography-mass spectrometry method for the determination of free amino acids as their dimethyl-tert-butylsilyl (TBDMS) derivatives in animal source food. J Agric Food Chem 60:2456–2463CrossRefGoogle Scholar
  19. Joint FAO/WHO/UNU expert consultation: Protein and amino acid requirements in human nutrition: report, World Health Organization, 1985 (WHO technical report series, no. 935)Google Scholar
  20. Kain RJ, Chen Z, Sonda TS, Abu-Kpawoh JC (2009) Study on the effects of enzymatic hydrolysis on the physical, functional and chemical properties of peanut protein isolates extracted from defatted heat pressed peanut meal flour (Arachis hypogaea L.). Pak J Nutr 8(6):818–825CrossRefGoogle Scholar
  21. Krumpochova P, Bruyneel B, Molenaar D, Koukou A, Wuhrer M, Niessen WM, Giera M (2015) Amino acid analysis using chromatography-mass spectrometry: an inter platform comparison study. J Pharm Biomed Anal 114:398–407. CrossRefGoogle Scholar
  22. Lars IN, Pia V, Jens OD (2004) Quantification of organic and amino acids in beer by 1H NMR spectroscopy. Anal Chem 76(16):4790–4798CrossRefGoogle Scholar
  23. Lestari D, Mulder W, Sanders J (2010) Improving Jatropha curcas seed protein recovery by using counter current multistage extraction. Biochem Eng J 50(1–2):16–23CrossRefGoogle Scholar
  24. Lestari D, Mulder WJ, Sanders JPM (2011) Jatropha seed protein functional properties for technical applications. Biochem Eng J 53(3):297–304CrossRefGoogle Scholar
  25. Liliana LL, Gloria DO, Cristian JM, Humberto HS (2013) Sequentially integrated optimization of the conditions to obtain a high-protein and low-antinutritional factors protein isolate from edible Jatropha curcas seed cake. ISRN Biotechnol 2013:1–7Google Scholar
  26. Line RN, Marianne NL, Michael JD, Jacob HN, Soren BN (2018) Effect of free cysteine on the denaturation and aggregation of holo α-lactalbumin. Int Dairy J 79:52–61CrossRefGoogle Scholar
  27. Makkar HPS, Becker K (2009) Jatropha curcas, a promising crop for the generation of biodiesel and value-added coproducts. Eur J Lipid Sci Technol 111(8):773–787CrossRefGoogle Scholar
  28. Makkar HPS, Francis G, Becker K (2008) Protein concentrate from Jatropha curcas screw-pressed seed cake and toxic and antinutritional factors in protein concentrate. J Sci Food Agric 88:1542–1548CrossRefGoogle Scholar
  29. Manyuchi MM, Mbohwa C, Muzenda E (2018) Bio stabilization of Jatropha Curcas Cake to bio fertilizers through vermicomposting. In: Proceedings of the international conference on industrial engineering and operations management, Paris, France, 26–27 July 2018, pp 9–13Google Scholar
  30. Martin AMM, Israel LM, Samuel RM (2010) Optimization of protein concentrate preparation from Bambara bean using Response Surface Methodology. J Food Process Eng 33:398–412Google Scholar
  31. Mati M, Staruch L, Soral M (2015) Use of NMR spectroscopy in the analysis of carnosine and free amino acids in fermented sausages during ripening. Chem Pap 69(10):1319–1324. CrossRefGoogle Scholar
  32. Monia SA, Siluana KTS, Gabriela R, Claudia BB, Marcel P, Luciano VG, de Daniel BF et al (2017) Free amino acid determination by GC-MS combined with a chemometric approach for geographical classification of bracatinga honeydew honey (Mimosa scabrella Bentham). Food Control 78:383–392CrossRefGoogle Scholar
  33. Oliyaei N, Ghorbani M, Moosavi-Nasab M, Sadeghimahoonak AR, Maghsoudloo Y (2017) Effect of temperature and alkaline pH on the physicochemical properties of the protein isolates extracted from the whole ungutted Lanternfish (Benthosema pterotum). J Aquat Food Prod Technol 26(10):1134–1143. CrossRefGoogle Scholar
  34. Qiaoyun CUI, Xinghong NI, Liang Z, Zheng TU, Jin L, Kang S, Xuan C, Xinghui LI (2017) Optimization of protein extraction and decoloration conditions for Tea residues. Horticul Plant J 3(4):172–176CrossRefGoogle Scholar
  35. Rhee KC, Cater CM, Mattil KF (1972) Simultaneous recovery of protein and oil from raw peanuts in an aqueous system. J Food Sci 37:90–93CrossRefGoogle Scholar
  36. Saha J, Chakraborty S, Deka SC (2016) A comparative study between Response Surface Methodology and genetic algorithm in optimization and extraction of leaf protein concentrate from Diplazium esculentum of Assam. Int J Biotechnol Wellness Ind 5:111–120CrossRefGoogle Scholar
  37. Selling GW, Hojilla-Evangelista MP, Evangelista R, Isbell T, Price N, Doll KM (2013) Extraction of proteins from pennycress seeds and press cake. Ind Crop Prod 41:113–119CrossRefGoogle Scholar
  38. Shaviklo AR, Rezapanah S, Motamedzadegan A, DamavandiKamali N, Mozafari H (2017) Optimum conditions for protein extraction from tuna processing by-products using isoelectric solubilization and precipitation processes. Iran J Fish Sci 16(2):774–792Google Scholar
  39. Sugiki T, Kobayashi N, Fujiwara T (2017) Modern technologies of solution nuclear magnetic resonance spectroscopy for three-dimensional structure determination of proteins open avenues for life scientists. Comput Struct Biotechnol J 15:328–339. CrossRefGoogle Scholar
  40. Tateda N, Matsuhisa K, Hasebe K, Kitajima N, Miura T (1998) High performance liquid chromatographic method for rapid and highly sensitive determination of histidine using postcolumn fluorescence detection with o-phthaldialdehyde. J Chromatogr B 718:235–241CrossRefGoogle Scholar
  41. Ugbogu AE, Akubugwo EI, Uhegbu FO, Chinyere CG, Ugbogu OC, Oduse KA (2014) Quality assessment profile of Jatropha curcas (L) seed oil from Nigeria. Int Food Res J 21(2):735–741Google Scholar
  42. Vilches AP, Norstrom SH, Bylund D (2017) Direct analysis of free amino acids by mixed-mode chromatography with tandem mass spectrometry. J Sep Sci 40(7):1482–1492. CrossRefGoogle Scholar
  43. Wu H, Wang Q, Ma T, Ren J (2009) Comparative studies on the functional properties of various proteins concentrate preparations of peanut protein. J Food Res Int 42:343–348CrossRefGoogle Scholar
  44. Yinuo Z, Yubao W, Haifeng W, Yueming W, Harinder PM, Jianxin L (2018) Nutritional value of detoxified Jatropha curcas seed cake protein isolates using rats as an animal model. Anim Nutr 4:429–434CrossRefGoogle Scholar
  45. You Shin S, Won-Jin Y, Jaeho H, Dongwon S, Kwang-Won L, Woo-Young L, Kwang-Il K et al (2013) Method validation of 16 types of structural amino acids using an automated amino acid analyzer. Food Sci Biotechnol 22(6):1567–1571CrossRefGoogle Scholar
  46. Yu J, Aahmedna M, Goktepe I (2007) Peanut proteins concentrate: production and functional properties as affected by processing. Food Chem 103:121–129CrossRefGoogle Scholar

Copyright information

© Islamic Azad University (IAU) 2019

Authors and Affiliations

  • S. Ahluwalia
    • 1
    Email author
  • R. Bidlan
    • 1
  • A. K. Shrivastav
    • 1
  • R. K. Goswami
    • 2
  • P. Singh
    • 3
  • J. G. Sharma
    • 1
  1. 1.Department of BiotechnologyDelhi Technological UniversityDelhiIndia
  2. 2.Department of ZoologyUniversity of DelhiDelhiIndia
  3. 3.Department of Mechanical EngineeringDelhi Technological UniversityDelhiIndia

Personalised recommendations