Moscow, Russian Federation
Moscow, Russian Federation
Moscow, Russian Federation
V.M. Gorbatov Federal Research Center for Food Systems of RAS
Russian Federation
Moscow, Russian Federation
preparations are obtained from plant materials by alkaline extraction (pH 6.0–12.0) followed by isoelectric precipitation. This article describes the effect of extraction and isoelectric precipitation parameters on protein yield from hydrolyzed hemp cake. Calculating the theoretical and experimental isoelectric point made it possible to improve the procedure and increase the yield. The study featured hemp cake of the Vera variety. A three-factor Box–Behnken design (15 experimental points) optimized the extraction parameters of pH, temperature, and time. The resulting regression model adequately described the process (R2 = 0.983) with the optimal extraction conditions as pH 11.2; 52°C; 3 h. Sodium chloride reduced extraction efficiency by 6.8–13.5% compared to the alkaline method. The optimal protein purity (92.6–93.1%) was achieved with isoelectric precipitation in the pH range of 4.4–4.6, which did not correspond to the modeled isoelectric point (6.95–7.22). The protein profile analysis of the isolated fraction demonstrated the predominance of edestin (~ 38 kDa) and albumin (~ 21 kDa). The resulting hemp protein isolate contained 89.53 to 92.28% protein, according to the Barnstein and Kjeldahl methods, respectively, which indicated a high-quality product. The use of alkaline extraction followed by protein precipitation in isoelectric point provided efficient protein isolation with a yield reaching 80% relative to its content in the raw material, containing 34.32 ± 3.43% crude protein.
Industrial hemp, protein extraction, isoelectric point, protein isolate, optimization, mathematical model, extraction degree, hemp protein profile, bioinformatics
1. Fang B, Peng Z, Chen B, Rao J. Unconventional sources of vegetable proteins: Technological properties. Current Opinion in Food Science. 2024;57:101150. https://doi.org/10.101 6/j.cofs.2024.101150
2. Hadnađev MS, Hadnađev TRD, Pojić MM, Šarić BM, Mišan AČ, et al. Progress in vegetable proteins isolation techniques: A review. Food and Feed Research. 2017;44(1):11–22. http://dx.doi.org/10.5937/FFR1701011H
3. Fang B, Chang L, Ohm J-B, Chen B, Rao J. Structural, functional properties, and volatile profile of hemp protein isolate as affected by extraction method: Alkaline extraction – isoelectric precipitation vs salt extraction. Food Chemistry. 2023;405:135001. https://doi.org/10.1016/j.foodchem.2022.135001
4. Serkov VA, Kabunina IV. Prospects of seed hemp for the Russian fat and oil industry. International Agricultural Journal. 2023;(2):188–191. (In Russ.) https://elibrary.ru/RJMTYI
5. Rizzo G, Storz MA, Calapai G. The role of hemp (Cannabis sativa L.) as a functional food in vegetarian nutrition. Foods. 2023;12(18):3505. https://doi.org/10.3390/foods12183505
6. Burton RA, Andres M, Cole M, Cowley JM, Augustin MA. Industrial hemp seed: From the field to value-added food ingredients. Journal of Cannabis Research. 2022;4(1):45. https://doi.org/10.1186/s42238-022-00156-7
7. Leonard W, Zhang P, Ying D, Fang Z. Hempseed in food industry: Nutritional value, health benefits, and industrial applications. Comprehensive Reviews in Food Science and Food Safety. 2020;19(1):282–308. https://doi.org/10.1111/1541-4337.12517
8. Banskota AH, Tibbetts SM, Jones A, Stefanova R, Behnke J. Biochemical characterization and in vitro digestibility of protein isolates from hemp (Cannabis sativa L.) by-products for salmonid feed applications. Molecules. 2022;27(15):4794. https://doi.org/10.3390/molecules27154794
9. Tang C-H, Ten Z, Wang X-S, Yang X-Q. Physicochemical and functional properties of hemp (Cannabis sativa L.) protein isolate. Journal of Agricultural and Food Chemistry. 20 06;54(23):8945–8950. https://doi.org/10.1021/jf0619176
10. Liu M, Toth JA, Childs M, Smart LB, Abbaspourrad A. Composition and functional properties of hemp seed protein isolates from various hemp cultivars. Journal of Food Science. 2023;88(3):942–951. https://doi.org/10.1101/2022.06.01.494437
11. Karabulut G, Feng H, Yemiş O. Physicochemical and antioxidant properties of industrial hemp seed protein isolate treated by high-intensity ultrasound. Plant Foods for Human Nutrition. 2022;77(4):577–583. https://doi.org/10.1007/s11130-022-01017-7
12. El-Sohaimy SA, Androsova NV, Toshev AD, El Enshasy HA. Nutritional quality, chemical, and functional characteristics of hemp (Cannabis sativa ssp. sativa) protein isolate. Plants. 2022;11(21):2825. https://doi.org/10.3390/plants11212825
13. Potin F, Lubbers S, Husson F, Saurel R. Hemp (Cannabis sativa L.) protein extraction conditions affect extraction yield and protein quality. Journal of Food Science. 2019;84(12) :3682–3690. https://doi.org/10.1111/1750-3841.14850
14. Aleksanochkin DI, Fomenko IA, Alekseeva EA, Chernukha IM, Mashentseva NG. Production of plant protein from seeds and cake of industrial hemp: Overview of processing methods for food industry. Food System. 2024;7(2):179–188. (In Russ.) https://doi.org/10.21323/2618-9771-2024-7-2-188-197
15. Nguet HTM. Extracting protein substances from soya white petals with aqueous solutions of mineral acids and alkalis. Advances in Chemistry and Chemical Technology. 2007;21(12):33–3 5. (In Russ.) https://elibrary.ru/QZMGNH
16. Mondor M, Hernández-Álvarez AJ. Processing technologies to produce plant protein concentrates and isolates. In: Manickavasagan A, Lim L-T, Ali A, editors. Plant protein foods. Cham: Springer International Publishing; 2022. pp. 61–108. https://doi.org/10.1007/978-3-030-91206-2_3
17. Mohanta TK, Khan A, Hashem A, Abd_Allah EF, Al-Harrasi A. The molecular mass and isoelectric point of plant proteomes. BMC Genomics. 2019;20(1):631. https://doi.org/10.118 6/s12864-019-5983-8
18. Ajibola CF, Aluko RE. Physicochemical and functional properties of 2S, 7S, and 11S enriched hemp seed protein fractions. Molecules. 2022;27(3):1059. https://doi.org/10.3390/molecules27031059
19. Dapčević-Hadnađev T, Dizdar M, Pojić M, Krstonošić V, Zychowski LM, et al. Emulsifying properties of hemp proteins: Effect of isolation technique. Food Hydrocolloids. 2019;89:912–920. https://doi.org/10.1016/j.foodhyd.2018.12.002
20. Ma S, Ye A, Singh H, Acevedo-Fani A. Heat-induced interactions between microfluidized hemp protein particles and caseins or whey proteins. Food Chemistry. 2025;463:141290. https://doi.org/10.1016/j.foodchem.2024.141290
21. Rehman M, Fahad S, Du G, Cheng X, Yang Y, et al. Evaluation of hemp (Cannabis sativa L.) as an industrial crop: A review. Environmental Science and Pollution Research. 2021;28(38):52832–52843. https://doi.org/10.1007/s11356-021-16264-5
22. Li A, Zheng J, Han X, Jiang Z, Yang B, et al. Health implication of lactose intolerance and updates on its dietary management. International Dairy Journal. 2023;140:105608. https://doi.org/10.1016/j.idairyj.2023.105608
23. Isnaini L, Estiasih T, Suseno SH, Lestari LA. The role vegetable proteins to stabilize emulsion: A mini review. IOP Conference Series: Earth and Environmental Science. 2021;924(1):012036. http://dx.doi.org/10.1088/1755-1315/924/1/012036
24. Zhang J. Protein-protein interactions in salt solutions. Protein-protein Interactions-computational and Experimental Tools. 2012;6:359–376. https://doi.org/10.5772/38056
25. Zhang Y, Roman L, Kirkensgaard JJK, Corredig M. Effect of salt extraction on composition, structure, and thermal properties of pea protein. Food Chemistry. 2025;144650. https://doi.org/10.1016/j.foodchem.2025.144650
26. Cabral EM, Poojary MM, Lund MN, Curtin J, Fenelon M, et al. Effect of solvent composition on the extraction of proteins from hemp oil processing stream. Journal of the Science of Food and Agriculture. 2022;102(14):6293–6298. https://doi.org/10.1002/jsfa.11979
27. Kozlowski LP. IPC–isoelectric point calculator. Biology Direct. 2016;11(1):55. https://doi.org/10.1186/s13062-016-0159-9
28. Kozlowski LP. IPC 2.0: Prediction of isoelectric point and pKa dissociation constants. Nucleic Acids Research. 2021;49(W1):W285–W292. https://doi.org/10.1093/nar/gkab295
29. Kaur N, Verma A, Girdhar M, Kumar A, Siddiqui MA, et al. Genome-wide analysis of the Cannabis sativa cytochrome P450 monooxygenase superfamily and uncovering candidate genes for improved herbicide tolerance. Frontiers in Plant Science. 2024;15:1490036. https://doi.org/10.3389/fpls.2024.1490036
30. Kruchinin AG, Bolshakova EI. Hybrid strategy of bioinformatics modeling (in silico): Biologically active peptides of milk protein. Food Processing: Techniques and Technology. 2022;52(1):46–57. https://doi.org/10.21603/2074-9414-2022-1-46-57
31. Potin F, Saurel R. Hemp seed as a source of food proteins. In: Crini G, Lichtfouse E, editors. Sustainable agriculture reviews 42: Hemp production and applications. Cham: Springer International Publishing; 2020. pp. 265–294. https://doi.org/10.1007/978-3-030-41384-2_9
32. Yano H, Fu W. Hemp: A sustainable plant with high industrial value in food processing. Foods. 2023;12(3):651. https://doi.org/10.3390/foods12030651
