Enzymatic synthesis of plant protein concentrates and their functional properties

specialists when developing formulations of protein-containing foods (meat, confectionery, etc.). In this study, optimal/rational parameters of biosynthesis of new pea, sunflower and wheat concentrates, with tyrosinase and microbial transglutaminase enzymes, belonging, respectively, to the class of oxidoreductases and transferases, have been developed in order to modify the functional properties of commercial protein preparations. At the same time, patterns of the effect of enzyme concentration, reaction duration, and the hydromodule on the mass fraction of amino nitrogen and soluble protein in the medium as effective indicators for monitoring the course of the reaction in the medium have been established. Compared to the control sample, the new protein concentrate obtained from dry wheat gluten, synthesized with the sequential addition of both types of enzymes has a 44% higher water-binding capacity and almost 2 times higher fat-binding capacity. The pea and sunflower concentrates prepared with separate addition of enzymes have a 24–56% higher water-binding capacity, 2.3–2.4 times higher foaming capacity and 1.6–5.7 times higher foam stability. The sunflower concentrate has increased fat-binding and fat-emulsifying capacity compared to the original concentrate. The results of the effect of tyrosinase and microbial transglutaminase on the functional properties of protein concentrates, despite their different principle of action, were practically the same. Therefore, to modify the properties, it is advisable to use also the enzyme tyrosinase instead of transglutaminase for the production of food products, taking into account the identified parameters of transglutaminase action on pea, sunflower proteins and wheat gluten.

1. Goncharova, N., Merzlyakova, N. (2021). Food shortages and hunger as a global problem. Food Science and Technology, 42(2), Article 70621. https://doi.org/10.1590/Fst.70621

2. Nechaev, A. P., Kochetkova, A. A., Kolpakova, V. V., Traubenberg, S. E., Vitol, I. S., Kobeleva, I. B. et al. (2024). Food chemistry. St. Petersburg: GIORD, 2024. (In Russian)]

3. Williams, R. A. (2021). Opportunities and сhallenges for the introduction of new food proteins. Annual Review of Food Science and Technology, 12, 75–91. https://doi.org/10.1146/annurev-food‑061220-012838

4. Kołodziejczak, K., Onopiuk, A., Szpicer, A., Półtorak, A. (2021). Meat analogues in the perspective of recent scientific research: A review. Foods, 11(1), Article 105. https://doi.org/10.3390/foods11010105

5. Heertje, I. (2014). Structure and function of food products: A review. Food Structure, 1(1), 3–23. https://doi.org/10.1016/j.foostr.2013.06.001

6. Kolpakova, V. V., Byzov, V. A. (2024). Functional characteristics and molecular structural modification of plant proteins. Review. Food Systems,7(3), 324–335. (In Russian)] https://doi.org/10.21323/2618-9771-2024-7-3-324-335

7. WHO/FAO. (2018). Driving commitment for nutrition within the UN Decade of Action on Nutrition: Policy brief. World Health Organization and Food and Agriculture Organization of the United Nations, 2018.

8. Lisitsyn, A. B., Zakharov, A. N., Iskakov, M. Kh., Aliev, M. S. (2014). Current state of the Russian market of soy and soy proteins. Vsyo o Myase, 4, 20–23. (In Russian)]

9. Нечаев, А.П., Дубцова, Г.Н., Колпакова, В.В. (1995). Белки пшеницы. Технология получения и применения (состояние, проблемы, пути развития). Известия высших учебных заведений. Пищевая технология, 1–2(224–225), 28–30. [Nechaev, A. P., Dubtsova, G. N., Kolpakova, V. V. (1995). Wheat proteins. Production technology and application (state, problems, ways of development). Izvestiya Vuzov. Food Technology, 1–2(224–225), 28–30. (In Russian)]

10. Day, L. (2011). Wheat gluten: Production, properties and application. Chapter in a book: Handbook of Food Proteins. Sawston, Cambridge: Woodhead Publishing, 2011. http://doi.org/10.1533/9780857093639.267

11. Kolpakova, V. V., Lukin, N. D., Gaivoronskaya, I. S. (2018). Interrelation of functional properties of protein products from wheat with the composition and physicochemical characteristics of their proteins. Chapter in a book: Global Wheat Production. London: IntechOpen, 2018. http://doi.org/10.5772/intechopen.75803

12. Kolpakova, V. V., Kulikov, D. S., Ulanova, R. V., Chumikina, L. V. (2021). Food and feed protein preparations from peas and chickpeas: Production, properties, application. Food Processing: Techniques and Technology, 51(2), 333–348. (In Russian)] http://doi.org/10.21603/2074-9414-2021-2-333-348

13. Gao, Z., Shen, P., Lan, Y., Cui, L., Ohm, J.-B, Chen, B, Rao, J. (2020). Effect of alkaline extraction pH on structure properties, solubility, and beany flavor of yellow pea protein isolate. Food Research International, 131(4), Article 09045. http://doi.org/10.1016/j.foodres.2020.109045

14. Shen, Y., Hong, S., Li, Y. (2022). Pea protein composition, functionality, modification, and food applications: A review. Advances in Food and Nutrition Research, 101, 71–127. http://doi.org/10.1016/bs.afnr.2022.02.002

15. Patil, N. (2023). Chickpea protein: A comprehensive review on nutritional properties, processing, functionality, applications, and sustainable impact. The Pharma Innovation Journal, 12(7), 3424–3434.

16. Funke, M., Loeffler, M., Winkelmeyer, C., Krayer, M., Boom, R., Weiss, J. (2022). Emulsifying properties of lentil protein preparations obtained by dry fractionation. European Food Research and Technology, 248, 381–391. https://doi.org/10.1007/s00217-021-03883‑y

17. Vogelsang-O’Dwyer, M., Petersen, I. L., Joehnke, M.S, Sørensen, J. C., Bez, J., Detzel, A. et al. (2020). Comparison of faba bean protein ingredients produced using dry fractionation and isoelectric precipitation: Techno-functional, nutritional and environmental performance. Foods, 9(3), Article 322. https://doi.org/10.3390/foods9030322

18. Kolpakova, V. V., Fan, C. K., Gaivoronskaya, I. S., Chumikina, L. V. (2023). Properties and structural features of proteins of native and modified concentrates from white and brown rice. Food systems, 6(3), 317–328. (In Russian)] https://doi.org/10.21323/2618-9771-2023-6-3-317-328

19. Östbring, K., Malmqvist, E., Nilsson, K., Rosenlind, I., Rayner, M. (2020). The effects of oil extraction methods on recovery yield and emulsifying properties of proteins from rapeseed meal and press cake. Foods, 9(1), Article 19. https://doi.org/10.3390/foods9010019

20. Dabbour, M., He, R., Ma, H., Musa, A. (2018). Optimization of ultrasound assisted extraction of protein from sunflower meal and its physicochemical and functional properties. Journal of Food Process Engineering, 41(5), Article e12799. https://doi.org/10.1111/jfpe.12799

21. Mondor, M., Hernández-Álvarez, A. J. (2022). Processing technologies to produce plant protein concentrates and isolates. Chapter in a book: Plant Protein Foods. London-Berlin: Springer Nature, 2022. https://doi.org/10.1007/978-3-030-91206-2_3

22. Joshi, V.K., Kumar, S. (2015). Meat analogues: Plant based alternatives to meat products — A review. International Journal of Food and Fermentation Technology, 5(2), 107–119. https://doi.org/10.5958/2277-9396.2016.00001.5

23. Kumar, P., Sharma, B., Kumar, R.R., Kumar, A. (2012). Optimization of the level of wheat gluten in analogue meat nuggets. Indian Journal of Veterinary Research, 21(1), 54–59.

24. Onwezen, M. C., Bouwman, E. P., Reinders, M. J., Dagevos, H. (2021). A systematic review on consumer acceptance of alternative proteins: Pulses, algae, insects, plant-based meat alternatives, and cultured meat. Appetite, 159, Article 105058. https://doi.org/10.1016/J.Appet.2020.105058

25. Xia, W., Botma, T.E., Sagis, L.M.C., Yang, J. (2022). Selective proteolysis of β-conglycinin as a tool to increase air-water interface and foam stabilising properties of soy proteins. Food Hydrocolloids, 130(3), Article 107726. https://doi.org/10.1016/j.foodhyd.2022.107726

26. Yolandani, Y., Ma, H., Li, Y., Liu, D., Zhou, H., Liu, X. et al. (2023). Ultrasoundassisted limited enzymatic hydrolysis of high concentrated soy protein isolate: Alterations on the functional properties and its relation with hydrophobicity and molecular weight. Ultrasonics Sonochemistry, 95, Article 106414. https://doi.org/10.1016/j.ultsonch.2023.106414

27. Di, A., Li, L. (2020). The effect of limited proteolysis by trypsin on the formation of soy protein isolate nanofibrils. Journal of Chemistry, 4–5, Article 185037. https://doi.org/10.1155/2020/8185037

28. Kolpakova, V. V., Chumikina, L. V., Arabova, L. I., Lukin, D. N, Topunov, А. F, Тitov, Е. I. (2016). Functional technological properties and electrophoretic composition of modified wheat gluten. Foods and Raw Materials, 4(2), 48–57. https://doi.org/10.21179/2308-4057-2016-2-48-57

29. Pourmohammadi, K., Elahe, A. (2021). Hydrolytic enzymes and their directly and indirectly effects on gluten and dough properties: An extensive review. Food Science and Nutrition, 9(12), 3988–4006. https://doi.org/10.1002/fsn3.2344

30. Kolpakova, V. V., Chumikina, L. V., Vasil’ev, A. V., Arabova, L. I., Topunov, A. F. (2014). Wheat gluten proteolysis by enzyme preparations of directional action. International Journal of Agronomy and Agricultural Research, 5(2), 72–86.

31. Merz, M., Kettner, L., Langolf, E., Appel, D., Blank, I., Stressler, T. et al. (2016). Production of wheat gluten hydrolysates with reduced antigenicity employing enzymatic hydrolysis combined with downstream unit operations. Journal of the Science of Food and Agriculture, 96(10), 3358–3364. https://doi.org/10.1002/jsfa.7515

32. Mika, M., Wikiera, A. (2024). Enzymatic hydrolysis as an effective method for obtaining wheat gluten hydrolysates combining beneficial functional properties with health-promoting potential. Molecules, 29(18), Article 4407. https://doi.org/10.3390/molecules29184407

33. Kieliszek, M., Misiewicz, A. (2014). Microbial transglutaminase and its application in the food industry. A review. Folia Microbiologica, 59(3), 241–250. https://doi.org/10.1007/s12223-013-0287‑x

34. Akbari, M., Razavi, S.H., Kieliszek, M. (2021). Recent advances in microbial transglutaminase biosynthesis and its application in the food industry. Trends in Food Science and Technology, 110, 458–469. https://doi.org/10.1016/j.tifs.2021.02.036

35. Kolotylo, V., Piwowarek, K., Kieliszek, M. (2023). Microbiological transglutaminase: Biotechnological application in the food industry. Open Life Science, 18(1), Article 20220737. https://doi.org/10.1515/biol‑2022-0737

36. Sorapukdee, S., Tangwatcharin, P. (2018). Quality of steak restructured from beef trimmings containing microbial transglutaminase and impacted by freezing and grading by fat level. Asian-­Australasian Journal of Animal Sciences, 31(1), 129–137. https://doi.org/10.5713/ajas.17.0170

37. Lesiow, T., Rentfrow, G. K., Xiong, Y. L. (2017). Polyphosphate and myofibrillar protein extract promote transglutaminase-mediated enhancements of rheological and textural properties of PSE pork meat batters. Meat Science, 128, 40–46. https://doi.org/10.1016/j.meatsci.2017.02.002

38. Santhi, D., Kalaikannan, A., Malairaj, P., Prubhu, A, (2017). Application of microbial transglutaminase in meat foods: A review. Critical Reviews in Food Science and Nutrition, 57(10), 2071–2076. https://doi.org/10.1080/10408398.2014.945990

39. Carballo, J., Ayo, J., Colmenero, F.J. (2006). Microbial transglutaminase and caseinate as cold set binders: Influence of meat species and chilling storage. LWT — Food Science and Technology, 39(6), 692–699. https://doi.org/10.1016/j.lwt.2005.03.020

40. Abou-Soliman, N. H. I., Sakr, S. S., Awad, S. (2017). Physico-chemical, microstructural and rheological properties of camel-milk yogurt as enhanced by microbial transglutaminase. Journal of Food Science and Technology, 54(6), 1616–1627. https://doi.org/10.1007/s13197-017-2593-9

41. Gharibzahedi, S. M. T., Koubaa, M., Barba, F. J., Greiner, R., George, S., Roohinejad, S. (2018). Recent advances in the application of microbial transglutaminase cross linking in cheese and ice cream products: A review. International Journal of Biological Macromolecules, 107(Pt B), 2364–2374. https://doi.org/10.1016/j.ijbiomac.2017.10.115

42. D’Alessandro, A. G., Martemucci, G., Faccia, M. (2021). Effects of microbial transglutaminase levels on donkey cheese production. Journal of Dairy Research, 88(3), 351–356. https://doi.org/10.1017/S0022029921000601

43. Darnay, L., Miklós, G., Lőrincz, A., Szakmár, K., Pásztor-Huszár, K., Laczay, P. (2022). Possible inhibitory effect of microbial transglutaminase on the formation of biogenic amines during Trappist cheese ripening. Food Additives and Contaminans: Part A, 39(3), 580–587. https://doi.org/10.1080/19440049.2021.2005831

44. Palmeira, K. R., Rodrigues, B. L., Gaze, L. V., Freitas, M. Q., Teixeira, C. E., Marsico, E. T. et al. (2014). Use of transglutaminase, soybean waste and salt replacement in the elaboration of trout (Oncorhynchus mykiss) meatball. International Food Research Journal, 21(4), 1597–1602.

45. Yuan, F., Lv, L., Li, Z., Mi, N., Chen, H., Lin, H. (2017). Effect of transglutaminasecatalyzed glycosylation on the allergenicity and conformational structure of shrimp (Metapenaeus ensis) tropomyosin. Food Chemistry, 219, 215–222. https://doi.org/10.1016/j.foodchem.2016.09.139

46. Bellido, G., Hatcher, D. W. (2011). Effects of a cross-linking enzyme on the protein composition, mechanical properties, and microstructure of Chinese-style noodles. Food Chemistry, 125(3), 813–822. https://doi.org/10.1016/j.foodchem.2010.08.008

47. Niu, M., Hou, G. G., Kindelspire, J., Krishnan, P., Zhao, S. (2017). Microstructural, textural, and sensory properties of whole-wheat noodle modified by enzymes and emulsifiers. Food Chemistry, 223, 16–24. https://doi.org/10.1016/j.foodchem.2016.12.021

48. Ogilvie, O., Roberts, S., Sutton, K., Larsen, N., Gerrard, J., Domigan, L. (2020). The use of microbial transglutaminase in a bread system: A study of gluten protein structure, deamidation state and protein digestion. Food Chemistry, 340. Article 127903. https://doi.org/10.1016/j.foodchem.2020.127903

49. Escamilla-García, M., Ríos-Romo, R. A., Melgarejo-Mancilla, A., Díaz-Ramírez, M., Hernández-Hernández, H. M., Amaro-Reyes, A. et al. (2020). Rheological and antimicrobial properties of chitosan and quinoa protein filmogenic suspensions with thyme and rosemary essential oils. Foods, 9(11), Article 1616. https://doi.org/10.3390/foods9111616

50. Uresti, R.M., Téllez-Luis, S.J., Ramírez, J.A., Vázquez, M. (2004). Use of dairy products and microbial transglutaminase to obtain low-salt fish products from filleting waste from silver carp (Hypophtalmichtis molitrix). Food Chemistry, 86(2), 257–262. https://doi.org/10.1016/j.foodchem.2003.09.033

51. Martínez, B., Miranda, J.M., Franco, C.M., Cepeda, A., Vázquez, M. (2011). Evaluation of transglutaminase and caseinate for a novel formulation of beef patties enriched in healthier lipid and dietary fiber. LWT — Food Science and Technology, 44(4), 949–956. https://doi.org/10.1016/j.lwt.2010.11.026

52. Giosafatto, C. V. L., Al-Asmar, A., Mariniello, L. (2018). Transglutaminase protein substrates of food interest. Chapter in a book: Enzymes in food technology. Singapore: Springer, 2018. https://doi.org/10.1007/978-981-13-1933-4_15

53. Jaros, D., Heidig, C., Rohm, H. (2007). Enzymatic modification through microbial transglutaminase enhances the viscosity of stirred yogurt. Journal of Texture Studies, 38(2), 179–198. https://doi.org/10.1111/j.1745-4603.2007.00093.x

54. Fenoll, L. G., Rodríguez-López, J. N., García-Sevilla, F., García-Ruiz, P. A., Varón, R., García-Cánovas, F. et al. (2001). Analysis and interpretation of the action mechanism of mushroom tyrosinase on monophenols and diphenols generating highly unstable o-quinones. Biochimica et Biophysica Acta (BBA) — Protein Structure and Mo‑ lecular Enzymology, 1548(1), 1–22. https://doi.org/10.1016/s0167-4838(01)00207-2

55. Ito, S., Sugumaran, M., Wakamatsu, K. (2020). Chemical reactivities of orthoquinones produced in living organisms: Fate of quinonoid products formed by tyrosinase and phenoloxidase action on phenols and catechols. International Journal of Molecular Sciences, 21(17), Article 6080. https://doi.org/10.3390/ijms21176080

56. Kumar, C. M., Sathisha, U.V., Dharmesh, S., Rao, A.G.A., Singh, S.A. (2011). Interaction of sesamol (3,4‑methylenedioxyphenol) with tyrosinase and its effect on melanin synthesis. Biochimie, 93(3), 562–569. https://doi.org/10.1016/j.biochi.2010.11.014

57. Arias, S., Amini, S., Horsch, J., Pretzler, M., Rompel, A., Melnyk, I. et al. (2020). Toward artificial mussel-glue proteins: Differentiating sequence modules for adhesion and switchable cohesion. Angewandte Chemie, International Edition, 59(42), 18495–18499. https://doi.org/10.1002/anie.202008515

58. Krüger, J. M., Börner, H. G. (2021). Accessing the next Generation of synthetic mussel-glue polymers via mussel-inspired polymerization. Angewandte Chemie, International Edition, 60(12), 6408–6413. https://doi.org/10.1002/anie.202015833

59. Fernandes, M. S., Kerkar, S. (2017). Microorganisms as a source of tyrosinase inhibitors: A review. Annals of Microbiology, 67, 343–358. https://doi.org/10.1007/s13213-017-1261-7

60. Virginia, B.A., Apetrei, C. (2023). Tyrosinase immobilization strategies for the development of electrochemical biosensors — A review. Nanomaterials, 13(4), Article 760. https://doi.org/10.3390/nano13040760

61. Semenov, G. V., Krasnova, I. S. (2021). Freeze-drying. Moscow: DeLi, 2021. (In Russian)]

62. Vanin, S. V., Kolpakova, V. V. (2007). Functional properties of dry wheat gluten of different quality. Izvestiya Vuzov. Food Technology, 1(296), 21–24. (In Russian)]

63. Kolpakova, V. V., Krikunova, L. N., Kononenko, V. V. (2001). Study of the possibility of producing protein preparations from differentiated fractions of rye and barley grain. Izvestiya Vuzov. Food Technology, 5–6(264–265), 35–39. (In Russian)]

64. Andreev, N. R., Kolpakova, V. V., Goldstein, V. G. (2018). To the question of profound triticale grain processing. Food Industry, 9, 30–33. (In Russian)]

65. Buchert, J., Ercili-Cura, D.E., Ma, H., Gasparetti, C., Monogioudi, E., Faccioet, G. al. (2010). Crosslinking food proteins for improved functionality. Annual Review of Food Science and Technology, 1(1), 113–138. https://doi.org/10.1146/annurev.food.080708.100841

66. Zimoch-Korzycka, A., Krawczyk, A., Król-Kilińska, Ż., Kulig, D., Bobak, Ł., Jarmoluk, A. (2024). Influence of мicrobial transglutaminase on the formation of physico-chemical properties of meat analogs. Foods, 13(24), Article 4085. https://doi.org/10.3390/foods13244085

67. Zo, S. M., Sood, A., Won, S. Y., Choi, S. M, Han, S. S. (2025). Structuring the future of cultured meat: Hybrid gel-based caffolds for edibility and functionality. Gels, 11(8), Article 610. https://doi.org/10.3390/gels11080610

68. Baugreet, S., Kerry, J. P., Brodkorb, A., Gomez, C., Auty, M., Allen P. et al. (2018). Optimisation of plant protein and transglutaminase content in novel beef restructured steaks for older adults by central composite design. Meat Science, 142, 65–77. https://doi.org/10.1016/j.meatsci.2018.03.024

69. Partanen, R., Torkkeli, M., Hellman, M., Permi, P., Serimaa, R., Buchert, J. et al. (2011). Loosening of globular structure under alkaline pH affects accessibility of β-lactoglobulin to tyrosinase-induced oxidation and subsequent cross-linking. Enzyme and Microbial Technology, 49(2), 131–138. https://doi.org/10.1016/j.enzmictec.2011.04.010

70. Madsen, M., Khan, S., Kunstmann, S., Aachmann, F. L., Ipsen, R., Westh, P. et al. (2022). Unaided efficient transglutaminase cross-linking of whey proteins strongly impacts the formation and structure of protein alginate particles. Food Chemistry: Molecular Sciences, 5, Article 100137. https://doi.org/10.1016/j.fochms.2022.100137

71. Nivala, O., Mäkinen, O.E., Kruus, K., Nordlund, E., Ercili-Cura, D. (2017). Structuring colloidal oat and faba bean protein particles via enzymatic modification. Food Chemistry, 231, 87–95. https://doi.org/10.1016/j.foodchem.2017.03.114

72. Marco, C., Pérez, G., Ribotta, P., Rosell, C.M. (2007). Effect of microbial transglutaminase on the protein fractions of rice, pea and their blends. Journal of the Science of Food and Agriculture, 87(14), 2576–2582. https://doi.org/10.1002/jsfa.3006

73. Tang, C. H., Sun, X., Yin, S. W., Ma, C. Y. (2008). Transglutaminase induced crosslinking of vicilin-rich kidney protein isolate: Influence on the functional properties and in vitro digestibility. Food Research International, 41(10), 941–947. https://doi.org/10.1016/j.foodres.2008.07.015

74. Queirós, R.P., Moreira, N., Pinto, C.A., Fidalgo, L.G., Saraiva, J.A., Lopes da Silva, J.A. (2024). Influence of high-pressure processing and microbial transglutaminase on the properties of pea protein isolates. Macromol, 4(2), 213–226. https://doi.org/10.3390/macromol4020011

75. Glusac, J., Isaschar-Ovdat, S., Fishman, A. (2020). Transglutaminase modifies the physical stability and digestibility of chickpea protein-stabilized oil-in-water emulsions. Food Chemistry, 315, Article 126301. https://doi.org/10.1016/j.foodchem.2020.126301

76. de Barros Soares, L.H., Albuquerque, P.M., Assmann, F., Ayub, M.A.Z. (2004). Physical properties of three food proteins treated with transglutaminase. Ciencia Rural, 34(4), 1219–1223. https://doi.org/10.1590/S0103–8478200400040003