Experimental studies of the enzymatic hydrolysis patterns of whey proteins have been carried out. Proteases of different origin (animal, plant and microbial) were selected and optimal hydrolysis parameters were selected.
The studies were performed with serum protein concentrate obtained by ultrafiltration with 80% mass fraction of protein (KSB-80 manufacturer “TechMolProm” (TM “Bios”). Enzyme preparations used: pepsin (LLC “Alsi”), papain PSM-400 (LLC “Alex”), protolade (SE “Enzym”). The degree of hydrolysis of the protein substances of the samples was evaluated by changing the concentration of nitrogen amine groups (NAG).
The patterns of hydrolysis of serum proteins were studied by the following parameters: concentration of enzyme and substrate, pH of the medium, temperature and duration of the process. It was found that NAG in the initial (control) solution of whey protein concentrate with a protein content of 20% is 32,67 mg / 100 g.
The dynamics of pepsin concentrations from (0,5 to 3)% at pH 2.0, papain and protolade – (1-6)% at pH 7.0 were studied. It was found that the optimal concentration of pepsin enzyme for the subsequent studies is 2,5% (NAG content is 56,0 mg / 100 g), papain – 4% (NAG – 67,67 mg / 100 g), Protolad – 5% (NAG – 102,67 mg / 100 g).
Studies of the effect of substrate concentration at a fixed enzyme concentration showed that the maximum accumulation of NAG (53,67 mg / 100 g) occurred in an environment with 25% protein and 2,5% pepsin; maximum NAG value (70 mg / 100 g) with 20% protein and 4% papain; maximum NAG value (102,67 mg / 100 g) with 20% protein and 5% Protolad enzyme.
Studies of changes in the degree of hydrolysis of serum peptide bonds by changes in the pH of the medium for pepsin from 1 to 6, for papain – (3-12), for Protolad – (4-12). It was found that NAG acquires the most value when using pepsin pH is 2.0 (NAG 53,67 mg / 100 g); when using papain or protolate pH 8.0 (NAG 84,0 mg / 100 g and 107,33 mg / 100 g, respectively).
The dependence of the proteolysis intensity of serum protein peptide bonds by the enzymatic preparations on the dynamics of temperature changes (from 20 to 90 °C) was investigated. Studies have shown that in the temperature range (20-50) °C with the participation of pepsin there is a steady increase of NAG from (39,67 to 53,67) mg / 100 g; papain – at (20-60) ° C NAG is from (44,33 to 88,67) mg / 100 g; The protolade – at (20–40) °C the NAG was 93,33 mg / 100 g, and at (60–70) ° C – 102,67 mg / 100 g.
The dependence of the degree of hydrolysis on the duration of hydrolysis was investigated. It has been found that the test enzymes provide the most intense hydrolysis in the first 30 min of the enzymatic process followed by a gradual increase in the NAG value over 180 min of hydrolysis. Thus, for pepsin, the content of NAG for (30–180) min increases from (42,00 to 60,67) mg / 100 g; for papain – from (67,67 to 95,67) mg / 100 g; Protolade – from (91,00 to 128,33) mg / 100 g.
Thus, it was found that obtaining a product with a high degree of hydrolysis is possible with the use of the enzyme preparation Protolad at a concentration of 5%, with a protein substrate concentration of 20%, with the medium should be alkaline, the pH value is 8.0, and the process temperature should be 60 °C.
Hydrolysis of whey proteins allows to obtain low molecular weight peptides and amino acids, which further ensure the production of flavors and aromatic substances. Research findings can be used in the development of natural flavoring additives.
Key words: whey, whey protein concentrate, enzymatic hydrolysis, pepsin, papain, Protolad.
- Onwulata C., Huth P. (2009). Whey processing, functionality and health benefits. Ames, Iowa: John Wiley & Sons.
- Hramcov A. G. (2011). Fenomen molochnoj syvorotki [The phenomenon of whey]. Saint Petersburg: Profession. [in Russian].
- Evdokimov I. A., Hramtsov A. G., Nesterenko P. G. (2008). Sovremennoe sostojanie i perspektivy pererabotki molochnoj syvorotki [Present state of milk whey processing]. Molochnaja promyshlennost’ [Dairy industry], 11, 36–43. [in Russian].
- Frolova N., Ukrayinets A. (2018). Development of methods of production in natural aromatic production. Ukrainian Food Journal, 7, 4, 692–702. DOI: 10.24263/2304-974X-2018-7-4-13.
- Kurbanova M. G., Razumnikova I. S., Prosekov A. Yu. (2010). Belkovye gidrolizaty s biologicheski aktivnymi peptidami [Protein hydrolyzates with biologically active peptides]. Molochnaja promyshlennost’ [Dairy industry], 9, 70–71. [in Russian].
- Cheison S. C., Zhang S. B., Wang Z., Xu S. Y. (2009). Comparison of a modified spectrophotometric and the pH-stat methods for determination of the degree of hydrolysis of whey proteins hydrolysed in a tangential-flow filter membrane reactor. Food Research International, 42, 91–97. DOI: 10.1016/j.foodres.2008.09.003.
- Doucet D., Otter D. E., Gauthier S. F., Foegeding E. A. (2003). Enzyme-induced gelation of extensively hydrolyzed whey proteins by Alcalase: peptide identification and determination of enzyme specificity. Journal of Agricultural and Food Chemistry, 51, 21, 6300–6308. DOI: 10.1021/jf026242v.
- Ghosh B. C., Prasad L. N., Saha N. P. (2017). Enzymatic hydrolysis of whey and its analysis. Journal of food science and technology, 54, 6, 1476–1483. DOI: 10.1007/s13197-017-2574-z.
- Pihlanto-Leppala A., Koskinen P., Piilola K., Tupasela T., Korhonen H. (2000). Angiotension I—converting enzyme inhibitory properties of whey protein digest: concentration and characterization of active peptides. Journal of Dairy Research, 67, 53–64. DOI: 10.1017/S0022029999003982.
- Schmidt D. G., Poll J. K. (1991). Enzymatic hydrolysis of whey proteins. Hydrolysis of a-lactalbumin and b-lactoglobulin in buffer solution by proteolytic enzymes. Netherlands Milk and Dairy Journal, 45, 225–240.
- Lodygin A. D., Khramtsov A. G., Donskoy N. S. (2010). Metody gidroliza syvorotochnyh belkov moloka [Methods of hydrolysis of whey milk proteins]. Sbornik nauchnyh trudov SevKavGTU. Serija «Prodovol’stvie» [Collection of scientific papers of SevKavSTU. Series “Food”], 6, 19–21. [in Russian].
- Donskoy N. S., Khramtsov A. G., Lodygin A. D. (2009). Isledovanie kinetiki fermentativnogo gidroliza syvorotochnyh belkov, podvergnutyh jelektofizicheskoj obrabotke [Investigation of the kinetics of enzymatic hydrolysis of whey proteins subjected to electrophysical processing]. Sbornik nauchnyh trudov SevKavGTU. Serija «Prodovol’stvie» [Collection of scientific papers of SevKavSTU. Series “Food”], 5, 28–31. [in Russian].
- Ostroumov L. A., Prosekov A. Yu., Babich O. O. (2008). Gidroliz koncentrata syvorotochnyh belkov jekzo- i jendopeptidazami [Hydrolysis of the whey protein concentrate with exo- and endopeptidases]. Molochnaja promyshlennost’ [Dairy industry], 12, 55–56. [in Russian].
- Prosekov A. Yu., Babich O. O. (2008). Osobennosti poluchenija smesi aminokislot iz belkov molochnoj syvorotki [Features of obtaining a mixture of amino acids from whey proteins.]. Aktual’nye problemy tehniki i tehnologii pererabotki moloka: sbornik nauchnyh trudov s mezhdunarodnym uchastiem. Barnaul: GNU Sibirskij NII syrodelija SO RASHN [Actual problems of machinery and technology for milk processing: a collection of scientific papers with international participation. Barnaul: GNU Siberian Research Institute of Cheesemaking SB RAAS], 5, 161–165. [in Russian].
- Tepel A. (1979). Himija i fizika moloka [Chemistry and physics of milk] (translation from German). Moscow: Food Industry. [in Russian].
- Borisova G. V., Novoselova M. V., Bondarchuk O. N., Malova Y. (2012) Vybor fermentnyh preparatov s cel’ju poluchenija gidrolizatov molochnoj syvorotki s nizkoj allergennost’ju [Schoice enzyme preparation to obtain whey hydrolyzate low allergenicity]. Fundamental’nye issledovanija [Basic research], 11, 5, 1164–1167. [in Russian].
- Tsyhankou V. G., Halavach T. N., Kurchenko V. P., Bondaruk A. M. (2015). Izuchenie peptidnogo sostava fermentativnogo gidrolizata koncentrata syvorotochnyh belkov korov’ego moloka s cel’ju razrabotki pishhevyh produktov dlja turistichesko-ozdorovitel’noj dejatel’nosti [Study of the peptide enzymatic hydrolysates of whey protein concentrate cow’s milk to develop foods for tourism and recreation]. Trudy BGTU. Lesnoe hozjajstvo [Proceedings of BSTU. Forestry], 1, 272–275. [in Russian].
- Kurbanova M. G. (2010) Fermentativnyj gidroliz belkov moloka s ispol’zovaniem razlichnyh proteaz [Enzymatic hydrolysis of milk proteins with various proteases use]. Vestnik Krasnojarskogo gosudarstvennogo agrarnogo universiteta [Bulletin of the Krasnoyarsk State Agrarian University], 1, 157–160. [in Russian].
- Sidorov Yu. I., Poznanska S. A, Novikov V. P. (2008). Rozroblennja tehnologії oderzhannja bіologіchno aktivnoї sumіshі amіnokislot z molochnoї sirovatki [Development of technology for obtaining biologically active mixture of whey amino acids]. Vіsnik Nacіonal’nogo unіversitetu «L’vіvs’ka polіtehnіka» [Bulletin of the National University “Lviv Polytechnic”], 622, 88–96. [in Ukrainian].
- Halavach T. N., Havrilenko N.V., Zhabanos N. К., Kurchenko V. P. (2008). Zakonomernosti gidroliza syvorotochnyh belkov jekzo- i jendoproteazami [Regularities of hydrolysis of whey proteins with exo- and endoproteases]. Trudy BGU [BSU Proceedings], 3, 1, 1–15. [in Russian].
- Kurbanova M. G., Shevyakova K. A. (2017). Analiz fermentnyh preparatov, osushhestvljaemyh proteoliz syvorotochnyh belkov [Analysis of enzyme preparations projected by proteolysis of serum proteins]. Vysokie intellektual’nye tehnologii v nauke i obrazovanii [High intellectual technologies in science and education], 103–105. [in Russian].
- Kalinichenko М. А., Telishevskaya L. Y. (2009). Opredelenie kineticheskih konstant gidroliza belkovyh substratov raznymi proteoliticheskimi preparatami [Determination kinetic constants of hydrolysis protein substances by different proteolytic preparates]. Rossijskij veterinarnyj zhurnal. Sel’skohozjajstvennye zhivotnye [Russian Veterinary Journal. Farm animals], 1, 42–44. [in Russian].