INTRODUCTIONThe development and subsequent quality assessmentof functional foods is one of the priorities of healthynutrition [1]. Functional foods with a programmedchemical composition can be fortified with importantnutrients and are suitable for various categories ofpopulation, e.g. athletes, lactating and pregnant women,senior citizens, children, etc. [2].However, priority goes to gastrointestinal andallergic patients and professional athletes. Their nutrition38Gavrilova K.V. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. Х–Хrequires scientific approaches, since their diet shouldcontain a complex of peptides and free amino acids, aswell as simple and complex carbohydrates [3].Foods fortified with proteins, especially thosecontaining essential amino acids, contribute to the rapidand effective recovery of muscle tissue after intensephysical exertion. Peptides and polypeptides are knownto accelerate metabolic processes, hormone production,and muscle tissue growth [4].Food intolerance and allergic reactions are anotherproblem of modern society. Some people are allergic toproducts that contain proteins of animal or plant origin.Hence, a new generation of food products with easyto-digest nutrients remains an important objective of foodindustry. Modern studies confirm that plant raw materials– and legumes in particular – are suitable for isolationand modification of proteins, short peptides, and aminoacids [5, 6]. These groups of compounds are widely usedas dietary supplements and ingredients for functionalproducts [7–9]. Pea protein has a better nutritional value,amino acid composition, and anti-nutrients than soybeans,beans, and other legumes [10, 11].There are many methods to isolate protein fromplant materials for subsequent hydrolysis [12–14].However, most of them remain inefficient for enzymatictransformations of heterogeneous substrate. Preliminarymechanical activation means that raw material has tobe processed in specially designed energy-stressedactivator mills. The procedure makes it possible tocontrol the reactivity of solid substrates. In addition, itincreases the speed and yield of water-soluble productsfor commercial purposes [15, 16].However, the process of enzymatic reactionsafter preliminary mechanical activation remainsunderstudied. A series of studies on the hydrolysis ofcellulose showed that the increase in specific surfacearea and the degree of crystallinity of the substrateaffected the rate and yield of enzymatic hydrolysis [17].In addition, it is important to study the elusive transferof mechanochemical processes from lignocellulose toprotein and starch.The research objective was to study the mechanicallyactivated hydrolysis of pea biomass, as well as toobtain a hydrolysate fortified with free amino acids andpeptides to be used in functional foods.STUDY OBJECTS AND METHODSThe experiment featured dry biomass of split peaseeds harvested in 2017. The peas corresponded withState Standard 6201-68*, Class I, and was produced byOOO ECO-PAK (Novosibirsk region, Russia). Beforethe experiment, the pea biomass was subjected to roughgrinding in a knife mill to the size of ≤ 2 mm. Theground biomass was vacuum-packed, stored at roomtemperature, and used for further experiments.* State Standard 6201-68. Polished pea. Specifications. Moscow:Standartinform; 2010. 3 p.Protosubtilin G3h was used as enzyme preparation(OOO Sibbiopharm, Berdsk, Russia). The complexwas chosen for its catalytic activity and availabilityfor further technological application. This industriallyavailable enzyme preparation contains a complex ofenzymes that consists of neutral and alkaline proteasesand glycosidases, i.e. ≈ 11000 U/g of protease, ≤ 150 U/gof xylanase, ≤ 200 U/g of β-glucanase, and ≤ 300 U/gof α-amylase [18]. Protosubtilin belongs to the groupof enzyme feed additives that are able to break downhigh-molecular proteins. This enzyme preparation isproduced by Bacillus subtilis.Gravimetric methods were used to assess moistureand ash content in the plant materials and processedproducts, respectively [19, 20].X-ray diffraction and thermal desorption of gaseswere employed to measure the degree of crystallinityand specific surface area according to the methodsdescribed in [17] and [21], respectively.The method described by Fadeeva et al. was usedto perform the elemental analysis that made it possibleto determine the quantitative protein content in thepeas. After that, the protein content was determinedusing the nitrogen content with conversion factor of6.25 according to the Kjeldahl method [22–24].The mass fraction of soluble substances wasdetermined by the method of exhaustive extraction in aSoxhlet extractor for 24 h. Distilled water was used asextractant. The yield of water-soluble substances wasmeasured according to the reduction of the mass afterthe extraction.The content of free amino acids was defined at theCentre of Mass Spectrometry Analysis (Institute ofChemical Biology and Fundamental Medicine). Anoptimised standard procedure was used as in [25]. Aset of isotope-labelled amino acids and acyl carnitinesNo. 55000 (Chromsystems Instruments & Chemicals,Germany) served as internal standards and solutions. AnAgilent-1200 chromatographic system with an Agilent6410 QQQ mass spectrometer (Agilent Technologies,USA) was employed as an HPLC-MS/MS system. Aquantitative analysis was performed in the mode ofmultiple reactions monitoring; the total analysis timewas 2.5 min. The obtained data were processed usingMassHunter v.1.3 software.The molecular weight of protein molecules wasmeasured using the Laemmli SDS PAGE procedure[26]. For pre-denaturation, proteins were treated with1.4-dithiothreitol at a 1:1 ratio. After that, they wereplaced in a thermoshaker at 95°C (Biosan, Latvia) for7 min. An Elf-4 power source was used to create electricfield (DNA-Technology, Russia). The concentrations ofpolyacrylamide in the concentrating and separating gelwere 4% and 18%, respectively. The pre-phoresis stagelasted 15 min. The current force was 15 mA, whileduring the phoresis stage it was 35 mA.To identify the zones of proteins after theelectrophoresis, they were stained with CoomassieR-250 pigment according to the procedure described by39Gavrilova K.V. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. Х–Х56%21%4%12%7%Grains and legumes Sugar beetSunflower PotatoVegetables5%10%4% 4%Dyballa et al. [27]. Protein markers were representedby Unstained protein MW marker (Thermo FisherScientific, USA) with protein molecular weight of14.5–116 kDa and Unstained protein ladder (ThermoFisher Scientific, USA) with protein molecular weight of5–250 kDa. MultiChrom-Planar programme processedthe mathematical data [28].The fractionation of the plant material wasconducted according to the method described in [29, 30].The initial crushed pea biomass was extracted inalkaline water. A 1M solution of sodium hydroxidewas added to the pre-ground pea biomass. The solutionconsisted of 2.5 ml of solution per 1 gram of biomass(pH 9.0). The suspensions were placed in a WSB-30water bath at 45°C and 180 rpm for 30 min (DAIHANScientific, Korea). After the extraction, the solubleportion was separated by centrifugation at 6000 rpm for20 min. The precipitate was used in the next extractioncycle under the same conditions. The extractedcomponents were precipitated with a threefold volume ofcooled ethanol and dried in a laboratory frost dryer Iney4 (Institute for Biological Instrumentation of the RussianAcademy of Sciences, Russia).After three extraction cycles (fraction No. 4), theinsoluble residue – a carbohydrate fraction – was washedtwice with chilled ethanol and dried under similarconditions.The mechanical activation of the plant materialwith enzymes was carried out in an RM-20 roller millactivator(5.5 kW), which was equipped with a watercooling device (Fig. 1). The pea biomass was mixedwith a dry enzyme preparation and processed in anactivator at a rotor speed of 1450 rpm. The mixture ofraw materials and enzymes was supplied automaticallyat a rate of 3 kg/h.The spray-drying was performed in a Mini SprayDryer B-290 (Büchi, Switzerland) in the followingconditions: nozzle temperature = 110°C, cyclonetemperature = 70°C, gas flow rate = 700 L/h, feed rate =5 mL/min.The enzymatic hydrolysis l asted 7 h a t 5 0°C.50 ml of distilled water was added to 15 g of initial ormechanically activated pea biomass with a certainamount of the enzyme preparation. Enzyme loadingequalled 0.5%–3%. Suspensions were thoroughly mixeduntil uniform. For enzymatic hydrolysis, the suspensionswere placed in a WSB-30 water bath (DAIHANScientific, Korea) at 50°C and 120 rpm. After enzymatichydrolysis, the supernatant was centrifuged at 6000 rpmfor 20 min. No enzyme preparation was added to thecontrol samples.RESULTS AND DISCUSSIONSuitable protein plant materials were selectedfor the mechanoenzymatic processing to be used infunctional, special, and therapeutic food products. Thephysical and chemical characteristics are given below.In addition, the selection was based on an analysis ofthe existing market for high-protein plant materials,state statistics, distribution of croppage, and percentageof various cultures in Russian regions. This approachmade it possible to identify raw material with suitablephysicochemical parameters, as well as to determine itsprospects in subsequent processing and implementation.Figure 2 shows a distribution diagram of croppagein Russia in 2017. The diagram was based on the dataobtained from the Federal State Statistics Service [31].Cereals and legumes clearly prevail over other cultures.Legumes are richer in protein than grains. An analysisof the distribution of croppage within the group ofleguminous crops showed that a large proportion (77%)belongs to peas (Fig. 3).Figure 1 Scheme of the roller type activator mill RM-20Plant raw materialRollsRotatorMechanically processed productStarterFigure 2 Croppage distribution in Russia56%21%4%12%7%Grains and legumes Sugar beetSunflower PotatoVegetables5%10%4% 4%40Gavrilova K.V. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. Х–ХThus, legumes proved to be the most advantageoussource of vegetable protein in Russia, especially peas,which contain about 25% of protein. In spite of thefact that soy contains up to 35% of protein, it was notconsidered in this study since it is rich in anti-nutritionalsubstances, Moreover, it has a low consumer loyalty,which cannot be ignored in product development [5–7,10, 32]. The protein content in peas varies greatlyaccording to genotypic characteristics and thecultivation conditions. Leguminous proteins are poorin methionine and cysteine. This is typical of plantproteins. For instance, grain crops are poor in lysineand threonine. However, the biological value of productsobtained from them can be fortified by a limiting aminoacid or other types of plant materials.The present research involved a comparativeanalysis of the protein content and amino acidcomposition together with its coefficient of imbalanceand functionality in high-protein plant raw materials.Peas demonstrated the highest functionality ratio ofamino acid composition (FRAAC) – 0.6, while soybeanshad 0.4 and beans and lentils had 0.3. This indicatedthat peas possessed the optimal ratio of amino acids ifcompared with reference chicken egg protein.Thus, pea biomass appeared to have a highnutritional value and a balanced amino acidcomposition, which made it an optimal research subject.Its physical and chemical patterns can subsequentlybe transferred to other types of biomass. The samplesobtained after fractionation (Fig. 4) and freeze dryingwere analysed for the protein content in the dry product.The results are presented in Table 1. A polyacrylamidegel electrophoresis defined the molecular weight of theproteins in the fractions.The obtained data are consistent with those alreadypublished Mession et al.: the pea biomass contained23–24.4% of protein and 48–60.3% of starch [33].Fractions No. 1 and 2 isolated from the biomass werefortified with proteins, while fraction No. 3 was fortifiedwith proteins and carbohydrates, and fraction No. 4 – withcarbohydrates.The electrophoregram (Fig. 5) shows that fractions1–3 contained proteins with molecular weight =5–135 kDa, which corresponded to molecules thatconsisted of 50–1350 amino acid residues. Thepredominating molecules were those with molecularweight = 24–135 kDa (240–1350 amino acidresidues). They were most likely to be sub-units of11S-globulins [34]. Both the elemental analysis and thegel electrophoresis showed that the content of proteinmolecules in fraction 4 was at the level of trace amounts.As proved by cellulose processing, enzymepreparation increases the efficiency of subsequentenzymatic hydrolysis, if added at the stage of mechanicchemicalprocessing [34]. The enzyme complexused in the present research had a suitable catalyticactivity profile and was cheaper than its analogues,such as proteases AP1, Alcalase, Savinase, Esperase,and Neutrase (Shandong Longda Bio-Products andNovozymes).A set of experiments made it possible to determinethe effect of the conditions of mechanical activationon the subsequent enzymatic hydrolysis. The peabiomass was subjected to mechanical activation 1)without enzymes and 2) with an insufficient amountFigure 3 Percentage ratio of the croppage of legumes inRussia21%Grains and legumes Sugar beetSunflower PotatoVegetables77%5%10%4% 4%PeasLentilsChick peasGrain vetch (Vicia L.) and vetch-prevailing mixesGrain forage lupine (Lupinus L.)0204060801000369120.60 0.90 1.40 2.10 3.15 4.75 7.20 10.90 16.50 24.90 37.70Differential distribution, %Fraction size, μmIntegral distribution, %21%Grains and legumes Sugar beetSunflower PotatoVegetables77%5%10%4% 4%PeasLentilsChick peasGrain vetch (Vicia L.) and vetch-prevailing mixesGrain forage lupine (Lupinus L.)0204060801000369120.60 0.90 1.40 2.10 3.15 4.75 7.20 10.90 16.50 24.90 37.70Differential distribution, %Fraction size, μmIntegral distribution, %Figure 4 Frozen fractionation products before freeze drying:1–4 are numbers of corresponding fractionsTable 1 Protein content in the initial raw material and in thefractionsSample Proteincontent, %Fraction content inthe raw material, %Raw material 24.3 –Fraction No. 1 97.1 19.0Fraction No. 2 86.7 6.5Fraction No. 3 45.7 0.4Fraction No. 4 Trace 74.121%Grains and legumes Sugar beetSunflower PotatoVegetables77%5%10%4% 4%PeasLentilsChick peasGrain vetch (Vicia L.) and vetch-prevailing mixesGrain forage lupine (Lupinus L.)0204060801000369120.60 0.90 1.40 2.10 3.15 4.75 7.20 10.90 16.50 24.90 37.70Differential distribution, %Fraction size, μmIntegral distribution, %41Gavrilova K.V. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. Х–Хof enzymes (1%) in relation to the substrate. Thesubsequent hydrolysis and complete extraction (Table 2)showed that the mechanical activation without enzymesbarely increased the yield of the subsequent hydrolysis.However, the mechanical activation with enzymesincreased the yield during subsequent hydrolysis from18% to 60%, i.e. by ≥ 3 timesThe results can be explained by the fact that asimultaneous activation of substrate and enzymesproduced mechanocomposite. The mechanocompositewas an intermediate solid-phase product with a highreactivity. In such mechanocomposites, enzyme particlesare distributed non-diffusively, or mechanically, over thesurface of the substrate, which was disordered duringthe activation process. Similar effects were observed inother cases of activation of food and non-food plant rawmaterials [35, 36]. When mechanocomposite is formed,it usually increases the rate and yield of the subsequentproteolytic and glycolytic processes. In this case, apreliminary chemical interaction preceded the mixing ofthe enzymes and the substrate. This interaction resultedfrom a significant increase in surface area, whichenlarged from 0.6 to1.9 m2/g, and an extra disorderingof the substrate structure, whose crystallinity decreasedfrom 25% to 14%.The conversion of enzymatic hydrolysis was studiedunder the same conditions, according to the substrate –enzyme ratio. The enzyme preparation was added in 0.5,1, 2, 2.5, and 3% (Table 3).Table 3 shows that the amount of water-solublesubstances increased, as the amount of enzymesincreased from 0.5% to 2%. The water-solublesubstances included reducing carbohydrates, which arelow molecular weight products of starch hydrolysis.When the load of the enzyme complex increased to2.5%–3%, the number of reaction products did notincrease. This might have been caused by the fact thatthe sorption sites of the substrate were completelyfilled with enzymes. The situation was fully consistentwith the idea that the heterogeneous stage of enzymatichydrolysis has a limiting effect.The polyacrylamide gel electrophoresis wasused to study the changes in the molecular weightduring the enzymatic hydrolysis. Figure 6 shows theelectrophoregram of the proteins contained in thehydrolysate 1–7 h after the hydrolysis. The data provethat the amount of the original protein moleculessignificantly decreased within 7 h. As a rule, proteinsdegrade within 2 h. The molecular weight of thedegradation products of the original polypeptideproteins revealed no significant changes after 4 h. AfterFigure 5 Electrophoregram (A) and MWD profilograms (B) of proteins in the fractions; 1, 2, and 3 – fraction numbers. FractionNo. 4 is not represented as it appeared to have no proteins in its composition116 kDa69 kDa45 kDa35 kDa25 kDa18 kDa14 kDaTable 2 Yield of water-soluble substances according to the processing conditionsExtraction fromthe initial rawmaterialExtraction from the productof mechanical activationwithout enzymesProduct after mechanicalactivation (withoutenzymes) and hydrolysisProduct after mechanicalactivation with 1% of enzymepreparation after hydrolysisYield of water-solublesubstances, %18.0 18.5 25.1 60.6Table 3 Yield of water-soluble substances and reducingcarbohydrates according to the amount of enzyme preparationEnzymepreparation, %Yield of water-solublesubstances, %Yield of reducingcarbohydrates, %0.0 25.8 1.50.5 38.7 4.01.0 55.4 7.62.0 78.5 16.32.5 78.5 16.33.0 78.5 16.342Gavrilova K.V. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. Х–Х7 h, there remained an insignificant amount of stablepolypeptides with a molecular weight of ≈ 20 kDa.These polypeptides were associated with the polypeptidechain of legumin, which is resistant to neutral andalkaline proteases [33].Thus, an enzymatic hydrolysis that exceeded 4 h isineffective, since the number of low molecular weightpolypeptides did not increase much after that time.Figure 6 Electrophoregram (A) and profilogram (B) of the molecular weight distribution of proteins in the hydrolysate after 0, 1, 2,3, 4, and 7 h69 kDa45 kDa35 kDa25 kDa18 kDaTable 4 Content of essential amino acids in the hydrolysateand in the control sampleAmino acid Amino acid content, μg/gPea biomass extract Hydrolysed pea biomassIle+Leu 515 10479Met 110 1320Phe 514 7681Val 441 3921Figure 7 Scanning electron microscopy of the hydrolysateafter spray-dryingμmFigure 8 Granulometric composition of the hydrolysate after spray-drying56%21%4%12%7%Grains and legumes Sugar beetSunflower PotatoVegetables77%5%10%4% 4%PeasLentilsChick peasGrain vetch (Vicia L.) and vetch-prevailing mixesGrain forage lupine (Lupinus L.)0204060801000369120.60 0.90 1.40 2.10 3.15 4.75 7.20 10.90 16.50 24.90 37.70Differential distribution, %Fraction size, μmIntegral distribution, %43Gavrilova K.V. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. Х–ХA mass-spectrometric analysis of amino acids wasperformed to study the low molecular weight productsof the enzymatic hydrolysis. Table 4 shows that thehydrolysis resulted in a significant increase in thenumber of essential amino acids in comparison with thecontrol sample obtained without enzymes.For the hydrolysates to be widely implemented,there have to be new ready-made food products withprolonged shelf life. Thus, a set of experiments onspray-drying had to be performed [37]. The spraydryingprocess can be easily scaled and is widely used infood industry to produce dry enzymes, foodstuffs, andunstable compounds [38–40].The product obtained by spray drying (Figs. 7and 8) had a monomodal particle size distribution.The main share belonged to spherical particles with adiameter of 5–20μм. The size was associated with thecharacteristics of the equipment: the nozzle opening was25μм in diameter.Most of the particles were concave, which made itpossible to describe the mechanism of drying. Initially,a powerful inward-directed deformation removed thesolvent from the surface of the drop. As a result, thereformed a layer of the product. The solvent diffusedthe layer of the dry product, after which the particledeformed and collapsed.In the control experiment, vacuum drying withoutsplashing the hydrolysate resulted in the formation ofa layer that was not dispersed into individual particles.An electron scanning microscopy of the ground product(Fig. 9) showed that it had a dense structure withoutpores. This confirms the spray-drying mechanism:the drying occurs on the surface, while the dry layercaptures the solvent, and a high mechanical tensiondeforms the particle, giving it a concave shape.CONCLUSIONThus, the paper featured the process of mechanicalactivation and subsequent enzymatic hydrolysis of peaproteins. The original pea biomass was described usingmodern chemical methods. The protein content was24.3%, and MWD was 5–135 kDa.The fractionation produced four fractions ofbiopolymers with various contents of protein andcarbohydrate molecules. The experiment made itpossible to define the optimal conditions for themechanical activation performed together withproteolytic enzymes. The enzymes were obtainedfrom the complex enzyme preparation ProtosubtilinG3x. When both the substrate and the enzymes weremechanically activated, it produced mechanocomposite.As a result, the specific surface area increased by3.2 times, while the crystallinity decreased by 2 times,which raised the yield of the subsequent enzymatichydrolysis from 18% to 61%.During hydrolysis, protein broke down within2 h, and there was almost no change after 4 h. Theexperiment detected non-hydrolysed protein moleculeswith a molecular weight of ≈ 20 kDa. They presumablycorresponded with legumin, which is resistant to neutraland alkaline proteases.The research involved an experiment on spraydryingof the obtained hydrolysates for their potentialuse as food components. The resulting product had amonomodal particle size distribution. The particles had aspherical shape with a diameter of 5–20 μ.CONFLICT OF INTERESTThe authors declare that there is no conflict ofinterest related to this article.ACKNOWLEDGEMENTSThe authors would like to express their deepestgratitude to V.I. Berezin, G.N. Nesterova, I.V. Ivanov,S.Yu. Abramov, and I.B. Orekhov (Institute of SolidState Chemistry and Mechanochemistry), A.G. Ogienkoand N.F. Beizel (Institute of Inorganic Chemistry), andL.N. Rozhdestvenskaya (Novosibirsk State TechnicalUniversity).FUNDINGThe research was funded by the Russian ScienceFoundation, Project No. 17-73-10223: ‘Processes ofmechanical activation and enzymatic hydrolysis of plantraw material polymers for obtaining low-molecularcomponents of functional foods’.