Conditions for making plant dispersions based on nature-like technologies

An objective of this study was to examine the possible use of sprouted grains of domestically selected legumes to produce dairy alternative products. The paper presents the results of a comprehensive assessment of the food properties of ‘Chishminskii 95’, ‘Chishminskii 229’, ‘Pamiati Hangildina’, ‘Iuldash’ pea varieties and ‘Nerussa’, ‘Lukeria’, ‘Omichka’ bean varieties of Russian selection. The work investigated the water absorption kinetics under different temperature conditions and established germination patterns for a model medium (drinking water treated with an electromagnetic field) being as follows: germination temperature is 18 ± 2 °C; the soaking period lasts from 5 to 8 hours (depending on the variety); the germination time from 0.54 0.62 to 0.70 0.81 days (depending on the variety). The work analyses their microstructure and changes in the germination process. The proteolytic activity of bean trypsin before and after germination has been proved to be lower than that of peas. At the same time, the proteolytic activity of trypsin after pea and bean grain germination increased in all samples. The grain digestibility as a result of germination has increased; the ratio of essential and non-essential amino acids has changed in favor of the former; the fractional composition of protein has changed to higher content of albumin and globulin and lower level of glutelin. The results of this study indicate that the most suitable varieties to produce vegetable milk are ‘Omichka’, ‘Lukeria’ beans and ‘Pamiati Hangildina’ peas.


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Currently, dairy alternatives are produced from soy. Far too little attention has been paid to products from legumes like peas and beans, and no previous study has investigated barley being a promising cereal crop for the industry. This indicates a need to identify the most suitable varieties for producing dairy alternatives by conducting a systematic, comprehensive assessment of pea and bean varieties of domestic selection.

Materials and Methods Materials and Methods Materials and Methods Materials and Methods
Protein digestibility was determined by treating the product sample with a hydrochloric acid solution of pepsin and keeping it in a thermostat at 42-45 °C for 16 hours under methodological guidelines for assessing the fodder quality and nutritional value (Central Research Institute Agrochemical Services for Agriculture, 2002). The resulting suspension was then filtrated, centrifugated and examined to determine the weight of the residual sample fraction non-hydrolyzed by pepsin M1.
The digestibility of protein concentrate (X1), %, was calculated by the equation (1): (1) where M1 is the weight of the residual sample fraction non-hydrolyzed by pepsin, g; m is the sample weight taken for testing, g.
The protein fraction composition was found by the Osborn method; the protein content and individual protein fractions were determined by burning extracts, followed by Kjeldahl nitrogen determination (Russian state standard GOST 10846-91, 1991).
The seed coat content was examined by soaking samples in hot distilled water, followed by removing the seed coats, drying them to a constant weight, weighing and calculating their percentage to the weight of unbroken seeds (Russian State Standard GOST 10843-76, 1976).
To assess the grain suitability for germination, its sprouting energy and property were detected (Russian state standard GOST 10968-88, 1988). The proportion of sprouted pea grains was found based on the instructions for the techno-chemical control in brewing production (Ministry of Agriculture of the Russian Federation, 1991). To do this, 100 grains were germinated four times on wet filter paper in Petri dishes in a thermostat at 19.5 ° C. Counting and removal of sprouted grains was carried out after 24 (N24), 48 (N48) and 72 (N72) hours of germination. Grains were considered sprouted when their embryo roots emerged. The proportion of sprouted grains (D) was determined by the equation (2) (Narciss, 2007;Russian state standard GOST 10968-88, 1988; Russian state standard GOST 12039-82, 1982): (2) where N24 is the number of grains sprouted after 24 hours; N48 is the number of grains sprouted after 48 hours; N72 is the number of grains sprouted after 72 hours.
The average germination time (Tav) was determined by the equation (3): (3) The sprouting index (Ispr) was calculated by the equation (4): The grain microstructure and the seed coat thickness were analyzed using a Micromed 3-20M scanning microscope with real-time image output to a PC screen using a video eyepiece. The magnification of the microscope was 1600 times.

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The active acidity (pH) of the studied varieties was determined by the potentiometric method using a Mettler Toledo SevenGo pH meter with a measurement error of 0.01 pH units. The samples were analyzed in three-fold repetition, with an accuracy of 0.01 pH units.
The weight of 1000 grains was found by the weight method (Russian state standard GOST 10842-89, 1989).
The proteases hydrolyzing N, a-benzoyl-DL-arginine-paranitroanilide (BAPNA, Sigma, USA) were determined by the Erlanger method with modifications (Solomintsev and Mogilny, 2009). The activity of the enzyme was expressed in optical units (arbitrary units of enzymatic activity, E).
The enzyme activity was expressed in optical unites (conditional units of enzyme activity, E). The activity of trypsin inhibitors was determined by the Hoffman-Weisblay method with modifications. The analysis was carried out similarly to the assessment of enzymatic activity; the buffer solution contained 1 mg/ml of trypsin.
To make a reliable evaluation of the proteolytic activity of enzymes, the samples were subjected to temperature exposure at t = 60 °C τ=20 min, to denature their enzymes, given that the total proteolytic activity is the total effect of proteolysis of the set of enzymes contained in the studied objects.
The urease activity was measured in pH units on a pH meter. The calculation was based on a phosphate buffer solution pH = 6.86, which changes due to the urease action on the urea contained in the solution (Russian state standard GOST 13979.9-69, 1969).
The amino-acid composition of pea and bean protein was determined in the scientific and educational centre (research laboratory) of the Kemerovo State University with a Kapel-105 M analyzer using the Kapel capillary electrophoresis system (Russian state standard GOST 31480 -2012GOST 31480 - , 2012. The content of water-soluble vitamins was measured by the spectrophotometric method using the capillary electrophoresis system "Kapel-105/105M" with subsequent detection on the Shimadzu UV-1800 spectrophotometer at the Scientific and Educational Center of the Kemerovo State University (Trineeva et al., 2017).
The results of experimental studies were subjected to statistical processing by parametric methods using standard MS Excel software Packages. Experiments had five replications as required by methods of statistical analysis.
Two temperature conditions were investigated to optimize the soaking parameters; these were 14 ± 2˚C (ordinary soaking) and 18 ± 2˚C (warm soaking). Trial No. 1 used a solvent model medium, drinking water of a centralized potable water supply system treated with an electromagnetic field of 15 mT induction (Table 1). Water was treated in an experimental laboratory installation of a solenoid type. The installation scheme is shown in Figure 1.   The studied pea and bean samples were hydrothermally treated (soaked) in a ratio (1:3) and kept in contact with solvents; the samples were kept there up to 16 hours (with water being replaced every 60 minutes to avoid grain contamination). Seeds were soaked in a model medium in thermostatically controlled cells at two temperature conditions: 14 ± 2˚С and 18 ± 2˚С. The experiment was carried out in three replications.
The experiment determined the minimum hydrothermal treatment (soaking) time when legume grains reached the maximum swelling and size enlargement (Butavin et al., 2019;Pankina and Borisova, 2016).
The quantitative measure of swelling (swelling degree) was found by the weighting method by the degree of swelling (α) from the equation (5): (5) α is the swelling degree during hydrothermal treatment (soaking); Mτ is the grain weight of the studied pea and bean varieties during hydrothermal treatment (soaking), g; Mo is the grain weight of the studied pea and bean varieties before hydrothermal treatment (soaking), g. Grain geometric characteristics (length (L), thickness (B), width (A) of peas and beans were measured using a calliper. 100 grains of each variety were taken for the measurements. The radius, arithmetic mean and geometric mean diameters, spherical shape, volume and area of the outer surface of the grains, the aspect ratio of the bean grains were calculated by standard equations (Danko, 2020;Kydyraliev, 2015).
The arithmetic mean grain diameter was calculated by the equation (6): = (6) The geometric mean grain diameter was found by the equation (7): where L is the length (mm), B is the thickness (mm), A is the width (mm). The bean grain sphericity (Bn), in %, was measured by the equation (8).
(8) The bean grain surface area (S), in mm 2 , was determined by the equation (9): The aspect ratio of bean grains (Ra), in %, was found using the equation (10): 6 (10) The pea grain sphericity (ψ) was determined by the equation (11): where Fsph is the surface area of the equivoluminar sphere; Fg is the area of the outer surface of the grain. The sphere surface area was determined by the equation (12): (12) where V is the volume of a single grain; r v is the radius calculated by the equation (13): = 0.62√ (13) The area of the outer surface of the grain was measured by the equation (14) (14) The grain volume was determined by the equation (15): (15) where k is a coefficient that considers the grain shape specifics (Apostolov et al., 2002). Seeds were sprouted in the original experimental device "Smart Sprouter "Rosinka", manufactured in the Omsk State Agrarian University, named after PA Stolypin. It is based on aeroponics technology (Algazin et al., 2016;Algazin et al., 2017). The installation can simulate a nature-like environment for sprouting legume grains based on automated germination programs (Rybchenko et al., 2022;Zolotov et al., 2022). The technological algorithm of the experiment was as follows. Seeds of the studied crops and varieties were placed on trays and subjected to hydrothermal treatment (soaking) under the established regime. Then the trays were placed into a sprouting container having fixed supports, impenetrable walls, a mesh bottom and a lid. Water vapour (aerosol) is supplied by an ultrasonic steam generator with automatic humidity control from 40 to 90%, which breaks water into fine dust and mixes it with air. As a result, seeds absorb moisture uniformly, and the air is enriched with oxygen. There are humidity and temperature sensors. Infrared emitters maintain the temperature and provide additional seed irradiation that warms seeds and biostimulates the germination process.

Results Results Results
Tables 2 and 3 show the morphometric characteristics of pea and bean grains. All the studied pea varieties had a bright yellow colour with cotyledons translucent through the seed coat. The 'Iuldash' pea variety demonstrated pronounced varietal differences. Its grain is smaller; the minimum weight of 1000 grains and the weight of 1000 grains on a dry-matter basis are 241.8 g; 216.7 g, respectively; it has the highest seed coat content, 9.85%, with a coat thickness of 0.2 microns. The 'Pamiati Hangildina' pea variety had the minimum seed coat content of 8.89% with a coat thickness of 0.2 microns and the maximum actual weight of 1000 grains and 1000 grains on a dry-matter basis 348.9 g; 314.3 g, respectively. The seed uniformity of all studied pea varieties is over 80%, except the 'Iuldash' pea variety (the average value is 77.66%). 7  Table 3. Table 3. Table 3. The 'Nerussa' bean grain is white, not large, with uniform linear dimensions (the length is less than 10 mm) compared to the 'Omichka' bean grain (white with grey strokes) and 'Lukeria' (black). The main grain fraction of the 'Omichka' and 'Lukeria' bean varieties is represented by a large grain with a spherical shape from 60.00 to 79.00% and a length from 11 to 12 mm. One unanticipated finding was that the 'Lukeria' and 'Omichka' bean grain is larger (but contains the smallest seed coat content of 10.21; 8.87%, respectively. The maximum value of this indicator of 11.01% was found in the 'Nerussa' variety.

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The pea and bean grains of all the studied legumes, except the 'Iuldash' pea variety, had a uniform, soft consistency. They were easily chewed and preserved the seed coat by the time they had sprouted. The 'Pamiati Hangildina' peas and the 'Omichka' beans showed the minimum cooking time. The best taste qualities were found in the 'Pamiati Hangildina' peas (4.8 points), 'Chishminskii 95' and 'Chishminskii 229' (4.5 points) and 'Omichka' (4.5 points), 'Lukeria' beans (4.3 points).
The grain microstructure of all varieties was also studied (Figures 2 and 3). The endosperm sections clearly show differences in structure.    Starch granules of 'Iuldash' peas and 'Nerussa' beans are loosely bound to the protein matrix, collected in complex combinations. It is due to the low protein content, insufficient to develop a continuous matrix structure. As a result, the protein has the form of granules. Cotyledons of 'Chishminskii 229' peas, 'Omichka' beans variety, are characterized by a loose microstructure, a fragile connection of starch granules with a protein matrix; most granules do not have protein interlayers. Grains of 'Chishminskii 95', 'Pamiati Hangildina' peas, and 'Lukeria' beans have a developed protein matrix with clearly spaced starch granules. The starch granules of the studied varieties have different shapes, sizes of the protein matrix and starch grains (Table 4). This difference was observed in soaking and germination patterns. Magnetic water treatment is an alternative to ion exchange, and its ease of use makes it an affordable, cost-effective, environmentally friendly technology (Alexandrov et al., 2017;Krasnova, 2018). The magnetic processing efficacy and practicality have been proven in dairy production technology (Fialkov and Kirgintseva, 2011;Fialkov and Kostyuchenko, 2012).
Using standard mathematical methods, regression equations and the R-squared value for the kinetic curves of hydrothermal treatment (soaking) of the studied legume varieties at a temperature of 14 ±2 °C and 18 ±2 °C were calculated (Table 5). Soaking time (X) ranged from 1 hour to 16 hours. As a result of a comprehensive analysis of the results obtained, the conditions for hydrothermal treatment (soaking) were determined, recommended for further use in the germination process: When using a temperature regime of 18 ±2 °C, the soaking time for 'Chishminskii 95', 'Chishminskii 229' pea grains is 6 hours, for 'Pamiati Hangildina' is 5 hours; the soaking time for 'Omichka' beans is 7 hours, for 'Lukeria' -8 hours, it is 1 hour less than at an identical temperature regime and model media of control No. 1, control No. 2 (Table 1).
Further studies showed that the maximum number of sprouted pea grains of 'Chishminskii 95', 'Chishminskii 229', 'Pamiati Hangildina' is 98.75%, the germination index is from 16.13 to 18.51, the minimum average germination time is from -0.54 to 0.62 days; the maximum number of sprouted bean grains of 'Omichka' and 'Lukeria' is 98.75%, the germination index is from 12.34 to 14.28, the minimum average germination time is from 0.70 to 0.81 days were observed when using the model medium (trial No.1).
The endosperm microstructure of the studied varieties was compared before and after germination. The changes are visible in Figures 5 and 6.  Figure 6. Figure 6. Figure 6. Photos of the bean grain microstructure: a -'Omichka' before germination; b -'Omichka' after germination; c -'Lukeria' before germination; d -'Lukeria' after germination There is visible disintegration of starch grains due to the diverse swelling of biopolymers. Changes in proteolytic activity as a result of germination were also studied. The data in Table 6 indicate differences between the samples (Russian state standard GOST 13979.9-69, 1969).

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Studies of the proteolytic activity of grains in the presence of trypsin showed that the proteolytic activity of trypsin in the studied varieties of peas is higher than in the bean grain. Considering that the value of the proteolytic activity of trypsin in the studied varieties depends on the suppressive activity of their inhibitors, it can be concluded: the higher the value of the proteolytic activity of trypsin in the studied varieties, the lower the inhibitory activity.
When summarizing the results of proteolytic (enzymatic) activity of bean and pea breeding varieties before and after germination, it was found that the proteolytic activity of trypsin in bean grains was lower compared to pea grain samples both dormant (native) and germinated. This indicates a higher inhibitory activity of both sprouted and native grains of the studied bean varieties compared to pea grain.
The proteolytic activity of trypsin at the pea and bean grain germination increases on average by 19.76 -46.42% and by 26.12 -48.45%, respectively. The activity of its own proteases weakens.
The results of the urease activity analysis of the studied pea and bean varieties before and after germination indicate that bean grains have a very low content of less than 0.01 pH while it is absent in all the pea varieties.
As a result of germination, the protein digestibility increases (Table 7) due to the transformations of nitrogenous substances. 'Pamiati Hangildina' peas and 'Omichka' beans had the maximum digestibility value both before and after germination.
A decrease in the content of fat, carbohydrates, and dietary fibre proteins indicates structural changes in the cell (Table 8).

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Considering the increased proteolytic activity of proteases in the germination process (Table 5), the legume protein is broken down to amino acids and becomes easily digestible, the content of amino acids increases.
A comparative analysis of the grain amino acid composition before and after germination is given in Table 9. Sprouting leads to a change in the concentration of individual amino acids relative to the total protein. It should be noted that the ratio of essential and non-essential amino acids changes in favour of the former. Thus, the relative concentration of lysine for beans and peas increases by 1.3 and 2.8 times (respectively), isoleucine-leucine by 2.6; 4.8 (respectively), phenylalanine by 1.3; 3.4 (respectively), valine by 3.9, 3.0 (respectively).
The fractional composition of the protein has also changed (Table 10). Albumin and globulin fractions have increased while the gluten fraction has reduced. The research findings convincingly show the structural changes in the grain of legumes due to germination, expressed in the higher protein availability and the increased phytochemical potential of legumes. In addition, the specific bean flavour, not always acceptable to potential consumers, practically disappears. It makes sprouted pea and bean grains advisable to use in the production of vegetable milk and other products of the dairy alternative's segment.

Discussion Discussion Discussion
The production of vegetable milk is based on water extraction, widely used in beverage production not only from legumes, but also from cereals, oilseeds and nuts with varying degrees of particle dispersion.
The technological process of producing vegetable milk from sprouted grain of the studied varieties consisted of a number of sequential operations (compilation of a hydromodule (sprouted grain: water), grinding and dispersing, extraction and filtration) (Veber et al., 2021).
The physico-chemical characteristics of vegetable milk from sprouted grain of the studied varieties according to the developed technology are given in Table 11, organoleptic indicators are presented in Table 12.  The taste is pleasant, sweetish with a malty aftertaste.
The taste is clean, sweetish, slightly pronounced bean smell and aroma, grassy The taste is sweetish, pronounced bean smell and aroma.
Color Pleasant, uniform throughout the mass of pale yellow.
White with a grayish tint.
Gray color with black tint.
A comparative analysis of the physico-chemical characteristics of vegetable milk with traditional products (pasteurized skimmed milk. 0.05 % fat and soy milk, 1.0% fat) revealed that vegetable milk, for example, made from sprouted peas of the 'Pamiati Hangildina' variety and beans of the 'Omichka' variety is superior (to pasteurized skimmed milk, 0.05% fat and soy milk, 1.0% fat) 1.2 and 1.6 times, respectively, in 16 terms of protein content; 1.6 and 1.1 times, respectively, in terms of dietary fiber content. The developed dispersions in comparison with soy milk are characterized by a reduced caloric content of 1.26 -1.35 times and have no significant difference in this indicator in comparison with pasteurized skim milk. It should be noted that the developed vegetable milk contains no allergens, GMOs and trans-isomers of fatty acids.

Conclusions Conclusions Conclusions
Summarizing the results obtained, it can be concluded that the proposed method of bean and pea soaking and germination can be successfully used for the vegetable milk production from peas and beans within the dairy alternatives' segment.
Thus, the findings on sprouting conditions for beans and peas of new breeding varieties and the biochemical changes occurring during the germination process provide evidence that dispersions based on sprouted grains of the studied crops can be used to make "dairy alternatives". Vegetable milk, having increased nutritional value and useful consumer properties can be used in the production of vega ice cream, fermented beverages, tofu (Patent No. 2782858, 2017), as well as in baking and to get products significantly superior in nutritional value to traditional ones. Ethical approval Ethical approval Ethical approval Ethical approval (for researches involving animals or humans) Not applicable.