A comparison of two hydrolysis parameter sets of sunflower and camelina seed cakes, and two methods for stabilizing protein hydrolysate

Abstract

Global vegetable oil production has a steady growth trend, specifically in Russia, where the production of vegetable oil is growing rapidly under the influence of domestic and external demand; this leads to the generation of enormous quantities of waste and by-products. Therefore, in recent years, new strategies have been proposed in order to dispose of waste and by-products of oilseed crops. This study highlights the enzymatic hydrolysis technique for obtaining protein hydrolysates from two by-products of the sunflower and camelina oil industries. The objective of this research was to compare the yield of the amino acids content at the end of a 5-h hydrolysis of sunflower cake (SFC) and camelina seed cake (CSC), using alkaline protease enzyme “Protozyme,” produced by the Torgovy Dom Biopreparat company, under two different hydrolysis conditions: pH (7–9) and temperature (40–45 °C) for the first hydrolysis parameter set, and pH (9–11) and temperature (45–50 °C) for the second one, using two enzyme-substrate ratios (w/v) (1:10 and 1:20). The study also compared the effectiveness of two methods for stabilizing protein hydrolysate: using sorbic acid and convection drying. Based on the results, it was found that, the optimal parameters for SFC hydrolysis are: an enzyme-substrate ratio (1:20) and hydrolysis conditions of (t: 45.0–50.0 °C and pH: 9–11) with an increase in the lysine, methionine, and tryptophan content of the finished hydrolysate by 55.4, 32.6, and 55.6%, respectively, compared to the control. For CSC hydrolysis the optimal parameters are the enzyme-substrate ratio (1,10) and hydrolysis conditions (t: 40.0–45.0 °C and pH: 7–9), with an increase in the lysine, methionine, and tryptophan content of the finished hydrolysate by 25.3, 51.9, and 41.4%respectively compared to the control. Also, it was found that, the most effective method for stabilizing hydrolysates was the addition of sorbic acid. Over 3 months of storage of SFC hydrolysate using sorbic acid, lysine and tryptophan contents decreased by only 3.2 and 7.1% respectively, while the methionine content remained constant and unchanged. The decrease in lysine content was 1%, while methionine and tryptophan contents remained stable over 3 months of storage of CSC hydrolysate.

1 Introduction

The world’s population continues to grow, and is expected to reach nearly 10 billion by 2050 (Obiero et al., 2019). Therefore, several escalating issues have emerged around the world, e.g., over-exploitation and mismanagement of natural resources, accumulation of waste in the food processing, also in food and feed manufacturing sector, and ineffective waste-management policies, that affect air, water, and soil, leading to land degradation, climate change, environmental degradation, and health hazards due to the persistence of hazardous materials (Gebrekidan et al., 2024). As the growing of global population, the demand for edible plant oils also on the rise, thus various cultivars of oil-bearing plants such as palm, soybean, rapeseed, camellia, corn sunflower etc. are widely grown worldwide, using agricultural machinery and high-tech methods (Zhou et al., 2020).

Oil extraction from these plants produces enormous quantities of by-products, that can be used as animal feed, biogas generation, or to extract biologically active substances, that can be used as nutritional supplements or medicines for humans and animals (Rashwan et al., 2023). Delays in processing these oil extraction byproducts, or insufficient processing facilities, cause significant environmental and economic damage. This is due to their high protein and fat content, which rapidly decomposes and becomes rancid when exposed to light or air and under bacterial influence, releasing odors and toxic compounds, rendering them unusable and thus increasing production costs.

Oilseed cakes are broadly classified into two categories; edible and inedible oilseed cake. Edible oilseed cake.is the residue resulting by pressing edible oilseeds, which is used to meet part of the nutritional requirements of both animals and humans. There are also oilseed cakes, which are produced by pressing inedible oilseeds and can be used partially to meet animal feed requirements, but not for human consumption, due to their content of antinutrients such as tannins, phytates, trypsin inhibitors, pepsin inhibitors, hemaglutinin, etc. However, heat treatment can deactivate some antinutrients, thus allowing their use as a food additive after passing allergy testing (Sunil et al., 2016).

Published literature has shown that the nutritional value of oilseed cakes is very rich in protein, making them a promising option for obtaining high-protein products or for obtaining active ingredients used in many food and pharmaceutical industries (Renzyaeva et al., 2020).

Oilseed cakes are rich in fiber and have a high concentration of non-starch polysaccharides such as cellulose and hemicellulose (Xylan), unsaturated fatty acids, proteins and biopeptides and contain various antioxidants, and vitamins (Renzyaeva et al., 2020; Sarkar et al., 2021; Sousa et al., 2024), It should be noted that its fat content varies depending on the method of oil extraction during pressing (Singh et al., 2022). Subsequent treatments also affect the ratio of insoluble and soluble fiber, carbohydrates, vitamins, and antinutrients and aim to maximize bioavailability. Therefore, to enhance the nutritional value of oilseed cakes, such as increasing protein availability and eliminating anti-nutrients, effective strategies have been adopted in recent years, such as heat treatments under pressure (extrusion), heating, cooking, fermentation, protein isolation, and hydrolysis (Goryunov and Feryabkov, 2024; Romanov, 2025; Belyshkina et al., 2025). Biologically active peptides with pharmaceutical applications can also be obtained; where peptides with antidiabetic activity (e.g., α-glucosidase inhibitor peptides) were isolated by applying improved enzymatic hydrolysis parameters to camellia cake (Zhang et al., 2023), which is a by-product of cold-pressing camelina oil extraction., and it is a nutritionally and biologically valuable material with a high protein content, and favorable amino acid profile, as well as a considerable fat content rich in alpha-linolenic acid, and it contains significant amounts of bioactive compounds (tocopherols, phenolic acids, flavonoids, and biopeptides) (Rakita et al., 2025).

Many reports indicate the widespread use of hydrolysis on oilseed cakes due to its effect on the physical and chemical properties of hydrolyzed proteins, as all hydrolyzed proteins showed better functional properties than the original protein. The water-holding capacity, foaming activity, and emulsifying activity of the hydrolysates increases with the increase in levels of hydrolysis (Demirhan and Özbek, 2013).

Sharikov and others have also demonstrated that, hydrolysis of extruded sunflower cake increased protein solubility by 3.8 times and the concentration of reducing sugars by 2 times compared to the original substrate (Sharikov et al., 2016).

In Russian Federation, the production of sunflower seed cake has been increasing every year and it is highest than all other countries (Bagul, 2024). After oil extraction, sunflower cake retains 1–23.6% residual oil, 29–43.4% protein, and 13%–36% fiber, highlighting its nutritional potential. (Grahovac et al., 2025). Therefore, current research focuses on applying hydrolysis to sunflower cake to yield highly digestible proteins and bioactive peptides.

2 Materials and methods

2.1 Materials

2.1.1 Oilseed cake

The winter Camelina variety (Camelina sativa L.), named “Adamas,” grown in the Volgograd region of the Russian Federation, and sunflower variety (Helianthus annuus L.), named “Status,” grown in the Krasnodar region of the Russian Federation, were used to obtain oilseed cakes for this study: sunflower cake (SFC), and camelina seed cake (CSC). The winter Camelina variety “Adamas,” has been developed by the Russian Research and Design Technological Institute of Sorghum and Corn, and the LLC Pokrovskoe Company, and listed in the State Register of the Russian Federation for Oilseed Crop Growing Regions since 2017, it is distinguished by its high seed fat content (43.9%–44.5%). The oil contains 2.94% erucic acid. The weight of 1,000 seeds is1.3–1.4 g (Zhuzhukin et al., 2015). The single-line sunflower hybrid “Status” has been developed by the Federal State Budgetary Scientific Center VNIIMK in 2020. Seed oil content was 48.0%, hull content was 28.7%, and 1,000-seed weight was 60.6 g (Gorbachenko et al., 2021).

The oilseed cakes were obtained from the vegetable oils industry after cold-press extraction, then they were dried and stored at 4 °C in darkness.

For the study, samples of high-protein sunflower cake with a residual fat content of at least 4.0% were obtained from the Russian company Agrosadovod (Moscow, Russia). Вefore grinding, the (SFC) samples were tested for key physical and chemical quality indicators in accordance with the state standard GOST 80-96 Sunflower cake, 2002. The protein and fiber content at the initial stage was 42.7 and 14.7%, respectively, according to the methods of estimating protein and fiber used in (GOST 13496.4, 2019) and (GOST 31675, 2012).

High-quality camelina seed cake (CSC) was supplied by company Russkoye seno (Moscow, Russia), Where they were dried under industrial drying conditions to a constant moisture content of no more than 11.5%. Samples of camelina cake contained protein 38.9% and fiber 10.1% at the initial stage, according to the methods of estimating protein and fiber used in (GOST 13496.4, 2019) and (GOST 31675, 2012).

2.1.2 The enzyme complex

The alkaline bacterial proteases of Bacillus licheniformis, under the trade name “Protozyme,” produced by the Torgovy Dom Biopreparat company (Serpukhov, Russia) was used in this study. This enzyme complex has the following properties: it is a bacterial alkaline protease with an activity of 50,000 u/g in conditions of pH: 5.5–11.0 and a temperature range of 25.0–70.0 °C, with an optimal temperature range of 55 °C to 65 °C and an optimal pH range of 6.0 to 10.0. Also it is a stable enzyme in the presence of chelating chemicals, such as EDTA and has a low susceptibility to tri-polyphosphoric acid salts.

2.2 Methods

2.2.1 Experimental design

A factorial design was chosen for the study, which was conducted in two stages. The first stage involved determining the effectiveness of the hydrolysis process parameters, based on the mass fraction of amino acids obtained in the final hydrolysate. The initial values of the amino acid contents of lysine, methionine, and tryptophan in the cakes collected for further biotechnological processing served as control samples at this stage. The final hydrolysates served as test samples.

The second stage involved determining the effectiveness of the hydrolysate stability methods, based on the preservation of the studied amino acids during storage. Sorbic acid and convection drying were involved as stability methods of the final hydrolysate. The content of studied amino acids in the hydrolysate samples was examined immediately after the hydrolysis process (control), during 1 month, and then during 3 months of storage (for stability testing samples).

To create the DoE (Design of Experiments) by using Excel, all factors were identified and their levels coded for example: The first factor was temperature, and the second factor is pH, each factor has two levels (low and high levels) etc.

2.2.2 Sample preparation

The sunflower and camelina seed cakes samples were subjected to preliminary heat treatment in a laboratory drying oven (ШСВ-200-01, Orionterm LLC, Russia) at a temperature of 100–110 °C for 30 min to inactivate some anti-nutrients and eradicate undesirable bacteria.

The enzyme complex was activated prior to hydrolysis according to the manufacturer’s recommendations.: 1000 ± 0.01 g of enzyme preparation and 15 L of distilled water (t: 30–35 °C, pH: 7–9), combined in a glass container and subjected to slow stirring until the enzyme powder was completely dissolved.

2.2.3 Enzymatic hydrolysis

Both sunflower and camelina seed cakes were mixed with the enzyme solution at the ratio of 1:10 or 1:20 and incubated for 5 h, under different temperature and pH conditions as per experimental design in Figure 1.

Figure 1

For a ratio of 1:10, the enzyme concentration in the solution (w/v) is 1.32%, and the dry cake concentration in the enzyme solution (w/v) is 52.4%.

For a ratio of 1:20, the enzyme concentration in the solution (w/v) is 1.57%, and the dry cake concentration in the enzyme solution (w/v) is 58.8%.

To obtain hydrolysate samples with different process parameters, the experimental conditions shown in the scheme Figure 1 were selected.

The prepared samples were placed in industrial thermostats with air convection at a speed of 0.5 m/s, then hydrolysis was carried out in two sets of parameters to compare the yield. In our study these parameters were temperature and pH conditions, also enzyme-substrate ratio. The temperature and pH ranges of (t: 40–45 °C, and pH: 7–9); were compared to the temperature and pH ranges of (t: 45–50 °C and pH: 9–11), using two enzyme-substrate ratios (1:10 and 1:20). The efficiency of the parameters was determined by the mass fraction of amino acids obtained in the finished hydrolysate. The hydrolysis process was carried out for 5 h. During the hydrolysis, the moisture content of mixture decreased by an average of 20.0 ± 1.0%.

2.2.4 Stability methods

Moisture and pH are crucial factors for the stability of hydrolyzed proteins; therefore, two techniques were employed to ensure the stability of the final product of the hydrolysis process (Dabbour et al., 2021; Belyshkina et al., 2025). Sorbic acid was added to some samples at the final hydrolysis stage at a rate of 0.2 g per 10.0 kg (first treatment samples), other ones were dried in a laboratory scale convection dryer at 45.0 ± 0.05 °C, air velocity of 0.8 m/s, and a relative humidity of no more than 55.0% for 3.5 h (second treatment samples). The exact amount of sorbic acid, calculated on a dry matter basis (w/w), was 1.82%. The stability of hydrolysates in the second method was achieved by using convection drying in a convection thermostat from the Optimum series (Omnislab, Russia). Hydrolysate samples were dried until a moisture content of 11%–12% was reached.

All hydrolysate samples were stored in hermetically sealed containers made of food-grade plastic in a dry, ventilated warehouse at t: 21–25 °C and a relative air humidity of 60%–65%.

2.3 Determination of amino acids

Quantitative determination of the amino acids was made after isolating the protein in the cakes and resulting hydrolysates on a Kapel-105 M capillary electrophoresis device from the Russian company Lumex, (Saint-Petersburg, Russia) for the methionine and lysine analysis (GOST 31480, 2012). For the analysis of tryptophan using high-performance chromatography HPLC, a chromatographic column with dimensions of 125 × 4 mm, with a C18 filler with a particle size of 3 μm with a standard eluent of 0.1% TFA in water was used, with fluorescence detection from (BiokhimMak ST, Germany) GOST 32201, 2013, (ISO 13904:2005). Control and test samples underwent the same treatment and analysis. The protein isolate from sunflower and camelina cake and hydrolysate met the requirements of GOST R 72353, 2025. To prepare for the protein fragments isolation, the following steps were taken: samples were ground into a particle of 0.1–0.2 mm. Extraction of the protein fraction was performed with a 10% aqueous sodium chloride (NaCl) solution, with immediate separation of the insoluble precipitate and subsequent filtration of the resulting extract. To precipitate the extract, the resulting mixture was treated with a 3–5% aqueous succinic acid solution. The next stage of sample preparation involved centrifugation to separate the resulting protein fraction, followed by washing with water.

2.4 Statistical analyses

Forty-two samples of each of the sunflower and camelina oil cakes were used. Samples for each cake was divided into three parts: a control and two parts that underwent enzymatic hydrolysis with two different sets of hydrolysis parameters.

Two replicates were carried out in each experiment. All data were analyzed by SPSS software. The level of statistical significance was set at 5%. Data were expressed as means with standard deviations. Student’s t-test was used to evaluate significant differences at p ≤ 0.05 between the means.

3 Results

The results of the amino acids contents (lysine, methionine and tryptophan) in the protein isolate hydrolysates of sunflower cake using enzyme-substrate ratios of 1:10 are presented in Table 1.

As is obvious from Table 1, that the use of an enzyme-substrate ratio of 1:10, with the specified parameters of the biotechnological process (t:40.0–45.0 °C/pH:7–9 and t:45.0–50.0 °C/pH:9–11) contributes to a reliable increase in the content of lysine, methionine and tryptophan in the finished hydrolysate.

When studying the lysine content in the control samples, it was found that the amount of this amino acid in 100 g of protein was 2.0 ± 0.07 g. Subjecting sunflower cake to enzymatic hydrolysis under conditions of t: 40.0–45.0 °C and pH: 7–9 contributes to a reliable increase in the lysine content by 15.0%, and ultimately amounted to 2.3 ± 0.09 g/100 g of protein. Carrying out hydrolysis under conditions of t:45.0–50.0 °C and pH:9–11, an increase in the lysine content in the finished hydrolysate by 27.9% relative to the initial value was established, and amounted to 2.6 ± 0.16 g/100 g protein. It should be noted that the significant difference in the lysine content in the composition of experimental samples of hydrolysates produced under the specified conditions is at least 13.5%.

The methionine content in sunflower cake was also determined (1.9 ± 0.12 g/100 g protein). When the sunflower cake was hydrolyzed under conditions of (t: 40.0–45.0 °C and pH: 7–9), it was observed a reliable increase in the methionine content in the finished hydrolysate by an average of 5.3%, which amounted to 2.0 ± 0.07 g/100 g protein. Samples of finished hydrolysates produced under the conditions of t: 45.0–50.0 °C and pH: 9–11, with an enzyme-substrate ratio of 1:10, contained methionine at the level of—2.3 ± 0.17 g/100 g protein, which is 21.1% higher than the initial value of the indicator. The difference in the values of the test samples was 15.4%.

The study found, that the tryptophan content of sunflower cake protein was 0.9 ± 0.08 g/100 g protein. Obtaining hydrolysate samples in conditions of t:40.0–45.0 °C and pH 7–9 resulted in a significant increase in tryptophan content by 11.1%, which is 1.0 ± 0.07 g/100 g protein. In the hydrolysate samples produced under conditions of t: 45.0–50.0 °C and pH: 9–11, with the specified enzyme-substrate ratio of 1:10, the tryptophan content was found to be 1.2 ± 0.14 g/100 g protein (p ≤ 0.05); which is 32.9% higher than the initial value. The difference in the values of the experimental samples was 20.5%.

As can be seen from the experimental results, the specified biotechnological parameters of the sunflower cake hydrolysis (t: 45.0–50.0 °C and pH: 9–1), in the specified enzyme-substrate ratio of 1:10, contribute to a more effective degree of hydrolysis, than the parameters of (t: 40.0–45.0 °C and pH: 7–9).

From the data presented in Table 2, noted that the use of an enzyme-substrate ratio of 1:20, with the specified parameters of the biotechnological process (t: 40.0–45.0 °C/pH: 7–9 and t: 45.0–50.0 °C/pH: 9–11) also contributes to a significant increase in the content of lysine, methionine and tryptophan in the composition of the finished hydrolysate.

The use of enzymatic hydrolysis under conditions of t: 40.0–45.0 °C and pH: 7–9, contributes to a reliable increase in the lysine content by 20.0%, and ultimately amounted to 2.4 ± 0.12 g/100 g of protein. When carrying out hydrolysis under conditions of t: 45.0–50.0 °C and pH: 9–11, an increase in the lysine content in the finished hydrolysate by 55.4% relative to the initial value was found, and amounted to 3.1 ± 0.17 g/100 g of protein (p ≥ 0.01). It should be noted that the reliable difference in the lysine content in the composition of the experimental samples of hydrolysates produced under the specified conditions is at least 29.2%.

The hydrolytic processing of the sunflower cake at t: 40.0–45.0 °C and pH: 7–9 contributed to a reliable increase in the methionine content in the finished hydrolysate by an average of 10.5%, which amounted to 2.1 ± 0.19 g/100 g of protein. Samples of finished hydrolysates produced under the conditions of t: 45.0–50.0 °C and pH: 9–11, with the specified enzyme-substrate ratio of 1:20, contained methionine at a level of 2.5 ± 0.13 g/100 g of protein (p ≤ 0.01), which is 32.6% higher than the initial value of the indicator. The difference in the values of the experimental samples was 19.1%.

Hydrolysate samples produced at 40.0–45.0 °C and pH 7–9 significantly increased tryptophan content by 22.2%, or 1.1 ± 0.08 g/100 g protein. Hydrolysate samples produced at 45.0–50.0 °C and pH 9–11, with an enzyme-to-substrate ratio of 1:20, yielded a tryptophan content of 1.4 ± 0.09 g/100 g protein (p ≤ 0.01), 55.6% higher than the initial value. The difference in values between the experimental samples was 27.3%. Based on the amino acid yields obtained from the hydrolysis of sunflower cake using two sets of parameters, our study proposes using this set of parameters. t: 45.0–50.0 °C and pH: 9–11 with an enzyme-substrate ratio of 1:20, which allow obtaining the maximum yield of amino acids.

The content of some essential amino acids (lysine, methionine, and tryptophan) in the protein isolated from CSC hydrolysates was determined.

The results of the analysis of the protein isolated from the hydrolysates obtained using parameters of (t: 40,0–45,0 °C/рН: 7–9 and t: 45,0–50,0 °C/рН: 9–11) with an enzyme-substrate ratio of 1:10, are presented in Table 3.

The results presented in Table 3 show that the use of an enzyme-to-substrate ratio of 1:10, with the specified biotechnological process parameters (t: 40.0–45.0 °C/pH: 7–9 and t: 45.0–50.0 °C/pH: 9–11), significantly increases the lysine, methionine, and tryptophan content of the finished CSC hydrolysate. By studying the lysine content of the original CSC, it was found that the amount of this amino acid in 100 g of protein was 1.62 ± 0.27 g/100 g of protein.

The use of enzymatic hydrolysis under conditions of t: 40.0–45.0 °C and pH: 7–9, contributes to a reliable increase in lysine content by 25.3% (and ultimately amounted to 2.03 ± 1.12 g/100 g of protein; p ≤ 0.01). When carrying out hydrolysis under conditions of (t: 45.0–50.0 °C and pH: 9–11), an increase in lysine content in the finished hydrolysate by 13.0% relative to the initial value was found, and amounted to 1.83 ± 0.77 g/100 g. It should be noted that the reliable difference in lysine content in the composition of experimental samples of hydrolysates produced under the specified conditions is at least 10.9%.

The methionine content in the camelina cake was also found to be 0.52 ± 0.21 g/100 g protein, when performing hydrolysis on CSC using parameters at t: 40.0–45.0 °C and pH: 7–9 contributed to a reliable increase in the methionine content in the finished hydrolysate by an average of 51.9%, which amounted to 0.79 ± 1.03 g/100 g protein (p ≤ 0.01). Samples of the finished hydrolysates produced under the conditions of t: 45.0–50.0 °C and pH: 9–11, with the specified enzyme-substrate ratio of 1:10, contained methionine at a level of 0.72 ± 0.29 g/100 g protein, which is 38.4% higher than the initial value. The difference in the values of the experimental samples was 9.7%.

The study found that the tryptophan content in the protein isolate of CSC was 0.29 ± 0.15 g/100 g protein. Obtaining hydrolysate samples under conditions of t: 40.0–45.0 °C and pH: 7–9 contributes to a reliable increase in tryptophan content by 41.4%, which is 0.41 ± 0.34 g/100 g protein (p ≤ 0.01). In hydrolysate samples produced under conditions of t: 45.0–50.0 °C and pH: 9–11, at the specified enzyme-substrate ratio of 1:10, the tryptophan content was found to be 0.39 ± 0.19 g/100 g protein, which is 34.5% higher than the initial value of the indicator. The difference in the values of the experimental samples was 5.1%.

As can be seen from the experimental results, the specified biotechnological parameters of hydrolysis of camelina cake (t: 40.0–45.0 °C and pH: 7–9), in the specified enzyme-substrate ratio of 1:10, contribute to a more effective degree of hydrolytic treatment than the parameters (t: 45.0–50.0 °C and pH: 9–11).

From the results presented in Table 4, the use of the enzyme-substrate ratio of 1:20, under the specified parameters of the biotechnological process (t: 40.0–45.0 °C/pH: 7–9 and t: 45.0–50.0 °C/pH: 9–11) contributes to a reliable increase in the content of lysine, methionine and tryptophan in the composition of the finished hydrolysate based on camelina cake. The use of enzymatic hydrolysis under the conditions of t: 40.0–45.0 °C and pH: 7–9, contributes to a reliable increase in the lysine content by 1.67% (and ultimately amounted to 1.89 ± 1.02 g/100 g protein; p ≤ 0.05). Hydrolysis at 45.0–50.0 °C and pH 9–11 resulted in a 4.9% increase in lysine content in the finished hydrolysate relative to the initial value, reaching 1.70 ± 1.07 g/100 g protein. It should be noted that the significant difference in lysine content between the experimental hydrolysate samples produced under these conditions was at least 11.2%. When using parameters during hydrolysis of CSC (t: 40.0–45.0 °C and pH: 7–9) contributed to a reliable increase in the methionine content in the hydrolysate by an average of 42.3%, which amounted to 0.74 ± 0.53 g/100 g protein (p ≤ 0.05). Samples of CSC hydrolysates produced under the conditions of t: 45.0–50.0 °C and pH: 9–11, at the specified enzyme-substrate ratio of 1:20, contained methionine at a level of 0.60 ± 0.31 g/100 g protein, which is 15.4% higher than the initial value of the indicator. The difference in the values of the experimental samples was 23.3%.

Obtaining hydrolysate samples under conditions of t: 40.0–45.0 °C and pH: 7–9 contributes to a reliable increase in tryptophan content by 37.9%, which is 0.40 ± 0.16 g/100 g protein (p ≤ 0.05). In hydrolysate samples produced under conditions of t: 45.0–50.0 °C and pH: 9–11, with the specified enzyme-substrate ratio of 1:20, the tryptophan content was found to be 0.31 ± 0.09 g/100 g protein, which is 6.9% higher than the initial value of the indicator. The difference in the values of the experimental samples was 29.3%.

Based on the obtained results of the amino acid content, it was established that in a comparative study of the efficiency of biotechnological parameters for the hydrolysis of CSC, it is necessary to highlight t: 40.0–45.0 °C and pH: 7–9 with an enzyme-substrate ratio of 1:10, which allow obtaining the maximum yield of essential amino acids in a short time.

The Figure 2 below shows the results of the effectiveness of the sorbic acid stability methods for SFC hydrolysates after the hydrolysis process, during 1 month of storage, and during 3 months.

Figure 2

By analyzing the charts presented in Figure 2, It was found, that the amino acid stability methods, used in the hydrolysate demonstrated varying degrees of effectiveness during storage for 1 month and 3 months. The addition of sorbic acid led to enhanced stability of lysine. Over 3 months of storage, lysine content decreased by only 3.2%, reaching 3.0 ± 0.09 g/100 g protein (p ≥ 0.05) relative to the initial value. Using sorbic acid to stabilize the SFC hydrolysate composition, we demonstrated its effectiveness in preserving methionine, with its content remaining unchanged during 3 months of storage. Tryptophan content, when using sorbic acid, also remained stable throughout the entire hydrolysate storage period, decreasing by only 7.1% (p ≥ 0.05) relative to the initial value.

By analyzing the results of the second stability method used with SFC hydrolysates, which is the convection drying method, it was found, that this method is less effective in stabilizing lysine than sorbic acid. Over 3 months of storage, lysine content decreased by 9.7%, reaching 2.8 ± 0.09 g/100 g protein (p ≤ 0.05) relative to the initial value.

The use of convection drying as a method for stabilizing the composition of SFC hydrolysates also showed lower efficiency for the preservation of methionine, the content of which changed by 12.0% during storage for 3 months and amounted to 2.2 ± 0.06 g/100 g protein (p ≤ 0.05). The tryptophan content, when using convection drying, also changed after the first and second periods of hydrolysate storage and decreased by 28.6% (1.0 ± 0.04 g/100 g protein; p ≤ 0.05) relative to the initial value.

Thus, the study concluded that, the greatest effectiveness in stabilizing amino acids in SFC hydrolysate was observed when using sorbic acid, and it was less effective when using the convection drying method.

The results of the effectiveness of the convection drying stability method for CSC hydrolysates after the hydrolysis process, during 1 month of storage, and during 3 months are show in the Figure 3.

Figure 3

For the camelina seed cake hydrolysates, different stability methods also showed varying levels of effectiveness, when comparing the content of the studied amino acids immediately after the camelina cake hydrolysis process, during 1 month and 3 months of preservation by sorbic acid addition and by convection drying. The results are shown in Figure 3. Thus, the analysis of the studied amino acid content showed that adding sorbic acid to CSC hydrolysate, helped stabilize lysine content. Over 3 months of storage, lysine content decreased by only 1.0%, reaching 20.1 ± 1.12 g/100 g protein (p ≥ 0.05) relative to the initial value. Our study also demonstrated that the use of sorbic acid as a stabilizer for hydrolysates of CSC is effective in maintaining methionine content for up to 3 months of storage, its content remaining unchanged during 3 months of storage. Tryptophan content also remained stable throughout the entire storage period of the hydrolysate when using sorbic acid. By studying the effectiveness of the convection drying stability method for CSC hydrolysates after the hydrolysis process, during 1 month of storage, and during 3 months, we found that this method was less effective in stabilizing lysine than sorbic acid. Over three months of storage, lysine content decreased by 4.4%, reaching 19.4 ± 1.01 g/100 g protein (p ≥ 0.05) relative to the initial value. The use of convection drying as a method for stabilizing the composition of hydrolysates of CSC also showed lower efficiency for the preservation of methionine, the content of which changed by 5.1% during storage for 3 months and amounted to 7.5 ± 0.77 g/100 g protein (p ≤ 0.05). The tryptophan content, when using convection drying, also changed after the first and second periods of hydrolysate storage and decreased by 17.1% (3.5 ± 0.24 g/100 g protein; p ≤ 0.05), relative to the initial value.

Thus, the study showed a high degree of efficiency of the stabilizing properties of sorbic acid method, compared to the method of conventional drying of CSC hydrolysates.

4 Discussion

The production of hydrolysates on an industrial scale requires the development of appropriate parameters for the bioconversion process in order to obtain high-protein value hydrolysates for using in the production of food and feed products. The use of a complex of proteolytic enzymes with a specific substrate ratio and under appropriate temperature and pH conditions allows for the breakdown of high molecular weight proteins into smaller protein fragments, thus increasing the nutritional value of the feedstock.

Hydrolysis cannot create additional carbon or nitrogen atoms to form new amino acids; it only breaks existing bonds. In fact, you might observe a slight increase in the relative concentration of some amino acids (such as lysine and methionine) compared to others when certain protein sources are hydrolyzed, because hydrolysis is breaking down proteins into smaller peptides (Brosnan et al., 2007) and exposing previously buried hydrophobic amino acid residues (including aromatic residues such tryptophan) (Liu et al., 2026). Consequently, proteins unfold to expose additional peptide cleavage and binding sites, facilitating enzyme-substrate interactions (Liu et al., 2026).

Unlike harsh acid hydrolysis, enzymatic hydrolysis preserves the original composition of amino acids because it does not cause the severe thermal degradation of sensitive amino acids like tryptophan or methionine and lysine. (Moktip et al., 2025). During hydrolysis, the protein (folded into complex 3D shapes.) unfolds, resulting in modifying of the native configuration and exposure of amino acids previously buried inside the intact protein, and may in addition induce amino acid modifications (Özogul et al., 2019; Mishra et al., 2024) exposes buried peptide bonds, previously blocked from analytical detection.

Based on the amino acid content obtained after the hydrolysis process, the optimal parameters for the hydrolysis process were determined for both sunflower and camelina seed cakes. The most effective hydrolysis for sunflower cakes was achieved using the following parameters: enzyme-substrate ratio is 1:20 and hydrolytic treatment conditions are (t: 45.0–50.0 °C and pH: 9–11).

Sunflower cake has a more crumbly and porous structure, with less densely packed cell walls than other types of cake. This is due to the low lignin content of the cell walls, the high mass fraction of hemicellulose, and the less dense packing of cellulose fibers (Sousa et al., 2023). Therefore, the cell walls of sunflower cake are more easily broken down by enzymes, accelerating the hydrolytic processing of this raw material. However, when reducing the concentration of the enzyme preparation, the parameters of the hydrolytic process must be maintained at the level of t: 45.0–50.0 °C and pH: 9–11, which allows create suitable working conditions for the protease enzyme in terms of temperature and acidity. The results of the amino acids obtained in the hydrolysate of camelina seed cake allow us to determine that the most effective biotechnological parameters for the hydrolysis process, which are: an enzyme-to-substrate ratio of 1:10 and hydrolytic treatment conditions (t: 40.0–45.0 °C/pH: 7–9).

During the hydrolytic treatment of CSC, the physico-mechanical properties of CSC structure is also taken into account, which requires more concentrated enzyme solutions. It was found that the cellular structures of CSC are denser for the following reasons: high lignin content; low bioavailability of cellulose fibers; high concentration of structural polysaccharides. For this reason, the cell walls of CSC are more resistant to enzymatic action, which slows the fermentation process compared to SFC, requiring an increase in the enzyme solution concentration. However, the hydrolysis conditions for CSC are milder than those for processing SFC. Compared to the SFC, the CSC contains less moisture and has a higher residual oil content, requiring mild operating conditions of fermentation to prevent oxidation and increase the free amino acids content. Based on the content of amino acids in the resulting hydrolysates, we found that stability of hydrolysates by adding sorbic acid demonstrates an effective method for preserving amino acids content during storage. In addition, numerous studies have shown that, sorbic acid has the following properties: antimicrobial activity, safety, solubility in semi-solid and highly viscous media, and biochemical compatibility with the hydrolysate, therefore, it is considered a good option for use (Nemes et al., 2020).

5 Conclusion

Based on the obtained results, it can be concluded that the efficient production of protein hydrolysates from various plant materials requires individually selected optimal technological conditions for the enzymatic hydrolysis for each biomaterial. Also, it was found that, the most effective method for stabilizing hydrolysates was the addition of sorbic acid. The study recommends using the optimal parameters for SFC hydrolysis are: an enzyme-substrate ratio (1:20) and hydrolysis conditions of (t: 45.0–50.0 °C and pH: 9–11). For CSC hydrolysis the optimal parameters are the enzyme-substrate ratio (1:10) and hydrolysis conditions (t: 40.0–45.0 °C and pH: 7–9). It also recommends using sorbic acid to stabilize protein hydrolysates and suggests further research into combining sorbic acid and convection drying methods for hydrolysate stabilization.

This study was conducted as part of a comprehensive research and development project to develop a device and technology for industrial-scale bioconversion. This work presents preliminary results on optimal conditions for hydrolysis of plant byproducts, with a view to their subsequent implementation in the production process.

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