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Article

Determination of the Optimal Biotechnological Parameters for Industrial Production of Protein Hydrolysates for Animal Feed

by
Marina Belyshkina
,
Tamara Kobozeva
,
Mikhail Zagoruiko
,
Oksana Serebryakova
,
Maisoon Shaaban
*,
Tatiana Ananeva
and
Igor Bashmakov
Federal State Budgetary Scientific Institution “Federal Scientific Agroengineering Center VIM”, 5. 1st Institutskiy Proezd Str., Moscow 109428, Russia
*
Author to whom correspondence should be addressed.
Fermentation 2025, 11(4), 209; https://doi.org/10.3390/fermentation11040209
Submission received: 17 February 2025 / Revised: 4 April 2025 / Accepted: 8 April 2025 / Published: 10 April 2025
(This article belongs to the Section Industrial Fermentation)
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Versions Notes

Abstract

:
The main disadvantages of using soybean oil extraction waste as a raw feed material are its high contents of fiber, fat, and anti-nutritional factors. Therefore, several processing methods such as extrusion and hydrolysis are used to overcome these disadvantages and increase the availability of high-quality proteins to animals from this by-product. This study is concerned with the hydrolysis of extruded soybean meal in the presence of bacterial alkaline proteases. The effects of various process parameters were investigated to determine the optimal process parameters for hydrolysis in terms of the total free amino acid and amine nitrogen contents. The experiment included two sets of parameters that were selected for comparison: the temperature and pH in ranges of t 45–50 °C, pH 8–11, compared to the temperature and pH ranges of t = 40–45 °C and pH 7–9, using three enzyme/substrate ratios (1:10, 1:20, and 1:30). The protein hydrolysate was stored for three months after it was treated with two different preservatives (sorbic acid and thymol). Based on the results, it was found that the total free amino acid content was higher when the temperature range was 45–50 °C, the pH range was 8–11, and sorbic acid was used as a preservative.

1. Introduction

Despite the wide range of feed technologies developed and implemented in Russia, the development of feed additive manufacturing biotechnologies remains the most dynamically developing industrial sector [1]. The importance of biotechnological developments for the needs of the feed industry is justified by the urgent need for economically profitable, safe, effective, and adapted technologies for the large-scale introduction of feed products. Such developments should be focused on simplifying the selection and preparation of rations, taking into account the relevant needs of farm animals and poultry, and of course, increasing efficiency in obtaining products [2,3].
The relevance of biotechnological trends in the development of feed production is also due to environmental factors that are based on the development of environmentally friendly consumer products [4,5,6]. The most important biotechnological method used in this regard is the hydrolysis method to obtain protein hydrolysates as a source of high-protein feedstuff.
As a protein source in feed, protein hydrolysate is a special product produced by processing high-molecular-weight raw materials to the level of peptides and polypeptides (low-molecular-weight materials and most easily digestible proteins) of animal and plant origin [7]. Protein hydrolysates have properties determined by regulating the parameters of the hydrolytic process and may have nutritional, therapeutic, antitoxic, and other properties. The raw materials for protein hydrolysate production can be various components of plant or animal origin or native plant components; however, the most effective and economically advantageous options are recyclable products: meal, cake, food waste, and others [8,9,10,11,12,13]. Soybean oil industry waste is the first choice for animal feed production in Russia due to its high availability and low price. However, the presence of anti-nutritional factors limits its use in animal feed, such as aquaculture feed. The concentrations of anti-nutritional factors can be reduced in the feedstuff by using various techniques, such as extrusion and fermentation, including hydrolysis [14].
The increasing interest in soybean meal as a raw material for the production of hydrolyzed feed components is growing due to the limited resources of animal proteins, which were previously actively used in the production of feed products. Soybean meal, as a by-product of oil manufacture, is characterized by an abundance of protein, lipid fractions, and biologically active components; therefore, it can become a full-fledged alternative for other protein feedstuffs, such as fish meal or blood meal in the diets of farm animals. Soybean protein contains well-balanced amino acids and is a good source of many essential amino acids [15,16]. Amino acid concentrates can be obtained through biotechnological processing of soybean meal. Various technologies are used for this purpose; the most common is enzymatic hydrolysis, but there are others like the application of a cavitation system in hydrolysis, solid-state fermentation, and so on [17,18]. Despite the sufficient prevalence of enzymatic hydrolysis in the production of feed amino acid concentrates, a number of production obstacles, such as complex and expensive production technology, instability of the composition of finished products, and short shelf life of product make it difficult to carry out efficient production [19,20]. Accordingly, the relevance of optimizing the parameters of hydrolysis and using preservatives for protein feed additives is due to a number of factors: long-term preservation of feed quality, reduction in product losses, and economic benefits. It should also be noted that current trends in feed production are aimed at finding the most environmentally friendly and safe preservatives [21,22]. The purpose of this study is to develop optimal parameters of the technology of production and preservation of protein hydrolysate based on extruded soybean meal (ESBM) in terms of the content of free amino acids [23].
The research objectives were to select and justify the options of biotechnological parameters for the production of protein hydrolysate based on extruded soybean meal; to carry out a comparative assessment of the obtained hydrolysates in terms of the content of total free amino acids, free amino group (amine nitrogen), and two essential amino acids: lysine and methionine; to compare the conservation methods used for the obtained hydrolysates during 3 months of storage; and to identify and justify the optimal parameters for the production of feed hydrolysate, based on the results of the studies in [24,25].

2. Materials and Methods

2.1. Extruded Soybean Meal (ESBM)

Extruded soybean meal, used as a substrate in our study, was obtained under the conditions of industrial oil production by the VitaKorm Company (Ulyanovsk, Russia). The meal was selected after processing soybeans in an oil press, which were then cooled and subjected to preliminary separation to remove impurities, followed by extrusion at t = 110–130 °C with a pressure of 35–40 atm. for 30 s. Then, the extruded soybean meal was cooled and ground to give a homogeneous composition to the dry mass.

2.2. The Enzyme Complex

In this study, the enzyme complex of alkaline bacterial proteases “Protozyme” produced by the Torgovy Dom “Biopreparat” company (Serpukhov, Russia) was used. The enzyme complex was obtained by culturing a natural strain of Bacillus licheniformis microorganisms isolated by directional selection, followed by purification and concentration. The optimal dosage for using this enzyme as a biotechnological agent was selected by laboratory and production tests. 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. The enzyme complex used is a stable enzyme in the presence of chelating chemicals, such as EDTA. It also has a low susceptibility to tri-polyphosphoric acid salts. These characteristics make it possible to use the enzyme complex in the production of feed ingredients using preservatives used in this study.

2.3. Obtaining Hydrolysate Samples Using Various Technological Modes

Samples of extruded soybean meal were subjected to hydrolytic treatment under different conditions (Figure 1) and then kept in thermostats for 12 h under the set conditions.
The enzyme–substrate ratio (hydro–module ratio) 1:10 (11.0 kg) was formed with 10 parts of dry ESBM (10.0 kg) and 1 part of an activated enzyme complex (1.0 kg).
The enzyme–substrate ratio (hydro–module) 1:20 (21.0 kg) was formed with 20 parts of dry ESBM (20.0 kg) and 1 part of an activated enzyme complex (1.0 kg).
The enzyme–substrate ratio (hydro–module) 1:30 (31.0 kg) was formed with 30 parts of dry ESBM (30.0 kg) and 1 part of an activated enzyme complex (1.0 kg).
After preparing the three previous solutions (1:10, 1:20, and 1:30), six experimental groups were formed, i.e., two groups from each of the three solutions, with 9 repeats per group.
The first, third, and fifth sample groups were subjected to the following hydrolysis process conditions: t = 40.0–45.0 °C and pH = 7.0–9.0. The second, fourth, and sixth sample groups were subjected to the following hydrolysis process conditions: t = 45.0–50.0 °C and pH = 8.0–11.0.

2.4. Selection and Application of Preservatives

The key aspects when choosing the type of preservatives for the hydrolysate are the spectrum of antimicrobial activity, safety, degree of solubility in semi-liquid media and in high-viscosity media, biochemical compatibility with the components of the hydrolysate, versatility, and the ratio of “efficiency-consumption concentration-price”. These tasks can be solved by chemical substances such as sorbic acid and thymol, due to the fact that organic acids (like sorbic acid) can be used to impair fungal growth [26,27] while thymol is a promising molecule in essential oils that are present in oregano and thyme, which has broad-spectrum antibacterial effects [28]. Based on their effectiveness and relatively low cost, sorbic acid and thymol were chosen as preservatives in this study [29]. Each hydrolysate sample from the six hydrolysate groups was preserved in two preservation methods for comparison by adding sorbic acid or thymol as follows: in the first method, sorbic acid solution (25%) was added at an addition rate of (0.2 g/10 kg), while in the second one thymol solution (25%) was added at a rate of (0.1 g/10 kg) for each of the six experimental groups, i.e., each group was divided into two treatments, each of which was subjected to a different preservation method, and dried to a moisture content of 10% ± 0.1%. Then all samples were stored for three months at 4–5 °C.

2.5. Free Amino Acid Content Analysis

Amino acids were determined by capillary electrophoresis, according to the Government Standard (GOST)32195-2013 (ISO 13903:2005) [30]. Based on the decomposition of the sample by hydrolysis with the conversion of amino acids into free forms, and then with the production of Phenylthiocarbamide (PTC) derivatives of amino acids, the resulting amino acid residue was separated and quantitatively determined by capillary electrophoresis system KAPEL®-205 from the company LUMEX® (Saint Petersburg, Russia). Hydrolysate samples were prepared for analysis by drying them to a moisture of 50% by mass, grinding them, and removing excess fat; then, a sample of 0.1–1.0 g was weighed, in which the nitrogen content should not be less than 100 mg. The samples were subjected to oxidative treatment using sodium di-sulfite and cooled to a temperature of 0 °C. After that, the mass with the set pH value was transferred to the chromatography tubes for the determination of amino acids.

2.6. Analysis of the Lysine and Methionine Contents in the Composition of the Obtained Hydrolysate

To determine the content of lysine and methionine amino acids, the standardized method GOST 32195-2013 (ISO 13903:2005) [30] was used. The method is based on the extraction of the material with dilute hydrochloric acid. The resulting amino acids and nitrogenous macromolecules are precipitated with sulfosalicylic acid and filtered. Then, the filtrate is subjected to ion-exchange chromatography, followed by the determination of the contents by photometric detection at a wavelength of 570 nm. A weight (1.0–5.0 g) of each hydrolysate sample was mixed with 100.0 cm3 of an extraction solution prepared on the basis of hydrochloric acid (HCl) and subjected to mechanical shaking for 60 min, settled, and then 10.0 cm3 of the resulting filler liquid was mixed with 5.0 cm3 of sulfosalicylic acid and mixed for 5 min. The resulting solution was filtered to remove sediment, and then its pH value was set to the value of 2.20 using a solution of sodium hydroxide (NaOH 25.0%), and mixed with citrate. Finally, chromatography was performed using standard titers of lysine and methionine, in accordance with the established methodology.

2.7. Statistical Analysis

Statistical data processing was performed using Student’s t-test, and the reliability of differences between the average values of each experimental group compared to the control group was assessed. The results were considered reliable upon reaching the level of significance of differences (p ≤ 0.05 or more) [31].

3. Results

3.1. Comparison of the Hydrolysis Parameter Conditions

The results of the determination of the total amount of free amino acids and amine nitrogen are presented in Table 1.
Six groups of hydrolases were produced using different hydrolysis conditions as follows: (1) sample: 1:10 under conditions of t = 40–45 °C and pH = 7–9; (2) sample: 1:10 under conditions of t = 45–50 °C and pH = 8–11; (3) sample: 1:20 under conditions of t = 40–45 °C and pH = 7–9; (4) sample: 1:20 under conditions of t = 45–50 °C and pH = 8–11; (5) sample: 1:30 under conditions t = 40–45 °C and pH = 7–9; and (6) sample: 1:30 under conditions t = 45–50 °C and pH = 8–11.
Experimental samples of protein hydrolysate produced under different technological conditions were examined for the content of total free amino acids and amine nitrogen. Based on the results, it can be seen that the values of the amine nitrogen content and the total free amino acids in the obtained hydrolysate samples showed a certain chromatographic pattern. Thus, when using an enzyme–substrate ratio of 1:10 for hydrolysis, it was found that the best conditions for this ratio were when temperature ranges of 45.0–50.0 °C were used in a buffered environment range of 8.0–11.0. When using these hydrolysis parameters of the ESBM substrate, it was noted that the amino acid content increased by 64.2%—0.182 ± 0.47 g/kg (p < 0.01) relative to the initial values of the control samples. However, the content of amine nitrogen did not change immediately after hydrolysis.
When the enzyme–substrate ratio of 1:20 was studied, it was found that a more significant change in the content of free amino acids and amine nitrogen occurred in samples of the fourth group that were subjected to hydrolysis at a temperature of 45.0 to 50.0 °C and in a buffer medium from 8.0 to 11.0. Under these conditions, the total free amino acid content increased by 61.5% relative to the initial values and reached a value of 0.195 ± 0.51 g/kg (p < 0.01), while the amino acid content in samples in the third group under temperature conditions increased from 40.0 to 45.0 °C with pH values in the range of 7.0–9.0. The content of amino acids increased by 57.5%—0.189 ± 0.37 g/kg (p < 0.01). The amine nitrogen content increased by 8.0% to 2.5 g/kg in both samples of the third and fourth groups.
When the enzyme–substrate ratio (1:30) was studied, the best hydrolysis conditions were found during fermentation at a temperature from 45.0 to 50.0 °C and in a buffer medium in a pH range of 8.0 to 11.0. Under these conditions, the amino acid content increased by 64.2% and reached a value of 0.187 ± 0.44 g/kg (p < 0.01). The content of amine nitrogen in the samples in the sixth group increased by 14.3% relative to the initial values, and amounted to 2.8 ± 0.37 g/kg (p < 0.05), whereas under the conditions in the fifth group when the temperature range was 40.0–45.0 °C and the pH range was 7.0–9.0, the increase in amino acids was at the level of 54.2%, which was 0.185 ± 0.27 g/kg (p < 0.01). Thus, based on the increase in total free amino acid concentration and amine nitrogen in the experimental groups, the best conditions for the manifestation of hydrolytic activity were in the temperature range of 45.0–50.0 °C and in a buffer medium with a pH range of 8.0–11.0.
The results of the content of essential amino acids (lysine and methionine) in the composition of ESBM before and after hydrolysis are presented in Table 2.
Analysis of the amino acid composition showed that the content of lysine and methionine in the ESBM was lower than in the hydrolysate samples.
This study found that when using an enzyme–substrate ratio of 1:10 for hydrolysis under different conditions, the best conditions were a temperature range of 45.0–50.0 °C and a pH range of 8.0–11.0. Under these process parameters of hydrolysis, it was found that the content of the amino acid lysine increased by 66.7%—0.070 ± 0.05 g/kg (p < 0.01) relative to the initial values of the control samples. The methionine content under these conditions was also increased by 73.3% and reached a value of 0.052 ± 0.05 g/kg (p < 0.01).
By studying the enzyme–substrate ratio of 1:20, it was found that a more significant change in the content of the amino acids lysine and methionine occurred in samples in the fourth group, which were subjected to hydrolysis at a temperature range of 45.0 to 50.0 °C and in a buffered medium with a pH range of 8.0–11.0. Under these conditions, the total lysine content increased by 69.1% relative to the initial values and reached a value of 0.071 ± 0.02 g/kg (p < 0.01), while the amino acid content in samples of the third group increased under heating temperature conditions from 40.0 to 45.0 °C and at pH values in the range of 7.0–9.0. The content of lysine increased by 64.3%—0.069 ± 0.02 g/kg (p < 0.05). The methionine content increased by 70.0% to a value of 0.051 ± 0.01 g/kg in the samples in the fourth group, while in samples in the third group, the content of this amino acid increased by 53.3% to a value of 0.046 ± 0.02 g/kg (p < 0.01).
When using the enzyme–substrate ratio of 1:30, the best hydrolysis conditions were when temperatures ranged from 45.0 to 50.0 °C and the pH ranged from 8.0 to 11.0. The lysine content increased by 64.3% and reached a value of 0.069 ± 0.07 g/kg (p < 0.01). The methionine content in the samples of the sixth group increased by 63.3% relative to the initial values and amounted to 0.049 ± 0.06 g/kg (p < 0.05), whereas in the conditions of the samples in the fifth group at a temperature of 40.0–45.0 °C and pH range of 7.0–9.0, the increase in methionine was at the level of 50.0%, which amounted to 0.045 ± 0.07 g/kg (p < 0.05). Thus, based on the increase in the lysine and methionine contents in the samples of all experimental variants of the enzyme–substrate ratio, the best conditions for achieving the greatest hydrolytic activity were when the temperature was between 45.0 and 50.0 °C and the pH value was between 8.0 and 11.0.

3.2. Evaluation of Preservation Methods

The changes in the total free amino acid content during storage of the hydrolysate samples in a ratio of 1:10 using different conservation methods are shown in Figure 2.
A comparative analysis of the changes in the amino acid content during storage of hydrolysate samples using different conservation methods revealed that the level of total free amino acid content varied depending on the conservation method.
Sorbic acid is an organic compound (trans, trans-2,4-hexadienoic acid) that is often used as a preservative in the food industry. It is a crystalline and colorless substance that is practically insoluble in water and easily sublimated with a chemical formula. Using sorbic acid as a preservative showed that the amino acid content decreased by only 0.001 g/kg after three months of storage. By conducting a comparative analysis of the hydrolysate samples with the control group (that did not contain preservatives), it was found that sorbic acid almost completely retained the properties of the hydrolysate, and the difference in values relative to the control samples was 0.55%. Also, a second group of hydrolysates prepared in an enzyme–substrate ratio of 1:10 was subjected to another preservative. As a natural preservative, thymol (2-isopropyl-5-methylphenol) was selected, which is used in various branches of technological activity, including in the food industry. When thymol was added as a preservative to the hydrolysate, the amino acid content decreased by 0.55% relative to the initial content after one month of storage and amounted to 0.181 g/kg. After three months of storage, the amino acid content in the hydrolysate decreased by 1.7% relative to the initial value and amounted to 0.178 g/kg (p ≥ 0.05).
It should be noted that storage of the hydrolysate prepared in an enzyme–substrate ratio of 1:10, without preservative, contributed to a decrease of 1.1%—0.179 g/kg (p ≥ 0.05 after a month of storage in the initial amino acid content of 0.181 g/kg (p ≥ 0.05) and a decrease of 2.21%—0.177 g/kg (p ≥ 0.05) after three months of storage. In comparison of the previous preservation methods with the control group, it was found that the highest preservation properties were in the samples to which sorbic acid was added.
A comparative analysis of the amino acid content changes during storage of hydrolysate samples that were prepared in an enzyme–substrate ratio of 1:20 using different conservation methods revealed that the level of total free amino acid content varied depending on the conservation method, as shown in Figure 3.
Studying the preservative properties of sorbic acid showed that the amino acid content decreased by only 0.002 g/kg after three months of storage. When conducting a comparative analysis of the control samples, it was found that sorbic acid almost completely retained the properties of the hydrolysate, and the difference in values relative to the control samples was 1.04%. When thymol was added as a preservative to the hydrolysate, the amino acid content decreased by 1.04% relative to the initial content after one month of storage and amounted to 0.192 g/kg (p ≥ 0.05). After three months of storage, the amino acid content in the hydrolysate decreased by 1.6% relative to the initial value and amounted to 0.189 g/kg (p ≥ 0.05). It should be noted that storage of the hydrolysate prepared in an enzyme–substrate ratio of 1:20, without the use of preservatives, contributed to a decrease of 1.6%—0.189 g/kg (p ≥ 0.05) in the initial amino acid content of 0.192 g/kg after a month of storage and a decrease of 3.7%—0.185 g/kg (p ≥ 0.05) after three months of storage. Thus, it can be seen that the preservative properties of sorbic acid at the specified concentration of amino acids and the enzyme–substrate ratio of 1:20 exhibit a higher degree of preservative properties. Likewise, when using the enzyme–substrate ratio of 1:30, it was noted that the type of preservative plays a major role in changes in the level of total free amino acid, as shown in Figure 4.
The preservative properties of sorbic acid showed that the amino acid content decreased by only 0.002 g/kg after three months of storage. When conducting a comparative analysis with the control samples, it was found that sorbic acid almost completely retained the properties of the hydrolysate, and the difference in values relative to the control samples was 1.1%. When thymol was added as a preservative to the hydrolysate, the amino acid content decreased by 0.5% relative to the initial content after one month of storage, amounting to 0.186 g/kg (p ≥ 0.05). After three months of storage, the amino acid content in the hydrolysate decreased by 1.6% relative to the initial value and amounted to 0.183 g/kg (p ≥ 0.05). It should be noted that storage of the hydrolysate prepared in an enzyme–substrate ratio of 1:30, without the use of preservatives, contributed to a decrease in the initial amino acid content that amounted to 0.186 g/kg (p ≥ 0.05) after a month of storage, which was a decrease of 1.6%—0.183 g/kg (p ≥ 0.05), and after three months of storage a decrease of 3.2%—0.180 g/kg (p ≥ 0.05). Also, in comparison, it was found that sorbic acid had the highest preservation properties of hydrolysates compared to thymol and the control group.
The changes in lysine content in the hydrolysate composition with different methods of preservation are presented in Table 3.
As is obvious from Table 3, the value of the lysine amino acid content in the hydrolysate varies within fairly narrow limits for different variants of the enzyme–substrate ratio. The analysis of hydrolysate, prepared in an enzyme–substrate ratio of 1:10, showed that the lysine content in the control samples was 0.070 g/kg. After a month, the lysine content decreased by 1.6% relative to the initial values and amounted to 0.068 g/kg. Storage of the hydrolysate without preservative for three months contributed to a decrease in the lysine content of 7.2% and amounted to 0.065 g/kg. When using thymol as a preservative, it was found that the decrease in lysine in the hydrolysate with an enzyme–substrate ratio of 1:10 after a month of storage was 1.4%—0.069 g/kg (p ≥ 0.05), and after three months of storage, its content decreased by 2.7% and amounted to 0.068 g/kg (p ≥ 0.05). The study of the preservation properties of thymol showed that when the hydrolysate (1:10) was stored for one month, there were no changes in the lysine content, whereas after three months of storage, the lysine content decreased by 1.4% relative to the initial value and amounted to 0.069 g/kg (p ≥ 0.05).
When studying the changes in the lysine content in a hydrolysate with an enzyme–substrate ratio of 1:20 with or without adding preservatives (thymol and sorbic acid), the following results were established.
The storage of the hydrolysate without the use of preservatives showed that after a month of storage the lysine content decreased by 1.6% (0.068 g/kg), and after three months of storage it decreased by 5.7% (0.066 g/kg) relative to the initial value of 0.070 g/kg. Whereas when thymol was added to the hydrolysate, the lysine content decreased by 1.4% after a month and amounted to 0.069 g/kg (p < 0.05), and after three months of storage, the content decreased by 4.3%—0.067 g/kg. Studying the preservative properties of sorbic acid, it was established that the lysine content did not change after a month of storage, and after three months the decrease was 1.4%—0.069 g/kg (p ≥ 0.05( relative to the initial value.
Also, storage of a control sample of hydrolysate with an enzyme–substrate ratio of 1:30, without the use of preservatives, showed a decrease in lysine of 4.4% (0.066 g/kg) after a month relative to the initial value of 0.069 g/kg. After three months of storage in these hydrolysate samples, the decrease in lysine was 8.7% (0.063 g/kg) relative to the initial values. The study of the preservative properties of thymol when added to the hydrolysate with an enzyme–substrate ratio of 1:30 showed that after a month, as well as three months of storage, the lysine content decreased and amounted to 0.067 g/kg (p ≥ 0.05). The addition of sorbic acid to the hydrolysate (1:30) contributed to a decrease in lysine content after three months by 1.4% and amounted to 0.068 g/kg (p ≥ 0.01).
Our results demonstrate that sorbic acid exhibits the best preservative properties when added to hydrolysate prepared with different enzyme–substrate ratios.
According to our results, thymol shows preservative properties to increase the safety and stability of hydrolysate, but less than what sorbic acid shows.
The changes in the methionine content in the composition of hydrolysate with different methods of preservation are presented in Table 4.
It is evident from Table 4 that when studying the changes in the amount of the essential amino acid methionine in hydrolysate samples prepared with different enzyme–substrate ratios, it was found that the difference was not significant. By analyzing the hydrolysate prepared at a ratio of 1:10 in the control group, it was found that the methionine content was 0.061 g/kg. After a month of storage without preservatives, the methionine percentage decreased by 3.3% and amounted to 0.059 g/kg. Storage of the hydrolysate without preservatives for three months contributed to a decrease in the methionine content of 4.9% and amounted to 0.058 g/kg. When using thymol as a preservative, it was found that the decrease in methionine in the hydrolysate with an enzyme–substrate ratio of 1:10 after a month of storage was 1.6%—0.060 g/kg (p ≥ 0.05), and after three months of storage, its content decreased by 3.3% and amounted to 0.059 g/kg (p ≥ 0.05). The study of the sorbic acid preservative properties showed that when storing the hydrolysate (1:10) for a month, as well as for three months, there were no changes in the content of methionine.
When studying the changes in the methionine content in the hydrolysate with an enzyme–substrate ratio of 1:20, it was noted that storage of the hydrolysate without the use of preservatives showed that after a month the value of the studied amino acid decreased by 3.2% (0.060 g/kg) and after three months it decreased by 4.8% (0.059 g/kg) relative to the initial value of 0.062 g/kg. Whereas when thymol was added to the hydrolysate, the methionine content decreased by 1.6% after a month and amounted to 0.061 g/kg (p ≥ 0.05), and after three months of storage, the decrease was 3.2%—0.060 g/kg (p ≥ 0.05). Studying the preservative properties of sorbic acid, it was determined that the methionine content did not change after a month of storage, and after three months the decrease was 1.6%—0.061 g/kg (p ≥ 0.01) relative to the initial value. Also, storage of a control sample of hydrolysate with an enzyme–substrate ratio of 1:30 without the use of preservatives showed a decrease in methionine content of 1.7% (0.059 g/kg) after a month relative to the initial value of 0.060 g/kg. After three months of storage in these hydrolysate samples, the decrease in methionine was 5.0% (0.057 g/kg) relative to the initial values. A study of the preservative properties of thymol when added to the hydrolysate with a ratio of 1:30 showed that after a month, as well as three months of storage, the value of the methionine content decreased by only 1.7% relative to the initial value and amounted to 0.059 g/kg (p ≥ 0.05). The addition of sorbic acid to the hydrolysate (1:30) contributed to the preservation of methionine content during the storage period. Thus, it can be noted that the addition of thymol to hydrolysate showed preservative properties, but the safety of the amino acids (methionine and lysine) was less than in samples compared with using sorbic acid, which exhibits the best preservative properties.

4. Discussion

There is a constant search for tools and methods to improve the production and quality of protein hydrolysates [18,19]. The enzymatic hydrolysis process can be designed in various ways. However, bacterial enzymatic hydrolysis is the most popular and commonly used process because it achieves several benefits, as it works to modify proteins, achieve multifunctionality, and increase their added value, and it digests anti-nutritional factors in raw materials, such as indigestible oligosaccharides [32]. Also, because of its low environmental cost, bacterial enzymatic hydrolysis is considered to be a green technology, and the quantity of enzymes used in the hydrolysis process is smaller than that of acids in chemical hydrolysis [33]. Moreover, many of these enzymes of bacterial origin operate under mild conditions, thus minimizing energy consumption and increasing the efficiency of hydrolysis [22]. Given their specificity, alkaline protease enzymes display high activity because they are dynamic in neutral to alkaline pH [34]. Parameter optimization for enzymatic hydrolysis and the development of methods for the long-term preservation of the protein hydrolysate are the most important directions for improving the technology of the feed industry. The use of proteolytic enzymes, as one of the effective enzyme groups, for the bioconversion of high-molecular-weight plant raw materials during the hydrolysis process, promotes the breakdown of complex proteins into simple protein fragments, such as peptides, amino acids, and free amino groups [35]. Proteolytic enzymes also facilitate the process of controlling the degree of hydrolysis of peptide bonds, and at the appropriate moment they can be deactivated and the reaction stopped [36]. The effectiveness of these enzymes is also due to the possibility of the alkaline proteolytic enzymes exhibiting stable activity at pH 8–11; this is the pH range of the reaction medium that allows the binding between the substrate and the enzyme, as the changes in pH change the electrical state of the free side functions of the amino acids in the peptide chain, especially those located at the level of the active site of the enzyme. These changes in electrical state (when the pH value moves away from the ideal value for the enzyme) lead to the loss of the distinctive shape of the active site of the enzyme, which hinders the fixation of the reaction material (substrate) and thus prevents the enzymatic reaction [23,36,37]. Also, when using a temperature range of 45–50 °C, the chemical reaction kinetics exhibits maximum hydrolytic activity. This hydrolysis activity is designed for a short reaction time but is sufficient to obtain a concentrated precipitate of amino acids. The high value of the free amino acid content resulting from enzymatic and chemical hydrolysis processes has been proven by a team of researchers at a leading animal nutrition institute. By comparing the resulting hydrolysates, they found that the percentage of free amino acids in the hydrolysates resulting from enzymatic hydrolysis of feed soybeans was 7.4% higher than that in the protein hydrolysates resulting from chemical hydrolysis [38]. In another study, Sokolov and Bolkhonov were able to achieve a high hydrolysis rate of 88% in soybean protein. But the enzymatic hydrolysis was performed on whole soybeans where the percentage of anti-nutritional factors is very high, which prevents complete absorption of biologically active compounds by the animal [39]. The resulting protein hydrolysates were successfully preserved using preservatives: sorbic acid and thymol. Sorbic acid is a well-known preservative in food and feed preservation and is widely used; it is added at a rate of 0.1–0.3% to protein hydrolysates used as pet feed [40]. This study showed that the use of sorbic acid as a preservative at a rate of 0.1% proved to be highly effective in preserving hydrolysates. It should also be noted experimentally that preservative properties were lower when thymol was used than that observed when sorbic acid was used.

5. Conclusions

Based on the results, it can be concluded that in all experimental variants of the enzyme–substrate ratio (1:10, 1:20, and 1:30), the best conditions for the manifestation of hydrolytic activity were a temperature range from 45.0 to 50.0 °C in a buffered medium with a pH range from 8.0 to 11.0. Such conditions are the best and they contributed to the efficient hydrolysis process and the production of free amino acids.
According to the content of methionine and lysine in the hydrolysate after three months of storage, it was established that sorbic acid and thymol exhibit preservative properties, but sorbic acid is more effective. The development of optimal parameters of hydrolysis technology will increase the yield and enable obtaining a protein product that is easily digestible and absorbed by the animal, which will increase the efficiency of feed utilization and reduce losses.

Author Contributions

Conceptualization, T.K. and M.B.; methodology, M.S. and O.S.; validation, M.S. and T.K.; formal analysis, M.Z. and I.B.; investigation, T.A., M.S. and O.S.; resources, I.B. and M.Z.; writing—original draft, M.B., T.K., O.S. and M.S.; supervision, M.B.; project administration, M.B., T.A., O.S., and M.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
ESBMExtruded soybean meal
GOSTGovernment Standard
CECapillary electrophoresis

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Fermentation 11 00209 g001
Figure 1. The scheme of this study and the numbering of the samples obtained.
Figure 1. The scheme of this study and the numbering of the samples obtained.
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Figure 2. Comparative dynamics of changes in the total free amino acid content during storage of hydrolysate samples in a ratio of 1:10 using different conservation methods.
Figure 2. Comparative dynamics of changes in the total free amino acid content during storage of hydrolysate samples in a ratio of 1:10 using different conservation methods.
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Figure 3. Comparative dynamics of changes in the total free amino acid content during storage of hydrolysate samples in a ratio of 1:20 using different conservation methods.
Figure 3. Comparative dynamics of changes in the total free amino acid content during storage of hydrolysate samples in a ratio of 1:20 using different conservation methods.
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Figure 4. Comparative dynamics of changes in the total free amino acid content during storage in hydrolysate samples prepared in a ratio of 1:30 using different conservation methods.
Figure 4. Comparative dynamics of changes in the total free amino acid content during storage in hydrolysate samples prepared in a ratio of 1:30 using different conservation methods.
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Table 1. The content of the total amount of free amino acids and amine nitrogen in samples with different hydrolysis conditions.
Table 1. The content of the total amount of free amino acids and amine nitrogen in samples with different hydrolysis conditions.
SamplesTotal Free Amino Acid Content, g/kg% to the
Initial Value		Amine Nitrogen Content, g/kg% to the Initial Value
The Initial Value0.120 ± 0.142.4 ± 0.19
1:101 (t = 40.0–45.0 °C; pH = 7.0–9.0)0.179 ± 0.41 *54.2 ± 0.322.4 ± 0.140
2 (t = 45.0–50.0 °C; pH = 8.0–11.0)0.182 ± 0.47 **64.2 ± 0.612.4 ± 0.170
1:203 (t = 40.0–45.0 °C; pH = 7.0–9.0)0.189 ± 0.37 **57.5 ± 0.422.5 ± 0.168.0 ± 0.21
4 (t = 45.0–50.0 °C; pH = 8.0–11.0)0.195 ± 0.51 **61.5 ± 0.572.5 ± 0.478.0 ± 0.23
1:305 (t = 40.0–45.0 °C; pH = 7.0–9.0)0.185 ± 0.27 **54.2 ± 0.332.7 ± 0.22 *12.5 ± 0.77
6 (t = 45.0–50.0 °C; pH = 8.0–11.0)0.187 ± 0.44 **64.2 ± 0.472.8 ± 0.37 *14.3 ± 0.51
Data are shown as mean ± SD; * p < 0.05, ** p < 0.01.
Table 2. Content of essential amino acids (lysine and methionine) in samples with different hydrolysis conditions.
Table 2. Content of essential amino acids (lysine and methionine) in samples with different hydrolysis conditions.
SamplesLysine, g/kg% to the Initial ValueMethionine, g/kg% to the Initial Value
The Initial Value0.042 ± 0.040.030 ± 0.09
1:101 (t = 40.0–45.0 °C; pH = 7.0–9.0)0.069 ± 0.05 *64.3 ±0.050.050 ± 0.05 *66.7 ± 0.05
2 (t = 45.0–50.0 °C; pH = 8.0–11.0)0.070 ±0.05 **66.7 ± 0.050.052 ± 0.05 **57.7 ± 0.05
1:203 (t = 40.0–45.0 °C; pH = 7.0–9.0)0.069 ± 0.02 *64.3 ± 0.020.046 ± 0.02 *53.3 ± 0.02
4 (t = 45.0–50.0 °C; pH = 8.0–11.0)0.071 ± 0.02 **59.2 ± 0.020.051 ± 0.01 **58.8 ± 0.02
1:305 (t = 40.0–45.0 °C; pH = 7.0–9.0)0.068 ± 0.07 *61.9 ± 0.070.045 ± 0.07 *50.0 ± 0.07
6 (t = 45.0–50.0 °C; pH = 8.0–11.0)0.069 ± 0.07 *60.9 ± 0.070.049 ± 0.06 *61.2 ± 0.06
Data are shown as mean ± SD; * p < 0.05, ** p < 0.01.
Table 3. The change in lysine content values in hydrolysate samples with different preservation methods during storage, g/kg.
Table 3. The change in lysine content values in hydrolysate samples with different preservation methods during storage, g/kg.
Preservative and Storage PeriodEnzyme–Substrate Ratio
1:101:201:30
ControlImmediately after preparation0.070 ± 0.070.070 ± 0.030.069 ± 0.09
In a month0.068 ± 0.070.068 ± 0.030.066 ± 0.04
In three months0.065 ± 0.090.066 ± 0.080.063 ± 0.07
ThymolIn a month0.069 ± 0.08 *0.069 ± 0.07 *0.067 ± 0.03 *
In three months0.068 ± 0.04 *0.067 ± 0.05 *0.067 ± 0.09 *
Sorbic acidIn a month0.070 ± 0.03 **0.070 ± 0.04 **0.069 ± 0.09 **
In three months0.069 ± 0.07 *0.069 ± 0.09 *0.068 ± 0.03 **
Data are shown as mean ± SD; * p < 0.05, ** p < 0.01.
Table 4. The changes in the methionine content in hydrolysate samples with different preservation methods during storage.
Table 4. The changes in the methionine content in hydrolysate samples with different preservation methods during storage.
Preservative and Storage PeriodEnzyme–Substrate Ratio
1:101:201:30
ControlImmediately after preparation0.061 ± 0.030.062 ± 0.070.060 ± 0.06
In a month0.059 ± 0.070.060 ± 0.090.059 ± 0.06
In three months0.058 ± 0.070.059 ± 0.070.057 ± 0.03
ThymolIn a month0.060 ± 0.04 *0.061 ± 0.03 *0.059 ± 0.07 *
In three months0.059 ± 0.03 *0.060 ± 0.07 *0.059 ± 0.09 *
Sorbic acidIn a month0.061 ± 0.09 **0.062 ± 0.04 **0.060 ± 0.07 **
In three months0.061 ± 0.07 **0.061 ± 0.09 *0.060 ± 0.03 **
Data are shown as mean ± SD; * p < 0.05, ** p < 0.01.
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Belyshkina, M.; Kobozeva, T.; Zagoruiko, M.; Serebryakova, O.; Shaaban, M.; Ananeva, T.; Bashmakov, I. Determination of the Optimal Biotechnological Parameters for Industrial Production of Protein Hydrolysates for Animal Feed. Fermentation 2025, 11, 209. https://doi.org/10.3390/fermentation11040209

AMA Style

Belyshkina M, Kobozeva T, Zagoruiko M, Serebryakova O, Shaaban M, Ananeva T, Bashmakov I. Determination of the Optimal Biotechnological Parameters for Industrial Production of Protein Hydrolysates for Animal Feed. Fermentation. 2025; 11(4):209. https://doi.org/10.3390/fermentation11040209

Chicago/Turabian Style

Belyshkina, Marina, Tamara Kobozeva, Mikhail Zagoruiko, Oksana Serebryakova, Maisoon Shaaban, Tatiana Ananeva, and Igor Bashmakov. 2025. "Determination of the Optimal Biotechnological Parameters for Industrial Production of Protein Hydrolysates for Animal Feed" Fermentation 11, no. 4: 209. https://doi.org/10.3390/fermentation11040209

APA Style

Belyshkina, M., Kobozeva, T., Zagoruiko, M., Serebryakova, O., Shaaban, M., Ananeva, T., & Bashmakov, I. (2025). Determination of the Optimal Biotechnological Parameters for Industrial Production of Protein Hydrolysates for Animal Feed. Fermentation, 11(4), 209. https://doi.org/10.3390/fermentation11040209

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