BIOTRANSFORMATION OF INORGANIC SELENIUM (Na₂SeO₃) AND ZINC (ZnSO₄) SALTS INTO ORGANIC SELENIUM (SELENOCYSTEINE) COMPOUNDS BY Lacticaseibacillus rhamnosus AND Lacticaseibacillus casei STRAINS

ABSTRACT

In this study, the tolerance of Lacticaseibacillus rhamnosus and Lacticaseibacillus casei strains to inorganic selenium (Na₂SeO₃) and zinc (ZnSO₄) salts, as well as their optimal concentrations, were determined. At the identified concentrations, the biotransformation capacity of the strains was investigated. The strains were incubated in MRS broth at 37 °C under aerobic conditions for 24 and 48 hours. The concentrations of Na₂SeO₃ and ZnSO₄ used in the study ranged from 0, 10, 20, 50, 75, and 100 ppm. The results showed that the inorganic salt of selenium exhibited high toxicity, with 20 ppm identified as the optimal concentration. In contrast, the strains demonstrated higher tolerance to the zinc salt, with 50 ppm determined as the optimal concentration. To assess the biotransformation activity of the strains, cultures were grown at the optimal concentration (20 ppm) in MRS broth under aerobic conditions for 48 hours, and the supernatant was analyzed using high-performance liquid chromatography (HPLC). The biotransformation efficiency of the strains was found to be within the range of 55–70%.

АННОТАЦИЯ

В данном исследовании была определена толерантность штаммов Lacticaseibacillus rhamnosus и Lacticaseibacillus casei к неорганическим солям селена (Na₂SeO₃) и цинка (ZnSO₄), а также установлены их оптимальные концентрации. При выявленных концентрациях исследована способность штаммов к биотрансформации. Культуры инкубировали в бульоне MRS при 37 °C в аэробных условиях в течение 24 и 48 часов. Концентрации Na₂SeO₃ и ZnSO₄, использованные в исследовании, составляли 0, 10, 20, 50, 75 и 100 ppm. Результаты показали, что неорганическая соль селена обладает высокой токсичностью, при этом оптимальной концентрацией была признана 20 ppm. В отличие от этого, штаммы проявили более высокую толерантность к соли цинка, для которой оптимальной концентрацией определено значение 50 ppm. Для оценки биотрансформационной активности штаммов культуры выращивали при оптимальной концентрации (20 ppm) в бульоне MRS в аэробных условиях в течение 48 часов, после чего супернатант анализировали методом высокоэффективной жидкостной хроматографии (ВЭЖХ). Установлено, что эффективность биотрансформации штаммов находилась в диапазоне 55–70 %.

Keywords: Lacticaseibacillus rhamnosus, Lacticaseibacillus casei, Na₂SeO₃, ZnSO₄, biotransformation, tolerance, optimal concentration, selenocysteine, HPLC.

Ключевые слова: Lacticaseibacillus rhamnosus, Lacticaseibacillus casei, Na₂SeO₃, ZnSO₄, биотрансформация, толерантность, оптимальная концентрация, селеноцистеин, ВЭЖХ.

Introduction

Zinc (Zn) and selenium (Se) are essential microelements required for the human body. Zinc, as a cofactor of numerous enzymes, plays a critical role in cell division, immune system function, and stabilization of the cell membrane. Selenium, on the other hand, is a structural component of antioxidant enzymes such as glutathione peroxidase and thioredoxin reductase, and participates in defense mechanisms against oxidative stress [1, 2]. The inorganic salt forms of zinc and selenium (ZnSO₄, ZnCl₂, Na₂SeO₃, and Na₂SeO₄) can exert toxic effects on cells at high concentrations. Therefore, investigating the tolerance of probiotic microorganisms to these salts and their ability to biotransform them has become an important scientific direction [3].

Lacticaseibacillus rhamnosus and Lacticaseibacillus casei strains are widely used as probiotics. Studying their tolerance to inorganic salts of zinc and selenium is of great significance in the fields of biotechnology and functional foods [4].

The aim of this study is to evaluate the tolerance of Lacticaseibacillus rhamnosus and Lacticaseibacillus casei strains to inorganic salts of zinc and selenium, as well as to assess their biotechnological and probiotic potential.

Materials and methods

The Lacticaseibacillus rhamnosus and Lacticaseibacillus casei strains used in this study were obtained from the Laboratory of Probiotic Microbiology and Biotechnology, Institute of Microbiology, Uzbekistan Academy of Sciences. The strains were incubated in MRS broth (HIMEDIA Laboratories Pvt., Russia) containing various concentrations of selenium (Na₂SeO₃) and zinc (ZnSO₄) salts, at 37 °C under aerobic conditions for 48 hours [5, 6].

After incubation, 100 µL of suspension from each sample was taken and plated onto pre-prepared MRS agar medium. For each Petri dish, 25 µL of medium was applied, and the bacteria were inoculated using the “lawn” culture method. Subsequently, the plates were incubated again at 37 °C under aerobic conditions for 48 hours [7].

At the end of the incubation period, the resulting colonies were counted, and the growth levels at different concentrations were compared. Based on these results, the tolerance limits and optimal growth concentrations of each strain for inorganic selenium and zinc salts were determined.

Proteins and peptides from the aqueous extracts of the samples were precipitated in centrifuge tubes. For this purpose, 1 mL of the sample was mixed with 1 mL (exact volume) of 20% trichloroacetic acid (TCA). After 10 minutes, the precipitate was separated by centrifugation at 8000 rpm for 15 minutes. From the supernatant, 0.1 mL was taken and lyophilized. The obtained hydrolysate was evaporated, and the dry residue was dissolved in a triethylamine–acetonitrile–water mixture (1:7:1) and dried. This procedure was repeated twice to neutralize the acid. Amino acid derivatives, phenylthiocarbamyl (PTC) compounds, were obtained by reaction with phenylisothiocyanate according to the method of Steven A. and Cohen D. The resulting amino acid derivatives were analyzed by high-performance liquid chromatography (HPLC) [8, 9].

HPLC conditions: Agilent Technologies 1200 chromatograph with DAD detector, Discovery 1. HS C18 column (75 × 4.6 mm).

2. Solvent A: 0.14 M CH₃COONa + 0.05% triethylamine (TEA), pH 6.4

3. Solvent B: acetonitrile (CH₃CN)

4. Flow rate: 1.2 mL/min

5. Detection wavelength: 269 nm

6. Gradient program (%B/min):

- 1–6% / 0–2.5 min
- 6–30% / 2.51–40 min
- 30–60% / 40.1–45 min
- 60–60% / 45.1–50 min
- 60–0% / 50.1–55 min

Table 1.

The desired concentrations were obtained by adding appropriate volumes of 1,000 ppm stock solutions of selenium and zinc salts to the MRS broth

Results and discussion

The growth characteristics of Lacticaseibacillus rhamnosus and Lacticaseibacillus casei strains in response to Na₂SeO₃ (selenite) and ZnSO₄ (zinc sulfate) salts were investigated. The strains were cultivated in MRS medium at various concentrations (10–100 ppm Na₂SeO₃ and 10–100 ppm ZnSO₄), and the resulting colonies were counted.

According to the results, Na₂SeO₃ exhibited high toxicity. At concentrations above 50 ppm, growth decreased sharply and the number of colonies declined. Optimal growth was observed at 20 ppm, where the colony count was close to that of the control (0 ppm). Thus, the tolerance threshold for Na₂SeO₃ was determined to be 20 ppm.

In contrast, the strains demonstrated greater tolerance to ZnSO₄. Growth was observed even up to 100 ppm, but optimal growth was recorded at 50 ppm. Although colony numbers decreased at 100 ppm, the degree of toxicity was significantly lower compared to Na₂SeO₃.

Overall, both strains showed higher resistance to ZnSO₄, whereas selenite exhibited toxic effects even at lower concentrations. These findings confirm the ability of L. rhamnosus and L. casei strains to biotransform microelements (evidenced by the formation of a red precipitate) and highlight their potential for effective probiotic application at optimal concentrations.

Figure 1. Growth performance of the strains in response to selenium and zinc salts

Figure 2. Tolerance characteristics of the strains to different concentrations of inorganic selenium salt

Figure 3. Tolerance characteristics of the strains to different concentrations of inorganic zinc salt

Table 1.

Growth indicators (CFU/ml) of the studied strains at different concentrations of inorganic salts

At the selected optimal concentration, the biotransformation efficiency of the strains was determined using the HPLC method. The results showed that the Lacticaseibacillus rhamnosus strain biotransformed 55–65% of inorganic selenium into organic selenium (seleno-cysteine). The Lacticaseibacillus casei strain demonstrated relatively higher efficiency, converting 65–70% of inorganic selenium into organic forms.

Figure 4. HPLC analysis of the supernatant fraction of Lacticaseibacillus rhamnosus strain for organic selenium

Figure 5. HPLC analysis of the supernatant fraction of Lacticaseibacillus casei strain for organic selenium

Conclusion. In this study, the tolerance and biotransformation capacity of Lacticaseibacillus rhamnosus and Lacticaseibacillus casei strains to Na₂SeO₃ and ZnSO₄ salts were evaluated. The results demonstrated that Na₂SeO₃ exhibited high toxicity, with an optimal concentration of 20 ppm, whereas the strains showed higher tolerance to ZnSO₄, with an optimal level of 50 ppm. HPLC analysis revealed that both strains were capable of converting up to 55–70% of inorganic selenite into organic selenium (selenocysteine), with L. casei displaying slightly higher efficiency.

These findings highlight the potential application of these probiotic strains in the development of selenium- and zinc-enriched functional foods and biotechnology. However, further studies are required to elucidate the underlying molecular mechanisms and to validate these results in biological models.

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