Bifidobacterium adolescentis strain 48 as potential neurobiotic

Abstract

FIELD: biotechnology; pharmacology; psychiatry.SUBSTANCE: strain Bifidobacterium adolescentis 48 (VKM Ac-2296), capable of synthesizing gamma-aminobutyric acid (GABA), a neurotransmitter involved in the inhibition processes of the central nervous system. The strain genome contains a significant number of genes that determine the immunomodulatory, neuromodulatory, and antioxidant potential of the strain. The strain can be used to produce biotherapeutic medicinal products and specialized food products used in the complex treatment and prevention of depressive disorders, moderate anxiety, and related diseases.EFFECT: invention includes species identification, description of properties, characterization of the sequenced genome and comparative analysis of the genetic potential of the strain, comparison of the nucleotide sequence of genes involved in the production and transport of GABA.1 cl, 5 dwg, 3 tbl

Description

[0001] Field of technology to which the invention relates

[0002] The invention relates to biotechnology, pharmacology, and medicine, particularly psychiatry, and concerns a bifidobacteria strain capable of producing gamma-aminobutyric acid, a known relaxant. The strain can be used to produce pharmaceuticals and dietary supplements used in the therapy and complex treatment of depression, anxiety, and other borderline psychiatric disorders. State of the Art

[0003] Bifidobacteria are important members of the human intestinal microbiota. Due to their positive impact on human health, bifidobacteria are considered probiotic microorganisms. They are considered generally regaded as safe (GRAS) and are widely used in functional foods, dietary supplements, and pharmaceuticals. Bifidobacterium-based products are increasingly used in combination treatments for the early stages of various diseases. The advantages of using probiotic bacteria and their cellular components as medicinal products include their relative harmlessness and physiological properties compared to chemical drugs, as well as the ability to select a specific drug—even individualized therapy—for the treatment of the same disease in different patients.

[0004] The development of microbe-targeted biologics for the treatment of depressive disorders and other psychiatric diseases has gained great popularity in the last decade. A promising approach in this direction is the use of natural products based on live probiotic bacteria that exhibit the ability to influence higher brain functions, in the presence of sufficient evidence confirming their positive effect on mental state. Clinical improvements in patients with depressive disorders have previously been shown after using such a therapeutic approach [Averina OV, Poluektova EU, Zorkina YA et al. Human Gut Microbiota for Diagnosis and Treatment of Depression. Int. J. Mol. Sci. 2024. V. 25. №11. e5782]. The term “psychobiotic” was introduced, which refers to probiotics, prebiotics and all biological effects that have a positive effect on mental health [Dinan TG, Stanton C, Cryan JFPsychobiotics: a novel class of psychotropic. Biol Psychiatry. 2013. Vol. 74. №10. Pp. 720-726]. The term neurobiotic can also be used with a broader interpretation. Preclinical evaluations show that certain bacterial strains have psychobiotic activity that reduces the symptoms of depression and anxiety. The effects may be mediated through the microbiota-gut-brain axis, affecting the functions of the vagus nerve, spinal cord, immune and neuroendocrine systems [Averina OV, Poluektova EU, Zorkina YA et al. Human Gut Microbiota for Diagnosis and Treatment of Depression. Int. J. Mol. Sci. 2024. Vol. 25. №11. e5782].

[0005] Bifidobacteria, as important representatives of the intestinal microbiota, are considered as promising neuropsychobiotics with antidepressant action. A number of bifidobacteria strains of various species have demonstrated the ability to reduce depression in animal models and in various clinical studies [Averina OV, Poluektova EU, Zorkina YA et al. Human Gut Microbiota for Diagnosis and Treatment of Depression. Int. J. Mol. Sci. 2024. V. 25. №11. e5782]. Among bifidobacteria, strains of the species Bifidobacterium adolescentis deserve special attention as active producers of gamma-aminobutyric acid (GABA). B. adolescentis 150, B. adolescentis PRL2019 and B. adolescentis HD17T2H are bifidobacteria strains that are effective GABA producers [Yunes RA, Poluektova EU, Vasileva EV et al. A multi-strain potential probiotic formulation of GABA-producing Lactobacillus plantarum 90sk and Bifidobacterium adolescentis 150 with antidepressant effects.Probiotics Antimicrob. Proteins. 2020. V.12. P. 973-979; Duranti, S., Ruiz L., Lugli G. A., et al. Bifidobacterium adolescentis as a key member of the human gut microbiota in the production of GABA. Sci. Rep.2020. V. 10. 14112].

[0006] B. adolescentis is one of the most common species of bifidobacteria in the human colon and is found in 60-80% of healthy adults in numbers up to 10 10cells / g feces [Gupta VK, Kim M., Bakshi U. et al. A predictive index for health status using species-level gut microbiome profiling. Nature Communications. 2020. Vol. 11. e 4635]. B. adolescentis can account for up to 5% of the total gut microbiota population of healthy adults [Noh SK, Lee KJ Fecal microbiota alterations and small intestinal bacterial overgrowth in functional abdominal bloating / distention / / J. Neurogastroenterol. and Motility. 2020. Vol. 26. P. 539-549.]. Lower numbers were found in children and the elderly [Ma T., Yao C, Shen X., et al. The diversity and composition of the human gut lactic acid bacteria and bifidobacterial microbiota vary depending on age. Applied Microbiology and Biotechnology. 2021. V. 105. P. 8427-8440]. Two studies of Italian centenarians (over 100 years) found significantly higher levels of B. adolescentis in feces compared to older (68-88 years) and younger adults [Rampelli, S., Soverini, M., D'Amico et al.Shotgun metagenomics of gut microbiota in humans with up to extreme longevity and the increasing role of xenobiotic degradation. mSystems. 2020. V. 5. e00124-20]. This species is evolutionarily adapted to the fermentation of dietary fiber, plant-derived glycans, and resistant starch and possesses an extensive set of enzymes for this process. The main product of complex carbohydrate metabolism is short-chain fatty acids (SCFA) acetate and lactate in a 3:2 ratio. Some B. adolescentis strains have been shown to exhibit a number of health-promoting properties in vitro and in rodent models, including enhanced intestinal barrier function, anti-inflammatory and immunoregulatory effects, and the production of neurotransmitters (GABA) and vitamins [Leser T., Baker A. Bifidobacterium adolescentis - a beneficial microbe. Beneficial Microbes. 2023. V. 14. P. 525-551].

[0007] γ-Aminobutyric acid (GABA; C4H9NO2) is a monocarboxylic alpha-amino acid that is widespread in nature. GABA is a key neurotransmitter involved in inhibitory processes in the mammalian central nervous system. Under the influence of GABA, energy processes in the brain are activated, respiratory activity of tissues increases, glucose utilization by the brain improves, and blood supply improves. Low levels or decreased GABA are associated with psychiatric and neurological disorders, including anxiety and depression [Distler MG, Plant LD, Sokoloff G. et al. Glyoxalase 1 increases anxiety by reducing GABA a receptor agonist methylglyoxal. J. Clin. Invest. 2012. V.122. P. 2306 2315; Padgett CL, Lalive AL, Tan KR et al. Methamphetamine-evoked depression of GABA receptor signaling in GABA neurons of the VTA. Neuron. 2012. Vol. 73. P. 978-989], insomnia [Gotesmann C. GABA mechanisms and sleep. Neuroscience. 2002. Vol. 111. P. 231-239], and epilepsy [Czapinski P., Blaszczyk B., Czuczwar SJ Mechanisms of action of antiepileptic drugs. Curr. Top.Med. Chem. 2005. V.5. P. 3-14]. Dysfunction of GABA signaling can lead to disruption of synaptogenesis and cognitive disorders, including Alzheimer's disease (AD). The action of GABA in the central nervous system (CNS) is realized through connections with specific GABAergic receptors (subdivided into GABA-A and GABA-B receptors), leading to inhibition of nerve impulses. Outside the nervous system, the GABAergic system has been described in various tissues and organs of the human body (intestine, stomach, pancreas, kidneys, lungs, liver and others).

[0008] Although causal relationships have not been established, a decrease in B. adolescentis abundance during dysbiotic disturbances in the colonic microbiota is observed in various mental illnesses, including depression [Kovtun AS, Averina O.V., Angelova IY et al. Alterations of the composition and neurometabolic profile of human gut microbiota in major depressive disorder. Biomedicines. 2022. V. 10. №9. e2162.] and is associated with inflammatory processes in the intestine. Today, B. adolescentis is considered a bacterial species associated with or even contributing to human health. B. adolescentis bacteria are highly stable colonizers in the human intestine, even when the rest of the microbiota is dynamic. A number of studies have shown that the oral administration of bifidobacteria to animals (mice and rats) relieves symptoms of anxiety and depressive-like behavior, and also reduces the level of stress hormones [Desbonnet L., Garrett L., Clarke G., et al. The probiotic B. infantis: An assessment of potential antidepressant properties in the rat. J Psychiatr Res. 2008. 43(2). P. 164-174; Sudo N., Chida Y., Aiba Y. et al. Postnatal microbial colonization programs the hypothalamic-pituitary-adrenal system for stress response in mice. J. Physiol. 2004. V.558 (Pt 1). P. 263-275].

[0009] Whole genome sequencing allows us to determine the presence of genes involved in the synthesis of neurotransmitters and other neuroactive compounds, genes controlling the production of potential immunomodulators (exopolysaccharides, pectins, SCFAs, etc.), genes controlling antioxidant production, xenobiotic detoxification genes, vitamin biosynthesis genes, allergen genes, and others in the genome of functionally useful strains. It also allows us to identify genes that determine pathogenicity and drug resistance localized on mobile genetic elements.

[0010] The use of bifidobacteria, which are natural human symbionts, as a source of neuromodulators, immunomodulators, and other active compounds offers several advantages over other therapeutic approaches. Unlike chemical pharmaceuticals, the use of live therapeutic products based on bifidobacteria allows for the continuous production of active compounds in physiological doses directly in the intestinal mucosa. We believe that the development of psychobiotics and neurobiotics (strains and preparations based on them) is highly timely and necessary. In Russia, it is desirable to use preparations based on Russian bifidobacteria strains adapted to the Russian population, taking into account the strain's origin (i.e., isolated from the gastrointestinal tract of people living in Russia). The strain being patented was isolated from the intestinal contents of a resident of the central region of Russia and is an active producer of GABA.

[0011] Disclosure of invention

[0012] The objective of the present invention is to select, using the method of comparative genomic analysis, among the strains of the species Bifidobacterium adolescentis isolated from the body of people living in the central region of Russia, a strain with the largest number of genes involved in the manifestation of psychobiotic properties and possessing the ability to actively synthesize and release GABA into the environment.

[0013] Strain selection

[0014] To select a strain for therapeutic purposes, complete genome sequencing is currently performed to conduct a comprehensive genomic and then functional analysis to identify the strain's genetic, functional, and adaptive advantages. Sequenced genomes of 10 complete assembly bifidobacteria strains of the species B. adolescentis, isolated from the feces of apparently healthy individuals from the Central region of Russia, were studied. All strains were obtained from the collection of the Laboratory of Microorganism Genetics of the N.I. Vavilov Institute of General Genetics of the Russian Academy of Sciences https: / / gisbb.minzdrav.gov.ru / open-microflora. The strains showed the ability to produce GABA in the culture medium in varying quantities [Danilenko V.N., Averina O.V. Dyachkova M.S. Strains Bifidobacterium adolescentis 150 and Bifidobacterium angulatum GT 102, synthesizing gamma-aminobutyric acid. Patent for invention No. 2614110, 2015].

[0015] To obtain the nucleotide sequence of the genomes, chromosomal DNA sequencing was performed using two platforms: Illumina NextSeq 1000 and PromethION (Example 1). The strains were grown in liquid MRS medium (HIMEDIA, India) supplemented with cysteine ​​to a final concentration of 0.5 g / L under anaerobic conditions in a HiAnaerobic System anaerobic jar (HiMedia) at 37°C for 24 hours. Genomic DNA from the strains' cells pelleted by centrifugation was isolated using a Wizard DNA Extraction kit (Promega Corporation, USA). DNA concentration and quality were determined using a Qubit 4 fluorometer and a Nanodrop ND-1000 (Thermo Fisher Scientific, USA). Library preparation for sequencing on the Illumina NextSeq 1000 platform was performed as described in Example 1. The resulting DNA libraries were sequenced on the NextSeq1000 platform (Illumina, USA) according to the manufacturer's recommendations. NextSeq1000 / 2000P2 reagent kits (200 cycles) were used for this purpose.The process included control of 2% PhiX emission.

[0016] DNA library preparation for sequencing using Nanopore Technologies is described in Example 1. Sequencing was performed on a PromethION sequencing instrument (Oxford Nanopore Technologies, UK). Reads were performed using Guppy version 6.5.7 and default parameters (guppy basecaller).

[0017] The nucleotide sequences obtained using two sequencing technologies were assembled de novo into a complete genome using the following genome assemblers: Unicyclerv 0.5.0, Flye 2.9.2-Ы786, and Pilonv 1.24. All assembled B. adolescentis genomes were deposited in the NCBI GenBank database under the project number (BioProject) PRJNA1217612.

[0018] The obtained genome sizes ranged from 2.06 to 2.51 Mb, with an average of 2.3±0.1 Mb. The average GC content was 59.4±0.1%, and each genome contained an average of 1831±162 CDS (CoDing Sequence). Table 1 lists the data for each genome. These data indicate that the genomic characteristics of the strains sequenced in this study are comparable to those of B. adolescentis genomes from the NCBI database.

[0019] Annotation of the sequenced assembled genomes allowed us to search for genes involved in the metabolism of compounds with anti-inflammatory, neuro- and immunomodulatory activities that correlate with depression, as well as adaptive and protective genes. To search for genes encoding enzymes involved in the metabolism of depression biomarker compounds, a catalog of orthologs was used [Kovtun AS, Averina OV, Angelova IY et al. Alterations of the composition and neurometabolic profile of human gut microbiota in major depressive disorder. Biomedicines. 2022. V. 10. №9. e2162], as well as a catalog of orthologs for genes involved in the metabolism of complex carbohydrates, vitamins, amino acids, alcohols, adhesion and communication genes, adaptive genes, and genes involved in stress protection. BLASTp was performed to search for sequence similarity. Sequences matching e-value=1 × 10 -5, with a sequence identity of 60% or higher, were considered homologous. Visualization of the obtained data was performed using the ggplot2 library of the R programming language (version 4.3.1) using the Rstudio environment (version 2023.06.2+561). The distribution data of genes from the catalogs in the studied genomes are presented in Figure 1. The comparative analysis included the B. adolescentis strain 703 B (former PRL2023) and the type strain of B. adolescentis ATCC15703 [Argentini C, Lugli GA, Tarracchini C. et al. Ecology- and genome-based identification of the Bifidobacterium adolescentis prototype of the healthy human gut microbiota. Appl. Envir. Microb. 2024. V. 90. №2. P. 1-21].

[0020] The data presented in Figure 1 indicate that basically all the genes encoding enzymes involved in the metabolic pathways of B. adolescentis, including carbohydrate, amino acid, vitamin and alcohol metabolism, are present in the genomes of the Russian strains, indicating that these strains have the potential to ferment plant-derived glycans and produce useful metabolites. Most of the genes found in all compared genomes are related to immune modulation: SCFA production, adhesion and colonization; stress resistance: toxin-antitoxin (TA) type 2 systems, WhiB transcription regulators; phage defense: CRTSPR / Cas systems, Wadjet antiphage system; and neuromodulatory activity: genes for the production and transport of GABA, amino acids and others, as well as those involved in the metabolism of vitamins, the metabolism of alcohols, the accumulation and absorption of iron, communication with other bacteria of the microbiota, and in epigenetic processes (Figure 1).Only the genome of B. adolescentis strain 48 contained all genes except those from one TA family (Figure 1). Therefore, this strain was selected as a potential psychobiotic (Example 2).

[0021] Determination of the species affiliation of the patented strain

[0022] Confirmatory determination of the species affiliation of the B. adolescentis 48 strain was carried out based on the nucleotide sequence of the 16S RNA gene, 1321 bp in length. Sequences of the 16S rRNA gene fragment from various strains of the following species: B. stercoris, B. ruminantium, B. dentium, B. kashiwanohense, B. pseudocatenulatum, and B. catenulatum were also used for comparison. Table 2 presents the sequence comparison data. As additional confirmation of the affiliation of the analyzed strain to the B. adolescentis species, an analysis of the pkb2 serine-threonine protein kinase gene sequence, which is specific for this species, was performed [Nezametdinova VZ, Zakharevich NV, Alekseeva MG et al. Identification and characterization of the serine / threonine protein kinases in Bifidobacterium. Archives of Microbiology. 2014. V.196. P. 125-136]. The length of the analyzed gene fragment was 1422 bp.Table 3 shows the nucleotide sequence homology of a portion of the serine-threonine protein kinase pkb2 gene from B. adolescentis 48 to homologues from GenBank. The strain was identified as Bifidobacterium adolescentis. Genomic characterization of D. adolescentis strain 48.

[0023] The genome of the patented B. adolescentis 48 strain has been deposited in GenBank under accession number SAMN46524648. The complete genome of B. adolescentis 48 consists of a single circular sequence, which was a 2,384,895 bp chromosome with a GC content of 59.35%. A total of 2,043 genes were identified in the genome, including 1,935 protein-coding genes, 73 RNA genes, and 35 pseudogenes. A full-length set of rRNA genes, including 5S, 16S, and 23S; 57 tRNA genes, and 3 non-coding RNA genes were identified. One CRISPR / Cas system was identified. Annotation of the complete genome sequence was performed using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP).

[0024] The B. adolescentis 48 strain was characterized in accordance with the biosafety requirements published in the Pharmacopoeial article “Probiotics for medical use” of the Ministry of Health of the Russian Federation (State Quality Standard of Medicines).

[0025] A passport has been developed for the strain, characterizing the full range of biological and functional (probiotic) properties. In accordance with the requirements for characterizing probiotic cultures, the strain was deposited in the All-Russian Collection of Microorganisms (VKPM) (Moscow) in 2025. Collection number: B. adolescentis 48 VKM Ac-2296.

[0026] Cultural and morphological characteristics of the strain

[0027] Morphological and tinctorial properties: Gram-positive, non-spore-forming, granular rods with thickened ends or bifurcations. When grown on the surface of MRS agar (HIMEDIA, India) with the addition of cysteine ​​to a final concentration of 0.5 g / L for 48 hours, round, convex, smooth, cream-colored (white) colonies with smooth edges, 0.5-1 mm in diameter, are formed. When cultivated in liquid MRS broth (HIMEDIA, India) with the addition of cysteine ​​to a final concentration of 0.5 g / L for 24 hours, a uniform turbidity of the medium occurs closer to the anaerobic zone, with the formation of a precipitate.

[0028] Method, conditions and composition of media for strain propagation

[0029] The strain is cultivated on liquid and agar MRS media (Himedia) with the addition of cysteine ​​to a final concentration of 0.5 g / l for 24-48 hours. The composition of the MRS medium per 1 liter in grams: proteose peptone - 10.0; meat extract - 10.0; yeast extract - 5.0; glucose - 20.0; polysorbate 80 - 1.0; ammonium citrate - 2.0; sodium acetate - 5.0; magnesium sulfate - 0.1; manganese sulfate - 0.05; dibasic potassium phosphate - 2.0. pH (at 25 ° C) 6.5 with the addition of sterile 0.05% L-cysteine-HCl. Cultivation is carried out under anaerobic conditions using an anaerobic jar and the GasPak+ system. Incubation temperature is 37°C. In MRS broth, the strain's productivity reaches 10 9 CFU / cm 3 The strain did not exhibit hemolytic, proteolytic, lecithinase, DNA or RNA activity.

[0030] Resistance (sensitivity) to antibiotics

[0031] The strain is resistant to the following antibiotics: streptomycin, neomycin, gentamicin, kanamycin, polymyxin, amikacin, bacitracin; the strain is sensitive to the following antibiotics: rifampicin, lincomycin, vancomycin, chloramphenicol, erythromycin, tetracycline, ampicillin.

[0032] Resistance is not associated with genes located on mobile genetic elements.

[0033] Products synthesized by the strain

[0034] When grown in a growth medium for bifidobacteria Bifidobacterium Broth (BB, HiMedia, India) with 1% sodium glutamate, the patented strain synthesizes and secretes GABA into the medium. The maximum amount of GABA under these conditions is detected after 48 hours of incubation and reaches 3090 μg / ml, which was about 30% conversion of the amount of sodium glutamate [Danilenko V.N., Averina O.V. Dyachkova M.S. Strains Bifidobacterium adolescentis 150 and Bifidobacterium angulatum GT 102, synthesizing gamma-aminobutyric acid. Patent for invention No. 2614110]. Glutamate decarboxylase genes in the patented strain.

[0035] The NCBI database contains information on the sequences of glutamate decarboxylases and the corresponding genes in various strains of bifidobacteria (www.ncbi.nlm.nih.gov).

[0036] The genome of the patented strain was found to contain genes involved in the synthesis and transport (secretion) of GABA: gadB encodes the enzyme glutamate decarboxylase, and gadC encodes an antiporter that transports glutamate and gamma-aminobutyric acid. An antiporter is part of a membrane transport system that allows one substance to pass through the membrane into the cell and another substance to pass out of the cell in the opposite direction.

[0037] The nucleotide sequence of gadB and gadC genes from the genome of the patented strain was aligned with the nucleotide sequence of similar genes of other strains of the B. adolescentis species from the NCBI database and a high degree of homology (99.78-99.89) was revealed.

[0038] Thus, the ability of B. adolescentis strain 48 to actively produce GABA, the absence of antibiotic resistance genes on its mobile elements, and the absence of protease and other undesirable activities make it suitable for use in medicinal formulations, including lyophilized preparations, capsules, liquid cultures, and other similar agents for the prevention and treatment of psychiatric disorders. This strain differs from all previously described GABA-producing B. adolescentis strains because it was isolated from humans living in the central region of the Russian Federation and possesses a broad set of functional genes that may contribute to its antidepressant properties.

[0039] Brief description of the drawings

[0040] Figure 1

[0041] Distribution of functional genes in the genomes of the compared Bifidobacterium adolescentis strains (the presence of the gene in the strain genome is shown in gray). Inclusion of genes encoding: *xylul o kinase, xylose isomerase, 1,4-beta-xylanase, beta-xylosidase, pullulanase type I, alpha-amylase, beta-glucosidase, alpha-galactosidase, glycogen-splitting enzymes; ** genes involved in the biosynthesis of arginine, threonine, tryptophan, glutamine, glutamate, phenylalanine, tyrosine, glycine, amino acid transport; *** genes encoding hemagglutinin, a protein of the hemolysin family that transports ferrous iron, iron permease of the FTRT family, iron transporter; **** genes encoding the Ig-like domain-containing protein (FN3), the DUF6466 family protein, the B-type Cna domain-containing protein, the G5 domain-containing protein, and the VWA domain-containing protein. Control strains 703B (does not produce GABA) and ATCC 15703 (type strain) from the GenBank, NCBI.

[0042] Figure 2

[0043] Sequence of the 16S rRNA gene fragment of bacteria of the species Bifidobacterium adolescentis, 1321 bp in length.

[0044] Figure 3

[0045] Sequence of a 1422 bp fragment of the species-specific serine-threonine protein kinase pkb2 gene of Bifidobacterium adolescentis.

[0046] Figure 4

[0047] Alignment of the gadB (glutamate decarboxylase) gene from the B. adolescentis 48 genome with the gene of B. adolescentis strain 22L (complete genome). The identity is 99%.

[0048] Figure 5

[0049] Alignment of the gadC gene (an antiporter that transports glutamate and gamma-aminobutyric acid) from the B. adolescentis 48 genome with the gene of B. adolescentis strain 22L (complete genome). The identity is 99%.

[0050] Examples on the selection characteristics and properties of the B. adolescentis strain 48 of the present invention

[0051] Example 1. Sequencing and whole-genome assembly of chromosomal DNA of Russian B. adolescentis strains

[0052] Ten B. adolescentis strains from the laboratory collection of the Institute of General Genetics of the Russian Academy of Sciences, isolated from the feces of healthy volunteers from the central region of Russia, were sequenced. Bifidobacteria were grown in MRS arape and MRS broth (Hi-Media, India) with the addition of L-cysteine ​​(0.5 g / L). Cultivation was carried out under anaerobic conditions (HiAnaerobic System MarkIII, AnaeroHi Gas Pack 3.5L; HiMedia, India) at 37°C for 18-24 hours. Genomic DNA from the strains precipitated by centrifugation was isolated using the Wizard DNA Extraction kit (Promega Corporation, USA). DNA concentration and quality were determined using a Qubit4 fluorimeter and Nanodrop ND-1000 (Thermo Fisher Scientific, USA).

[0053] Library preparation for sequencing on the Illumina NextSeq 1000 platform was performed using the KAPAHyper Plus kit (Roche, Switzerland) according to the manufacturer's protocol. A library was prepared using 100 ng of isolated DNA and purified using KAPAHyper Pure beads (Roche, Switzerland). Library size distribution and quality were assessed using a high-sensitivity DNA chip (Agilent Technologies). Libraries were quantitatively analyzed using the Quant-iT kit (Thermo Fisher Scientific, USA). DNA libraries were sequenced on the NextSeq1000 platform (Illumina, USA) according to the manufacturer's recommendations. For this purpose, the NextSeq1000 / 2000P2 reagent kits (200 cycles) were used. A 2% PhiX release control was included in the process.Libraries for sequencing using Nanopore Technologies technology were prepared using the SQK-LSK109 sequencing kit, the SQK-LSK109 ligation kit, the EXP-NBD114 extension kit, and the built-in barcoding kit. Sequencing was performed on a PromethION sequencing instrument (Oxford Nanopore Technologies, UK). Reads were performed using Guppy version 6.5.7 with default parameters (guppy basecaller). Nucleotide sequences obtained using both sequencing technologies were assembled de novo into a complete genome using the following genome assemblers: Unicyclerv 0.5.0, Flye 2.9.2-b1786, and Pilonv 1.24. All 10 assembled B. adolescentis genomes were deposited in the NCBI GenBank database under the project number (BioProject) PRJNA1217612.

[0054] The obtained genome sizes ranged from 2.06 to 2.51 Mb, with an average of 2.3±0.1 Mb. The average GC percentage was 59.4±0.1%, and each genome contained an average of 1831±162 CDS (CoDing Sequence). Table 1 lists the data for each genome. The data in the table indicate that the genomic characteristics of the strains sequenced in this study are comparable to those of the genomes in the NCBI database.

[0055]

[0056] Example 2. Selection of B. adolescentis strain 48

[0057] The selection of the strain as a result of comparative genomic analysis was carried out among 11 genomes of Russian B. adolescentis strains with a known nucleotide sequence (Table 1).

[0058] Genome annotation allowed us to search for genes involved in the metabolism of compounds with anti-inflammatory, neuro- and immunomodulatory activities that correlate with depression, as well as adaptive and protective genes. To search for genes encoding enzymes involved in the metabolism of depression biomarker compounds, we used the ortholog catalog [Kovtun AS, Averina OV, Angelova IY et al. Alterations of the composition and neurometabolic profile of human gut microbiota in major depressive disorder. Biomedicines. 2022. V. 10. №9. e2162], as well as a catalog of orthologs for genes involved in the metabolism of complex carbohydrates, vitamins, amino acids, alcohols, adhesion and communication genes, adaptive genes, and genes involved in stress protection. BLASTp was performed to search for sequence similarity. Sequences matching with e-value = 1 × 10 -5, with a sequence identity of 60% or higher, were considered homologous. Visualization of the obtained data was performed using the ggplot2 library of the R programming language (version 4.3.1) using the Rstudio environment (version 2023.06.2+561). The most common B. adolescentis strain 703 B (former PRL2023) (not producing GABA) and the least common B. adolescentis strain ATCC15703 (type) were used as a control [Argentini C., Lugli GA, Tarracchini C. et al. Ecology- and genome-based identification of the Bifidobacterium adolescentis prototype of the healthy human gut microbiota. Appl. Envir. Microb. 2024. V. 90. №2. P. 1-21].

[0059] Figure 1 shows the gene distribution in the studied genomes. Comparative analysis indicates that virtually all genes encoding proteins involved in B. adolescentis metabolic pathways, including carbohydrate, amino acid, vitamin, and alcohol metabolism, are present in the genomes of the Russian strains. This indicates that these strains possess the potential to ferment plant-derived glycans and produce useful metabolites, as do most B. adolescentis strains in the NCBI database. The majority of genes found in all compared genomes are associated with characteristics inherent to probiotics: immune modulation (synthesis of short-chain fatty acids (SCFA)), adhesion and colonization, stress resistance (toxin-antitoxin (TA) systems, WhiB transcription regulators), protection from phages (CRISPR / Cas systems, Wadjet anti-phage system), and neuromodulatory activity (production of GABA, amino acids, etc.).), vitamin metabolism, alcohol metabolism, iron accumulation and absorption, communication with other bacteria of the microbiota, participation in epigenetic processes (Figure 1). Only the genome of the B. adolescentis 48 strain contains genes encoding the MobC relaxosome (for DNA conjugation), gas vesicle protein, and immunoglobulin domain-containing protein (for adhesion and immune modulation) (Figure 1). The genome of the B. adolescentis 48 strain contains a number of genes that are rarely found in other genomes. These are genes encoding the VirD4-like conjugation protein, nitrate reductase (production of nitric oxide), isochorismatase (biosynthesis of a group of siderophores), a protein of the NlpC / P60 family (adhesion and immunomodulation), a set of genes for the synthesis of thiamine (vitamin B1), nicotinamidase for the formation of nicotinic acid (vitamin B3) (Figure 1).

[0060] Example 3. Determination of the species of Bifidobacterium adolescentis strain 48

[0061] The 16S rRNA gene fragment sequence was used to identify the species. The length of the analyzed 16S rRNA gene fragment was 1321 bp. The nucleotide sequence of the fragment is shown in Figure 2. BLASTp was performed to identify sequence similarities. The results of comparison with the 16S rRNA gene sequences of other known strains available in the NCBI database are presented in Table 2. This table also presents the results of comparison of the analyzed 16S rRNA gene fragment of Bifidobacterium adolescentis with sequences from various strains of the following species: Bifidobacterium ruminantium, Bifidobacterium dentium, Bifidobacterium kashiwanohense, Bifidobacterium pseudocatenulatum, Bifidobacterium catenulatum. Their sequences were highly homologous to the target sequence.

[0062] To further confirm the affiliation of the analyzed strain with the species Bifidobacterium adolescentis, the pkb2 serine-threonine protein kinase gene sequence was analyzed. The length of the analyzed fragment was 1422 bp (Figure 3). BLASTp was used to identify sequence similarities. The results of comparison with the prb2 gene sequences of other known strains available in the NCBI database are presented in Table 3.

[0063]

[0064]

[0065] Example 4. Characteristics of Bifidobacterium adolescentis strain 48

[0066] The assembled genome of B. adolescentis strain 48 as a complete genome has been deposited in the NCBI GenBank database and is available as Biosample ID - S AMN46524648.

[0067] The complete genome of B. adolescentis strain 48 consists of a single circular sequence, which was a 2,384,895 bp long chromosome with a GC content of 59.35%. A total of 2043 genes were identified in the genome, including 1935 protein-coding genes, 73 RNA genes, and 35 pseudogenes. A full-length set of rRNA genes was identified, including 5S, 16S, and 23S; 57 tRNA genes; and 3 non-coding RNA genes. One CRISPR / Cas system was identified. Annotation of the complete genome sequence was performed using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP). The strain has the accession number of deposition in the All-Russian Collection of Microorganisms VKM Ac-2296.

[0068] The strain is cultivated on liquid and agar MRS media (Himedia) with the addition of cysteine ​​to a final concentration of 0.5 g / l for 24-48 hours. The composition of the MRS medium per 1 liter in grams: proteose peptone - 10.0; meat extract - 10.0; yeast extract - 5.0; glucose - 20.0; polysorbate 80 - 1.0; ammonium citrate - 2.0; sodium acetate - 5.0; magnesium sulfate - 0.1; manganese sulfate - 0.05; dibasic potassium phosphate - 2.0. pH (at 25 ° C) 6.5. The medium must be sterilized for 15 minutes at 121 ° C at 1 atm. Cool to 45-50°C and add sterile L-cysteine-HCl to 0.05%.

[0069] Cultivation is carried out under anaerobic conditions using an anaerobic aerostat and a GasPak+ system (HiAnaerobic System Marklll, AnaeroHi Gas Pack 3.5L; HiMedia, India). Incubation temperature is 37°C.

[0070] When grown on agar medium, the strain forms white, glossy, convex colonies with smooth edges, 0.5-1 mm in diameter. When cultivated in liquid MRS medium for 24 hours, the medium becomes uniformly cloudy near the anaerobic zone, with sediment forming. In MRS broth, the strain's productivity reaches 10 9 CFU / cm 3 According to the characteristics, the cells of the strain are gram-positive, non-motile, have the form of granular rods with thickenings at the ends or bifurcations, and do not form spores.

[0071] This species (genus) is not listed in the classification of microorganisms by pathogenicity groups in SanPiN 1.2.3685-21 "Hygienic Standards and Requirements for Ensuring the Safety and (or) Harmlessness of Environmental Factors for Humans," meaning it can be used as a probiotic strain. The strain is stored in cryovials in Trypticase Soy Broth (Bio Rad USA) with the addition of 10% glycerol at -80°C.

[0072] The strain did not exhibit hemolytic, proteolytic, lecithinase, DNA, or RNA activities. Therefore, it can be used in the production of probiotics. The strain is resistant to the following antibiotics: streptomycin, neomycin, gentamicin, kanamycin, polymyxin, amikacin, and bacitracin; the strain is sensitive to the following antibiotics: rifampicin, lincomycin, vancomycin, chloramphenicol, erythromycin, tetracycline, and ampicillin. Resistance is not associated with genes located on mobile genetic elements.

[0073] The B. adolescentis strain exhibits uniform morphological, tinctorial, and cultural properties without obvious signs of dissociation. It lacks the ability to form mucous colonies and capsules. The cells actively ferment carbohydrates, producing primarily acetic and lactic acids in a molar ratio of 3:2.

[0074] The cells of the strain do not produce catalase, do not form indole and hydrogen sulfide, do not produce phenol, and do not form ammonia from arginine.

[0075] The lyophilized strain B. adolescentis 48 is stored at a temperature of plus (6±8)°C.

[0076] When grown in Bifidobacterium Broth (BB, HiMedia, India) growth medium for bifidobacteria containing 1% monosodium glutamate, the patented strain synthesizes and secretes GABA into the medium. The maximum amount of GABA under these conditions is detected after 48 hours of incubation and reaches 3090 μg / ml, which is approximately 30% of the conversion of monosodium glutamate.

[0077] Patents - analogs

[0078] Bifidobacterium adolescentis 150 and Bifidobacterium angulatum GT 102 strains synthesizing gamma-aminobutyric acid. Patent for invention RU 2614110, 2015 Danilenko V.N., Averina O.V., Dyachkova M.S.

[0079] Lactobacillus plantarum and Lactobacillus brevis strains synthesizing gamma-aminobutyric acid. Patent for invention RU 2575625 C1, 20.02.2016. Danilenko V.N., Yunee R.A., Poluektova E.U.

[0080] A pharmaceutical composition for the treatment of inflammatory diseases of the intestinal mucosa based on the Lactobacillus brevis strain 47f, exhibiting local anti-inflammatory activity. Patent for invention RU 2675110 C1, December 17, 2018. Marsova M.V., Klimina K.M., Abilev S.K., Poluektova E.U., Danilenko V.N.

[0081] Probiotic composition for the treatment and prevention of pathological conditions arising from dysregulated interactions of the gut-brain axis. Patent for invention RU 2831469, December 9, 2024. Specialists from the Federal State Budgetary Institution "Center for Clinical and Practical Support" of the Federal Medical and Biological Agency of Russia.

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