Metabolite composition of probiotic strain lactobacillus paracasei B-11821

. Lactobacillus paracasei B-11821 is a probiotic strain of microorganisms, for which previous studies have shown pronounced antagonistic and adhesive activity, a high level of acid formation and manufacturability. The characteristics of the metabolite profile of the promising probiotic strain can significantly expand the scope of its application in the medical, pharmaceutical and food industries. Using high-performance liquid chromatomass spectrometry and gas-liquid chromatography, it was established that this strain is an active producer of essential amino acids: (isoleucine (1.920 mg/mL), phenylalanine (0.963 mg/mL), valine (0.840 mg/mL), leucine (0.561 mg/mL), methionine (0.369 mg/mL), threonine (0.233 mg/mL), histidine (0.055 mg/mL), lysine (0.008 mg/mL)); as well as a number of replaceable amino acids (tyrosine (0.622 mg/mL), alanine (0.438 mg/mL), proline (0.278 mg/mL), glycine (0.111 mg/mL), glutamine (0.015 mg/mL), asparagine (0.014 mg/mL), serine (0.008 mg/mL), arginine (0.005 mg/mL)). The strain is also an active producer of organic acids (lactic (16.2 mg/ml), acetic (0.9 mg/ml) and gamma-aminobutyric (0.3 mg/ml) GABA, propionic (0.02 mg/ml) acids. The obtained results can be used for development based on the strain Lactobacillus paracasei B-11821 probiotic preparations, functional food products, technologies for synthesis of lactic and gamma-aminobutyric acids.


Introduction
Probiotic preparations based on lactobacilli are very effective in preventing and treating intestinal infections and have been actively used in medical and veterinary practice for more than a century.The Food and Drug Administration (FDA, USA) has assigned lactobacilli the status of GRAS, that is, absolutely harmless microorganisms to humans and animals [1].Consequently, all metabolites produced by lactobacilli during the cultivation process are also considered absolutely safe.
Metabolites are metabolic products released by lactobacilli during cultivation.They are the main intermediaries in realizing the beneficial effects of biological products and functional nutrition products.The variety of released metabolites from the point of view of chemical structure determines the multifaceted mechanisms of the beneficial effects of lactobacilli on both the local intestinal microflora and the body as a whole.Many studies have shown that lactobacilli secrete various organic acids, alcohols, hydrogen peroxide, bacteriocins, due to which their antagonistic effect is manifested against pathogenic and conditionally pathogenic microorganisms [2, 3].The vitamins and amino acids secreted by lactobacilli, including essential ones, have a beneficial effect on metabolism and replenish their deficiency.
Lactic acid bacteria are capable of synthesizing gamma aminobutyric acid (GABA), which is the most important inhibitory neurotransmitter of the human central nervous system, improves metabolic processes in the brain, helps remove toxic metabolic products, which generally results in reduced stress levels, improved sleep, memory and attention.According to numerous studies, GABA is a cytoprotective antihypoxant and antioxidant, has antitumor, anti-inflammatory, antihypertensive and analgesic properties, and stimulates the immune system [4, 5].The precursor to GABA is monosodium glutamate, which is added to the nutrient medium for cultivating lactobacilli.
The purpose of this work is to isolate and characterize metabolites of the probiotic strain Lactobacillus paracasei B-11821.
The object of the study is the metabolites of the culture liquid of the strain Lactobacillus paracasei B-11821.
Determination of amino acid composition by high-performance liquid chromatography-mass spectrometry.Experimental samples of the supernatant liquid were diluted 50 times with distilled water.Identification and determination of amino acid concentrations in the sample were carried out using a tandem liquid chromatography-mass spectrometer «LCMS-8040» (SHIMADZU, Japan).MRM (Multiple Reaction Monitoring) detection mode with electrospray ionization and triple quadrupole MS/MS analyzer.The substances were separated on a Zorbach EclipseXDB-C18 column (2.1 mm x 150 mm, 3.5 µm), thermostated at a temperature of 35ºC, with an eluent flow rate of 0.25 ml/min.The volume of the injected sample was 3 μl.Elution mode -binary gradient: phase A -1000 ml of ultrafine water with 1 ml of formic acid, phase B -acetonitrile, initial content 10% from 0 min, increase to 70% within 6 min, hold at 70% for 10 min.Ionizing voltage 3.5 kV, collision energy (collision gas -argon) 35 eV.Interface temperature 400°C, desolvation line temperature 250°C.Gas flow rate -atomizer (nitrogen) 3 l/min, gas flow rate -dryer (nitrogen) 15 l/min.
Determination of short-chain fatty acids by gas-liquid chromatography.Experimental samples of the supernatant liquid were diluted 10 and 100 times with distilled water.Identification and determination of the concentration of acids in the samples was carried out using a gas chromatograph «GC-2010» (SHIMADZU, Japan).The device is equipped with a capillary quartz column CP-Wax 58 FFAP CB with a length of 25 m and an internal diameter of 0.2 mm with a polar stationary phase based on modified polyethylene glycol with a thickness of 0.3 μm.Detector -flame ionization, universal detection mode.Analysis conditions: detector temperature 300°C, carrier gas -helium; flow through the column 1.1 ml/min, column thermostat temperature: initial 65°C, rise at a rate of 10°C/min to 250°C, hold at 250°C for 10 minutes, injector temperature 200°C, injection mode -Split (with flow division), division factor -10, injected sample volume 1 µl.
Methodology for determining the content of lactic and gamma-aminobutyric acids in the culture liquid using high-performance liquid chromatography-mass spectrometry.Identification and determination of acid concentrations in samples was carried out using a tandem liquid chromatography mass spectrometer «LCMS-8040» (SHIMADZU, Japan) with a three-quadrupole system.Dosed sample volume 3 µl, column thermostat temperature 35°C, mobile phase flow rate 0.25 ml/min, elution mode -binary gradient: phase A -water, phase B -400 ml of ultrafine water with 100 mg of ammonium formate: initial content 10% from 0 min, increase to 70% for 6 min, hold at 70% for 10 min.Ionization type -electrospray (positive polarity for GABA and negative polarity for lactic acid).Ionizing voltage 3.5 kV, collision energy (collision gas -argon) 35 eV.Interface temperature 400°C, desolvation line temperature 250°C.Gas flow rate -atomizer (nitrogen) 3 l/min, gas flow rate -dryer (nitrogen) 15 l/min.Data acquisition modes: Q3 SIM at selected m/z 89, MRM (transition 104 → 69).
The antagonistic activity of lactobacilli against pathogenic and conditionally pathogenic strains is influenced by the level of production of lactic acid, as well as a number of other organic acids.The results of determining the concentration of organic acids in the studied culture liquid are presented in Table 2.  1 5.10±0.25 Gamma-aminobutyric 2 4.30±0.20Gamma-aminobutyric 3 0.08±0.004 1 -when adding 1% monosodium glutamate to the nutrient medium 2 -when adding 1.5% monosodium glutamate to the nutrient medium 3 -without added monosodium glutamate The results of the study show that the strain under study is an active producer of lactic acid (16.2 mg/ml).The limiting acid production level of L. paracasei B-11821 is high and amounts to 340 Turner degrees.This allows for the accumulation of relatively high concentrations of lactic acid in the culture fluid.Lactobacilli as producers of lactic acid are currently actively used in food technologies.A promising direction is also the use of biosynthetic lactate in the production of bidegradable plastic.The studied acetic and propionic acids were synthesized by the strain in significantly smaller quantities.Lactobacilli are an active producer of GABA; moreover, GABA production increases significantly when monosodium glutamate is added to the nutrient medium at a concentration of 1%, which is its precursor.Higher concentrations of monosodium glutamate led to a decrease in the level of GABA synthesis.To assess the dynamics of GABA concentration during incubation of L. paracasei B-11821, samples of the culture liquid were taken every 6 hours.The results of the study are presented in Table 3 and Figure 1.As can be seen in the graph of Figure 1, lactobacilli begin to actively synthesize GABA in the stationary phase of culture growth; after 30 hours of incubation, the concentration of GABA reaches a maximum value of 5.88 mg/ml and then begins to decrease.This should be taken into account when developing metabolite preparations enriched with GABA.

Conclusion
Metabolites of the probiotic strain Lactobacillus paracasei B-11821 were isolated and characterized.This strain is an active producer of all essential amino acids and a number of non-essential ones.The highest concentrations of isoleucine (1.920 mg/ml), phenylalanine (0.963 mg/ml), valine (0.840 mg/ml), tyrosine (0.622 mg/ml), leucine (0.561 mg/ml), alanine (0.438 mg/ml), methionine (0.369 mg/ml), proline (0.278 mg/ml), threonine (0.233 mg/ml), glycine (0.111 mg/ml) were detected.Histidine (0.055 mg/ml), glutamine (0.015 mg/ml), asparagine (0.014 mg/ml), lysine (0.008 mg/ml), serine (0.008 mg/ml), arginine (0.005 mg/ml) are contained in less quantity.It was also shown that the culture liquid of the strain Lactobacillus paracasei B-11821 contains high concentrations of lactic (16.2 mg/ml), acetic (0.9 mg/ml) and gamma-aminobutyric (0.3 mg/ml) acids.An assessment of the dynamics of GABA concentration showed that its maximum amount accumulates after 30 hours of cultivation.The data obtained are relevant for the development of probiotic preparations based on the strain Lactobacillus paracasei B-11821, functional food products, technologies for the synthesis of lactic acid for the purpose of producing bioplastics, as well as technologies for the production of biosynthetic GABA and preventive medications based on it, for example, psychobiotics.

Table 1 .
Content of amino acids in the culture liquid of Lactobacillus paracasei B-11821

Table 2 .
Content of organic acids in the culture liquid of the strain Lactobacillus paracasei B-11821

Table 3 .
Concentration of GABA in the culture liquid at various stages of growth of L. paracasei B-11821