Introduction

One of the important tasks of modern crop production is to increase the resistance of valuable agricultural crops to the influence of abiotic stress factors (soil drought, frost, salinity, heavy metals, UV radiation, herbicides, and flooding) and biotic stress factors (phytopathogens, phytoviruses) [1, 2]. The increase in their intensity leads to an increase in the level of reactive oxygen species (ROS), which are aggressive stress agents. Exceeding the generation of oxidants over the cell’s ability to eliminate them leads to hyperoxidation—oxidative stress [3]. Oxidative stress determines the state in which the “prooxidant-antioxidant” balance is disturbed in the cell, which leads to hydroxylation of nucleic acids, protein denaturation, lipid peroxidation, and apoptosis [4].

Among the members of the Poaceae family, barley (Hordeum vulgare L.) is one of the most economically important grain crops [5], since this cereal is grown in countries whose climatic conditions differ dramatically [6].

In recent years, the increase of anomalies in the environment leads to the accumulation of oxidants in the organism of plants. The high damaging capacity of ROS, the initiators of phytostress, poses a threat to important biomolecules (DNA, proteins, enzymes, lipids, etc.) of plant cells. The redox-homeostasis of phytoobjects is supported by a complex of antioxidants (AN). The balanced operation of high- and low-molecular protectors during different phases of oxidative stress plays an important role in maintaining the viability of plants. However, prolonged exposure to oxidants disrupts redox cycles [7].

Thus, the functioning of the plant organism’s defense system largely depends on the concentration of oxidants. First, high levels of ROS damage protective enzymes (catalase, peroxidase, superoxide dismutase), which leads to a decrease in their activity. Second, the action of stress agents depletes the pool of low-molecular-weight antioxidants. As a result, these disturbances in the normal work of AN lead to the death of plant cells [8].

One of the stabilization ways of redox homeostasis in the plant organism is their mutually beneficial relationships with rhizosphere microorganisms [9]. Such microbes include representatives of the genera Bacillus, Pseudomonas, Azospirillum, Azotobacter, Klebsiella, Enterobacter, Alcaligenes, Arthrobacter, Burkholderia, Serratia, and others [10, 11]. Plant growth-promoting rhizobacteria (PGPR) are of interest in the creation of biological plant protection agents, as they are characterized by the formation of long-term protection of the macroorganism against various stress factors [12]. PGPR contribute to the development of stress tolerance in phytoobjects through various mechanisms [13]. One of which is the activation of the synthesis of various antioxidant compounds in the plant organism [14].

Among them, compounds of phenolic nature (Ph-OH), in particular phenolcarboxylic acids and flavonoids, are of particular interest. These secondary metabolites play an important role in plant metabolism. However, their main mechanisms are related to direct and indirect antioxidant action. That is, Ph-OH acts as electron donors for oxidants, inactivate free radicals, and chelate metal ions that initiate ROS formation reactions [15]. And they can also indirectly activate antioxidant enzymes and inhibit enzymes that induce pro-oxidant effects [16].

In the crop sector of Ukraine, Mongolia, and Canada, barley is one of the dominant cereal crops. The most promising varieties in these countries include Virazh (Ukraine), Burkhant (Mongolia), and Copeland (Canada). They are widely used in production and consumption, and therefore have a high research value.

Accordingly, the main goal of the presented work was to compare the differences that were founded in the composition and content of free and bound phenolic compounds in different barley varieties, the seeds of which were exposed to the action of hydrogen peroxide and post-treatment with the nanocomposite complex bacterial preparation Azogran. This study is important in understanding the role of complex microbial preparations in modern agricultural biotechnologies.

Materials and methods

Research objects

Bacterial strains

  • Bacillus subtilis IMV B-7023 was isolated from black soil (Cherkasy region, Ukraine). The strain is supported at the Depositary of the Zabolotny Institute of Microbiology and Virology, NAS of Ukraine. This strain is a component of the nanocomposite complex bacterial preparation Azogran for crop production [17].

  • Azotobacter vinelandii IMV B-7076 was isolated from the rhizosphere of sugar beet in the Department of Microbiological processes on solid surfaces, Zabolotny Institute of Microbiology and Virology, NAS of Ukraine. This strain is a component of the nanocomposite complex bacterial preparation Azogran for crop production [18].

    • Nanostructured mineral bentonite is a variety of minerals of the montmorillonite group. We used bentonite from the Dashukovsky deposit (Cherkasy region). The size of bentonite nanoparticles was 28.92–99.21 nm;

    • Grain seeds of spring barley: Virazh (Ukraine), Burkhant (Mongolia), Copeland (Canada) varieties;

    • Nanocomposite complex bacterial preparation Azogran. The bacteria that make up the biological product are active producers of amino acids, enzymes, organic acids, antibiotics, phytohormones, vitamins, phenolic compounds, and other biologically active components [19].

The cultivation conditions of bacteria

B. subtilis IMV B-7023 was cultivated in modified glucose-mineral liquid nutrient medium [20], g L−1: (NH4)2SO4—0.5; MgSO4 × 7H2O—0.3; NaCl—0.3; KCl—0.3; CaCO3—5.0; MnSO4 × 7H2O—0.001; FeSO4 × 7H2O—0.001; glucose—10.0; sodium glycerophosphate—2.0; distilled water—1.0 L; pH 7.0–7.2.

The number of viable cells (colony-forming units (CFU)) was determined by the method of seeding a suspension of bacteria on a potato agar medium from serial tenfold dilutions. The composition of potato agar medium [21], g L−1: peeled potatoes—200.0; CaCO3—0.2; MgSO4 × 7H2O—0.2; agar–agar—15.0; tap water—1 L; pH 6.8–7.2.

A. vinelandii IMV B-7076 was cultivated in liquid Burke’s medium [22], g L−1: K2HPO4 × 3H2O—0.64; KH2PO4—0.16; NaCl—0.2; MgSO4 × 7H2O—0.2; CaSO4 × 2H2O—0.05; Fe2(SO4)3—0.005; FeSO4 × 7H2O—0.003; Na2MoO4 × 2H2O—0.001; sucrose—20.0; distilled water—1.0 L; pH 7.0–7.2.

The number of viable cells (colony-forming units (CFU)) was determined by the method of seeding a suspension of bacteria on an Ashby’s solid nutrient medium [22], g L−1: sucrose—20.0; K2HPO4 × 3H2O—0.2; MgSO4 × 7H2O—0.2; NaCl—0.2; K2SO4—0.1; CaCO3—5.0; distilled water—1.0 L. In this medium was added the 1 mL of microelement solution (according to Fedorov) of the following composition: H3BO3—5.0; (NH4) 2MoO4 × 2H2O—5.0; ZnSO4 × 7H2O—0.2; KJ—0.5; NaBr—0.54; Al2(SO4)3 × 18H2O—0.3; distilled water—1.0 L; pH 7.2–7.3.

Cultivation of the studied bacterial strains in liquid nutrient media was carried out on rotary shakers (n = 240 rpm) in Erlenmeyer flasks with a volume of 750 mL (100 mL of medium) or in microbiological test tubes into which 20 mL of the medium was added. A daily culture of bacteria was used as an inoculum. The number of viable cells was determined by seeding bacterial suspensions from serial tenfold dilutions onto agar media. After cultivating the inoculations at a temperature of + 28 ± 1 °C, the colonies (colony-forming units, CFU) were counted on the surface of the agar medium in the dilution where their number ranged from 30 to 300.

The design of peroxide stress

Plants of three barley cultivars were grown under greenhouse conditions (the temperatures around 18 °C during day and 14 °C at night, a photoperiod of 12 h and 50–70% humidity). The seed material previously was subjected to different treatment options:

  • Seeds were soaked in sterile distilled water (H2O) during 1 h;

  • Seeds were bacterized with 3 ml of nanocomposite complex bacterial preparation Azogran for 1 h (Nano-CP);

  • Seeds were treated with 33% hydrogen peroxide for 30 min. (H2O2);

  • Seeds were exposed to 33% hydrogen peroxide (30 min.) and bacterized with 3 ml of nanocomposite complex bacterial preparation Azogran for 1 h (H2O2 + Nano-CP).

The seeds of each barley variety were sowed in 4 replications, 50 seeds per row.

Extraction of free and bound phenolic compounds from different varieties of barley

Plants were selected in phase of stem elongation and dried at room temperature (22 °C) without access to direct light to constant weight. The samples were ground to a powder-like state by a SaturnST-CM1031 electric coffee grinder (220–240 V, 50 Hz, China). A sample (1 g) was took from each variant and divided into two parts of 0.5 g each:

  • First part (extraction of the free phenolic compounds): a sample (0.5 g) of each of the samples of barley plants was placed in round-bottom flasks under reflux and extracted twice with methanol (CH3OH) (50 mL per sample) at a water bath (67.4 °C) during 2 h. The total volume of the extract (100 mL) was filtered through No. 1 filter paper in the Buchner funnel. The resulting filtrate was evaporated to dryness on an IR-1M2 rotary evaporator (Production Association Khimlaborpribor, USSR). The dry residue was redissolved in 2 mL of methanol and analyzed by HPLC;

  • Second part (extraction of the hydrolysis fraction of phenolic compounds): a weighed portion (0.5 g) of each of the samples of barley plants was introduced into round-bottom flasks under reflux and was subjected to acid hydrolysis by adding 30 mL of a mixture of 2 M HCl:CH3OH (1:1). The presence of methanol prevents the destruction of some phenolic acids [23].

Hydrolysis was carried out at 90 °C for 2 h. The hydrolysates were filtered through No. 1 filter paper in the Buchner funnel. The extraction was repeated three times by ethyl acetate (CH3COOC2H5) (30 mL per sample) during 30 min. The extracts were evaporated to dryness on an IR-1M2 rotary evaporator (Production Association Khimlaborpribor, USSR). The dry residue was redissolved in 2 mL of methanol and analyzed by HPLC. The content of bound phenolic compounds was determined from the difference between the amount of total and free polyphenols in the hydrolysis fraction.

HPLC analysis of phenolic acids

High-performance liquid chromatography (HPLC) (Agilent 1200, USA) was used to assess the composition of phenolic acids extracted from the barley samples. Methanol (CH3OH) (A) and 0.1% formic acid (H-COOH) in water (B) were used as the mobile phase. The settings for the elution gradient were as follows: 0 min—A (25%): B (75%); 25 min—A (75%): B (25%); 27 min—A (100%): B (0%); 35 min—A (100%): B (0%). Separation was carried out on a Zorbax SB-Aq column (4.6 mm × 150 mm, 3.5 µm) (Agilent Technologies, USA), flow rate was 0.5 mL/min, thermostat temperature was 30 °C, injection volume was 4 µL. Detection was carried out using a diode array detector with signal registration at 250 nm and 275 nm and fixation of absorption spectra in the range of 210–700 nm [24]. Identification and quantitative analyses were carried out using standard solutions of phenolic compounds: gallic acid, 4-hydroxyphenylacetic acid (4-HPAA), chlorogenic acid, caffeic acid, syringic acid, p-coumaric acid, trans-ferulic acid, sinapic acid, trans-cinnamic acid. The values are expressed as µg g−1 DW (dry weight).

HPLC analysis of flavonoids

High-performance liquid chromatography (HPLC) (Agilent 1200, USA) was used to assess the composition of flavonoids extracted from the barley samples. Acetonitrile (A) and 0.1% formic acid in water (B) were used as the mobile phase. The settings for the elution gradient were as follows: 0 min—A (30%): B (70%); 20 min—A (70%): B (30%); 22 min—A (100%): B (0%); 30 min—A (100%): B (0%). Separation was carried out on a Zorbax SB-C18 column (4.6 mm × 150 mm, 3.5 µm) (Agilent Technologies, USA), flow rate was 0.25 mL/min, thermostat temperature was 30 °C, injection volume was 4 µL. Detection was carried out using a diode array detector with signal registration at 280 nm and 365 nm and fixation of absorption spectra in the range of 210–700 nm [25,26,27]. Identification and quantitative analyses were carried out using standard solutions of flavonoids: rutin, quercetin-3-b-glucoside, naringin, neohesperidin, quercetin, naringenin, kaempferol, luteolin, apigenin. The values are expressed as µg g−1 DW.

Statistical analysis

Microsoft Excel (Microsoft Corporation, USA) was used to analyze the data on the average of the three replicates (± SE) obtained from the three independent experiments. Differences were compared with the statistical significance at a P level less than 0.05 (P < 0.05) [28]. Tukey’s test was used to determine significant (P < 0.05) differences between the samples.

Results and discussion

Composition and content of the free phenolic acids of different barley varieties

Modern agricultural biotechnologies are closely related to microbial biopreparations for crop production, in particular those of complex action. They are one of the important components of ecological agriculture [29, 30]. The mechanism of formation of plants stress tolerance with the help of biological preparations is closely related to the metabolites of bacteria included in their composition. A number of these compounds are considered as triggers that start a cascade of plant-specific biosynthetic processes that increase resistance to the damaging effects of oxidants [31, 32]. One of these processes can be the activation of the production of phenolic compounds in the organism of phytoobjects [14].

We established significant differences in the qualitative and quantitative composition of phenolic compounds in the plants of the studied varieties of barley, the seeds of which were subjected to hydrogen peroxide (H2O2) treatment and post-treatment with Azogran.

The 4-hydroxyphenylacetic, chlorogenic, caffeic, syringic, benzoic, p-coumaric, trans-ferulic, sinapic and trans-cinnamic acids were identified in the variant with the treatment of seeds of different varieties of barley with distilled sterile water (dH2O). Their highest content was observed in the free fraction of phenolic compounds obtained from Copeland variety barley plants—1488 µg g−1 DW (Fig. 1, Table 1). No significant differences in the quantitative composition were found in the extracts obtained from plants of two other varieties of barley (Table 1).

Fig. 1
figure 1

HPLC analysis of phenolic acids in the free fraction that was obtained from Copeland variety barley plants after seed treatment with sterile distilled water

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Table 1 Composition and content of phenolic acids in the free fraction that was obtained from plants of different barley varieties
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Whereas, the content of phenolic acids in the free fraction obtained from Virazh variety barley plants increased significantly when seeds were treated with the nanocomposite complex bacterial preparation Azogran. Accordingly, the concentration of sinapic acid increased by 97.9%, trans-cinnamic acid by 106.9%, syringic acid by 116.4%, caffeic acid by 117.9%, benzoic acid by 123.9%, 4-HPAA by 124.6%, chlorogenic acid by 158.8%, p-coumaric acid by 230.2%, compared to the sample where the seeds were treated with sterile distilled water (Table 1).

As for the other varieties, only in the Copeland variety plants, the content of sinapic acid was increased by 1.6%, syringic acid by 8.8%, caffeic acid by 13.9%, and trans-ferulic acid by 104.2%, compared to the variant where barley seeds were treated with dH2O (Table 1). The concentration of phenolic acids in Burkhant variety plants practically did not differ from this sample where seeds were treated with dH2O. No changes in the qualitative composition of these compounds in the plants of the studied barley varieties were recorded (Table 1).

The results obtained on the effect of PGPR on the levels of phenolic acids in plants were consistent with a number of other studies. In particular, the content of these compounds in the Piper betle L. after inoculation with Serratia marcescens NBRI1213 bacteria [33] and in the Tagetes minuta after inoculation with Pseudomonas fluorescens WCS417r and Azospirillum brasilense [34] was increased. Singh et al. [35] reported that mono- or complex inoculation of chickpea seeds with Ps. fluorescens Pf4 and Ps. aeruginosa Pag strains activated the synthesis of gallic, ferulic, and chlorogenic acids in plants.

Under the influence of an aggressive stress agent (H2O2) on barley seeds, the total content of phenolic acids in the free fraction obtained from Copeland variety and Burkhant variety plants decreased by 15.4% and 78.9%, compared to the variant where the seeds were treated with sterile distilled water. In particular, in the Burkhant variety plants, the concentration of chlorogenic acid decreased by 138.71 µg g−1 DW, caffeic acid by 175.52 µg g−1 DW, benzoic acid by 139.00 µg g−1 DW, trans-ferulic acid by 41.34 µg g−1 DW, and sinapic acid by 132.74 µg g−1 DW, respectively. And in the Copeland variety plants, the level of chlorogenic acid decreased by 39.04 µg g−1 DW, caffeic acid by 45.50 µg g−1 DW, benzoic acid by 116.54 µg g−1 DW, and sinapic acid by 57.80 µg g−1 DW, respectively. It was also found that syringic acid was not identified in methanol extracts obtained from plants of these barley varieties (Fig. 2, Table 1).

Fig. 2
figure 2

HPLC analysis of phenolic acids in the free fraction that was obtained from barley plants of the Burkhant (A) and Virazh (B) varieties after treatment of seeds with the stress agent hydrogen peroxide

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Elguera et al. [36] found that at growing of Lepidium sativum in the presence of cadmium chloride [Cd(II)], the content of chlorogenic, ferulic, and caffeic acids in the free fraction of phenolic acids obtained from plant leaves decreased. Increased levels of ROS can lead to disruption of the secondary-metabolite biosynthesis (in particular phenolic compounds) whose structural skeleton consists of carbon atoms [37].

However, the treatment of seeds of the barley Virazh variety with hydrogen peroxide increased the level of phenolic acids (free fraction) by two times in plants compared to the variant (dH2O) (Table 1). Stress tolerance and biological activity of cereal crops significantly depend on their variety [38,39,40].

At post-treatment with Azogran for seeds of the tested cereal varieties, the concentration of phenolic acids (free fraction) increased only in plants of barley Burkhant variety by 27.24 µg g−1 DW, compared to stressed plants of the same variety. Especially the level of sinapic acid increased by 34.1%. This phenolic compound is a powerful antioxidant. Its antiradical activity (ARA) is significantly higher than that of ferulic acid [41, 42]. Chiappero et al. [43] found that inoculated with Ps. fluorescens WCS417 r and B. amyloliquefaciens GB03 drought-stressed Mentha piperita plants activated the synthesis of phenolic compounds. Post-treatment of stressed seeds of two other barley varieties with biological preparation Azogran did not have a pronounced stimulating effect on the content of phenolic carboxylic acids of the free fraction in their plants. Thus, for barley Virazh variety, an increase in caffeic acid by 5.7% was observed, and in plants of Copeland variety, the content of 4-HPAA increased by 15.0% and trans-ferulic acid by 79.9%, compared to the stressed variant (Table 1). These phenolic compounds effectively inactivate such stress agents as hydrogen peroxide and hydroxyl radical [44, 45]. Their high antioxidant activity depends on the presence of hydroxyl and methoxy groups in the chemical structure [46]. All phenolic carboxylic acids, including syringic acid (SA), were identified in the qualitative composition (Table 1). The antioxidant potentials of SA and caffeic acid are very similar [47]. And the high ARA of this phenolic compound is due to the presence of two methoxy groups attached to the aromatic ring at positions 3 and 5 [48, 49]. Thus, post-treatment with the nanocomposite complex bacterial preparation Azogran of hydrogen peroxide-stressed seeds of different barley varieties activated the synthesis of phenolic acids with powerful antioxidant and antiradical properties in plants.

Composition and content of the bound phenolic acids of different barley varieties

The qualitative and quantitative content of phenolic acids in the hydrolysis fraction obtained from plants of different barley varieties differed significantly from their presence in the free fraction and depended on the variant of seed treatment.

It was found that the hydrolyzed fraction of phenolic compounds obtained from plants of the Burkhant barley variety, whose seeds were soaked in sterile distilled water, contains syringic, benzoic, p-coumaric, trans-ferulic, sinapic, and trans-cinnamic acids (Table 2). In addition to these phenolic acids, 4-HPAA and caffeic acids were found in the plants of Virazh barley variety, but syringic acid was absent. The quantitative content of each of the phenolic acids in the bound fraction was the highest compared to the other two varieties (Fig. 3B, Table 2). In the hydrolyzed fraction of phenolic acids from plants of Copeland barley variety, all phenolic acids were present, except for gallic acid (Fig. 3C, Table 2).

Table 2 Composition and content of phenolic acids in the bound fraction that was obtained from plants of different barley varieties
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Fig. 3
figure 3

HPLC analysis of phenolic acids in the hydrolyzed fraction that was obtained from plants of Virazh (B) and Copeland (C) barley varieties after soaking of seeds in sterile distilled water

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At inoculation of seeds of the Burkhant barley variety with the nanocomposite complex bacterial preparation Azogran, in the bound fraction of phenolic acids obtained from plants, a high level of caffeic acid (3,4-dihydroxycinnamic acid) was found—89.88 µg g−1 DW (Table 2). This compound is an ortho-dihydroxyphenol with a powerful antioxidant potential and can inactivate the highly aggressive hydroxyl radical [50]. The product of the methylation reaction of caffeic acid is ferulic acid, which, together with p-coumaric acid, initiates the synthesis of lignin [51, 52]. This natural polymer provides plant resistance to various abiotic stressors [53].

In the bound fraction obtained from plants of Virazh barley variety, after inoculation seeds with Azogran, high content of caffeic acid (239.82 µg g−1 DW) and benzoic acid (264.40 µg g−1 DW) was detected (Table 2).

The treatment of seeds with 33% H2O2 negatively affected both on qualitative and on quantitative composition of phenolic acids in the bound fraction obtained from plants of the three tested barley varieties. In particular, in plants of Burkhant barley variety, the concentration of benzoic acid decreased by 145.40 µg g−1 DW, p-coumaric acid by 3.14 µg g−1 DW, trans-ferulic acid by 55.90 µg g−1 DW, sinapic acid by 6.60 µg g−1 DW, and trans-cinnamic acid by 9.88 µg g−1 DW, compared to plants of the same variety grown from seeds soaked in sterile distilled water (Table 2).

In plants of the Virazh barley variety, only the content of sinapic acid decreased by 55.60 µg g−1 DW compared to the sample which seeds were soaked in the sterile distilled water (Table 2).

Hydrogen peroxide had the most negative effect on the content of phenolic acids in the bound fraction in plants of Copeland barley variety. Accordingly, the concentration of caffeic acid decreased by 76.18 µg g−1 DW, benzoic acid by 64.70 µg g−1 DW, p-coumaric acid by 32.08 µg g−1 DW, trans-ferulic acid by 13.60 µg g−1 DW, sinapic acid by 92.44 µg g−1 DW, and trans-cinnamic acid by 7.78 µg g−1 DW, compared to the variant which seeds were soaked only in sterile distilled water. Also, 4-HPAA, chlorogenic, and syringic acids were not identified for this variety (Fig. 4, Table 2).

Fig. 4
figure 4

HPLC analysis of phenolic acids in the hydrolyzed fraction that was obtained from plants Copeland barley variety after treatment of seeds with a stress agent—hydrogen peroxide

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Thus, the effect of peroxide stress on phenolic acids in the bound fraction of all three tested barley varieties differed significantly. However, the concentration of caffeic, benzoic, trans-ferulic, and sinapic acids decreased most sharply (Table 2).

Similar results were obtained by different groups of scientists in the study of the profile of phenolic compounds in different plant species under abiotic stress. In scientific papers [54, 55], it was showed that Vitis vinifera plants grown under cold stress conditions had decreased levels of esters and glycosides of the caffeic, ferulic, and p-coumaric acids compared to the control. According to a study by Kovacik et al. [56], among the 14 identified phenolic acids in the leaf rosette of Matricaria chamomilla, the content of chlorogenic and caffeic acids decreased sharply under NaCl stress. The decrease in the content of phenolic acids may be the result of inhibition by stress agents of the activity of enzymes involved in their biosynthesis [57].

Post-treatment of seeds of the different barley varieties with nanocomposite complex bacterial preparation Azogran caused an increase in the content of only some phenolic acids in stressed plants. Accordingly, in the bound fraction obtained from barley plants of the Burkhant variety, the concentration of chlorogenic acid increased by 14.22 µg g−1 DW and benzoic acid by 50.76 µg g−1 DW, compared to plant varieties where seeds were treated only with hydrogen peroxide (Table 2). Chlorogenic acid (CGA) is a phenolic derivative with a unique chemical structure that is a combination of caffeic and quinic acids. This allows CGA to effectively neutralize R in plant cells [58, 59]. Benzoic acid, in turn, ensures the resistance of phytoobjects to various abiotic and biotic stresses: drought, cold [60], phytopathogenic fungi [61].

In the same fraction from plants of the Virazh barley variety, the concentration of 4-HPAA increased by 32.58 µg g−1 DW, trans-ferulic acid by 3.36 µg g−1 DW, and sinapic acid by 4.40 µg g−1 DW compared to the variant where seeds are treated with a stress agent only (Table 2). 4-Hydroxyphenylacetic acid, in addition to being an antioxidant [62], effectively inhibits the development of some phytopathogenic microbes: Fusarium culmorum 50536, Fusarium solani 50666, Alternaria alternate 16765 [63]. The mechanism of antioxidant action of the ferulic acid is difficult and is aimed at inhibiting the surge of ROS and neutralizing free radicals in living cells. Also, this phenolic compound is responsible for chelation of protonated metal ions (Cu (II) and Fe (II)), the initiators of the Fenton reaction [64, 65], the product of which is a highly reactive hydroxyl radical. Ferulic acid not only converts free radicals (R·) into neutral molecules, but also inhibits enzymes that catalyze R· generation [66]. No significant changes were found in the content of phenolic acids in the bound fraction obtained from plants of Copeland barley variety. In addition, syringic acid was identified, which was absent in barley plants that developed from stressed seeds (Table 2). Tiwari et al. [67] reported that inoculation of wheat (Triticum aestivum) seeds with Bacillus pumilus under salt stress conditions promoted the accumulation of syringic acid in plants.

Composition and content of the free flavonoids of different barley varieties

In plants of barley varieties, whose seeds were subjected to different treatments, the content of flavonoids in free and bound fractions was determined. As a result, significant differences were found.

The free fraction of flavonoids extracted from plants Burkhant barley variety, whose seeds were treated with sterile distilled water, contained rutin, quercetin-3-β-glycoside, quercetin, and luteolin (Fig. 5A, Table 3). The pronounced antioxidant, anti-inflammatory, and antitumor properties of quercetin and luteolin are due to the high similarity of their chemical structure. Only the presence of a hydroxyl group in the quercetin molecule at position 3 distinguishes these two flavonoids [68].

Fig. 5
figure 5

HPLC analysis of flavonoids in the free fraction that was obtained from barley plants of Burkhant (A), Virazh (B), and Copeland (C) varieties after seed treatment with sterile distilled water

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Table 3 Composition and content of flavonoids in the free fraction that was obtained from plants of different barley varieties
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In the same fraction obtained from plants Virazh barley variety, in addition to the above flavonoids, neohesperidin was identified. The total content of these compounds was the highest and amounted to 30.72 µg g−1 DW (Fig. 5B, Table 3). Only two flavonoids were detected in Copeland plants—quercetin-3-β-glycoside and quercetin (Fig. 5C, Table 3). The flavonoid content is an important indicator of the antioxidant potential of plants and also determines the health benefits of functional foods [69].

The treatment of seeds of different barley varieties with the nanocomposite complex bacterial preparation Azogran increased the concentration of flavonoids in the free fraction obtained from plants of Burkhant and Virazh varieties by 9.52 µg g−1 DW and 11.64 µg g−1 DW, respectively, compared to the previous variant. The study [70] showed that inoculation of the roots of two broccoli varieties with Paraburkholderia graminis PHS1, P. hospita mHSR1, and P. terricola mHS1 strains contributed to the accumulation of secondary metabolites, including flavonoids, in plants.

In addition, naringin was identified in Burkhant plants (Fig. 6, Table 3). This flavanone is a glycoside of naringenin and is able to effectively inactivate hydroxyl and superoxide radicals, thus protecting DNA from oxidative stress [71, 72].

Fig. 6
figure 6

HPLC analysis of flavonoids in the free fraction that was obtained from Burkhant barley plants after seed treatment with nanocomposite complex bacterial preparation Azogran

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Under the action of 33% hydrogen peroxide on barley seeds, the synthesis of flavonoids in the free fraction in barley plants of the Burkhant variety was significantly reduced. Among the previously detected flavonoid compounds, only quercetin-3-β-glycoside was identified—4.30 µg g−1 DW. Glycosylated flavonoids with a catechol group at the 3′–4′ position are characterized by a high antioxidant potential [73]. A number of experimental studies [74,75,76] have shown that the concentration of quercetin glycosides in different plant species remained high in response to various abiotic stresses.

While for barley variety Virazh, an increase in the concentration of some flavonoids in the free fraction was observed. In particular, the content of quercetin increased by 6.13 μg/g DW and luteolin by 9.15 μg/g DW, compared to plants of the same variety, whose seeds were treated with sterile distilled water (Fig. 7, Table 3). On the one hand, the impact of stress factors on plants can impair their ontogeny and productivity in general, and on the other hand, it can activate the metabolism of important biologically active compounds [40].

Fig. 7
figure 7

HPLC analysis of flavonoids in the free fraction obtained from Burkhant barley plants after seed treatment with the stress agent hydrogen peroxide

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No significant changes in the quantitative and qualitative content of flavonoids in the free fraction of the barley variety Copeland were found.

Post-treatment of the stressed seed material of the studied cereal crop with the biological product Azogran had no significant effect on the level of flavonoids in the free fraction in plants of all three barley varieties (Table 3).

Composition and content of the bound flavonoids of different barley varieties

In barley samples, the content of flavonoids in the bound fraction was checked and significant differences between varieties were found. The concentration of flavonoids in the bound fraction was higher than in the free fraction. It should be noted that in plants of different varieties of blue Highland barley, the content of flavonoids in the free fraction significantly exceeded the content of bound flavonoids [77]. While, in buckwheat, wheat, rice, corn, and oats, flavonoids prevailed in the bound fraction [78]. At treatment seeds with sterile distilled water, the total content of these compounds in plants of Burkhant variety was 190.52 µg g−1 DW, Virazh variety was 47.76 µg g−1 DW, and Copeland variety was 20.55 µg g−1 DW. Differences were also found in the qualitative composition. Rutin, quercetin-3-β-glycoside, quercetin were identified in barley plants of Burkhant variety; quercetin, kaempferol in Virazh variety; quercetin-3-β-glycoside, quercetin in Copeland variety (Fig. 8; Table 4). This difference is related to the genotype of each of the barley varieties under study. For example, Xi-Juan with co-authors [77] found that in blue Highland barley plants, naringenin and hesperidin predominated in the bound fraction of flavonoids. While Kim with co-authors [79] showed that in colored barley, the main flavonoid was myricetin.

Fig. 8
figure 8

HPLC analysis of flavonoids in the hydrolysis fraction obtained from barley plants of Burkhant (A), Virazh (B), and Copeland (C) varieties after treatment of their seeds with sterile distilled water

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Table 4 Composition and content of flavonoids in the bound fraction that was obtained from plants of different barley varieties
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Treatment of seeds with nanocomposite complex bacterial preparation was accompanied by an increase in the concentration of flavonoids only in plants of the Virazh variety to 125.27 µg g−1 DW (Table 4). Ali et al. [80] found that the treatment of Arabidopsis thaliana with the microbial preparation Soil Builder™-AF increased the induction of the transcriptional profile of genes of the phenylpropanoid pathway, which contributed to the accumulation of flavonoids in the leaves of plants.

The stimulating effect of Azogran on the qualitative and quantitative composition of flavonoids in the bound fraction in plants of the other two varieties was not detected. In particular, for Burkhant variety, a decrease in flavonoid content by 170.69 µg g−1 DW was recorded compared to the variant in which the seeds were treated with sterile distilled water (Table 4). This may be due to the specifics of the development of each of the studied cereal varieties, when their seeds were treated with the nanocomposite complex bacterial preparation Azogran. The effect of PGPR on phenylpropanoid pathways is associated with the stimulation or inhibition of plant growth at a certain phase of their development. That is, if rhizobacteria activate growth, then the biosynthesis of flavonoids is inhibited and, conversely, with a decrease in growth, the level of these phenolic compounds in plants increases [81]. We conducted our research with barley plants in the tube stage. This is one of the most critical periods in the ontogeny of cereal spiked crops. This phase is characterized by the formation of flowers in the spikelets and active growth of the spikelet. That is, this is the transition from the vegetative to the generative phase of cereal crop development [82]. In barley plants of Burkhant and Copeland varieties, whose seeds were inoculated with Azogran, this transition was very slow. They were still growing quite actively, which may have influenced the decrease in flavonoid levels.

Under the action of hydrogen peroxide on the seed of the Virazh variety, the content of flavonoids in the bound fraction decreased by 29.36 µg g−1 DW, compared to plants whose seeds were treated with the nanocomposite complex bacterial preparation Azogran. Treatment of barley grain of Burkhant variety with this stress agent stimulated the synthesis of naringin in plants, its concentration was 19.66 µg g−1 DW, while the content of other flavonoids in the bound fraction decreased (Table 4). Hydrogen peroxide had a stimulating effect only on the flavonoid complex of Copeland plants. In addition, neohesperidin was identified. The increase in the flavonoid content may be due to the ability of H2O2 to regulate the expression of the genes of phenylalanine ammonia lyase, chalcone synthase, and stilbene synthase, which are involved in the synthesis of plant flavonoids [83, 84].

Post-treatment of stressed barley seeds with Azogran had the most positive effect on the flavonoid complex of the bound fraction of plants of the Virazh variety. Accordingly, a high content of quercetin-3-β-glycoside—36.78 µg g−1 DW and quercetin—42.63 µg g−1 DW was found (Table 4). For the other two varieties, this effect was not observed. Ayuso-Calles with co-authors [85] showed that in lettuce inoculated with Rhizobium laguerreae bacteria, which developed under salt stress, the content of flavonoids was slightly reduced compared to inoculated plants growing under normal conditions. Such effects may have different causes. First, it is the type of microorganisms-inoculants. Zapata-Sufientes et al. [86], at studying the effect of Pseudomonas paralactis, Sinorhizobium meliloti, and Acinetobacter radioresistens on the flavonoid content of cucumbers, found that S. meliloti bacteria contribute to the greatest accumulation of these compounds in the fruits of these plants. Secondly, it is the plant variety. According to a study by Jeon et al. [81], treatment of two broccoli varieties, Malibu and Coronado, with the epiphytic rhizobacterium Paraburkholderia led to a greater accumulation of flavonoid glycosides only in Malibu plants. Whereas Zaferanchi et al. [87] pointed out an insignificant concentration difference of flavonoids in marigold plants of Isfahan double flower and Isfahan single flower varieties, the seeds of which were inoculated with PGPR (Azotobacter sp.145PI and Azospirillum sp.AC49I). And third, it is the level of influence of the stressor on the plant, the higher it is, the greater the imbalance of redox homeostasis and other biochemical processes in the cells.

Conclusion

The large amplitude of variation of phenolic carboxylic acids and flavonoids indicates the specificity of the interaction of different barley varieties with the bacteria components of Azogran and their different responses to the effect of the preparation under conditions of peroxide stress. For barley variety Virazh, higher results were obtained in studying the effect of the stress agent and nanocomposite complex bacterial preparation Azogran on the qualitative and quantitative content of phenolic compounds in its plants. Since the selection of this variety and its agricultural technology is carried out in Ukraine, it is more adapted to the climatic conditions and soil microbial community of this country. At that time, the Burkhant (Mongolia) and Copeland (Canada) barley varieties were first grown in Ukraine. However, the proposed treatment of native and post-treatment of stressed seeds of these barley varieties with a nanocomposite complex bacterial preparation helped to activate the synthesis of a complex of phenolic compounds in their plants.