Findings

Electron beam irradiation (eBeam), being an inexpensive, environmentally friendly, and time-efficient alternative to traditional thermal decontamination technology, has the potential for use in food processing technologies to improve food quality and reduce the risk of microbial contamination of food products (Ravindran and Jaiswal 2019). Probiotics are defined as live cells which, when administered in adequate amounts, benefit the host’s health. Some of them (including lactobacilli probiotics) also possess antagonistic potential against pathogens (Pepoyan et al. 2018a, Pepoyan et al. 2018c)and are used in food production for control of foodborne pathogens (Mattila-Sandholm et al. 2002). The combined use of eBeam (50–100 Gy) and Lactobacillus rhamnosus Vahe (potential probiotic) and Lactobacillus acidophilus DDS®-1 (Lacto-G, a marketed synbiotic formulation) cells was previously suggested by our group for quality improvement and packaging practices (Pepoyan et al. 2019). While no significant changes in cell surface hydrophobicity (CSH) were found after the 150-Gy eBeam irradiation, an increase in biofilm-formation (BF) ability was shown for L. rhamnosus Vahe and L. acidophilus DDS®-1 (0.22 ± 0.03 vs. 0.149 ± 0.02 and 0.218 ± 0.021 vs. 0.17 ± 0.012, respectively) (Pepoyan et al. 2019). The evaluation of radiation dose-response effects revealed that L. rhamnosus Vahe is more resistant to 50–150-Gy irradiation than L acidophilus DDS®-1. D10 value of L. rhamnosus Vahe was defined as the radiation dose (Gy) required to reduce the number of CFU by one Log10. This (218 Gy) was determined by calculating the negative reciprocal of the slope of the linear regression curve (Manvelyan et al. 2019). Taking into account the use of low-dose eBeam in different food processing technologies and possible changes in antagonistic activities against pathogens after irradiation, we further investigate the impact of 50–150-Gy eBeam irradiation on the anti–Klebsiella pneumoniae activity of the probiotic L. rhamnosus Vahe.

The putative probiotic L. rhamnosus Vahe was isolated from the feces of a healthy infant (Pepoyan et al. 2018b). A multidrug-resistant clinical isolate of K. pneumoniae was obtained from the Armenian National Agrarian University culture collection. Bacterial strains were cultured in de Man, Rogosa, and Sharpe (MRS) broth and on MRS agar (Thermo Scientific™, UK). When required, Oxoid™ Endo Agar (Thermo Scientific™, UK) and VITEK® 2 compact (BioMerieux, France) were used for the identification of bacterial cells.

AREAL, a laser-driven photocathode RF gun-based electron accelerator, was used to irradiate lactobacilli cells (Tsakanov et al. 2016). A bacterial suspension was prepared in phosphate buffered saline (2 ml; 1.5 × 108 CFU/ml) from overnight-grown cell cultures, immediately before irradiation. Bacteria were irradiated in glass vessels, which allows for the scattering on the background of absorption to be ignored. A detailed description of the parameters and conditions of irradiation is given in Pepoyan et al. (2019): radio frequency (RF) high voltage, 117 kV; RF phase, − 82°; pulse repetition rate, 12 Hz; solenoid current, 9.7/47 A/V; dipole current, 4/9 A/V; corrector magnet (X | Y), 2.5/7.3 A/V (RF system); beam charge (C-IN/FC-OUT), 440/55 pC; beam energy, 3.6 MeV; laser pulse duration, 0.42 ps; mass of the samples, 3.2 g; dose, 50–150 Gy; time (mm/ss), 3 min 7 s, 4 m in23 s, and 6 min 35 s.

After irradiation, an 0.1 ml suspension of L. rhamnosus Vahe cells was transferred into 0.9 ml of fresh MRS broth and incubated for 24 h at 37 °C. The cells were then removed by centrifugation at 4200g for 15 min, and the supernatant was sterilized using 0.22 μm Millipore filters (Millex-GV, Sigma-Aldrich). The inhibitory activity of the lactobacilli cell free supernatant (CFS) against K. pneumoniae was initially evaluated by observing the changes in optical density (OD600) of the pathogen’s suspension after 24-h incubation at 37 °C using the biochemical analyzer (STAT FAX 3300, Awareness Technology). Colony-forming units (CFU) were determined after 24-h incubation by plating on MRS agar. To describe the growth characteristics of K. pneumoniae treated with the cell free supernatants of irradiated and non-irradiated lactobacilli, Verhulst’s function was used (Gasparyan et al. 2013; Pepoyan et al. 2017):

$$ X=\frac{\left(A-C\right)}{\left(1+{10}^{\alpha +\beta \times t}\right)}+C $$
(function 1)

where X is the optical density at time t; A is the asymptote, maximal optical density; C is the initial value of optical density; t is the total cultivation time; and α and β are kinetic parameters that define the shape, point of inflection, and slope of the curve.

The inhibitory activity of lactobacilli CFS against K. pneumoniae was also evaluated by the agar well diffusion method. In this case, 0.1 ml of irradiated and non-irradiated suspensions of L. rhamnosus Vahe was transferred onto MRS agar plates. Plates were incubated for 24 h at 37 °C, and one colony from each plate was transferred into 0.9 ml MRS broth and grown at 37 °C for 24 h. Then cultures were centrifuged at 4200g for 15 min and the supernatants were harvested. Supernatants were sterilized using 0.22-μm Millipore filters. The pathogen was propagated overnight in MRS broth. Then the pathogenic bacteria were streaked on the surface of Mueller-Hinton agar. Wells were made (6 mm diameter) on the surface of the streaked agar. The CFSs from the culture of L. rhamnosus Vahe were placed in the wells (100 μl) and the plates were then incubated for 24 h at 37 °C. A clear zone of inhibition (≥ 6 mm in diameter) was defined as positive result.

Statistical processing of the data was performed using the Mann-Whitney and Student t test, as well as the Chi-square test (Excel 2010). The probability P < 0.01 was considered as statistically significant. All experiments were performed in duplicate twice.

The growth kinetics of K. pneumoniae treated with the CFS of irradiated L. rhamnosus Vahe cells are presented in Fig. 1. The non-treated pathogen’s OD600 reached 1.58 ± 0.09 after a 24-h incubation in MRS broth. However, the pathogen’s growth was inhibited in the presence of the untreated L. rhamnosus Vahe CFS (OD600 = 0.14 ± 0.06). When the pathogen was treated with the irradiated L. rhamnosus Vahe CFS, its OD600 reached 0.13 ± 0.08 (50-Gy irradiated cells), 0.22 ± 0.12 (100 Gy), and 0.68 ± 0.05 (150 Gy), respectively. The antagonistic effect of the CFS was much less pronounced after irradiation of lactobacilli with a higher dose (150 Gy), which can be explained by the number of viable lactobacilli after irradiation, as described earlier in our assessment of dose-response effects of 50–150-Gy eBeam irradiation (Manvelyan et al. 2019). The results shown in Fig. 1 also indicate a decrease in the number of viable lactobacilli in correlation with an increase in eBeam radiation doses.

Fig. 1
figure 1

Anti–K. pneumoniae effects of cell free supernatants from the irradiated and non-irradiated L. rhamnosus Vahe (24-h incubation). CFS, cell free supernatant; CFU, colony-forming unit

Full size image

Modeling the growth of microbial populations serves as a tool for predicting changes in artificial and natural biocenoses. The use of mathematical models to determine the characteristics of bacterial growth can serve as one of the criteria for the intelligent use of probiotics. Verhulst’s equation was previously used to describe the growth phases of gut Escherichia coli isolates (Gasparyan et al. 2013) and to evaluate and quantify the beneficial effects of the probiotic strain L. acidophilus INMIA 9602 Er 317/402 on patients with familial Mediterranean fever (Pepoyan et al. 2017).

Taking into account the importance of evaluating the specific growth rate in comparative studies (Berneyet et al. 2006), the specific growth rate of pathogens was also calculated. The data obtained and calculations utilizing the Verhulst equation demonstrated the inhibitory effect of the lactobacilli’s CFS on the growth parameters of K. pneumonia cells (Fig. 2). These changes, independent of changes in the pH of the growth medium (data not shown), refer to the preparatory (lag), logarithmic, and stationary phases of the growth of the pathogen (Fig. 2). Control (non-treated) K. pneumoniae cells had a significantly higher maximum specific growth rate (μmax) and achieved a greater total biomass compared with K. pneumoniae cells treated with the CFS of L. rhamnosus Vahe cells (the coefficient of determination of R2 was 0.9955 and 0.8829, respectively) (Fig. 2). The duration of the preparatory phase, including the phase of growth inhibition, when there is no growth, and the phase of accelerated growth, when the growth rate reaches its maximum (before the logarithmic growth phase), was more than twice as high in the CFS-treated groups compared with that in the control group of K. pneumoniae. At the same time, there were no statistically significant differences in these characteristics of growth of the pathogen after the addition of the CFSs of irradiated and non-irradiated L. rhamnosus Vahe (Fig. 2).

Fig. 2
figure 2

Growth kinetics according to Verhulst’s model: Function 1 and specific growth rate of control K. pneumoniae cells (a) and K. pneumoniae cells (b) treated with the CFS of 150-Gy electron beam irradiated and none-irradiated L. rhamnosus Vahe.X, the optical density at time t; A, maximal optical density; C, the initial value of the optical density; t, the total cultivation time; α and β, kinetic parameters that define the shape, point of inflection, and slope of the curve; μ, specific growth rate

Full size image

The evaluation of the effect of CFS on activity against K. pneumoniae was also conducted by the agar well diffusion method. The results showed no statistical differences in the antagonistic effects of the CFS derived from non-irradiated and irradiated cells, when the bacterial titers of L. rhamnosus Vahe in the suspensions were the same. Also, 85 ± 5% of wells with CFSs from the control (untreated) and irradiated cells produced ≥ 6-mm inhibition zone. The anti–K. pneumoniae activity of neutralized (pH 7.0 ± 0.01) CFS did not differ much from that of non-neutralized CFS.

Lactobacilli produce a wide range of antibacterial compounds, including weak organic acids (lactic acid and acetic acid), hydrogen peroxide, and proteinaceous compounds such as bacteriocins (Giri et al. 2009). The anti–K. pneumoniae activity of the CFS from the putative probiotic strain L. rhamnosus Vahe is reported here and can be explained by antibacterial compounds that are naturally produced by the lactobacilli.

Currently, X-ray and eBeam technologies аrе used to eliminate microbial pathogens (i.e., cold pasteurization) or (in higher doses) to sterilize food ingredients (Pillai 2016). They can also be used at very low doses for phytosanitary treatment, which eliminates insects and pests on agricultural products (Pillai 2016). Gosiewski et al. (2016) reported that 3–50-Gy doses of irradiation have a neutral effect on viability of lactobacilli, while our study has shown that 50–150-Gy eBeam irradiation decreases the viability of the novel potential probiotic strain L. rhamnosus Vahe. At the same time, the strain’s antagonistic potential was not affected.

Thus, the obtained results suggest that 150-Gy eBeam-irradiated cells of probiotic L. rhamnosus Vahe produce metabolite(s) with an anti–K. pneumoniae effect, similar to the effects of non-irradiated lactobacilli cells. The combined use of eBeam (5–100 Gy) and L. rhamnosus Vahe might be suggested for possible use in different scenarios in the healthcare and food industries where inhibition of undesired microorganisms is required.