Bacterial Strains and Growth Conditions
Lactobacillus helveticus 34.9 was used as the producer of antibacterial activity, while Lactobacillus delbrueckii subsp. bulgaricus LMG 6901T was used as the indicator strain. Both the producer and the indicator strain were maintained at -80°C in MRS broth (de Man et al. 1960), in the presence of 25% (v/v) of glycerol as a cryoprotectant. Before use, they were propagated twice, for 24h, in MRS broth, at 37°C.
Antibacterial activity assessment
Cell free culture supernatant (CFCS) was obtained from fresh L. helveticus 34.9 culture, by centrifugation (13000 x g, 10 min, 4 ºC). Antibacterial activity was assayed by both agar spot (De Vuyst et al. 1996), and agar well diffusion (Balouiri et al. 2018) methods. Briefly, ten ml of the CFCS were spotted onto fresh lawns (soft agar media, 0.7 % (w/v) agar) containing 100 ml of the indicator culture. Alternatively, 50 ml of the CFCS were added to wells cutted into agar media previously inoculated with the indicator. Plates were then incubated for 24h to allow the growth of the indicator strain. The presence of clear inhibition zones around the spots/wells was recorded as a positive result.
Evidence of bacteriocin production
In order to establish the nature of the inhibitory compound, several additional tests were done. Firstly, to remove the antibacterial activity of organic acids, the pH of CFCS was adjusted to 6.5-7.0, before testing. Secondly, the influence of several proteolytic enzymes, such as: tripsin (Sigma-Aldrich Chemie GmbH, Germany), pronase E (Merck KGaA, Darmstadt, Germany), proteinase K (Carl Roth, Karlsruhe, Germany), and pepsin (Sigma-Aldrich), on the antibacterial activity was investigated. Enzymes were dissolved in 0.2 M sodium phosphate buffer (pH 7.5), except pepsin, which was dissolved in 0.2 M HCl/KCl buffer (pH 2.0) and were added to the CFCS, at a final concentration of 1 mg/ml. Supernatants were incubated with proteinase K for 2h at 55°C, and with other enzymes for 2h at 37°C, before being tested. The CFCS diluted with the above mentioned buffers, and enzymes solutions without CFCS were used as positive and negative controls, respectively.
Quantitative assay of bacteriocin activity
Serial twofold dilutions in water, of the bacteriocins samples, were spotted (10 μl) onto fresh lawns of the indicator strain, or added (50 μl) to wells cutted in the inoculated agar medium (as described above). The activity was defined as the reciprocal of the highest dilution for which a clear inhibition zone could be still observed, and was expressed in activity units (AU) per millilitre of sample.
Antibacterial Activity under Stress Conditions
Previous results showed an increase of the antibacterial activity of L. helveticus 34.9 when grown under different stress conditions, such as an initial pH of 4.5, or in the presence of low concentrations of NaCl or bile salts (BS) in the growth medium (Stefan et al. 2018). In the present study, the bacteriocin producing strain was cultivated under more stressful conditions, such as higher concentrations of bile salts (Sigma-Aldrich, up to 2%), or under combined stresses, as they are presented in Table 1. The antibacterial activity was quantitatively assayed, using L. delbrueckii subsp. bulgaricus 6901T as indicator strain.
Bacteriocin isolation and characterization
The bacteriocin produced by L. helveticus 34.9 was concentrated and extracted as described before (Zamfir et al. 1999). Briefly, the strain was inoculated (2% inoculum) in 500 ml MRS broth supplemented with 2% NaCl and with an initial pH of 4.5, and incubated overnight at 37°C. CFCS was neutralized with 10N NaOH and treated with 40% ammonium sulphate, with gentle stirring, to precipitate the proteins. Afterwards, the mixture was centrifuged (13,000 x g, 10 min, 4 °C) and the pellicle was resuspended in 5 mM potassium phosphate buffer, pH 6.5 (crude bacteriocin). The bacteriocin was finally extracted with 15 volumes of chloroform: methanol (2:1, v/v). After 2h of incubation at 4 °C, the suspension was centrifuged (13,000 x g, 15 min, 4°C) and the precipitate was resuspended in ultrapure water.
Characterization was further done using the crude bacteriocin sample.
Antibacterial inhibition spectrum
To determine the activity spectrum of L. helveticus 34.9 bacteriocin, several bacterial strains were used as indicator, including other LAB, some bacterial pathogens, and a strain of Halobacillus hunanensis, namely 5Hum, previously isolated from the degraded wall of a Romanian monastery (Cojoc et al. 2019). Table 2 shows a list of the indicator strains and the growth conditions used for each of them. The antibacterial activity of the crude bacteriocin was assayed using the well diffusion method. The diameters of the inhibition zones around the wells were measured, as an indication of the strength of the antibacterial activity.
Heat sensitivity and pH stability of the bacteriocin
Crude bacteriocin was subjected to different pH and thermal treatments. Untreated samples were used as controls and the antimicrobial activities were quantitatively determined against L. delbrueckii subsp. bulgaricus LMG 6901T by agar spot method.
The pH stability was tested by adjusting the pH of the crude bacteriocin from 2 to 10 (using sterile 1M HCl or 1M NaOH) with increments of two units, and allowing the samples to stand for one hour at room temperature before testing the inhibitory activity.
To determine the heat stability of the antibacterial activity, independent incubations of the crude bacteriocin were carried on at 60°C, and 100°C, and the activity was tested after 15, 30, and 60 min of treatment.
Molecular Mass Determination
Tricine sodium dodecylsulphate-polyacrylamide gel electrophoresis (Tricine-SDS-PAGE, Schägger and von Jagow 1987) was carried on in a Biometra Minigel Twin (Biometra, Germany) to estimate the molecular mass of the produced bacteriocin. CFCS, crude bacteriocin, and bacteriocin extracted with chloroform/methanol mixture, were used for the run. Electrophoresis was conducted at a constant voltage of 30 V in the stacking gel, and 90 V in the separating gel. For the bioassay, the gel was washed during 5 h with sterile ultrapure water and overlaid with a top layer of soft MRS agar (0.7% agar) inoculated with the indicator strain, L. delbrueckii subsp. bulgaricus LMG 6901T. The broad range molecular weight markers (10-225 KDa, Promega, USA) were stained with Coomasie Brilliant Blue (Carl Roth GmbH, Germany).
Mode of action
Bactericidal effect of the bacteriocin
Cells from 24h cultures of the sensitive strains, L. delbrueckii subsp. bulgaricus LMG 6901T and H. hunanensis 5Hum, were washed with sterile potassium phosphate buffer (pH 6.5) and suspended in the same buffer to get about 7-8 log CFU/ml. In case of 5Hum strain, the buffer was supplemented with 10% NaCl, to minimize the osmotic stress. Crude bacteriocin was filter sterilized and serially diluted before being added to the cells suspensions. Final activities of about 6400, 3200, and 1600 AU/ml (determined against L. delbrueckii subsp. bulgaricus LMG 6901T) were used. Samples were maintained at 37°C. At various time intervals, viable cell counts (CFU/ml) were determined. All experiments were done in triplicate. Untreated cells served as controls.
Bacterial survival rate (%) was calculated according to the formula
Survival rate (%) = (N/N0)x100, where
N represents log10 colony forming units per ml suspension,
N0 represents log10 colony forming units per ml at time zero.
Growth inhibition of the sensitive cells
Crude bacteriocin was filter-sterilized and added to MRS and MH broth, respectively (final activities between 1600 and 6400 AU/ml). Each medium was further inoculated (5%) with the corresponding sensitive strain. The growth was monitored by hourly measurements of the OD at 600nm, using a FLUOstar Omega microplate reader (BMG-Labtech GmbH, Germany). Cultures without bacteriocins served as controls.
Scanning electron microscopy
The impact of the bacteriocin produced by L. helveticus 34.9 on the sensitive cells’ morphology was observed by scanning electron microscopy (SEM), using a previously described protocol (Zamfir et al. 2016). Cells of L. delbrueckii subsp. bulgaricus LMG6901T and H. hunanensis 5Hum were collected by centrifugation from the exponentially growing cultures, washed and treated for 24h with the bacteriocin (final concentration of 6400 AU/ml). Cellular suspensions without bacteriocin served as controls. After centrifugation (10,000 x g, 10 min, 4°C), cells were fixed for 2h at 4°C with 4% glutaraldehyde in 0.1 M potassium phosphate buffer (pH 7.4). Water was then extracted from the samples using a graded series of ethanol: 50-75-100%, for 15 min in each bath. The last baths were in ethanol/acetone (1/1) and acetone 100%. A drop of each sample was spotted on a microscopic blade, air-dried and coated with gold. Cells were visualized with a JSM-6610LV scanning electron microscope (Jeol).
Statistical Analyses
All the experiments were done in triplicate or quadruplicate. The results were processed using Microsoft Excel software (Microsoft Corp.) and are expressed as mean value ± SD (standard deviation).