Two different MRS media were used throughout the study. Normal MRS medium (MRS_5.6, Merck), which was prepared according to the instructions from the supplier. An acidified MRS medium (MRS_4.3), where pH was lowered to 4.3 using hydrochloric acid. The final concentration of hop compounds (iso-α-acids) in stress experiments was 55.2 μM unless otherwise indicated, which was obtained by addition of a stock solution of 30% (w/w) iso-α-acids in an aqueous solution of potassium (Hopsteiner, New York, NY, USA). Sodium acetate buffer (50.0 mM, pH 4.3, with 0.1 M glucose) was used for experiments where growth was not wanted. A final concentration of either 2.9 μM manganese (corresponding to levels in pilsner lager beer) (Behr and Vogel, 2009) or 265.0 μM manganese (as in MRS), was obtained by addition of a 26.5 mM MnSO₄ stock solution to sodium acetate buffer.

A total of six L. brevis strains isolated from beer were included in the present study (Table 1). A preculture was started by inoculating 10 mL MRS_5.6 from a frozen stock culture, and incubated at 30°C overnight. The preculture was subcultured into fresh MRS_5.6 (inoculation concentration 1%) at 30°C. After 16 h, optical density (OD₆₀₀) of each strain was adjusted with fresh MRS_5.6 to 1.0, corresponding to approximately 5 × 10⁸ CFU/mL.

Hundred microliter standardized culture was inoculated into 10 mL of the following media MRS_5.6 (control), MRS_5.6 with hop compounds (MRS_5.6+H), MRS_4.3 and MRS_4.3 with hop compounds (MRS_4.3+H), separately. The growth curves were assessed in 96-wells microplates by measuring OD₆₀₀ for every 12 h using a Varioskan^TM Flash (Thermo Fisher Scientific Oy, Finland) until 168 h. Each well of a microplate was added 200 μL suspension. Microplates were sealed with parafilm (Sigma–Aldrich) to minimize loss of volume and incubated at 30°C until measurement. For each strain, the average OD₆₀₀ value was calculated from 16 wells.

To investigate viability in individual cells, cells were stained with SYTO 13 (Molecular Probes, Thermo Fisher scientific) and Propidium Iodide (PI, Molecular Probes, Thermo Fisher scientific). 1 mL of an overnight culture was incubated with SYTO 13 (final concentration 10.0 μM) and PI (final concentration 15.0 μM) simultaneously at 30°C for 30 min in the dark. The suspensions were subsequently diluted 20 times to an approximate concentration of 10⁷ cells/mL with saline solution (NaCl, 0.9%, w/w). All dual stained cell suspensions were kept on ice until further analysis.

For pHi measurement, cells were stained with 5(6)-Carboxy-2, 7-dichlorofluorescein diacetate succinimidyl ester (CDCFDA-SE, 3.9 mM, Molecular Probes, Thermo Fisher scientific). The CDCFDA-SE staining procedure used in this study was modified from Shabala et al. (2006). Briefly, 1mL standardized culture was harvested by centrifugation (10,000 × g, 5 min), and the pellet was re-suspended in 980 μL phosphate buffered saline (PBS, 50.0 mM, pH 7.4) with 10 μL 1.0 M glucose and 10 μL CDCFDA-SE. The cell suspension was incubated at 30°C in the dark for 30 min.

A standardized culture (200 μL) was inoculated in 20 mL MRS_4.3 and MRS_4.3+H, separately, and incubated at 30°C. A 1 mL sample was taken every 6 h until 48 h and then every 24 h until 96 h. Cells were centrifuged at 10,000 × g for 5 min and the pellets were re-suspended in 0.5 mL saline solution and stained with SYTO13/PI as described above. Subsequently, samples were analyzed by FM and/or FCM. For FCM, a positive control (fresh overnight culture, live cells) was stained with SYTO 13 and a negative control (dead cells) was stained with PI. For the negative control, preliminary results using 70% ethanol to kill all the cells, but this method caused cell aggregation in FCM analysis. Therefore, a combination of 552 μM hop compounds and 20% ethanol (v/v) was used to obtain a fully permeabilized population of single cells, where almost 100% cells were stained with PI, which was confirmed by FM.

Cells from overnight culture were stained with CDCFDA-SE, centrifuged at 10,000 × g for 5 min and the pellet was re-suspended in 1mL sodium acetate buffer. Subsequently, the suspension was divided into two tubes, centrifuged at 10,000 × g for 5 min and re-suspended into 500 μL sodium acetate buffer with and without hop compounds. Images were acquired after 1, 12, and 24 h by FM. Samples were kept at 30°C in the dark until image acquisition.

To construct a calibration curve for pHi, stained cells were exposed to 70% ethanol for 30 min to achieve fully permeabilized cells. Aliquots of permeabilized cells were then re-suspended in sodium acetate buffer with pH 4.3, 4.7, 5.0, 5.5, and 6.0. Since all six strains exhibited similar ratio values at a given pH, it was decided to construct one standard curve. The average ratio of at least 100 cells for each pH value was plotted against the Ratio _{(488/435 nm)}, and the standard error of the mean (SEM) was calculated (Figure 1).

1.5-mL standardized culture was centrifuged at 10,000 × g for 5 min and re-suspended in 1.5 mL of acetate buffer. For viability assessment, the suspension was divided into three tubes, followed by centrifugation (10,000 × g, 5 min), and the pellets were re-suspended in acetate buffer with hop compounds, acetate buffer with hop and 2.9 μM manganese, as well as acetate buffer with hop and 265.0 μM manganese, respectively. Cell suspensions were incubated at 30°C for 2.5 h, stained with SYTO 13/PI for 0.5 h, and analyzed by FCM. For pHi measurement, the suspension was first stained with CDCFDA-SE, then divided into three tubes, centrifuged at 10,000 × g for 5 min and the pellets were re-suspended in the three solutions described above. Cell suspensions were incubated at 30°C for 3 h before being analyzed by FM.

OD₆₀₀ of all strains were measured in MRS_4.3 and MRS_4.3+H with and without addition of ethanol (4.6%, v/v), according to the method described above. For viability assessment, 2 mL standardized culture was centrifuged at 10,000 × g for 5 min and the pellet was re-suspended in 2 mL acetate buffer. The suspension was divided into four tubes, followed by centrifugation (10,000 × g, 5 min) and the pellets were re-suspended in acetate buffer with and without hop compounds and ethanol. Cell suspensions were incubated at 30°C for 2.5 h, stained with SYTO 13/PI for 0.5 h, and then analyzed by FCM.

For viability assessment, a 63× Plan-Apochromat, N.A 1.4 (Zeiss) was used. The green fluorescence (SYTO 13) was recorded by excitation of 470–490 nm and emission of 515–565 nm. The red fluorescence (PI) was recorded by excitation of 515–565 nm and emission of 610–680 nm.

For pHi measurement, the method was the same as previously described (Shabala et al., 2006). Briefly, the two excitation wavelengths were 435 and 488 nm, and emission was collected by a 515–565 nm bandpass filter. Images were acquired by using the Metamorph 7 software (Universal imaging Corp., West Chester, PA, USA) and analyzed with the free image analysis software ImageJ [version 1.48; National Institutes of Health (NIH), Bethesda, MD, USA¹].

Samples were counted and analyzed by a BD FACS Jazz Cell Sorter with a 488 nm argon ion laser. Green fluorescence emitted from SYTO 13 stained cells was collected with bandpass filter 530 ± 20 nm, whereas the red fluorescence emitted form PI stained cells was collected with bandpass filter 692 ± 20 nm. Events were collected by triggering on the side scatter channel. A 1 mL cell suspension was added into a plastic tube and 100.000 events per sample were recorded. Data were collected and stored in BD FACS Software sorter software (BD Biosciences, USA). The collected data were analyzed with the FlowJo_V10 (Tree Star, Inc. USA). For subpopulation assessments, four quadrants were defined clockwise starting from the top-left one as Q1, Q2, Q3, and Q4 on the dot plot images. For cell size comparisons, the histogram of the forward scatter channel (FSC) was used.

When growth experiments were performed in several replicates, the average and the standard deviation were calculated. In the FCM experiments, 100.000 cells were analyzed to obtain a large sample, and the proportions of subpopulations were generated by FlowJo_V10 software. In the intracellular pH experiments, the calibration curve from section “Sample Preparation for pHi Measurement” was used to calculate the pHi. Subsequently, the average of at least 20 individual cells was calculated, and the individual cells were manually ascribed to predefined pHi intervals.

Growth curves of six strains were obtained under normal conditions, under low pH stress, under hop compounds stress as well as under the combined stress condition (Figure 2). All six isolates grew well in normal MRS_5.6, with G430 reaching the highest OD₆₀₀ (Figure 2E). In MRS_4.3, five strains had a slightly prolonged lag phase, but with the same growth rate and the same final OD₆₀₀. Only G430 was significantly inhibited by the low pH alone (Figure 2E). In contrast, all the bacteria exhibited poor growth under hop stress. The combined stress was even more inhibitory. In MRS_4.3+H, JK09--, G430 and Q were so inhibited that they did not produce any change in optical density during the experiment (168 h), and these strains were consequently designated as hop sensitive strains. JK09, HF01 and A were comparatively tolerant to hop compounds in MRS_4.3+H, and these strains were designated as hop tolerant strains. However, the lag phase and growth rate of the hop tolerant strains were dramatically affected, particularly for strain A. As the antibacterial effect of hop compounds was mostly pronounced at pH 4.3, a typical pH for beer, pH 4.3 was used for further investigations.

Viable cells could easily be distinguished from membrane damaged cells by dual-staining with SYTO 13 (green fluorescence) and PI (red fluorescence) using FM. At the beginning of the experiment (T = 0 h), most cells of both JK09 and JK09-- showed bright green fluorescence (Figures 3A,D). After incubation in MRS_4.3+H for 24 h, fuzzy, weak red fluorescent clusters of cells could be observed for both strains, and some cells even appeared unstained (Figures 3B,E). But most fluorescent cells of JK09 were labeled green (Figure 2B) while most cells of JK09-- were red (Figure 3E). After 48 h, the same pattern could be observed with the exception that most of the clusters were not stained with any fluorescent dye (Figures 3C,F).

In order to investigate the viability of L. brevis under hop compounds stress at a single cell level, stained cells were additionally analyzed with flow cytometer. Four different quadrants were recognized, which represents cells in different physiological states. Quadrant 1 (Q1) is the subpopulation of viable cells with intact membranes that only stained with SYTO 13, Q2 represents an intermediate subpopulation which corresponds to viable cells but with damaged membranes, Q3 describes the subpopulation of dead cells, which stained strongly with PI, Q4 consists of weakly stained cells after hop treatment (Figure 4A).

The proportion of viable cells for all the strains without hop compounds treatment were above 90% until 72 h followed by a small reduction after 96 h (results not shown). The hop compounds caused an obvious reduction in viable cells in the first 24 h in all strains, especially for the sensitive strains (Figures 4 and 5). The viable proportions of JK09, HF01, and A dropped to 48, 35, and 20%, respectively, after 24 h exposure (Figures 4B and 5; Supplementary Figure S1), most of the cells were situated in Q3 and/or Q4 instead (Figure 4B; Supplementary Figure S1). However, the proportion of viable cells of these three strains increased markedly during the next 24 h, where the viable proportion of JK09 and HF01reached the same level as in the control experiments after 48 h (Figures 4C and 5; Supplementary Figure S1). On the contrary, the proportion of viable cells in the hop sensitive strains JK09--, G430 and Q continued to decrease after 24 h, and there were almost no viable cells detected after 48 h (Figures 4E,F and 5; Supplementary Figure S1). The proportion of viable cells did not change from 48 to 96 h for any of the strains (Figure 5).

The histogram of the FSC from the FCM analysis reveals the distribution of cell sizes after 48 h (Figure 6). Some of the strains revealed a homogenous distribution of cell sizes in the control experiments, e.g., JK09 and Q (Figures 6A,F), where other strains exhibited a more heterogeneous distribution, e.g., HF01 and JK09-- (Figures 6B,D). The histogram of hop stressed cells shifted toward lower values for all the strains, which indicates that the average cell size was reduced, compared to the control cells, and this tendency was more pronounced for the three sensitive strains.

Changes in the intracellular pH during 24 h of hop stress in acetate buffer (pH 4.3) were determined by fluorescence microscopy. For clarity, three subpopulations were created according to the pH points of the calibration curve. Cells with 4.3 ≤ pHi < 4.7 have a small or absent ΔpH, which correspond to the population of damaged or dead cells, cells with 4.7 ≤ pHi < 5.0 have a small but noticeable ΔpH, and represent the intermediate population, whereas cells with pHi ≥ 5.0 have a large ΔpH and are regarded as viable cells.

After 1 h, the average pHi of all six strains in acetate buffer without hop compounds was 5.3 ± 0.1, with all of the individual cells exhibiting a pHi ≥ 5.0 (Figure 7A). The only exception was JK09--, which had a minor percentage (10%) in the intermediate population. There was a slight reduction in average pHi for all strains over time, but G430 showed the biggest pHi reduction. Under hop stress for 1 h, a sharp drop in pHi was observed for all six strains, where all cells reached the detection limit of pH 4.3 (Figure 7B). During the following 23 h, JK09, HF01, A as well as JK09-- had an obvious increase in pHi. For HF01, the average pHi value after 24 h reached 5.0, and with 55% of the individual cells exhibiting a pHi ≥ 5.0. On the other hand, the average pHi value for Q and G430 was fluctuating around 4.3, with almost all cells in the damaged/dead subpopulation (4.3 ≤ pHi < 4.7) throughout the experiment.

The impact of manganese on the viability of L. brevis was tested in acetate buffer with hop compounds using FCM (Figure 8A). After 3 h of exposure to hop compounds, the number of viable cells in acetate buffer without manganese was decreased. There is no clear trend that separated the hop tolerant strains from the hop sensitive strains. After supplementation with the level of Mn²⁺ found in pilsner lager beer (2.9 μM Mn²⁺), there was a tiny increase in the viable and/or intermediate populations of all strains except JK09--. When Mn²⁺ addition was increased to the level found in MRS (265.0 μM), the percentage of viable cells increased significantly in all strains with the largest relative change in strain Q.

As shown in Figure 8B, the pHi of all six strains after 3 h exposure was around 4.3, the same value as after 1 h exposure (Figure 7B). 2.9 μM Mn²⁺ had limited effect on pHi for most of the strains except JK09, where 40% shifted to the intermediate population. At high level of Mn²⁺ (265.0 μM), a very significant increase in the average pHi was observed in all strains, with 100% individual cells exhibiting a pHi ≥ 4.7, except in JK09--, where 55% individual cells had pHi < 4.7.

Ethanol (4.6% v/v) inhibited the growth of both hop tolerant strain HF01 and hop sensitive strain Q, as demonstrated by the OD₆₀₀ values after 96 h incubation (Figure 9A). This reduction was less pronounced than the effect of hop addition, but the combined addition of hop and ethanol did not show any additional reduction in OD₆₀₀. In fact, the hop tolerant strain grew faster in the combined addition than hop alone, with the largest difference in OD₆₀₀ at 96 h (Figure 9A). This phenomenon was also observed in the other two hop tolerant strains (results not shown). The two strains, HF01 and Q, were subsequently analyzed by FCM, and the damaged subpopulations after 3 h showed that the combination of hop and ethanol at this stage was more lethal than hop alone (Figure 9B).