The Effect of High Pressure on Levilactobacillus brevis in Beer—Inactivation and Sublethal Injury

Abstract

Beer, with its low pH, presence of hop acids, alcohol content, and limited nutrient availability, presents a hostile environment for most bacteria. However, Levilactobacillus brevis remains a significant spoilage organism in the brewing industry. This study examines the impact of high hydrostatic pressure (HHP) on the inactivation and sublethal injury of Lb. brevis KKP 3574 in beer and wort. The results indicate that applying HHP at 400 MPa for 5 min effectively inactivates Lb. brevis, achieving up to a 7 log CFU/mL reduction in bacterial counts in beer, with no detectable sublethal injuries in beer samples. In contrast, in 10% wort, a sublethal injury level of 1.1 log CFU/mL was observed following the same HHP treatment. Furthermore, this study reveals a differential response of Lb. brevis cells depending on their growth phase; cells in the logarithmic growth phase are more susceptible to HHP, showing greater reduction in viability compared to those in the stationary phase. The survival dynamics of sublethally injured cells during refrigerated storage are also explored, with no regeneration observed in beer samples treated at pressures of 400 MPa or higher. These findings underscore the potential of HHP as a robust method for enhancing the microbiological safety and stability of beer while minimizing the risk of spoilage due to sublethally injured bacterial cells. This study provides crucial insights into optimizing HHP parameters to ensure product quality in the brewing industry.

1. Introduction

Beer, the oldest and most frequently consumed alcoholic beverage in the world [1,2,3], is defined as a fermented beverage made from malted grain, typically barley, along with hops, yeast, and water [4]. Beer exhibits microbiological stability due to several factors such as high alcohol content (0.5–10%), bitter hop acids (approximately 17–55 ppm iso-α-acids), CO₂ (approximately 0.5%), sulfur dioxide, and low levels of dissolved oxygen (<0.3 ppm) [5,6,7,8].

A wide range of microorganisms spoiling beer range from Gram-positive and Gram-negative bacteria to fungi, molds, and yeasts [1,5,6,8,9]. Among spoiling bacteria, lactic acid bacteria (LAB) are responsible for 70% of beer spoilage incidents, adversely affecting its sensory properties. Their spoiling potential depends on the different environmental factors, such as the time and temperature in which they are found. To thrive in the challenging conditions of beer, bacteria must develop adaptation mechanisms. The hop concentration is one of the major stress factors for bacteria. The ability of LAB to spoil beer is related to their tolerance to ethanol and acids, mainly iso-acids from hops which have an antibacterial effect. The lactobacilli genus is very diverse, with different ecotypes; some exhibit intrinsic tolerances and stress responses that allow survival under the influence of antimicrobials in beer. Insight into the LAB mechanism of resistance to hop compounds is necessary for understanding and estimating the level of risk involved in spoiling the final product. According to other researchers [1,10,11,12], hop tolerance within species cannot be predicted based on cell or colony morphology, growth pH, carbohydrate metabolism, manganese requirement, superoxide sensitivity, or cellular protein expression. Studies indicate that hop compounds cause cell membrane damage, decrease intracellular pH, and reduce the size and number of LAB. Additionally, only a small subpopulation of hop-tolerant strains can maintain membrane integrity when exposed to hops at low pH. The cell wall of beer-spoiling LAB exhibits galactosylation of teichoic acid glycerol, which impedes the penetration of hop acids into the cell. Beer spoilage strains have a higher amount of lipoteichoic acid in their cell walls. Furthermore, these strains show increased ATP and ATPase activity. LAB species resistant to antibacterial bitter hop compounds can give the beer a bitter taste [13]. The most frequently detected LAB in beer spoilage are Levilactobacillus brevis [14], a Gram-positive, catalase-negative, nonsporulating, and heterofermentative species of LAB. Commonly found in various fermented foods and beverages, such as sauerkraut, kimchi, and beer, Lb. brevis plays a significant role in fermentation processes due to its ability to convert sugars into lactic acid, contributing to the flavor, texture, and preservation of these products [15,16]. This species is highly diverse, with different ecotypes exhibiting intrinsic tolerances and stress responses that enable their survival despite the antimicrobials present in beer [1,14,17].

High hydrostatic pressure (HHP) is an innovative food and beverage preservation method that offers numerous benefits regarding food quality and safety, meeting the stringent requirements of food regulatory authorities. In recent years, HHP has been recognized as one of the most significant food processing innovations of the past century [18,19,20]. HHP has been widely adopted in the food industry for preserving fruit and vegetable products, dairy products, meats, fish, and seafood. The main quality characteristics of the wheat beer, such as original extract, ethanol content, pH, and bitterness, except for color, are almost unaffected by HHP as opposed to thermal pasteurization. HHP treatment increases beer’s foaming and haze characteristics. Despite its extensive use, HHP-treated alcoholic beverages, such as beer and wine, have not yet been introduced to the global market [21,22]. The antimicrobial effect of HHP is primarily attributed to two mechanisms: the mechanical destruction of cell walls and damage to genetic material, which significantly reduces vegetative microorganisms, including spoilage and pathogenic species. Numerous studies have documented the destructive effects of HHP on the cell membranes of microorganisms and the intracellular changes it induces [23,24,25,26].

Not all cells in a population have identical resistance to stress factors, with HHP being considered a stressor in food processing. Consequently, a heterogeneous population is generated, comprising cells with varied physiological states [27,28,29]. Depending on the component affected by the pressure, the effect can range from minor, repairable damage to lethal outcomes [30]. Lethal injuries (inactivated cells), being irreversible and irreparable, inevitably result in cell death. Bacterial inactivation after any treatment refers to the reduction or elimination of bacteria through applied methods. The effectiveness of a treatment in inactivating bacteria is typically quantified by measuring the reduction in the number of viable bacterial cells. Sublethal injury in bacterial cells leads to functional disorders, which may either be temporary or permanent. Bacterial cells enter a phase of growth stagnation and remain viable. Such injuries result in reduced growth rates or the inability of cells to grow in standard laboratory conditions. Sublethally injured cells can activate adaptive responses to cope with the stress, allowing them to remain alive, although they may not be easily detectable. Given favorable conditions, these cells can repair themselves and begin to proliferate in food, posing a significant risk to food safety. Therefore, after regenerating and adapting to new conditions, these cells can become metabolically active. Therefore, the presence of sublethally damaged bacterial cells is a critical concern, potentially undermining the effectiveness of food preservation methods. For that reason, it is important to quantify them after treatment to assess the effectiveness and safety of the treatment process [27,28,29,31].

Sublethally injured microorganisms in food are particularly significant for products preserved using non-thermal technologies [31]. This situation presents a risk, as these cells may recover and regain their ability to grow during storage, potentially leading to spoilage and serious public health threats [32]. Nevertheless, issues regarding the safe use of HHP, including potential hazards such as the induction of sublethal injuries in cells, are still under review by the European Food Safety Authority (EFSA) [33].

The growth phase of microorganisms at the time of HHP treatment can significantly influence the extent of microbial inactivation and the occurrence of sublethal injuries. Cells in the log phase, which are metabolically active and rapidly dividing, are generally more susceptible to HHP due to their less robust cellular structures [34]. In contrast, cells in the stationary phase, having developed various stress resistance mechanisms, tend to show greater resilience to high pressure. This resilience can lead to higher rates of sublethal injury rather than complete inactivation. Elucidation of the relationship between the growth phase of Lb. brevis and its response to HHP is pivotal for optimizing processing parameters to ensure microbial safety and product quality.

This study delves into the survival dynamics and sublethal injuries experienced by Levilactobacillus brevis in beer and wort under high-pressure treatment, offering insights necessary for improving microbial control and enhancing the quality and safety of beer. Additionally, since LAB have evolved to possess the ability to grow in the presence of hops, we wanted to analyze the response of microorganisms to HHP-induced stress by assessing its impact on the growth and viability of cells.

2. Materials and Methods

2.1. Bacterial Identification and Culture Conditions

Levilactobacillus brevis KKP 3574 strain was isolated from spoiled beer, as previously described by Bucka-Kolendo et al., 2023 [1]. The genetic affiliation was confirmed through 16S rDNA sequencing and examination of proteomic mass spectra profiles using the MALDI-TOF MS technique [35]. The 16S rDNA sequence was deposited in the GenBank NCBI database under accession number OK287283. The strain was also deposited in the Collection of Industrial Microorganisms (KKP) at the Microbiological Resource Centre, Prof. Wacław Dąbrowski Institute of Agricultural and Food Biotechnology—State Research Institute in Warsaw, Poland.

First, cryopreserved strain culture was added to 10 mL of MRS broth (DeMan, Rogosa, and Sharpe, MerckMillipore, Burlington, MA, USA/Merck KGaA, Darmstadt, Germany) and incubated overnight at 30 °C. Next, 100 µL of pure culture was added to 10 mL of fresh broth medium and incubated under the same conditions to reach the logarithmic or stationary phase. Then, bacterial cultures were centrifuged (4000× g, 10 min, 4 °C) (Rotina 380R Hettich Instruments, Tottlingen, Germany). Subsequently, the supernatants were removed, and the sedimented cells were resuspended in phosphate-buffered saline (PBS, pH 7.2) (MerckMillipore, Burlington, MA, USA/Merck KGaA, Darmstadt, Germany) and centrifuged once more. This washing procedure was repeated three times. Finally, model suspensions of the tested bacteria were prepared in PBS at a 1:9 (v/v) ratio. The initial concentration of the inoculum was above 8 log CFU/mL.

2.2. Model Suspensions and Beers

Two commercial regional beers and 10% wort were used in this study. The full characteristics of beers are described in

Table 1. Full characterization of used beers and wort.

Name	Viena Lager Beer	Pale Lager Beer	10% Wort
Type	unfiltered full beer	pasteurized, light beer	semi-product for beer production
Alcohol, % (m/m)	4.62 ± 0.16	4.31 ± 0.15	-
Alcohol, % (v/v)	5.91 ± 0.16	5.45 ± 0.15	-
Apparent Extract, % (w/w)	2.88 ± 0.09	<0.50	-
Real Extract, % (w/w)	4.99 ± 0.06	2.03 ± 0.03	-
Original wort Extract, % (m/m)	13.85 ± 0.14	10.46 ± 0.11	10.00 ± 0.2
Bitterness (International Bitterness Units—IBUs)	20.0	20.4	-

. The 10% wort (pH 4.8 ± 0.2) was prepared as an aqueous solution of Malt extract broth (MerckMillipore, Burlington, MA, USA). Just before HHP treatment, the abovementioned medium and beverages were inoculated with bacterial suspensions, in a volume of 10:1 (mL) in a final concentration of 7–8 log CFU/mL. Next, they were transferred into sterile polyethylene tubes (Sarstedt, Newton, NC, USA) in 4 mL portions. Each pressure process was performed in replications for two parallel samples.

2.3. HHP Device and Process Parameters

Pressurization was conducted using high-pressure single vessel apparatus U4000/65 (Unipress equipment, Warsaw, Poland) with a maximum operating pressure of 600 MPa, a volume of approximately 0.95 L, and a theoretically operable temperature range of −10 °C to +80 °C. The pressure-transmitting fluid was distilled water and polypropylene glycol (1:1, v/v). The samples were subjected to a pressure of 300 MPa, 400 MPa, and 500 MPa for 5 min, and the pressurization times reported do not include the come-up and come-down times. The process was carried out at room temperature. The temperature was measured in the chamber, and the increase during pressurization was 6 °C/500 MPa.

2.4. Analytical Methods

The number of total viable cells was determined using the pour plate method in MRS agar medium according to PN ISO 15214: 2002 [36], while the number of noninjured cells in the population was determined using MRS agar medium with 2% NaCl. The first medium is a non-selective one that allows the growth of all viable cells in the sample. The second is a selective medium that contains NaCl, impairing the microorganisms’ ability to self-repair. The 2% concentration of NaCl was estimated in the laboratory as the maximum concentration that did not change the morphology and number of unstressed Lb. brevis. The plates were incubated in anaerobic conditions for 72 ± 3 h at 30 °C. The effectiveness of HHP treatment was measured by bacterial inactivation. The inactivation of bacterial cells after HHP was calculated based on the number of bacteria before (N0) and after (N) treatment obtained on non-selective MRS agar and expressed as log (CFU/mL) according to the following Equation (1):

$$\mathrm{I}\mathrm{n}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}=\log {\displaystyle \frac{{\mathrm{N}}_0}{{\mathrm{N}}_{\mathrm{t}}}}$$

(1)

where

• N₀—the number of bacteria before the HHP treatment (control sample)
• N_t—the the number of bacteria after the HHP treatment

Sublethal injury (log ratio) was calculated as the difference between cell numbers expressed as log (CFU/mL) on non-selective MRS agar (NA) and selective MRS agar + 2% NaCl (SA), according to the following Equation (2):

$$\mathrm{S}\mathrm{u}\mathrm{b}\mathrm{l}\mathrm{e}\mathrm{t}\mathrm{h}\mathrm{a}\mathrm{l} \mathrm{i}\mathrm{n}\mathrm{j}\mathrm{u}\mathrm{r}\mathrm{y}(\log \mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o})=\log {\displaystyle \frac{{\mathrm{N}\mathrm{A}}_{\mathrm{t}}}{{\mathrm{S}\mathrm{A}}_{\mathrm{t}}}}$$

(2)

where

• NA [CFU/mL]—number of colonies obtained on non-selective MRS agar
• SA [CFU/mL]—number of colonies obtained on selective MRS agar + 2% NaCl
• t—time period

The inactivation and sublethal injury were tested immediately after the treatment and subsequently after 7 and 14 days of refrigerated storage.

2.5. Analysis of Growth Inhibition across a Range of Hop Concentrations

To determine the resistance to hop, the growth kinetics of the Levilactobacillus brevis KKP 3574 strain was assessed using the Bioscreen C Pro automated growth curve analysis system (Oy AB Ltd., Growth Curves, Turku, Finland), as per Kiousi et al. [2]. London Ale beer, containing 5.79% alcohol (v/v) and 43.6 IBU, was used to prepare a 40 IBU starting concentration. This was then diluted to create 5, 10, 20, and 30 IBU solutions for the hop resistance analysis. The analyses were performed in accordance with standard methods of the European Brewer Convention (EBC) and Mitteleuropäische Brautechnische Analysenkommission (MEBAK) methods.

An 18 h pre-cultivation was performed, followed by adjusting the culture to an OD of 0.5. Subsequently, 50 µL of the 0.5 McF microbial culture (10⁷ CFU/mL) was inoculated into MRS broth (Merck KGaA, Darmstadt, Germany) and added to wells containing 250 µL of the medium, as shown in

Table 2. Composition of media at different bitterness levels.

	5 IBU	10 IBU	20 IBU	30 IBU	Beer 43.6 IBU	Control
MRS broth concentrate (2×)	50%	50%	50%	-	-	50%
MRS broth concentrate (4×)	-	-	-	25%	-	-
Water	12.5%	25%	-		-	50%
Beer (40 IBU)	37.5%	25%	50%	75%	-	-
Beer (43.6 IBU)	-	-	-	-	100%	-

. The growth was monitored over 72 h at 30 °C with hourly OD600 measurements. Each medium variant was tested in quintuplicate. Non-inoculated MRS broth and media with varying hop concentrations served as negative controls. Table 2 outlines the bitterness concentration scheme for evaluating hop resistance.

After the determination of the growth curve, a Gompertz curve was fitted to the data using the LabPlot 2.9.0 program (KDE).

$${\mathrm{L}}_{\mathrm{t}}=\mathrm{A}+\mathrm{C}\times {\mathrm{e}}^{{-\mathrm{e}}^{-\mathrm{B}\times \left(\mathrm{t}-\mathrm{D}\right)}}$$

(3)

where

• L_t—OD at time t;
• t—time (h);
• A—asymptotic OD value as t decreases indefinitely;
• B—relative growth rate at D;
• C—the asymptotic amount of growth that occurs as t increases indefinitely;
• D—time at which the absolute growth rate is at its maximum (h).

The maximum growth rate µ_max was determined based on the Gompertz model.

$${\mathrm{\mu }}_{\mathrm{m}\mathrm{a}\mathrm{x}}={\displaystyle \frac{\mathrm{B}\times \mathrm{C}}{\mathrm{e}}}\left[{\mathrm{h}}^{-1}\right]$$

(4)

The change in optical density (ΔOD) was determined based on the difference between OD_max and OD_min.

$$\mathsf{\Delta }\mathrm{O}\mathrm{D}={\mathrm{O}\mathrm{D}}_{\mathrm{m}\mathrm{a}\mathrm{x}}-{\mathrm{O}\mathrm{D}}_{\mathrm{m}\mathrm{i}\mathrm{n}}$$

(5)

where

• OD_max—the highest value of optical density observed during the process
• OD_min—the lowest value of optical density observed during the process

2.6. Statistical Analyses

Statistical analyses were performed using one-way ANOVA to assess the differences among groups. To check for normality, the Shapiro–Wilk test was employed. Tukey’s HSD test (α = 0.05) was subsequently used for post hoc comparisons. Data are presented as mean ± standard deviation (SD). The analyses were conducted using Statistica 14.0 (TIBCO Software, Palo Alto, CA, USA).

3. Results

3.1. Growth Inhibition across a Range of Hop Concentrations

The maximum growth rate (μ) for Levilactobacillus brevis KKP 3574 shows a clear dependence on bitterness concentration (

Table 3. Results for growth rate coefficients (μ) and optical density difference (ΔOD) for Levilactobacillus brevis KKP 3574.

Medium	μ	ΔOD
Control	0.171 ± 0.003 e	1.696 ± 0.006 e
5 IBU	0.252 ± 0.004 f	1.829 ± 0.006 f
10 IBU	0.168 ± 0.004 d	1.611 ± 0.011 d
20 IBU	0.101 ± 0.004 c	1.189 ± 0.029 c
30 IBU	0.035 ± 0.001 b	0.555 ± 0.038 b
Beer 43.6 IBU	0.018 ± 0.001 a	0.480 ± 0.016 a

All data are presented as the mean ± SD, n = 5. Lowercase letters indicate statistically significant differences for μ and ΔOD between medium variants.

). The highest maximum growth rate was observed for the medium with 5 IBU (0.252 ± 0.004), indicating that KKP 3574 grows most efficiently at this level of bitterness. As the IBU concentration increases above 5, the maximum growth rate gradually decreases, reaching the lowest value for beer with 43.6 IBU (0.018 ± 0.001). These results indicate that high levels of bitterness have a strong inhibitory effect on the growth of KKP 3574.

A similar trend was observed in the optical density difference (ΔOD). The highest ΔOD was recorded for the medium with 5 IBU (1.829 ± 0.006), which indicates the highest activity of Lb. brevis KKP 3574 at this bitterness concentration. As the IBU concentration increases, ΔOD decreases, with the lowest value observed for a beer with 43.6 IBU (0.480 ± 0.016). The low ΔOD at high IBU concentrations confirms the inhibitory effect of hop compounds on the growth of Lb. brevis KKP 3574.

While higher concentrations of hops restrict the growth of Lb. brevis KKP 3574, it is noteworthy that the strain grows better in a medium with 5 IBU than in the control medium, in terms of both maximum growth rate and ΔOD. This suggests that low concentrations of hops have a positive effect on the growth of KKP 3574, enhancing its activity compared to a hop-free medium. The optimal growth is observed at 5 IBU, where both maximum growth rate and ΔOD are at their highest, indicating a beneficial influence of mild bitterness on Lb. brevis KKP 3574.

3.2. The Influence of Pressurization Conditions on Survivability and Sublethal Injuries

The effects of pressure on the inactivation and sublethal injuries of Levilactobacillus brevis KKP 3574 in untreated and high-pressure-treated samples of 10% wort, Viena Lager Beer, and Pale Lager Beer are shown in

Table 4. The level of inactivation of Levilactobacillus brevis KKP 3574 after HHP treatment.

Pressure [MPa]/Time [min]	10% Wort	Pale Lager Beer	Viena Lager Beer
	log [CFU/mL]
300/5	0.52 ± 0.07 aA	1.08 ± 0.23 aB	2.96 ± 0.08 aC
400/5	5.03 ± 0.27 bA	6.99 ± 0.49 bB	6.96 ± 0.54 bC
500/5	7.07 ± 0.75 cA	7.77 ± 0.86 cB	7.59 ± 0.32 cC

All data are presented as the mean ± SD, n = 4. Lowercase letters indicate statistically significant differences in the level of inactivation between different HHP treatment parameters within the same medium variant. Uppercase letters indicate differences in the level of inactivation between different media under the same HHP treatment parameters.

and

Table 5. The level of sublethal injuries of Levilactobacillus brevis KKP 3574 after HHP treatment.

Pressure [MPa]/Time [min]	10% Wort	Pale Lager Beer	Viena Lager Beer
	log [CFU/mL]
300/5	0.24 ± 0.02 aB	0.06 ± 0.00 aA	0.04 ± 0.01 bA
400/5	1.09 ± 0.03 bB	0.07 ± 0.01 aA	0.01 ± 0.00 aA
500/5	0.18 ± 0.00 aB	0.33 ± 0.01 bC	0.01 ± 0.00 aA

All data are presented as the mean ± SD, n = 4. Lowercase letters indicate statistically significant differences in the level of inactivation between different HHP treatment parameters within the same medium variant. Uppercase letters indicate differences in the level of inactivation between different media under the same HHP treatment parameters.

. In this experiment, cells from the stationary phase were used. The reduction in Lb. brevis in 10% wort after applying a pressure of 300 MPa for 5 min was 0.52 log [CFU/mL] (p < 0.05) while extending the pressure up to 400 MPa resulted in the reduction of 5.03 log [CFU/mL]. Pressurization under 500 MPa led to a significant decrease in the Lb. brevis population, and the reduction was 7.7 log [CFU/mL]. The presence of sublethally injured cells in the population was observed only after applying 400 MPa pressure for 5 min. The level of sublethally injured cells in the Lb. brevis population was 1.01 [CFU/mL] (p < 0.05) (Table 5). Changes within the population of Lb. brevis suspended in Viena Lager and Pale Lager beers were observed (Table 4). After 300 MPa/5 min HHP treatment, the number of viable cells decreases by 1.08 and 2.96 [CFU/mL], respectively. The results obtained for Lb. brevis suspended in beers demonstrated that treatment at 400 MPa for 5 min greatly affected bacterial viability. The bacterial inactivation in both beers was above 6.9 log [CFU/mL]. Further enhancement of the pressure (up to 500 MPa) caused greater inactivation. No sublethally injured cells were observed after applying pressures of 300, 400, and 500 MPa for 5 min in both beers.

3.3. Effect of Storage on Survival and Regeneration of HHP-Sublethally Injured Lb. brevis KKP 3574

The impact of refrigerated storage on the survival of Lb. brevis KKP 3574 population in untreated and high-pressure-treated samples of 10% wort, Viena Lager Beer, and Pale Lager Beer are shown in Figure 1, Figure 2 and Figure 3. In this experiment, cells from the stationary phase were used. The pressures of 300 MPa, 400 MPa, and 500 MPa for 5 min were used. The number of Lb. brevis KKP 3574 in Vienna Lager and Pale Lager beers in control samples (unpressurized) decreased during 14 days of refrigerated storage by 1.07 and 1.45 [CFU/mL], while in 10% wort broth, the reduction was not significant (p ≥ 0.05).

Survival rates of bacteria suspended in 10% wort treated with 300 MPa, after 14 days of refrigerated storage (Figure 1), showed 1.39 log [CFU/mL] inactivation. It was observed that the number of the bacterial population treated with HHP under 400 MPa after 7 days of storage significantly decreased by 1.08 [CFU/mL] in comparison to the initial HHP-treated viable cell counts. Subsequently, the extension time of storage had no significant effect on further inactivation. On the 7th day of storage, HHP-treated bacterial cells under 500 MPa were not detected. There was no significant recovery of sublethally injured cells during the 14 days of refrigerated storage in all tested samples. As for the beer samples (Figure 2 and Figure 3), the inactivation of bacterial cells treated with 300 MPa after 14 days of storage did not exceed 1.2 log [CFU/mL]. No growth of HHP-treated Lb. brevis KKP 3574 under 400 MPa and 500 MPa was observed in both tested beers after 7 days of storage. The changes in the sublethal injuries of bacterial cells in Vienna Lager during 14 days of storage were not statistically significant (p ≥ 0.05). In turn, in Pale Lager beer, the level of sublethally injured cells in the bacterial population increased between 7 and 14 days of refrigerated storage.

3.4. Effect of Pressure on the Survivability and Sublethal Injuries of Lb. brevis KKP 3574 Cells from Different Growth Phases

To investigate the effect of HHP on the survivability and sublethal injuries of Lb. brevis KKP 3574 depending on the growth phase, the pressure of 300 MPa for 5 min was chosen. To this, the 8–9 log [CFU/mL] Lb. brevis KKP 3574 cells from a 26 h culture in the logarithmic growth phase and a 40 h culture in the stationary phase were used.

In the non-pressurized control sample during the logarithmic growth phase, a statistically significant (p < 0.05) sublethal injury level of 0.74 log [CFU/mL] was observed. After pressurization at 300 MPa for 5 min, the reduction in Lb. brevis KKP 3574 cells suspended in 10% wort was 1.78 log [CFU/mL] in the logarithmic phase and 0.52 log [CFU/mL] in the stationary phase (both p < 0.05). Pressurization increased the level of sublethal injuries to 1.46 log [CFU/mL] in the logarithmic phase, while no significant sublethal injuries were observed in the stationary phase, as indicated in Figure 4.

In the Vienna Lager beer, no sublethally injured cells were observed in the stationary phase control sample. However, in the logarithmic growth phase control, sublethally injured cells were detected at a statistically significant level of 0.74 log [CFU/mL]. After pressurization, a decrease of 1.08 log [CFU/mL] in the stationary phase cell number was observed, leading to the appearance of sublethally injured cells. The reduction in the bacterial count following pressurization was as high as 3.98 log [CFU/mL] in the stationary phase culture and 1 log [CFU/mL] in the logarithmic growth phase culture (Figure 5).

In Pale Lager beer, no sublethally injured cells were observed in the control samples from the stationary phase. However, in the logarithmic growth phase, a statistically significant level of sublethally injured cells was detected at 0.74 log [CFU/mL]. After pressurization, the reduction in cell count was 2.96 log [CFU/mL] in the stationary phase, and no sublethally injured cells survived. In the logarithmic growth phase, the bacterial population decreased by as much as 6.73 log [CFU/mL] after applying a pressure of 300 MPa. The lack of statistically significant differences between the selective and non-selective media in this phase indicates that these pressurization conditions effectively eliminated all sublethally injured cells, which were present before pressurization (Figure 6).

3.5. Effect of Storage Time on the Survivability and Sublethal Injuries of Lb. brevis KKP 3574 Cells Subjected to Pressurization in the Stationary and Logarithmic Growth Phases

Lb. brevis KKP 3574 cell samples in the 10% wort and Vienna and Pale Lager beers were refrigerated and stored for 14 days to observe the effect of storage time on the survivability and sublethal injuries depending on the growth phase under the pressure of 300 MPa for 5 min.

In 10% wort, for non-pressurized control samples of Lb. brevis KKP 3574, a decrease in the bacterial count of 0.45 log [CFU/mL] was observed for cells in the stationary phase, and no sublethally injured cells were detected after 2 weeks of refrigerated storage. In the logarithmic phase, a decrease in cell count of 1.15 log [CFU/mL] was noticed after 7 days of storage. The level of sublethal injuries decreased by 0.63 log [CFU/mL] after 7 days and increased by 0.82 log [CFU/mL] after 14 days (Figure 7).

After 14 days of refrigerated storage, the number of HHP-treated bacterial populations from the stationary phase decreased by 1.39 log [CFU/mL], while the changes in the level of sublethal injuries during storage were not statistically significant (p ≥ 0.05). However, cells from logarithmic growth phase cultures decreased by 0.91 log [CFU/mL]. The number of sublethally injured cells increased with prolonged storage time, from 1.46 log [CFU/mL] immediately after pressurization to 2.06 log [CFU/mL] after 7 days of storage and to 2.13 log [CFU/mL] after 2 weeks.

A decrease in the bacterial number of 1.07 log [CFU/mL] was observed for control samples in the stationary phase suspended in Vienna Lager beer cells, and no sublethally injured cells were detected. In turn, for cells in the logarithmic growth phase, a decrease in population of 2.6 log [CFU/mL] was observed after 14 days of storage. The level of sublethal injuries decreased by 0.63 log [CFU/mL] after 7 days and increased by 0.82 log [CFU/mL] after 14 days (Figure 8).

The number of HHP-treated bacterial population Lb. brevis KKP 3574 cells from the stationary phase culture decreased by 1.14 log [CFU/mL], while the changes in the level of sublethal injuries during storage were not statistically significant (p ≥ 0.05). Cells from logarithmic growth phase cultures decreased by 2.6 log [CFU/mL]. The level of sublethal injuries during storage changed as follows: immediately after pressurization, it was 1 log; one week later, it was 0.94 log [CFU/mL]; and after 2 weeks, it increased to 2.3 log [CFU/mL].

After 2 weeks of storage, a decrease of 1.45 log [CFU/mL] for cells in the stationary phase was observed for control samples of Pale Lager beer, while sublethal injured cells were not detected. In the case of cells in the logarithmic growth phase, a decrease in the bacterial count of 1.15 log [CFU/mL] was noticed. The level of sublethal injuries decreased by 0.63 log [CFU/mL] after 7 days and increased by 0.82 log [CFU/mL] after 14 days (Figure 9).

Pressurized samples of cells in the stationary phase presented a decrease in the number by 0.53 log [CFU/mL] after 2 weeks of storage (p < 0.05). The level of sublethal injuries was 0.44 log [CFU/mL] after one week and 0.9 log [CFU/mL] after 2 weeks. In turn, for the Lb. brevis KKP 3574 cells in the logarithmic growth phase, there was no growth on media after that time.

4. Discussion

The data from our study indicate a clear relationship between the bitterness level and the growth of Levilactobacillus brevis KKP 3574. The maximum growth rate (µ) and optical density difference (ΔOD) were highest at 5 IBU, suggesting that mild bitterness enhances microbial activity. As the level of bitterness increased beyond this point, both µ and ΔOD significantly decreased, demonstrating an inhibitory effect of higher bitterness levels.

These findings are consistent with the existing literature on the impact of hop compounds and bitterness on microbial growth in fermented beverages. Research shows that hop acids, primarily responsible for the bitterness in beer, have antimicrobial properties that can inhibit the growth of various bacteria. This is particularly relevant in the brewing industry, where specific microbial activities are either promoted or suppressed to achieve desired product characteristics.

A study by Suzuki [6] emphasizes that high levels of hop-derived bitterness can significantly inhibit microbial growth, which aligns with the observed decrease in Lb. brevis KKP 3574 growth at higher bitterness levels. This antimicrobial effect is beneficial for preventing spoilage and ensuring the microbiological stability of beer during storage and distribution.

Interestingly, the enhancement of Lb. brevis KKP 3574 growth at 5 IBU suggests that low concentrations of hop compounds might stimulate microbial activity, possibly by providing mild stress that triggers adaptive responses beneficial for growth. This phenomenon has been noted in other studies, where sublethal levels of antimicrobial agents can induce stress responses that enhance microbial resilience and activity. The optimal growth at 5 IBU indicates a beneficial influence of mild bitterness, which may enhance the overall fermentation process and contribute positively to the sensory qualities of the final product.

For instance, Dawan and Ahn [37] discuss how bacterial stress responses to sublethal antimicrobial levels can trigger adaptive mechanisms that improve microbial resilience and activity. Similarly, Beskrovnaya et al. [38] highlight that sublethal stress conditions can induce robust stress responses in microorganisms, promoting survival and adaptation which might explain the optimal growth of Lb. brevis KKP 3574 at 5 IBU.

Research on the potential of preserving beer using high hydrostatic pressure (HHP) technology began 20 years ago [11]. These studies have focused on evaluating the physicochemical properties, such as color, foam stability, bitterness, and ethanol content [39,40], as well as the sensory attributes of beer. Additionally, they explore the effectiveness of HHP in inactivating microorganisms that spoil beer [41,42].

In this study, it was confirmed that the degree of inactivation of Lb. brevis KKP 3574 increases with the increase in the set pressure. Applying a pressure of 300 MPa for 5 min resulted in the inactivation of Lb. brevis in wort samples at a level of 0.52 log [CFU/mL]. In beer samples, the inactivation ranged from 1.08 to 2.96 log [CFU/mL]. These results indicate that the chemical composition significantly influences the effectiveness of inactivating Lb. brevis KKP 3574.

Gänzle et al. [41] applied a pressure of 300 MPa at 20 °C for 5 min to Lactobacillus plantarum (currently Lactiplantibacillus plantarum) introduced into model beer samples. After 3 h of storage at 10 °C, the number of viable cells decreased by 90%, and those that survived had sublethal injuries. After 27 h of storage at 10 °C, the number of Lp. plantarum cells was below the detection level [22,41]. These findings were similar to those obtained for the Lb. brevis KKP 3574 strain in Vienna Lager beer, where the inactivation was 1.08 log [CFU/mL]. As Bucka-Kolendo et al. [1] noticed, the medium and strain type leverage the inactivation of Lp. plantarum cells during the pressurization process, and the baroprotective effect of wort and the compounds of used beer were pivotal for the survivability of bacteria.

Applying a pressure of 400 MPa for 5 min resulted in the inactivation of Lb. brevis KKP 3574 at levels ranging from 5.03 log [CFU/mL] to 6.99 log [CFU/mL], depending on the medium used. The achieved level of inactivation in beer samples demonstrates the practical application potential of these process parameters. A synergistic bactericidal effect between HHP and beer ingredients, such as alcohol and hop acids, was observed. Notably, during 14 days of refrigerated storage of the beer samples, no changes in the number of Lb. brevis were detected.

In the studies by Yin et al. [42] and Santos et al. [43], lactic acid bacteria showed no growth during 84 days of storage at 20 °C in unfiltered wheat beer preserved under 400 MPa for 15 min, 500 MPa for 10 min, and 600 MPa for 5 min. However, it should be noted that the initial level of these bacteria was low at 1.2 log [CFU/mL]. A reduction of Lb. brevis by 5 log [CFU/mL] was possible after applying a pressure of 400 MPa for 30 s at 40 °C [22,43]. Applying 400 MPa for 12 min to beer with Lp. plantarum resulted in a 6 log [CFU/mL] inactivation [44].

Differences between the results of our study and those obtained by Yin et al. [42] and Fischer et al. [11] may arise from variations in the duration of the pressurization process and temperature. Similar results to those obtained by Fischer et al. [11] were achieved in this study after pressurization at 400 MPa for 5 min with a 10% wort sample containing Lb. brevis KKP 3574, where bacterial inactivation was 5.03 log [CFU/mL]. This inactivation was achieved by conducting the HHP process at room temperature, whereas Fischer et al. [11] used a temperature of 40 °C resulting in comparable inactivation in a shorter time. However, increasing the process temperature may adversely affect the sensory properties of beer.

A difference can also be observed between the results obtained in this study and the research by Ulmer et al. [44], where applying 400 MPa for 12 min resulted in a 6 log [CFU/mL] inactivation for Lp. plantarum in beer. In this study, a similar level of inactivation for Lb. brevis KKP 3574 was achieved despite a shorter pressurization time.

Ulmer et al. [44] achieved a 6 log [CFU/mL] inactivation of Lp. plantarum cells in beer after applying a pressure of 500 MPa for 2 min. These results differ from those obtained in this study, where applying a pressure of 500 MPa for 5 min resulted in Lb. brevis KKP 3574 inactivation ranging from 7.77 to 7.59 log [CFU/mL] in Pale Lager Beer. These differences could be attributed to the longer process duration and the use of a different Lactobacillus species. The achieved level of inactivation indicates the potential for applying such process parameters for industrial beer preservation using HHP.

Scientists have explored preserving beer using high pressure, introducing strains of Lp. plantarum and Lb. brevis exhibiting high hop tolerance [22,43]. L. plantarum and Lb. brevis treated with 200 MPa did not show a significant decrease in cell numbers; however, after several hours of storage, total inactivation of L. plantarum cells was observed, while highly hop-tolerant Lb. brevis cells survived. Applying 200 MPa for 60 min to beer with Lp. plantarum resulted in a 6 log [CFU/mL] inactivation [44]. Pressurizing beer samples at 350 MPa during 56 days of storage inhibited the growth of lactic acid bacteria [11,39]. Applying 600 MPa for 5 min to preserve beer samples resulted in no lactic acid bacteria growth on the MRS medium during 49 days of storage [11,40]. Additionally, applying 600 MPa at 15 °C resulted in the total inactivation of Lp. plantarum cells [11,44,45]. In turn, Gervilla et al. [46] studied the effect of HHP (100 to 500 MPa for 15 in.) on the inactivation of Lb. helveticus CECT 414 inoculated in Ringer solution at different temperatures (4, 25, and 50 °C). Pressurizations at low temperature (4 °C) produced greater inactivation on Lb. helveticus CECT 414 than at room temperature (25 °C). Moreover, Lactobacillus helveticus showed greater resistance to HHP treatments than did another bacteria used in the same study (P. fluorescens, E. coli, or L. innocua).

Our study also revealed significant differences in the inactivation of Lb. brevis KKP 3574 cells between the logarithmic and stationary growth phases following the application of 300 MPa pressure. Immediately after pressurization, the reduction in bacterial numbers in the logarithmic growth phase ranged from 1.78 to 6.73 log [CFU/mL], depending on the medium. This reduction was substantially greater than that observed in the stationary phase, where the decrease ranged from 0.52 to 2.96 log [CFU/mL]. During two weeks of refrigerated storage, Lb. brevis KKP 3574 samples in the logarithmic growth phase exhibited further reductions in bacterial numbers, ranging from 0.91 log [CFU/mL] to 2.25 log [CFU/mL], ultimately leading to total inactivation. In contrast, the inactivation of cells in the stationary phase during storage was more limited, with reductions ranging from 0.53 to 1.39 log [CFU/mL].

In this study, sublethal injuries were detected immediately after pressurization, with the highest level observed at 1.1 log [CFU/mL] after applying 400 MPa for 5 min in 10% wort samples. Throughout 14 days of refrigerated storage, no regeneration of these sublethally injured cells was noted. Specifically, in cells from the logarithmic growth phase, sublethal injury levels varied depending on the medium, ranging from 0.44 to 2.3 log [CFU/mL]. In contrast, cells in the stationary phase exhibited lower sublethal injury levels, ranging from 1.00 to 1.46 log [CFU/mL]. Notably, in non-pressurized control samples, sublethal injury levels were recorded at 0.74 log [CFU/mL].

While sublethal injuries induced by high hydrostatic pressure (HHP) have been well documented in various microorganisms, such as Listeria sp., Escherichia coli, Salmonella sp., and Staphylococcus aureus [20,47,48,49,50,51,52,53], there are limited data regarding these effects on lactic acid bacteria (LAB) in beer. Ulmer et al. (2002) [10] found that HHP treatment at 500 MPa for 5 min at 15 °C did not inactivate Lp. plantarum in model beer; however, sublethal injuries were detected, particularly at pH levels of 6 and 7. At pH values below 5, more than a 5 log reduction was observed. Fischer et al. (2006) [11] supported these findings, showing that Lp. plantarum (which is moderately hop tolerant) experienced greater inactivation at lower pH values, achieving a 5 log reduction.

Sublethal injuries were especially prominent during short pressurization times. For example, treatment at 300 MPa and 20 °C for 60 min, followed by storage at pH 6.5, resulted in only a 20% decrease in colony-forming units. However, storage at lower pH levels (5.0 and 4.0) led to a much more significant reduction of 90% to 95%. Gänzle et al. (2001) [40] also demonstrated that HHP at 300 MPa for 5 min reduced Lp. plantarum survival by 90% in model beer containing 50 ppm hop extract, 5% ethanol, or both. Furthermore, 90% of the surviving cells were sublethally injured. Over the course of 48 h at 10 °C, these injured cells were completely inactivated and undetectable. The presence of 50 ppm hop extract resulted in the sublethal injury of nearly all cells within just 3 h, with viable cell counts dropping by 90% and ultimately falling below detection levels after 27 h of storage.

5. Conclusions

This research demonstrated the significant potential of high hydrostatic pressure (HHP) as an effective method for inactivating Lb. brevis KKP 3574, a known beer spoilage bacterium, in various beer matrices. This study highlighted that the degree of bacterial inactivation is strongly dependent on the pressure level applied, with higher pressures resulting in greater reductions in bacterial viability. Specifically, pressures of 400 MPa and 500 MPa were particularly effective in achieving a substantial reduction in bacterial counts, with no observed regeneration of sublethally injured cells during refrigerated storage. The influence of the medium composition was also evident, with beer matrices providing a more challenging environment for bacterial survival compared to wort, likely due to the synergistic effects of alcohol content, hop acids, and other beer constituents. This finding underscores the importance of considering the specific characteristics of the product when optimizing HHP parameters for microbial control. Moreover, this study revealed differential responses between bacteria in the logarithmic and stationary growth phases, with cells in the logarithmic phase being more susceptible to pressure-induced inactivation and sublethal injuries. This indicates the necessity of considering the bacterial growth phase when designing HHP treatment protocols to ensure maximum efficacy. In summary, this research provides valuable insights into the use of HHP as a non-thermal preservation method for beer, offering a viable solution for enhancing microbial stability while maintaining product quality. Future research should focus on scaling up the process and evaluating the sensory impact of HHP-treated beers to facilitate its commercial application.