1. Introduction
Beer is the most widely consumed alcoholic beverage in the world. The concentration of alcohol (ranging from 0.5 to 10%), bitter hop acids (estimated range of iso-α-acids from 17 to 55 ppm), the presence of 0.5% CO
2, sulfur dioxide, and also dissolved oxygen deficiency (<0.3 ppm) suggests that beer is a microbiologically stable beverage [
1,
2,
3]. It is also poor in nutrients as the fermentative activity of brewer’s yeast almost depletes them [
1,
2,
4]. The microbial stability is achieved by using heat treatment, which often leads to quality deterioration. However, high-pressure processing (HPP) effectively inactivates vegetative microorganisms without the influence of thermal treatment [
5]. HPP can be used for liquid products and solids with high moisture content. Studies have shown that the main advantage of applying high hydrostatic pressure (HHP) is inactivation of undesirable microorganisms, improvement in microbiological safety, and preservation of the organoleptic properties of beer and wine, which can extend shelf life [
5,
6,
7,
8,
9]. Since beer production requires high starch content in barley malt due to the mashing process in which the starch is saccharified to produce fermented sugar, it turned out that pressure can effectively improve enzymatic saccharification during the malting process under appropriate conditions [
10]. HHP treatment did not affect wheat beer’s main quality characteristics, including original extract, ethanol content, pH, and bitterness, and increased the beer’s foaming and haze characteristics [
11]. Therefore, HHP may be a promising nonthermal method for wheat beer production without affecting the original characteristics [
12].
The presence of microorganisms in beer, which causes spoilage, adversely affects the sensory properties of this beverage. Some microorganisms tolerate beer parameters and lead to changes in beers, like turbidity, sedimentation, acidity, sometimes with a diacetyl flavor [
1,
2], and unpleasant odor caused by compounds such as butyric acid, caproic acid, and hydrogen sulfide [
13]. In a recent review, the methods for detecting and identifying beer-spoiling microorganisms were summarized by Oldham and Held, 2023 [
14]. Beer spoilage microorganisms range from Gram-positive and Gram-negative bacteria to fungi, including wild yeasts and molds. The presence of LAB in breweries can be harmful, where
Lactobacillus and
Pediococcus are the most common contaminants in beer, accounting for 60–90% of all spoiling [
2,
15].
LAB shows different beer spoilage capacities, and the response of individual strains to hop compounds differs [
16]. Some
Lactobacillus can grow extensively and spoil almost any type of beer [
2], while others do not. This genus is very diverse, and some members exhibit intrinsic tolerances and stress responses that allow them to survive in a harsh environment like beer [
5,
17]. Moreover, the spoilage potential of
Lactobacillus must be determined in a short time to take preventive measures against this contamination. How quickly spoilage occurs depends on time and temperature. To develop in such difficult conditions, bacteria have to develop adaptive mechanisms [
3], as intraspecific differences in hop tolerance cannot be predicted by differences in cell or colony morphology, growth pH, carbohydrate metabolism, manganese requirements, superoxide sensitivity, or cellular protein expression [
6,
18,
19]. This could be attributed primarily to their acquired ability to grow in the presence of hops. Studies show that hop compounds cause membrane damage, a decrease in intracellular pH, and a reduction in the size and number of
Lp. plantarum [
6,
20] or
Lv. brevis cells [
5,
21]. In addition, only a small subpopulation within hop-tolerant strains retains membrane integrity when exposed to hops at low pH. These cells have been shown to act as ionophores of a mobile carrier, which causes a decrease in intracellular pH and an increase in the concentration of divalent cations, particularly Mn
2C [
22], and contributes to growth. In addition, a large amount of Mn
2C increased the viability of cells on hops [
21]. The cell wall of LABs spoiling beer exhibits galactosylation of glycerol teichoic acid, which hinders the penetration of hop acids into the cell. The amount of lipoteichoic acid in the bacterial cell wall is higher in beer-spoiling strains. In these strains, ATP and ATPase activity increases [
17,
19]. Microscopic observations indicate that the LABs in beer are shaped like shorter sticks, suggesting that the smaller cell surface area benefits defense mechanisms [
1,
2].
The microbial response to stress conditions like the hop compound was found to be explained using tools like single-cell analysis [
21].
Figure 1 illustrates the hop-related mechanisms of bacterial inhibition in beer. Research is being undertaken to determine which genes in
lactobacillus are responsible for the bacteria’s ability to spoil beer [
5,
23,
24,
25]. Detection of marker sequences is essential for better risk assessment in the brewing industry [
19]. Beer spoilage could be mainly correlated to the genes responsible for tolerance to hop compound [
13,
15] cases [
17,
26].
In lactobacilli, hop resistance genes have been identified on plasmids
horA,
horC, and
hitA [
2,
13,
28,
29,
30], but their presence or expression does not always correlate with the ability of LABs to grow in beer [
31]. The
hitA,
horA, and
horC genes are not found in a consistent combination in beer spoilage bacteria [
2,
13,
17,
32,
33]. Most likely, other as of yet uncharacterized products of genes are present on specific plasmids responsible for beer spoilage. These novel gene products may function well with plasmid-encoded HorA, HorC, and HitA [
26]. In
Lp. plantarum and
Lv. brevis resistance to hop compounds, HorA activity and ATP-binding multidrug resistance transporter (ABC), conferring resistance to hop compounds, were detected [
6,
32].
The potential risk of the presence of adapted cells to stress is crucial during beer processing. The physiology of strains that survive HHP simulates one of the resistant cells under other various stresses [
34]. Currently, research is focused on analyzing the response of microorganisms to HHP-induced stress by assessing its impact on the structure, metabolism, growth, and viability of cells [
5,
35]. Under the influence of HHP, the cell membrane’s fluidity decreases, leading to a decrease in transmembrane transport and loss of flagellum motility. The membrane is usually the first cell elements to be damaged by high pressure [
36]. Other studies show that high pressure inhibits the synthesis of ATP in microorganisms and can also activate or deactivate the enzyme, denature functional proteins, and lead to a reduction in proton flow, reducing intracellular pH [
34]. Specific gene regulation for stress resistance mechanisms involves accumulating significant amounts of heat shock proteins (HSPs) in the cell [
37]. Transfer or elimination of regulatory genes related to pressure resistance affects the pressure tolerance of a strain [
38]. The stress response HPP uses subsets of other responses rather than evoking a specific reaction to HPP. As a part of the cross-regulation mechanism in HHP, the expression of genes regulated with regulons CtsR and HrcA were analyzed [
23]. Studies have shown that the relative amount of mRNA of many genes involved in the stress mechanism can result from selective transcription or mRNA stability under HHP.
This study investigated the tolerance to different hop concentrations and the response to HHP treatment of the beer-spoiling strain KKP 3573 in vitro. Furthermore, whole-genome sequencing and annotation were performed to determine the strain’s phylogenomic and genomic characteristics, focusing on annotated genes involved in spoiling and viability during stresses, including HHP.
3. Results and Discussion
3.1. Beer-Spoiling Ability of Lp. plantarum KKP 3573
This study aimed to investigate the growth rate of the KKP 3573 strain in different media variants containing varying concentrations of beer and iso-α acids (IBU) (
Table 3). The growth rate was monitored by measuring the increased optical density (Δ
OD) of the cultures over time.
The findings indicated that the growth rate of strain KKP 3573 was dependent on the medium variant, with a decrease in the growth rate observed as the concentration of IBU increased (
Table 3,
Figure 2). This pattern was consistent across most media variants with the 30 IBU and beer with 43.6 IBU variants showing significant differences from the control. However, the 5 IBU variant was an exception to this trend, as the growth rate was significantly higher for strain KKP 3573 in this variant than the control (MRS broth).
This study also revealed that increasing the concentration of beer led to a decline in the growth rate and the number of cells, indicating a negative impact of hop, alcohol, and/or other beer ingredients on cell growth. This effect was observed in the 20 IBU, 30 IBU, and pure beer variants, where significantly decreased ΔOD values were counted.
Adding a small amount of beer with a concentration of 5 IBU stimulated growth for strain KKP 3573, suggesting a positive effect of low beer concentration on cell growth.
3.2. Influence of HHP on Lp. plantarum Strain Survivability
In 10% wort, the decrease in cell number (CFU/mL) after applying the pressure of 300 MPa for 5 min was 1.62 log for Lp. plantarum KKP 3573. The pressure of 400 MPa/5 min resulted in a significant cell inactivation in the range of 6.38 log and, at the same time, increasing the pressure to 500 MPa caused total inhibition of the strain.
In the Vienna Lager beer, after applying 300 MPa for 5 min, the cell number decreased, with a higher number for the Lp. plantarum strain KKP 3573 of 4.51 log (CFU/mL) being observed; applying 400 MPa and 500 MPa resulted in total inhibition. In Pale Lager beer, for the KKP 3573 strain, total inhibition was observed for all three pressures (300 MPa, 400 MPa, 500 MPa).
The inactivation of
Lp. plantarum cells in the pressure process was influenced by the strain type and the medium (
Figure 3). The highest inactivation was found in Pale Lager beer, which indicates that this type of beer can be relatively easily preserved with HHP. During high-pressure treatment, the baroprotective effect of wort and compounds in unfiltered Vienna Lager beer on
Lp. plantarum cells was observed.
The Spearman Rank Order Correlations revealed interesting findings for pressures up to 400 MPa. The analysis showed that the alcohol content is not significantly correlated with the quantity of microorganisms present. However, there is a significant correlation between the extract content and the number of microorganisms.
Furthermore, the analysis indicated that a higher extract content was positively correlated with increased microbial survivability. This finding suggests that a higher extract content in the samples was associated with a higher microbial survival rate under the specified pressure conditions. This observation could indicate that the nutrients or other factors present in the extract may have provided a more favorable environment for the microorganisms, allowing them to thrive and survive better. The premise of these results is consistent with the relative gene expression results shown by Bucka-Kolendo et al., 2021 [
24].
3.3. The Impact of HHP on MALDI-TOF MS Identification
Strains after growth in optimal conditions (unpressured) and after being subjected to a pressure of 300 MPa for 5 min (pressurized) were analyzed with MALDI-TOF MS. The obtained mass spectra profiles (MSP) were investigated (
Figure 3), and attained identification was compared with previously obtained phylogenetic affiliation.
The MALDI-TOF MS analysis identified the unpressured strain as an
Lv. brevis species, with an average score of 2.33. According to the manufacturer Brucker, scores in the 2.30–3.00 range indicate a high probability of species identification. The result was confirmed with
16S rDNA gene sequence analysis, identifying the strain as an
Lv. brevis species with 99.88% gene sequence similarity [
39]. The pressurized strain was identified as
Lp. plantarum, with a score of 2.21, reflecting probable identification at the species level according to the producer (2.00–2.29). Additionally, identification based on the housekeeping gene phenylalanyl-tRNA synthase α subunit (
pheS) sequence was performed. This method identified the strain as
Lp. plantarum with 99.23% similarity.
Based on the MALDI-TOF MS analysis, a stress factor, such as HHP, can impact the changes in the protein profile. As is visible in
Figure 4, when the mass spectra profiles for the unpressurized strain KKP 3573 and the strain treated with 300 MPa/5 min were compared, the different protein profiles from their protein mass fingerprinting analyses showed changes between the unpressured and pressured strain. Different protein profiles correlated with different identification for this strain when performed with MALDI-TOF MS.
This misinterpretation led to performing whole-genome sequencing (WGS) to obtain the reliable and validated result of the strain phylogenetical affiliation.
3.4. Whole-Genome Sequencing, Gene Annotation, and Phylogenomic Analysis of Strain Lp. plantarum KKP 3573
Whole-genome sequencing and assembly were performed to determine the genetic identity of the strain of interest. The chromosome of KKP 3573 has a length of 3.29 Mbp and GC content of 44.39% (
Figure 5,
Table 4), indicative of its classification as an
Lp. plantarum strain. The WGS of the strain also contains two plasmids (repUS64 and rep28), as revealed using plasmidSPADES and PlasmidFinder 2.0 (
Table S1). The
Lp. plantarum KKP 3573 genome contains 39 insertion elements and three intact prophage regions (
Table 5), while extensive regions in the genome resulted from horizontal gene transfer (
Figure 5). Moreover, the strain does not contain CRISPR arrays or CAS proteins. Finally, the strain does not code for virulence factors or transferable antibiotic resistance genes.
The annotated genes of the strain were further categorized into 19 clusters of orthologous groups using EggNOG. The two most represented groups are carbohydrate metabolism and transport (E) and transcription (K), followed by amino acid metabolism and transport (E) (
Table 5). Of note, the majority of genes possess unknown functions (19%). Furthermore, the most represented COG category in the
Lp. plantarum pangenome is replication and repair (L), followed by groups G and K. Furthermore, predicted proteins were assigned to 206 KEGG pathways, organized into 24 functional categories (
Figure 5). Most proteins are assigned to the “carbohydrate metabolism” category (216 proteins), followed by the “amino acid metabolism” (135 proteins) and “membrane transport” (113 proteins) functional categories. Concerning the KEGG pathway assignment, most annotated proteins are involved in “carbohydrate metabolism” (228 proteins) or “genetic information processing” (198 proteins).
The ANI and phylogenetic relationships with other lactobacilli were determined using established algorithms to validate the phylogeny of the novel strain. Strain
Lp. plantarum KKP 3573 presents high genomic similarity with other species members (>98.9%), validating its classification in the
Lp. plantarum species. Accordingly, phylogenetic analysis based on the WGS of the strain showed that it clusters with other members of the species (
Figure 6).
3.5. Lp. plantarum KKP 3573 Possesses Genes Involved in Tolerance to Stress and the Beer-Spoiling Phenotype
Several genes involved in the strain’s capacity to persist in environmental stress conditions were annotated in the WGS, as shown in
Table 6. More specifically,
Lp. plantarum KKP 3573 possesses the
atpABCDEFGH cluster coding for a F0-F1 ATPase and the gene
yvgP coding for a sodium–proton antiporter, conferring tolerance to low pH. Furthermore, several proteins involved in heat and cold shock resistance were annotated in the genome of the novel strain. These genes may belong to the HSP20 family or are multichaperone systems that ensure cellular integrity and recovery after exposure to extreme temperatures. The strain also carries genomic features indicative of osmotic shock tolerance, including
grpE and the
opuABCD cluster. Accordingly, genes involved in oxidative stress response and oxygen tolerance (
nox,
gpo, and
tpx) were annotated in the WGS of
Lp. plantarum KKP 3573. High-pressure resistance is conferred via a multitude of mechanisms, correlated with high transcription or activity levels of proteins involved in heat shock response and SOS response triggered by environmental stresses that result in DNA damage [
64,
65]. In this context,
Lp. plantarum KKP 3573 contains the machinery that could be used to ensure viability during HHP, including
dnaK and
lon. Additionally, the strain contains genes for
ctsR and
hrcA, two transcriptional regulators, that were shown to be involved in HHP response.
The beer matrix is a hostile niche for bacterial growth due to the presence of hop bitters. In this context, multiple genes involved in the export of bitters were annotated. More specifically, the strain carries three copies of the mntH gene coding for an H(+)-stimulated, divalent metal cation uptake system that regulates the detoxification of hop bitters. Additionally, a full cluster for unsaturated fatty acids biosynthesis was identified. Gene fabZ is thought to be involved in the capacity of strains to withstand the beer microenvironment. All genes involved in hop resistance are chromosomally encoded.
The beer-spoiling phenotype can be attributed to several phenotypic properties of bacteria. EPS production and biofilm formation mainly contribute to the phenotypic changes related to beer spoilage. In this context, genes involved in EPS production (
epsB) and biofilm formation (
luxS) were annotated in the chromosome of the strain. Finally, the capacity of strains to produce antimicrobial metabolites could negatively affect matrix microbiota, ultimately influencing the organoleptic characteristics of fermented beverages. The use of BAGEL4 and consecutive comparative genomic analyses resulted in the identification of two plantaricin clusters. More specifically, the
Lp. plantarum KKP 3573 strain carries complete clusters for the production of the two-peptide, class II plantaricins EF and JK (
Figure S1). The mature core peptides of the clusters present 100% sequence identity and structural conservation with other family members and with functionally characterized peptides produced by
Lp. plantarum C11 (
Table S2). Additionally, biosynthetic pathways for the production of the small antimicrobial molecules were identified.
Lp. plantarum KKP 3573 carries the biosynthetic machinery for secretion of L-lactate (FMN-dependent L-lactate dehydrogenase) and of hydrogen peroxide (NADH oxidases, multicopper oxidase) (
Table 6).
Next, we sought to determine the possible detrimental effects of strain consumption on the host’s health. The strain
Lp. plantarum KKP 3573 does not contain virulence genes or genes involved in the production of hemolysins. Accordingly, it does not carry transferable antimicrobial resistance genes. However, the strain may be resistant to vancomycin, as it carries chromosomally encoded
vanH and
vanY genes. Furthermore, the capacity of the strain to code for biogenic amines was examined in silico. Biogenic amines are derived from the catabolism of proteins [
66]. Enzymes involved in the formation of biogenic amines are amino acid deiminases and decarboxylases.
Lp. plantarum KKP 3573 does not code for these enzymes, and it therefore may not be able to produce these detrimental compounds in situ.