Levilactobacillus brevis, a heterofermentative lactic acid bacterium, co-exists with Lactiplantibacillus pentosus in commercial-scale cucumber fermentations brined with 1.06 M (6%) sodium chloride (NaCl) and is usually detected from days 7 to 14 post-tanking (Pérez-Díaz et al., 2017). Cucumber fermentations that host Lvb. brevis were found to suffer from bloater defect, the formation of hollow cavities inside the fruits, as the result of an increment in internal pressure by carbon dioxide production (CO₂) (Etchells et al., 1968; Zhai et al., 2018). The ability of Lvb. brevis to produce carbon dioxide from the heterofermentation of glucose and fructose prompted such an association (Etchells et al., 1968; Zhai et al., 2018). However, more recent studies indicate that bloater defects develop in cucumber fermentation well before Lvb. brevis reaches maximum densities and can occur in its absence (Zhai and Pérez-Díaz, 2020). Understanding that Lvb. brevis is not the primary contributor to the CO₂-mediated bloater defect creates opportunities to exploit its metabolic versatility as an adjunct or starter culture for cucumber fermentation given its competitiveness in such a habitat.

The diversion of a portion of the carbon from sugars to acetic acid via heterofermentation instead of solely lactic acid formation from homofermentation may present technological advantages for the retention of cucumber tissue firmness and enhancement of flavor in pickling. For instance, pure cultures of the heterofermentative lactic acid bacteria Lvb. brevis produced less acid and firmer fermented olives (Etchells et al., 1974; Portilha-Cunha et al., 2020). Moreover, desirable flavors form in fermented vegetables from the combination of volatile compounds derived from the vegetable and those that are naturally produced by microbes. To date, more than 200 volatile compounds have been identified in fermenting cucumber or commercial tankyard brines (Zhou and McFeeters, 1998; Marsili and Miller, 2000; Johanningsmeier and McFeeters, 2011), but the relative contributions of fermentation microbiota are not known. Early work showed that cucumber fermented with pure cultures of Lvb. brevis was perceived as more aromatic and less acidic than those fermented with Lpb. plantarum, which were more acidic, less bitter, and had more raw cucumber flavor (Aurand et al., 1965). None of the pure cultures tested could independently reproduce the flavor profile of the indigenous ferments, and there was significant strain variation within each species (Aurand et al., 1965). Considering such findings, it is relevant to investigate the indigenous diversity of heterofermentative lactic acid bacteria such as Lvb. brevis in industrial-scale cucumber fermentation.

Inclusion of Lvb. brevis in starter cultures for vegetable fermentation may also contribute to greater long-term microbial stability. Utilization of a tripartite starter culture of Lpb. pentosus, Lvb. brevis, and Lentilactobacillus buchneri in Cucumber Juice Medium (CJM) resulted in microbial stability at 60 days of storage (Ucar et al., 2020a). Lvb. brevis was found to utilize xylose and trehalose in CJM (Ucar et al., 2020a). Both these sugars are intrinsic to cucumber fermentation (Ucar et al., 2020a) and are depleted during anaerobic spoilage along with other trace mono- and disaccharides (Johanningsmeier and McFeeters, 2015). Lvb. brevis was also found to utilize the cellulose-derived disaccharide gentiobiose in CJM, which may be relevant should this sugar become available in cucumber fermentation (Ucar et al., 2020b). The putative ability of Lvb. brevis to utilize xylose, trehalose, and gentiobiose is hypothesized to aid in precluding spoilage microbes from deriving energy for growth in cucumber fermentation, thus conferring microbial stability.

While Lvb. brevis spontaneously co-dominates in commercial cucumber fermentations brined with 6% sodium chloride (Pérez-Díaz et al., 2017), most members of this species are sensitive to infection by indigenous bacteriophages (Lu et al., 2012). It is presumed that, given the sensitivity to bacteriophages and the fact that Lvb. brevis is a slow grower in cucumber fermentations compared to Lpb. plantarum and Lpb. pentosus, only a fraction of the acetic acid and carbon dioxide formed are contributed by such a bacterium (Pérez-Díaz et al., 2017).

Lack of an understanding of the intrinsic biodiversity of Lvb. brevis in commercial cucumber fermentation impedes the identification of adjunct starter cultures that may aid in enhancing quality, flavor, and microbial stability. Thus, this study was designed to screen the intrinsic biodiversity of Lvb. brevis autochthonous to commercial fermentations to enable the selection of genetically and phenotypically unique strains as adjunct starter culture candidates. A collection of 131 Lvb. brevis isolated from commercial fermentations in the seasons of 2010 in North Carolina and 2012 in Minnesota (Pérez-Díaz et al., 2017) was analyzed to define genetic biodiversity using rep-PCR-(GTG)₅. The screening of the ability of 99 isolates from North Carolina to ferment cucumber juice was done under varied conditions of pH, temperature, and salt concentration. The genome sequences of representative Lvb. brevis were subjected to comparative genome analysis to understand putative biodiversity among isolates from a common habitat relative to allochthonous strains. Four out of the fourteen Lvb. brevis isolates selected for comparative genome analysis that represent unique phylogenetic clusters were used for phenotype microarrays using an Omnilog system to study carbohydrate utilization.

Rep-PCR-(GTG)₅ is considered the technique with the best discrimination power for intraspecies biodiversity among lactic acid bacteria (Gevers et al., 2001; Kostinek et al., 2005; Anekella and Pérez-Díaz, 2020). Contrary to Lpb. plantarum and Lpb. pentosus, the Lvb. brevis rep-PCR-(GTG)₅ patterns suggest limited genetic diversity (Panagou et al., 2008; Pino et al., 2018; Pérez-Díaz et al., 2021), which agrees with reports made by Tamang et al. (2005) for varied lactobacilli. Lpb. plantarum is known for its nomadic lifestyle, and its genome reflects diversity and plasticity resulting from its ability to adapt to various habitats (Martino et al., 2016). Lvb. brevis, however, presents a prolonged doubling time in cucumber fermentation (Pérez-Díaz et al., 2017) and is associated with specific niches, such as the human microbiota and spoilage of fermented foods such as beer (Feyereisen et al., 2019).

Seven and nine clusters were identified in the dendrograms generated with the results of the rep-PCR-(GTG)₅ and the fermentation ability assay, respectively, with divergent isolates distribution (Figures 1, 2). The pH data from the fermentation ability assay indicated an enhancement in acid production by Lvb. brevis at the higher temperature (30°C) in CJM. Figure 2 indicates that most of the Lvb. brevis cultures were able to perform well under theoretically optimal conditions of 6% NaCl, initial pH 5.4, and 30°C as evidenced by the primarily green third column from the left in the Hierarchical Cluster Analysis dendrogram. The most robust isolates for adjunct cultures were grouped together in clusters 2 and 4, which were comprised of 36 Lvb. brevis cultures that demonstrated the ability to acidify CJM across all conditions (Figure 2). The fermentation ability data also suggest that potential synergies between the three variables tested were more useful in screening for robust adjunct culture candidates. It is inferred that the lack of performance of selected Lvb. brevis under sub-optimal conditions responds to their inability to adapt to multiple stresses, population densities, and/or growth conditions.

Understanding the divergence in the clustering derived from rep-PCR-(GTG)₅ and the fermentation ability assay, we selected fourteen isolates that represent each of the clusters for whole genome sequencing. The availability of such genome sequences in public databases was announced by Page et al. (2022). Differences in the incidence of CRISPR loci and horizontal gene transfer in favor of the Lvb. brevis strains reinforce the concept of microbial adaptation to habitats (Martino et al., 2016). The number of uncharacterized genes is significantly lower in the autochthonous Lvb. brevis genomes as compared to the allochthonous strains (Figure 4), suggesting the need for specialization in cucumber fermentations is relatively lower. ANI scores suggest that differences at the genome level exist among Lvb. brevis isolated from varied fermentation habitats and that one strain prevails in cucumber fermentation.

Contrary to the allochthonous Lvb. brevis ATCC 14869, the autochthonous strain was found to be unable to metabolize citric acid. While citric acid is central to the tricarboxylic acid cycle and aerobic respiration, it is absent in immature cucumbers (McFeeters et al., 1982). Lactic acid bacteria can use it to generate alternative products such as acetate, ethanol, lactate, succinate, and acetoin in exchange for a proton motive force or ATP production by substrate-level phosphorylation (Gänzle, 2015). The allochthonous Lvb. brevis ATCC14869 strain produces the characteristic aroma associated with diacetyl production on the testing medium and CJM (data not shown). Further studies are needed to determine whether the absence of citrate lyase in the autochthonous Lvb. brevis is detrimental to competitive growth as a starter culture.

The autochthonous Lvb. brevis utilized sugars associated with plant systems such as cereals, vegetables, and fruits (Buckenhüskes, 1993, 1997; Jones et al., 1999; Dumville and Fry, 2003; Bolarinwa et al., 2014; Lei et al., 2022) (Figure 6), as well as gentiobiose, a disaccharide intrinsic to cucumber (Ucar et al., 2020a). Fructose, glucose, xylose, and trehalose are present in cucumber fermentation (Ucar et al., 2020a,b), and their concentrations decline prior to or during spoilage led by Lentilactobacillus buchneri (Johanningsmeier and McFeeters, 2015). Fructose and glucose are preferably removed by lactobacilli in cucumber fermentation, including Lvb. brevis. Together, these observations suggest that a mixed culture of autochthonous and allochthonous Lvb. brevis may aid in preventing spoilage by removing not only gentiobiose but also trehalose and xylose post-primary fermentation.

The identification of putative genes coding for the multiple components of Pdu microcompartments in all the Lvb. brevis genomes agrees with the observed ability of the species to utilize the substrates in the phenotyping assay using the Omnilog (Figure 8). 1-2-propanediol and propionic acid are produced in cucumber fermentation spoilage by Le. buchneri (Stefanie et al., 2001; Johanningsmeier and McFeeters, 2013). The diol is converted to propionaldehyde via a coenzyme B₁₂-dependent diol dehydratase in Pdu microcompartments, which in turn is catabolized to 1-propanol and propionic acid (Liu et al., 2007). Such a pathway generates ATP, regenerates NAD + H⁺, and produces propionyl-CoA for central metabolism (Liu et al., 2007). The observations that all Lvb. brevis tested could metabolize 1,2-propanediol by expressing the putative Pdu microcompartment cluster suggest that such a compound may be key to the ability of the bacterium to colonize. Further studies are needed to define the conditions needed for Lvb. brevis to utilize the diol and to understand the advantages associated with its utilization within the fermentation habitat.

It is relevant to mention that Lvb. brevis isolates capable of utilizing the antifungal preservative sorbic acid, such as 30.2.29 and ATCC367, have limited application as starter cultures in industrial cucumber fermentation. Sorbic acid has been used for decades in pickling in the United States as a processing aid to control the undesired growth of yeast and molds, particularly on the surface of brines contained in open-top tanks (Etchells et al., 1961). The preservative has been proven useful in enabling industrial cucumber fermentation without sodium chloride (Pérez-Díaz et al., 2022). The effectiveness of the preservative is limited during long-term storage by its removal, presumably by microbial activity. Thus, starter cultures capable of utilizing the preservatives would be detrimental to the long-term stability of cucumber fermentations brined with low salt.

Our data suggest that polysorbates typically added to food products could positively impact the growth of the autochthonous Lvb. brevis (Figure 6). The mechanism by which such an effect is exerted and its relevance to vegetable fermentation need further studies. However, an enhancement of biofilm formation by Lvb. brevis and other lactobacilli in the presence of the surfactants could augment the probiotic content of fermented vegetable products, as in fermented olives (Benítez-Cabello et al., 2020). It is understood that the surfactants and/or deriving degradation products can influence bacterial growth and the ability to form biofilm in various ways (Nielsen et al., 2016).

The ability of Lvb. brevis to utilize amino sugars led us to inquire if Lvb. brevis can degrade amino sugars derived from cucumber tissue during long-term storage of the fermented fruit, as observed for the non-starter lactic acid bacterium, Lactobacillus wasatchensis, in aged Cheddar cheese (Lauret et al., 1996; Oberg et al., 1996; Plumbridge and Vimr, 1999; Uehara and Park, 2004). On the other hand, the polymeric pectin, which is also a component of plant cell walls, was only utilized by Lvb. brevis 14.2.10 in the PM02 plate. Because pectin degradation is associated with fermented cucumber tissue softening (Walter et al., 1985; McFeeters, 1992), this isolate would have limited application as a starter culture for vegetable fermentation.

Interestingly, Lvb. brevis 3.2.41 utilizes gamma-amino butyric acid, which is formed during indigenous cucumber fermentation (Fideler Moore, 2021) and may contribute to its health-promoting properties. Therefore, Lvb. brevis 3.2.41 would need further evaluation prior to use as an adjunct culture.

A genetically distinct Lvb. brevis prevails in cucumber fermentation, robustly utilizes glucose and fructose under varied conditions of pH, temperature, and salt, and presents distinct metabolic activities as compared to allochthonous strains. Such an autochthonous Lvb. brevis strain was isolated from two distinct, geographically distant fermentations and is genotypically defined by the absence of genes that encode for citrate lyase. We postulate that mixed cultures of allochthonous and autochthonous Lvb. brevis may outcompete spoilage microbes by utilizing key sugars intrinsic to cucumber. Members of the species encode for a complete and putative Pdu microcompartment cluster that theoretically enables the production of energy in significant ways.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary material.

IP-D contributed to the scientific approach, experimental design, data generation and interpretation, comparative proteomic and genomics analyses, supervision, project management, and the writing and editing of the manuscript. LM-S executed the Rep-PCR-(GTG)₅ analysis, the corresponding data processing and reviewed the manuscript. SJ contributed the data analysis for the fermentation ability assay and Venn diagram and edited the manuscript. All authors contributed to the article and approved the submitted version.