Traditionally, the applications of lactic acid bacteria (LAB) have been primarily associated with the production of fermented foods, such as vegetables, meats, dairies, and beverages (Zannini et al., 2016). Moreover, modern biotechnologies and bioengineering have transformed many LAB species, e.g., Lactococcus lactis (Song et al., 2017) and Lactiplantibacillus plantarum (Mathiesen et al., 2008), into cell factories to produce bioactive molecules (van Tilburg et al., 2019). Stress physiology of LAB has been extensively studied to improve related industrial practices as well as to expand the knowledge in bacteriology. Understandably, as a self-imposed stress, acidic stress is often gradually introduced to the bacterial culture in company with the proliferation of LAB and the accumulation of lactic acid (LA). These altered culture environments are likely harmful to LAB and could eventually stagnate culture growth and related bacterial activities (Liu et al., 2015; Papadimitriou et al., 2016). Therefore, the utilization of LAB in both fermented food production and engineered industrial fermentation requires robust acid tolerance in featured bacteria strains (Wu et al., 2014). The connection between acidic resistance of LAB and the presence of metabolizable carbohydrates have been previously established. Previous research (Corcoran et al., 2005) demonstrated that the presence of metabolizable carbohydrates could enhance the survival of Lactobacilli, although different LAB strains displayed different level of survival enhancement with the supplementation of glucose. Another study (Gullón et al., 2014) showcased the improvement in acidic stress resistance of targeted LAB with different xylooligosaccharides (XOS) which possess potential for symbiotic product development. Also, since the broad adoption of bioinformatic methods, many studies verified that LAB could exert probiotic activities to improve overall host gut health and combat chronic diseases (Toumi et al., 2014; Pandey et al., 2015; Saez-Lara et al., 2015). Notably, colonization of LAB relies on their passage through upper gastrointestinal (GI) track to reach lower GI track in viable state, thus it is necessary for LAB to survive extreme acidic gastric environment to manifest their probiotic functionalities (Bezkorovainy, 2001; Klu and Chen, 2015). In summary, acid tolerance of LAB is of great importance to their applications.

Pediococcus pentosaceus is a gram-positive LAB belongs to the family of Lactobacillaceae. It has been recognized as an important species in fermented foods, such as fermented beverage, meats, vegetables, dairy products, and wine (Todorov and Dicks, 2005; Shin et al., 2008; Osmanagaoglu et al., 2010; Vidhyasagar and Jeevaratnam, 2013). P. pentosaceus was also proven to possess host health improvement effects, like producing beneficial bacteriocin (Porto et al., 2017) and cholesterol decreasing effect (Ilavenil et al., 2016). Whole genome sequencing of P. pentosaceus has been previously conducted and the study revealed that P. pentosaceus is closely related phylogenetically to members of the Lactobacillaceae family, such as Lactiplantibacillus plantarum and Levilactobacillus brevis (Makarova et al., 2006). Universal transcriptome sequencing analysis revealed that functional oligosaccharide culture environments, such as fructooligosaccharides (FOS), konjac mannooligosaccharides (KMOS), and XOS, could systematically regulate the global gene expression of P. pentosaceus and extensively change the transcriptomic profile (Han et al., 2021). This study demonstrated these functional oligosaccharides could up-regulated acid tolerance-related gene clusters in P. pentosaceus, such as arginine deiminase system.

Here, these functional oligosaccharide supplementations were comprehensively evaluated on viable population growth and survival of P. pentosaceus under LA or acetic acid (AA) environments. Then global transcriptome sequencing and follow-up gene expression analysis was employed to explore underlying gene expression mechanisms.

When applied as industrial fermentation factories or in fermented foods, LAB inevitably encounter organic acid stresses, which are primarily originated from LA (Wang et al., 2018). Many methodologies have been employed to combat the challenges induced by LA stress as well as to improve innate LA resistance in LAB, such as intracellular microenvironment and cell membrane engineering, adaptive evolution, and extracellular protective supplementation (Zhang et al., 2012; Wu et al., 2014; Bucka-Kolendo and Sokołowska, 2017). Also, owing to their broad utilization and perceived host beneficial effects in foods (Mathur et al., 2020), modern applications of LAB in foods request sustainable bacterial survivability and viability in both food matrix and host GI track, which, unsurprisingly, ask for solid acid resistance from the feature LAB strains. The connection between acidic resistance of LAB and the presence of metabolizable carbohydrates have been previously established (Corcoran et al., 2005). P. pentosaceus has long been engineered and applied as bacterial factory to produce valuable substances (Wu et al., 2004; Sun et al., 2019). More and more recent studies have illustrated that P. pentosaceus could exert comprehensive host beneficial effects, e.g., antioxidant activities (Zhang et al., 2020), hyperlipidemia relief (Wang et al., 2020), and gut microbiota restoration (Hao et al., 2021). Moreover, the combination between beneficial bacteria (probiotic) and fermentable non-digestible dietary carbohydrate (prebiotic) could manifest synergistic health improvement effects, thus has been attracted much attention in novel functional food manufacturing (Swanson et al., 2020).

The results in this study demonstrated that LA and AA possessed similar growth retard and population viability reduction capability on P. pentosaceus strains (Figures 1, 2 and Supplementary Figures 1, 2), although the effectiveness may vary owing to their different acidic levels. In agreement with previous research (Corcoran et al., 2005), the addition of utilizable carbohydrates increased the organic acid resistance for all surveyed groups, whereas XOS exhibited the highest population protective effects when pH is low (pH = 4.2). This finding is rather interesting when consider that previous study found XOS was the least metabolizable carbohydrate for P. pentosaceus when pH value was close to neutral (Han et al., 2021). Further experiments highlighted that the viability protective effect of XOS is independent from its monosaccharide component, whereas xylose did not achieve comparable results (Figures 2B,D and Supplementary Figure 2). The authors propose that the combination between XOS and P. pentosaceus may have application potential in both fermented food and industrial fermentations.

High-throughput sequencing and its everlasting advancement has empowered researchers to conducted high-resolution global transcriptome studies (Reiter, 2021). RNA-seq pipeline in this study reliably captured the mRNA expression profile. The experiment was designed to discover the LA stress caused transcriptional regulations as well as the regulations induced glucose and XOS under LA stress. As presented, LA stress imposed extensive gene regulations in P. pentosaceus, whereas fewer genes were mobilized in glucose and XOS utilization under LA conditions (Figures 3, 4). Total of 259 genes were significantly up- or down expressed in LAC compared to NC, which accounted 16.5% of 1572 mRNA coding sequences (CDs). As a self-imposed stress which is often wielded by the LAB to achieve ecological competitiveness, P. pentosaceus displayed dynamic transcriptional regulation capacities to LA stresses, although prolonged exposure could also lead to cell death (Papadimitriou et al., 2016). Furthermore, glucose repressed the expression of 83 genes but promoted only 2, this finding aligns with general knowledge that glucose tends to suppress multiple metabolic pathways in LAB. this can be attributed to the fact that glucose is highly metabolizable that only requires the mobilization of limit gene repertoires (Gänzle and Follador, 2012; Goh and Klaenhammer, 2015). On the contrary, XOS supplementation resulted in the up-expression of 19 genes.

The pathways illustrated in detail consist of the most highly regulated genes in XOS metabolism of P. pentosaceus (Figures 5, 6). Interestingly, phosphate ABC transporter pstSCAB is heavily down-regulated in LA stress and further down-regulated in glucose and XOS environment (Figure 5C). The regulation of this transporter is often up-expressed during phosphate insufficiency or starvation (Vuppada et al., 2018), but not in LA stress, apparently. The expression of manXYZ operon, responsible for multiple of sugar transportations (Okochi et al., 2007), is higher expressed in LA stress and XOS culture, but significantly down-regulated in glucose-supplied LA stress compared to LAC (Figure 5D). When XOS is supplied to LA stressed P. pentosaceus, the pathways that carry out the biofunction of xylose isomerization and metabolism (Figure 6A) were significantly up-regulated for energy production, presumably. The XOS catabolic pathway in LAB has been previously proposed, XOS (degree of polymerization: 2−6) were carried by ABC transporters and hydrolyzed either by endo-1,4-β-xylanase and β-xylosidase to release xylose which further participates monosaccharide metabolism (Goh and Klaenhammer, 2015). Notably, the glycolysis unit 1 (Figure 6B) that included two fructose phosphorylases, T256_00805 and T256_00800, was significantly down-regulated by LA stress, while glycolysis unit 2 (T256_05730 and T256_05735) was up-regulated by LA stress and further up-expressed with the addition of XOS. Also, the ADS, previously known to be involved in acidic environment response (Liu et al., 2008), was mildly down-regulated by LA stress alone, but apparently further down-regulated by the addition glucose. This finding is expected since glucose, as an efficient energy supplier, often suppress the expression of this secondary ATP producing pathway (Hitzmann et al., 2013).

Regulation of gene expression is of great importance for overall bacterial fitness and whether can respond to environmental stresses at a high pace is truly a matter of life and death to bacterial cells (Browning and Busby, 2016). This study selectively evaluated the gene transcription of 15 genes during the 90-min of incubation time to profile the regulation pattern of the LA stress, glucose and XOS to P. pentosaceus. Firstly, when neither LA stress nor no additional carbohydrate is supplied in the mMRS culture, initial transcriptional response (often up-expression) for the bacterial still occurred at 15 min and then fadeaway after 90 min. This phenomenon may be owing to the adopted culture re-introduction methodology for the transcriptome experiment since all medium remained separated from their aimed bacterial cells prior to time 0. Moreover, glucose supplementation could rapidly achieve its transcriptional regulation effect (often suppression) as the expression level often remain stable for glucose post the 15-min time point. This finding could be attributed to the high glucose uptake efficiency in bacteria (Jahreis et al., 2008). Furthermore, from the transcriptional promoter classification, it can be inferred that the expression of the genes was not solely decided by these promoters, whereas only the LacI family has similar gene transcriptional patterns observed in their downstream polycistrons (malP and poxD). It is also worth noting that very similar patterns were discovered between gene duos with and without a transcriptional regulator (Supplementary Table 4), such as fruK (DeoR) and treR, manX (GntR) and aceF, and glpK (LysR), and ackA, which implying RNA polymerase-centered transcriptional regulation, instead of promoter-centered regulation, is the predominant transcription regulatory mode in LA stress response and respective carbohydrate metabolisms.

When applied as environmental stresses, LA and AA possessed similar growth stagnate effect and eventually lead to cell death in P. pentosaceus. The supplementation of carbohydrate and functional oligosaccharides, especially XOS, could protect bacterial culture and retain the culture viability. RNA-seq analysis revealed extensive global transcriptional regulation caused by LA stress while XOS supplementation maintained most regulations. However, glucose suppressed numerous genes when supplied as energy source under LA stress. Follow-up RT-qPCR survey illustrated that RNA polymerase-centered transcriptional regulation should be considered as the primary gene regulation approach for P. pentosaceus under LA stress. Alone with previous literatures, this study reiterates that the combinations between P. pentosaceus and functional oligosaccharides has the potential to be applied under acidic environment, e.g., dairy foods and acidic fermentations. Also, when LAB, like P. pentosaceus, are applied in fermentation cultures, the carbohydrate formulation should be thoughtfully evaluated and modified to ensure desirable fermentation results.

Underlying RNA sequencing data in FASTQ format have been deposited into NCBI database and are available under the Bio Project accession number of PRJNA678704.

DH carried out the investigation and drafted the original manuscript. QY contributed to the methodology and designed the experiments. JL analyzed the data and revised the manuscript. ZJ supervised the project and reviewed the manuscript. SY supervised the experiments and edited the manuscript. All approved the submitted version of the article.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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