Effects of Temperature Shock on Viability and Stress-Related Gene Expression in Pediococcus acidilactici, a Probiotic Lactic Acid Bacteria

Probiotics are live microorganisms that can confer health benefits when consumed in sufficient amounts. However, probiotics are often subjected to various temperature stresses during their processing and storage, which may lead to undesirable loss of viability. Pediococcus acidilactici, a species of lactic acid bacteria, is a promising probiotic candidate due to its ability to produce the antimicrobial peptide pediocin. Their response to temperature-related stress, especially at the molecular level, is still poorly understood. This study investigated the effect of shocks at various temperatures on the viability and stress-related gene expression of P. acidilactici. There was no significant reduction in the viability of P. acidilactici after temperature shock for 5 minutes at -80°C, 4°C, and 60°C compared to the control at 30°C (Log 9.2-9.3 CFU/mL), while there was a significant reduction in the culture subjected to 75°C (Log 6.17) and 90°C (0), both for 5 mins. RT-qPCR analysis showed no significant differences in the expression of groEL, a heat shock response gene, in P. acidilactici subjected to -80°C, 4°C, and 60°C compared to 30°C, although possible gDNA contamination might occur. These results suggest that P. acidilactici potentially has good survival when subjected to heat-based food processing for probiotics product development.


Introduction
Probiotics are defined by the WHO/FAO as "live microorganisms that, when administered in adequate amounts, confer a health benefit on the host" [1].The most well-known probiotics belong to the Lactic Acid Bacteria (LAB) group, with Lactobacillus and Bifidobacterium as representative genera [2].However, other species such as Pediococcus acidilactici have also been researched as potential probiotics.P. acidilactici has been extensively studied for its application in aquaculture, including fish [3] and shrimp [4], but recent research suggests its potential for human consumption as well [5].P. acidilactici has desirable properties such as tolerance toward acidic conditions [6], adhesion to intestinal epithelial cells [7], and production of useful compounds such as pediocin [8] and gamma-aminobutyric acid [9], making it a potential probiotic.
To provide health benefits, probiotics need to be present in sufficient amounts in the gut.The International Dairy Federation (IDF) recommends a minimum concentration of Log 7 Colony Forming Units/gram (CFU/g) in food products to ensure these benefits [10].However, some recommendations suggest that a minimum concentration of Log 6-7 CFU/g of probiotics in the gut is necessary for health benefits, which would require a higher concentration of probiotics in the food product, potentially reaching up to Log 8-9 CFU/g [11].
The consumption of probiotics typically involves ingestion, either through supplements or incorporation into food products.However, before reaching the consumer, these products must undergo 1255 (2023) 012068 IOP Publishing doi:10.1088/1755-1315/1255/1/012068 2 various processing and storage, posing challenges for probiotic survival.Probiotics must be able to withstand various stresses, from manufacturing to digestion [12,13].One significant stress is temperature, which can occur during manufacturing and storage.Extreme temperature stress may arise during processes such as freeze drying, spray drying, or pasteurization, while cold stress can occur during chilling [12].To ensure the viability of probiotics, it is essential to develop and optimize strategies to mitigate the effects of these stresses.
There have been several studies investigating the effects of temperature stress on P. acidilactici, including those using processes such as spray drying [5] and freeze-drying [14].However, it is important to note that temperature stress is not the only factor that can affect the survival of probiotics during these processes.Other factors, such as the protective agents used in the drying process, may also play a role in the viability of the probiotics.Tirta et al. (2023) reported that during spray drying of P. acidilactici using whey protein and gum arabic as the encapsulation matrix, cell viability was significantly affected by the inlet temperature but not by the wall material ratios [15].
While many studies have investigated the effects of various food manufacturing processes that involve temperature stress on probiotics, there have been few studies on P. acidilactici, especially those that involve exposing P. acidilactici to temperature stress without prior treatment, such as encapsulation.Furthermore, no study has been conducted to evaluate the effect of temperature shock on P. acidilactici at the molecular level, such as by analyzing gene expression to detect the expression of relevant stress response proteins, such as heat-shock proteins (HSPs) during temperature shock conditions.GroEL and DnaK, each representing the chaperone families Hsp60 and Hsp70, are HSPs which play a crucial role in protein folding even under normal growth conditions [16].However, their significance increases in stress conditions.These HSPs bind to the hydrophobic surfaces of unfolded proteins as they facilitate proper folding of substrate polypeptides with the help of co-chaperones (GroES and DnaJ-GrpE) and ATP hydrolysis [16].GroEL, particularly in its nucleotide-free form, has the ability to strongly bind to a diverse range of partially folded proteins [17].GroEL is typically upregulated during heat stress but may be unaffected or even downregulated by cold stress [18].
Understanding how temperature shocks affect P. acidilactici culture might help to develop strategies to ensure their optimal viability during processing and, thus, maximize their potential health benefits.Therefore, this study aims to investigate the effect of various temperature shocks on the viability of P. acidilactici culture.In addition, this study also aims to investigate the stress-related molecular response of P. acidilactici culture, specifically by investigating the expression level of the groEL gene in response to the temperature shock.

Materials and methods
2.1.Culture maintenance P. acidilactici used in this study was obtained from the Food & Nutrition Culture Collection (FNCC) at Universitas Gadjah Mada (UGM).The culture was kept in De Man, Rogosa, and Sharpe (MRS) broth (Merck) or Agar (Merck) at 30°C and identified using Gram staining and 16s rRNA sequencing to a third-party provider (PT.Genetika Science Indonesia).The culture was stored at 4°C and sub-cultured in MRSB every 1-2 weeks, with two sub-cultures needed for cultures stored for more than 2 weeks before being used in the experiments.Maintenance culture was made by inoculating 2.5 mL of the previous liquid culture into 25 mL of fresh MRS broth and incubated at 30°C for 18 hours before storing.Cryo stocks were made by mixing 600 μL of liquid culture with 400 μL of 50% glycerol and stored at -80°C until further use.

Temperature shock treatment
The temperature shock treatment of the culture was performed as follows.An overnight culture was prepared by inoculating 2.5 mL of the maintenance culture into 25 mL fresh MRS broth and then incubated at 30°C for 18h until late-log phase as measured by OD600 readings.Subsequently, 10 mL of this culture (referred to as the treatment culture) was centrifuged at 3500 rpm, 25°C, for 10 minutes (Eppendorf 5810R Centrifuge) and the supernatant was discarded.The pellet was then mixed with 10 mL of pre-cooled/pre-heated media depending on the treatment.Media was pre-cooled at 4°C for the -80°C and 4°C treatments, while for 30°C, 60°C, 75°C, and 90°C, media was pre-heated in a water bath until it reached the target temperature.The cultures were then incubated for 5 minutes at the target temperature.Samples were taken for analysis after incubation.For the 30°C, 60°C, 75°C, and 90°C treatment, the temperature was monitored using a using a temperature sensor on a tube containing MRSB media that was incubated with the culture, For the -80°C treatment, after 5 minutes of incubation, the culture was thawed by incubating it in 30°C for maximum of another 5 minutes.Samples of treatment cultures before temperature treatment (before analysis of OD600) were also taken and analyzed to ensure a similar starting amount of bacteria before each temperature treatment.

Culture viability testing
After temperature shock treatment, viability testing was immediately performed by taking 100 μL of the treated culture and serially diluting it with 900 μL of 0.9% NaCl.The dilutions ranged from 10 -1 to 10 -

8
, and samples were plated using the Miles & Misra method [19], with 10 μL dropped onto MRS agar.CFU counts were performed after incubating the agar in an inverted position at 30°C for 24-48 hours, and results were expressed as log CFU/mL.Each treatment was performed in biological triplicates with one drop counts as one technical replicate.Plate counting was also performed using samples from the overnight culture to ensure a consistent starting amount for all treatments and replicates.

Gene expression analysis
Samples that were selected for this section were samples that have no significant differences in viability before and after temperature shock treatment based on the result of culture viability testing (Section 2.3).

RNA extraction.
RNA extraction was performed immediately after the samples underwent temperature shock treatment using the GeneJET RNA Purification Kit (Thermo Fisher Scientific) following the manufacturer's instructions with necessary adjustments.Briefly, 1-1.5 mL of samples were transferred to sterile 1.5 mL centrifuge tubes and centrifuged at 12,000 g for 2 minutes (Eppendorf 5424 Centrifuge).The supernatant was discarded, and if 3 mL samples were required for extraction, an additional 1.5 mL sample was added to the tubes containing the pellet, followed by another round of centrifugation where the supernatant was discarded.The pellet was then resuspended in a 100 µL TE buffer supplemented with 0.4 mg/mL Lysozyme (Vivantis) and incubated for 5 minutes at 25°C using a thermal heating block (Thermomixer Comfort).The resuspended cells were then mixed by vortexing for up to 15 s or until a homogenous mixture was formed, followed by the addition of 300 µL of lysis buffer supplemented with 14.2 M Beta-mercaptoethanol.Then, 180 µL of absolute ethanol was added, mixed by resuspension, and transferred to the purification column provided by the kit, which was inserted into a collection tube.
The purification tube was centrifuged at 12,000 g for 1 minute, and the flowthrough was discarded.The purification tube was then placed in a new 2 mL collection tube, and 700 µL of Wash Buffer 1 was added, followed by centrifugation at 12,000 g for 1 minute.The flowthrough was discarded, and 600 µL of Wash buffer was added to the purification column, which was centrifuged similarly to the previous step.After discarding the flowthrough, 250 µL of Wash buffer 2 was added to the purification tube, followed by centrifugation at 12,000 g for 2 minutes.The purification tube was then inserted into an RNAse-free 1.5 mL centrifuge tube provided by the kit, and 100 µL of nucleasefree water was added to the purification column.The tube was then centrifuged at 12,000 g for 1 minute to elute the RNA, and the purification column was discarded, leaving about 100 µL of eluted RNA in the centrifuge tube.Samples were stored at -80°C until further analysis.

RNA Concentration, purity, and verification.
The purity of the extracted RNA was evaluated by measuring the A260/A280 ratio using Nanodrop Lite (Thermo Fisher Scientific).RNA concentration was also determined using the same machine, which was programmed to detect RNA.The 'blank' used for the measurement was nuclease-free water from the extraction kit.RNA integrity was assessed using agarose gel electrophoresis.A 1% agarose gel was prepared with 1X TAE buffer and 10,000 SYBR Safe DNA Gel Stain (Thermo Fisher Scientific) was added to the agarose during casting.Electrophoresis was conducted using a Mini Sub Cell GT system (BioRad) at 75 V for 1 hour with a 1 kb DNA ladder as a marker.The resulting electrophoresis image was visualized using the G:BOX Chemi XRQ (Syngene) Gel imaging system.

DNAse digestion.
DNAse digestion was performed to remove possible gDNA contamination from the RNA samples.The RNA samples were treated with DNAse I reagent (New England Biolabs) following the manufacturer's instructions.For 100 µL reactions, 20 µL (<10 µg RNA) RNA was mixed with 10 µL DNAse I reaction buffer (10X), 1 µL DNAse I, and 69 µL nuclease-free water in a sterile 0.2 mL PCR tube on ice.The reactions were then incubated at 37°C for 10 minutes using a SimpliAmp Thermal Cycler (Thermo Fisher Scientific).After the incubation, 1 µL of 0.5 M EDTA was added to each reaction to stop the reaction.The mixture was then incubated at 75°C for 10 minutes using SimpliAmp Thermal Cycler (Thermo Fisher Scientific) to disable the DNAse.The samples were stored at -80°C until further use.
2.4.4.cDNA synthesis.cDNA synthesis was performed using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) following the manufacturer's protocol.Thawing of the reagents and reaction setup were performed on ice.Briefly, the reactions for a 20 µL reaction volume were set up as follows: 10 µL DNAse-digested RNA, 1 µL random hexamer primer, 1 µL nuclease-free water, 4 µL 5x reaction buffer, 1 µL Riboblock RNAse inhibitor (20 U/µL), 2 µL 10µM dNTP Mix, and 1 µL RevertAid M-MuLV RT (200 U/µL).The mixture was gently mixed by resuspension, followed by brief centrifugation using a spin-down centrifuge.The mixture was then incubated for 5 minutes at 25°C, followed by 60 minutes of incubation at 45°C.The reaction was terminated by incubation for 5 minutes at 70°C using a SimpliAmp Thermal Cycler (Thermo Fisher Scientific).The resulting cDNA samples were stored at -20°C until further use.

RT-qPCR
RT-qPCR was performed using Quantinova SYBR Green PCR kit (Qiagen).The reagents and samples were thawed on ice prior to use.The sequences of ldh and groEL primers (Integrated DNA Technologies, Singapore) used are available in Table 1.R: TTCAACGACTGCAACTAAGTCC RT-qPCR reactions were prepared as follows for a 20 µL reaction volume: 2 µL cDNA, 10 µL 2X SYBR Green Master Mix, 1 µL forward primer (14 µM), 1 µL reverse primer (14 µM), and 6 µL nuclease-free water.For quality control, additional reactions included a no template control (NTC) in which cDNA template was replaced by water and a negative control in which cDNA template was replaced by DNAse-digested RNA samples.Samples for the negative control were tested using ldh primers.The reactions were run on a Rotor Gene Q (Qiagen) instrument using the settings specified in Table 2. Three runs were performed, each containing one biological replicate of the samples.After the PCR process was completed, the products were verified using agarose gel electrophoresis.The agarose gel electrophoresis utilized 2% agarose with a 100 bp ladder as a marker.Electrophoresis was run using the same parameters as described in Section 2.4.2.Initial data analysis, such as Cq values, was performed using Qrex software v.1.1.0.4.The Cq values obtained were normalized using the dynamic tube option and noise slope correction.The data were further analyzed using the Livak/2 -∆∆Ct method in Microsoft Excel 2010.The RT-qPCR analysis was performed using three biological replicates, with each biological replicate utilizing technical duplicates for the RT-qPCR process.∆∆Ct normalization was performed using the average ∆Ct of control samples per run.

Statistical analysis
The statistical analysis for viability testing and gene expression analysis was performed using GraphPad Version 8.0.1 for Windows 10.Ordinary one-way analysis of variance (ANOVA) was performed to evaluate the results of viability testing, with a p-value < 0.05 indicating significant differences.Tukey's multiple comparisons test was used to evaluate significant differences between the means of the results.

3.
Results and discussion

Culture viability testing
The viability of P. acidilactici under different temperature shocks is presented in Figure 1.ANOVA analysis before the shocks revealed that the initial cultures had similar amounts of bacteria (log 9.3-9.4CFU/mL).ANOVA and Tukey's post hoc analysis showed no significant differences in viability (log 9.2-9.3CFU/mL) for the cultures exposed to -80°C, 4°C, 30°C (control), and 60°C for 5 minutes.However, exposure to 75°C for 5 minutes significantly reduced P. acidilactici viability to log 6.17 ± 0.15 CFU/mL, and no viable cultures were detected after exposure to 90°C for 5 minutes.
To the best of our knowledge, previous studies have not examined how both thermal and cold shock in various durations affect P. acidilactici viability.Despite our results showing that exposure to -80°C, 4°C, 30°C (control), and 60°C for 5 minutes did not affect P. acidilactici viability, a longer duration might yield a different outcome.Halim et al. (2017) exposed encapsulated P. acidilactici to various temperatures for 1 hour, including 60°C, and found no viable cultures left regardless of the protective matrix used [14].However, their exposure duration was longer than in this study, so P. acidilactici may still lose viability if exposed to 60°C for a longer time.Ahmad et al. (2019) exposed encapsulated P. acidilactici to temperatures up to 70°C for 5 minutes with nanoparticles that did not provide thermal protection, and the culture remained viable [22].However, at 60°C for 15 minutes, viability was significantly reduced, suggesting that P. acidilactici viability may decrease if exposed to 60°C for longer than 5 minutes.This study indicated that P. acidilactici culture can survive heat shock of 60°C for 5 minutes or less without a significant reduction in viability.This suggests that the probiotic may be able to withstand processing techniques such as spray drying, which typically use an outlet temperature of 60°C or less.This outlet temperature has been reported in spray drying studies on other probiotics [23].The results of this study provide insight into what to expect when using carrier formulation for processes like spray drying.However, exposure to 75°C and 90°C for 5 minutes significantly reduced, if not eliminated, the bacteria.These temperatures closely resemble the outlet temperature of spray drying with high inlet temperature settings.Therefore, proper carrier formulation is crucial to reduce loss of viability during the process.
The viability of P. acidilactici at 60°C for 5 minutes suggests that it could be a possible pretreatment parameter before high-temperature processes like spray drying.Previous studies have shown that mild heat shock before stressful procedures can increase probiotic viability compared to those that were not adapted.For example, Zhang et al. (2016) reported that a Lactobacillus salivarius culture that had been given a 15-minute heat treatment at 50°C had higher survivability after spray drying with an outlet temperature of 98-100°C with a significantly lower viability reduction after spray drying (3.91 ± 0.38 Log CFU/g reduction) compared to the non-adapted culture (4.54 ± 0.40 Log CFU/g reduction) [24].Another study by Paéz et al. (2012) found that a 15-minute mild heat treatment of 52°C increased the survivability of Lactobacillus casei and Lactobacillus plantarum after spray drying, with significantly lower death rates of 0.16 log CFU/mL and 0.49 log CFU/mL respectively, compared to their non-heat-treated counterparts which had death rates of 0.85 log CFU/mL and 0.95 log CFU/mL, respectively [25].The increased survivability might be attributed to the upregulation of heat shock protein expression during the pre-treatment.Capozzi et al. (2011) reported that in Lactobacillus acidophilus, heat shock protein 16 (hsp16) was expressed the most after exposure to 53°C for 15 minutes [26].Therefore, it is recommended to investigate longer durations of heat shock between 50-60°C to find the optimum pre-treatment condition for P. acidilactici.Gene expression analysis on the suspected upregulated heat shock proteins could help analyze the effect of such pre-treatment, which can subsequently be tested against exposure to higher temperatures.
On cold and freezing shocks, this study found that there was no significant reduction in culture viability for P. acidilactici after exposure to 4°C and -80°C for 5 minutes.This coincides with previous findings that suggest cold stress above 0°C does not kill the culture [12].However, the short duration of the shock may not have been enough to show a meaningful change, and longer durations should be investigated.For example, Song et al. (2014) found that subjecting L. plantarum to 1 hour of cold shock at 5°C resulted in a slower initial growth rate compared to control bacteria when grown at 37°C [Ref. 27].However, both cultures had similar growth rates after 9 hours of incubation at 37°C.Since a previous review suggested that cold shock might also increase the expression of heat shock proteins [12], it is worth analyzing the heat shock gene expression of P. acidilactici after 5-minute exposure at 4°C.The findings might help indicate whether the culture survives low-temperature processes such as freeze-drying [27] or other types of stress [28].Our results also align with previous reports using freezedrying, where one freeze-thaw cycle at -80°C for longer periods of up to 24 h did not reduce the viability of the P. acidilactici culture [29] However, further studies using longer freezing durations or more freeze-thawing cycles are recommended to better understand how P. acidilactici responds to freezing conditions.
There are limitations in this study that should be considered.One limitation is that the viability of P. acidilactici may vary depending on the growth phase it is in when exposed to stress.For example, if the culture is exposed to stress in the mid-exponential phase or mid-stationary phase, the viability may differ as the metabolism pattern during these phases is also different.Laakso et al. (2011) found that protein abundance in Lactobacillus rhamnosus GG varied over time, with DnaK being more abundant in mid-exponential growth whereas GroEL production is increasing over time [30].Also, in this study, the culture was maintained through constant sub-culturing, which may increase the chances of mutation occurring in the bacteria.These mutations could potentially affect how the culture responds to temperature shock, although this was not yet confirmed in this study.Overall, the viability results are applicable but should be considered alongside these limitations.

Gene expression analysis
Four temperature treatments were selected for gene expression analysis based on the results from Section 3.1.The control group was set at 30°C, while -80°C, 4°C, and 60°C were included as they showed no significant differences in viability to the control group.This analysis aimed to determine whether high viability was due to stress-related gene expression or other mechanisms.Figure 2 shows the relative expression of the groEL gene at each temperature compared to the control.The groEL gene expression is normalized to the ldh gene expression as the reference.ANOVA and Tukey's post hoc analysis indicated no significant difference between the treatment and control groups.The results demonstrate that cold and freezing treatment for 5 minutes did not cause significant change in groEL expression.Previous studies have shown that most bacteria do not usually induce or even downregulate groEL when exposed to cold stress [19] so this might explain our findings for P. acidilactici.Liu et al. (2020) reported that heat shock proteins such as GroES and DnaK were downregulated in L. plantarum when exposed to cold stress [31].Cold stress mainly induces small heat shock proteins (sHSPs), such as hsp18.5 and hsp19.3 reported in L. plantarum [14].Although GroEL expression is not reportedly induced by cold stress, it may still contribute to probiotic survival if the protein is present in abundance.This was demonstrated by a previous study that found a GroESLoverproducing Lactobacillus paracasei had better survivability in freeze-drying [32].GroESL is a chaperone complex with GroEL units forming a double-ring, barrel-like structure (for the unfolded protein to enter) and GroES as the lids [33].However, bacterial stress response is a complex system involving multiple genes and pathways.Therefore, whether other genes are also unaffected needs to be further investigated, preferably using high-throughput methods such as transcriptomics and proteomics.It is also possible that the stress duration might not have been long enough to induce any observable changes in groEL expression.
The result for groEL gene expression in the culture exposed to 60°C for 5 minutes, however, does not match previous studies.It was expected that under heat conditions, probiotics would express or upregulate heat shock proteins like GroEL.However, previous research suggested that a heat shock response could still be expressed at lower temperatures and durations for other probiotics like L. acidophilus [26].This could mean that 60°C for 5 minutes may not be enough to induce a heat stress response in P. acidilactici as indicated by the same level of groEL expression.Another possibility is other genes such as dnaK, rather than groEL, may be the primary gene upregulated in response to heat stress for P. acidilactici.For example, Rezzonico et al. (2007) reported that in Bifidobacterium longum, its dnaK gene was expressed at higher level compared to groEL during heat stress [34].
The negative control reactions in the RT-qPCR revealed possible gDNA contamination (Figure 3), which can produce false positive or aberrant results.This is because the amplification steps during RT-PCR can occur for both cDNA and gDNA templates.Moreover, this might affect the assumption that the treatment did not induce groEL-related stress response in P. acidilactici.Strategies to minimize gDNA contamination include designing primers that target exon flanking long introns or exon-exon junctions [35], but this is not applicable for P. acidilactici since bacteria does not have introns.The gDNA contamination is possibly due to the DNAse I digestion not working properly, which can affect the accuracy of the cDNA synthesis step and the RT-qPCR results.This could lead to difficulty in determining if cDNA was successfully synthesized and may cause the Cq value obtained (data not shown) to primarily come from gDNA contamination instead of cDNA if it was synthesized at all.
Another plausible error in this experiment includes primer-dimers indicated by the gel electrophoresis result of the NTC from the RT-qPCR (Figure 3.3), suggesting further optimization may be needed.Primer dimer might be caused by an excess primer in the reaction mix.Systematic errors due to pipetting errors may also be possible [36], but gDNA contamination is likely a bigger problem.The high standard deviation of T60 may indicate an outlier, and low RNA amount extracted (an average of 7.73 ± 0.37 ng/µL for 3 samples) for T60 may contribute to the high standard deviation because the extracted RNA may become more sensitive to error or loss.More biological replicates and further optimization is recommended to obtain better data for future experiments.The RT-qPCR data in this study were analyzed using the Livak method, which assumes similar PCR efficiency between the reference gene and target gene.However, the PCR efficiency may differ, leading to less accurate results.To correct this, a standard curve should have been constructed to calculate the PCR efficiency of each primer pair, which can be considered when analyzing relative gene expression [37].
Improvement could also be made regarding the reference gene selection.Selecting reference genes based on a small study of a few reference gene candidates that have been tested for their stability in the experimental conditions for the experiment is recommended [38].However, in this study, only ldh (lactate dehydrogenase) was used as the reference gene based on a previous study [20].This could impact the accuracy of the Cq normalization for the target genes in experimental conditions.NTC includes all RT-qPCR component mix except the template was replaced with nuclease free water.The negative control reactions include all RT-qPCR component mix except the template was replaced with DNAse digested RNA from respective treatment (T-80°C, T4°C, T30°C, and T60°C).

Conclusion
In this study, the viability of P. acidilactici cultures after a 5-minute temperature shock was evaluated.A temperature shock of -80°C, 4°C, and 60°C had no significant effect on the viability of P. acidilactici, with a maintained viability of around log 9.2-9.3CFU/mL.However, heat shocks of 75°C and 90°C significantly reduced the viability of P. acidilactici, with a reduction to log 6.17 and 0 log CFU/mL, respectively.The gene expression analysis of groEL for samples subjected to temperature shocks of -80°C, 4°C, and 60°C for 5 minutes showed no significant differences in viability compared to control (30 o C).These results suggested that P. acidilactici might have a good survival when subjected to heatbased food processing.However, further confirmation is needed due to possible gDNA contamination during experiments.To further investigate the effect of temperature stress on P. acidilactici, exploring more parameters is strongly suggested.Longer duration of cold stress at -80°C and 4°C should be explored, and for heat shock, longer duration or lower temperature should be tested.These conditions could be analyzed further using gene expression or proteomics analysis.It could also be investigated whether mild temperature shock can improve culture survival in harsher conditions.Further optimization of the RT-qPCR experiment is necessary to obtain more conclusive results.

Figure 1 .
Figure 1.Viability of P. acidilactici culture before and after being treated with 5-minute heat shock at -80°C, 4°C, 30°C, 60°C, 75°C, and 90°C (Stripped bar represents culture before treatment and solid color bar represents after treatment).The error bar represents the standard deviation of 3 biological replicates (n = 3) with the exception of T90 which only had 2 biological replicates (n = 2).Different superscript stars (* & **) represent significant differences of p < 0.0001 according to ANOVA and Tukey's multiple comparison test.

Figure 2 .
Figure 2. Relative Gene Expression of groEL gene of P. acidilactici treated with temperature shock of 5 minutes at -80°C, 4°C, 30°C, and 60°C.Gene Expression levels were normalized with the ldh and are relative to T30 as the control group.The error bar represents the standard deviation of three biological replicates (n = 3).

Table 1 .
Primer sequences used for this study.