*o f events in cell region* # *o f events in bead region* <sup>×</sup> # *o f beads per test* <sup>∗</sup> *test volume* <sup>×</sup> *dilution f actor* <sup>=</sup> *concentration o f cell population* (1)

\* This value was found on the vial of BD Liquid Counting Beads and could vary from lot to lot.

In the case of *Lactobacillus plantarum*, changes in cell morphology under the influence of acrylamide were noted, which manifested in the form of cells with twice or several times stronger FL1 signal. Analysis of microscopic preparations stained by the Gram method confirmed that they were *Lactobacillus plantarum* cells appearing individually (bacillus), in pairs (diplobacillus), or in the form of chains (streptobacillus).

#### *2.7. Statistical Analysis*

All experiments were carried out in 5 replicates, and results are expressed as mean ± standard deviation (SD). When the impact of acrylamide on bacterial cell number was assessed, one-way analysis of variance (ANOVA) with Tukey's honest significant difference (HSD) posthoc test was used to compare mean values and determine the significance of differences. The Brown–Forsythe test was used to verify the hypothesis of homogeneity of variances, while Shapiro–Wilk test was used to test the normality of distribution. A *p*-value < 0.05 was considered statistically significant. This part of statistical analysis was carried out in Dell Statistica (Data Analysis Software System, version 13, 2016, software.dell.com). Two-way ANOVA in a mixed model was used to assess data from flow cytometry, which means the interrelationship of two independent variables (incubation times and acrylamide concentrations) with a dependent variable (% of particular cell types), using IBM SPSS Statistics for Windows (2017, Version 25.0; IBM Corp., Armonk, NY, USA). Bonferroni posthoc test was used, and the differences were considered significant when *p*-value < 0.05.

#### **3. Results**

#### *3.1. Impact of Acrylamide on LAB Growth on Solid Medium*

The model medium used for experiments was low nitrogen and low carbon; therefore, the growth of *Lactobacillus* was significantly limited compared to the MRS medium. Acrylamide added to such medium did not show bactericidal or bacteriostatic activity against tested bacteria from the *Lactobacillus* genus even in very big concentrations, not reported in food (Table 1).


**Table 1.** Impact of acrylamide on the growth of *Lactobacillus* strains on solid medium.


**Table 1.** *Cont.*

Scale: ++++ very intense growth (colonies cover whole surface creating lawn plates); +++ intense growth (too many colonies to count, but they are distinguishable); ++ good growth (30–300 colonies/plate); + only a few colonies (<30 colonies/plate); – no growth. AA, acrylamide.

Moreover, it was surprising that AA could stimulate the growth of *L. plantarum* and probiotic strain *L. acidophilus* LA-5 at a concentration of 1000 μg/mL, while *L. brevis* and *L. lactis* sp. *lactis* growth was more intense compared to control in the presence of 500 μg and 1000 μg of acrylamide per mL. The acrylamide concentration used in that part of the study was much higher than that detected in food. The concentrations of acrylamide reported in the literature vary from <10 to even 80,920 μg/kg, with the highest levels in potato chips, French fries, roasted coffee, and coffee extract [44,47–50]. Considering the quantities of particular foodstuffs we consume each day, it has been estimated that total AA uptake varies from 0.3 to 1.4 μg per kg body weight per day [48], depending on the age group (high consumption of coffee in adults) and eating habits. In particular cases, it can reach up to 5 μg/kg/day [5].

The obtained results suggested that some lactic acid bacteria probably could utilize acrylamide as a source of carbon and nitrogen if they lack in the environment (medium). The possibility of acrylamide degradation (not binding) by LAB has been suggested by the results of a study conducted on rats fed with acrylamide 3 h after consumption of four species of *Bifidobacterium*. A significant reduction in the degree of liver damage [51] has been observed. Other studies have demonstrated that in portions of potatoes prepared for French fries subjected to 15 min of fermentation before frying, the AA level is reduced by 90% [52]. However, these results only confirm that lactic acid bacteria can utilize substances that are precursors of acrylamide for their own use; the possibility of AA degradation by LAB has not been studied.

Another strategy to reduce acrylamide formation in bread was proposed by Nachi et al. [53] by using selected lactic acid bacteria strains for dough fermentation. When the LAB was used to inoculate sourdough, the acrylamide concentration in the bread was reduced. This was due to the lower pH of the LAB-inoculated sourdough after fermentation for 16 h compared to the spontaneous sourdough (using only baker's yeast). The acidification was accompanied by a significant increase in the concentration of reducing sugars, which were then used as electron acceptors by LAB and reduced to mannitol. The lack of sugar and low pH prevented the Maillard reaction. The most pronounced reduction of acrylamide formation (by 84.7%) was obtained in bread made with *Pediococcus acidilactici* strain S16.

#### *3.2. Impact of AA on Lactic Acid Bacteria Concentration in Medium*

Three LAB strains were chosen for further experiments: *L. brevis, L. plantarum,* and probiotic strain *L. acidophilus* LA-5. The bacteria concentration was measured by flow cytometry immediately and after 24 h and 48 h of acrylamide addition at various concentrations (Figure 1). The number of bacteria cells in the medium was determined according to Equation (1) using the number of events in the bacteria and bead regions and expressed as cell number per 1 mL.

**Figure 1.** Impact of acrylamide (AA) concentration in medium (0, 7.5, 15, 30, and 100 μg/mL) on bacterial cell number in culture (cells/mL) determined by cytometric method immediately after AA addition and after 24 h and 48 h incubation. (**A**) *L. acidophilus LA-5,* (**B**) *L. brevis,* (**C**) *L. plantarum*. Values in graphs with different letters differ from each other at the level of *p* < 0.05 (Tukey's HSD test).

Already at 0 h, some influence of AA on the number of bacteria in the limiting medium could be seen. It should be recalled that preparing cells for cytometric analysis takes about 2 h from the addition of AA to the medium, so the bacteria have time to change their metabolism and can already start using AA as a source of carbon or nitrogen. The presence of AA in the medium resulted in a decrease in *L. brevis* number after 24 h and 48 h incubation, but not significantly correlated with the AA concentration used (Figure 1B). For *L. acidophilus* LA-5, differences in population size compared to controls and between AA doses were not statistically significant (Figure 1A). The initial increase in cell number in the sample with 7.5 μg AA might have resulted from the use of small amounts of AA, but the concentration was too low to guarantee adequate conditions for bacterial growth and multiplication over a longer period of time. After 24 h, very large fluctuations in culture were reported, but after 48 h, the observed differences were not statistically significant, except for incubation in the presence of 15 μg/mL AA, when LA-5 was lower than in other samples.

These results suggested that *L. acidophilus* LA-5 was sensitive to AA because of decreased cell numbers, which would be consistent with the results of studies on other bacteria. However, these studies have used a medium-low in nitrogen and carbon, and not, as in most other studies, an optimal medium for LAB growth. Therefore, the LAB number also decreased in the medium without the addition of AA. This environment is, therefore, ideal for assessing the impact of AA in the absence of other, more absorbable sources of carbon and nitrogen. In milk, lactic acid bacteria use casein as a source of amino acids, thanks to having appropriate proteolytic enzymes [54]. Gene encoding the cell-wall bound proteinase (PrtP) is only found on the chromosome of *L. acidophilus*; neither *L. plantarum* nor *L. brevis* [55] has it. Also, some peptidases are unique to individual species. The presence of these enzymes, however, is important primarily in the environment typical for these microorganisms (milk) and affects the rate of multiplication of individual bacteria due to various assimilation possibilities of proteins available in the environment, as well as the final effect of the fermentation process, including the resulting secondary metabolites.

In the medium used in this experiment, the only source of carbon and nitrogen was 0.45% of peptone obtained as enzymatic meat tissue hydrolysate, while, in MRS, usually about 2.5% of nitrogen compounds and 2% glucose are present. Furthermore, *L. acidophilus* and *L. brevis* possess all three known LAB peptide transport systems: the di/tripeptide Dpp and DtpT systems and the oligopeptide Opp system [55]. LAB are auxotrophs relative to amino acids, and, depending on the species, they can synthesize only a few amino acids, while the others must be provided with the medium. This means that a medium-low in protein and amino acids will quickly become a factor that limits bacterial growth because bacterial growth and multiplication require the assimilation of substrates to supply the cell with the necessary energy, carbon, and nitrogen to build new structures. That is why the ability to degrade acrylamide to be used as a source of nitrogen (released by NH4 <sup>+</sup> amidase) and carbon was so important in this experiment.

It should be recalled that in this part of the study, all cells were counted: those that were alive and able to function properly and further divide, those that were damaged and whose metabolism was temporarily switched to repair the damage, and dead cells that had not yet broken down.

The situation differed in the case of *L. plantarum* (Figure 1C). After 24 h in the control medium without AA, an increase in the number of bacteria was observed; however, by analyzing their morphology, it was clear that this correlated with the grouping type in which these cells occurred. In the initial population (0 h), 92.55 ± 0.08% of cells appeared as single bacilli and 7.41 ± 0.08% as diplobacilli, while no streptobacilli were observed. After 24 h, the number of cells in culture without AA increased slightly; however, this was mainly due to the fact that the diplobacilli split into single cells. A lack of division meant that after another 24 h, 99% of the population were single rods, and their numbers significantly decreased compared to the initial value.

#### *3.3. Acrylamide Impact on LAB Viability*

Cell concentration and viability were measured by flow cytometry immediately and 24 h and 48 h after acrylamide addition at various concentrations. Populations of dead, injured, and alive bacteria were discriminated based on fluorescence signal after staining with thiazole orange (FL1) and propidium iodide (FL2) provided in the assay. The concentrations of various cell populations were determined using counting beads.

First, it was checked whether incubation time, regardless of acrylamide concentration, had a significant impact on the percentages of specific cell types (main effect of incubation time). All tested main effects of incubation time were statistically significant, except for dead cells of *Lactobacillus brevis* (Table 2). The viability of *L. brevis* increased after AA addition, while the percentage of injured cells was significantly diminished. Contrary to that was the viability of *Lactobacillus acidophilus* LA-5. The highest percentage of live cells was at the beginning of the experiment, while the lowest was after 24 h. Tracking changes in the percentage of injured cells, it appeared that some were repaired, and after 48 h, they were considered to be fully viable. Moreover, some dead cells underwent autolysis, and the cell content released in the medium was utilized by survivors. Lactic acid bacteria are characterized by differentiated autolytic activity, but the process allows them to eliminate weak or impaired cells from the population [56]. In the mentioned study, *L. plantarum* strains were autolyzed more than other LAB strains; however, the authors did not test the autolytic activity of *L. brevis* or *L. acidophilus* [56]. It is well known that some lactic acid bacteria can undergo enzymatic cleavage of cell wall peptidoglycans by peptidoglycan hydrolases present in the bacterial cells and that the autolysis depends on factors, such as carbon source, temperature, osmotic concentration, and pH [56]. It has also been demonstrated that N-acetylmuramidase has a critical function in *Lactobacillus bulgaricus* autolysis [57] as one of the major degraders of the cell wall.


**Table 2.** The main effect of incubation time: impact of time on percentage of specific cell types, regardless of AA concentration.

a, b, c—Means indicated with different letters differ from each other at the level of *p* < 0.05 (Bonferroni test). Values given are mean ± SD of the percentage of cells of a certain type.

In our experiment, the percentage of live *L. plantarum* cells was significantly lower after 48 h than at the beginning or after 24 h. Moreover, significant differences in *L. plantarum* morphology were observed. After 48 h of incubation in the presence of acrylamide, fewer cells were present in the form of single bacilli, while the amounts of diplobacilli and streptobacilli were significantly increased (Table 2 and Figure 2). Such an effect was not observed in other LAB strains.

**Figure 2.** Impact of acrylamide concentration on the morphology of *Lactobacillus plantarum*. (**A**) 0 h, (**B**) 24 h, (**C**) 48 h.

Then, it was tested whether the acrylamide concentration, regardless of the time of incubation, had a significant effect on the percentage of specific cell types (alive, injured, dead) or *L. plantarum* morphology (main effect of acrylamide concentration). The ANOVA results are presented in Table 3 and posthoc tests in Table 4.

**Table 3.** The main effect of acrylamide concentration: influence of acrylamide concentration on the percentage of occurrence of certain cell types (expressed as arithmetic mean (SD)), regardless of the incubation time.


a, b, c—Means with different letters differ from each other at the level of *p* < 0.05 (Bonferroni test).

The analysis showed that the main effect of acrylamide concentration did not occur in the *L. brevis* strain, which meant that in this case, acrylamide (regardless of the incubation time) had no effect on their viability. The AA impact was observed in other tested strains and when the morphology of *L. plantarum* was taken into account. Posthoc analysis showed that acrylamide significantly increased the percentage of alive cells of *L. acidophilus* LA-5 strain, but this was only observed at a concentration of 30 μg/mL and was accompanied by a significant decrease in the number (percentage) of injured cells. In the *L. plantarum* strain, acrylamide at each concentration significantly reduced viability while also significantly increasing the number of injured cells. In addition, morphological examination of *L. plantarum* showed a decrease in the proportion of single cells (bacilli), mainly in favor of increasing their frequency in pairs (diplobacilli). The number of cells in the form of chains (streptobacillus) also increased, but to a lesser extent.

Finally, the interaction effects (simultaneous impact) of incubation time and acrylamide concentration were tested. Acrylamide significantly increased the viability of *L. acidophilus* LA-5 cells at a concentration of 30 μg/mL after 24 h incubation and at 0, 30, and 100 μg/mL after 48 h, when compared to the model medium without acrylamide (Table 5). The increase in alive cells was

mainly accompanied by a reduction in injured cells, rather than dead ones. In turn, the viability of *L. acidophilus* LA-5 decreased at an acrylamide concentration of 15 μg/mL after 48 h.


**Table 4.** The main effect of acrylamide concentration: posthoc Bonferroni test.

\* In a morphological study, percentages of bacillus, diplobacillus, and streptobacillus cells were compared.

**Table 5.** The interaction effect of incubation time and concentration of acrylamide on the viability of *Lactobacillus acidophilus* LA-5 (Bonferroni test).


In the case of *Lactobacillus brevis*, acrylamide at each concentration decreased the percentage of injured cells after 24 h and 48 h incubation (compared to control), although a statistically significant reduction in the percentage of alive bacteria was observed only after 48 h at 100 μg/mL (Table 6). All other changes were not statistically significant.


**Table 6.** The interaction effect of incubation time and concentration of acrylamide on the viability of *Lactobacillus brevis* (Bonferroni test).

After 24 h and 48 h of incubation in the presence of acrylamide at concentrations higher than 7.5 μg/mL, reduced viability of *L. plantarum* cells was observed, while the number of injured cells increased compared with medium without acrylamide (Table 7).

**Table 7.** The interaction effect of incubation time and concentration of acrylamide on the viability of *Lactobacillus plantarum* (Bonferroni test).


We observed that the morphology of one of the tested bacteria, *Lactobacillus plantarum,* was significantly influenced by acrylamide. Based on the fact that cells with both twofold and several times stronger FL1 fluorescence signal (thiazole orange) appeared in the population, we concluded that acrylamide did not inhibit or even stimulate the division of *L. plantarum* but blocked cell separation; hence bacteria in the form of diplobacilli and streptobacilli were present in the population. This conclusion was confirmed by microscopic preparations. A statistically significant reduction in the number of single rods (bacilli) in the presence of AA in amounts of 7.5 and 15 μg/mL after 24 h incubation and in all AA concentrations after 48 h was demonstrated compared to the medium without acrylamide. This was mainly accompanied by a significant increase in the number of cells found in pairs (diplobacilli) and to a much lower extent in chains (streptobacilli). For each analyzed concentration of AA, this increase was especially significant after 48 h, reaching even ~50% at 30 μg/mL (Table 8).


**Table 8.** The interaction effect of incubation time and concentration of acrylamide on the morphology of *Lactobacillus plantarum* (Bonferroni test).

#### **4. Discussion**

In this study, we demonstrated that the tested lactic acid bacteria strains were tolerant of acrylamide even at high concentrations (up to 1 g/mL). Moreover, the growth of *Lactobacillus plantarum*, *L. lactis* sp. *Lactis,* and *L. brevis,* as well as probiotic strain *L. acidophilus* LA-5, was more intense in the presence of acrylamide at high concentration than in medium with limited accessibility of carbon and nitrogen compounds. The obtained results suggested that: (1) acrylamide had no toxic impact on LAB; (2) some lactic acid bacteria probably could utilize acrylamide as a source of carbon and nitrogen if they lack in the environment/medium. Of course, fermented milk beverages and the human gut cannot be considered nutrient-poor environments, as the availability of easily digestible food for bacteria is large, but the possibility of using acrylamide by lactic acid bacteria might be beneficial for both bacteria and the human intestine where the LAB reside.

Our results proved that acrylamide not only influenced the number of lactic acid bacteria but also their viability. The impact of acrylamide on LAB viability depended on both the AA concentration and the bacteria species. First of all, when the impact of incubation time on bacterial viability was analyzed, all the main effects were statistically significant, except the percentage of dead cells of *Lactobacillus brevis*. Secondly, the main effect of acrylamide concentration on the percentage of alive, injured, and dead cells was not observed only in *L. brevis*. This suggested that *L. brevis* was less sensitive to acrylamide

among the tested bacteria strains, and it was confirmed in the further analysis as almost all observed differences were not statistically significant.

The posthoc tests showed that acrylamide caused a significant increase in the percentage of alive cells of probiotic strain *L. acidophilus* LA-5 at an AA concentration of 30 μg/mL compared to the cultures without AA. This increase was mainly accompanied by a reduction in the number of injured cells rather than dead ones. On the other side, acrylamide reduced the viability of *L. plantarum* cells after 24 h and 48 h incubation at each AA concentration except 7.5 μg/mL, simultaneously increasing the amount of injured cells. Moreover, we observed a strong influence of acrylamide (especially at a concentration of 30 μg/mL) on the morphology of bacteria only in *L. plantarum*. Based on the fact that cells with both twofold and several times stronger FL1 fluorescence signal (thiazole orange) appeared in the population, we concluded that acrylamide had no impact on the division of *L. plantarum,* but at the same time, it inhibited cell separation, as cells in the form of diplobacilli and streptobacilli were present in the population (confirmed in microscopic preparations). This suggested that in this case, acrylamide could have a harmful or even mutagenic impact on *L. plantarum*.

It is known that many proteins and hydrolytic enzymes are involved in the proper growth and division of bacteria. Various enzymes participate in turnover (remodeling) of peptidoglycan, and their proper activity and specificity are critical, as bacterial division requires both localized hydrolysis and de novo biosynthesis of the peptidoglycan layer. For example, amidase and glucosaminidase displaying murein hydrolase activity are necessary for the generation of the equatorial ring on the staphylococcal cell surface and complete cell division and separation [58]. *Escherichia coli* division requires the activity of amidases—AmiA, AmiB, and AmiC [59]. It is important that muralytic enzymes distinguish elements of peptidoglycan of specific species. Generally, these enzymes are secreted into the surrounding medium, so they need to distinguish between the cell walls of other species and their own. It seems likely that the targeting mechanisms of murein hydrolases employ species-specific receptors for either physiological cell-wall turnover or the bacteriolytic killing of competing microorganisms [58,60,61]. Most Gram-positive bacteria contain a structurally similar peptidoglycan layer [62]. Thus, targeting of muralytic enzymes cannot be achieved by simple enzyme-substrate interactions but requires specific surface receptors [63]. For example, choline within teichoic acid moieties serves as a receptor for the LytA enzyme of *Streptococcus pneumoniae* [64]. A mutant of *S. pneumoniae* showing complete deletion in the lytA gene coding for N-acetylmuramyl-L-alanine amidase has been isolated. It shows a normal growth rate, and the most remarkable biological consequences of the absence of amidase are the formation of short chains (six to eight cells) and the absence of lysis in the stationary phase of growth. In our study, *L. plantarum* morphology changed in the presence of acrylamide, and bacteria started to form diplobacilli and streptobacilli. It is possible that acrylamide reacted with the active site of muralytic amidases and, therefore, blocked cell separation during division.

Different influence of AA on *Lactobacillus* species tested in the study could also be caused by the diversity of their teichoic acid (TA) structure. Teichoic acids in lactic acid bacteria consist of poly(ribitol phosphate) polymers with attached glucose, D-alanine, and/or glycerol molecules, among others [43]. Their structure is highly variable; thus, even closely related strains can differ in their ability to bind toxins. This is coincident with our results, showing that *L. brevis* was less and *L. plantarum* most sensitive to acrylamide among tested LAB strains. Serrano-Nino et al. [43] proved a significant correlation between the binding percentage of acrylamide and the content of some constituents of cell wall TAs. They proposed that H-bonds could occur between the carbonyl oxygen and the amino group (NH ··· OC) between adjacent acrylamide and D-alanine attached to the ribitol. Moreover, the amine group of D-alanine might react with acrylamide units by means of a Michael addition, while hydrogen bonds might also occur between carbonyl (C=O) oxygens of acrylamide and the hydroxyl groups of glucose residue or glycerol phosphate substituents attached to the poly (ribitol phosphate) chain. Moreover, they demonstrated that acrylamide binding to teichoic acids in *Lactobacillus* was irreversible.

The role of teichoic acids in cell division and morphogenesis has been investigated in some bacteria species, and it appears that wall teichoic acids (WTAs) are involved in elongation of bacteria, while lipoteichoic acids (LTAs) participate in the cellular division [65]. By obtaining the mutants of *L. plantarum,* it has been revealed that WTAs are not essential for survival, but they are required for proper cell elongation and cell division [66]. Therefore, the reaction of acrylamide with teichoic acids could impede division and cause that *L. plantarum* remains in the form of chains and diplobacillus.

Studies of Zhang [67] showed that the ability of acrylamide binding also depended on the peptidoglycan structure. The peptidoglycan of *L. plantarum* (strain 1.0065) had the highest affinity for AA binding (87.14%), whereas peptidoglycans of *L. casei* ATCC393 and *L. acidophilus* KLDS1.0307 showed lower affinity (75.50% and 56.75%, respectively). This binding ability of *L. plantarum* positively correlated with the carbohydrate content in peptidoglycan and the contents of four amino acids (alanine, aspartic acid, glutamic acid, and lysine). Additionally, it was demonstrated that C–O (carboxyl, polysaccharides, and arene), C=O amide, and N–H amines groups were involved in the AA binding.

Analyzing the interaction of acrylamide with peptidoglycan, one should take into account the differences in the structure of cell wall stem peptides. The amino acid sequence of stem peptide involved in linking glycan chains in LAB peptidoglycan is L-Ala–D-Glu–X–D-Ala. The third amino acid (X) is a diamino acid, which in LAB usually is L-Lys (e.g., *L. lactis* and most lactobacilli), but can also be meso-diaminopimelic acid (mDAP) (e.g., in *L. plantarum*) or L-ornithine (e.g., in *L. fermentum*) [62]. Peptidoglycan with mDAP is typical for Gram-negative bacteria, and in such cell walls, a direct cross-connection between neighboring stem peptides takes place (the mDAP in position 3 of one peptide chain binds to D-Ala in position 4 of another chain). In lactic acid bacteria with Lys-type peptidoglycan, an additional interpeptide bridge made of one D-amino acid (e.g., D-Asp or D-Asn in *L. lactis*, *L. casei,* and most lactobacilli) is included [62]. It means that the structure of *L. plantarum* is unusual among LAB peptidoglycans, and it is different from the structure of other tested species. Additionally, this bacterium is characterized by a unique process among bacteria—O-acetylation of peptidoglycan [66]—which has an impact on *L. plantarum* autolysis. O-acetylation of N-acetylglucosamine (GlcNAc) inhibits the N-acetylglucosaminidase Acm2 (which is required for the ultimate step of cell separation of daughter cells), while O-acetylation of N-acetylmuramic acid (MurNAc) has been shown to activate autolysis through the activity of the N-acetylmuramoyl-L-alanine amidase LytH [68]. It is possible that acrylamide interacts with the mentioned enzymes (amidases) and hence influences cell division and separation. In our study, we observed that in the presence of AA, the *L. plantarum* morphology was changed, i.e., the percentage of cells in pairs or chains increased. It is worth mentioning that in *L. plantarum,* almost all the mDAP side chains are amidated. Defects of mDAP amidation in the *L. plantarum* mutant strain strongly affect the growth and cell morphology, causing filamentation and long-chain formation, suggesting that mDAP amidation may play a critical role in controlling the septation process [69]. Further studies are needed to explain whether acrylamide interacts with the amidation of mDAP or the activity of muralytic amidases. It is also possible that the presence of AA in low-carbon and low-nitrogen medium induces the synthesis of other amidases necessary for acrylamide degradation to acrylic acid and ammonia, but also able to cleave the amide bound in mDAP, influencing cell morphology.

The impact of acrylamide on LAB morphology should also be discussed in terms of the importance of bacterial aggregation on their functioning. First of all, bacterial aggregation (auto-aggregation) may facilitate biofilm formation by favoring bacterial attachment to surfaces or other microbes (co-aggregation). It also implicates better survival of LAB in the gut. Some studies indicate that biofilms are a stable point in a biological cycle that includes initiation, maturation, maintenance, and dissolution. According to O'Toole et al. [70], microbe development involves changes in form and function that play prominent roles in the life cycle of the organism, and biofilm formation is a prominent part of the lifestyle of microbes. Moreover, bacteria seem to initiate the development of biofilm in response to specific environmental conditions, such as nutrient availability. It has been proposed that the starvation response pathway can be subsumed as a part of the overall biofilm development cycle [70]. Secondly, when growing in biofilm, organisms become more resistant to higher deliverable

levels of antibiotics or other antimicrobial compounds compared to single "suspended" cells [71]. The last matter is that aggregation and co-aggregation among bacteria are important in the prevention of colonization of surfaces by pathogens. It has been proved that some lactic acid bacteria are also able to control biofilm formation by pathogens and can, therefore, prevent the colonization of food-borne pathogens [72]. It is true, for example, for some *Lactobacillus plantarum* strains showing an aggregation phenotype [73].

#### **5. Conclusions**

In conclusion, we can assume that the tested strains of lactic acid bacteria found in the human digestive tract or in fermented milk drinks are tolerant to high concentrations of acrylamide (up to 1 g/mL). Some show better growth in medium with AA than in medium with limited carbon and nitrogen sources, suggesting the possibility that they use AA for their own metabolism. Of course, in the digestive tract, especially in the initial sections of the intestine, there is sufficient availability of easily digestible food, but the possibility of using AA is beneficial for both the lactic acid bacteria and the human in whose intestine the LAB resides.

Moreover, we can assume that eating AA-containing products with a properly functioning microbiota will be less harmful to human organs than previously thought. It is also good information for producers of food (e.g., yogurt) with the addition of AA-containing ingredients, such as roasted coffee, almond or nuts, muesli, baked biscuits, or cornflakes because it should not negatively affect the microorganisms necessary for their production.

**Author Contributions:** Conceptualization, K.P. and A.D.-C.; methodology, K.P., T.T., and A.D.-C.; formal analysis, K.P., T.T., and A.D.-C.; investigation, K.P.; resources, A.D.-C.; writing—original draft preparation, K.P., T.T., and A.D.-C.; writing—review and editing, K.P. and A.D.-C.; visualization, K.P. and T.T.; supervision, A.D.-C.; project administration, A.D.-C.; funding acquisition, A.D.-C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by financial means on science in the years 2017–2020 as the research project 2016/21/B/BN9/01171 funded by the National Science Center (Krakow, Poland).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


73. García-Cayuela, T.; Korany, A.M.; Bustos, I.; Gómez de Cadiñanos, L.P.; Requena, T.; Peláez, C.; Martínez-Cuesta, M.C. Adhesion abilities of dairy *Lactobacillus plantarum* strains showing an aggregation phenotype. *Food Res. Int.* **2014**, *57*, 44–50. [CrossRef]

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
