The Degradation of Acrylamide by Microorganisms Present in Food Products

Acrylamide (AA) present in food is considered a harmful compound for humans, but it exerts impact on microorganisms too. The aim of the study was to evaluate the impact of AA (at conc. 0-10 µg/mL) on the growth of bacteria (Leuconostoc mesenteroides, Lactobacillus acidophilus LA-5) and yeasts (Saccharomyces cerevisiae, Kluyveromyces lactis var. lactis), which naturally occur in food products. Moreover, we decided to verify whether these microorganisms could decompose acrylamide. Our results proved that AA can stimulated the growth of L. acidophilus and K. lactis. We have also reported, to the best of our knowledge for the first time, that probiotic strain of bacteria L. acidophilus LA-5 is able to degrade AA by amidase production and hence can utilize AA as a source of carbon and nitrogen if they lack in the environment. The conducted Response Surface Methodology indicated that pH as well as incubation time and temperature significantly influenced the amount of ammonia released from acrylamide by the bacteria. Concluding, our studies suggest that some strains of bacteria present in milk fermented products can exert additional beneficial impact by acrylamide degradation and preventing against its harmful impact on human body and other members of intestinal microbiota.


Introduction
Changes in the standard of living, eating habits and the increased awareness of the impact of nutrition on the human health resulted in more and more consumers that pay attention not only to the nutritional value of food products. The consumers are increasingly convinced that food affects their health, enables the prolongation of life and well-being, and may also prevent non-transmissible chronic diseases [1]. Hence, each year we can find more types of the so-called functional food on the market. It is estimated that 60 % -80 % of functional products available on the market are probiotics [2]. Dairy products contain a variety of lactic acid bacteria (LAB) and yeasts, which are responsible for the organoleptic and health-promoting properties of these products. Among the products containing many microorganisms, first of all distinguish we can natural yoghurts, kefirs, ripening cheeses, fermented and acidophilic milk and koumiss. The microorganisms present in them are bacteria strains belonging mainly to the species Lactobacillus bulgaricus, Lactobacillus acidophilus, Bifidobacterium lactis, and Streptococcus thermophilus, and less often to other species e.g., Bifidobacterium longum, B. bifidum, B. breve, B. infantis, Lactobacillus casei, L. rhamnosus, L. acidophilus, L. lactis, L. gasseri, L. brevis, L. salivarius, L. paracasei, L. plantarum, and Propionibacterium freudenreichii. Fermented milk drinks also contain yeasts of various types and species, e.g., Kluyveromyces marxianus (Candida kefyr), K. lacits, Saccharomyces cerevisiae, S. exiguus, S. omnisporus [1].
It is known that the human gastrointestinal tract can be colonized by even 1200 various species of microorganisms, most residing in the colon, where they are detected most frequently and reach 10 12 bacteria per gram of intestine content [3]. The intestinal such as Escherichia coli, Enterococcus faecalis, Bacillus clausii, Helicobacter pylori [6] indicates the possible existence of an undiscovered mechanism of acrylamide degradation in human gut.
In our previous work we demonstrated that acrylamide could influence the viability of beneficial intestinal bacteria from Lactobacillus genus [6]. First of all we demonstrated that the tested lactic acid bacteria strains were tolerant to acrylamide even at high concentrations (up to 1 g/mL) and the growth of Lactobacillus plantarum, L. brevis, and Lactococcus lactis subsp. lactis, as well as probiotic strain Lactobacillus acidophilus LA-5, was more intense in the presence of AA at high concentration than in medium with limited accessibility of carbon and nitrogen compounds. It was concluded that acrylamide had no toxic impact on LAB, and that some strains probably could utilize acrylamide as a source of carbon and nitrogen if they lacked in the environment/medium. Moreover, the viability of bacteria was modulated by AA, the viability of probiotic strain L. acidophilus LA-5 increased, that of L. plantarum decreased, while L. brevis was the less sensitive.
Basing on the above, the aim of the present study was to check whether the microorganisms naturally occurring in milk products (lactic acid bacteria and yeasts), which growth was stimulated by acrylamide, are able to amidase production and acrylamide degradation.

Bacteria and Yeasts Preparation
All microorganisms were cultured 24 h before experiments. Yeasts were grown at 28 °C in Sabouraud broth supplemented with chloramphenicol (100 mg/L) in 100 mL flasks on the rotary shaker (Orbit 1000, Labnet International Inc., Edison, NJ, USA) at 120 rpm. Bacterial strains were grown in MRS broth at 37 °C (L. acidophilus LA-5, S. salivarius subsp. thermophilus) and 30 °C (L. mesenteroides subsp. cremoris). Just before experiments the cultures were centrifuged for 15 min at 194 × g (MPW-35JR centrifuge, MPW MED Instruments, Warsaw, Poland) and washed twice with sterile water to remove traces of growth media. The resulting pellets were resuspended in MRD so as to obtain the bacterial/fungal suspensions containing 10 7 cfu/mL. For that purpose, the optical density of microorganisms solution was measured by Den-1B densitometer (Biosan, Latvia) and expressed in McFarland units. The bacteria and yeast concentrations were calculated from the relationship established as described below.

Measurement of Optical Density of Bacterial Suspension: Calibration
To tubes containing 5 mL of sterile MRS medium, a volume of 0.1 mL of 24-h liquid bacterial culture was added, the contents were mixed, and the tubes were incubated for 24 h at the optimum temperature for the tested strain. After incubation, bacterial cultures were centrifuged at 194 × g for 15 min (MPW-35JR centrifuge, MPW MED Instruments, Warsaw, Poland), and the supernatant was discarded. The pellets were rinsed by mixing with 5 mL of sterile distilled water followed by centrifugation (parameters as above). The resulting pellets were resuspended in sterile water so as to obtain an optical density of the bacterial suspensions equal to McFarland standard 1.0 (using a Den-1B densitometer, Biosan, Latvia). Then, serial 10-fold dilutions were made in sterile water, and 1 mL of subsequent dilution was spread over the surface of the MRS medium (in triplicate). After 72 h of incubation at an optimal temperature, bacterial colonies were counted, and the mean bacterial cell density (expressed in cfu/mL) from 3 replicates was calculated for each tested strain. The relationship between the optical density of McFarland = 1 and bacterial cell density was determined. Exactly the same experiments were done for tested yeasts strains, except that Sabouraud broth and Sabouraud agar were used instead of MRS, and the incubation temperature was 28 °C.
The relationships obtained for individual strains were as follows (the optical density value that corresponds to the cell concentration of 10 7 cfu/mL): L. acidophilus LA-5 (0.22), L. mesenteroides (0.03), S. thermophilus (0.03), K. lactis (1.23) and S. cerevisiae (7.89). In the case of S. thermophilus and L. mesenteroides optical density was firstly adjusted to 0.3 and then diluted 10-fold.

The impact of AA concentration on microbial growth
The impact of acrylamide on tested microorganisms was determined in liquid cultures. Control samples were prepared by mixing 8 mL of MRD, 1 mL of microbial suspension (10 7 cfu/mL) and either 1 mL of sterile water (negative control, Cneg) or 1 mL of MRD (positive control, Cpos). Test samples contained 8 mL of MRD, 1 mL of microbial suspension (10 7 cfu/mL) and 1 mL of adequate AA "stock" solution so that the final concentration of acrylamide was 2.5, 5.0, 7.5 and 10.0 µg/mL. Therefore, it was possible to track how growing amount of carbon and nitrogen source influence the microbial growth (starting from the lower amount in negative control, through the samples with growing AA concentration up to positive control). Experimental variants were incubated at temperatures optimal for the growth of each tested strain, i.e. 28 °C (both yeast strains), 30 °C (L. mesenteroides), 37 °C (LA-5 and S. thermophilus). Separate sets of experimental variants were prepared for each time interval (0, 24 h and 48 h) for each microorganism. The number of microorganisms was enumerated by the spread plate method on MRS agar (for LAB) or YM agar (yeast), wherein 1 mL of 10-fold serially diluted samples were inoculated on the plates. Plates were incubated at optimal temperatures for 72 h and colonies were counted. The experiment was performed in 5 replicates.

Assessment of the Impact of pH and Temperature on Microbial Growth in the Presence of AA
Basing on the results of experiment described in section 2.5., where the presence of acrylamide seemed to stimulate the growth of L. acidophilus LA-5 (at 7.5 µg/mL) and K. lactis (at 10 µg/mL), these two microorganisms were selected for further studies. A set of tubes with MRD with appropriate AA concentration (causing the highest growth stimulation) was prepared and pH in the media was adjusted with 0.1 M HCl or 0.1 M Na2CO3 ranging from 2.0 to 9.0. Then 1 mL of microbial suspension (10 7 cfu/mL) prepared as described in section 2.3. was added to 9 mL of MRD with corrected pH, incubated and enumerated as above. Controls for each strain had constituted of 9 mL of MRD (without AA) adjusted to certain pH value inoculated with 1 mL of microbial suspension. They were prepared to exclude pH conditions that inhibited microbial growth. Then pH range 4-9 was selected for K. lactis and pH range 5-8 for L. acidophilus LA-5.
After selecting the optimum pH for microbial growth, each strain was incubated at such pH and at three different temperatures: 4 °C (corresponding to the cooling conditions), 20 °C (room temperature) and 37 °C (the temperature of human body, e.g., in the human intestine). Enumeration of microorganisms was carried out as above. Each experiment was performed in 5 replicates.

Assessment of the Microorganism's Ability to Acrylamide Degradation
On the basis of the above experiments the microorganism which growth was stimulated by acrylamide had been chosen to check whether they are able to degrade acrylamide by amidase production (the ammonia released during such reaction can be detected by commercial kit). The experiments were carried out in MRD with the addition of acrylamide at concentration of 7.5 µg/mL for L. acidophilus LA-5 and 10.0 µg/mL for K. lactis. Medium was inoculated with 1 mL of microbial suspension prepared from 24-h culture (the final amount of microorganisms was 10 6 cells/mL) and incubated for 48 h at 37 °C (bacteria) or 28 °C (yeast). After 24 h and 48 h of incubation the medium was collected and the ammonia concentration was determined spectrophotometrically at 340 nm using Ammonia Assay Kit (cat. AA0100, Sigma Aldrich, St. Louis, USA). Control samples contained 9 mL of MRD without acrylamide inoculated with 1 mL of the same microbial suspension as test sample. The ammonia concentration in test samples was compared to the control. Each experiment was performed in 5 replicates.

Response Surface Methodology (RSM)
The Response Surface Methodology (RSM) can be used to evaluate the relative significance of several factors in the presence of complex interactions. In our study, RSM was carried out according to the procedure described by Lenth [18] and it was applied to evaluate the effects of 3 various parameters on acrylamide degradation rate measured as the concentration of ammonia released by amidase. The experimental design was based on Box-Behnken design matrix. Seven experimental runs were carried out and the experimental design, variables, coded and decoded levels, and responses are listed in Table  3 (section 3. Results and Discussion). We considered 3 variables that proved to be significant for the growth of Lactobacillus acidophilus LA-5 which were: pH, time and temperature. The variable that was measured as the outcome was the concentration of ammonia (µg/mL). Obtained model was prepared according to the equation (1), which expresses the relationship between the predicted response and independent variables in coded values: where Y is the estimated response, β0 , βi , βii and βij are the regression coefficients for intercept, linearity, square and interaction, respectively, while Xi and Xj are the independent coded variables.

Basic Statistics and Principal Component Analysis (PCA)
All experiments described in the current paper were carried out in 5 replicates. The results are shown as arithmetic mean ± standard deviation (SD). The normality of distribution was assessed by Shapiro-Wilk test and one-way variance analysis (ANOVA) with Tukey's honest significant difference (HSD) posthoc test was used to compare mean values and determine the significance of differences. A p-value < 0.05 was considered statistically significant.
The Principal Component Analysis (PCA) with varimax rotation was applied to assess correlations among variables. All statistical analyses were carried out using R: A language and environment for statistical computing, version 3.5.0 (Foundation for Statistical Computing, Vienna, Austria, 2015). ANOVA was carried out using "lm" function and Tukey's test was done using "HSD.test" function in the "agricolae" package. PCA was carried out after each experiment for various acrylamide concentrations considering bacteria and yeast separately; pH and temperatures. We considered those variables as scores and numbers of microorganisms determined at particular time of collecting samples as loadings. The PCA was carried out in "psych" package [19]. Cortest-Bartlett test was carried out using "cortest.bartlett" function in "psych" package as well. The data demonstrated normal distribution so its transformation was not necessary. Strong correlations between loads and scores were considered when values obtained in correlation matrix exceeded 0.3 or -0.3. Based on the basic statistics and results obtained in the PCA we selected optimum conditions for continuing experiments.

The Impact of Various Acrylamide Concentrations on Microbial Growth
In the current paper we decided to verify whether yeast and/or bacteria, which naturally occur in food products could decompose acrylamide and therefore limits its negative impact on human body. Therefore we selected microorganisms which can naturally occur in fermented milk products (2 strains of LAB and 2 strains of yeasts) and one bacterial strain that demonstrate probiotic properties (L. acidophilus LA-5).
The lactic acid bacteria S. thermophilus did not growth in the MRD, the medium in which the carbon and nitrogen source are very limited (only 0.45% of bacteriological peptone), regardless of the absence or presence of AA. In turn, L. mesenteroides DSM 20343 showed weak growth in MRD, the number of cells decreased in all experimental variants (both with and without acrylamide) after 24 h and remained constant till 48 h ( Table 1). Although differences between negative and positive control were not statistically significant, it can be seen that additional 1 mL of MRD (in positive control) enhanced the L. mesenteroides growth, suggesting that bacteria were ready to quickly utilize any additional source of carbon and nitrogen. This also means that not only MRD did not provide enough nutrients for the basal metabolism of L. mesenteroides, but also that AA was not used as an additional carbon/nitrogen source by these bacteria. Contrary to L. mesenteroides, the number of L. acidophilus LA-5 cells was increasing with the concentration of acrylamide, reaching the maximum at 7.5 µg of AA/mL and it was significantly higher than all other tested variants, including positive control (Table 1). Therefore, L. acidophilus LA-5 has been chosen for further experiments.
When S. cerevisiae DSM 70478 was considered, it was shown that after 24 h yeast number was equal between controls and experimental variants. This suggests that the presence of AA did not affect S. cerevisiae growth and indicates that the tested yeast are not able to assimilate AA. Moreover, the number of yeast cells decreased drastically after 48 h in all tested variants (to values below 3 × 10 4 cfu/mL), indicating that the majority of nutrients present in MRD have already been exhausted (Table 1) and cells started to die. This would also mean that the MRD was the only source of available nutrients required for tested strain. On the contrary, the growth of other tested yeast, Kluyveromyces lactis var. lactis DSM 70799, was enhanced when AA concentration in culture equaled 10 µg/mL and reached the values similar to those of positive control. This suggests that AA was utilized by K. lactis as additional source of carbon and nitrogen, so this strains was selected for further tests too.  The same letters below the standard deviations within the results for particular microorganism indicate the lack of statistical differences at p < 0.05. Cpos -positive control (9 mL of Maximum Recovery Diluent inoculated with 1 mL microbial suspension), Cneg -negative control (8 mL of Maximum Recovery Diluent inoculated with 1 mL of microbial suspension and 1 mL of sterile water). For DSM 70478 the number of yeast colonies after 48 h was <30 on the plate or they were not detected at all. So the results are presented as < 3.0 ± 0.0 (the lowest sample dilution spread on the plate with YM medium was 10 -3 ).
It was only possible to carry out PCA for the results that were obtained in the experiment investigating the responses of both tested yeast strains to various AA concentrations. In all other tested cases, the results of Cortest-Bartlett test did not allow to continue PCA. Based on PCA, in the case of S. cerevisiae and K. lactis we observed negative correlation between the number of cells of those strains determined at time "0"and after 24 h, respectively ( Figure 1A, B and C). It took place because the number of cells remained constant within 24 h for S. cerevisiae, while it increased for the latter yeast. Another negative correlation occurred for K. lactis after at time "0"and 48 h. It is that initially the number of fungi was the same in all variants, while it varied after 48 h depending on the AA concentration -it was greatest in positive control (Cpos) and when AA concentration was 10 µg of AA/mL (Table 1). We also noted positive correlations between cells concentration (cfu/mL) after 24 h and 48 h for S. cerevisiae -the decrease took place regardless AA dose. Another negative correlation (1C) indicated that AA concentration of 5 µg/mL was not sufficient to sustain K. lactis growth after 48 h.

The Impact of pH and Temperature on Microbial Growth in the Presence of Acrylamide
Basing on the results from first experiment, for further analysis describing the impact of pH and temperature on the microorganisms growth in the presence of AA only Kluyveromyces lactis var. lactis and Lactobacillus acidophilus LA-5 were selected.
Firstly, we chose pH range that occurs within human digestive tract (2-9) and verified how it affects microbial growth without the addition of AA. We noted that selected bacterium strain was not affected at pH from 5 to 8, while the growth of fungal strain was unaffected from pH 4 to 9 (data not shown). Due to those findings we continued experiments by adding acrylamide to the MRD adjusted to particular pH values. The acrylamide concentration that was used for this experiment was selected based on the results presented above (Table 1) as the AA concentration that most strongly stimulated the growth of the test microorganisms, i.e. 7.5 µg/mL for L. acidophilus and 10 µg/mL for K. lactis.
We demonstrated that after 24 h at pH 4, 6 and 7 the presence of AA in medium did not affect yeast number, however, at pH 7 it was slightly higher (lack of statistical significance) in the variant that included AA than in control ( Table 2). The growth of yeast at pH 8 and 9 was significantly reduced when compared to conditions closer to optimal. In turn, at pH 4 the presence of AA allowed maintaining similar number of cells after 24 and 48 h, while in control sample the yeast concentration decreased significantly after 48 h. At pH 5, the addition of AA caused slower fungal growth after 24 h, but after 48 h there were no statistically significant differences between control and MRD with AA (Table 2). When the growth of probiotic L. acidophilus was analyzed, the greatest growth at pH 6 in the presence of AA was demonstrated, especially after 48 h ( Table 2). It must be highlighted that at all tested pH values the addition of acrylamide caused the increase of bacterial cells in comparison to controls. The same letters below the arithmetic means for each microorganism indicate the lack of statistical differences at p < 0.05. Abbreviations: AA -acrylamide present in medium at concentration of 10 µg/mL (yeast) or 7.5 µg/mL (bacteria); Control -control prepared by adding 1 mL of microbial suspension to 9 mL of MRD adjusted to adequate pH.; NA -not analyzed.
For determining the impact of temperature on the growth of tested microorganisms we chose pH 7 for K. lactis var. lactis and pH 6 for probiotic bacteria. Three different temperatures were analyzed: 4 ℃ (representing the temperature of the storage of fermented milk beverages), 20 °C (the mean room temperature) and 37 °C (the temperature of human body). We demonstrated that the most optimal temperature for microbial growth was 20 ℃ for yeast and 37 ℃ for bacterium (Table 3), however the differences between culture with and without acrylamide were not statistically significant, except for test carried out at 37 ℃ after 48 h (Table 3). In both cases the growth was better in the AA presence. Table 3. The impact of temperature on the growth of Kluyveromyces lactis var. lactis and Lactobacillus acidophilus LA-5 in the presence of acrylamide (AA) (n=5) from acrylamide by amidase) was slightly (but statistically significant) higher in medium inoculated with LA-5 (after 24 h) than in control medium without bacteria. In case of K. lactis var. lactis the ammonia concentration in samples with addition of 10 µg/l AA did not differ significantly from the control samples (data not shown).
There are several ways to reduce the acrylamide content in the food products. For example, the formation of acrylamide can be reduced by selecting the appropriate vegetable varieties (poor in AA precursors like asparagine and reducing sugars), by limiting the time and temperature during thermal processing, as well as by using the appropriate frying media or antioxidants [7,20]. It is also possible to use the lactic fermentation before frying, e.g., Lactobacillus plantarum fermentation reduced the simple sugars content in potato rods, which allow to decrease the AA formation in French fries [21]. It was also reported that application of selected strains of LAB or the usage of enzymes that catalyze deamination of asparagine and glutamine (L-asparaginase and L-glutaminase, respectively) allowed for a significant decrease of AA formation [22][23][24]. Esfahani et al. [25] proved that the reduction of acrylamide content of whole-wheat breads can be obtained by combining lactobacilli and yeast in sourdough fermentation, because those microorganisms use AA precursors as the source of carbon and nitrogen. It has also been reported that acrylamide can be decomposed under the influence of microbial amidases resulting in the release of ammonia and acrylic acid, which might be then transformed in various pathways to β-hydroxypropionate, propionate, lactate or CO2, however, the studies were focused on environmental bacteria species [26][27][28]. Figure 2. The ability to amidase synthesis by L. acidophilus LA-5 assessed by the measurement of ammonia concentration in medium without or with 7.5 µg/mL of acrylamide after 48 h of incubation with L. acidophilus LA-5 at 37 °C. The same letters next to means indicate the lack of statistical differences. Significance level: * p < 0.05, ** p < 0.01, and *** p < 0.001.
The ability to amidases synthesis has been already demonstrated in various bacteria, including LAB. For example, the synthesis of N-acetylmuramoyl-L-alanine amidase, involved in the degradation of peptidoglycan in the bacterial cell wall [29] was reported in Lactobacillus sakei, as well as the synthesis of N-acetylmuramidase with a key role in autolysis was demonstrated for Lactobacillus bulgaricus [30]. Amidases with activity of murein hydrolase are produced by staphylococci and are necessary for the generation of the equatorial ring on the cell surface and complete cell division and separation [31]. Similarly, the division of Escherichia coli depends on the activity of amidases AmiA, AmiB and AmiC [15]. However, there are no published papers demonstrating that amidases produced by lactic acid bacteria can utilize acrylamide as a substrate for deamination. Our results for the first time report that probiotic strain Lactobacillus acidophilus LA-5 has the ability to degrade acrylamide and then used the ammonia released by amidase as a source of nitrogen, while the rest of molecule (acrylic acid or its metabolites) as a carbon source.
Our studies revealed therefore that some LAB strains can exert beneficial impact on human organisms after intestine colonization, not only by their well-known impact on digestion and maintenance of proper gastrointestinal balance, but also by acrylamide degradation and preventing against its harmful impact on human body.

Response Surface Methodology
Since we demonstrated that only Lactobacillus acidophilus LA-5 was a putative amidase producer, that strain was selected for the experiments involving RSM. We considered variables that significantly influenced the growth of mentioned bacterium in the presence of acrylamide: pH (X1), temperature (X2) and time (X3). The response in the model was ammonia concentration (µg/mL) measured at particular experimental time. For the first run of experiments for central point we decided to apply the following values: pH 6.0, temperature 20 ℃ and time 24 h ( Table 4). As increments we selected the following values: 1 for pH, 17 for temperature and 24 for time. We demonstrated that only first order model explains investigated phenomenon with satisfactory reliability (R 2 = 0.9807 and adjusted R 2 = 0.9663). After the first and second run of experiments we have moved center points to: pH 6.78, temperature 33 °C, and time 48 h. This operation caused significant decrease in R 2 and adjusted R 2 (0.02244 and -0.7107, respectively for experimental run 3; 0.02441 and -0.3303, respectively for experimental run 4, Table 4), therefore we concluded that first order model could be reliable only in the range of pH 6 to 7, temperature 20 to 37 ℃ and time 24 to 48 h. When we tested if the data could fit to the second order model it was proved to fail, because R 2 and adjusted R 2 were too low (0.6914 and 0.4084, respectively for experimental run 5, Table 4). Those figures did not improve when we changed central points to: pH 6.67, temperature 34.7 ℃, and time 75 h.
At the end, the validation of the model built with RSM was carried out within pH 6 -7, temperature 20 to 37 °C and time 24 to 48 h (Table 5).

Conclusions
Acrylamide present in food product can exert both positive and negative impact on intestinal microbiota. Fortunately, some bacteria strains, including beneficial LAB, are resistant to the presence of this toxic substance, and what's more important, they can use AA as an additional source of carbon and nitrogen in a situation where the environment (medium) lacks them. To the best of our knowledge, we showed for the first time, that probiotic strain of bacteria Lactobacillus acidophilus LA-5, often added to yogurts by food producers, has the ability to degrade acrylamide and used the ammonia released by amidase as a source of nitrogen, while the rest of molecule (acrylic acid or its metabolites) as a carbon source. The analysis of variance and RSM indicated that pH as well as incubation time and temperature significantly influenced the amount of ammonia released from acrylamide by bacteria. Our studies suggest that some strains of bacteria present in milk fermented products can exert beneficial impact on human organisms after intestine colonization, not only by their well-known impact on digestion and maintenance of proper gastrointestinal balance, but also by acrylamide degradation and preventing against its harmful impact on human body and other members of intestinal microbiota.
Further studies are required to show whether the ability of acrylamide degradation is carried out by intestinal bacteria in the colon milieu, where the concentration of nutrients is high. Moreover, the impact of acrylamide on the metabolism pathways of lactic acid bacteria should be also checked.