3.2. Biopreservation of Chocolate Mousse: Determination of the Survival of Lactobacilli and Pathogenic Bacteria during Storage at Temperatures of 4 ± 2 °C and 20 ± 2 °C
In order to estimate the capacity of Lb. helveticus 2/20 cells to protect chocolate mousse from contamination by either S. aureus ATCC 25923 or E. coli ATCC 25922, MRS broth and chocolate mousse inoculated with either free or encapsulated Lb. helveticus 2/20 cells were artificially contaminated with either S. aureus ATCC 25923 or E. coli ATCC 25922 cells.
Monitoring of Lb. helveticus 2/20 and E. coli ATCC 25922 populations and of the total titratable acidity of chocolate mousse and MRS broth during storage at 4 ± 2 °C and 20 ± 2 °C.
The changes in the dimensionless biomass in the chocolate mousse with free or encapsulated
Lactobacillus helveticus 2/20 cells during storage at 4 ± 2 °C were similar to those in MRS broth at the same temperature (
Figure 2). In the case of the chocolate mousse, a slight increase in the biomass during the first 7 days of refrigerated storage was observed. Then, there was a slight decrease in the cell number or retained growth.The initial
Lb. helveticus 2/20 concentration in the chocolate mousse samples was 10
8 cfu/g. Interestingly, even after 21 days storage at 4 ± 2 °C, chocolate mousse always had a
Lb. helveticus 2/20 population exceeding 10
6 cfu/g, which is the minimal population of probiotic bacteria in fermented milk according to French regulation [
31].
The data presented in the figures show that MRS broth did not support the growth of both free and encapsulated lactobacilli and
E. coli ATCC 25922 under refrigerated storage conditions (4 ± 2 °C), likely due to the low temperature (
Figure 3). A gradual reduction in the number of LAB (inoculated as free or encapsulated cells) with comparable death rate constants—0.012 h
−1 for lactobacilli in the samples with free cells and 0.009 h
−1 for lactobacilli in the samples with encapsulated cells—was observed from the very beginning of the storage period (
Figure 3). A similar trend was observed in the control sample with
E. coli ATCC 25922, which was expressed in the gradual reduction in the pathogen cells from the very beginning of the storage period with a death rate constant of 0.025 h
−1, and the concentration of viable
E. coli ATCC 25922 cells decreased to about 10
7 cfu/mL at the end of the storage period, which significantly exceeded the minimum infectious concentration of active cells of the pathogen (10
5 cfu/mL). However, a concentration of active lactobacilli cells of about 10
7 cfu/mL for the samples inoculated with free cells and about 10
8 cfu/mL for the ones inoculated with encapsulated cells was maintained in the MRS broth medium up to the end of the storage period. The lower death rate and the higher concentration of active lactobacilli cells (by one logarithmic unit) in the samples inoculated with encapsulated cells was due to the protective effect of the immobilization matrix on LAB. A decrease in the concentration of active lactobacilli cells from the beginning of the process with the same death rate of 0.017 h
−1 in the mixed populations of free or encapsulated lactobacilli and
E. coli ATCC 25922 was observed, and the final concentration of active lactobacilli was about 10
7 cfu/mL. However, for
E. coli ATCC 25922 in the mixed populations with free or encapsulated lactobacilli, complete reduction in the active cells of the pathogen was observed with comparable death rates, 0.062 h
−1 for the mixture with free lactobacilli cells and 0.067 h
−1 for the mixture with encapsulated lactobacilli cells (
Figure 3). Other authors examined the antimicrobial activity of a number of LAB against
E.coli during storage at different temperatures including refrigerated storage. The present observations are consistent with Aguilar et al. (2010) and Fooladi et al. (2014) ones [
32,
33].
Slight growth in the chocolate mousse samples inoculated only with free or encapsulated lactobacilli cells and the control of
E. coli ATCC 25922 until the 7th day from the beginning of the storage under refrigerated conditions (4 ± 2 °C) was observed (
Figure 2). In the controls with only free or encapsulated lactobacilli, the maximum growth rates were comparable, 0.0033 h
−1 and 0.0038 h
−1, respectively, while for the control of
E. coli ATCC 25922, the maximum specific growth rate was 0.0034 h
−1. The maximum concentration of active cells of the lactobacilli controls achieved on the 7th day was comparable and was about 10
10 cfu/g, while that of the pathogen control was about 10
9 cfu/g. A reduction in lactobacilli and
E. coli ATCC 25922 cells in the controls after the 7th day was observed, and at the end of the storage period, the concentration of active lactobacilli cells in the chocolate mousse samples (inoculated with only free or encapsulated lactobacilli cells) and the
E. coli ATCC 25922 control was the same and was about 10
7 cfu/g. Once again, the concentration of active pathogen cells remained high and exceeded the minimum infective concentration. The death rate constants for the lactobacilli controls (inoculated with only free or encapsulated lactobacilli cells) were 0.039 h
−1 and 0.046 h
−1, respectively, and that of the pathogen was 0.038 h
−1. In the mixed populations of free or encapsulated lactobacilli and
E. coli ATCC 25922 in chocolate mousse (food matrix), there was also an increase in the active lactobacilli cells up to day 7 with the maximum specific growth rates being comparable to those of the respective controls—0.0030 h
−1 for the lactobacilli number in the samples inoculated with free lactobacilli cells and
E. coli ATCC 25922, and 0.0037 h
−1 for the lactobacilli number in the variant inoculated with encapsulated lactobacilli cells and
E. coli ATCC 25922. The achieved concentration of active lactobacilli in the mixed population was about 10
9 cfu/g. A reduction in the lactobacilli number in the mixed population after the 7th day of storage was observed, and this trend was similar to the controls, and at the end of the storage period, the concentration of free lactobacilli in the mixed population reached about 10
9 cfu/g, and that in the mousse inoculated with encapsulated lactobacilli cells was about 10
7 cfu/g. The death rate constants were 0.021 h
−1 and 0.035 h
−1, respectively. A reduction in viable pathogen cells from the very beginning of the storage period with a death rate constant of 0.062 h
−1 was observed in the mixed population of
E. coli ATCC 25922 and free lactobacilli cells. At the end of the storage period, 1 cfu/g was determined. In the mixed population of
E. coli ATCC 25922 and encapsulated lactobacilli cells, the concentration of active pathogen cells was maintained until the 7th day, after which their reduction with a death rate constant of 0.069 h
−1 began, the death rate constant being comparable to that in the presence of the free
Lb. helveticus 2/20 cells. Once again, 1 cfu/g of the pathogen was detected at the end of the storage process, which can be considered as complete elimination of the pathogen by LAB in the food matrix. In the mixed populations of
E. coli ATCC 25922 and
Lb. helveticus 2/20, it was observed that after 14 days of refrigerated storage, lactobacilli (inoculated as free or encapsulated cells) managed to reduce the population of active pathogen cells to values close to the minimum infectious concentration, about 10
6 cfu/g, after which almost complete inactivation of
E. coli ATCC 25922 by lactobacilli was observed (
Figure 2).
A continuous increase in the concentration of viable lactobacilli cells in the mixed population of free LAB and
E. coli ATCC 25922 in the chocolate mousse stored at 20 ± 2 °C was observed a concentration of active cells of about 10
10 cfu/g at the end of the storage period was measured (
Figure 4). Maximum concentration of lactobacilli in the mixed population of encapsulated LAB and
E. coli ATCC 25922 in chocolate mousse was reached after 5 days of storage and was about 10
9 cfu/g, and it remained constant until the end of the storage period. The maximum specific growth rates of lactobacilli, inoculated as free or encapsulated cells, in the mixed populations with the pathogen in MRS broth were slightly reduced and remained relatively high and close to those of the controls, 0.023 h
−1 and 0.021 h
−1, respectively. A reduction in active pathogen cells from the beginning of the storage period, with relatively high death rates of 0.216 h
−1 and 0.234 h
−1, respectively, in the mixed population of
E. coli ATCC 25922 and
Lb. helveticus 2/20 (inoculated as free or encapsulated cells) in chocolate mousse stored at 20 ± 2 °C, was observed (
Figure 4).
Unlike at 4 °C, a storage temperature of 20 ± 2 °C allowed the growth of microorganisms in MRS broth (
Figure 5). In the lactobacilli controls (inoculated only with free or encapsulated
Lb. helveticus 2/20 cells), there was a continuous increase in the active cells population in the food matrix with maximum specific growth rates of 0.026 h
−1 and 0.029 h
−1, respectively. Populations of active lactobacilli cells were about 10
9–10
10 cfu/g at the end of the storage period in both chocolate mousse controls inoculated with lactobacilli. An increase in the concentration of viable pathogen cells up to the 5th day from the beginning of the storage with a maximum growth rate of 0.051 h
−1 was observed following chocolate mousse contamination with
E. coli ATCC 25922 alone. The concentration of active cells of the pathogen reached about 10
12 cfu/g, after which it remained constant until the end of the storage period. Comparatively, the concentration of active lactobacilli cells in both lactobacilli MRS broth controls increased up to the 5th day of storage, with a maximum growth rate of 0.039 h
−1 and 0.026 h
−1, respectively (
Figure 5). These values were close to those of the respective chocolate mousse samples stored at 20 ± 2 °C, which showed that the mousse was a favorable growth environment and ensured the preservation of a high titer of beneficial probiotic microflora. An increase in the concentration of active pathogen cells up to the 5th day of storage at room temperature in the
E. coli ATCC 25922 control inoculated in MRS broth was also observed. It reached about 10
10 cfu/mL, after which this value remained constant until the end of the storage period. The maximum growth rate of the pathogen in MRS broth at 20 ± 2 °C was 0.053 h
−1, which was close to that obtained in the chocolate mousse stored at 20 ± 2 °C (0.051 h
−1) (
Figure 4). This confirmed the conclusion that chocolate mousse food matrix was a suitable environment for pathogen growth, which would lead to their rapid growth above minimum infectious concentration of
E. coli ATCC 25922, when stored at room temperature.
A similar trend was observed in the mixed populations of
Lb. helveticus 2/20 (inoculated as free or encapsulated cells) and
E. coli ATCC 25922 in MRS broth at 20 ± 2 °C: there was a continuous increase in the concentration of active lactobacilli cells, and the maximum specific growth rates were close to those of the respective lactobacilli controls, 0.033 h
−1 and 0.023 h
−1, respectively (
Figure 5). A reduction in the viable pathogen cells from the beginning of the storage period, with a relatively high death rate constant of 0.144 h
−1 in the mixed population of
E. coli ATCC 25922 and
Lb. helveticus 2/20 (inoculated as free or encapsulated cells) in MRS broth at storage temperature of 20 ± 2 °C, was observed (
Figure 5).
The possibility of predicting the final concentrations of active cells of LAB or pathogens in chocolate mousse at different storage temperatures in the range from 4 ± 2 °C to 20 ± 2 °C is quite an interesting opportunity. Indeed, predictive microbiology mathematical models allow us to predict the evolution of microorganisms in foods and are increasingly used to ensure the microbiological safety of foods in complementarity with existing quality assurance systems [
34]. For this purpose, the activation energy of growth of the lactobacilli in the mixed populations was calculated (according to equations 3 and 4) from the data on the maximum growth rates of lactobacilli, inoculated as free or encapsulated cells, and
E. coli ATCC 25922 in the chocolate mousse samples stored at both storage temperatures (
Table 3).
The activation energies of lactobacilli growth in the mixed populations with
E. coli ATCC 25922 were comparable to those of the lactobacilli controls, which showed that the presence of the pathogen and its metabolites did not lead to
Lb. helveticus 2/20 using additional energy for its growth and development, as well as for the processes related to its competition with the pathogen, i.e., competition for substrate consumption, synthesis of substances with antimicrobial action and its adaptation to the metabolites secreted by the pathogen (
Table 3). This characterizes
Lb. helveticus 2/20 as a microorganism with strong antimicrobial properties against the tested pathogenic strain
E. coli ATCC 25922. When the activation energy and the pre-exponential multiplier are known, the maximum specific growth rate of lactobacilli alone (in the chocolate mousse controls inoculated with free or encapsulated
Lb. helveticus 2/20 cells), of
E. coli ATCC 25922 in the pathogen control or of both microorganisms in the mixed populations in chocolate mousse at different temperatures in the range from 4 ± 2 °C to 20 ± 2 °C, can theoretically be calculated according to the following equation:
where
N0 is the initial biomass concentration, cfu/g or mL.
The pH of the chocolate mousse samples, stored under refrigeration conditions (4 ± 2 °C) and at room temperature (20 ± 2 °C) was also monitored (
Figure 6,
Figure 7,
Figure 8 and
Figure 9).
There was a slight change in the pH of the chocolate mousse samples during the storage period (
Figure 6 and
Figure 9). The change was less significant until the 7th day in the chocolate mousse samples with free
Lb. helveticus 2/20 cells stored at 4 ± 2 °C (
Figure 6), while in the samples stored at room temperature, the changes in the pH during the storage period were very significant, the reduction was 2 pH units (
Figure 8). LAB such as
Lb. helveticus are known to consume glucose and produce lactic acid when growing in glucose-containing media such as MRS broth [
14], while
E. coli has been reported to produce acetic acid in such media [
15]. This would explain the pH reduction during the first 7 days of storage of the chocolate mousse with encapsulated lactobacilli cells at 4 ± 2 °C (
Figure 6) and of the chocolate mousse with free or encapsulated lactobacilli cells at 20 ± 2 °C (
Figure 8). The titratable acidity of the complex nutrient medium varied in the range of 50 °T to 90 °T for all MRS broth samples stored under refrigeration conditions (
Figure 7). The intensive growth of the free or encapsulated cells of
Lb. helveticus 2/20 inhibited the pathogen growth from the very beginning of the co-cultivation, reducing the concentration of viable pathogenic cells below the threshold of detection of the method for enumeration (
Figure 5).
The data from the experimental studies unequivocally show that the chocolate mousse food matrix, inoculated with free or encapsulated
Lb. helveticus 2/20 cells, preserved both the viability of lactobacilli and the pathogenic microorganisms (
Figure 4). In the food matrix stored at 20 ± 2 °C, high concentrations of living cells of both the lactobacilli and the pathogen remained. The titratable acidity of MRS broth varied from 50 °T to 300 °T. Thus, the conditions for the growth of pathogenic bacteria were detrimental and likely contributed to the observed reduction in the viable pathogen counts (
Figure 9).
- B.
Biopreservation of chocolate mousse variants with Lb. helveticus 2/20. Monitoring of lactobacilli and S. aureus ATCC 25923 populations, pH, and titratable acidity of chocolate mousse and MRS broth during storage at 4 ± 2 °C and 20 ± 2 °C.
Similar studies were performed with the Gram-positive pathogen strain,
S. aureus ATCC 25923 (
Figure 10 and
Figure 11).
MRS broth at 4 ± 2 °C did not support the growth of
S. aureus ATCC 25923 or
Lb. helveticus 2/20 (
Figure 11). A gradual reduction in the concentration of viable
S. aureus ATCC 25923 cells with a death rate constant of 0.029 h
−1 from the beginning of the storage period in MRS broth at 4 ± 2 °C was observed; at the end of the storage period, the number of viable pathogen cells was about 10
4 cfu/mL. Complete reduction in the active cells of
S. aureus ATCC 25923 with death rate constants of 0.057 h
−1 and 0.055 h
−1, respectively, in its mixtures with free or encapsulated
Lb. helveticus 2/20 cells was observed. Reduction in
Lb. helveticus 2/20 counts in the mixed populations of the strain (inoculated as free or encapsulated cells) and
S. aureus ATCC 25923 in MRS broth at 4 ± 2 °C was established. The lactobacilli death rate constants were 0.01 h
−1 and 0.02 h
−1, respectively, their populations being about 10
6–10
8 cfu/mL at the end of the storage period (
Figure 11).
The chocolate mousse food matrix maintained the growth of
S. aureus ATCC 25923 and
Lb. helveticus 2/20 to some extent (
Figure 10). In the control sample inoculated with
S. aureus ATCC 25923 alone, an increase in the number of active cells to about 10
10 cfu/g by day 7 of the storage period, at a maximum growth rate of 0.0008 h
−1 was observed. Then, a reduction in the pathogen active cells followed with a death rate constant of 0.049 h
−1 and at the end of the storage period the concentration of pathogen living cells reached 10
5 cfu/g. The growth of the pathogen in the food matrix at storage temperature of 4 ± 2 °C during storage could have been associated, on one hand, with the synthesis of staphylococcal enterotoxins and on the other hand there was no complete reduction in
S. aureus ATCC 25923 cells, which were preserved above the level of minimum infectious concentration by the end of the storage period. A constant concentration of active cells of
S. aureus ATCC 25923 until the 7th day of storage in the mixed populations of the pathogen and
Lb. helveticus 2/20, inoculated as free or encapsulated cells, in the food matrix at 4 ± 2 °C was observed. Then, a reduction in the pathogen cells population with the same death rate constants (0.052 h
−1) for its both mixtures with lactobacilli (inoculated as free or encapsulated cells) began, and at the end of the storage period, no more viable cells of the pathogen were detected (
Figure 10).
There was a slight increase in and retention of a relatively constant population of active lactobacilli cells in the chocolate mousses samples inoculated with free or encapsulated cells until day 7, which was characterized by maximum specific growth rates (0.0004 h
−1) for the samples inoculated with free or encapsulated
Lb. helveticus 2/20 cells, in the mixed population of
Lb. helveticus 2/20 and
S. aureus ATCC 25923 in chocolate mousse (
Figure 10). This maximum specific growth rate constant characterized the equilibrium state between the newly formed cells and the dead cells in the population. The concentration of active lactobacilli cells in the mixed population was 10
10 cfu/g. A reduction in the lactobacilli number in the mixed population, with death rate constants of 0.028 h
−1 and 0.021 h
−1, respectively, for the chocolate mousse samples inoculated with free or encapsulated lactobacilli in their mixtures with
S. aureus ATCC 25923 after the 7th day of storage, was observed. At the end of the storage period, the lactobacilli concentration in the mixed population was about 10
7 cfu/g for the samples inoculated with free lactobacilli cells and about 10
8 cfu/g for the samples inoculated with encapsulated lactobacilli cells (
Figure 10).
In the chocolate mousse pathogen control stored at 20 ± 2 °C, there was an increase in the concentration of active cells with a maximum specific growth rate of 0.023 h
−1, and at the end of the storage period, a maximum concentration of active pathogen cells of 10
10 cfu/g was determined (
Figure 12). A steady decrease in the concentration of active pathogen cells with a death rate of 0.252 h
−1 was observed, and no viable
S. aureus cells were found at the end of the storage period in the mixed population of
S. aureus ATCC 25923 and free
Lb. helveticus 2/20 cells. While pathogen population remained constant up to the 3rd day of storage, a reduction in its population with a death rate of 0.248 h
−1 was then observed in the mixed population of the test microorganism and encapsulated
Lb. helveticus 2/20 cells in chocolate mousse at 20 ± 2 °C. The pathogen cell concentration at the end of the storage period was about 10
3 cfu/g, which was two logarithmic units lower than the minimum infective concentration. The slower reduction and the higher residual population of active cells of
S. aureus ATCC 25923 in its mixed population with encapsulated lactobacilli cells was likely due to the slower diffusion of antimicrobials through the immobilization matrix and probably the higher resistance of
S. aureus ATCC 25923 to substances with antimicrobial activity in comparison with
E. coli ATCC 25922.
Free and encapsulated LAB in a mixed population with
S. aureus ATCC 25923 retained their ability to grow in chocolate mousse at a storage temperature of 20 ± 2 °C with relatively high growth rates of 0.021 h
−1 for free cells and 0.023 h
−1 for encapsulated cells (
Figure 12). At the end of the storage period, the population of active LAB cells in the mixed populations with
S. aureus ATCC 25923 reached values of up to about 10
10 cfu/g. These high values of the final population of active lactobacilli cells indicated that they were not affected by the presence of
S. aureus ATCC 25923 (
Figure 12).
MRS broth supported the growth of the studied microorganisms at 20 ± 2 °C, unlike at 4 ± 2 °C (
Figure 11 and
Figure 13, respectively). In MRS broth at 20 ± 2 °C, there was an increase in the concentration of active
S. aureus cells with a relatively high maximum specific growth rate of 0.060 h
−1 until the 5th day of the storage period, and the population of active cells reached 10
11 cfu/mL, and it remained relatively constant until the end of storage (
Figure 13). In the mixed population of
S. aureus ATCC 25923 and free or encapsulated
Lb. helveticus 2/20 cells in MRS broth, there was a continuous decrease in the population of active pathogen cells from the very beginning of the storage with death rate constant values of 0.149 h
−1 and 0.154 h
−1, respectively, and no viable pathogen cells were detected at the end of the process (
Figure 13). The complete reduction in the active
S. aureus cells in the mixed population with encapsulated
Lb. helveticus 2/20 cells in contrast to that in the respective chocolate mousse was likely due to the lower viscosity of MRS broth, which is associated with better molecular and convective diffusion of the substances with antimicrobial action synthesized by
Lb. helveticus 2/20 in the medium. In addition, MRS broth has a lower buffering capacity than chocolate mousse, and accordingly,
S. aureus cells were exposed to a lower pH and thereby a higher antimicrobial effect of lactic acid produced (
Figure 11 and
Figure 13).
In the mixed population of free
Lb. helveticus 2/20 cells and
S. aureus ATCC 25923 in MRS broth at 20 ± 2 °C, an increase in the population of active lactobacilli cells up to day 5 with a maximum specific growth rate of 0.035 h
−1 was observed. It reached 10
10 cfu/mL and remained relatively constant until the end of the storage period (
Figure 13). In the mixed population of encapsulated
Lb. helveticus 2/20 cells and
S. aureus ATCC 25923 in MRS broth stored at 20 ± 2 °C, an increase in the number of active lactobacilli cells up to day 3 with a maximum specific growth rate of 0.026 h
−1 was observed, and the final population was 10
12 cfu/mL. A reduction in active cells population was then observed, but it remained higher than 10
11 cfu/mL until the end of the storage period (
Figure 13).
The activation energy of growth and the pre-exponential multiplier in the Arrhenius equation for
Lb. helveticus 2/20 and
S. aureus ATCC 25923 when cultured alone or in a mixed population in chocolate mousse were calculated (
Table 4).
The activation energies of growth and the pre-exponential multipliers for
Lb. helveticus 2/20 inoculated as free or encapsulated cells in its mixed population with
S. aureus ATTC 25923 were significantly higher than those of the respective lactobacilli controls (
Table 4). This indicated that the presence of
Lb. helveticus 2/20 and its metabolites had an effect on the lactobacilli themselves, and it was associated with
Lb. helveticus 2/20 conducting additional activity and spending more energy in order to grow and develop, and also to suppress the pathogen growth and to adapt to the metabolites secreted by
S. aureus ATCC 25923. Higher activation energy of growth for lactobacilli in the mixed population of encapsulated
Lb. helveticus 2/20 cells and
S. aureus ATCC 25923 (182.7 kJ/mol) than that in the mixed population of free
Lb. helveticus 2/20 cells and
S. aureus ATCC 25923 (171.7 kJ/mol) was observed (
Table 3). This was probably due to secreted metabolites by
S. aureus ATCC 25923 that pass through the immobilization matrix and concentrate in the microenvironment around the LAB, which is necessary in order to activate additional enzyme systems to eliminate their action, which in its turn is associated with more energy expenditure.
The activation energies of death of
S. aureus ATCC 25923 and
E. coli ATCC 25922 in the chocolate mousse food matrix as well as the pre-exponential multiplier in the Arrhenius equation were also calculated (
Table 5).
S. aureus ATCC 25923 was characterized by a slightly higher activation energy of death compared to E. coli ATCC 25922, which is consistent with the observation that S. aureus ATCC 25923 is more resistant to the presence of lactobacilli and their antimicrobial metabolites than E. coli ATCC 25922. This once again confirmed that Lb. helveticus 2/20 from the mixed population of Lb. helveticus 2/20 and S. aureus ATCC 25923 will need to spend more energy to completely reduce the amount of S. aureus ATCC 25923 cells, compared to the energy required for the complete reduction in E. coli ATCC 25922.
With these parameters, it is possible to determine the death rate and the time for complete reduction in pathogen cells at different storage temperatures ranging from 4 ± 2 °C to 20 ± 2 °C.
A pH change in the first days following the preparation of the chocolate mousse samples preserved with free or encapsulated
Lb. helveticus 2/20 cells, contaminated with
S. aureus ATCC 25923 and stored at 4 ± 2 °C, was observed (
Figure 14). During further storage, a relatively constant pH value was maintained until the end of the storage period. For the chocolate mousse samples containing only free or encapsulated
Lb. helveticus 2/20 cells (controls), the pH values were 5.99 and 6.3 and remained within these limits throughout the storage period. A slight increase in the pH was observed in the mixtures of free or encapsulated cells of
Lb. helveticus 2/20 and
S. aureus ATCC 25923. The pH of the chocolate mousse samples with free
Lb. helveticus 2/20 cells and
S. aureus ATCC 25923 increased from 6.0 to 6.3 during the storage period, and for the chocolate mousse samples with encapsulated
Lb. helveticus 2/20 cells and
S. aureus ATCC 25923, it increased from 5.9 to 6.2 (
Figure 14).
When the pathogen was co-cultivated with
Lb. helveticus 2/20, the change in the acidity of the medium was from 40 °T to 90 °T. This made the conditions for the growth of
S. aureus ATCC 25923 more detrimental. Together with the substances with antimicrobial action secreted in the medium by
Lb. helveticus 2/20 cells, this likely contributed to complete reduction in the living pathogen cells observed after 7 days (
Figure 15).
A decrease in chocolate mousse samples pH only with free or encapsulated
Lb. helveticus 2/20 cells, stored at a temperature of 20 ± 2 °C during the storage period, was observed (
Figure 16). The pH decreased from pH = 6 to pH = 4.2 in both samples. A similar pH decrease was observed in the chocolate mousse variants with a mixed population of free or encapsulated
Lb. helveticus 2/20 cells and
E. coli ATCC 25922 or
S. aureus ATCC 25923 (
Figure 16).
The curves reflecting the changes in the acidity of the complex nutrient medium (MRS broth) for the co-cultures of
Lb. helveticus 2/20 and
S. aureus ATCC 25923 at 20 ± 2 °C looked different (
Figure 17). The pathogenic staphylococci grew poorly in MRS broth. The slight change in the pH of the medium also supported the high concentration of viable
S. aureus ATCC 25923 cells. In the co-cultivation of the pathogen with
Lb. helveticus 2/20, the change in the acidity of the medium from 50 °T to 300 °T was significant. This made the conditions for the growth of
S. aureus ATCC 25923 more detrimental. Together with the substances with antimicrobial action secreted in the medium, this is likely the reason for the radical reduction in the living pathogen cells population (
Figure 17).
Sensorial analysis of both lactobacilli control samples (with free or encapsulated Lb. helveticus 2/20 cells) immediately after preparation has been performed, and it showed that there was no influence of the added probiotic strain on the taste, aroma and overall acceptance of the chocolate mousse variants by the consumers (unpublished data).