3.1. Thermal death parameters of LAB species in RSM emulsions.
The number of survivors during heat treatment at 62.5 ⁰C, in RSM emulsions and RSM emulsions + 0.2% E.C., for S. thermophilus, L. lactis subsp. lactis var. diacetylactis, L. rhamnosus and L. delbrueckii subsp. bulgaricus, are shown in Fig. 1. Decimal reduction times (D values) are shown in Fig. 2. S.thermophilus presented the highest thermal resistance, and allowed better observation of changes by E.C. effect, so further trials were focused on this species.
3.2. Thermal death parameters of S. thermophilus at different temperatures.
D and Z values corresponding to the linear section of the thermal death curves of S. thermophilus at 62.5 ⁰C, 65 ⁰C and 68 ⁰C for various emulsions are shown in Table 2. It was found that the thermal resistance of bacterial cells decreased in the absence of fat with the presence of E.C. In emulsions prepared with some type of fat, the bactericidal effect of the emulsifier decreased, regardless of the type of fat used. Furthermore, the addition of emulsifier also modified the shape of the thermal death curve, resulting in a combination of two straight lines represented by I (the linear section closest to time zero) and II, respectively. Analysis of variance showed that the D values obtained for the RSM emulsions were the highest; there was no significant difference between the D and Z values obtained for the RSM + 0.02% E.C.+ fat emulsions; and there was a significant difference in the D values, but not in the Z values for the RSM + 0.02% C.E. emulsions.
Table 2
D values at different temperatures for S. thermophilus in RSM emulsions with different lipid composition, pH 6.6. RSM. (Mean of quadruplicate essays).
Temp (⁰C)
|
Linear
|
RSM
|
RSM + CE
|
RSM + CE
|
RSM + CE
|
RSM + CE
|
RSM + CE
|
|
region
|
|
|
+PO
|
+MO
|
+AMF
|
HPKO
|
60
|
I
|
327.6
|
111.7
|
300.2
|
343.1
|
312.8
|
329.4
|
62.5
|
I
|
59.1
|
4
|
47.5
|
47.6
|
49.2
|
47.8
|
|
II
|
|
28.6
|
|
|
|
|
65
|
I
|
11.1
|
1.8
|
8.7
|
8.1
|
8.4
|
9
|
|
II
|
|
6.5
|
|
|
|
|
68
|
I
|
1.4
|
0.4
|
0.9
|
0.7
|
0.8
|
0.8
|
Z values
|
|
3.4
|
3.4
|
3.2
|
2.9
|
3.1
|
3.2
|
3.3. Influence of the bacterial strain used for the thermal experiments.
Table 3 shows the D values obtained during heat treatment in RSM emulsions of two different strains of S. thermophilus, codes TA054 and TA060. The analysis of variance showed that, for the same bacterial strain, there was not a significant difference in the D values obtained from different emulsions. However, strain TA060 presented higher thermal resistance than strain TA054. The results highlighted the fact that D values are specific to the bacterial strain used. Therefore, strain TA060 was used for the rest of the experiments.
Table 3. D values for S. thermophilus strains TA054 and TA060. (Mean of quadruplicate essays; s.d.=standard deviation)
Strain
|
Temp
(⁰C)
|
SMP + CE
+PO
|
s.d.
|
SMP + CE
+MO
|
s.d.
|
SMP + CE
+AMF
|
s.d.
|
SMP + CE
+HPKO
|
s.d.
|
TA054
|
62.5
|
11.1
|
⁺ 0.46
|
12.2
|
⁺ 0.93
|
11.5
|
⁺ 0.29
|
11.2
|
⁺ 0.36
|
TA054
|
65
|
3.8
|
⁺ 0.49
|
3.1
|
⁺ 0.27
|
3.2
|
⁺ 0.14
|
3.4
|
⁺ 0.10
|
TA060
|
62.5
|
47.5
|
⁺ 1.90
|
47.6
|
⁺ 2.22
|
49.2
|
⁺ 1.13
|
47.8
|
⁺ 1.01
|
TA060
|
65
|
8.7
|
⁺ 0.15
|
8.1
|
⁺ 0.30
|
8.4
|
⁺ 0.20
|
9.0
|
⁺ 0.21
|
3.4. Emulsions containing different monoacylglycerol concentration.
Figure 3A shows the thermal death curves of S. thermophilus at 62.5 ⁰C in RSM emulsions containing monomyristin (C14:0), monopalmitin (C16:0) and monostearin (C18:0) at the same concentration as found in C.E. All monoacylglycerol fractions affected the thermal resistance to some extent. At the concentration of monomyristin present in C.E. (0.0057%), only a slight bactericidal effect additional to that of temperature could be observed. Monopalmitin showed the greatest contribution to thermal inactivation, showing that at concentrations as low as 0.079% in the RSM emulsion, it was able to reduce the D value at 62.5 ⁰C from 59.1 min to 0.3 min. Monostearin also showed a bactericidal effect in combination with temperature, although it was significantly less than that achieved by monopalmitin.
The combined effect of monoacylglycerols on the thermal inactivation of S. thermophilus is shown in Fig. 3B. It was observed that the combination of monoacylglycerols did not increase the lethality of the process. On the contrary, the bactericidal effect was greater for pure monopalmitin than for the mixtures, indicating a possible competition in their mode of action. The thermal death curves of the treatments at different concentrations of monomyristin, monopalmitin and monostearin are shown in Fig. 4. The amount of monomyristin required to promote a significant change in the thermal resistance of the culture was much lower than that of monopalmitin or monostearin. The lethality curves indicated that a concentration as low as 0.013% of monomyristin could increase the lethality of the heat treatment by a factor of 2.
As for monopalmitin, it was necessary to have a concentration higher than 0.02% to have a similar effect. In the case of monostearin, the same concentration of 0.02% showed a smaller increase in heat treatment lethality. Analysis of variance showed that the presence of monomyristin at concentrations below 0.02% significantly increased heat treatment lethality progressively. Higher concentrations increased the bactericidal effect to the point where no survivors could be detected at any time. In the case of monopalmitin, concentrations lower than 0.04% increased lethality progressively, and maximum lethality was reached at a concentration of 0.04%. At higher concentrations, no further changes were observed. Finally, monostearin showed a much smaller effect on heat treatment lethality. Concentrations below 0.04% increased lethality faster than concentrations above this value.
The results confirmed that the shorter the monoacylglycerol chain, the better bactericidal effect can be observed. Undoubtedly, the bactericidal effect originally observed by the presence of E.C., attributed to monopalmitin, was due to the higher amount of this acylglycerol in the E.C.
3.5. Effect of the presence of protein on the bactericidal action of monoacylglycerols.
To determine whether the bactericidal activity exhibited by monoacylglycerols in RSM emulsions was affected by the presence of protein, thermal death curves of S. thermophilus in emulsions with peptonized water (PW), instead of RSM, were obtained for different concentrations of monoacylglycerols. The relationship between D values at 62.5 ⁰C and monoacylglycerol concentrations in PW emulsions is shown in Fig. 5. It could be observed that the required concentration of monoacylglycerols to observe the bactericidal effect was much lower compared to RSM emulsions. Traces of monomyristin (i.e., 0.001%) reduced the D value from 19.8 min to almost zero. For monopalmitin, the maximum bactericidal effect was reached at a concentration of 0.005%. Concentrations above this level did not significantly increase the lethality of the heat treatment. With respect to monostearin, the maximum bactericidal effect was reached at a concentration of 0.02% and the subsequent addition of monoacylglycerol did not increase the lethality of the process. These results confirmed the lower bactericidal effect of monostearin with respect to the other monoacylglycerols. On the other hand, monostearin showed a significantly greater bactericidal effect when used together with the heat treatment.
According to these observations, the concentration of monoacylglycerol required to optimize the heat treatment was higher in RSM emulsions than in PW emulsions, due to the presence of milk proteins, which probably reduced the interactions of monoacylglycerols with bacterial cells (Glass & Johnson, 2004). These findings contrast with those reported by Zhang et al. (2009) for monolaurin, who reported that the antibacterial efficacy of monolaurin remained unchanged in the presence of protein.
3.6 Effect of pH on the synergistic effect of monoacylglycerols with temperature.
The determination of thermal death parameters in PW emulsions was very useful because it allowed adjusting the pH to lower values that were not possible to reach in RSM emulsions without causing casein precipitation. Thus, thermal death curves of S. thermophilus in PW emulsions at pH 6.6 and 5.5 were obtained for two different temperatures.
The relationship between D values, pH and temperature is shown in Table 4. Analysis of variance showed that significantly lower D values were achieved at lower pH. In addition, there was a significant difference in D values for each temperature change.
Table 4
D values at different pH values for S. thermophilus in PW emulsions with different lipid composition, temperatures 60⁰C and 65⁰C. (Mean of quadruplicate essays)
pH
|
Temp
(⁰C)
|
PW
|
PW + CE
|
PW + CE
+PO
|
PW + CE
+MO
|
PW + CE
+AMF
|
PW + CE
+HPKO
|
|
60
|
75.8
|
26.6
|
60.4
|
46.9
|
62.4
|
58.0
|
6.6
|
62.5
|
23.8
|
4.8
|
11.5
|
8.8
|
11.2
|
11.8
|
|
65
|
4.1
|
1.5
|
2.6
|
2.3
|
2.8
|
2.9
|
|
68
|
0.5
|
0.3
|
0.4
|
0.4
|
0.4
|
0.4
|
Z value
|
|
3.6
|
4.2
|
3.7
|
3.9
|
3.7
|
3.7
|
R2
|
|
0.999
|
0.996
|
1.000
|
0.999
|
0.999
|
0.999
|
|
60
|
37.1
|
15.8
|
21.7
|
17.3
|
25.6
|
26.2
|
5.5
|
62.5
|
10.4
|
4.7
|
5.3
|
5.4
|
6.0
|
6.9
|
|
65
|
1.6
|
1.6
|
1.7
|
1.7
|
1.7
|
1.8
|
|
68
|
0.7
|
0.4
|
0.4
|
0.5
|
0.5
|
0.5
|
Z value
|
|
4.8
|
5.1
|
4.9
|
5.3
|
5.1
|
4.7
|
R2
|
|
0.926
|
1.000
|
0.999
|
0.998
|
1.000
|
1.000
|
The above results showed the synergistic effect of monoacylglycerols (monopalmitin, monostearin, and monomyristin) in any heat treatment. On the other hand, once the E.C. composition was known, it was corroborated that there were no changes in the heat resistance of the bacteria as a result of the presence of diacylglycerols or because of the presence of fatty acids.
The addition of anhydrous milk fat or any vegetable fat reduced the bactericidal effect of monoacylglycerols. At concentrations of 15% and 30% fat content, bacterial heat resistance increased significantly, which agrees with the protective effect of fats observed by other authors (Yang et al., 2020). All fats had the same protective effect in RSM emulsions.
Observations in RSM emulsions indicated that the bactericidal effect was greater for pure monoacylglycerols than for mixtures, indicating a possible competition in their mechanism of action. However, the monomyristin-monopalmitin-monostearin mixture showed a significant inhibitory effect in combination with heat treatment in RSM emulsions at concentrations as low as 0.01% for monomyristin, 0.02% for monopalmitin and 0.04% for monostearin. These findings agree with those reported by Zhang et al. (2018) for the combination of monomyristin and monolaurin, which caused cell lysis at high doses as a result of their combined action. Garcia et al. (2007) also reported the bactericidal action of monocaprylin in combination with acetic acid, for the inactivation of Listeria monocytogenes.
Derived from the results, it could be argued that the enhancing effect on thermal inactivation of bacteria was not caused by a heat-enhanced heat injury of bacterial cells, but by a heat-enhanced chemical inactivation. That is, chemical inactivation by potentiation of the interaction of monoacylglycerols with the bacterial cell wall and cell membrane seems to be the most likely mechanism. This seems to agree with that reported by Yoon et al. (2018), who pointed out that the lytic behavior of fatty acids and monoacylglycerols on the membrane derives from their amphipathic properties, which can lead to membrane destabilization and pore formation, with consequent inhibition of bacterial cell growth (bacteriostatic action) or cell death (bactericidal action). In addition, fatty acids have the potential to disrupt the electron transport chain by binding to electron transporters or altering membrane integrity, as well as interfering with oxidative phosphorylation by decreasing the membrane potential and proton gradient (Garret and Grisham, 2021).
Another possible mechanism is that the fatty acids that are part of monoacylglycerols can directly inhibit enzymes present in the membrane such as glucosyltransferase, presumably because their molecular structure is similar to that of fatty acids, so they could also associate with other proteins that are part of the membrane (Desbois et al., 2010). Finally, an additional mechanism derives from the fact that monoacylglycerols are surfactant compounds. In this regard, Furukawa et al. (2005) reported that the addition of a surfactant to bacterial suspensions prevents the formation of microbial clumps by heat treatment and increases the inactivation rate.