3.1. Thermal Inactivation of S. montevideo CICC21588 in Undiluted or Diluted Tahini
S. montevideo CICC21588 was selected to be further used in tahini samples due to its most desiccation and thermal resistance in undiluted tahini among four strains of serotypes according to results of pre-experiments (data not shown). The thermal inactivation curves of
S. montevideo CICC21588 in undiluted or diluted tahini with a heating rate of 5 °C/min in the HBS under different temperatures are shown in
Figure 2. D-, δ- and
p-values obtained from first-order kinetic and Weibull distribution models for describing the thermal inactivation behavior are listed in
Table 1. The larger coefficient of determination (
R2 = 0.973–0.996) and lower root mean square deviation (RMSE = 0.078–0.279) of the Weibull model compared to those (
R2 = 0.775–0.906, RMSE = 0.435–0.913) of the first-order kinetic model indicated that the Weibull model was more suitable to describe the thermal inactivation behavior of
S. montevideo CICC21588 in undiluted and diluted tahini. In undiluted tahini,
δ-values were 10.6, 5.21 and 3.84 at 75 °C, 85 °C and 95 °C, respectively. The high thermal resistance of
Salmonella was reported in undiluted sample due to its extremely low a
w (0.256) and high fat content (52.6%). He et al. [
29] also found minimum times to achieve 1-log reduction of
S. enterica serotypes in peanut butter (a
w 0.2, fat 49%) at 90 °C were 6.32–12.08 min based on the Weibull model. Krapf and Gantenbein-Demarchi [
30] studied the thermal inactivation of
Salmonella in dark chocolate (a
w 0.3–0.5, fat ≥ 18%) and found that the D-value at 90 °C was 25 min. In diluted tahini,
δ-values were 16.52, 4.38 and 0.92 at 56 °C, 58 °C and 60 °C, respectively.
δ-value decreased with the increase of temperature and a
w, similar to results obtained by Gautam, Govindan, Gänzle and Roopesh [
31].
In undiluted tahini (a
w = 0.256),
p-values of the Weibull model were less than one under three thermal treatments of 75 °C, 85 °C and 95 °C. The inactivation curves were concave-upward, as shown in
Figure 2a, in accordance with previous reports, such as thermal inactivation of
Salmonella in peanut butter [
32], black peppercorns, pecans, almonds [
33] and pet food pellets [
31]. According to these reports, thermal inactivation curves of pathogens in low moisture foods (LMFs) usually exhibit concave-upward, indicating that pathogens are inactivated directly without a large damage accumulation and the survival populations become increasingly resistant to thermal stress.
As shown in
Figure 2b, a shoulder was observed for the inactivation curves of
S. montevideo CICC21588 in diluted tahini by heating at 56 °C, 58 °C and 60 °C. The concave-downward thermal inactivation curves also appear in foods with high water content, such as
Salmonella inactivation in whole liquid egg [
34],
Escherichia coli O157:H7 inactivation in orange juice [
35] or apple juice [
28] and
Listeria monocytogenes inactivation in semi-skim milk [
36]. These results indicated that bacteria cells were firstly resistant to the sublethal temperatures but continuous heating made the injured cells difficult to survive.
3.2. The Sublethal Injured Cells in Thermal Treatments in Undiluted and Diluted Tahini
Figure 3 presents the levels of sublethal injured
S. montevideo CICC21588 cells in undiluted or diluted tahini by thermal treatments. For undiluted tahini, as shown in
Figure 3A–C, statistically significant differences of the population grown on the TSA, XLD and TSA-NaCl were only observed at the thermal treatments of 75 °C for 0 min and 95 °C for 0 min (
p < 0.05), indicating that cell membrane damages occurred under these treatments. However, the injured cell populations were less than 1 log CFU/g. The similar result was also obtained by Lee et al. [
37], who found that steam treatment (93 °C) inactivated
Salmonella enteritidis on the raw shelled almonds without causing significant sublethal injury.
For diluted tahini, a large number of injured cells were produced under the thermal treatments of 56 °C, 58 °C and 60 °C, as shown in
Figure 3a–c. The heat treatments of 56 °C for 18 min, 58 °C for 6 min and 60 °C for 1 min caused more than 3 log CFU/g injured cells. In addition, the lower population grown on the TSA-NaCl than on the XLD after three temperature treatments for each time indicated that the thermal stress caused more damage to cytoplasmic membrane than outer membrane of
S. montevideo CICC21588. It has been reported that a mild thermal stress (54–60 °C) causes a large number of injured cells of
Escherichia coli O157:H7 in fruit juice, however, more outer membrane damages are observed than cytoplasmic membrane damages, which is inconsistent with the results in this study possibly due to the different strain used [
28,
35].
That more sublethal injured cells caused by thermal stress in diluted tahini than in undiluted tahini corresponds to the thermal inactivation curves as described in
Section 3.1,
S. montevideo CICC21588 populations were inactivated directly by relatively high temperatures in undiluted tahini without causing significant injured cells and curves were concave-upward. While in diluted tahini, a damage accumulation was observed at the beginning of heating and the inactivation curves were concave-downward.
3.3. Inhibitory Activity against Salmonella of EOs, ε-PL, Tea Polyphenol or Organic Acid
Table 2 and
Table 3 show the inhibitory activity against
Salmonella of EOs, ε-PL, tea polyphenol and organic acid using the oxford cup assay. For EOs, as listed in
Table 2, tea tree, eucalyptus, peppermint, garlic and turmeric oils exhibited less inhibitory activity or even no effect against four
Salmonella serotypes compared to oregano, cinnamon and thyme oils. Furthermore, oregano oil exhibited the strongest antimicrobial activity with inhibition zones of 21.38–24.00 mm.
For water-soluble antimicrobial agents, as presented in
Table 3, ε-PL and tea polyphenol also had the inhibitory activity against
Salmonella with inhibition zones of 16.25–23.50 mm and 13.00–18.50 mm at the concentration of 10%, respectively.
Organic acids, such as citric acid and lactic acid, are widely used in the food industry to control pathogens or regulate acid flavor, generally regarded as safe [
38]. From
Table 3, citric acid and lactic acid both exhibited significant inhibitory effect against
Salmonella with inhibition zones of 25.75–27.70 mm and 24.00–27.20 mm at the concentration of 10%, respectively. The antimicrobials effect of lactic acid was slightly smaller than that of the citric acid. Therefore, oregano oil, ε-PL, tea polyphenol and CA were further selected to control
S. montevideo CICC21588 in undiluted and diluted tahini according to the inhibition zones.
3.4. Effect of Oregano Oil, ε-PL and CA on the Viability of S. Montevideo CICC21588 in Undiluted and Diluted Tahini
As shown in
Figure 4a,
S. montevideo CICC21588 population was significantly reduced by 3.61 log CFU/g in undiluted tahini with 3% oregano oil after 7 days at 25 °C, compared to 2.17 log CFU/g in the control (
p < 0.05). After 7 days at 4 °C, 1.53 and 1.47 log CFU/g reductions were observed in undiluted tahini with 1% and 3% oregano oil, respectively, compared to 0.67 log CFU/g in the control (
p < 0.05). This result indicated that 3% oregano oil could effectively inactivate
Salmonella in undiluted tahini without bringing water into samples, causing no influence on its shelf life. However, 3% oregano oil influenced the flavor of tahini and might not be accepted by consumers.
In addition, results showed that the
Salmonella population in undiluted tahini declined faster at higher temperatures during the storage period of 7 d, in accordance with previous research related to LMFs [
39]. Park, Oh and Kang [
40] found that the survival of
S. Tennessee cells in peanut butter was significantly lower at 22 °C, compared to 4 °C during the storage period of 14 d. The possible reason probably is that the metabolism of bacteria at the relatively low temperature is lower than that in the high temperature, which helps
Salmonella to maintain biological activity.
The addition of ε-PL and CA may bring water into undiluted tahini, which might accelerate the oxidative rancidity of fat, influencing its shelf life. Therefore, it is necessary to ensure the original concentrations of ε-PL and CA high. From
Figure 4c, 0.2–0.4% ε-PL had no effect on the
Salmonella reduction during the storage period of 7 days at 4 °C or 25 °C (
p > 0.05). A reduction of 1.42 log CFU/g was observed in undiluted tahini with 0.5% CA after 7 days at 4 °C, compared to the control of 0.73 log CFU/g (
p < 0.05). This result indicated that 0.5% CA only caused less than 1 log CFU/g extra reduction compared to the control after 7 days of storage period. Although Al-Nabulsi et al. [
41] have found more pronounced bactericidal effect against
Salmonella typhimurium in undiluted tahini by CA compared to our results, but the amount of water brought into tahini is not shown in their study, which would influence the bactericidal effect.
The inhibitory effects of oregano oil, ε-PL and CA against
S. montevideo CICC21588 in diluted tahini during the storage period of 7 days at 4 °C or 25 °C are shown in
Figure 5 and
Table 4. At 25 °C, the bacteria grown from 6.19 log CFU/g up to the maximum population of 9.30 log CFU/g at 24 h, then slightly decreased to 8.63 log CFU/g at 52 h and 8.70 log CFU/g at 72 h in diluted tahini without any antimicrobials, indicating that 50% (
w/w) diluted tahini provided a suitable environment for
Salmonella growth, posing a great threat to consumer health. After 7 days at 25 °C, samples without antimicrobials suffered deterioration caused by
Bacillus subtilis originally existed in tahini (characterized by 16S rRNA gene sequencing), influencing the
Salmonella enumeration. During the storage period of 7 days at 4 °C,
S. montevideo CICC21588 population remained stable, about 6.19–6.40 log CFU/g in diluted tahini without antimicrobials.
As shown in
Figure 5A and
Table 4, the addition of 2–3% oregano oil to diluted tahini slowed down the growth of
S. montevideo CICC21588 and slightly reduced the population at 4 h. 3% oregano oil could inhibit the growth up to 7 h. After 2 or 3 days at 25 °C, the population reached levels of 8.99 or 8.67 log CFU/g with 2% and 3% oregano oil, respectively. While after 7 days at 4 °C, 2–3% oregano oil significantly reduced
S. montevideo CICC 21588 population by 0.70–3.57 log CFU/g (
p < 0.05) as listed in
Table 4. Furthermore, diluted tahini samples with 1–3% oregano oil did not suffer deterioration, suggesting that oregano oil could inhibit the growth of
Bacillus subtilis.Results also showed that oregano oil was more effective in diluted tahini than in undiluted tahini at 4 °C. In undiluted tahini, 1.47 Log CFU/g reduction was observed with 3% oregano oil compared to 0.67 log CFU/g reduction in the control, while 3.57 log CFU/g reduction in diluted tahini compared to no reduction in the control after 7 days at 4 °C. Diluted tahini used in this study had less fat, more water content and better fluidity than undiluted tahini. It is reported that increased fat content has a negative effect on the antimicrobial efficacy of EOs in model foods, such as peanut paste [
42], soft cheese with different fat content [
43] or model media containing different concentrations of sunflower oil [
44]. In addition, water content is also a nonnegligible factor because water facilitates the movement of antimicrobial compounds to the target site in the bacterial cell [
42].
As shown in
Figure 5B and
Table 4, the antimicrobial activity of ε-PL in diluted tahini depended on its concentration at 25 °C. ε-PL inhibited the growth of
S. montevideo CICC21588 for 4 h with 0.05% ε-PL and for 72 h with 0.5% ε-PL. However, after 7 days at 25 °C, the populations reached the maximum numbers of approximately 9 log CFU/g. While at 4 °C,
S. montevideo CICC21588 population remained stable for 72 h, about 6 log CFU/g with the addition of 0.05–0.5% ε-PL (
Figure 5b) and after 7 days, the population was reduced to 4.59 and 3.68 log CFU/g with 0.4% and 0.5% ε-PL, respectively (
Table 4). Furthermore, diluted tahini samples with 0.05–0.5% ε-PL did not suffer deterioration, suggesting that ε-PL could effectively inhibit the growth of
Bacillus subtilis. Previous researches have reported the antimicrobial activity of ε-PL in various foods. The addition of 1.5% ε-PL extended lag time (11.61 h) of
S. typhimurium compared to the control (5.56 h) in pork at 24 °C [
45]. Chang et al. [
20] found that 0.1–1% ε-PL reduced the
S. typhimurium population by 0.68–4.93 log CFU/g in roast beef after 7 days at 4 °C. However, some researchers found that ε-PL could exhibit excellent effect at particularly low concentrations. Geornaras and Sofos [
46] found that 0.01% ε-PL showed pronounced effect against
S. typhimurium in TSAYE on day 6 at 4 °C. Furthermore, Geornaras, Yoon, Belk, Smith and Sofos [
47] also demonstrated that 0.04% ε-PL reduced the
S. typhimurium populations to low levels even below the detection limit in fat-free/whole fat milk, beef, bologna, ice and vegetables at 12 °C on day 6 and the antimicrobial activity was influenced by food composition.
As reported by previous studies, ε-PL has been widely used to control pathogens in high water foods but rarely in LMFs. In this study, ε-PL did not exhibit any antimicrobial activity in undiluted tahini (
Figure 4b), possibly due to the limited movement of antimicrobial compounds to the target site in the bacterial cell and the protective effect of high fat content [
20].
As shown in
Figure 5C and
Table 4, 0.05–0.5% CA concentration dependently slowed down the growth rate of
S. montevideo CICC21588 in diluted tahini at 25 °C within 24 h due to the decrease of pH value (pH 5.53-4.95). The population reached to similar levels of 8.85–9.06 log CFU/g at 52 h in the absence or presence of CA. However, after 7 d, samples with CA suffered deterioration and influenced the
Salmonella enumeration, indicating that 0.05–0.5% CA had no antibacterial effect on
Bacillus subtilis. While during the period of 7 days at 4 °C, 0.3% and 0.5% CA had no antimicrobial activity against
S. montevideo in diluted tahini and kept stable populations of about 6 log CFU/g (
Figure 5c,
Table 4). Osaili et al. [
48] also found that 0.4–0.8% CA had no significant effect on the
S. typhimurium in eggplant dip samples during storage of 15 days at 4, 10 and 21 °C. In tabbouleh salad, 1% CA exhibited no inhibition against
S. typhimurium at 21 °C but was effective at 4 °C or 10 °C after 7 days [
49].
In addition, CA exhibited immediate antimicrobial effect against
Salmonella in water or food surface possibly due to the lack of protective composition [
50,
51]. For example, 0.5% CA caused 1.26 log CFU/mL reduction of
Salmonella in washing water of lettuce residues within 15 min of contact [
52].
3.5. Synergistic Effects of Thermal Inactivation and Antimicrobials in Undiluted or Diluted Tahini
As shown in
Figure 6, oregano oil, ε-PL or CA concentration dependently raised the thermal (56 °C for 8 min) inactivation of
S. montevideo CICC 21588 in diluted tahini. However, the combined treatment of 0.05–0.4% tea polyphenols and heating at 56 °C for 8 min exhibited no synergistic effect on the
S. montevideo CICC 21588 inactivation (
p > 0.05).
Figure 7 shows the inactivation curves of
S. montevideo CICC21588 of heating combined with 1% oregano oil or 0.4% ε-PL or 0.3% CA.
Table 5 further shows the
δ- and
p-values of the Weibull model for thermal inactivation in combination with antimicrobials. The
δ-values at 56 °C combined with 1% oregano oil, 0.4% ε-PL or 0.3% CA were 3.98 min, 4.20 min and 4.92 min, respectively, while the
δ-value was 16.46 min by heating alone at 56 °C. Furthermore, the
δ-values at 58 °C combined with these antimicrobials were also significantly smaller than that of heating alone at 58 °C (
p < 0.05), indicating that oregano oil, ε-PL and CA all significantly reduced the thermal resistance of
S. montevideo CICC21588 in diluted tahini.
From
Table 6, no synergistic effect was observed at 75 °C with 2% oregano oil. The addition of water-soluble antimicrobials raised the a
w of tahini samples, which influenced the thermal resistance of
Salmonella [
31]. The a
w increased from 0.256 of original sample to 0.283 or 0.335 of samples with 0.3% CA or 0.4% ε-PL. Therefore, the a
w of control group samples was adjusted to the same with antimicrobials treated samples. Although the
δ-values at 75 °C with 0.3% CA or 0.4% ε-PL significantly decreased as compared to the respective control (
p < 0.05), significant differences between the population reductions were only observed at 75 °C for 20 min (
p < 0.05) and no significant difference at 75 °C for 40–100 min (
p > 0.05), indicating that the addition of CA and ε-PL in undiluted tahini slightly reduced the thermal resistance of
S. montevideo CICC21588 but the synergistic effects were far lower than those in diluted tahini samples.
The different synergistic effects obtained between undiluted and diluted tahini may be due to the lower sublethal injured cells caused by thermal treatments in undiluted tahini compared to that in diluted tahini (
Figure 3). Antimicrobials could inactivate the sublethal injured cells caused by heating more easily than intact cells through enhanced access to target cell structures [
28]. In addition, the synergistic effect may also be the result of the cumulative stresses occurring over a short period, causing multiple damages of bacteria cells so that losing the ability of the bacteria to recover [
23]. For example, EOs have been reported to inactivate pathogens through alternating the fatty acid profile of cell membrane, disrupting the cytoplasmic membrane, and reducing the proton-motive force (PMF) [
53]. ε-PL could also alter the integrity and permeability of cell membranes of
Escherichia coli O157:H7 [
54]. For organic acids, their undissociated form can easily pass through the bacterial membrane and dissociate, resulting in the reduced pH value of the internal cell, finally leading to the cell death [
55]. Furthermore, synergistic effects of heating and antimicrobials were widely observed in food surface, juice, milk, various meat products or other foods with high water content but rarely in oily, low a
w paste-like foods, such as tahini, peanut paste or nut paste, due to the limited effect in these foods. Espina et al. [
35] reported that the combination treatment of orange essential oil or (+)-limonene and mild heat had a synergistic bactericidal effect on
E. coli O157:H7 in orange juice. Sun-Ah et al. [
56] found that superheated steam combined with lactic acid could be used to inactivate food pathogens on cantaloupe surface. Oregano essential oil and CA also raised thermal sensitivity of
Listeria monocytogenes in sous-vide salmon [
57].