The microbial safety and stability of minimally processed foods are based on the application of combined preservative factors. Since microorganisms are able to develop adaptive networks to survive under conditions of stress, food safety may be affected, and therefore understanding of stress adaptive mechanisms plays a key role in designing safe food processing conditions. In the present study, the viability and the sublethal injury of
Food manufactures and consumers demand additive-free, fresher, and full tasting food products while maintaining high standards of microbiological safety. The use of natural antimicrobial systems for preservation of foods could accomplish this demand. Although essential oil components are used as flavourings in the food industry, nowadays they represent a highly interesting source of natural antimicrobials for food preservation due to their antimicrobial and antioxidative activity [
As sublethal injury is supposed to be related to the higher sensitivity of survivors to stress conditions after treatment, the success of a combined treatment should be correlated with the degree of sublethal injury caused by the hurdles in the bacterial population [
The microbial safety and stability of most minimally processed foods are based on application of combined preservative factors of which (mild) heating is the most common preservation technique in use these days. Bacteria have evolved adaptive networks to face the challenges of changing environments and to survive under conditions of stress [
So far, the combined effect of carvacrol, thymol, and mild heat treatments on the viability, sublethal injury, and the protein expression profile of
Carvacrol (Fluka Chemie AG, Buchs, Switzerland) and thymol (Sigma Aldrich Chemie, Steinheim, Germany) stock solutions were held in 95% ethanol at 4°C.
Cultures in stationary phase, grown at 37°C, were harvested and concentrated by centrifugation (3500 ×g, 10 min, at 4°C) and resuspended in TSBYE. Treatments with only essential oil compounds were carried out through exposures of
To determine the loss of viability caused by a treatment, untreated and treated cell suspensions were serially diluted and plated on the surface of an appropriate count medium. TSAYE was used as nonselective agar medium in the enumeration of viable
Survival curves of
Since all survival curves had the same tail-shape, a single
Data were analyzed using Statgraphics Plus 5.1 software (Statistical Graphics Corp., Rockville, MD). One-way analysis of variance (ANOVA) for the parameters derived from the survival experiments was used to establish significant differences between the different treatments.
Total cellular protein extractions were performed as described by Wouters et al. [
2D-electrophoresis was performed, as described [
After electrophoresis, the gels were stained with MALDI TOF compatible silver nitrate using a Plus One Silver Staining Kit (Amersham Biosciences), as described by Shevchenko et al. [
Spots were manually excised from stained gels and sent for the digestion, the analysis by MS/MS, and the database searching to the Proteomics Lab of the Centro Nacional de Biotecnología (CNB-CSIC, Madrid, Spain). The digestion protocol used was based on Shevchenko et al. [
Recovery in TSAYE | Recovery in TSAYE-SC | |||||
---|---|---|---|---|---|---|
|
RMSE |
|
|
RMSE |
| |
Thymol | 15.58 | 0.226 | 0.893 | 11.02 | 0.259 | 0.901 |
Carvacrol | 12.79 | 0.249 | 0.898 | 11.07 | 0.252 | 0.904 |
Thymol + carvacrol | 8.74 | 0.334 | 0.878 | 3.76 | 0.393 | 0.907 |
55°C | 0.80 | 0.655 | 0.926 | 0.43 | 0.944 | 0.911 |
55°C + thymol | 0.25 | 0.276 | 0.993 | 0.16 | 0.447 | 0.988 |
55°C + carvacrol | 0.25 | 0.319 | 0.990 | 0.14 | 0.419 | 0.989 |
55°C + thymol + carvacrol | 0.18 | 0.332 | 0.992 | 0.10 | 0.813 | 0.966 |
Both thymol and carvacrol inactivated about one log cycle of the initial population of
Survival curves of
When both antimicrobials were added together, approximately additive results were reached, since one log cycle was inactivated within the first 10 min of exposition and a
Mild heat (55°C) was much more effective in inactivating
Combinations of heat with either thymol or carvacrol led again to synergistic results with
When antimicrobials and heat are combined, synergistic results are usually obtained [
All the treatments applied caused some injury in the population of
Percentage of injury in
Time (min) | 55°C | 55°C + thymol | 55°C + carvacrol | 55°C + thymol + carvacrol |
---|---|---|---|---|
5 | 76.27 ± 11.60aA | 92.76 ± 3.25aB | 98.19 ± 0.59aC | 99.53 ± 0.14aD |
10 | 79.78 ± 12.21aA | 97.25 ± 1.58bB | 99.16 ± 0.72abBC | 99.79 ± 0.06bC |
15 | 90.74 ± 2.43aA | 99.12 ± 0.36bB | 99.44 ± 0.48bBC | 99.82 ± 0.05bC |
20 | 97.33 ± 2.58bA | 99.24 ± 0.56bA | 99.75 ± 0.25bA | 99.89 ± 0.12bcA |
30 | 98.39 ± 0.69bA | 99.04 ± 1.02bAB | 99.87 ± 0.20bB | ≥99.99cB |
The synergistic effects of combined heat and antimicrobials have been explained in terms of inactivation of heat-injured cells when the antimicrobials are present in the heating medium [
However, in our case, when heat was combined with antimicrobials additional injured population was shown, as evidenced by recovery in presence of NaCl of the survivors of this combined treatments (Table
To gain an overview of the proteins induced upon mild heat treatment alone (55°C) and combined with essential oils compounds (55°C and carvacrol 0.3 mM and thymol 0.3 mM), 2D-electrophoresis was used. On gels containing cell-free extracts of control and treated/stressed
Induction factora of proteins of
Spot number | 55°C | 55°C + EOs |
---|---|---|
1 | 2.0 | 2.1 |
2 | −4.3 | −14.1 |
3 | −4.9 | −4.8 |
4 | 3.2 | 2.4 |
5 | −2.1 | −3.9 |
6 | 3.1 | 2.2 |
7 | 2.7 | 2.9 |
8 | 2.5 | 2.8 |
9 | −1.7 | −2.5 |
10 | 2.1 | 1.6 |
11 | −1.5 | −2.1 |
12 | −1.8 | −2.0 |
13 | 8.4 | 6.4 |
14 | 2.0 | 3.3 |
15 | 1.4 | 2.0 |
16 | 18.5 | 12.7 |
17 | 11.2 | 9.2 |
18 | 4.5 | 4.3 |
19 | 2.0 | 1.9 |
20 | 3.5 | 2.1 |
21 | −14.7 | −8.8 |
22 | −3.3 | 0.0 |
23 | 0.0 | −12.3 |
24 | 4.2 | 3.9 |
25 | 2.0 | 2.2 |
26 | 5.1 | 2.0 |
27 | 2.0 | 2.1 |
28 | 2.0 | 2.2 |
29 | 6.0 | 5.0 |
30 | 9.1 | 5.3 |
31 | −1.6 | −2.7 |
32 | 2.2 | 2.4 |
33 | −7.7 | −3.4 |
34 | 8.5 | 5.0 |
35 | 9.1 | 5.3 |
36 | 2.1 | 1.6 |
37 | 2.7 | 1.7 |
38 | 7.8 | 10.8 |
39 | 2.6 | 2.0 |
40 | 2.0 | 2.6 |
41 | 1.2 | 2.1 |
42 | 3.3 | 7.6 |
43 | −2.0 | −2.2 |
44 | 2.3 | 2.0 |
45 | 2.0 | 3.3 |
46 | −2.0 | −2.0 |
47 | 18.5 | 12.7 |
48 | 10.2 | 11.2 |
49 | −9.6 | −1.6 |
50 | −2.5 | −1.6 |
51 | 2.3 | 2.5 |
52 | −13.5 | −7.6 |
53 | 2.0 | 3.1 |
54 | 2.5 | 2.0 |
aNormalized value in treated gel/normalized value in control gel.
Identification of proteins upregulated in
Spot number | Protein identity | Accession number | Mascot score | Biological function |
---|---|---|---|---|
18 | Glyceraldehyde-3-phosphate dehydrogenase | gi/16804497 | 728 | Metabolic processes |
20 | 3-Bisphosphoglycerate-independent phosphoglycerate mutase | gi/441475375 |
141 | Metabolic processes |
24a | Translation elongation factor Ts | gi/47014632 | 262 | Protein synthesis |
24b | Molecular chaperone DnaK | gi/16803513 | 105 | Protein folding |
28 | Lactate dehydrogenase | gi/185497273 | 128 | Metabolic processes |
32 | Triosephosphate isomerase | gi/16804495 | 601 | Metabolic processes |
34 | Rod shape-determining protein MreB | gi/16803588 | 93 | Determination of bacterial cytoskeleton |
35 | PTS mannose transporter subunit IIAB | gi/16802144 | 117 | Regulation of metabolic and transcriptional processes |
37 | Cysteine synthase | gi/16802269 | 191 | Metabolic processes |
38 | ATP-dependent Clp protease proteolytic subunit | gi/16804506 | 187 | Protease activity |
41 | Transcription elongation factor GreA | gi/735685227 |
198 | Protein synthesis |
44 | Hypothetical protein lmo2511 | gi/16804549 | 345 | Stress response |
45 | 50 ribosomal protein L10 | gi/685938168 | 105 | Protein synthesis |
47a | Hypothetical protein lmo1580 | gi/16803620 | 204 | Stress response |
47b | Universal stress protein | gi/46907811 | 204 | Stress response |
51a | Regulatory protein SpoVG | gi/16802242 | 180 | Cell division |
51b | 50S ribosomal protein L7/L12 | gi/16802297 | 110 | Protein synthesis |
53a | Cochaperonin GroES | gi/16804108 | 116 | Protein folding |
53b | Major cold-shock protein homolog CspB | gi/1864167 | 105 | Stress response |
53c | 50S ribosomal protein L12 | gi/786164 | 58 | Protein synthesis |
2D-electrophoresis of extracts of stationary phase
Control
55°C
CTT
The results obtained in the current study revealed that
In bacterial cells heat causes damage to macromolecular cell components such as proteins and DNA. Carvacrol and thymol show the same mechanism of action due to their similar chemical structure. Both EOs seem to increase cytoplasmic membrane permeability [
In our study, the treatment applied induced the overexpression in
Cell metabolism is essential for energy generation, DNA replication, and cell division. Overexpression of proteins involved in energy metabolism could be an attempt to compensate for partially impaired energy generation caused by stressing treatments interacting with the bacterial cytoplasmic membrane. In fact, our results showed that stress treatments induced the expression in
In our study, an overproduction of proteins of stress response, like the major cold-shock-protein CspB, was observed when treatments were applied. The same protein was reported to have been induced in
The phenotypic and genotypic robustness of
Our results show that a combined treatment of moderate heat with antimicrobials (carvacrol and/or thymol) brings synergistic effects. Both heat and antimicrobials cause some degree of injury in the cells surviving moderate individual or combined treatments. Stress response in these injured cells involves overexpression of proteins implicated, among other functions, in stress response, metabolism, and protein refolding. This stress response could result in enhanced survival, compromising the safety of foods preserved by combined processes.
The authors declare that there is no conflict of interests regarding the publication of this paper.
This research was financially supported by the Ministry of Science and Technology of the Spanish Government and European Regional Development Fund (ERDF) through Projects AGL-2010-19775 and AGL2013-48993-C2-1-R.