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Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. Bibliography

Aims: The aim of the study was to investigate the combined antimicrobial action of the plant-derived volatile carvacrol and high hydrostatic pressure (HHP).

Methods and Results: Combined treatments of carvacrol and HHP have been studied at different temperatures, using exponentially growing cells of Listeria monocytogenes, and showed a synergistic action. The antimicrobial effects were higher at 1°C than at 8 or 20°C. Furthermore, addition of carvacrol to cells exposed to sublethal HHP treatment caused similar reductions in viable numbers as simultaneous treatment with carvacrol and HHP. Synergism was also observed between carvacrol and HHP in semi-skimmed milk that was artificially contaminated with L. monocytogenes.

Conclusions: Carvacrol and HHP act synergistically and the antimicrobial effects of the combined treatment are greater at lower temperatures.

Significance and Impact of the Study: The study demonstrates the synergistic antimicrobial effect of essential oils in combination with HHP and indicates the potential of these combined treatments in food processing.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. Bibliography

The Gram-positive facultative anaerobic bacterium Listeria monocytogenes is an environmental pathogen responsible for the occurrence of listeriosis, a disease of humans and animals (Cox 1989). Listeria monocytogenes is found in many food products and has been associated with food-borne outbreaks and sporadic cases of listeriosis, mostly in North America and Europe (Cox 1989; Vasseur et al. 1999). It is regarded as a major problem because of the mortality of the disease it causes, particularly in the unborn child and neonates. Listeria monocytogenes is able to proliferate at refrigeration temperatures (Vasseur et al. 1999) and can survive mild preservation treatments. These features make it difficult to eliminate this micro-organism from foods.

Conventional thermal processing is a reliable method of reducing the viable numbers of L. monocytogenes in food. However, heat treatment often causes a reduction in the nutritional quality of food products. Therefore, novel mild preservation techniques and new combinations of preservative treatments have been introduced in food processing. One such food processing technique is high hydrostatic pressure (HHP) treatment. High hydrostatic pressure-treated foods have been commercially available since 1990 in Japan and since 1996 in Europe and the USA (Knorr et al. 1998). This technique offers major benefits to the food industry compared with heat treatment, because its adverse effects on product characteristics such as taste, flavour or vitamin content are reduced (Smelt 1998). Furthermore, as a result of the instant distribution of pressure, it is effective throughout the food product, independent of size and geometry (Smelt 1998).

The antimicrobial effect of HHP was demonstrated as early as the end of the 19th century in experiments performed by Hite (1899). The HHP treatment of micro-organisms leads to a number of changes in the cell. Pressures in the range of 20–50 MPa inhibit cell division more than cell growth, causing single cells to form long filaments (Zobell et al. 1963). Pressure affects motility (Kitching 1957) and, in Escherichia coli, it was demonstrated that DNA synthesis stops at around 50 MPa, protein synthesis around 58 MPa and RNA synthesis around 77 MPa (Yayanos and Pollard 1969). Relatively moderate pressures affect a variety of cellular processes and result mostly in sublethal injury of bacteria, whereas at higher pressures the cellular membrane appears to be the primary site of damage and a rapid increase in the death rate occurs (Morita 1975; Hoover et al. 1989; Kalchayanand et al. 1998). However, the exact mechanisms of cellular damage by HHP have not been elucidated and may be complex (Iwahashi et al. 1993; Rönner 1998).

Although HHP treatment can effectively reduce the viable numbers of bacteria in food products, it may adversely alter the texture and colour of certain foods (Cheftel 1995). The intensity of pressures required to inactivate micro-organisms might be reduced in the presence of antimicrobial compounds, since moderate pressurization or short exposures can cause sublethal injury to bacterial cells, making them more susceptible to antibacterial compounds such as plant-derived volatiles (Adegoke et al. 1997). A suitable compound for combined use with HHP is carvacrol, a plant-derived flavour compound known for its antimicrobial activity (Ultee et al. 1998; Smid and Gorris 1999). Its hydrophobic nature allows for accumulation in the bacterial cytoplasmic membrane, where it can elicit several toxic effects that may eventually lead to cell death (Sikkema et al. 1994; Ultee et al. 1999). In this study, the effects of a simultaneous combined treatment of carvacrol (2, 2·5 or 3 mmol l−1) and HHP (150, 200, 250 or 300 MPa) at 1, 8 or 20°C on the viability of exponentially grown cells of L. monocytogenes were investigated. In addition, the viability of this strain was evaluated on the application of pressure followed by carvacrol treatment. Finally, the most effective combined treatment was applied, using semi-skimmed milk artificially contaminated with L. monocytogenes, to study the loss of viability of this organism in a food matrix.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. Bibliography

Micro-organism and growth conditions

Listeria monocytogenes Scott A (Department of Food Science, Wageningen Agricultural University, The Netherlands) was used throughout this study. The stock culture was kept at −80°C in 15% (v/v) glycerol. The stock cultures were transferred to 9 ml sterile Brain Heart Infusion (BHI) broth (Oxoid, UK) and incubated at 30°C overnight. Subsequently, a 0·3% (v/v) inoculum of L. monocytogenes was added to 100 ml BHI broth. Cultures were then incubated in a shaking incubator (160 rev min−1) at 8°C and harvested at mid-exponential phase (O.D.660 0·1–0·4).

Carvacrol and high hydrostatic pressure treatment

Listeria monocytogenes was harvested at an O.D.660 of 0·1–0·4 by centrifugation (10 000 g, 10 min, 8°C). The cells were washed twice in 50 mmol l−1 N-[2-acetamido]-2-aminoethanesulphonic acid (ACES buffer; Sigma-Aldrich, Steinheim, Germany), pH 7·0. The pellet was resuspended in ACES buffer to an O.D.660 of 0·1 and 10-ml aliquots transferred into sterile plastic tubes (Greiner, Kremsmünster, Austria). Exposure of cells to HHP was performed in ACES buffer in order to maintain the pH at 7·0 during HHP treatment (Smelt and Hellemons 1998). Carvacrol (Fluka Chemie AG, Buchs, Switzerland) was added to the cell suspensions to final concentrations of 2, 2·5 or 3 mmol l−1 using a carvacrol stock solution of 1 mol l−1 in ethanol. In control experiments, equivalent amounts of ethanol [maximum 0·1% (v/v)] were added to cells. No adverse effects of ethanol on the survival of L. monocytogenes were observed. Suspensions were placed in sterile plastic stomacher bags (Seward, London, UK) that were sealed while avoiding excess of air bubbles. These pouches were submerged in glycol, which acted as the hydrostatic fluid medium in the press (Resato, Roden, The Netherlands). Subsequently, cell suspensions were exposed to pressures of 150, 200, 250 or 300 MPa in a High Pressure unit (Resato) at 1, 8 or 20°C for 20 min. The viability of L. monocytogenes was determined before and after pressure treatment. Decimal dilutions of samples were prepared in peptone-physiological salt (1·5 g l−1 peptone and 8·5 g l−1 NaCl) and plated in triplicate onto BHI agar (1·2% w/v agar). Plates were incubated at 30°C for 5 d.

Application of high hydrostatic pressure followed by addition of carvacrol

In accordance with the above protocol, cell suspensions were placed in plastic pouches in the absence or presence of carvacrol (2 mmol l−1) and subjected to a pressure treatment of 200 MPa at 20°C for 20 min. After the pressure treatment, 2 mmol l−1 carvacrol was added to cell suspensions that were pressurized in the absence of carvacrol. After 20 min, decimal dilutions of all samples were made in peptone buffer and the viability of L. monocytogenes determined.

Effect of combined treatment on the viability of Listeria monocytogenes in semi-skimmed milk

Cells were grown and harvested as described in the above protocol, suspended in semi-skimmed milk (Coberco, Arnhem, The Netherlands) and left for 15 min to adjust to the new medium before the addition of 3 mmol l−1 carvacrol. Viable numbers of cells were determined and the suspensions transferred to pouches which were sealed and pressurized at 300 MPa for 20 min at 1°C, representing the most effective inactivation treatment in ACES buffer. Subsequently, viabilities were determined in pressurized or non-pressurized suspensions in the presence or absence of carvacrol. Samples were plated in triplicate and incubated at 30°C for 5 d.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. Bibliography

Combined effect of high hydrostatic pressure and carvacrol on viability of Listeria monocytogenes

The reductions in viable numbers [log (initial cfu ml−1) − log (final cfu ml−1)] of mid-exponential phase cells of L. monocytogenes grown at 8°C were assessed using different combinations of pressure treatments (0, 150, 200, 250 or 300 MPa) and concentrations of carvacrol (0, 2, 2·5 or 3 mmol l−1). Pressurization was performed at 1, 8 or 20°C.

The reductions in viable numbers of L. monocytogenes after a 150-MPa pressure treatment in the absence of carvacrol were only 0·2 and 0·3 log units on pressurization at 1 and 20°C, respectively (Figs 1 and 2). At increasing pressures, the reductions were more substantial, indicating a dose–effect correlation: at 300 MPa, 2 log unit reductions in viable counts were observed at 1 and 20°C. Overall, pressurization at 1°C (Fig. 1) was slightly more effective in reducing the viable counts of L. monocytogenes than that at 20°C (Fig. 2) or 8°C (similar to effects at 20°C; data not shown).

image

Figure 1.  Combined effect of different concentrations of carvacrol (0, 2, 2·5 and 3 mmol l−1) and pressures (0, 150, 200, 250 and 300 MPa) at 1 °C on exponentially growing cells of Listeria monocytogenes. Cells were grown in brain heart infusion broth at 8 °C with shaking (160 rev min−1), harvested and maintained in N-[2-acetamido]-2-aminoethanesulphonic acid buffer, pH 7·0. The detection limit was a 6 log reduction in the viable counts. Experiments were performed in duplicate and error bars represent the S.D.

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image

Figure 2.  Combined effect of different concentrations of carvacrol (0, 2, 2·5 and 3 mmol l−1) and pressures (0, 150, 200, 250 and 300 MPa) at 20 °C on exponentially growing cells of Listeria monocytogenes grown in brain heart infusion broth at 8 °C with shaking (160 rev min−1), harvested and maintained in N-[2-acetamido]-2-aminoethanesulphonic acid buffer, pH 7·0. Experiments were performed in duplicate and error bars represent the S.D.

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The antimicrobial effect of carvacrol on L. monocytogenes was higher at increasing carvacrol concentrations. Reductions in viable numbers increased from 0·2 to 0·6 log units, using 2 or 3 mmol l−1 carvacrol at 1°C, respectively (Fig. 1). At 20°C, carvacrol treatment was more effective; the reductions in viable numbers increased from 0·3 to 1·8 log units at 2 or 3 mmol l−1 carvacrol, respectively (Fig. 2). Results at 8°C (data not shown) were similar to those obtained at 20°C (Fig. 2).

Combined treatments with carvacrol and HHP resulted in significantly greater reductions in viable counts of L. monocytogenes compared with the reductions caused by carvacrol or HHP alone (Figs 1 and 2). At 1°C, more than 5 log reductions in viable counts were realized by applying pressures of 250 MPa in combination with 2·5 or 3 mmol l−1 carvacrol or 300 MPa in combination with 2, 2·5 or 3 mmol l−1 carvacrol. At 20°C, similar effects were observed, although less pronounced; the reductions in viable numbers varied from 3·5 to 5 log units for the above-mentioned treatments. Overall, the applied temperature during pressurization in the presence of carvacrol was an important factor, the reductions in viable numbers of L. monocytogenes on combined treatment being higher at 1°C (Fig. 1) than at 20°C (Fig. 2) or 8°C (data not shown).

In additional experiments, the combined action of HHP and the plant-derived antimicrobial compound thymol against L. monocytogenes was investigated. Pressure treatments of 150, 200, 250 or 300 MPa at 1, 8 or 20°C and thymol concentrations of 2, 2·5, or 3 mmol l−1 were applied (data not shown). Similarly to carvacrol, thymol was more effective in reducing the viable numbers of L. monocytogenes at increasing concentrations. Combined treatments of thymol and high pressures were more effective than applying thymol or pressure treatments alone and reductions in the viable counts were highest at 1°C (data not shown).

Effect of addition of carvacrol after high hydrostatic pressure treatment

To investigate whether the synergistic effect of the simultaneous application of carvacrol and HHP could also be achieved by HHP treatment followed by the addition of carvacrol, mid-exponential phase cells of L. monocytogenes were suspended in ACES buffer (pH 7·0), first subjected to 200 MPa for 20 min at 20°C and subsequently to 2 mmol l−1 carvacrol for 20 min. This combined treatment resulted in an approximate 1 log reduction in viable numbers of L. monocytogenes (Fig. 3), which was not significantly different from the reduction after simultaneous application of 200 MPa for 20 min at 20°C in the presence of 2 mmol l−1 carvacrol (0·9 log) (Fig. 3). Separate treatments had no effect on the viability of cells (Fig. 3).

image

Figure 3.  Log reduction in the viable counts of Listeria monocytogenes after treatment with (1) high pressure at 200 MPa; (2) 2 mmol l−1 carvacrol; (3) a combination of high pressure (200 MPa) and carvacrol (2 mmol l−1) and (4) 2 mmol l−1 carvacrol after high pressure treatment of 200 MPa. All treatments were performed for 20 min at 20 °C in N-[2-acetamido]-2-aminoethanesulphonic acid buffer, pH 7·0. Values are means of triplicate measurements. Bars represent the S.D. (n=3)

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Effectiveness of combined pressure treatment with carvacrol in a food matrix

Mid-exponential phase cells of L. monocytogenes grown at 8°C were suspended in milk as a model food substrate and subjected to separate or combined treatments with carvacrol (3 mmol l−1) or pressure (300 MPa for 20 min at 1°C). The application of 3 mmol l−1 carvacrol did not reduce the viable numbers of L. monocytogenes, while a 2·3 log reduction in viable counts was observed after the pressure treatment alone (Fig. 4). Combined treatment of pressure and carvacrol was most effective and resulted in a 3·2 log reduction in the viable counts. In the absence of any treatment, no increase in the viable numbers of L. monocytogenes in milk, at 20°C for 20 min, was observed.

image

Figure 4.  Log reduction in the viable counts of Listeria monocytogenes after treatment with (1) 3 mmol l−1 carvacrol; (2) no treatment; (3) high pressure at 300 MPa and (4) a combination of high pressure (300 MPa) and carvacrol (3 mmol l−1). Treatments were applied in semi-skimmed milk at 1 °C for 20 min. Values are means of triplicate measurements. Bars represent the S.D. (n=3)

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. Bibliography

In the present study, we demonstrated that the viable numbers of L. monocytogenes are significantly more reduced by a combined treatment of HHP and carvacrol than by separate treatments with HHP or carvacrol. Mid-exponentially grown cells of L. monocytogenes were subjected to different combinations of high pressures and carvacrol concentrations and pressurization was performed at 1, 8 or 20°C. At all of these temperatures, the reductions in viable counts of L. monocytogenes after combined treatments were significantly higher than the sum of the reductions caused by the individual treatments, suggesting a synergistic effect. The cellular targets at which carvacrol and high pressures act may account for this synergism. Carvacrol is a lipophilic agent that is believed to preferentially insert into the cytoplasmic membrane (Sikkema et al. 1994). In the Gram-positive bacterium Bacillus cereus, carvacrol causes increased permeability of the membrane for cations such as H+ and K+ (Ultee et al. 1999). The dissipation of the ion gradients leads to impairment of essential processes in the cell and finally to cell death (Sikkema et al. 1994; Ultee et al. 1999). High hydrostatic pressure treatment is also believed to cause damage to the cell membrane (Morita 1975). This is probably related to the increase in the melting temperature of lipids (triglycerides) by more than 10°C per 100 MPa. Thus, membrane lipids present in a liquid state at room temperature will crystallize under high pressure, resulting in changes in the structure and permeability of the cell membrane (Cheftel 1995). The observed synergistic effect may, therefore, be related to this common cellular target.

High hydrostatic pressure treatment in the absence of carvacrol, followed by exposure to carvacrol, caused reductions in viable numbers of L. monocytogenes similar to a simultaneous combined treatment. These findings suggest that HHP treatment alone causes a sublethal injury to part of the L. monocytogenes population. These cells can recover on non-selective media (Kalchayanand et al. 1998); however, the addition of carvacrol to these injured cells (at concentrations that are not lethal to untreated cells) do not allow for recovery and cause cell death. Similarly, it was demonstrated that combined treatments of L. monocytogenes with thymol and HHP effectively reduce the viable numbers of this organism in a synergistic way (data not shown). In line with our observations, Adegoke et al. (1997) have previously shown that the plant volatiles α-terpinene and (R)-(+)-limonene enhanced the antimicrobial effects of HHP by preventing the recovery of pressure-injured Saccharomyces cerevisiae cells.

The loss of viability of L. monocytogenes due to combined treatment with carvacrol (or thymol) and HHP was higher at 1°C than at 8 or 20°C. It has been reported that the pressure resistance of micro-organisms is highest at pressurization temperatures of 15–30°C and decreases significantly at higher or lower temperatures (Arroyo et al. 1999). The decreased resistance at high or low pressurization temperatures may be due to changes in the membrane structure and fluidity, through weakening of hydrophobic interactions and crystallization of phospholipids (Cheftel 1995). In our study, higher reductions in viable numbers of L. monocytogenes were observed after combined treatment at 1 than at 20°C. The observation of MacDonald (1992) that less fluid membranes are more sensitive to HHP might explain the observed effects on L. monocytogenes cultured at 8°C, since the membrane fluidity of these cells is lower at 1 than at 20°C.

The synergistic effect of carvacrol and HHP also occurred in milk, showing that the combined treatment is effective in a model food system. However, the effect of carvacrol combined with HHP was at least two orders of magnitude lower in milk (3·2 log reduction) compared with buffer (> 6 log reduction). These results are in agreement with reports that the effectiveness of the essential oils can be decreased in foods due to the presence of components, such as proteins and fats, which immobilize and inactivate the essential oil components (Smid and Gorris 1999). Separate pressure treatments (300 MPa) in milk or buffer were equally effective in reducing the viability of L. monocytogenes. We did not observe a protective effect of milk against HHP compared with buffer, as has previously been reported by Styles et al. (1991). This discrepancy might be related to the lower fat content in the semi-skimmed milk that was used in our study. In short, this study shows that plant-derived volatiles, such as carvacrol and thymol, can be effectively employed in food preservation, especially in combination with other mild treatments such as HHP. These essential oil compounds can play an important role in minimally processed foods, by reducing the intensity of HHP treatment (or of other treatments) enabling its economically attractive exploitation.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. Bibliography

A.K.K. was financially supported by the State Scholarships Foundation of Greece (IKY). The technical assistance of Paul G.M. Teunissen is greatly appreciated.

Footnotes
  1. Present address: NIZO Food Research, PO Box 20, 6701 BA Ede, The Netherlands

  2. Present address: NV Organon, PO Box 20, 5340 BH Oss, The Netherlands.

Bibliography

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. Bibliography
  • 1
    Adegoke, G.O., Iwahashi, H. & Komatsu, Y. (1997) Inhibition of Saccharomyces cerevisiae by combination of hydrostatic pressure and monoterpenes. Journal of Food Science 62 , 404405.
  • 2
    Arroyo, G., Sanz, P.D. & Préstamo, G. (1999) Response to high pressure, low temperature treatment in vegetables: determination of survival rates of microbial populations using flow cytometry and detection of peroxidase activity using confocal microscopy. Journal of Applied Microbiology 86 , 544556.DOI: 10.1046/j.1365-2672.1999.00701.x
  • 3
    Cheftel, J.C. (1995) Review: High pressure, microbial inactivation and food preservation. Food Science and Technology International 1 , 7590.
  • 4
    Cox, J.L. (1989) A perspective on Listeriosis. Food Technology 43 , 5259.
  • 5
    Hite, B.H. (1899) The effect of pressure in the preservation of milk. Bulletin of West Virginia University of Agriculture Experiment Station Morgantown 58 , 1535.
  • 6
    Hoover, D.G., Metrick, C., Papineau, A.M., Farkas, D.F. & Knorr, D. (1989) Biological effects of high hydrostatic pressure on food microorganisms. Food Technology 43 , 99107.
  • 7
    Iwahashi, H., Fujii, S., Obuchi, K., Kaul, S.C., Sato, A. & Komatsu, Y. (1993) Hydrostatic pressure is like high temperature and oxidative stress in the damage it causes to yeast. FEMS Microbiology Letters 108 , 5358.
  • 8
    Kalchayanand, N., Sikes, A., Dunne, C.P. & Ray, B. (1998) Factors influencing death and injury of foodborne pathogens by hydrostatic pressure-pasteurisation. Food Microbiology 15 , 207214.DOI: 10.1006/fmic.1997.0155
  • 9
    Kitching, J.A. (1957) Effects of high hydrostatic pressures on the activity of flagellates and ciliates. Journal of Experimental Biology 34 , 494510.
  • 10
    Knorr, D., Heinz, V., Lee, D.-U., Schlüter, O. & Zenker, M. (1998) High pressure processing of foods: introduction. In Proceedings of VTT Symposium Fresh Novel Foods by High Pressure ed. Autio, K. pp. 9–20. Technical Research Centre of Finland (VTT), Espoo, Finland.
  • 11
    MacDonald, A.G. (1992) Effects of high hydrostatic pressure on natural and artificial membranes. In High Pressure and Biotechnology ed. Balny, C., Hayashi, R., Heremans, K. and Masson, P. pp. 67–74. Paris: INSERM and John Libbey.
  • 12
    Morita, R.Y. (1975) Psychrophilic bacteria. Bacteriological Reviews 39 , 144167.
  • 13
    Rönner, U. (1998) Resistance of microorganisms exposed to high pressure. In Proceedings of VTT Symposium Fresh Novel Foods by High Pressure ed. Autio, K. pp. 39–46. Technical Research Centre of Finland (VTT), Espoo, Finland.
  • 14
    Sikkema, J., De Bont, J.A.M. & Poolman, B. (1994) Interactions of cyclic hydrocarbons with biological membranes. Journal of Biological Chemistry 269 , 80228028.
  • 15
    Smelt, J.P.P.M. (1998) Recent advances in the microbiology of high pressure processing. Trends in Food Science and Technology 9 , 152158.DOI: 10.1016/S0924-2244(98)00030-2
  • 16
    Smelt, J.P.P.M. & Hellemons, C. (1998) High pressure treatment in relation to quantitative risk assessment. In Proceedings of VTT Symposium Fresh Novel Foods by High Pressure ed. Autio, K. pp. 27–38. Technical Research Centre of Finland (VTT), Espoo, Finland.
  • 17
    Smid, E.J. & Gorris, L.G.M. (1999) Natural antimicrobials for food preservation. In Handbook of Food Preservation ed. Rahman, M.S. pp. 285–308. New York: Dekker.
  • 18
    Styles, M.F., Hoover, D.G. & Farkas, D.F. (1991) Response of Listeria monocytogenes and Vibrio parahaemolyticus to high pressure. Journal of Food Science 56 , 14041407.
  • 19
    Ultee, A., Gorris, L.G.M. & Smid, E.J. (1998) Bactericidal activity of carvacrol towards the food-borne pathogen Bacillus cereus. Journal of Applied Microbiology 85 , 211218.
  • 20
    Ultee, A., Kets, E.P.W. & Smid, E.J. (1999) Mechanisms of action of carvacrol on the food-borne pathogen Bacillus cereus. Applied and Environmental Microbiology 65 , 46064610.
  • 21
    Vasseur, C., Baverel, L., Hébraud, M. & Labadie, J. (1999) Effect of osmotic, alkaline, acid or thermal stresses on the growth and inhibition of Listeria monocytogenes. Journal of Applied Microbiology 86 , 469476.DOI: 10.1046/j.1365-2672.1999.00686.x
  • 22
    Yayanos, A.A. & Pollard, E.C. (1969) A study of the effects of hydrostatic pressure on macromolecular synthesis in Escherichia coli. Journal of Biophysics 9 , 14641482.
  • 23
    Zobell, C.E. & Cobert, A.B. (1963) Filament formation by Escherichia coli at increased hydrostatic pressures. Journal of Bacteriology 87 , 710719.