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Abstract

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

Aims: To investigate which components of milk increase the heat resistance of Salmonella senftenberg 775W, and to explore the mechanisms that could be involved in this protective effect.

Methods and Results: The heat resistance of Salm. senftenberg was determined in a specially designed resistometer in several heating media. The molecules responsible for the thermal protective effect of milk were in the protein fraction, even in the < 3000 Da ultrafiltrate. The protective effect was lost when whey was demineralized. The former protective effect was restored when calcium or magnesium was added. Milk components protected cell envelopes of Salm. senftenberg from heat damage.

Conclusions: The protein fraction and divalent cations were responsible for the protective effect of milk. The whole protective effect on Salm. senftenberg was not the result of the addition of the protective effect of each component, but the result of a synergistic effect of some of them interacting.

Significance and Impact of the Study: This work could be useful for improving food preservation and hygiene treatments. It also contributes to our knowledge of microbial physiology.


INTRODUCTION

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

It is well known that bacterial thermal tolerance changes with heating media (Hansen and Riemann 1963; Tomlins and Ordal 1976; Doyle and Mazzotta 2000; Murphy et al. 2000). It has been suggested (Hansen and Riemann 1963) that these changes could be due to pH and/or water activity differences. However, some authors (Condón and Sala 1992) have demonstrated that micro-organisms can show different heat resistances in several media of the same pH. It has also been demonstrated that micro-organisms suspended in media of the same water activity, achieved by adding different solutes, can show different thermal sensitivities (Baird-Parker et al. 1970; Goepfert et al. 1970; Corry 1974). Therefore, it could be concluded that chemical components, regardless of pH and water activity, could protect bacterial cells against heat treatments.

Most chemical compounds added to the heating media can affect microbial thermal tolerance (Hansen and Riemann 1963; Blackburn et al. 1997; Casadei et al. 2001; Mañas et al. 2001), but the mechanism of action in some complex systems, such as food, is still unclear. The addition of salts and soluble carbohydrates generally increases bacterial heat resistance, and it has been suggested that this increase could be due to bacterial dehydration (Hansen and Riemann 1963; Tomlins and Ordal 1976). However, most foods contain only low percentages of these compounds and high water activities, and therefore, this mechanism would not explain the protective effect of many of them (Tomlins and Ordal 1976).

The addition of lipids to the heating medium can also increase bacterial heat resistance (Ababouch et al. 1987; Fain et al. 1991; Ahmed et al. 1995; Kaur et al. 1998). This could be due to the dehydration of cells immersed in the lipoid phase (Jay 1992; Ahmed et al. 1995). However, cells in the water phase would not be protected and survival curves would show a biphasic inactivation rate. The occurrence of tails in survival curves obtained in foods containing lipids has been reported (Ababouch et al. 1987; Kaur et al. 1998), but this mechanism could not explain the protective effect on the whole bacterial population which is sometimes observed in foods (Fain et al. 1991; Ahmed et al. 1995).

Proteins and peptides also protect micro-organisms against heat inactivation (Hansen and Riemann 1963; Moats et al. 1971; Tomlins and Ordal 1976). The mechanisms by which these components increase heat resistance are not well known. The electrostatic interaction between micro-organisms and some peptides could induce bacterial aggregation (Ng and Garibaldi 1975). Survival curves are usually drawn by plotting the number of colony-forming units (cfu) vs heating time. Aggregates will form a colony while a single cell remains alive; therefore, the microbial inactivation and the survival counts (cfu) could follow different kinetics. This could lead to the appearance of tails and upward concavities in the survival curves (Hansen and Riemann 1963) and the overestimation of microbial heat resistance. However, aggregation should decrease the initial counts and would not be able to explain the straight survival curves observed by different authors (Hansen and Riemann 1963; Moats et al. 1971; Craven and Blankeship 1983) in several foods.

Microbial heat resistance is usually higher in foods than in buffers, but the mechanisms by which foods protect bacterial cells against heat are still unknown. Foods are very complex chemical systems in which most components (salts, sugars, protein, fats etc.) may affect microbial thermal tolerance, hence the difficulty in studying the protective mechanism of all interacting components. Furthermore, these investigations require a high number of heat-resistance determinations, by the multi-point method, to draw survival curves and to understand the heat inactivation kinetics of micro-organisms.

Cow’s milk could be a useful model for investigating the mechanisms by which foods protect micro-organisms against heat. Its chemical composition is well known and fractions could be obtained by contrasting techniques. Furthermore, published data (Hansen and Riemann 1963; Moats et al. 1971; Tomlins and Ordal 1976; Doyle and Mazzotta 2000) have demonstrated the protective effect of milk on most bacterial species investigated. It is difficult to compare the magnitude of the effect observed by different authors as data have been obtained by different methods. From the results obtained by this research group in similar experimental conditions, it was concluded that the heat protective effect of milk was higher on Salmonella senftenberg than on other Salmonella spp. (Mañas 1999), and on Yersinia enterocolitica and Listeria monocytogenes (Pagán 1997).

The aim of this work was to investigate which components of milk increased the heat resistance of Salm. senftenberg 775W, and to explore the mechanisms that could be involved in this protective effect.

MATERIALS AND METHODS

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

Bacterial culture and media

The strain of Salmonella senftenberg 775W (ATCC 43845) used in this investigation was supplied by the Spanish Type Culture Collection and was maintained on slants of Nutrient Agar (NA; Biolife) at 2–4°C.

Subcultures were prepared by inoculating, with one single colony from a plate, test tubes containing 5 ml sterile Nutrient Broth (NB; Biolife) and incubating overnight at 37°C. Using these subcultures, 250 ml Erlenmeyer flasks containing 50 ml sterile NB were inoculated to a concentration of 106 cells ml–1 and incubated under agitation (130 rev min–1; Selecta, Rotabit, Spain) at 37°C for 29 h. These cultures, which had attained the stationary growth phase and maximum heat resistance (data not shown), were stored at 4°C until use. Storage did not change the heat resistance of the bacterial cells during the time in which this investigation was carried out (data not shown).

Heating media

McIlvaine citrate phosphate buffers of pH 7·7 and 6·7 (Dawson et al. 1974), simulate milk ultrafiltrate (SMUF; Jenness and Koops 1962) and simulate milk ultrafiltrate without divalent cations (SMUF-W; Barach et al. 1976), were used as reference heating media. SMUF is a water solution containing the same salts at the same concentration as are present in cow’s milk. UHT whole and skimmed milk (Sali, Zaragoza, Spain), pasteurized whole milk (Sali) and several fractions of milk were also used as heating media. In some experiments, calcium chloride (Panreac), magnesium chloride (Panreac), lactose (Difco) and β-lactoglobulin (Sigma) were added to the heating media.

The whey was obtained from pasteurized milk. Fat was removed by centrifugation at 2000 g for 15 min at 4°C, and the casein fraction by acidification to pH 4·6 and centrifugation at 2000 g for 15 min at 4°C. Milk ultrafiltrate was obtained from milk whey by filtration through 3000 Da filters (Amicon, Denver, CO, USA). In some experiments, divalent cations were withdrawn from the whey by incubation with 20 mmol l–1 ethylenediaminetetraacetic acid disodium salt (EDTA; Panreac) for 20 min, and extraction by dialysation against SMUF-W as described by Barach et al. (1976).

Heat-resistance determinations

Heat treatments were carried out in a specially-designed resistometer as already described (Raso et al. 1998). Once the temperature had attained stability, 0·2 ml of an adequately-diluted cell suspension was injected into the 23 ml treatment chamber containing the heating medium. After inoculation, 0·2 ml samples were collected at different heating times and immediately pour-plated. For each heat-resistance determination, at least eight samples were taken at different heating times. Plates were incubated for 24 h at 37°C. Previous experiments had shown that longer incubation times did not influence survivor counts (data not shown). After incubation, cfu were counted with an improved Image Analyser Automatic Counter (Protos, Analytical Measuring Systems, Cambridge, UK) as described elsewhere (Condón et al. 1996). NA was usually used as recovery medium. The effect of heat treatment on the outer membrane integrity was examined by comparing survival counts in NA and in NA with 0·15% bile salts (Biolife) added. This concentration of bile salts did not decrease counts of untreated cells (data not shown).

Heat-resistance parameters

Dt-values (time in min for a 10-fold reduction in survival counts) were calculated from the straight portion of survival curves. Survival curves were drawn by plotting the logarithm of the number of surviving micro-organisms vs heating time. Coefficients of variation of D-values among replicates were, using this methodology, always lower than 10% (Raso et al. 1998).

The decimal reduction time curves (DRTC) were obtained by plotting the log of the Dt-values vs treatment temperature, and z-values (°C increase in treatment temperature for Dt-value to decrease 10-fold) were calculated from the slope.

Correlation coefficients (r0) and 95% confidence limits (C.L.) were calculated by the appropriate statistical package (StatView SE + GraphicsTM, Abacus Concepts Inc., Berkeley, CA, USA). The statistical significance (P≤ 0·05) of differences between D- and z-values was tested as described by Steel and Torrie (1960).

RESULTS AND DISCUSSION

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

Table 1 shows decimal reduction time values at 63°C of Salm. senftenberg 775W in UHT whole milk and in citrate phosphate buffer at the same pH (6·7). The heat resistance in whole milk (D63=1·2 min) was threefold higher than that obtained in buffer (0·37 min). However, survival curves in milk showed longer lag phases before the killing began (Fig. 1). Therefore, the protective effect of milk against heat is higher than that obtained by comparing decimal reduction time values. The occurrence of shoulders has been related (Tomlins and Ordal 1976; Condón et al. 1996; Pagán et al. 1997) to the high heat resistance and heat damage repair capability of cells after heating. Overall, these results confirmed that other factors, regardless of the pH in food, increase Salm. senftenberg 775W heat resistance.

Table 1.   Heat resistance of Salmonella senftenberg 775W in different media* Thumbnail image of
image

Figure 1.  Survival curves at 63°C of Salmonella senftenberg 775W in whole milk (●) and in citrate-phosphate buffer of pH 6·7 (▴)

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Although it seemed unlikely that water activity (aw) was the factor that increased the heat resistance of Salm. senftenberg 775W in milk (aw > 0·99), the influence of salts and soluble carbohydrate concentration was also studied. The D63-values of Salm. senftenberg 775W in SMUF, in SMUF without divalent cations (SMUF-W) and in SMUF with 4·8% added lactose (Table 1) were the same as those obtained in citrate phosphate buffer (P≤ 0·05). The results showed that neither salts nor soluble carbohydrate content were responsible for the thermal protective effect of milk. It has been reported that divalent cations could increase microbial heat resistance stabilizing ribosomes (Tolker-Nielsen and Molin 1996) or the cell envelopes (Bender and Marquis 1985). The present results demonstrated that divalent cations did not protect Salm. senftenberg against heat, as heat resistance in SMUF and in SMUF-W was the same.

It has been reported that high fat concentration in foods increases bacterial heat resistance (Ababouch et al. 1987; Fain et al. 1991; Ahmed et al. 1995; Kaur et al. 1998). However, Salm. senftenberg 775W showed the same D63-values in whole and in skimmed UHT milk (Table 1). Survival curves in whole milk did not show upward concavities, as other authors have observed in foods containing lipoid phases (Ababouch et al. 1987; Kaur et al. 1998). It is possible that the fat content was so low (3·5%) that the number of cells immersed in the lipoid phase and consequently, more heat resistant, could not be detected. Overall, these results discounted any contribution of fat to the observed protective effect of milk on the whole Salm. senftenberg 775W population.

It has been demonstrated (Moats et al. 1971; Tomlins and Ordal 1976) that proteins, peptides and amino acids added to the heating media can also increase microbial heat resistance. In an attempt to identify the milk components involved in the protective effect, skimmed milk was fractionated by acidification and the whey was used as heating medium. When the pH of the whey was restored to the original value (6·7) by adding sodium hydroxide, the heat resistance of Salm. senftenberg 775W in this whey was the same (P≤ 0·05) as that observed in whole or skimmed milk (Table 1). This demonstrated that protective components were present in the whey. However, when the pH of the whey was restored by adding the McIlvaine buffer components instead of sodium hydroxide, the heat resistance was lower (Table 1). It seemed that sodium phosphate and/or citric acid restrained the protective effect of the whey components. It is well known that phosphates are chelators (Dawson et al. 1974), and it has been reported that divalent cations can increase bacterial heat resistance (Tolker-Nielsen and Molin 1996). The results of this experiment suggested that divalent cations were involved in the protective effect. However, as discussed above, the heat resistance of Salm. senftenberg 775W was the same in SMUF, SMUF-W and in citrate-phosphate buffer (Table 1). The results of the two experiments appear to be contradictory.

To examine any contribution of divalent cations to the protective effect of milk by indirect mechanisms, the heat resistance of Salm. senftenberg 775W in dialysed EDTA-treated whey (without divalent cations), and in this medium with added calcium chloride (10 mmol l–1) or magnesium chloride (10 mmol l–1), was investigated. Figure 2 shows survival curves of Salm. senftenberg in the three media, and demonstrates that the dialysation of whey withdrawing divalent cations reduced the protective effect of whey. The addition of calcium or magnesium chloride restored the former protective effect. These results indicated that although divalent cations in water solutions did not increase the heat resistance of Salm. senftenberg (Table 1), its presence in whey was necessary for the other components to protect bacterial cells against heat (Fig. 2). Furthermore, the results demonstrated that the effect of divalent cations was non-specific, as the heat resistance observed in media with added calcium or magnesium was the same.

image

Figure 2.  Survival curves at 63°C of Salmonella senftenberg 775W in whey (●), whey without divalent cations (▮), and in this medium with 10 mmol l–1 calcium chloride (◆) or 10 mmol l–1 magnesium chloride (▴) added

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Lee and Goepfert (1975) observed that cells of Salm. typhimurium heated in a medium containing polyamines showed high heat resistance. The protective effect was even observed in media without polyamines when cells had been previously suspended at room temperature with these compounds before heat treatment. In the present study, a similar effect was observed. Cells suspended in milk at 48°C for 5 s and thermally treated at 63°C in SMUF had the same heat resistance (P≤ 0·05) as native cells thermally-treated in milk (Table 1). However, when the cells suspended in milk before heating were thermally-treated in SMUF-W (without divalent cations), the heat resistance was only slightly higher than that of native cells heated in McIlvaine pH 6·7 buffer (Table 1). These results indicated that the protective effect of milk whey components was reversible and could be lost during heat treatment, depending on the presence of divalent cations in the heating medium.

The time/temperature at which cells were suspended in milk before thermal treatment determined the protective effect. Cells suspended in milk at room temperature for 20 min did not increase the D63 in SMUF up to the value attained when they were suspended at 48°C for 5 s before heating in SMUF (Table 1). At higher temperatures, the protective effect should have been acquired even more quickly. If microbial heat inactivation had happened faster than the occurrence of the thermal protection, as should have happened at very high heating temperatures, the same Dt-value would have been expected in buffer and in milk. Figure 3 shows DRTC of Salm. senftenberg 775W in whole milk and in McIlvaine buffer, and illustrates that the z-value in both media (5·0°C) was the same (P≤ 0·05). Therefore, it can be concluded that the protection exerted by milk components at 73°C occurred faster than 0·01 min at least, as this was the minimum decimal reduction time included in Fig. 3.

image

Figure 3.  Decimal reduction time curves of Salmonella senftenberg 775W heated in milk (●) and in neutral McIlvaine buffer (▴)

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As demonstrated above, divalent cations were indirectly involved in the protective effect of other milk whey components. As the major protein of whey is β-lactoglobulin, in a first approach, the effect of this protein on the heat resistance of Salm. senftenberg 775W was explored. The results (Table 1) showed that this protein, suspended in SMUF, did not protect Salm. senftenberg 775W against heat to the same degree as milk or whey (Table 1). Therefore, other whey components must be involved. When Salm. senftenberg 775W was suspended in milk whey ultrafiltrate (< 3000), its heat resistance was the same (P≤ 0·05) as that observed in whole milk (Table 1). This indicated that components of low molecular weight (< 3000 Da) were involved in the protective effect of milk.

Moats et al. (1971) found that peptide solutions protected Salmonella anatum against heat more than protein solutions did. These authors postulated that peptides complex with heat-sensitive molecules in the cell, increasing their stability to heat. The lower protective effect of proteins and other macromolecules should be expected, since they will be unable to pass through cell envelopes binding heat-sensitive sites within the cell. The present results also demonstrated the higher thermal protective effects of low molecular weight milk whey components, but they suggested that the protective effect was related, most probably, to a surface phenomenon, as divalent cations were necessary for the protective effect of the other whey components. Furthermore, cells attained thermal protection very quickly and this effect was reversible, depending on the divalent cation present in the heating medium. It has been demonstrated that cell envelopes are sometimes key targets in heat inactivation (Beuchat 1978) and perhaps milk components could stabilize them against heat.

To explore the mechanism by which milk protected Salm. senftenberg against heat, a new experiment was carried out. The outer membrane is the most external layer of the cell envelopes in Gram-negative bacteria, and acts as an efficient permeability barrier against macromolecules and hydrophobic substances such as bile salts (Nikaido and Vaara 1985). Heat treatments can remove divalent cations and lipopolysaccharide molecules from this structure (Hurst 1984), progressively disrupting the outer membrane which first becomes permeable to hydrophobic compounds (Nikaido and Vaara 1985; Vaara 1992) and finally leads to bacterial inactivation. If divalent cations acted by binding milk components to the outer membrane of Salm. senftenberg 775W, stabilizing it against heat, then the percentage of surviving cells sensitized to hydrophobic substances, after heat treatments of the same lethality, should be higher when cells are heated in SMUF-W (without divalent cations) instead of in milk. To check this hypothesis, the integrity of the outer membrane of Salm. senftenberg 775W after heating was evaluated by comparing the survival counts in NA and in NA with bile salts added. As shown in Fig. 4, whereas more than a two log reduction was observed in NA with bile salts when cells had been heated in SMUF-W, less than a half log reduction was sensitized when cells had been heated in milk. This demonstrated that milk components protected the outer membrane of Salm. senftenberg 775W against heat injury. This mechanism could also explain the occurrence of long shoulders in the survival curves observed in milk (Figs 1 and 2), as the presence of shoulders has been related to heat damage and repair mechanisms (Condón et al. 1996; Pagán et al. 1997).

image

Figure 4.  Percentage of Salmonella senftenberg 775W cells non-sensitive to bile salts after being heated in SMUF-W (61°C; 1·5 min) and in milk (63°C; 2·5 min). Data in the figure are the mean of three experiments

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It would be very interesting to know whether the mechanism described here is exclusive to milk, or common to other foods. Table 2 includes some heat-resistance data obtained with the same methodology in liquid whole egg and related media. As shown in the Table, liquid whole egg protected Salm. senftenberg against heat more than milk. The D63 in egg was fivefold higher than that obtained in phosphate citrate buffer at the same pH. The addition of egg fat to the buffer did not increase the thermal tolerance of Salm. senftenberg. The addition of McIlvaine components restrained the protective effect of liquid egg. These results seem to indicate that the mechanisms by which egg components protect Salm. senftenberg are similar to those proposed for milk. However, more investigations are needed.

Table 2.   Heat resistance of Salmonella senftenberg 775W in different media* Thumbnail image of

It would also be interesting to know whether this mechanism of protection is common to most bacterial species, or is only characteristic of abnormally high heat-resistant micro-organisms, such as Salm. senftenberg 775W. Overall, this investigation has demonstrated that, at least in some cases, the whole protective effect of some foods on micro-organisms is not the result of the addition of the protective effect of each component, but the result of a synergistic effect of some of the components interacting.

Acknowledgements

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

Thanks are given to the Diputación General de Aragón for a scholarship granted to P.M.

References

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