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Baroprotection of vegetative bacteria by milk constituents: A study of Listeria innocua
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International Dairy Journal 17 (2007) 104–110 www.elsevier.com/locate/idairyj
Baroprotection of vegetative bacteria by milk constituents: A study of Listeria innocua Elaine P. Blacka,b, Thom Huppertzb, Gerald F. Fitzgeralda,b, Alan L. Kellyb, a
Department of Microbiology, University College Cork, Ireland Department of Food and Nutritional Sciences, University College Cork, Ireland
b
Received 24 June 2005; accepted 11 January 2006
Abstract The baroprotective effect of milk constituents on Listeria innocua 4202 treated at 350 or 500 MPa for 5 min was examined. Highpressure (HP) treatment of L. innocua 4202 (1 109 cfu mL 1) resulted in complete inactivation in simulated milk ultra-filtrate (SMUF), a 5.0 log reduction in phosphate-buffered saline and a 2.9 log reduction in milk. The addition of micellar casein to SMUF increased survival of the bacterium by 3 logs, compared with SMUF alone, but the protective effect was negated if the minerals associated with the casein micelles were removed. The colloidal minerals calcium (30 mM), magnesium (5 mM), citrate (10 mM) and phosphate (20 mM), suspended in SMUF increased survival by 3.3, 1.7, 3.3 and 3.5 logs, respectively. The buffering capacity of the suspending medium was found to be a key factor in microbial baroresistance. Buffering by phosphate and citrate in milk may protect microorganisms against changes in pH during HP treatment, whereas the divalent cations calcium and magnesium may protect cell membranes against HP. r 2006 Elsevier Ltd. All rights reserved. Keywords: High pressure; Milk; Listeria innocua; Colloidal calcium phosphate
1. Introduction High-pressure (HP) processing can potentially deliver food products that meet many of the demands of today’s consumer, i.e., high-quality, minimally-processed foods that are microbiologically safe and have a long shelf-life. The efficacy of HP-induced inactivation of microorganisms depends on a range of factors, related to the microorganism itself, the conditions of treatment and to the medium in which the microorganisms are suspended. While some of these factors have been identified, fundamental understanding of the mechanisms of baroresistance of microorganisms is still poorly developed. Factors that affect HPinduced inactivation of microorganisms include the applied pressure, treatment time and temperature (Patterson, Margey, Mills, Simpson, & Gilmour, 1997), the bacterial strain (Alpas, Kalchayanand, Bozoglu, Sikes, Dunne, & Ray, 1999; Benito, Ventoura, Casadei, Robinson, & Corresponding author. Tel.: +353 21 4903405; fax: +353 21 4270001.
E-mail address: [email protected] (A.L. Kelly). 0958-6946/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2006.01.009
Mackey, B. 1999), cell density (Furukawa, Noma, Shimoda, & Hayakawa, 2002), growth phase (Karatzas & Bennik, 2002; McClements, Patterson, & Linton, 2001; Pagan & Mackey, 2000), growth temperature (McClements et al., 2001; Simpson & Gilmour, 1997) and suspending medium (Palou, Lopez-Malo, Barbosa-Canovas, Welti-Chanes, & Swanson, 1997; Patterson, Quinn, Simpson, & Gilmour, 1995; Simpson & Gilmour, 1997; Smiddy et al., 2005). To date, much research has focused on HP-induced microbial inactivation in buffer systems, but when such data are compared with those obtained in food systems, levels of inactivation are not always in agreement (Palou et al., 1997; Patterson et al., 1995; Smiddy et al., 2005; Styles, Hoover, & Farkas, 1991). Microbial baroresistance is usually higher in low-acid food products than in buffer systems (Simpson & Gilmour, 1997), but the mechanism(s) by which food products protect bacterial cells are not fully understood. The complex chemistry of individual foods is difficult to mimic in a buffer system, as a range of components and factors contribute to, or antagonize, baroprotection, e.g., water activity, pH (Jordan, Cascual,
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Bracey, & Mackey, 2001; Wouters, Glaasker, & Smelt, 1998), the presence of antimicrobial compounds (Black, Kelly, & Fitzgerald, 2005; Masschalck, Garcia-Graells, Van Haver, & Michiels, 2000) or additives (Shearer, Dunne, Sikes, & Hoover, 2000), as well as individual food constituents, such as fat, proteins, solutes (Molina-Hoppner, Doster, Vogel, & Ganzle, 2004) and minerals (Cheftel, 1995; Hauben, Bernaerts, & Michiels, 1998; Van Opstal, Vanmuysen, & Michiels, 2003). Bovine milk is a useful model for investigating the protective effect of foods against pressure, because its chemical composition is well known and many recent studies have added to our knowledge of the effects of HP treatment on the constituents of milk (Huppertz, Kelly, & Fox, 2002). Milk exerts a strong protective effect, compared to buffer systems, when Listeria monocytogenes or E. coli are subjected to HP (Patterson et al., 1995; Simpson & Gilmour, 1997; Styles et al., 1991); previous studies have suggested that the presence of calcium ions (Hauben et al., 1998) or solutes (Simpson & Gilmour, 1997) may confer protection, but the specific baroprotective nature of milk is poorly understood. In the present study, the constituents of milk such as lactose, minerals, whey proteins and casein were examined for a possible baroprotective effect on L. innocua. The casein micelle was of particular interest as a number of potential protective agents (e.g. calcium) are associated with it. The dry matter of casein micelles consists of 94% protein plus 6% minerals; these minerals, predominantly calcium, phosphate, citrate and magnesium, are collectively referred to as colloidal calcium phosphate (CCP; Fox, 2003). The objective of the study was to identify the individual constituent or constituents of milk that provide a specific baroprotective effect on L. innocua, and thereby gain understanding of the mechanisms of protection. 2. Materials and methods 2.1. Preparation of test media Skimmed pasteurized bovine milk was obtained from CMP Dairies (Cork, Ireland). CCP-adjusted milk samples were prepared by adjusting the pH of the milk to 4.6 (CCP free), 5.0, 5.5 or 6.0 (CCP depleted by 85%, 40% or 5%, respectively) or 8.0 or 9.0 (CCP enriched by 30% or 60% respectively) followed by dialysis against 2 20 volumes of bulk milk for 48 h at 5 1C. Simulated milk ultrafiltrate (SMUF) was prepared as described by Jenness and Koops (1962). SMUF contains Na, K, Ca, Mg, PO4, Cl, citrate, SO4 or CO2 at a level of 18.3, 39.4, 9.0, 3.2, 11.6, 32.4, 9.6, 1.0 or 2.2 mmol L 1, respectively. Phosphocasein (Dairy Products Research Centre, Moorepark, Fermoy, Ireland), sodium caseinate (Dairygold Co-op., Mitchelstown, Ireland), b-lactoglobulin, a-lactose, tris-hydroxymethiomethylamine (Tris), calcium chloride, magnesium chloride, trisodium citrate and dipotassium hydrogen phosphate (all from Sigma Chemical
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Table 1 Suspending media used for HP treatment of L. innocua 4202 Test media
Description
PSM SMUF SMUF-PhosCN
Pastuerized skim milk Simulated milk ultrafiltrate SMUF containing 25 or 30 g L 1 added phosphocasein SMUF containing 25 g L 1 added sodium caseinate Phosphate-buffered saline adjusted to pH 6.6 SMUF containing 45 or 60 g L 1 added lactose SMUF containing 3 g L 1 added b-lactoglobulin SMUF containing 5 mM added MgCl2 SMUF containing 30 mM added CaCl2 SMUF containing 10 mM added Na3Citrate SMUF containing 20 mM added K2HPO4 0.1 M Tris-hydroxymethiomethylamine (Tris) adjusted to pH 6.6 Tris containing 5 mM added MgCl2 Tris containing 30 mM added CaCl2
SMUF-NaCN PBS SMUF-Lac SMUF-b-lg MUF-Mg SMUF-Ca SMUF-Cit SMUF-P Tris Tris-Mg Tris-Ca
Corp., St. Louis, USA) were added to milk, 0.1 m Tris or SMUF, as outlined in Table 1. 2.2. Bacterial strains, culture conditions and suspending media L. innocua 4202 was obtained from the University College Cork culture collection and grown on Tryptic Soy Agar supplemented with 0.5% yeast extract (TSA-YE) at 37 1C. Working cultures were prepared by transferring one colony from a TSA-YE plate to Tryptic Soy Broth containing 0.5% yeast extract (TSB-YE) and grown overnight to stationary phase (1 109 cfu mL 1) prior to HP treatment. The cells were centrifuged at 1677 g for 5 min and resuspended at a level of 1 109 cfu mL 1 in a range of test media (Table 1). 2.3. Determination of the level of total or colloidal calcium and phosphate in milk The level of calcium in milk or its ultracentrifugal supernatant (100,000 g for 60 min at 20 1C) was determined as outlined by Huppertz et al. (2005). The level of inorganic phosphate in milk or its ultracentrifugal supernatant was determined as outlined by IDF (1990). Levels of colloidal calcium or phosphate were calculated by subtracting the level of calcium or phosphate in the ultracentrifugal supernatant from that in the milk, after correction for the volume of the ultracentrifugal supernatant. 2.4. HP treatment Samples of L. innocua were suspended in test media and dispensed into 1 3 cm sterile polyethylene bags, heat-sealed while excluding as much air as possible,
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double-packaged in larger bags and vacuum-packed. Samples were HP-treated in a Stansted Fluid Power IsoLab 900 High Pressure Food Processor (Stansted Fluid Power Ltd., Stansted, Essex, UK), using a 90:10 mixture of ethanol and castor oil as the pressure-transmitting medium, at 350 or 500 MPa for 5 min. HP treatment was carried out at 20 1C, by thermostatically controlling the vessel of the HP unit. Adiabatic heating on compression resulted in an increase in temperature of the processing fluid of approximately 15 1C at 500 MPa; most of this heat dissipated during the holding time of 5 min. A control sample was held at atmospheric pressure and 20 1C for the duration of treatment. All experiments were replicated in triplicate throughout the entire study, and results presented are means of data from three replications, with standard deviations where appropriate.
3.2. Influence of CCP on HP-induced inactivation of L. innocua Partial or complete removal of micellar minerals from milk, had a large effect on the HP-induced inactivation of L. innocua; complete inactivation of L. innocua was observed in milk containing o15% of its original micellar calcium and phosphate content, and the level of survival of L. innocua progressively increased with increasing CCP content of the milk, up to its original content (Fig. 1). Further increasing the CCP content of milk gave no further baroprotection to L. innocua. The principal constituent minerals of CCP, i.e., phosphate, citrate, calcium and magnesium, were also added to SMUF individually, up to levels commonly found in milk (20, 10, 30 or 5 mmol L 1, respectively). All four minerals provided substantial baroprotection to L. innocua (Fig. 2). The strongest
2.5. Enumeration of viable cells Appropriate decimal dilutions of L. innocua suspensions were spread-plated or pour-plated immediately after pressure treatment on TSA-YE plates and incubated for 48 h at 37 1C. The results shown are the means and standard deviations of data from triplicate independent experiments. 2.6. Studies on membrane protection To assess protection of cell membranes by milk constituents, samples of L. innocua suspended in Tris, Tris-Ca or Tris-P (Table 1) and treated at a sublethal pressure of 350 MPa were plated on TSA-YE agar supplemented with nisin (100 IU mL 1) as described by Hauben, Wuytack, Soontjens, & Michiels (1996) or bile salts (0.3%), as described by Manas, Pagan, Sala, & Condon (2001).
Table 2 Survivala of L. innocua 4202 suspended in test media and treated at 500 MPa for 5 min at 20 1C Test Mediab
Survivalc (Log cfu mL 1)
Initial innoculum PBS PSM SMUF SMUF-PhosCN (25 g L 1) SMUF-PhosCN (30 g L 1) SMUF-NaCN (25 g L 1) SMUF-Lac (45 g L 1) SMUF-Lac (60 g L 1) SMUF-b-lg (3 g L 1)
8.90 3.8970.09 5.9870.37 o1.0 2.5070.45 3.5070.08 o1.0 o1.0 o1.0 o1.0
a Data are means (7standard deviation) of results of triplicate experiments. b Refer to Table 1 for abbreviations. c Limit of detection—1.0 log cfu mL 1.
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3.1. Influence of milk constituents on HP-induced inactivation of L. innocua After HP treatment at 500 MPa for 5 min, the level of surviving L. innocua in milk was 5.98 log, 2 log units higher than that in PBS (3.89 log), whereas a reduction to an undetectable level was observed in SMUF (Table 2). The apparent protective effect of milk was further investigated by using SMUF as a base medium to which constituents of milk were added individually, to determine their specific influence on HP-induced inactivation of L. innocua. Adding lactose (45 or 60 g L 1), b-lactoglobulin (3 g L 1) or sodium caseinate (25 g L 1) to SMUF prior to HP treatment did not influence HP-induced inactivation of L. innocua (Table 2), whereas adding phosphocasein (25 or 30 g L 1) provided baroprotection of 3 logs.
Survival (Log cfu mL-1)
3. Results 6
4
2
0 0
20 40 60 80 100 120 140 160 Colloidal calcium or phosphate as % of value in control milk
Fig. 1. Influence of the level of micellar calcium (K) or micellar phosphate (J) in milk on the survival of L. innocua 4202 (initial level of 1 109 cfu mL 1) following treatment at 500 MPa for 5 min. Dashed line indicates detection limit of L. innocua 4202.
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8 Survival (Log cfu mL-1)
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6
4
2
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6
4
2 NS
0
0 +Calcium
+ Citrate + Magnesium + Phosphate
Milk
Fig. 2. Influence of calcium (30 mM), magnesium (5 mM), phosphate (10 mM) or citrate (20 mM), added to simulated milk ultrafiltrate, on survival of L. innocua 4202 (initial level of 1 109 cfu mL 1) following treatment at 500 MPa for 5 min. NS denotes samples where no survival was detected. The dashed line indicates the detection limit of L. innocua 4202. Values are means of data from triplicate independent experiments, with the standard deviation indicated by vertical error bars.
protective agents were calcium, phosphate and citrate, providing 43 log protection compared with control SMUF; magnesium, added at a level of 5 mM, gave a lower level of protection (1.7 log). Increasing the concentration of magnesium in SMUF to 30 mM gave similar protection to that provided by calcium (30 mM; data not shown). 3.3. Influence of buffering capacity on HP-induced inactivation of L. innocua in milk Survival of L. innocua following treatment at 500 MPa for 5 min when suspended in 0.1 m Tris-HCl (pH 6.6), a pressure-stable buffer (Morild, 1981; Quinlan & Reinhart, 2005), was 5.2 log, compared with 3.4 log in PBS (pH 6.6), which is less pressure-stable (Morild, 1981; Quinlan & Reinhart, 2005). This suggests that maintaining a constant pH under pressure reduced inactivation of this bacterium. To test this hypothesis, Tris-HCl (0.1 mol L 1) was added to milk and it was found that this increased the protection of L. innocua, by 41.5 log (Fig. 3). Adding calcium (30 mM), phosphate (20 mM), magnesium (5 mM) or citrate (10 mM) to 0.1 m Tris-HCl buffer, pH 6.6, did not further increase the already strong baroprotective effect of Tris (Fig. 4). 3.4. Protection of the L. innocua against sub-lethal injury L. innocua suspended in Tris-HCl buffer or in Tris-HCl supplemented with calcium or phosphate was subjected to a sublethal pressure of 350 MPa for 5 min at 20 1C. The effect of this pressure treatment was examined by comparing growth of L. innocua following this pressure treatment on TSA-YE or TSA-YE containing either 0.3% bile salts
PBS
Tris
Milk
Milk + Tris
Fig. 3. Survival of L. innocua 4202 (initial level of 1 109 cfu mL 1), suspended in phosphate-buffered saline, 0.1 M Tris-HCl, milk or milk containing 0.1 M added Tris-HCl (all pH 6.6), following treatment at 500 MPa for 5 min. The dashed line indicates the detection limit of L. innocua 4202. Values are means of data from triplicate independent experiments, with the standard deviation indicated by vertical error bars.
10
8 Survival (Log cfu mL-1)
SMUF
6
4
2
0 Tris-HCl
+Calcium
+ Citrate + Magnesium + Phosphate Milk
Fig. 4. Influence of micellar minerals added to Tris-HCl on survival of L. innocua 4202 (initial level of 1 109 cfu mL 1) following treatment at 500 MPa for 5 min. The dashed line indicates the detection limit of L. innocua 4202. Values are means of data from triplicate independent experiments, with the standard deviation indicated by vertical error bars.
or nisin (500 IU mL 1). The concentrations of bile salts or nisin used did not decrease numbers of untreated cells (data not shown). When calcium was present in the suspending medium during HP treatment, it protected the cells by 1 log from bile salts or nisin contained in the plating medium. If calcium was added to cells following HP treatment, no protection was observed (data not shown). This suggests that calcium protects L. innocua during pressure treatment rather than acting post pressure treatment in some way. The presence of phosphate in the suspending medium conferred no protection to the effects of bile or nisin (Fig. 5).
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Survival (Log cfu mL-1)
8
6
4
2
0 HP
HP + Bile Salts
HP + Nisin
Fig. 5. Influence of calcium ( ) or phosphate (&) on survival of L. innocua 4202 (initial level of 1 109 cfu mL 1) following treatment at 350 MPa for 5 min and plating on bile-salts- or nisin-supplemented agar. The black bar (’) represents the control sample plated on TSA-YE without additions. The dashed line indicates the detection limit of L. innocua 4202. Values are means of data from triplicate independent experiments, with the standard deviation indicated by vertical error bars.
4. Discussion Data presented in this communication indicate a strong protective effect of milk constituents against HP-induced inactivation of L. innocua 4202; other authors have previously commented on the strong protection milk confers to other microorganisms against HP (Dogan & Erkmen, 2004; Garcia-Graells, Masschalck, & Michiels, 1999; Hauben et al., 1998; Solomon & Hoover, 2004; Styles et al., 1991). However, little was known previously about either the specific protective agents in milk or the mechanisms by which they protect. Initial experiments were performed to identify the most likely constituents of milk to impart resistance to L. innocua against HP. It has been previously reported that the presence of solutes such as sugars at high levels, i.e., 410%, increases microbial baroresistance (Palou et al., 1997; Van Opstal et al., 2003); therefore, it was perhaps not surprising that SMUF containing lactose, the most abundant sugar in milk, at concentrations similar to, or higher than, that in milk, i.e. 4.5% or 6%, did not provide baroprotection to L. innocua (Table 2). Furthermore, no baroprotection of L. innocua was observed when the whey protein b-lactoglobulin was added to SMUF (Table 2). Adding 25 or 30 g L 1 micellar casein (phosphocasein) to SMUF provided 2.5 or 3.5 logs protection, respectively, but when casein was added to SMUF in non-micellar form (sodium caseinate), complete HP-induced inactivation of L. innocua was observed (Table 2). These results suggest that, while the protective effect of milk lies, at least partially, in the casein fraction of milk, it cannot be attributed to the proteins themselves; rather, it is likely that such protection is provided by the minerals associated with the micelle, as these are the only constituents present
in phosphocasein, but absent from sodium caseinate (Mulvihill & Ennis, 2003). The minerals calcium, phosphate, citrate and magnesium, are collectively known as CCP. CCP exists predominantly in the form of amorphous nanoclusters with a core of 3 nm, around which a shell of caseins is attached through phosphoseryl–calcium interactions (De Kruif & Holt, 2003). Calcium, the main cation in CCP, is present at a level of 30 mM in milk, of which 70% is the form of CCP; calcium has been shown previously to confer baroprotection to E. coli in Tris-HCl buffer, as has magnesium (Hauben et al., 1998), also a constituent of CCP. Removal of CCP from milk by dialysis was shown to completely diminish its baroprotective capabilities (Fig. 1); L. innocua was completely inactivated by HP when suspended in CCP-free milk, while protection against HPinduced inactivation increased with CCP content of the milk (Fig. 1). It is unlikely that minerals can confer baroprotection in their amorphous form, i.e., in the form of CCP nanoclusters, but the solubility of minerals is generally increased under HP, due to the electrostrictive effect of separate charges (Isaacs, 1981), i.e., the arrangement of water molecules around ions is more structured and dense than around uncharged ion pairs. To counteract the HP-induced reductions in volume of the system, the more dense organization of water molecules around ions, and thus ionization, is favoured. Hence, HP treatment results in increased solubility of calcium phosphate, as measured by Huppertz, Kelly, and De Kruif (unpublished data); furthermore, Keenan, Hubbard, Mayes, & Tier (2003) observed that the level of non-micellar phosphate in milk increases under HP. An increased level of minerals in the serum phase of milk may thus provide baroprotection to microbes therein. Further studies were performed to identify if baroprotection of L. innocua is provided by all, or only specific, micellar minerals. Fig. 2 illustrates that adding additional calcium, magnesium, citrate or phosphate (in the form of calcium chloride, magnesium chloride, tri-sodium citrate or di-potassium hydrogen phosphate, respectively) to SMUF individually allowed between 1.7 and 3.5 log survival of L. innocua after treatment at 500 MPa, suggesting that the baroprotective effect of CCP is not related to a single mineral constituent, but is, in fact, exerted by all micellar minerals studied. One mechanism by which the micellar minerals may influence HP-induced inactivation of L. innocua is through affecting the buffering capacity of milk. Citrate, and in particular, phosphate, provide considerable buffering capacity to the milk serum (Lucey, Hauth, Gorry, & Fox, 1995; Singh, McCarthy, & Lucey, 1997). Treatment at 1000 MPa reduces the pH of water by 1.0 unit (Marshall & Frank, 1981), so it is likely that the pressure applied in the present study (500 MPa) also reduces milk pH considerably; Molina-Gutierrez, Stippl, Delgado, Ganzle, and Vogel (2002) calculated the pH of milk serum to be reduced by one full unit at 300 MPa, although experimental proof for this extensive reduction in pH was not reported.
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Exposure to low pH combined with HP can increase inactivation synergistically (Alpas, Kalchayanand, Bozoglu, & Ray, 2000; Linton, McClements, & Patterson, 1999). While cells were very pressure-sensitive in SMUF, TrisHCl and milk gave 3.9 and 6.0 log protection, respectively. Tris is a pressure-stable buffer (Morild, 1981; Quinlan & Reinhart, 2005) which, when added to milk, increased its protective effect on L. innocua (Fig. 3) possibly by counteracting HP-induced reductions in pH. The micellar minerals citrate and phosphate have good buffering capacities (Lucey et al., 1995; Singh et al., 1997) and may therefore partially protect the suspended bacteria from the HP-induced reduction in milk pH. Addition of calcium, phosphate or citrate to Tris-HCl gave little additional protection to L. innocua against HP (Fig. 4). The main target of HP-induced inactivation of bacteria is the cell membrane (Pagan & Mackey, 2000); to study the potential protective influence of micellar minerals against HP-induced damage to the cell membrane, L. innocua was treated at a sub-lethal pressure (350 MPa), which weakens the cell membranes (Black et al., 2005), thereby sensitizing the cells to the effects of membrane-active agents, e.g., bile salts or the bacteriocin nisin. Nisin, active against Grampositive bacteria, acts by creating pores in the cellular membrane, which results in the loss of intracellular ions and disruption of the cells pH gradient and proton motive force, thereby leading to cell death (Jack, Tagg, & Ray, 1995). Bile salts cause increased permeability of the membrane and increased trans-membrane flux of divalent cations (Begley, Gahan, & Hill, 2005). The acquired sensitivity of bacteria to low levels of either bile or nisin in the plating medium following HP treatment may suggest sub-lethal injury (Hauben et al., 1996), which may be associated with damage of the cell membrane. When calcium (in the form of CaCl2) was added to the suspending medium during HP treatment, L. innocua appeared more resistant to nisin or bile salts in the plating medium (Fig. 5), indicating either a general protective effect or a specific baroprotective effect of added calcium. When calcium was added to the bacterial suspension after HP treatment, no protective effect was observed (data not shown), strongly suggesting that the protection provided by calcium is specifically baroprotective. Divalent cations, e.g., Ca2+ or Mg2+, have previously been shown to protect E. coli during HP treatment (Hauben et al., 1998). The protective effect, however, could not be attributed to stabilization of the outer membrane of E. coli. L. innocua, a Gram-positive bacterium, does not possess an outer membrane; therefore, Ca2+ may play a role in the stabilization of the cytoplasmic membrane during HP treatment in both Gram-positive and Gram-negative bacteria. Further work is needed to determine the exact nature of this protection. 5. Conclusion The results obtained in this study suggest that a combination of HP-induced solubilization of CCP, with a
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concomitant increase in the buffering capacity of milk, and stabilization of the membrane by divalent cations, may protect L. innocua against HP-induced inactivation. Further research is required, however, to confirm these hypotheses and to determine whether these mechanisms also apply to other vegetative bacterial cells in milk. Acknowledgments This research was funded by the Food Institutional Research Measure (FIRM), which is administered by the Irish Government under the National Development Plan 2000–2006, and through the NUI Travelling Studentship in Food Science and Technology awarded to E. P. Black. References Alpas, H., Kalchayanand, N., Bozoglu, F., & Ray, B. (2000). Interactions of high hydrostatic pressure, pressurization temperature and pH on death and injury of pressure-resistant and pressure-sensitive stains of foodborne pathogens. International Journal of Food Microbiology, 60, 33–42. Alpas, H., Kalchayanand, N., Bozoglu, F., Sikes, A., Dunne, C. P., & Ray, B. (1999). Variation in resistance to hydrostatic pressure among strains of food-borne pathogens. Applied and Environmental Microbiology, 65, 4248–4251. Begley, M., Gahan, C. G. M., & Hill, C. (2005). The interaction between bacteria and bile. FEMS Microbiology Reviews, 29, 625–651. Benito, A., Ventoura, G., Casadei, M., Robinson, T., & Mackey, B. (1999). Variation in resistance of natural isolates of Escherichia coli 0157 to high hydrostatic pressure, mild heat and other stresses. Applied and Environmental Microbiology, 65, 1564–1569. Black, E. P., Kelly, A. L., & Fitzgerald, G. F. (2005). The combined effect of high pressure and nisin on inactivation of microorganisms in milk. Innovative Food Science and Emerging Technologies, 6, 286–292. Cheftel, J. C. (1995). Review: High pressure, microbial inactivation and food preservation. Food Science and Technology International, 1, 75–90. De Kruif, C. G., & Holt, C. (2003). Casein micelle structure, functions and interactions. In P. F. Fox, & P. L. H. McSweeney (Eds.), Advanced dairy chemistry, Volume 1: Proteins (3rd ed.) (pp. 233–276). New York, USA: Kluwer Academic/Plenum Publishers. Dogan, C., & Erkmen, O. (2004). High pressure inactivation kinetics of Listeria monocytogenes inactivation in broth, milk, and peach and orange juices. Journal of Food Engineering, 62, 47–52. Fox, P. F. (2003). Milk proteins: General and historical aspects. In P. F. Fox, & P. L. H. McSweeney (Eds.), Advanced dairy chemistry, Volume 1: Proteins (3rd ed.) (pp. 1–48). New York, USA: Kluwer Academic/ Plenum Publishers. Furukawa, S., Noma, S., Shimoda, M., & Hayakawa, I. (2002). Effect of initial concentration of bacterial suspensions on their inactivation by high hydrostatic pressure. International Journal of Food Science & Technology, 37, 573–577. Garcia-Graells, C., Masschalck, B., & Michiels, C. W. (1999). Inactivation of Escherichia coli in milk by high hydrostatic pressure treatment in combination with antimicrobial peptides. Journal of Food Protection, 62, 1248–1254. Hauben, K. J. A., Bernaerts, K., & Michiels, C. W. (1998). Protective effect of calcium on inactivation of Escherichia coli by hydrostatic pressure. Journal of Applied Microbiology, 85, 678–684. Hauben, K. J. A., Wuytack, E. Y., Soontjens, C. C. F., & Michiels, C. W. (1996). High-pressure transient sensitisation of Escherichia coli to lysozyme and nisin by disruption of outer-membrane permeability. Journal of Food Protection, 59, 350–355.
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Huppertz, T., Kelly, A. L., & Fox, P. F. (2002). Effects of high pressure on constituents and properties of milk: A review. International Dairy Journal, 12, 561–572. Huppertz, T., Zobrist, M. R., Uniacke, T., Upadhyay, V. K., Fox, P. F., & Kelly, A. L. (2005). Effects of high pressure treatment on some constituents and properties of buffalo milk. Journal of Dairy Research, 72, 226–233. IDF. (1990). Determination of total phosphorus content (Spectrometric method). Standard 42B. Brussels, Belgium: International Dairy Federation. Isaacs, N. S. (1981). Liquid phase high pressure chemistry. Chichester: John Wiley & Sons. Jack, R. W., Tagg, J. R., & Ray, B. (1995). Bacteriocins of Gram-Positive Bacteria. Microbiological Reviews, 59, 171–200. Jenness, R., & Koops, J. (1962). Preparation and properties of a salt solution which simulates milk ultrafiltrate. Netherlands Milk Dairy Journal, 16, 153–164. Jordan, S. L., Cascual, C., Bracey, E., & Mackey, B. M. (2001). Inactivation and injury of pressure-resistant strains of Escherichia coli O157 and Listeria monocytogenes in fruit juices. Journal of Applied Microbiology, 91, 463–469. Karatzas, K. A. G., & Bennik, M. H. J. (2002). Characterization of a Listeria monocytogenes Scott A isolate with high tolerance towards high hydrostatic pressure. Applied and Environmental Microbiology, 68, 3183–3189. Keenan, R. D., Hubbard, C. D., Mayes, D. M., & Tier, C. M. (2003). Role of calcium phosphate in the high-pressure-induced gelation of milk. In E. Dickinson, & T. Van Vliet (Eds.), Food colloids, biopolymers and materials (pp. 109–118). Cambridge, UK: The Royal Society of Chemistry. Linton, M., McClements, J. M. J., & Patterson, M. F. (1999). Inactivation of Escherichia coli O157:H7 in orange juice using a combination of high pressure and mild heat. Journal of Food Protection, 62, 277–279. Lucey, J. A., Hauth, B., Gorry, C., & Fox, P. F. (1995). The acid–base buffering properties of milk. Milchwissenshaft, 48, 268–272. Manas, P., Pagan, R., Sala, F. J., & Condon, S. (2001). Low molecular weight milk whey components protect Salmonella senftenberg 775W against heat by a mechanism involving divalent cations. Journal of Applied Microbiology, 91, 871–877. Marshall, W. L., & Frank, E. U. (1981). Ion product of water substance, 0–1000 1C, 1–10000 bars. New International Formulation and 1st background. Journal of Physical and Chemical Reference Data,, 10, 295–304. Masschalck, B., Garcia-Graells, C., Van Haver, E., & Michiels, C. W. (2000). Inactivation of high pressure resistant Escherichia coli by lysozyme and nisin under high pressure. Innovative Food Science and Emerging Technologies, 1, 39–47. McClements, J. M. J., Patterson, M. F., & Linton, M. (2001). The effect of growth stage and growth temperature in high hydrostatic pressure inactivation of some psychrotrophic bacteria in milk. Journal of Food Protection, 64, 514–522. Molina-Gutierrez, A., Stippl, V., Delgado, A., Ganzle, M. G., & Vogel, R. F. (2002). In situ determination of the intracellular pH of Lactococcus lactis and Lactobacillus plantarum during pressure treatment. Applied and Environmental Microbiology, 68, 4399–4406. Molina-Hoppner, A., Doster, W., Vogel, R. F., & Ganzle, M. (2004). Protective effect of sucrose and sodium chloride for Lactococcus lactis
during sublethal and lethal high-pressure treatments. Applied and Environmental Microbiology, 70, 2013–2020. Morild, E. (1981). The theory of pressure effects on enzymes. Advances in Protein Chemistry, 34, 93–166. Mulvihill, D. M., & Ennis, M. P. (2003). Functional milk proteins: Production and utilization. In P. F. Fox, & P. L. H. McSweeney (Eds.), Advanced dairy chemistry Volume 1: Proteins (3rd ed.) (pp. 1175–1228). New York, USA: Kluwer: Academic/Plenum Publishers. Pagan, R., & Mackey, B. (2000). Relationship between membrane damage and cell death in pressure-treated Escherichia coli cells: Differences between exponential- and stationary-phase cells and variation among strains. Applied and Environmental Microbiology, 66, 2829–2834. Palou, E., Lopez-Malo, A., Barbosa-Canovas, G. V., Welti-Chanes, J., & Swanson, B. G. (1997). Effect of water activity on high hydrostatic pressure inhibition of Zygosaccharomyces bailii. Letters in Applied Microbiology, 24, 417–420. Patterson, M. F., Margey, D. M., Mills, G., Simpson, R., & Gilmour, A. (1997). The effect of high hydrostatic pressure treatment on microorganisms in foods. In K. Heremans (Ed.), High pressure research in the biosciences and biotechnology (pp. 269–272). Leuven, Belgium: Leuven University Press. Patterson, M. F., Quinn, M., Simpson, R., & Gilmour, A. (1995). Sensitivity of vegetative pathogens. In D. A. Ledward, D. E. Johnston, R. G. Earnshaw, & &A. M. P. Hastings (Eds.), High pressure processing of foods (pp. 47–63). Nottingham, England: Nottingham University Press. Quinlan, R. J., & Reinhart, G. D. (2005). Baroresistant buffer mixtures for biochemical analysis. Analytical Biochemistry, 341, 69–76. Shearer, A. E. H., Dunne, C. P., Sikes, A., & Hoover, D. G. (2000). Bacterial spore inhibition and inactivation in foods by pressure, chemical preservatives and mild heat. Journal of Food Protection, 63, 1503–1510. Simpson, R. K., & Gilmour, A. (1997). The effect of high hydrostatic pressure on Listeria monocytogenes in phosphate-buffered saline and model food systems. Journal of Applied Microbiology, 83, 181–188. Singh, H., McCarthy, O. J., & Lucey, J. A. (1997). Physico-chemical properties of milk. In P. F. Fox (Ed.), Advanced dairy chemistry, Volume 3: Lactose, water, salts and vitamins (2nd ed.) (pp. 469–518). New York, USA: Chapman & Hall. Smiddy, M., O’Gorman, L., Sleator, R. D., Kerry, J. P., Patterson, M. F., Kelly, A. L., & Hill, C. (2005). Greater high-pressure resistance of bacteria in oysters than in buffer. Innovative Food Science & Emerging Technologies, 6, 83–90. Solomon, E. B., & Hoover, D. G. (2004). Inactivation of Campylobacter jejuni by high hydrostatic pressure. Letters in Applied Microbiology, 38, 505–509. Styles, M. F., Hoover, D. G., & Farkas, D. F. (1991). Response of Listeria monocytogenes and Vibrio parahaemolyticus to high hydrostatic pressure. Journal of Food Science, 56, 1404–1407. Van Opstal, I., Vanmuysen, S. C. M., & Michiels, C. W. (2003). High sucrose concentration protects E. coli against high pressure inactivation but not against high pressure sensitisation to the lactoperoxidase system. International Journal of Food Microbiology, 88, 1–9. Wouters, P. C., Glaasker, E., & Smelt, J. P. P. M. (1998). Effects of high pressure on inactivation kinetics and events related to proton efflux in Lactobacillus plantarum. Applied and Environmental Microbiology, 64, 509–514.
International Dairy Journal 17 (2007) 104–110 www.elsevier.com/locate/idairyj
Baroprotection of vegetative bacteria by milk constituents: A study of Listeria innocua Elaine P. Blacka,b, Thom Huppertzb, Gerald F. Fitzgeralda,b, Alan L. Kellyb, a
Department of Microbiology, University College Cork, Ireland Department of Food and Nutritional Sciences, University College Cork, Ireland
b
Received 24 June 2005; accepted 11 January 2006
Abstract The baroprotective effect of milk constituents on Listeria innocua 4202 treated at 350 or 500 MPa for 5 min was examined. Highpressure (HP) treatment of L. innocua 4202 (1 109 cfu mL 1) resulted in complete inactivation in simulated milk ultra-filtrate (SMUF), a 5.0 log reduction in phosphate-buffered saline and a 2.9 log reduction in milk. The addition of micellar casein to SMUF increased survival of the bacterium by 3 logs, compared with SMUF alone, but the protective effect was negated if the minerals associated with the casein micelles were removed. The colloidal minerals calcium (30 mM), magnesium (5 mM), citrate (10 mM) and phosphate (20 mM), suspended in SMUF increased survival by 3.3, 1.7, 3.3 and 3.5 logs, respectively. The buffering capacity of the suspending medium was found to be a key factor in microbial baroresistance. Buffering by phosphate and citrate in milk may protect microorganisms against changes in pH during HP treatment, whereas the divalent cations calcium and magnesium may protect cell membranes against HP. r 2006 Elsevier Ltd. All rights reserved. Keywords: High pressure; Milk; Listeria innocua; Colloidal calcium phosphate
1. Introduction High-pressure (HP) processing can potentially deliver food products that meet many of the demands of today’s consumer, i.e., high-quality, minimally-processed foods that are microbiologically safe and have a long shelf-life. The efficacy of HP-induced inactivation of microorganisms depends on a range of factors, related to the microorganism itself, the conditions of treatment and to the medium in which the microorganisms are suspended. While some of these factors have been identified, fundamental understanding of the mechanisms of baroresistance of microorganisms is still poorly developed. Factors that affect HPinduced inactivation of microorganisms include the applied pressure, treatment time and temperature (Patterson, Margey, Mills, Simpson, & Gilmour, 1997), the bacterial strain (Alpas, Kalchayanand, Bozoglu, Sikes, Dunne, & Ray, 1999; Benito, Ventoura, Casadei, Robinson, & Corresponding author. Tel.: +353 21 4903405; fax: +353 21 4270001.
E-mail address: [email protected] (A.L. Kelly). 0958-6946/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2006.01.009
Mackey, B. 1999), cell density (Furukawa, Noma, Shimoda, & Hayakawa, 2002), growth phase (Karatzas & Bennik, 2002; McClements, Patterson, & Linton, 2001; Pagan & Mackey, 2000), growth temperature (McClements et al., 2001; Simpson & Gilmour, 1997) and suspending medium (Palou, Lopez-Malo, Barbosa-Canovas, Welti-Chanes, & Swanson, 1997; Patterson, Quinn, Simpson, & Gilmour, 1995; Simpson & Gilmour, 1997; Smiddy et al., 2005). To date, much research has focused on HP-induced microbial inactivation in buffer systems, but when such data are compared with those obtained in food systems, levels of inactivation are not always in agreement (Palou et al., 1997; Patterson et al., 1995; Smiddy et al., 2005; Styles, Hoover, & Farkas, 1991). Microbial baroresistance is usually higher in low-acid food products than in buffer systems (Simpson & Gilmour, 1997), but the mechanism(s) by which food products protect bacterial cells are not fully understood. The complex chemistry of individual foods is difficult to mimic in a buffer system, as a range of components and factors contribute to, or antagonize, baroprotection, e.g., water activity, pH (Jordan, Cascual,
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Bracey, & Mackey, 2001; Wouters, Glaasker, & Smelt, 1998), the presence of antimicrobial compounds (Black, Kelly, & Fitzgerald, 2005; Masschalck, Garcia-Graells, Van Haver, & Michiels, 2000) or additives (Shearer, Dunne, Sikes, & Hoover, 2000), as well as individual food constituents, such as fat, proteins, solutes (Molina-Hoppner, Doster, Vogel, & Ganzle, 2004) and minerals (Cheftel, 1995; Hauben, Bernaerts, & Michiels, 1998; Van Opstal, Vanmuysen, & Michiels, 2003). Bovine milk is a useful model for investigating the protective effect of foods against pressure, because its chemical composition is well known and many recent studies have added to our knowledge of the effects of HP treatment on the constituents of milk (Huppertz, Kelly, & Fox, 2002). Milk exerts a strong protective effect, compared to buffer systems, when Listeria monocytogenes or E. coli are subjected to HP (Patterson et al., 1995; Simpson & Gilmour, 1997; Styles et al., 1991); previous studies have suggested that the presence of calcium ions (Hauben et al., 1998) or solutes (Simpson & Gilmour, 1997) may confer protection, but the specific baroprotective nature of milk is poorly understood. In the present study, the constituents of milk such as lactose, minerals, whey proteins and casein were examined for a possible baroprotective effect on L. innocua. The casein micelle was of particular interest as a number of potential protective agents (e.g. calcium) are associated with it. The dry matter of casein micelles consists of 94% protein plus 6% minerals; these minerals, predominantly calcium, phosphate, citrate and magnesium, are collectively referred to as colloidal calcium phosphate (CCP; Fox, 2003). The objective of the study was to identify the individual constituent or constituents of milk that provide a specific baroprotective effect on L. innocua, and thereby gain understanding of the mechanisms of protection. 2. Materials and methods 2.1. Preparation of test media Skimmed pasteurized bovine milk was obtained from CMP Dairies (Cork, Ireland). CCP-adjusted milk samples were prepared by adjusting the pH of the milk to 4.6 (CCP free), 5.0, 5.5 or 6.0 (CCP depleted by 85%, 40% or 5%, respectively) or 8.0 or 9.0 (CCP enriched by 30% or 60% respectively) followed by dialysis against 2 20 volumes of bulk milk for 48 h at 5 1C. Simulated milk ultrafiltrate (SMUF) was prepared as described by Jenness and Koops (1962). SMUF contains Na, K, Ca, Mg, PO4, Cl, citrate, SO4 or CO2 at a level of 18.3, 39.4, 9.0, 3.2, 11.6, 32.4, 9.6, 1.0 or 2.2 mmol L 1, respectively. Phosphocasein (Dairy Products Research Centre, Moorepark, Fermoy, Ireland), sodium caseinate (Dairygold Co-op., Mitchelstown, Ireland), b-lactoglobulin, a-lactose, tris-hydroxymethiomethylamine (Tris), calcium chloride, magnesium chloride, trisodium citrate and dipotassium hydrogen phosphate (all from Sigma Chemical
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Table 1 Suspending media used for HP treatment of L. innocua 4202 Test media
Description
PSM SMUF SMUF-PhosCN
Pastuerized skim milk Simulated milk ultrafiltrate SMUF containing 25 or 30 g L 1 added phosphocasein SMUF containing 25 g L 1 added sodium caseinate Phosphate-buffered saline adjusted to pH 6.6 SMUF containing 45 or 60 g L 1 added lactose SMUF containing 3 g L 1 added b-lactoglobulin SMUF containing 5 mM added MgCl2 SMUF containing 30 mM added CaCl2 SMUF containing 10 mM added Na3Citrate SMUF containing 20 mM added K2HPO4 0.1 M Tris-hydroxymethiomethylamine (Tris) adjusted to pH 6.6 Tris containing 5 mM added MgCl2 Tris containing 30 mM added CaCl2
SMUF-NaCN PBS SMUF-Lac SMUF-b-lg MUF-Mg SMUF-Ca SMUF-Cit SMUF-P Tris Tris-Mg Tris-Ca
Corp., St. Louis, USA) were added to milk, 0.1 m Tris or SMUF, as outlined in Table 1. 2.2. Bacterial strains, culture conditions and suspending media L. innocua 4202 was obtained from the University College Cork culture collection and grown on Tryptic Soy Agar supplemented with 0.5% yeast extract (TSA-YE) at 37 1C. Working cultures were prepared by transferring one colony from a TSA-YE plate to Tryptic Soy Broth containing 0.5% yeast extract (TSB-YE) and grown overnight to stationary phase (1 109 cfu mL 1) prior to HP treatment. The cells were centrifuged at 1677 g for 5 min and resuspended at a level of 1 109 cfu mL 1 in a range of test media (Table 1). 2.3. Determination of the level of total or colloidal calcium and phosphate in milk The level of calcium in milk or its ultracentrifugal supernatant (100,000 g for 60 min at 20 1C) was determined as outlined by Huppertz et al. (2005). The level of inorganic phosphate in milk or its ultracentrifugal supernatant was determined as outlined by IDF (1990). Levels of colloidal calcium or phosphate were calculated by subtracting the level of calcium or phosphate in the ultracentrifugal supernatant from that in the milk, after correction for the volume of the ultracentrifugal supernatant. 2.4. HP treatment Samples of L. innocua were suspended in test media and dispensed into 1 3 cm sterile polyethylene bags, heat-sealed while excluding as much air as possible,
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double-packaged in larger bags and vacuum-packed. Samples were HP-treated in a Stansted Fluid Power IsoLab 900 High Pressure Food Processor (Stansted Fluid Power Ltd., Stansted, Essex, UK), using a 90:10 mixture of ethanol and castor oil as the pressure-transmitting medium, at 350 or 500 MPa for 5 min. HP treatment was carried out at 20 1C, by thermostatically controlling the vessel of the HP unit. Adiabatic heating on compression resulted in an increase in temperature of the processing fluid of approximately 15 1C at 500 MPa; most of this heat dissipated during the holding time of 5 min. A control sample was held at atmospheric pressure and 20 1C for the duration of treatment. All experiments were replicated in triplicate throughout the entire study, and results presented are means of data from three replications, with standard deviations where appropriate.
3.2. Influence of CCP on HP-induced inactivation of L. innocua Partial or complete removal of micellar minerals from milk, had a large effect on the HP-induced inactivation of L. innocua; complete inactivation of L. innocua was observed in milk containing o15% of its original micellar calcium and phosphate content, and the level of survival of L. innocua progressively increased with increasing CCP content of the milk, up to its original content (Fig. 1). Further increasing the CCP content of milk gave no further baroprotection to L. innocua. The principal constituent minerals of CCP, i.e., phosphate, citrate, calcium and magnesium, were also added to SMUF individually, up to levels commonly found in milk (20, 10, 30 or 5 mmol L 1, respectively). All four minerals provided substantial baroprotection to L. innocua (Fig. 2). The strongest
2.5. Enumeration of viable cells Appropriate decimal dilutions of L. innocua suspensions were spread-plated or pour-plated immediately after pressure treatment on TSA-YE plates and incubated for 48 h at 37 1C. The results shown are the means and standard deviations of data from triplicate independent experiments. 2.6. Studies on membrane protection To assess protection of cell membranes by milk constituents, samples of L. innocua suspended in Tris, Tris-Ca or Tris-P (Table 1) and treated at a sublethal pressure of 350 MPa were plated on TSA-YE agar supplemented with nisin (100 IU mL 1) as described by Hauben, Wuytack, Soontjens, & Michiels (1996) or bile salts (0.3%), as described by Manas, Pagan, Sala, & Condon (2001).
Table 2 Survivala of L. innocua 4202 suspended in test media and treated at 500 MPa for 5 min at 20 1C Test Mediab
Survivalc (Log cfu mL 1)
Initial innoculum PBS PSM SMUF SMUF-PhosCN (25 g L 1) SMUF-PhosCN (30 g L 1) SMUF-NaCN (25 g L 1) SMUF-Lac (45 g L 1) SMUF-Lac (60 g L 1) SMUF-b-lg (3 g L 1)
8.90 3.8970.09 5.9870.37 o1.0 2.5070.45 3.5070.08 o1.0 o1.0 o1.0 o1.0
a Data are means (7standard deviation) of results of triplicate experiments. b Refer to Table 1 for abbreviations. c Limit of detection—1.0 log cfu mL 1.
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3.1. Influence of milk constituents on HP-induced inactivation of L. innocua After HP treatment at 500 MPa for 5 min, the level of surviving L. innocua in milk was 5.98 log, 2 log units higher than that in PBS (3.89 log), whereas a reduction to an undetectable level was observed in SMUF (Table 2). The apparent protective effect of milk was further investigated by using SMUF as a base medium to which constituents of milk were added individually, to determine their specific influence on HP-induced inactivation of L. innocua. Adding lactose (45 or 60 g L 1), b-lactoglobulin (3 g L 1) or sodium caseinate (25 g L 1) to SMUF prior to HP treatment did not influence HP-induced inactivation of L. innocua (Table 2), whereas adding phosphocasein (25 or 30 g L 1) provided baroprotection of 3 logs.
Survival (Log cfu mL-1)
3. Results 6
4
2
0 0
20 40 60 80 100 120 140 160 Colloidal calcium or phosphate as % of value in control milk
Fig. 1. Influence of the level of micellar calcium (K) or micellar phosphate (J) in milk on the survival of L. innocua 4202 (initial level of 1 109 cfu mL 1) following treatment at 500 MPa for 5 min. Dashed line indicates detection limit of L. innocua 4202.
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8 Survival (Log cfu mL-1)
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6
4
2
107
6
4
2 NS
0
0 +Calcium
+ Citrate + Magnesium + Phosphate
Milk
Fig. 2. Influence of calcium (30 mM), magnesium (5 mM), phosphate (10 mM) or citrate (20 mM), added to simulated milk ultrafiltrate, on survival of L. innocua 4202 (initial level of 1 109 cfu mL 1) following treatment at 500 MPa for 5 min. NS denotes samples where no survival was detected. The dashed line indicates the detection limit of L. innocua 4202. Values are means of data from triplicate independent experiments, with the standard deviation indicated by vertical error bars.
protective agents were calcium, phosphate and citrate, providing 43 log protection compared with control SMUF; magnesium, added at a level of 5 mM, gave a lower level of protection (1.7 log). Increasing the concentration of magnesium in SMUF to 30 mM gave similar protection to that provided by calcium (30 mM; data not shown). 3.3. Influence of buffering capacity on HP-induced inactivation of L. innocua in milk Survival of L. innocua following treatment at 500 MPa for 5 min when suspended in 0.1 m Tris-HCl (pH 6.6), a pressure-stable buffer (Morild, 1981; Quinlan & Reinhart, 2005), was 5.2 log, compared with 3.4 log in PBS (pH 6.6), which is less pressure-stable (Morild, 1981; Quinlan & Reinhart, 2005). This suggests that maintaining a constant pH under pressure reduced inactivation of this bacterium. To test this hypothesis, Tris-HCl (0.1 mol L 1) was added to milk and it was found that this increased the protection of L. innocua, by 41.5 log (Fig. 3). Adding calcium (30 mM), phosphate (20 mM), magnesium (5 mM) or citrate (10 mM) to 0.1 m Tris-HCl buffer, pH 6.6, did not further increase the already strong baroprotective effect of Tris (Fig. 4). 3.4. Protection of the L. innocua against sub-lethal injury L. innocua suspended in Tris-HCl buffer or in Tris-HCl supplemented with calcium or phosphate was subjected to a sublethal pressure of 350 MPa for 5 min at 20 1C. The effect of this pressure treatment was examined by comparing growth of L. innocua following this pressure treatment on TSA-YE or TSA-YE containing either 0.3% bile salts
PBS
Tris
Milk
Milk + Tris
Fig. 3. Survival of L. innocua 4202 (initial level of 1 109 cfu mL 1), suspended in phosphate-buffered saline, 0.1 M Tris-HCl, milk or milk containing 0.1 M added Tris-HCl (all pH 6.6), following treatment at 500 MPa for 5 min. The dashed line indicates the detection limit of L. innocua 4202. Values are means of data from triplicate independent experiments, with the standard deviation indicated by vertical error bars.
10
8 Survival (Log cfu mL-1)
SMUF
6
4
2
0 Tris-HCl
+Calcium
+ Citrate + Magnesium + Phosphate Milk
Fig. 4. Influence of micellar minerals added to Tris-HCl on survival of L. innocua 4202 (initial level of 1 109 cfu mL 1) following treatment at 500 MPa for 5 min. The dashed line indicates the detection limit of L. innocua 4202. Values are means of data from triplicate independent experiments, with the standard deviation indicated by vertical error bars.
or nisin (500 IU mL 1). The concentrations of bile salts or nisin used did not decrease numbers of untreated cells (data not shown). When calcium was present in the suspending medium during HP treatment, it protected the cells by 1 log from bile salts or nisin contained in the plating medium. If calcium was added to cells following HP treatment, no protection was observed (data not shown). This suggests that calcium protects L. innocua during pressure treatment rather than acting post pressure treatment in some way. The presence of phosphate in the suspending medium conferred no protection to the effects of bile or nisin (Fig. 5).
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10
Survival (Log cfu mL-1)
8
6
4
2
0 HP
HP + Bile Salts
HP + Nisin
Fig. 5. Influence of calcium ( ) or phosphate (&) on survival of L. innocua 4202 (initial level of 1 109 cfu mL 1) following treatment at 350 MPa for 5 min and plating on bile-salts- or nisin-supplemented agar. The black bar (’) represents the control sample plated on TSA-YE without additions. The dashed line indicates the detection limit of L. innocua 4202. Values are means of data from triplicate independent experiments, with the standard deviation indicated by vertical error bars.
4. Discussion Data presented in this communication indicate a strong protective effect of milk constituents against HP-induced inactivation of L. innocua 4202; other authors have previously commented on the strong protection milk confers to other microorganisms against HP (Dogan & Erkmen, 2004; Garcia-Graells, Masschalck, & Michiels, 1999; Hauben et al., 1998; Solomon & Hoover, 2004; Styles et al., 1991). However, little was known previously about either the specific protective agents in milk or the mechanisms by which they protect. Initial experiments were performed to identify the most likely constituents of milk to impart resistance to L. innocua against HP. It has been previously reported that the presence of solutes such as sugars at high levels, i.e., 410%, increases microbial baroresistance (Palou et al., 1997; Van Opstal et al., 2003); therefore, it was perhaps not surprising that SMUF containing lactose, the most abundant sugar in milk, at concentrations similar to, or higher than, that in milk, i.e. 4.5% or 6%, did not provide baroprotection to L. innocua (Table 2). Furthermore, no baroprotection of L. innocua was observed when the whey protein b-lactoglobulin was added to SMUF (Table 2). Adding 25 or 30 g L 1 micellar casein (phosphocasein) to SMUF provided 2.5 or 3.5 logs protection, respectively, but when casein was added to SMUF in non-micellar form (sodium caseinate), complete HP-induced inactivation of L. innocua was observed (Table 2). These results suggest that, while the protective effect of milk lies, at least partially, in the casein fraction of milk, it cannot be attributed to the proteins themselves; rather, it is likely that such protection is provided by the minerals associated with the micelle, as these are the only constituents present
in phosphocasein, but absent from sodium caseinate (Mulvihill & Ennis, 2003). The minerals calcium, phosphate, citrate and magnesium, are collectively known as CCP. CCP exists predominantly in the form of amorphous nanoclusters with a core of 3 nm, around which a shell of caseins is attached through phosphoseryl–calcium interactions (De Kruif & Holt, 2003). Calcium, the main cation in CCP, is present at a level of 30 mM in milk, of which 70% is the form of CCP; calcium has been shown previously to confer baroprotection to E. coli in Tris-HCl buffer, as has magnesium (Hauben et al., 1998), also a constituent of CCP. Removal of CCP from milk by dialysis was shown to completely diminish its baroprotective capabilities (Fig. 1); L. innocua was completely inactivated by HP when suspended in CCP-free milk, while protection against HPinduced inactivation increased with CCP content of the milk (Fig. 1). It is unlikely that minerals can confer baroprotection in their amorphous form, i.e., in the form of CCP nanoclusters, but the solubility of minerals is generally increased under HP, due to the electrostrictive effect of separate charges (Isaacs, 1981), i.e., the arrangement of water molecules around ions is more structured and dense than around uncharged ion pairs. To counteract the HP-induced reductions in volume of the system, the more dense organization of water molecules around ions, and thus ionization, is favoured. Hence, HP treatment results in increased solubility of calcium phosphate, as measured by Huppertz, Kelly, and De Kruif (unpublished data); furthermore, Keenan, Hubbard, Mayes, & Tier (2003) observed that the level of non-micellar phosphate in milk increases under HP. An increased level of minerals in the serum phase of milk may thus provide baroprotection to microbes therein. Further studies were performed to identify if baroprotection of L. innocua is provided by all, or only specific, micellar minerals. Fig. 2 illustrates that adding additional calcium, magnesium, citrate or phosphate (in the form of calcium chloride, magnesium chloride, tri-sodium citrate or di-potassium hydrogen phosphate, respectively) to SMUF individually allowed between 1.7 and 3.5 log survival of L. innocua after treatment at 500 MPa, suggesting that the baroprotective effect of CCP is not related to a single mineral constituent, but is, in fact, exerted by all micellar minerals studied. One mechanism by which the micellar minerals may influence HP-induced inactivation of L. innocua is through affecting the buffering capacity of milk. Citrate, and in particular, phosphate, provide considerable buffering capacity to the milk serum (Lucey, Hauth, Gorry, & Fox, 1995; Singh, McCarthy, & Lucey, 1997). Treatment at 1000 MPa reduces the pH of water by 1.0 unit (Marshall & Frank, 1981), so it is likely that the pressure applied in the present study (500 MPa) also reduces milk pH considerably; Molina-Gutierrez, Stippl, Delgado, Ganzle, and Vogel (2002) calculated the pH of milk serum to be reduced by one full unit at 300 MPa, although experimental proof for this extensive reduction in pH was not reported.
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Exposure to low pH combined with HP can increase inactivation synergistically (Alpas, Kalchayanand, Bozoglu, & Ray, 2000; Linton, McClements, & Patterson, 1999). While cells were very pressure-sensitive in SMUF, TrisHCl and milk gave 3.9 and 6.0 log protection, respectively. Tris is a pressure-stable buffer (Morild, 1981; Quinlan & Reinhart, 2005) which, when added to milk, increased its protective effect on L. innocua (Fig. 3) possibly by counteracting HP-induced reductions in pH. The micellar minerals citrate and phosphate have good buffering capacities (Lucey et al., 1995; Singh et al., 1997) and may therefore partially protect the suspended bacteria from the HP-induced reduction in milk pH. Addition of calcium, phosphate or citrate to Tris-HCl gave little additional protection to L. innocua against HP (Fig. 4). The main target of HP-induced inactivation of bacteria is the cell membrane (Pagan & Mackey, 2000); to study the potential protective influence of micellar minerals against HP-induced damage to the cell membrane, L. innocua was treated at a sub-lethal pressure (350 MPa), which weakens the cell membranes (Black et al., 2005), thereby sensitizing the cells to the effects of membrane-active agents, e.g., bile salts or the bacteriocin nisin. Nisin, active against Grampositive bacteria, acts by creating pores in the cellular membrane, which results in the loss of intracellular ions and disruption of the cells pH gradient and proton motive force, thereby leading to cell death (Jack, Tagg, & Ray, 1995). Bile salts cause increased permeability of the membrane and increased trans-membrane flux of divalent cations (Begley, Gahan, & Hill, 2005). The acquired sensitivity of bacteria to low levels of either bile or nisin in the plating medium following HP treatment may suggest sub-lethal injury (Hauben et al., 1996), which may be associated with damage of the cell membrane. When calcium (in the form of CaCl2) was added to the suspending medium during HP treatment, L. innocua appeared more resistant to nisin or bile salts in the plating medium (Fig. 5), indicating either a general protective effect or a specific baroprotective effect of added calcium. When calcium was added to the bacterial suspension after HP treatment, no protective effect was observed (data not shown), strongly suggesting that the protection provided by calcium is specifically baroprotective. Divalent cations, e.g., Ca2+ or Mg2+, have previously been shown to protect E. coli during HP treatment (Hauben et al., 1998). The protective effect, however, could not be attributed to stabilization of the outer membrane of E. coli. L. innocua, a Gram-positive bacterium, does not possess an outer membrane; therefore, Ca2+ may play a role in the stabilization of the cytoplasmic membrane during HP treatment in both Gram-positive and Gram-negative bacteria. Further work is needed to determine the exact nature of this protection. 5. Conclusion The results obtained in this study suggest that a combination of HP-induced solubilization of CCP, with a
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concomitant increase in the buffering capacity of milk, and stabilization of the membrane by divalent cations, may protect L. innocua against HP-induced inactivation. Further research is required, however, to confirm these hypotheses and to determine whether these mechanisms also apply to other vegetative bacterial cells in milk. Acknowledgments This research was funded by the Food Institutional Research Measure (FIRM), which is administered by the Irish Government under the National Development Plan 2000–2006, and through the NUI Travelling Studentship in Food Science and Technology awarded to E. P. Black. References Alpas, H., Kalchayanand, N., Bozoglu, F., & Ray, B. (2000). Interactions of high hydrostatic pressure, pressurization temperature and pH on death and injury of pressure-resistant and pressure-sensitive stains of foodborne pathogens. International Journal of Food Microbiology, 60, 33–42. Alpas, H., Kalchayanand, N., Bozoglu, F., Sikes, A., Dunne, C. P., & Ray, B. (1999). Variation in resistance to hydrostatic pressure among strains of food-borne pathogens. Applied and Environmental Microbiology, 65, 4248–4251. Begley, M., Gahan, C. G. M., & Hill, C. (2005). The interaction between bacteria and bile. FEMS Microbiology Reviews, 29, 625–651. Benito, A., Ventoura, G., Casadei, M., Robinson, T., & Mackey, B. (1999). Variation in resistance of natural isolates of Escherichia coli 0157 to high hydrostatic pressure, mild heat and other stresses. Applied and Environmental Microbiology, 65, 1564–1569. Black, E. P., Kelly, A. L., & Fitzgerald, G. F. (2005). The combined effect of high pressure and nisin on inactivation of microorganisms in milk. Innovative Food Science and Emerging Technologies, 6, 286–292. Cheftel, J. C. (1995). Review: High pressure, microbial inactivation and food preservation. Food Science and Technology International, 1, 75–90. De Kruif, C. G., & Holt, C. (2003). Casein micelle structure, functions and interactions. In P. F. Fox, & P. L. H. McSweeney (Eds.), Advanced dairy chemistry, Volume 1: Proteins (3rd ed.) (pp. 233–276). New York, USA: Kluwer Academic/Plenum Publishers. Dogan, C., & Erkmen, O. (2004). High pressure inactivation kinetics of Listeria monocytogenes inactivation in broth, milk, and peach and orange juices. Journal of Food Engineering, 62, 47–52. Fox, P. F. (2003). Milk proteins: General and historical aspects. In P. F. Fox, & P. L. H. McSweeney (Eds.), Advanced dairy chemistry, Volume 1: Proteins (3rd ed.) (pp. 1–48). New York, USA: Kluwer Academic/ Plenum Publishers. Furukawa, S., Noma, S., Shimoda, M., & Hayakawa, I. (2002). Effect of initial concentration of bacterial suspensions on their inactivation by high hydrostatic pressure. International Journal of Food Science & Technology, 37, 573–577. Garcia-Graells, C., Masschalck, B., & Michiels, C. W. (1999). Inactivation of Escherichia coli in milk by high hydrostatic pressure treatment in combination with antimicrobial peptides. Journal of Food Protection, 62, 1248–1254. Hauben, K. J. A., Bernaerts, K., & Michiels, C. W. (1998). Protective effect of calcium on inactivation of Escherichia coli by hydrostatic pressure. Journal of Applied Microbiology, 85, 678–684. Hauben, K. J. A., Wuytack, E. Y., Soontjens, C. C. F., & Michiels, C. W. (1996). High-pressure transient sensitisation of Escherichia coli to lysozyme and nisin by disruption of outer-membrane permeability. Journal of Food Protection, 59, 350–355.
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