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Keywords:

  • high-pressure;
  • milk;
  • Mycobacterium avium ssp. paratuberculosis;
  • pasteurization

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Aims:  To determine the effect of high pressures alone and in conjunction with pasteurization on the viability of two strains of Mycobacterium avium ssp. paratuberculosis (Map).

Methods and Results:  Map in a milk matrix was subjected to 400, 500 and 600 MPa with and without pasteurization (72°C for 15 s) and plated onto Herrold’s egg yolk medium (HEYM) and Middlebrook 7H10 (7H10) agar, both containing antibiotic supplements. Medium 7H10 was found to give a significantly (P < 0·001) better recovery than HEYM. A significantly greater (P < 0·001) reduction in viable numbers was observed using 500 MPa (mean log reduction of 6·52) compared with 400 MPa (mean log reduction of 2·56) and between 400 MPa and control (no applied pressure) for 10 min treatments. A treatment time of 10 min resulted in significantly (P < 0·001) fewer survivors than 5 min. Low numbers of survivors were still detected when pressure treatment at 400 and 600 MPa was combined with subsequent pasteurization.

Conclusions:  The use of high-pressure was effective in reducing viable numbers of Map but even when combined with pasteurization there were still survivors, albeit when high inoculum levels of Map were used.

Significance and Impact of the Study:  To the authors’ knowledge the work reported here represents the first study of the efficacy of high-pressure treatments alone and in combination with pasteurization to kill Map. The results indicate that further research is warranted before more commercial-scale studies are commissioned.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Mycobacterium avium ssp. paratuberculosis (Map) is the known cause of Johne’s disease in both domestic and wild ruminants and their predators (Beard et al. 1999, 2001a,b) and has been implicated as a cause of Crohn’s disease in humans (Naser et al. 2004). Crohn’s disease is a chronic inflammatory bowel disorder that commonly affects the terminal ileum but can occur in any part of the gastrointestinal tract from mouth to anus. At present there is no recognized cure, but sufferers can experience periods of remission. However, the quality of life of the sufferer and their immediate family is low (Irvine 1996). The British Government considered the evidence of a causal link between Map and Crohn’s disease sufficiently strong to advocate the precautionary principle and attempt to minimize exposure of the public to this organism (Rubery 2001).

The organism is excreted directly into milk in low numbers but in much higher numbers in the faeces of infected animals, particularly those showing overt clinical signs of Johne’s. Even with good hygiene some of the infected faeces inevitably gain access to raw milk. There is evidence (Grant et al. 1996) that Map can survive commercial pasteurization of milk (UK legal minimum 71·7°C for 15 s). The finding that extending the holding time rather than increasing the pasteurization temperature was effective in killing Map in a laboratory study using spiked milk samples (Grant et al. 1999) prompted much of the UK dairy industry to voluntarily modify their pasteurization conditions accordingly. However, more recent work by Grant et al. (2005), using spiked milk and a pilot-scale pasteurizer has shown that under certain conditions even the extended holding time is insufficient to kill Map, albeit when unrealistically high numbers were present in the raw milk.

There is a need therefore to investigate alternative processing strategies, either to replace pasteurization or to be used in conjunction with it to ensure milk is free of viable Map. Stabel (2003) reviewed some of the methods available for postharvest intervention in dairy processing which included pasteurization, pulsed electric fields, high hydrostatic pressure with mild heat, irradiation, fermentation and antimicrobials. In this review, specific examples of bacteria on which data on high-pressure had been generated included Escherichia coli O157:H7 and Staphylococcus aureus. There is clearly an absence of published information on the effect of high-pressure, one such alternative processing strategy, on Map suspended in a milk matrix.

In the work reported here two strains of Map were spiked into sterilized whole milk and subjected to high-pressure treatments (400, 500 and 600 MPa for up to 10 min at 20°C) with and without a subsequent laboratory heat pasteurization treatment. This, it was hoped, would provide sufficient data to determine whether further research was warranted, at a more commercial scale, into the use of high pressures as a realistic replacement for or as an adjunct to milk pasteurization.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Map strains

Two Map strains were tested: a type strain NCTC 8578 (obtained from the National Collection of Type Cultures, London, UK) and strain 806R, originally isolated from pasteurized cows’ milk (Grant et al. 2002; kindly provided by Dr I. Grant, School of Agriculture, Food and Land Use, Queen’s University of Belfast, Belfast, Northern Ireland, UK).

Preparation of inoculum for high-pressure/pasteurization treatment

Each strain was cultured in Middlebrook 7H9 broth medium containing 10% (vol/vol) Middlebrook OADC (oleic acid-albumin-dextrose-catalase; Becton Dickinson Ltd, Oxford, UK), 0·5% (vol/vol) Tween 80 (Sigma, Dorset, UK) and 0·0002% (wt/vol) mycobactin J (Synbiotics Europe SAS, Lyon, France) from 6-week-old (37°C) colonies grown on Herrold’s egg yolk medium (HEYM) containing 2 μg of mycobactin J per millilitre (Synbiotics Europe SAS). Duplicate culture broths (100 ml), for each strain, were incubated (with agitation, 100 rev min−1) at 37°C for 8–10 weeks. Cells were harvested by centrifugation (4000 g, 20 min) and resuspended in 5 ml of phosphate-buffered saline (PBS; Oxoid, Basingstoke, UK). Cell suspensions were agitated with a Vortex Genie 2 (Scientific Industries Inc., Bohemia, NY, USA) for 3 min to disperse large clumps of Map and added to 1800 ml of sterile whole milk – a bottled UHT product which is subsequently pressure/temperature treated (Pure Milk, Delamere Dairy, Cheshire, UK). This mixture was subsequently subdivided into 250-ml aliquots for high-pressure and/or pasteurization treatments. Inoculated samples were pressurized within 1 h of mixing. It is recognized that a longer mixing/equilibration time may have allowed the organism longer to adapt to their new menstruum and possibly influence the effect of subsequent treatments but 1 h was chosen for logistical reasons. The sterile milk matrix was tested for the presence of Map on the test media discussed next.

High-pressure treatment

The 250-ml aliquots of Map-spiked milk were packaged in a polyethylene/polyamide pouch (Somerville Packaging, Lisburn, UK) and sealed using a heat sealer (RS Components, Corby, Northants, UK), excluding as much air as possible. The samples were then prepared for pressure treatment by placing inside a larger polyethylene/polyamide bag and vacuum-packing using a vacuum packager (Henkelmans, Hertogenbosch, Netherlands). This was repeated so that the pouch was sealed in two outer packs.

Pressure treatment was carried out using a Stansted Food Lab 900 high-pressure isostat, capable of operating at 900 MPa (Stansted Fluid Power Ltd., Stansted, UK). The internal diameter of the pressure chamber was 70 mm and the height of the chamber was 203 mm. The pressure transmission fluid was 90% water : 10% Cooledge oil (Castrol, UK) and the temperature increase because of adiabatic heating was approximately 3°C per 100 MPa. The pressure come-up rate was approximately 200 MPa per min and the pressure release time was approximately 2 min. The milk was pressure treated at 400, 500 or 600 MPa for 5 or 10 min at 20°C. A 250-ml batch of milk was held at ambient pressure (0·1 MPa) at 20°C as an untreated control.

High-temperature short time pasteurization

A sterile (i.e. previously autoclaved) Franklin high-temperature short time pasteurization (HTST) pasteurising unit was placed in a Grant Type SB3 water bath operating at 72 ± 0·1°C and allowed to equilibrate to temperature for 5 min. A 250-ml aliquot of inoculated milk was poured into the unit via the inlet funnel and the milk heated for a total of 70 s (55 s come-up time + 15 s treatment time) after which the entire apparatus was transferred to a water bath attached to a thermostatic circulator (6°C). Once cooled, the heat-treated milk was decanted from the Franklin plates and decimal dilutions prepared as described next. In cases where a combined pressure and thermal treatment of the inoculated milk was performed, the latter was performed approximately 30 min after the former, with samples taken between treatments.

Microbiological analysis

All microbiological analyses were performed within 1 h of pressure or pasteurization treatment. Following pressurization and/or pasteurization, samples were vigorously shaken and 1-ml aliquots of treated spiked milks were decimally diluted in maximum recovery diluent (MRD; Oxoid). Where low numbers of Map survivors were expected, cells in a 10-ml aliquot from the treated sample were concentrated by centrifugation (4000 g, 20 min) and resuspended in 1 ml of MRD.

Appropriate diluents of all samples were inoculated (200 μl) and spread onto deep (40 ml) plates of each of the following media: HEYM supplemented with an antibiotic cocktail containing vancomycin (8·4 μg ml−1), amphotericin B (16·8 μg ml−1) and nalidixic acid (25 μg ml−1) (all Sigma) (HEYM-VAN); 7H10-PANTA agar consisting of Middlebrook 7H10 agar base (Becton Dickinson) supplemented with 10% (vol/vol) Middlebrook OADC enrichment, 2·0% (vol/vol) reconstituted PANTA PLUS antibiotic supplement (polymyxin, amphotericin B, nalidixic acid, trimethoprim and azlocillin; Becton Dickinson) and 0·5% vol/vol glycerol. A 0·5-ml aliquot of appropriate diluents was also injected into vials of BACTEC 12B (Becton Dickinson) radiometric medium. All culture media were supplemented with 2 μg ml−1 of mycobactin J. Agar plates were sealed with parafilm to minimize the drying of agar during incubation. All media were incubated at 37°C for up to 12 weeks and colonies typical of Map on the agar medium were counted. The plates were incubated for a further 6 weeks (18 weeks in total) but no further colonies were detected. Antibiotic supplements were used because the excessive number of manipulations required for each sample, including packaging, transport to and from the high-pressure facility and coupled with the protracted incubation period made the chance of contamination unacceptably high. A representative number of typical and atypical colonies were confirmed by Ziehl-Neelsen acid-fast staining and IS900 PCR (Moss et al. 1992). BACTEC vials were read regularly on a BACTEC 460 TB instrument (Becton Dickinson) and growth index values recorded.

Statistical analysis

anova analysis was performed with Genstat Release 6·1 (PC/Windows 2000).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

As the highest pressure used, i.e. 600 MPa resulted in only two samples with surviving Map above the limit of detection (1 CFU ml−1) they were not included in the statistical analysis. Pressures of 500 MPa resulted in a significantly greater reduction (P < 0·001) in viable numbers compared with 400 MPa with the latter also producing significantly greater reduction (P < 0·001) compared with the control (no pressure applied; Table 1). Overall no significant difference (P = 0·611) in response to pressure was found between the strains (806R, NCTC 8578). Combining results for both strains the mean log reductions in viable numbers at 400 MPa for 5 and 10 min and 500 MPa for 5 and 10 min were 0·85, 2·56, 5·03 and 6·52, respectively. Duration of treatment was shown to have a significant effect (P = 0·048) with 10 min consistently resulting in greater reductions in viable numbers (Table 1). Pasteurization, even without the application of high-pressure, resulted in two samples with detectable survivors in 7H10 medium (Table 1). Additionally survivors were still detected after treatment at both 400 and 600 MPa followed by pasteurization. The instances of recovery were greatest on the 7H10 medium (Table 1). In each of these instances Map was also detectable using BACTEC 12B medium.

Table 1.   Impact of high pressure alone and in combination with high-temperature short time (HTST) pasteurization on inactivation of Mycobacterium avium ssp. paratuberculosis (Map) in milk
Pressure/pasteurization TreatmentAverage* log10 CFU 10 ml−1 (±SD)
Middlebrook 7H10/PANTAHEYM/VAN
NCTC 8578806RNCTC 8578806R
  1. *Duplicate experiments performed for each strain at each treatment.

  2. †Where no SD value is given only a single count was recorded above the minimum detection level.

  3. ‡No count recorded above minimum threshold level.

  4. HEYM/VAN, Herrold’s egg yolk medium/vancomycin; PANTA, polymyxin, amphotericin B, nalidixic acid, trimethoprim and azlocillin; SD, standard deviation.

No pressure applied (control)7·43(0·13)7·77(0·15)6·35(0·21)7·40(0·00)
400 MPa 5 min6·63(0·32)6·87(0·52)4·60(2·04)5·38(0·76)
400 MPa 10 min5·22(2·38)4·87(0·66)4·52(2·24)3·72(2·28)
400 MPa 5 min + past2·08† 2·69 <1·0 2·84 
500 MPa 5 min3·49(0·54)2·79 2·74(1·61)1·87(1·23)
500 MPa 10 min1·85 1·48 2·72 <1·0 
500 MPa 5 min + past<1·0‡(0·00)<1·0 <1·0 <1·0 
600 MPa 5 min<1·0(0·00)<1·0 <1·0 <1·0 
600 MPa 10 min1·30 1·0 <1·0 <1·0 
600 MPa 5 min + past1·8 <1·0 <1·0 <1·0 
HTST pasteurization only<1·0 3·47(0·81)<1·0 <1·0 

Sealed Petri plates rather than slopes of the respective media, used in previous heat resistance studies with Map in this laboratory, were employed and enabled a more accurate viable count to be obtained. Using this method the 7H10 medium gave significantly greater recovery (P < 0·001) than HEYM after high-pressure processing for both strains tested (Table 1).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Map has been shown to survive pasteurization heat treatments applied to milk (Grant et al. 1998), although the lethality of the heat process was originally calculated using the closely related Mycobacterium bovis and Coxiella burnetii as reference organisms (Zall 1990). It has been suggested that one of the contributory factors to this apparent heat resistance is Map’s propensity to form large clumps, with the cells at the periphery protecting those at the centre of such clumps (Rowe et al. 2000). High-pressure acts in an isostatic manner so that all parts of the food, and presumably cells within clumps, are subjected to the same pressure at exactly the same time (Patterson 2005). This, it was considered, would nullify, to some extent, effects of the clumping phenomenon and make all Map cells equally susceptible to such a process. It was also hoped that as high pressures exert their effect principally on the cytoplasmic membranes of bacteria (Paul and Morita 1971) any contribution to resistance imposed by the thick hydrophobic cell wall of Map would also be reduced.

In the work reported here stationary rather than exponential growth phase cells were used for two main reasons. First, it is known that bacteria in the environment remain mainly in the stationary growth phase (Kolter 1999) and hence the likelihood is that Map-infected raw milk would be contaminated with stationary phase cells. Second, stationary phase bacterial cells are more resistant to high pressures than exponential phase cells (Pagan and Mackey 2000) and hence the responses observed would represent a worst case scenario.

The significant improvement in Map recovery on 7H10 medium compared with HEYM following high-pressure treatment in this study is in agreement with Lopez-Pedemonte et al. (2006) who also found better recovery of two Map strains on agar-solidified 7H9 medium compared with HEYM after high-pressure processing.

As the highest pressure used, i.e. 600 MPa without heat treatment resulted in only two samples with surviving Map above the limit of detection (1 CFU ml−1) they were not included in the statistical analysis. The observation in the work reported here that 500 MPa resulted in a significantly greater reduction in viable numbers than 400 MPa compared with the control (Table 1) is not in agreement with Lopez-Pedemonte et al. (2006) who found a significant difference (P < 0·05) in viable Map after 500 MPa but not 400 MPa compared with their control (0·1 MPa). In addition, Lopez-Pedemonte et al. (2006) found overall (300, 400, 500 MPa) a significant difference (P < 0·0006) between the strains (3644/02, ATCC 19698), although complicated by a strong interaction between strain and culture medium. However, with the strains used in the work reported here (806R, NCTC 8578) there was no overall significant difference (= 0·611). This may be because of the small number of strains used, as variations in pressure difference between strains of the same species is common (Patterson 2005).

The log reduction in viable numbers of 6·52 after treatment at 500 MPa for 10 min was higher than the 4 log reduction obtained by Lopez-Pedemonte et al. (2006). This may be because of strain differences and differences in the operating characteristics of the high-pressure rigs used in each case. As many authors (Meylan et al. 1996; Grant et al. 1996, 2001; Gao et al. 2002) estimate that bulk raw milk can contain 104–105 Map cells per millilitre results reported here that a pressure of at least 500 MPa would be required to reliably treat raw milk in the absence of an additional pasteurization stage.

The fact that duration of treatment had a significant effect (Table 1) indicates that both severity of high pressure and duration of treatment represent two of the variables that should be considered when optimizing processing conditions. Another factor that should be considered, but was not addressed in this study, is the temperature during pressure treatment, as both mild heat and reduced temperatures may enhance the lethality of pressure treatment (Patterson 2005).

The observation that even with the highest pressure employed (600 MPa) followed by pasteurization did not ensure complete destruction of Map, albeit when high inoculum levels were used, indicates that more data are required before a more definitive conclusion regarding the lethality of high pressure alone or in conjunction with pasteurization can be reached and more commercial-scale studies performed.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The authors wish to thank Dr D. Kilpatrick of the Biometrics Branch of the Agri-Food Biosciences Institute (AFBI) for the statistical analysis and Mrs. J. Johnston (AFBI) for excellent technical assistance.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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