Pasteurization of milk: the heat inactivation kinetics of milk-borne dairy pathogens under commercial-type conditions of turbulent flow.

This is the first study to report kinetic data on the survival of a range of significant milk-borne pathogens under commercial-type pasteurization conditions. The most heat-resistant strain of each of the milk-borne pathogens Staphylococcus aureus, Yersinia enterocolitica, pathogenic Escherichia coli, Cronobacter sakazakii (formerly known as Enterobacter sakazakii), Listeria monocytogenes, and Salmonella was selected to obtain the worst-case scenario in heat inactivation trials using a pilot-plant-scale pasteurizer. Initially, approximately 30 of each species were screened using a submerged coil unit. Then, UHT milk was inoculated with the most heat-resistant pathogens at ~10(7)/mL and heat treated in a pilot-plant-scale pasteurizer under commercial-type conditions of turbulent flow for 15s over a temperature range from 56 to 66°C and at 72°C. Survivors were enumerated on nonselective media chosen for the highest efficiency of plating of heat-damaged bacteria of each of the chosen strains. The mean log(10) reductions and temperatures of inactivation of the 6 pathogens during a 15-s treatment were Staph. aureus >6.7 at 66.5°C, Y. enterocolitica >6.8 at 62.5°C, pathogenic E. coli >6.8 at 65°C, C. sakazakii >6.7 at 67.5°C, L. monocytogenes >6.9 at 65.5°C, and Salmonella ser. Typhimurium >6.9 at 61.5°C. The kinetic data from these experiments will be used by the New Zealand Ministry of Agriculture and Forestry to populate the quantitative risk assessment model being developed to investigate the risks to New Zealand consumers from pasteurized, compared with nonpasteurized, milk and milk products.

Inactivation of Bacillus spores in reconstituted skim milk by combined high pressure and heat treatment.

AIMS: To determine the resistance of a variety of Bacillus species spores to a combined high pressure and heat treatment; and to determine the affect of varying sporulation and treatment conditions on the level of inactivation achieved. METHODS AND RESULTS: Spores from eight Bacillus species (40 isolates) were high pressure-heat treated at 600 MPa, 1 min, initial temperature 72 degrees C. The level of inactivation was broad (no inactivation to 6 log10 spores ml(-1) reduction) and it varied within species. Different sporulation agar, high pressure equipment and pressure-transmitting fluid significantly affected the response of some isolates. Varying the initial treatment temperature (75, 85 or 95 degrees C) shifted the relative order of isolate high pressure-heat resistance. CONCLUSIONS: The response of Bacillus spores to combined high pressure-heat treatment is variable and can be attributed to both intrinsic and extrinsic factors. The combined process resulted in a high level of spore inactivation for several Bacillus species and is a potential alternative treatment to traditional heat-only processes. SIGNIFICANCE AND IMPACT OF THE STUDY: Sporulation conditions, processing conditions and treatment temperature all affect the response of Bacillus spores to the combined treatment of high pressure and heat. High levels of spore inactivation can be achieved but the response is variable both within and between species.

Effect of turbulent-flow pasteurization on survival of Mycobacterium avium subsp. paratuberculosis added to raw milk.

A pilot-scale pasteurizer operating under validated turbulent flow (Reynolds number, 11,050) was used to study the heat sensitivity of Mycobacterium avium subsp. paratuberculosis added to raw milk. The ATCC 19698 type strain, ATCC 43015 (Linda, human isolate), and three bovine isolates were heated in raw whole milk for 15 s at 63, 66, 69, and 72 degrees C in duplicate trials. No strains survived at 72 degrees C for 15 s; and only one strain survived at 69 degrees C. Means of pooled D values (decimal reduction times) at 63 and 66 degrees C were 15.0 +/- 2.8 s (95% confidence interval) and 5.9 +/- 0.7 s (95% confidence interval), respectively. The mean extrapolated D72 degrees C was <2.03 s. This was equivalent to a >7 log10 kill at 72 degrees C for 15 s (95% confidence interval). The mean Z value (degrees required for the decimal reduction time to traverse one log cycle) was 8.6 degrees C. These five strains showed similar survival whether recovery was on Herrold's egg yolk medium containing mycobactin or by a radiometric culture method (BACTEC). Milk was inoculated with fresh fecal material from a high-level fecal shedder with clinical Johne's disease. After heating at 72 degrees C for 15 s, the minimum M. avium subsp. paratuberculosis kill was >4 log10. Properly maintained and operated equipment should ensure the absence of viable M. avium subsp. paratuberculosis in retail milk and other pasteurized dairy products. An additional safeguard is the widespread commercial practice of pasteurizing 1.5 to 2 degrees above 72 degrees C.

Nucleotide sequence and analysis of pWC1, a pC194-type rolling circle replicon in Lactococcus lactis.

A 2.8-kb cryptic plasmid showing no homology to either pFX3 (rolling circle, pE194-type) or pCI305 (theta-type) lactococcal replicons was identified in Lactococcus lactis subsp. cremoris 2204. The plasmid, pWC1, was compatible with both pCI3340 (a pCI305 derivative) and pFX3 in L. lactis subsp. cremoris 2204. Sequence analysis of pWC1 showed one major ORF encoding a protein with a deduced size of 316 amino acids (aa). Database comparisons showed that the protein was distinct from the pFX- and pCI-type replication proteins (less than 21% aa identity), but shared significant homology (up to 57% aa identity) with the replication proteins from a different group of rolling circle plasmids (pC194-type) commonly found in gram-positive bacteria. A pC194-type rolling circle plasmid has not been previously described in L. lactis. Further sequence analysis showed a conserved double-stranded origin of replication in pWC1 preceded by a large (118-bp) direct repeat. The chloramphenicol-resistance gene from pC194 was inserted into a nonessential region of pWC1 to give pCP12. The host range of pCP12 included Streptococcus thermophilus, Enterococcus faecalis, and Staphylococcus aureus, but not Escherichia coli. Both pCP12 and to a lesser extent pWC1 generated single-stranded DNA (ssDNA) in L. lactis. A possible single-stranded origin of replication was identified by sequence analysis of pWC1 and by comparing levels of ssDNA produced by pCP12 deletion derivatives. The pWC1 replicon may be a useful addition to other replicons currently available for vector construction.

Expression of a beta-galactosidase gene from Clostridium acetobutylicum in Lactococcus lactis subsp. lactis.

A beta-galactosidase gene from Clostridium acetobutylicum NCIB 2951 was expressed after cloning into pSA3 and electroporation into derivatives of Lactococcus lactis subsp. lactis strains H1 and 7962. When the clostridial gene was introduced into a plasmid-free derivative of the starter-type Lact. lactis subsp. lactis strain H1, the resulting construct had high beta-galactosidase activity but utilized lactose only slightly faster than the recipient. beta-galactosidase activity in the construct decreased by over 50% if the 63 kb Lac plasmid pDI21 was also present with the beta-galactosidase gene. Growth rates of Lac+ H1 and 7962 derivatives were not affected after introduction of the clostridial beta-galactosidase, even though beta-galactosidase activity in a 7962 construct was more than double that of the wild-type strain. When pDI21 was electroporated into a plasmid-free variant of strain 7962, the recombinant had high phospho-beta-galactosidase activity and a growth rate equal to that of the H1 wild-type strain. The H1 plasmid-free strain grew slowly in T5 complex medium, utilized lactose and contained low phospho-beta-galactosidase activity. We suggest that beta-galactosidase expression can be regulated by the lactose phosphotransferase system-tagatose pathway and that Lact. lactis subsp. lactis strain H1 has an inefficient permease for lactose and contains chromosomally-encoded phospho-beta-galactosidase genes.

Genetic analysis of a lactococcal plasmid replicon.

The sequence and genetic organization was determined of the 2508 bp lactococcal portion of pFX2, which was derived from a cryptic Lactococcus lactis subsp. lactis plasmid and used as the basis for construction of a series of lactococcal vectors. A lactococcal plasmid plus origin and two replication protein-coding regions (repA and repB) were located. RepA has a helix-turn-helix motif, a geometry typical of DNA-binding proteins. RepB shows a high degree of homology to the plasmid replication initiation proteins from other gram-positive bacteria and Mycoplasma. The transcribed inverted repeat sequence between repA and repB could form an attenuator to regulate pFX2 replication. Up-stream of the ori site, and in a region which was non-essential for replication, a 215 bp sequence identical to the staphylococcal plasmid pE194 and carrying the RSA site was identified. The genetic organization of this lactococcal plasmid replicon shares significant similarity with pE194 group plasmids.

Construction of a family of lactococcal vectors for gene cloning and translational fusion.

A family of stable lactococcal vectors have been constructed based on the pFX1 replicon using either the alpha fragment or the complete Escherichia coli lacZ gene as a selective marker. These vectors also incorporate multiple cloning sites and examples are given of their use for gene cloning and translational fusion studies in lactococci.

Molecular cloning and expression of a proteinase gene from Lactococcus lactis subsp. cremoris H2 and construction of a new lactococcal vector pFX1.

The 6.5 kb HindIII DNA fragment of the Lactococcus lactis subsp. cremoris H2 plasmid pDI21 was cloned into Escherichia coli POP13 with lambda NM1149, and also directly into Lactococcus lactis subsp. lactis 4125 using a newly-constructed broad host-range vector pFX1. Proteinase was expressed in both transformed organisms. The proteinase resembles a PI type since it preferentially degraded beta-casein. The restriction map of the 6.5 kb proteinase gene fragment has minor differences from those of published plasmid proteinase genes. High-efficiency electroporation with pFX1 provides a direct approach for gene cloning in lactococci.

Plasmid linkage of the D-tagatose 6-phosphate pathway in Streptococcus lactis: effect on lactose and galactose metabolism.

The three enzymes of the D-tagatose 6-phosphate pathway (galactose 6-phosphate isomerase, D-tagatose 6-phosphate kinase, and tagatose 1,6-diphosphate aldolase) were absent in lactose-negative (Lac-) derivatives of Streptococcus lactis C10, H1, and 133 grown on galactose. The lactose phosphoenolpyruvate-dependent phosphotransferase system and phospho-beta-galactosidase activities were also absent in Lac- derivatives of strains H1 and 133 and were low (possibly absent) in C10 Lac-. In all three Lac- derivatives, low galactose phosphotransferase system activity was found. On galactose, Lac- derivatives grew more slowly (presumably using the Leloir pathway) than the wild-type strains and accumulated high intracellular concentrations of galactose 6-phosphate (up to 49 mM); no intracellular tagatose 1,6-diphosphate was detected. The data suggest that the Lac phenotype is plasmid linked in the three strains studied, with the evidence being more substantial for strain H1. A Lac- derivative of H1 contained a single plasmid (33 megadaltons) which was absent from the Lac- mutant. We suggest that the genes linked to the lactose plasmid in S. lactis are more numerous than previously envisaged, coding for all of the enzymes involved in lactose metabolism from initial transport to the formation of triose phosphates via the D-tagatose 6-phosphate pathway.

Proteinase activity in slow lactic acid-producing variants of Streptococcus lactis.

Variants of Streptococcus lactis that produce lactic acid slowly in milk were isolated by inducing plasmid loss in the wild type at 39 to 40 C. Such strains had lost most of their surface-bound proteinase activity and were designated prt(-). The specific proteinase activities of S. lactis C10 prt(+) whole cells and solubilized cell walls were 7 and 18 times, respectively, those of the prt(-) strain, but spheroplast lysates of prt(+) and prt(-) strains contained similar proteinase activity. S. lactis H1 showed a similar relative distribution of activity between prt(+) and prt(-) cellular fractions, although the overall level was lower. The limited growth in milk, characteristic of prt(-) strains, can be explained in terms of their low surface-bound proteinase activity.