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Aerobic spore-forming bacteria in bulk raw milk: factors influencing the numbers of psychrotrophic, mesophilic and thermophilic Bacillus spores

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Psychrotrophic, mesophilic and thermophilic spore concentrations of Bacillus spp. were determined on a weekly basis in bulk raw milk samples obtained from a central processing facility, over one calendar year. These data were correlated with concentrations of metal ions, free amino acids and somatic cell counts obtained from the same samples, as well as local meteorological mean temperature and relative humidity measurements relating to the same test period. A heat treatment of 80°C for 20 min followed by the addition of L-alanine to the milk at 0.1% w/v and incubation at 55°C for 7 days gave optimal recovery conditions for thermophilic spores. Free amino acids, metal ions, somatic cell counts, temperature, and relative humidity measurements were each significantly correlated with the recovery of spores of Bacillus spp. from bulk raw milk, although no single factor was shown to demonstrate a consistent effect with the psychrotrophic, mesophilic and thermophilic spore groups studied.
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  Vol 55, No 2May 2002International Journal of Dairy Technology ORIGINAL RESEARCH *Author for correspondence. E-mail: david.mccleery@dardni.gov.uk © 2002 Society of Dairy Technology 100 BlackwellScience,LtdOxford,UKInternationalJournalofDairyTechnology1364-727XSocietyofDairyTechnology2002May2002552OriginalresearchVol55No2May2002 Aerobic spore-forming bacteria in bulk raw milk: factors influencing the numbers of psychrotrophic, mesophilic and thermophilic  Bacillus  spores JAMES T M MCGUIGGAN, 2  DAVID R MCCLEERY, 1,2 * ALAN HANNAN 2  and ARTHUR GILMOUR  1,2 1  Department of Food Science (Food Microbiology), Queen’s University Belfast and 2  Department of Agriculture and Rural  Development for Northern Ireland, Newforge Lane, Belfast, Northern Ireland   Psychrotrophic, mesophilic and thermophilic spore concentrations of Bacillus  spp. were determined ona weekly basis in bulk raw milk samples obtained from a central processing facility, over one calendar  year. These data were correlated with concentrations of metal ions, free amino acids and somatic cell counts obtained from the same samples, as well as local meteorological mean temperature and relativehumidity measurements relating to the same test period. A heat treatment of 80 °   C for 20 min followed bythe addition of L-alanine to the milk at 0.1% w/v and incubation at 55 °   C for 7 days gave optimal recoveryconditions for thermophilic spores. Free amino acids, metal ions, somatic cell counts, temperature, and relative humidity measurements were each significantly correlated with the recovery of spores of Bacillus  spp. from bulk raw milk, although no single factor was shown to demonstrate a consistent effect with the psychrotrophic, mesophilic and thermophilic spore groups studied. Keywords Bulk raw milk, Interaction with milk components, Meteorological factors, Recovery of  Bacillus  spp. * Authorforcorrespondence.E-mail:david.mccleery@dardni.gov.uk INTRODUCTIONAerobic spore-forming bacteria of the genus  Bacillus  are commonly present in raw milk. Their spores survive pasteurization and subsequentlygerminate, outgrow, and multiply. They have beenresponsible for the spoilage of pasteurized milk and milk products 1–3  and UHT products. 4,5 Indeed, the presence of  Bacillus  spp. in foodsthat contain milk or milk products is also undesir-able because a number of species have been impli-cated in food-borne disease. 6,7    Bacillus cereus  wasincriminated in a large food poisoning outbreak attributed to pasteurized milk  8  and WHO figuresindicate that 5–10% of reported food-borne dis-ease is caused by this organism. 9 The dairy industry is interested in strategieswhereby the numbers of spore-formers in rawmilk can be controlled or reduced. 10  Germinationfollowed by a heat treatment to kill the resultantvegetative cells is one method that has been postu-lated. 1  However, there are many factors that could  possibly influence the germination and outgrowthof  Bacillus  spores in a complex medium such asraw milk and may therefore limit the desired effectof such a treatment. Nutrient germinants such asamino acids 11–14  and unsaturated fatty acids, 15 levels of some indigenous antibacterial factorssuch as lysozyme, lactoferrin and lactoperoxi-dase, 15–17  somatic cells 18  and metal ions 19  have been suggested as factors influencing the ger-mination of  Bacillus  species. Additionally, further effects may result from storage temperature, heattreatment 10,20  and even antagonism between thesporeformers themselves. 21 Such factors can act alone or in combination and may either stimulate or inhibit germination. 11–13,22 Indeed, a compound that causes germination in one  Bacillus  species may have little effect on a differentspecies. 23  Additional parameters influencing fluc-tuations in the levels of  Bacillus  spp. may be thenature of the milk produced as influenced by thestage of lactation and the dietary intake of the cow.Although each of these factors may have aneffect on the numbers and types of  Bacillus  spp.occurring in raw milk, their individual relative sig-nificance is unclear. Moreover, spoilage problemscaused by spore-forming bacteria are more preva-lent at certain times of the year. 1 McGuiggan et al  . 24  previously examined factorsinfluencing the recovery of psychrotrophic, mes-ophilic and thermophilic spores of  Bacillus  spp.from bulk raw milk and developed methods for their optimal detection and isolation. The aim of   © 2002 Society of Dairy Technology 101 Vol 55, No 2May 2002 the study reported here was to apply these devel-oped recovery methods in an investigation of theinteractions between raw milk components, envi-ronmental factors and the recovery of spores of   Bacillus  spp. Consequently, free amino acids, metalions, somatic cells counts, meteorological temper-ature and relative humidity measurements were eachcompared with the recovery of psychrotrophic,mesophilic and thermophilic spores of  Bacillus spp. from bulk raw milk, over a 1-year period.MATERIALS AND METHODS Collection of raw milk  Fifty samples of raw milk (2.0-L volumes) werecollected on a weekly basis over one calendar year January–December (excluding weeks 6 and 51)from a bulked source at a local milk processing plant and were maintained at chill temperature(< 5 ° C) during transfer to the laboratory. The milk was taken from a source (usually the balance tank)as close to the pasteurizer as possible. All sampleswere tested within 2 h of sampling. Preparation of skimmed milk medium, detection of inhibitory substances in raw milk and preparation of L-alanine solution Ultra high temperature (UHT) skimmed milk wasdispensed in appropriate volumes (9 or 10 mL)and L-alanine solutions (10% w/v) were prepared as previously described by McGuiggan et al  . 24 Detection of inhibitory substances in raw and skimmed milk was performed using the miniatur-ized Oxoid (AIM) test. 25 Recovery of psychrotrophic, mesophilic and thermophilic spores of  Bacillus  spp. from bulk raw milk  Sample treatment  A most probable number technique (MPN) wasused to enumerate  Bacillus  spores in raw milk,as previously documented. 24  Briefly, raw milk samples were thoroughly mixed before 10-mLvolumes were individually dispensed into five steriletest tubes, 1-mL aliquots were individually added to five test tubes containing 9 mL of the prepared sterile skimmed milk medium and similarly0.1-mL volumes were added to 10 mL of the sterileskimmed milk medium. Where spore numberswere expected to be high, further decimal dilutionsof the raw milk samples were examined by adding0.1 mL of the appropriate dilution(s) (peptonediluent) to five test tubes containing 10 mL of ster-ile skimmed milk. Tubes were then heat treated at80 ° C for a holding time of 10 min (psychrotrophicand mesophilic spore counts only) or 20 and 30 min (thermophilic spore counts). One hundred microlitres of L-alanine solution (10% w/v) wereadded to each tube after heat treatment, to give afinal concentration of 0.1% w/v L-alanine in thesample. A duplicate set of MPN tubes withoutL-alanine was included for the enumeration of thermophilic spores. Test tubes were incubated  before sampling, as shown in Table 1. Psychrotrophicspores were enumerated by incubating the MPNtubes at 6.5 ° C for 15 days; mesophilic spore popu-lations were detected using an incubation temper-ature of 30 ° C for 15 days; and thermophilic sporecounts were performed by incubating the tubes at55 ° C for 7 days. After incubation, each MPN tubewas thoroughly mixed and 10 µ L were transferred to a slope of milk plate count agar (mPCA; Oxoid CM 325) and incubated under the same conditionsas those that had been used for the srcinal MPNtubes to confirm growth, as summarized in Table 1. Spore confirmation Confirmation for the presence of spores was car-ried out by subculturing from mPCA onto sporula-tion agar  26  and incubating at 25 ° C overnight.Samples that showed no signs of growth after thistime were reincubated and examined after a further 24 h. Smears were examined for the presence of spores using phase contrast microscopy. 27 Somatic cell count A subsample of each raw milk sample was trans- ported (at 4–7 ° C), on the same day as sampling,to the laboratories of the United Dairy Farmers,Antrim Road, Belfast. The number of somatic cells per mL of milk was determined within 4 h of  Table 1 Heat treatments, incubation temperatures and times used for the recovery of psychrotrophic, mesophilic and thermophilic  Bacillus  spores from bulk raw milk by an MPN method and subsequent confirmation on milk plate count agar slopes    Holding time(minutes at 80 °   C) Incubation   temperature (  °   C)Incubation time (days) Psychrotrophic spores106.515Mesophilic spores103015Thermophilic spores20, 30557  Vol 55, No 2May 2002 © 2002 Society of Dairy Technology 102receipt using a Fossomatic instrument [Foss Elec-tric (Ireland) Ltd, Dublin]. Metal ions concentration Triplicate 25-mL aliquots of each raw milk samplewere combined with an equal volume of 20% (w/v)trichloroacetic acid (TCA) before incubating atroom temperature for 30 min with mixing by inver-sion at 5-min intervals. The samples were centri-fuged at 2300  g   for 15 min and each supernatantwas analysed in triplicate for copper, iron and manganese using a Perkin-Elmer Model AA 5000atomic absorption spectrophotometer (Perkin-Elmer, England) in accordance with the manu-facturer’s instructions. Free amino acids concentrations Duplicate milk samples were defatted by centri-fuging at 130  g   for 10 min. Defatted milk (5 mL)was mixed with 25 mL of a 1% v/v picric acid solution to precipitate the protein and centrifuged for 20 min at 1000  g  . The supernatant was passed through a prepared ‘Amberlite IRA-410’ ionexchange resin column using 0.02 N HCl and distilled water aliquots (Pharmacia). The eluentobtained was rotary evaporated with two water washings to dryness and dissolved in 1.0 mL of alithium citrate buffer solution (pH 2.2; Pharmacia)containing a known concentration of the internalstandard ‘norleucine’. Eighty microlitres of thesetest solutions were used for cation exchange chro-matographic separation of the constituent aminoacids on an ‘LKB 4400 amino acid analyser’(Amersham Pharmacia Biotech, UK). Resultswere calculated using a ‘Trivector, Trilab 3’ com- puting integrator. The free amino acids detected arelisted in Table 3. Temperature and relative humidity measurement Temperature ( ° C) and relative humidity (%) datawere obtained for the corresponding 1-year period of milk sampling from The Meteorological Office,Belfast Weather Centre, 1 College Square East,Belfast BT1 6BQ. These data comprised weeklymean values of combined daily observations fromeight stations, within a 20-mile radius of the milk  processing centre. Statistical analyses Analysis of variance was carried out according tothe two methods ×  two durations of heat factorialdesign used. This tested for the main effects of themethods used to enumerate thermophilic spores(with and without L-alanine) and the duration of heat treatment with associated interactions. Cor-relation coefficients were calculated to test for relationships between the somatic cell counts,temperature, relative humidity, free amino acidsand metal ions concentrations with the recovery of the three test groups of  Bacillus  spores isolated from bulk raw milk.RESULTS AND DISCUSSION Recovery method for thermophilic spores In a previous study by McGuiggan et al  ., 24  two of the developed protocols demonstrated equivalentoptimal recoveries for thermophilic  Bacillus  spp.isolated from raw milk, leading to an inability bythese researchers to recommend a single definitivemethod for the recovery of such spores. Conse-quently, in the study reported here, these recovery protocols for thermophilic spores were further evaluated by applying them in parallel to a larger number of raw milk samples over a longer time period.In the study reported here, the effect of the dura-tion of the heat treatment (80 ° C) and the presenceof L-alanine in the recovery of thermophilic  Bacil-lus  spp., were each found to influence the numbersof spores recorded from individual raw milk samples (Table 2). The addition of L-alanine wasshown to enhance the recovery of these spores.Indeed, the highest mean recoveries of ther-mophilic spores, throughout the course of theexperiment, were obtained using a 20-min heattreatment in the presence of L-alanine and conse-quently, unless otherwise stated, the data presented here were obtained using this method. Psychrotrophic spores The concentrations of psychrotrophic spores of   Bacillus  spp. recovered from raw milk, over the 1-year test period, ranged from 0.02 to 3.5 spores/mL(Figure 1) with an overall mean level of 0.8 spores/mL. Although variation in these recovery rates wasevident throughout the test period, with very lowlevels being measured from weeks 18–26 (May– June), this pattern did not appear to be related tometeorological temperature and relative humiditydata or to somatic cells (all  P   > 0.05; Table 3).These data are comparable with those reported  by McKinnon and Pettipher, 28  who recorded similar  Table 2 Effect of the duration of heat treatment and addition of L-alanine on the recovery of thermophilic spores of  Bacillus  spp. from bulk raw milk     Duration of heat treatment at 80 °   C (min)With L-alanineWithout L-alanine 203.982.02302.942.51Average 3.462.27  Figures are spores/mL, mean of 47 replications Standard error of the mean (SEM) = 0.416  © 2002 Society of Dairy Technology 103 Vol 55, No 2May 2002 spore concentrations in creamery silos, and alsoto data from raw milk samples reported by Meer  et al  . 2  In contrast, however, Sutherland and Murdoch 21  detected psychrotrophic species only inthe summer–autumn months and Phillips and Griffiths 29  recovered higher numbers of psychro-trophic spores in creamery silos during the summer– autumn months than in the winter.It is possible that the variations between theresults reported in these studies and the data pre-sented here may be related to differences in themethods used to recover the spore populations. It isimportant to note that the method used in the studyreported here was developed to optimize the recov-ery of psychrotrophic  Bacillus  spp. from milk  24 and may therefore demonstrate greater sensitivityin comparison with other procedures. Moreover,regional differences in husbandry practices mayhave led to disparities between these studies because Slaghuis et al  . 30  suggested that ‘pasturing Figure 1 Recovery of spores of psychrotrophic  Bacillus  spp. from bulk raw milk over one calendar year. Table 3 Correlation between the concentration of free amino acids, metal ions, meteorological data, somatic cell counts and the recovery of spores of  Bacillus  spp. isolated from bulk raw milk     Raw milk component  Psychrotrophic  spores Mesophilic  sporesThermophilic  spores *alanine0.415  b − 0.039  − 0.356  b β -alanine  − 0.359  b 0.364  b 0.048arginine  − 0.0080.537 c − 0.102aspartic acid   − 0.1430.0820.497 c ethanolamine0.0480.1440.089glutamic acid0.234  − 0.271 a − 0.104glycine0.011  − 0.1870.323 a histidine  − 0.200  − 0.111  − 0.195isoleucine  − 0.0340.161  − 0.256 a leucine  − 0.0750.033  − 0.239 a lysine  − 0.0380.266 a − 0.175ornithine  − 0.169  − 0.0560.001 phenylalanine  − 0.1470.349  b − 0.050 phosphoethanolamine  − 0.068  − 0.1260.272 a  proline0.0550.532 c − 0.238 a serine0.144  − 0.150  − 0.057taurine  − 0.432 c 0.268 a 0.329  b threonine0.070  − 0.033  − 0.044tyrosine  − 0.0380.227  − 0.195urea0.0380.430 c 0.018valine  − 0.0170.376  b 0.000Copper   − 0.0320.115  − 0.117Manganese  − 0.1980.1200.017Iron  − 0.267 a 0.006  − 0.028Relative humidity†0.028  − 0.0100.174Temperature†  − 0.0590.212  − 0.208Somatic cell count  − 0.1010.424  b − 0.268 a Data presented are correlation coefficients relating recovery of groups of  Bacillus  spp. with listed milk component concentrations, measured in the same samples *See Table 1 for recovery methods used to enumerate different groups of  Bacillus  spp. from bulk raw milk (thermophilic spores were recovered using a heat treatment of 80 ° C for 20 min followed by the addition of 0.1% w/v L-alanine and incubation at 55 ° C for 7 days)†Weekly mean values of combined daily meteorological observations from eight weather stations within a 20-mile radius of the milk processing centre a Statistically significant relationship where  P    ≤  0.05  b Statistically significant relationship where  P    ≤  0.01 c Statistically significant relationship where  P    ≤  0.001

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