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High temperature, short time pasteurization temperatures inversely affect bacterial numbers during refrigerated storage of pasteurized fluid milk

      Abstract

      The grade A Pasteurized Milk Ordinance specifies minimum processing conditions of 72°C for at least 15 s for high temperature, short time (HTST) pasteurized milk products. Currently, many US milk-processing plants exceed these minimum requirements for fluid milk products. To test the effect of pasteurization temperatures on bacterial numbers in HTST pasteurized milk, 2% fat raw milk was heated to 60°C, homogenized, and treated for 25 s at 1 of 4 different temperatures (72.9, 77.2, 79.9, or 85.2°C) and then held at 6°C for 21 d. Aerobic plate counts were monitored in pasteurized milk samples at d 1, 7, 14, and 21 postprocessing. Bacterial numbers in milk processed at 72.9°C were lower than in milk processed at 85.2°C on each sampling day, indicating that HTST fluid milk-processing temperatures significantly affected bacterial numbers in fluid milk. To assess the microbial ecology of the different milk samples during refrigerated storage, a total of 490 psychrotolerant endospore-forming bacteria were identified using DNA sequence-based subtyping methods. Regardless of processing temperature, >85% of the isolates characterized at d 0, 1, and 7 postprocessing were of the genus Bacillus, whereas more than 92% of isolates characterized at d 14 and 21 postprocessing were of the genus Paenibacillus, indicating that the predominant genera present in HTST-processed milk shifted from Bacillus spp. to Paenibacillus spp. during refrigerated storage. In summary, 1) HTST processing temperatures affected bacterial numbers in refrigerated milk, with higher bacterial numbers in milk processed at higher temperatures; 2) no significant association was observed between genus isolated and pasteurization temperature, suggesting that the genera were not differentially affected by the different processing temperatures; and 3) although typically present at low numbers in raw milk, Paenibacillus spp. are capable of growing to numbers that can exceed Pasteurized Milk Ordinance limits in pasteurized, refrigerated milk.

      Key words

      Introduction

      In the United States, Bacillus spp. and Paenibacillus spp. have been identified as the biological barriers currently limiting HTST pasteurized fluid milk shelf life (
      • Fromm H.I.
      • Boor K.J.
      Characterization of pasteurized fluid milk shelf-life attributes.
      ;
      • Durak M.Z.
      • Fromm H.I.
      • Huck J.R.
      • Zadoks R.N.
      • Boor K.J.
      Development of molecular typing methods for Bacillus spp. and Paenibacillus spp. isolated from fluid milk products.
      ;
      • Huck J.R.
      • Woodcock N.H.
      • Ralyea R.D.
      • Boor K.J.
      Molecular subtyping and characterization of psychrotolerant endospore-forming bacteria in two New York State fluid milk processing systems.
      , 2008). Bacillus spp. and Paenibacillus spp. are capable of forming heat-resistant spores that can survive HTST pasteurization (
      • Collins E.B.
      Heat resistant psychrotrophic organisms.
      ;
      • Huck J.R.
      • Woodcock N.H.
      • Ralyea R.D.
      • Boor K.J.
      Molecular subtyping and characterization of psychrotolerant endospore-forming bacteria in two New York State fluid milk processing systems.
      ), and some strains are able to germinate and grow at refrigeration temperatures, ultimately causing spoilage of processed products (
      • Washam C.J.
      • Olson H.C.
      • Vedamuthu E.R.
      Heat-resistant psychrotrophic bacteria isolated from pasteurized milk.
      ;
      • Huck J.R.
      • Sonnen M.
      • Boor K.J.
      Tracking heat-resistant, cold-thriving fluid milk spoilage bacteria from farm to packaged product.
      ). Because Bacillus spp. and Paenibacillus spp. spores are ubiquitously present in nature, it is difficult to exclude them from the milk supply. Representatives from both genera have been isolated from dairy farms (
      • Crielly E.M.
      • Logan N.A.
      • Anderton A.
      Studies on the Bacillus flora of milk and milk products.
      ;
      • Sutherland A.D.
      • Murdoch R.
      Seasonal occurrence of psychrotrophic Bacillus species in raw milk, and studies on the interactions with mesophilic Bacillus spp.
      ;
      • Lukasova J.
      • Vyhnalkova J.
      • Pacova Z.
      Bacillus species in raw milk and in the farm environment.
      ;
      • Huck J.R.
      • Sonnen M.
      • Boor K.J.
      Tracking heat-resistant, cold-thriving fluid milk spoilage bacteria from farm to packaged product.
      ), processing plants (
      • Lin S.
      • Schraft H.
      • Odumeru J.A.
      • Griffiths M.W.
      Identification of contamination sources of Bacillus cereus in pasteurized milk.
      ;
      • Huck J.R.
      • Woodcock N.H.
      • Ralyea R.D.
      • Boor K.J.
      Molecular subtyping and characterization of psychrotolerant endospore-forming bacteria in two New York State fluid milk processing systems.
      ), and pasteurized packaged products (
      • Griffiths M.W.
      • Phillips J.D.
      Incidence, source and some properties of psychrotrophic Bacillus spp. found in raw and pasteurized milk.
      ;
      • Lin S.
      • Schraft H.
      • Odumeru J.A.
      • Griffiths M.W.
      Identification of contamination sources of Bacillus cereus in pasteurized milk.
      ;
      • Douglas S.A.
      • Gray M.J.
      • Crandall A.D.
      • Boor K.J.
      Characterization of chocolate milk spoilage patterns.
      ;
      • Fromm H.I.
      • Boor K.J.
      Characterization of pasteurized fluid milk shelf-life attributes.
      ;
      • Durak M.Z.
      • Fromm H.I.
      • Huck J.R.
      • Zadoks R.N.
      • Boor K.J.
      Development of molecular typing methods for Bacillus spp. and Paenibacillus spp. isolated from fluid milk products.
      ;
      • Huck J.R.
      • Woodcock N.H.
      • Ralyea R.D.
      • Boor K.J.
      Molecular subtyping and characterization of psychrotolerant endospore-forming bacteria in two New York State fluid milk processing systems.
      ).
      In recent years, both food safety concerns and the desire to extend fluid milk shelf life have prompted many dairy processors to increase HTST pasteurization temperatures above the minimum conditions specified by the Pasteurized Milk Ordinance (72°C for 15 s;
      Pasteurized Milk Ordinance
      Revision.
      ;
      • Gandy A.L.
      • Schilling M.W.
      • Coggins P.C.
      • White C.H.
      • Yoon Y.
      • Kamadia V.V.
      The effect of pasteurization temperature on consumer acceptability, sensory characteristics, volatile compound composition, and shelf-life of fluid milk.
      ). Anecdotal reports from multiple milk processors of higher bacterial numbers and more rapid spoilage in fluid milk products after an increase in processing temperatures prompted this investigation of the effects of commonly applied HTST pasteurization temperatures on aerobic plate counts (APC) in refrigerated fluid milk products. To test the hypothesis that higher HTST processing temperatures result in higher bacterial numbers in fluid milk products during refrigerated storage, the objectives of this study were 1) to determine the effect of processing temperature on APC in milk samples that had been processed under a range of commonly applied HTST temperatures and held under refrigerated storage conditions (6°C); and 2) to monitor the microbial ecology of pasteurized products processed at each temperature during storage.

      Materials and Methods

      Experimental Design

      To measure the effects of HTST pasteurization temperatures on APC in pasteurized fluid milk, 4 independent batches of raw 2% fat homogenized milk were each processed for 25 s at one of the following temperatures: 72.9, 77.2, 79.9, or 85.2°C. A complete block design was used to allow all combinations of position in the processing order (first, second, third, or fourth) for each processing temperature (72.9, 77.2, 79.9, or 85.2°C) to ensure that each temperature held a different position in processing in each replicate.

      Pasteurization of Raw Milk

      Raw bovine milk from approximately 300 cows in Hartford, New York, was pooled, and representative samples were collected and transported to the Cornell University Food Processing and Development Laboratory (Ithaca, NY). The raw milk was cold-separated into cream and skim fractions with a DeLaval separator (Model 590; DeLaval, Poughkeepsie, NY), and then held refrigerated at 5°C for up to 72 h. On the day of processing, raw skim milk and cream were blended to make 2% fat milk. Approximately 5 gallons (18.93 L) of 2% fat raw milk was added to a jacketed steam kettle, heated to 60°C, and then homogenized with 2 passes through a Gaulin APV homogenizer (Model 200 E; Gaulin, Everett, MA). After homogenization, the milk was pasteurized at 72.9, 77.2, 79.9, or 85.2°C for 25 s as described by
      • Ma Y.
      • Barbano D.M.
      Milk pH as a function of CO2 concentration, temperature, and pressure in a heat exchanger.
      . Milk samples (2 L) pasteurized at each temperature were collected aseptically and held on ice until all processing treatments were completed. Immediately postprocessing, 8 aliquots of approximately 200 mL were poured from each of the 2-L containers into sterile 250-mL screw-capped Pyrex bottles. The pasteurized milk samples were held at 6°C for up to 21 d.

      Microbiological Testing of Milk Samples

      To determine the microbiological quality of the raw milk before pasteurization, a sample was taken from the standardized 2% fat milk before heating and homogenization. Milk samples were assessed for total APC, coliform count, psychrotrophic bacteria count (PBC), and laboratory pasteurized count (LPC) according to the Standard Methods for the Examination of Dairy Products (
      • Frank J.F.
      • Yousef A.E.
      Tests for groups of microorganisms
      ), except that the samples for APC, PBC, and LPC tests were spread-plated onto brain heart infusion agar (BHI; Difco, BD Diagnostics, Franklin Lakes, NJ). Preliminary incubation counts were performed on raw 2% fat milk samples according to the Standard Methods for the Examination of Dairy Products (
      • Richardson G.K.
      Preliminary incubation for raw milk.
      ), except that the raw 2% fat samples were spread-plated onto BHI agar and incubated at 32°C for 24 h. For the mesophilic spore count (MSC) and psychrotrophic spore count (PSC) tests, milk samples were heated at 80°C for 12 min, and then the milk was cooled rapidly, incubated overnight at 6°C, and spread-plated the following day on BHI agar, with subsequent incubation at 32°C for 24 h (for the MSC) or at 6°C for 10 d (for the PSC).
      Two aliquots of milk that had been processed at each temperature were tested for APC at d 1, 7, 14, and 21 postpasteurization. Samples were serially diluted in PBS (Weber Scientific, Hamilton, NJ), spread-plated onto BHI agar, and incubated at 32°C for 24 h. On d 1, plating was performed by spreading 1 mL of milk sample over 5 plates to allow bacterial enumeration in samples with low bacterial counts.

      Statistical Analyses

      A mixed model was used to analyze the APC data (JMP Version 7.0; SAS Institute Inc., Cary, NC). For all analyses, log-transformed bacterial count data were used as a response. The model included temperature of processing and time of refrigerated storage of pasteurized milk as independent fixed variables. Replicate and temperature were random variables in the model. Temperature was a between-replicate effect and day was a within-replicate effect. Models were tested for an interaction between time and temperature. When an effect was significant, multiple comparisons were done with a Tukey correction. Distribution of bacterial subtypes was analyzed in JMP using a chi-square test for independence.

      Bacterial Isolation

      For each milk sample, bacterial colonies present on BHI that had been plated at d 0, 1, 7, 14, and 21 postprocessing were visually examined and a colony representative of each distinct morphology present on 1 plate that had been used for colony enumeration for each sample was chosen for isolation and later identification. Typically, 5 to 10 colonies per sample were selected and streaked for purity on BHI agar. Purified isolates were characterized for gram reaction using a 3-step gram stain kit (Becton, Dickinson and Co., Sparks, MD) and subsequently frozen at −80°C in 15% glycerol.

      rpoB Sequencing

      Species identification and subtyping of Bacillus spp. and Paenibacillus spp. isolates were performed by determining the DNA sequence of a 632-nucleotide fragment of the rpoB gene, as described previously by
      • Huck J.R.
      • Hammond B.H.
      • Murphy S.C.
      • Woodcock N.H.
      • Boor K.J.
      Tracking spore-forming bacterial contaminants in fluid milk processing systems.
      . This method was selected for bacterial identification and differentiation purposes because it allows for phylogenetic characterization of Bacillus and Paenibacillus isolates in addition to subtype identification, and because it is more economical than most banding pattern-based subtyping methods such as ribotyping or pulsed-field gel electrophoresis (
      • Kabuki D.Y.
      • Kuaye A.Y.
      • Wiedmann M.
      • Boor K.J.
      Molecular subtyping and tracking of Listeria monocytogenes in Latin-style fresh-cheese processing plants.
      ). The software DnaSP version 4.0 (
      • Rozas J.
      • Rozas R.
      DnaSP version 3: An integrated program for molecular population genetics and molecular evolution analysis.
      ) was used for rpoB allele assignment, and different allelic types (AT) were assigned to gene sequences that differed from each other by 1 or more nucleotides. Isolates with different AT were considered to represent different molecular subtypes.

      16S Ribosomal DNA Sequencing

      Although the majority of isolates collected here could be characterized to genus and species through phylogenetic analyses by using the existing rpoB sequence database (
      • Durak M.Z.
      • Fromm H.I.
      • Huck J.R.
      • Zadoks R.N.
      • Boor K.J.
      Development of molecular typing methods for Bacillus spp. and Paenibacillus spp. isolated from fluid milk products.
      ;
      • Huck J.R.
      • Hammond B.H.
      • Murphy S.C.
      • Woodcock N.H.
      • Boor K.J.
      Tracking spore-forming bacterial contaminants in fluid milk processing systems.
      ,
      • Huck J.R.
      • Woodcock N.H.
      • Ralyea R.D.
      • Boor K.J.
      Molecular subtyping and characterization of psychrotolerant endospore-forming bacteria in two New York State fluid milk processing systems.
      ,
      • Huck J.R.
      • Sonnen M.
      • Boor K.J.
      Tracking heat-resistant, cold-thriving fluid milk spoilage bacteria from farm to packaged product.
      ), 16S ribosomal DNA (rDNA) sequencing 1) was used to confirm the genus or species identification, or both, of rpoB AT that had not been identified in any of our previous studies, and 2) was used for those isolates we could not identify by rpoB sequencing. Specifically, 1 isolate representing each newly identified rpoB allelic type was characterized by sequencing the 3′ end of the 16S rDNA, as described previously by
      • Huck J.R.
      • Hammond B.H.
      • Murphy S.C.
      • Woodcock N.H.
      • Boor K.J.
      Tracking spore-forming bacterial contaminants in fluid milk processing systems.
      . Final partial 3′ 16S rDNA sequences were used for similarity searches against the National Center for Biotechnology Information nucleotide sequence database, using the Blast Local Alignment Search Tool (
      • McGinnis S.
      • Madden T.L.
      BLAST: At the core of a powerful and diverse set of sequence analysis tools.
      ). Genus or species assignments, or both, for a specific 16S rDNA sequence were based on the top matches returned by the search with the Blast Local Alignment Search Tool.

      Results

      Microbiological Analysis of Raw Milk

      To assess the microbiological quality of the raw milk before pasteurization, a sample of the 2% fat raw milk was collected immediately after the skim and cream portions had been blended, but before heat tempering and homogenization on each processing day. A complete listing of log-transformed bacterial counts for each replicate is presented in Table 1. Mean raw 2% fat milk APC from the 4 replicates was 4.19 log cfu/mL, and ranged from 3.91 (replicate 4) to 4.98 log cfu/mL (replicate 1). The mean MSC was 1.67 log cfu/mL, and ranged from 0.85 (replicate 3) to 2.88 log cfu/mL (replicate 1). On 2% fat raw milk samples, we observed a mean LPC of 1.37 log cfu/mL, a mean preliminary incubation count of 4.25 log cfu/mL, a mean coliform count of 1.48 log cfu/mL, a mean PBC of 3.57 log cfu/mL, and no growth (<1 spore/mL) for the PSC (Table 1).
      Table 1Log-transformed bacterial numbers from 2% fat raw milk used in 4 independent replicates
      APC = aerobic plate count; MSC = mesophilic spore count; LPC = laboratory pasteurized count; PI = preliminary incubation count; CC = coliform count; PBC = psychrotrophic bacteria count. In all 4 replicates, psychrotrophic spore count analyses yielded no colonies, indicating the presence of less than 1 spore capable of growth at 6°C per milliliter of raw milk sample.
      ReplicateAPCMSCLPCPICCPBC
      14.982.881.954.901.633.18
      23.941.361.455.662.043.36
      33.920.851.002.191.184.18
      43.911.591.08>5.4021.08>4.40
      Estimated counts were not included in mean or standard deviation calculations; bacteria from these samples were “too numerous to count.”
      Mean4.191.671.374.251.483.57
      SD0.530.860.481.820.380.53
      1 APC = aerobic plate count; MSC = mesophilic spore count; LPC = laboratory pasteurized count; PI = preliminary incubation count; CC = coliform count; PBC = psychrotrophic bacteria count. In all 4 replicates, psychrotrophic spore count analyses yielded no colonies, indicating the presence of less than 1 spore capable of growth at 6°C per milliliter of raw milk sample.
      2 Estimated counts were not included in mean or standard deviation calculations; bacteria from these samples were “too numerous to count.”

      Microbiological Analysis of Pasteurized Milk

      Total aerobic bacterial numbers for the HTST pasteurized milk samples that had been stored at 6°C are shown in Figure 1. For d 1, 7, 14, and 21, the mean bacterial numbers for milk that had been processed at 72.9°C were lower than the mean numbers for the milk that had been processed at 85.2°C. In general, as pasteurization temperatures increased, mean aerobic bacterial plate counts also increased. At d 1 and 7 postpasteurization, the mean APC for the milk processed at the 3 highest temperature treatments were similar and all were higher than the mean for the milk processed at 72.9°C.
      Figure thumbnail gr1
      Figure 1Aerobic plate counts (APC) for 2% fat milk that had been pasteurized at 1 of 4 different temperatures (72.9, 77.2, 79.9, or 85.2°C) and held at 6°C for up to 21 d. Data represent mean APC for milk processed at each temperature from 4 independents replicates. Bars indicate mean ± 1 SD for each treatment.
      Overall, results from statistical analyses of our data showed a significant effect of temperature (P = 0.03) and day postpasteurization (P < 0.001) on bacterial numbers in pasteurized milk. The interaction between temperature and day was tested and found to be insignificant (P = 0.82). On d 1 postprocessing, mean APC ranged from 1.69 log cfu/mL for milk pasteurized at 72.9°C to 2.18 log cfu/mL for milk pasteurized at 79.9°C. Mean APC were slightly higher at d 7 postprocessing relative to the mean counts at d 1. At d 7, the mean APC for milk processed at 72.9°C was 1.74 log cfu/mL, whereas the mean APC for milk processed at 85.2°C was 2.30 log cfu/mL. Between d 7 and 14, the average increases in APC were 2.33, 2.66, 2.95, and 3.06 log cfu/mL for milk processed at 72.9, 77.2, 79.9, and 85.2°C, respectively. On d 21, the mean APC for milk processed at 72.9°C was 5.79 log cfu/mL (approximately 616,000 cfu/mL), whereas the mean APC for milk processed at 85.2°C was 7.10 log cfu/mL (approximately 12,590,000 cfu/mL). Mean APC for milk processed at 72.9°C was significantly lower (P < 0.05) than for milk processed at 85.2°C on each sampling day.

      Bacterial Species Distribution as Determined by rpoB and 16S rDNA Subtyping Data

      A total of 564 isolates were collected in the 4 replicates from both the raw and pasteurized product. Of those, 478 isolates were determined by rpoB sequencing to belong to genera characterized as gram-positive spore-forming bacteria (i.e., Bacillus and Paenibacillus). The remaining 86 isolates, which were not typable by the rpoB sequencing method, were identified by sequencing the 3′ end of the 16S rRNA gene. Among these 86 isolates, 45 represented gram-negative bacteria and 41 represented gram-positive bacteria. Table 2 includes the genus distribution of the bacterial isolates identified (by 16S and rpoB sequencing) in this study. All gram-negative bacteria were isolated from raw milk, and the majority (31 isolates) were Pseudomonas spp. The other gram-negative genera identified included Stenotrophomonas (6 isolates), Acinetobacter (3 isolates), Klebsiella (2 isolates), Pantoea (2 isolates), and Comamonas (1 isolate). The most frequently isolated gram-positive bacteria included Bacillus (258 isolates) and Paenibacillus (232 isolates). Other gram-positive genera identified were Enterococcus, Micrococcus, Staphylococcus, Streptococcus, Aerococcus, Brevibacillus, Corynebacterium, and Macrococcus.
      Table 2Genus distribution of bacterial isolates from raw and heat-treated milk samples
      GenusIsolates from raw milk,
      Raw milk isolates were obtained from multiple tests performed on 2% raw milk, including aerobic plate counts, mesophilic spore counts, and laboratory pasteurized counts.
      no.
      Isolates from pasteurized milk, no.Total
      Gram negative
      Pseudomonas31031
      Stenotrophomonas606
      Acinetobacter303
      Klebsiella202
      Pantoea202
      Comamonas101
       Total45045
      Gram positive
      Bacillus
      Of 258 Bacillus isolates, 249 were identified by rpoB sequencing, and 9 isolates were identified by 16S ribosomal DNA (rDNA) sequencing.
      29229258
      Paenibacillus
      Of 232 Paenibacillus isolates, 229 were identified by rpoB sequencing, and 3 isolates were identified by 16S rDNA sequencing.
      2230232
      Enterococcus4610
      Micrococcus077
      Staphylococcus156
      Streptococcus112
      Aerococcus101
      Brevibacillus011
      Corynebacterium011
      Macrococcus101
       Total39480519
      Total84480564
      1 Raw milk isolates were obtained from multiple tests performed on 2% raw milk, including aerobic plate counts, mesophilic spore counts, and laboratory pasteurized counts.
      2 Of 258 Bacillus isolates, 249 were identified by rpoB sequencing, and 9 isolates were identified by 16S ribosomal DNA (rDNA) sequencing.
      3 Of 232 Paenibacillus isolates, 229 were identified by rpoB sequencing, and 3 isolates were identified by 16S rDNA sequencing.
      Overall, 96 rpoB AT were differentiated among the 478 spore-forming bacterial isolates, indicating considerable genetic diversity among even the closely related microbes that were present in this milk-production and milk-processing system. An additional 9 Bacillus and 3 Paenibacillus isolates that did not yield rpoB products were identified by 16S rDNA sequencing. Of the isolates obtained from pasteurized milk, 229 were identified as Bacillus and 230 as Paenibacillus. Table 3 shows the distribution of Bacillus and Paenibacillus isolates by pasteurization temperature and day postpasteurization. There was a significant association between genus identified and day postpasteurization (P < 0.001). As shown in Table 4 Table 4 , at d 0, 1, and 7, the number of Bacillus isolates collected was significantly higher than that of Paenibacillus (P < 0.001). Of 280 isolates characterized at d 0, 1, or 7 postpasteurization, 242 were characterized as Bacillus. The 38 remaining isolates were characterized as Paenibacillus. However, the number of Paenibacillus isolates collected was significantly higher than the number of Bacillus isolates at d 14 and 21 postpasteurization (P < 0.001). Of 210 isolates collected at d 14 or 21 postpasteurization, 194 were characterized as Paenibacillus. There was no significant association between genus isolated and pasteurization temperature (P = 0.82; chi-square analysis), suggesting that the genera were not differentially affected by the different processing temperatures.
      Table 3Distribution of isolates obtained from pasteurized milk and characterized as Bacillus spp. or Paenibacillus spp.
      Genus
      Pasteurization temperature, °CDay postpasteurizationBacillus
      Of 229 Bacillus isolates, 222 were identified by rpoB sequencing, and 7 isolates were identified by 16S ribosomal DNA (rDNA) sequencing.
      Paenibacillus
      Of 230 Paenibacillus isolates. 227 were identified by rpoB sequencing, and 3 isolates were identified by 16S rDNA sequencing.
      72.91392
      7217
      14322
      21023
      All days6354
      77.21314
      7198
      14327
      21125
      All days5464
      79.91290
      7206
      14318
      21031
      All days5255
      85.21321
      7228
      14224
      21424
      All days6057
      Total
      Isolates obtained from raw milk or raw milk receiving heat treatments (laboratory pasteurized counts or mesophilic spore counts) are not included in these distributions.
      229230
      1 Of 229 Bacillus isolates, 222 were identified by rpoB sequencing, and 7 isolates were identified by 16S ribosomal DNA (rDNA) sequencing.
      2 Of 230 Paenibacillus isolates. 227 were identified by rpoB sequencing, and 3 isolates were identified by 16S rDNA sequencing.
      3 Isolates obtained from raw milk or raw milk receiving heat treatments (laboratory pasteurized counts or mesophilic spore counts) are not included in these distributions.
      Table 4Proportion of endospore-forming isolates collected from raw and heat-treated milk by day postpasteurization and by heat treatment (i.e., raw, laboratory pasteurization, mesophilic spore count, pasteurization)
      Isolates characterized, no. (%)
      Percentages are calculated based on the number of isolates given in each row.
      Day postpasteurization (heat treatment)Isolates characterized, no.Bacillus spp.Paenibacillus spp.
      0 (raw milk, none)22 (100)0 (0)
      1 (laboratory pasteurization)1110 (91)1 (9)
      1 (mesophilic spore count)1817 (94)1 (6)
      1 (pasteurized)
      Isolates were obtained from milk that had been pasteurized at all 4 experimental temperatures.
      138131 (95)7 (5)
      7 (pasteurized)
      Isolates were obtained from milk that had been pasteurized at all 4 experimental temperatures.
      11182 (74)29 (26)
      14 (pasteurized)
      Isolates were obtained from milk that had been pasteurized at all 4 experimental temperatures.
      10211 (11)91 (89)
      21 (pasteurized)
      Isolates were obtained from milk that had been pasteurized at all 4 experimental temperatures.
      1085 (5)103 (95)
      Total490258 (53)232 (47)
      1 Percentages are calculated based on the number of isolates given in each row.
      2 Isolates were obtained from milk that had been pasteurized at all 4 experimental temperatures.

      Subtype Patterns Across Replicates and Temperature Treatments

      One important outcome of this study was the further insight obtained regarding the diversity of microbes that pose a barrier to the extension of pasteurized milk shelf life. In the present study, only 8 of the 96 AT identified (4 Bacillus and 4 Paenibacillus) were isolated both from multiple replicates and more than 10 times (Table 5). The most frequently isolated Bacillus AT were AT1, AT6, AT20, and AT158. Allelic type 1 and AT6 were identified as Bacillus licheniformis, whereas AT20 and AT158 were identified as Bacillus pumilus and Bacillus cereus, respectively. The most commonly isolated AT of the genus Paenibacillus were AT13, AT15, AT25, and AT27. Research on Paenibacillus spp. is just emerging; thus, the majority of isolates that classify to this genus have not yet been speciated.
      Table 5Common
      Allelic types represented by ≥10 isolates shown; allelic types with ≤10 isolates included 50 isolates classified as Bacillus and 149 isolates classified as Paenibacillus.
      molecular subtypes of endospore-forming bacterial isolates obtained from raw and pasteurized milk samples
      Genus, speciesAllelic typeTotal isolated, no.Replicate (no. of isolates per replicate)Sample day postpasteurization (no. of isolates)
      Total numbers of isolates obtained on d 0, 1, 7, 14, and 21 were 13, 150, 107, 100, and 108, respectively.
      Bacillus spp.
      B. licheniformis1271 (4)0 (5)
      2 (14)1 (16)
      3 (6)7 (6)
      4 (3)14 (0)
      21 (0)
      B. licheniformis6131 (1)0 (0)
      2 (3)1 (5)
      3 (4)7 (7)
      4 (5)14 (1)
      21 (0)
      B. pumilus20101 (3)0 (1)
      2 (2)1 (8)
      3 (4)7 (1)
      4 (1)14 (0)
      21 (0)
      B. cereus1581491 (49)0 (0)
      2 (36)1 (92)
      3 (29)7 (52)
      4 (35)14 (5)
      21 (0)
      Paenibacillus spp.
      Paenibacillus spp.13131 (0)0 (0)
      2 (6)1 (0)
      3 (4)7 (1)
      4 (3)14 (4)
      21 (8)
      Paenibacillus spp.15251 (11)0 (0)
      2 (8)1 (0)
      3 (3)7 (2)
      4 (3)14 (11)
      21 (12)
      Paenibacillus spp.25161 (12)0 (0)
      2 (0)1 (0)
      3 (3)7 (1)
      4 (1)14 (12)
      21 (3)
      Paenibacillus spp.27261 (7)0 (0)
      2 (5)1 (0)
      3 (2)7 (4)
      4 (12)14 (13)
      21 (9)
      1 Allelic types represented by ≥10 isolates shown; allelic types with ≤10 isolates included 50 isolates classified as Bacillus and 149 isolates classified as Paenibacillus.
      2 Total numbers of isolates obtained on d 0, 1, 7, 14, and 21 were 13, 150, 107, 100, and 108, respectively.
      Certain Bacillus and Paenibacillus subtypes were found in samples collected from at least 3 replicates, indicating the persistence of these particular subtypes in the milk-production and milk-processing system. For example, AT1 (B. licheniformis) was isolated 4, 14, 6, and 3 times in replicates 1, 2, 3, and 4, respectively. Furthermore, AT1 was isolated 5 times from raw milk, 16 times on d 1 postpasteurization, and 6 times on d 7 postpasteurization. Allelic type 1 was not obtained from any milk on d 14 or 21 postpasteurization. The most commonly isolated Bacillus allelic type was AT158 (B. cereus). Allelic type 158 was isolated 149 times and was found in all 4 replicates. All AT158 isolates were collected from pasteurized samples; 92 were isolated on d 1, 52 were isolated on d 7, 5 were isolated on d 14, and 0 were isolated on d 21. Allelic type 158 was isolated 49, 36, 29, and 35 times in replicates 1, 2, 3, and 4, respectively. When comparing the relationship between AT and pasteurization temperature that had been applied, no pattern was observed (P = 0.85; chi-square analysis; Table 6). The most common Bacillus isolate, AT158, was found 33, 36, 39, and 41 times in the 72.9, 77.2, 79.9, and 85.2°C temperature treatments, respectively. Allelic type 158 was the only Bacillus isolate found solely in pasteurized products. Bacillus AT1, AT6, and AT20 were isolated from raw milk samples receiving MSC or LPC heat treatments (from samples that had not been homogenized or pasteurized) 9, 2, and 4 times, respectively (Table 6). The presence of AT158 isolates in pasteurized samples and not in raw samples suggests the source of AT158 is likely the processing environment. This hypothesis is further supported by the observation that although AT158 was not found in raw milk, it was most frequently isolated from pasteurized milk at d 1 postprocessing and that it decreased in isolation frequency with increasing refrigerated storage time, suggesting that this strain is not particularly successful at reproducing under refrigerated storage conditions.
      Table 6Relationship between heat treatment and allelic type (AT) isolated
      Bacillus allelic typePaenibacillus allelic type
      Heat treatmentAT1AT6AT20AT158Other AT
      “Other AT” represent combined AT, each found fewer than 10 times.
      Isolates without AT
      9 Bacillus were identified by 16S ribosomal DNA (rDNA) because they did not amplify with rpoB PCR; therefore, no AT are available for these isolates.
      AT13AT15AT25AT27Other AT1Isolates without AT
      Three Paenibacillus were identified by 16S rDNA because they did not amplify with rpoB PCR; therefore, no AT are available for these isolates.
      Raw milk
      Raw milk isolates were obtained from different tests performed on 2% fat raw milk, including aerobic plate counts, mesophilic spore counts, and laboratory pasteurized counts.
      9240122000020
      72.9°C761331422469312
      77.2°C50236926956380
      79.9°C14339501516420
      85.2°C510411034745361
      Total271310149509132516261493
      1 “Other AT” represent combined AT, each found fewer than 10 times.
      2 9 Bacillus were identified by 16S ribosomal DNA (rDNA) because they did not amplify with rpoB PCR; therefore, no AT are available for these isolates.
      3 Three Paenibacillus were identified by 16S rDNA because they did not amplify with rpoB PCR; therefore, no AT are available for these isolates.
      4 Raw milk isolates were obtained from different tests performed on 2% fat raw milk, including aerobic plate counts, mesophilic spore counts, and laboratory pasteurized counts.
      The common Paenibacillus subtypes identified in this study were AT13, AT15, AT25, and AT27 (Table 5). The most frequently isolated Paenibacillus subtype was AT27, which was isolated 26 times and was found in all 4 replicates. Allelic type 27 was isolated 7, 5, 2, and 12 times in replicates 1, 2, 3, and 4, respectively. All AT27 isolates were collected from pasteurized samples at d 7, 14, or 21 postpasteurization; 4 were isolated on d 7, 13 were isolated on d 14, and 9 were isolated on d 21. When comparing the relationship between AT and pasteurization temperature that had been applied, no pattern was observed (P = 0.85; chi-square analysis; Table 6). The most common Paenibacillus isolate, AT27, was found 9, 6, 6, and 5 times in the 72.9, 77.2, 79.9, and 85.2°C temperature treatments, respectively. None of the common Paenibacillus isolates was obtained from the raw milk, MSC heat treatment, or LPC heat treatment. These results suggest that the Paenibacillus spp. that are present in high numbers at d 14 and 21 postprocessing are likely to be initially present in raw milk at numbers below the detection limit of 1 spore/mL. They also may be introduced from the processing environment.

      Discussion

      Higher HTST Pasteurization Temperatures Result in Higher Numbers of Spore-Forming Spoilage Organisms During Refrigerated Storage

      Psychrotolerant microorganisms present a considerable challenge to the dairy industry because fluid milk is stored at temperatures permissive of the growth of these organisms. Currently, Pseudomonas spp. represent the most predominant psychrotolerant gram-negative bacteria contributing to milk spoilage, but these microbes are heat sensitive and usually do not survive pasteurization (
      • Cousin M.A.
      Presence and activity of psychrotrophic microorganisms in milk and dairy products: A review.
      ) unless present in raw milk at very high numbers (
      • Griffiths M.W.
      • Phillips J.D.
      Effect of aeration on extracellular enzyme synthesis by psychrotrophs growing in milk during refrigerated storage.
      ). Typically, such bacteria enter milk through postpasteurization contamination at the filling machine (
      • Eneroth S.
      • Christiansson A.
      • Brendehaug J.
      • Molin G.
      Critical contamination sites in the production line of pasteurised milk, with reference to the psychrotrophic spoilage flora.
      ;
      • Ralyea R.D.
      • Wiedmann M.
      • Boor K.J.
      Bacterial tracking in a dairy production system using phenotypic and ribotyping methods.
      ). In recent years, many commercial fluid milk processors have reduced postpasteurization contamination through implementation of improved filler and packaging technology to produce milk that does not result in consumer complaints at 15 to 25 d postpasteurization (
      • Carey N.R.
      • Murphy S.C.
      • Zadoks R.N.
      • Boor K.J.
      Shelf lives of pasteurized fluid milk products in New York State: A ten-year study.
      ). When gram-negative postpasteurization contaminants are excluded from fluid milk-processing systems, the presence of psychrotolerant endospore-forming spoilage bacteria, particularly Bacillus and Paenibacillus spp., are the next biological barrier to extending the shelf life of HTST milk (
      • Meer R.R.
      • Baker J.
      • Bodyfelt F.W.
      • Griffiths M.W.
      Psychrotrophic Bacillus spp. in fluid milk products: A review.
      ;
      • Ralyea R.D.
      • Wiedmann M.
      • Boor K.J.
      Bacterial tracking in a dairy production system using phenotypic and ribotyping methods.
      ;
      • Fromm H.I.
      • Boor K.J.
      Characterization of pasteurized fluid milk shelf-life attributes.
      ;
      • Durak M.Z.
      • Fromm H.I.
      • Huck J.R.
      • Zadoks R.N.
      • Boor K.J.
      Development of molecular typing methods for Bacillus spp. and Paenibacillus spp. isolated from fluid milk products.
      ;
      • Huck J.R.
      • Hammond B.H.
      • Murphy S.C.
      • Woodcock N.H.
      • Boor K.J.
      Tracking spore-forming bacterial contaminants in fluid milk processing systems.
      ,
      • Huck J.R.
      • Sonnen M.
      • Boor K.J.
      Tracking heat-resistant, cold-thriving fluid milk spoilage bacteria from farm to packaged product.
      ).
      Heat treatment survival and subsequent outgrowth of psychrotolerant endospore-forming spoilage microbes are problematic for mildly heated refrigerated food products such as dairy products and pasteurized vegetable purees (
      • Carlin F.
      • Guinebretiere M.H.
      • Choma C.
      • Pasqualini R.
      • Braconnier A.
      • Nguyen-The C.
      Spore-forming bacteria in commercial cooked, pasteurised and chilled vegetable purées.
      ;
      • Guinebretiere M.H.
      • Berge O.
      • Normand P.
      • Morris C.
      • Carlin F.
      • Nguyen-The C.
      Identification of bacteria in pasteurized zucchini purées stored at different temperatures and comparison with those found in other pasteurized vegetable purees.
      ). Specifically, in milk-processing systems, the spores of psychrotolerant endospore-forming spoilage bacteria can survive HTST pasteurization, germinate, and grow at refrigeration temperatures (
      • Collins E.B.
      Heat resistant psychrotrophic organisms.
      ). Psychrotolerant spore-forming bacteria have been isolated from silage (
      • te Giffel M.C.
      • Wagendorp A.
      • Herrewegh A.
      • Driehuis F.
      Bacterial spores in silage and raw milk.
      ), pasture (
      • Slaghuis B.A.
      • te Giffel M.C.
      • Beumer R.R.
      • André G.
      Effect of pasturing on the incidence of Bacillus cereus spores in raw milk.
      ), soil (
      • Christiansson A.
      • Bertilsson J.
      • Svensson B.
      Bacillus cereus spores in raw milk: Factors affecting the contamination of milk during the grazing period.
      ), and fecal material (
      • Labots H.
      • Hup G.
      • Galesfoot T.E.
      Bacillus cereus in raw and pasteurized milk. III. The contamination of raw milk with Bacillus cereus spores during its production.
      ); hence, they are widely present in milk-production systems. Spores have also been found in raw milk (
      • Shehata T.E.
      • Collins E.B.
      Isolation and identification of psychrotrophic strains of Bacillus from milk.
      ;
      • Crielly E.M.
      • Logan N.A.
      • Anderton A.
      Studies on the Bacillus flora of milk and milk products.
      ;
      • te Giffel M.C.
      • Wagendorp A.
      • Herrewegh A.
      • Driehuis F.
      Bacterial spores in silage and raw milk.
      ;
      • Huck J.R.
      • Hammond B.H.
      • Murphy S.C.
      • Woodcock N.H.
      • Boor K.J.
      Tracking spore-forming bacterial contaminants in fluid milk processing systems.
      ) and pasteurized milk (
      • Griffiths M.W.
      • Phillips J.D.
      Incidence, source and some properties of psychrotrophic Bacillus spp. found in raw and pasteurized milk.
      ;
      • Lin S.
      • Schraft H.
      • Odumeru J.A.
      • Griffiths M.W.
      Identification of contamination sources of Bacillus cereus in pasteurized milk.
      ;
      • Douglas S.A.
      • Gray M.J.
      • Crandall A.D.
      • Boor K.J.
      Characterization of chocolate milk spoilage patterns.
      ;
      • Fromm H.I.
      • Boor K.J.
      Characterization of pasteurized fluid milk shelf-life attributes.
      ;
      • Durak M.Z.
      • Fromm H.I.
      • Huck J.R.
      • Zadoks R.N.
      • Boor K.J.
      Development of molecular typing methods for Bacillus spp. and Paenibacillus spp. isolated from fluid milk products.
      ;
      • Huck J.R.
      • Woodcock N.H.
      • Ralyea R.D.
      • Boor K.J.
      Molecular subtyping and characterization of psychrotolerant endospore-forming bacteria in two New York State fluid milk processing systems.
      ).
      Application of DNA sequence-based subtyping strategies has helped identify transmission pathways of the spore-forming Bacillus and Paenibacillus spp. from dairy farms, tank trucks, and plant storage silos to pasteurized milk (
      • Huck J.R.
      • Hammond B.H.
      • Murphy S.C.
      • Woodcock N.H.
      • Boor K.J.
      Tracking spore-forming bacterial contaminants in fluid milk processing systems.
      ,
      • Huck J.R.
      • Sonnen M.
      • Boor K.J.
      Tracking heat-resistant, cold-thriving fluid milk spoilage bacteria from farm to packaged product.
      ), indicating that initial contamination of raw milk with these microbes can occur at the farm. Clearly, however, the potential exists for contamination to occur at various points in the production continuum (
      • Huck J.R.
      • Sonnen M.
      • Boor K.J.
      Tracking heat-resistant, cold-thriving fluid milk spoilage bacteria from farm to packaged product.
      ). To this point, B. cereus AT158 was isolated 149 times from 4 independent batches of pasteurized milk and across each of the 4 processing temperatures. Notably, we found AT158 only in processed milk, indicating that this microbe was most likely introduced into the milk in the processing environment. In support of this notion, further investigation of the microbial ecology of the processing environment used in this study revealed the presence of a diverse collection of endospore-forming bacteria, including an isolate with 99.8% sequence similarity with AT158 (M. L. Ranieri, unpublished data).
      Ironically, the heat treatment used to destroy vegetative bacterial cells can also activate bacterial spores, actually enhancing spore germination and outgrowth (
      • Meer R.R.
      • Baker J.
      • Bodyfelt F.W.
      • Griffiths M.W.
      Psychrotrophic Bacillus spp. in fluid milk products: A review.
      ). In the present study, HTST pasteurization temperatures were shown to inversely affect postprocessing APC of fluid milk products after storage at 6°C for 21 d. The lower APC for milk pasteurized at 72.9°C may be due to reduced activation of the spore population resident in the fluid milk relative to spore activation achieved at the higher temperatures. Supporting this hypothesis, at d 1, the 79.9°C temperature treatment had the highest mean APC (2.18 log cfu/mL), consistent with a previous report of optimal spore germination after an 80°C heat treatment (
      • Moran L.
      • Rowe M.T.
      • Hagan J.A.
      The effect of various heat activation treatments on fast, intermediate and slow germinating spores of Bacillus spp.
      ). At d 21 postpasteurization, however, the mean APC was highest for milk that had been treated at 85°C. These results suggest that thermosensitive factors intrinsic to the milk, to the spores, or both influence spore germination and outgrowth in fluid milk systems. Such factors could include differential availability to bacteria of milk-based nutrients as a function of heat treatment, possible destruction of indigenous antibacterial factors at higher processing temperatures, and possible pro- or antagonistic interactions between spore formers that are influenced by heat (
      • McGuiggan J.T.M.
      • McCleery D.R.
      • Hannan A.
      • Gilmour A.
      Aerobic spore-forming bacteria in bulk raw milk: Factors influencing the numbers of psychrotrophic, mesophilic and thermophilic Bacillus spores.
      ). One example of a naturally occurring antimicrobial factor in milk is the lactoperoxidase system. Previous work has indicated that the lactoperoxidase system is sensitive to temperature, possibly contributing to the improved storage quality of milk pasteurized at 72°C for 15 s relative to milk pasteurized at 80°C for 15 s (
      • Barrett N.E.
      • Grandison A.S.
      • Lewis M.J.
      Contribution of the lactoperoxidase system to the keeping quality of pasteurized milk.
      ).

      Paenibacillus Is the Predominant Spoilage Organism Present at d 14 and 21 Postprocessing

      In the present study, the predominant organisms isolated at d 0 (raw samples), 1, and 7 were Bacillus, whereas the predominant microbes isolated at d 14 and 21 were Paenibacillus. None of the common Bacillus AT (i.e., AT1, AT6, AT20, or AT158) was isolated at d 21 postpasteurization, and none of the common Paenibacillus AT (i.e., AT13, AT15, AT25, or AT27) was isolated from raw or d-1 samples. These results indicate that Paenibacillus spp. are generally present in raw milk at very low numbers or are entering milk through the processing environment.
      The shift in the predominant population of endospore-forming spoilage organisms from Bacillus spp. to Paenibacillus spp. over the product shelf life in HTST pasteurized milk has been observed previously.
      • Huck J.R.
      • Sonnen M.
      • Boor K.J.
      Tracking heat-resistant, cold-thriving fluid milk spoilage bacteria from farm to packaged product.
      found that 76.2% of isolates collected from pasteurized milk on d 12 of shelf life or beyond were Paenibacillus spp., whereas Bacillus spp. constituted 87.0% of environment and raw milk isolates. Additionally,
      • Fromm H.I.
      • Boor K.J.
      Characterization of pasteurized fluid milk shelf-life attributes.
      found a greater proportion of Paenibacillus spp. than Bacillus spp. in commercial fluid milk samples at d 17 of shelf life. These results suggest that Paenibacillus spp. are better able to grow in milk at 6°C than the Bacillus spp. that constitute the predominant spore-former microflora in raw milk. In the present study, the relative percentage of Bacillus spp. isolated decreased from 95, 74, and 11% to 5% on d 1, 7, 14, and 21 postprocessing, respectively, whereas the percentage of Paenibacillus spp. isolated increased from 5, 26, and 89% to 95% on d 1, 7, 14, and 21, respectively. These trends were consistent for each temperature treatment applied in the study, indicating that the heat treatments within the range studied (72.9 to 85.2°C) did not preferentially affect a subpopulation of the endospore-forming bacteria commonly present in milk.
      The fact that Paenibacillus was not isolated from raw milk yet was found as the predominant spoilage organism at 21 d postpasteurization across all 4 temperature treatments may explain why the microbiological tests currently used to assess raw milk quality do not effectively predict shelf-life performance of HTST pasteurized fluid milk (N. Woodcock, Cornell University, Ithaca, NY, personal communication). Because of the predominance of Paenibacillus spp. during product storage at refrigerated temperatures, targeting them for control or elimination appears to be an important goal for improving the quality and extending the shelf life of processed milk. Three of the 4 common Paenibacillus AT (AT13, AT15, and AT27) identified in the present study matched isolates previously reported by
      • Huck J.R.
      • Sonnen M.
      • Boor K.J.
      Tracking heat-resistant, cold-thriving fluid milk spoilage bacteria from farm to packaged product.
      as isolated from multiple stages of the milk-processing continuum. Allelic type 15 and AT27 were identified in the dairy farm environment, tank trucks, plant storage silos, and pasteurized milk (
      • Huck J.R.
      • Sonnen M.
      • Boor K.J.
      Tracking heat-resistant, cold-thriving fluid milk spoilage bacteria from farm to packaged product.
      ). Allelic type 13 was isolated from pasteurized milk and from the dairy farm environment. The fact that these common Paenibacillus isolates have been found throughout the processing continuum and that they are rarely isolated in pasteurized milk at d 1 postprocessing suggests that they are likely present in raw milk at low numbers. Representatives of these common AT will be excellent candidates for future investigation of Paenibacillus spp. growth characteristics, of the response of Paenibacillus spp. to heat treatments, and, most important, of Paenibacillus spp. as targets for the development of sensitive and specific assays needed to enable detection of these microbes at low levels. Additionally, the collection and identification of more isolates across a broader sample of farms and processing plants will further improve our understanding of Paenibacillus diversity and the persistence of specific subtypes in the environment.

      Conclusions

      High temperature, short time pasteurization temperatures can affect APC present in processed milk during refrigerated storage. Specifically—and counterintuitively—higher bacterial numbers were found in milk that had been processed at higher temperatures. The predominant bacterial genera isolated from the milk samples did not differ by heat treatment, suggesting that the heat treatments within the range studied (72.9 to 85.2°C) did not preferentially affect a subpopulation of the endospore-forming bacteria present. We conclude that the endospore-forming psychrotolerant bacteria present in milk grow more effectively in pasteurized milk after a higher heat treatment. Additionally, Paenibacillus spp., which are likely present at <1 spore/mL of raw milk, are capable of growing to numbers that can exceed the Pasteurized Milk Ordinance limit of 20,000 cfu/mL for grade A pasteurized milk, illustrating the need for a comprehensive strategy to limit the entry of endospore-forming bacteria into milk systems. Ultimately, new processing methods may need to be used to physically remove psychrotolerant endospore-forming bacteria from raw milk. Microfiltration represents one possible processing tool for removal of bacterial spores, with the goal of extending milk shelf life (
      • Elwell M.W.
      • Barbano D.M.
      Use of microfiltration to improve fluid milk quality.
      ). Continued efforts to improve the bacteriological quality of pasteurized milk will require an increased emphasis on limiting bacterial and spore contamination along the farm-to-table continuum.

      Acknowledgments

      This project was supported by the New York State Milk Promotion Advisory Board through the NY State Department of Agriculture and Markets—New York State dairy farmers committed to the production of high-quality dairy products. The authors thank Esref Dogan, Robert Kaltaler, and Mark Newbold (Department of Food Science, Cornell University, Ithaca, NY) for their assistance as well as Martin Wiedmann (Department of Food Science, Cornell University, Ithaca, NY) for helpful conversations and critical review of the manuscript, and staff members of the Cornell University Milk Quality Improvement Program for technical support.

      Supplementary data

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