1. Monitoring Endospores and Endospore-Forming Bacterial Populations During Commercial Skim Milk Powder Production C. Murillo, C. Kitts, and R. Jiménez-Flores EBI and DPTC California Polytechnic State University. Introduction  Milk powder is an important commodity on a national and international level and a key ingredient in many food products.  Confectionaries  Infant formulas  Baked goods  Endospores and thermoduric bacteria survive pasteurization and spray drying to become a concern for milk powder producers.  Endospores inhabit powder in “dormant” state, becoming active upon reconstitution.  Can cause proteolysis, lipolysis, sensory defects, and produce toxins.  Bacillus spp. are of particular interest because they are the predominant organisms that determine the keeping quality of pasteurized milk and milk products.  Critical points for the proliferation of Bacillus spp. and their relationship to the microbial population throughout milk powder processing is required for improvements in milk powder quality. Project Goals  Count spore-formers, and viable bacteria during commercial, low-heat skim milk powder production throughout production runs at the following points: –Milk storage silo –Cream separator –Evaporator (final effect) –Powder port  Follow the abundance of the predominant bacteria and spore formers using Terminal Restriction Fragment (TRF) patterns RawSkimCondensedPowder 0-2 hrs X X X X Sampling Strategy 8-10 hrs X X X X 15-19 hrs X X X X 23-27 hrs X X X X 30-35 hrs X X X X 40-42 hrs X X X X Space Time 3 processing plants 2 visits per plant 20 - 24 samples per visit Making Terminal Restriction Fragments Bacterial DNA Extracted Raw Skim Condensed Powder 16S rDNA PCR- Amplified Fluorescent Primer w/ Fluorescent Primer 5’ Denatured (94°C) Annealed (46.5°C) Extended (72°C) 16S rDNA Digested w/ HaeIII or DpnII Microorganism Types Identified and Followed with TRF Data  Bacillus spp. were clearly indicated. –Most common during skimming, condensing, and drying; low in raw milk. B. subtilis group were found in samples at all plants and during all visits. B. cereus group were found in samples at all plants at least some of the time.  Three types of rumen bacteria (e.g. Prevotella) were found along with 8 different Proteobacteria types. –Rumen bacteria and Proteobacteria were prominent in raw milk and decreased with processing. Bacillus spp. at Each Processing Step Over Time and Averaged Across all Plants and Visits 10 3 10 4 10 5 10 6 10 7 10 8 10 9 0 – 28 - 1015 – 1923 – 2728 – 3540 – 42 Time Into Process (hrs) CFU/g total solids Raw Skim Condensed Dry Conclusions – Spore formers  Bacillus spp. appeared to thrive in the separator or very near it.  Critical increases in spore levels at the separator appeared to result in spore increases in the downstream powder.  Peaks in separator spore levels generally occurred around 15- 19 hours of processing. –As early as 8 hours for Plant 1.  Even as the raw milk became cleaner toward the end of a run, contamination levels in the powder did not revert to their initial level. Conclusions – Other Bacteria  Possible fecal contamination was indicated by the presence of rumen bacteria, e.g. Prevotella spp. found in all plants. –Levels of these organisms were sensitive to pasteurization. –Equipment fouled despite cleaner incoming raw milk, possibly by a biofilm in or near the separator.  Possible soil contamination was indicated by the presence of Proteobacteria. – Levels of these organisms responded in the same way to pasteurization as the Prevotella spp. (data not shown) and they also appeared to accumulate in or near the separator. Milk Powder Processing Flow Pre-warmCooling Skim Storage Pre-heating 1 Pre-heating 2 Pasteurization Condense Storage Spray Drying Sifting Raw Milk 4°C Separation 50°C 54°C7°C 58°C 68°C 76°C Condensing (MVR &/or TVR) 71°C54°C 50°C 230°C Sample Fluorescence Intensity Fragment size (nucleotides) Bacillus spp. Prevotella spp. Sample 1 Sample 2 Fragment Separation and Detection by CGE + − DNA Fragments 15 kV POP6 Separation Polymer in 61cm x 50  m Capillary @ 60 o C 1x Buffer (EDTA) ABI 310 Genetic Analyzer 9.9 mW Laser Detection Organism ID – Peak Matching Across Enzyme Digests using PCA e.g. Plant 2 Principal Component 1 Principal Component 2 -0.4-0.20.00.20.4 -0.4 -0.2 0.0 0.2 0.4 Con1 Con3 Con4 Dry1 Dry2 Dry3 Dry4 Dry5 Dry6 Raw1 Raw2 Raw3 Raw4 Raw5 Raw6 Sep1 Sep2 Sep3 Sep4 Sep5 Sep6 -5*10^505*10^5 -5*10^5 0 5*10^5 H225 H271 H272 H166 Principal Component 1 Principal Component 2 -0.4-0.20.00.20.4 -0.4 -0.2 0.0 0.2 0.4 Con1 Con2 Con4 Dry1 Dry2 Dry3 Dry4 Dry5 Dry6 Raw1 Raw2 Raw3 Raw4 Raw5 Raw6 Sep1 Sep2 Sep3 Sep4 Sep5 Sep6 -5*10^505*10^5 -5*10^5 0 5*10^5 D264 D238 D265 D232 HaeIIIDpnII Hae166 & Dpn265 Proteobacteria Hae272 & Dpn265 Bacillus subtilis group Hae272 & Dpn265 Bacillus cereus group Hae225 & Dpn238 Prevotella ruminicola Bacillus subtilis group (Hae272 & Dpn265) and Bacillus cereus group(Hae273 & Dpn267) 0 10 20 30 40 50 60 70 RSCDSCDSCDSCDSCDD 0-2 hrs 8 -10 hrs 15 -19 hrs23-27 hrs 28 -35 hrs 40 – 42 hrs Processing Point and Time Relative Percentage H272 H273 D265 D267 And using Percent of Total Bacteria e.g. Plant 1 Prevotella spp. at Each Processing Step from Each Plant and Visit, Averaged Over Processing Time 10 2 10 3 10 4 10 5 10 6 10 7 10 8 1A1B2A2B3A3B Plant and Visit CFU/g total solids Raw Skim Condensed Dry Abstract The microflora of milk powder consists of a wide array of microorganisms of which special attention is given to Bacillus endospores given their role in defining commercial quality standards. Bacillus endospores survive pasteurization and spray drying and remain dormant in the powder. Once the powder is reconstituted, endospores germinate unpredictably, and through their enzymatic activity become detrimental to product quality. The objectives of this study were to 1) enumerate mesophilic and thermophilic endospore populations during commercial, skim milk powder production, and 2) characterize the microbial ecology of this process using Terminal Restriction Fragment (TRF) patterns of 16S rRNA genes, and 3) compare the changes in bacterial populations during processing of skim milk powder. Our approach was to observe changes bacterial populations during commercial operations, following the same lot of raw milk throughout the processing run. Fluid, condensed and powdered skim milk samples were collected periodically from three commercial milk powder facilities for a period of two years. Sampling points included the raw milk silo, separator, evaporator, and spray dryer. Microbial evaluation was normalized based on total solids. Every sample was evaluated for total aerobic plate count and mesophilic and thermophilic endospore counts. For TRF patterns, community DNA was extracted, amplified by PCR using 16S rRNA probes, and digested with an endonuclease, either HaeIII or DpnII. Endospore formers were predominant in condensed and powdered milk, and tended to increase in the powder with increasing processing time. In raw milk mesophilic and thermophilic endospores ranged from < 25CFU/g to 70 CFU/g and < 25CFU/g to 100 CFU/g, respectively. In powder they ranged from < 25CFU/g to 103 CFU/g and < 25CFU/g to 105 CFU/g, respectively. Both types of endospore counts from skim milk showed an increasing trend with run time and eventually rendered the powder out of the 103 CFU/g limit. There was a strong correlation of spore counts with season in all samples. In commercial samples TRF patterns successfully described microbial populations and a drastic change was observed between raw and powder milk for most runs. A particular case is presented for the illustration of the usefulness of this molecular technique in following a single species of microorganism through a milk process. Sponsors: