Spore concerns and testing in dairy: Reducing variability and establishing standards
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1 Spore concerns and testing in dairy: Reducing variability and establishing standards NICOLE MARTIN MILK QUALITY IMPROVEMENT PROGRAM DEPARTMENT OF FOOD SCIENCE, CORNELL UNIVERSITY
2 Overview What are they and why do we care Raw to finished product contaminants Testing methods matter
3 Spores: Defense strategies for harsh environmental conditions Endospores are formed under stressful environmental conditions Allow bacteria to persist in environments that would otherwise be lethal Heat (pasteurization) Drying (powders) Radiation Sanitizers Under favorable conditions, spores return to a metabolically active state
4 Sporulation and germination The key to the spore challenge in dairy foods
5 Fluid Milk Dairy relevant sporeforming bacteria Psychrotolerant Sporeformers Paenibacillus Viridibacillus Bacillus weihenstephanenis
6 Cheese Dairy relevant sporeforming bacteria Anaerobic Butyric Acid Bacteria (BAB) Clostridium tyrobutyricum C. butyricum C. sporogenes Historical association with high levels of BAB in silage raw milk used for certain cheeses in Switzerland cannot come from cows fed silage Penalties used to regulate BAB in raw milk used for cheesemaking in the Netherlands
7 Dairy Powder Dairy relevant sporeforming bacteria Mesophilic and Thermophilic Sporeformers Bacillus licheniformis Geobacillus Anoxybacillus
8 Sporeforming bacteria- Farm to finished product contaminants "Fromagerie fabrication du cantal, Ruynes-en-Margeride" by Jean-Pierre - Flickr: Fromagerie : fabrication du cantal, Ruynes-en-Margeride. Licensed under CC BY-SA 2.0 via Wikimedia Commons -
9 Sporeforming bacteria in dairy products
10 Sporeforming bacteria in dairy products
11 Distribution (%) Mesophilic spores represent the highest proportion of sporeforming bacteria in raw milk 100% 80% 60% < 0.03 spores/ml > 0.03 to < 10 spores/ml 40% > 10 spores/ml 20% 0% MSC TSC Spore Enumeration Method
12 Log cfu/ml Spores are present in low levels in raw milk PSC MSC TSC HHR TSC STSE BAB -1.5 Bulk Tank Milk
13 Bacillus licheniformis accounts for nearly half of spores in bulk tank raw milk 9% 11% 4% 4% 2% 5% 16% 49% B. licheniformis Other Other Bacillus species B. pumilus B. subtilis B. cereus group B. clausii Paenibacillus spp.
14 Sources of sporeforming bacteria Soil Feed Bedding
15 Sources of sporeforming bacteria Manure Drinking water
16 Sources of sporeforming bacteria Air Equipment swab Filter sock Individual cow milk Teat swab Towel Wash water Bulk tank milk
17 Sporeforming bacteria- Farm to finished product contaminants "Fromagerie fabrication du cantal, Ruynes-en-Margeride" by Jean-Pierre - Flickr: Fromagerie : fabrication du cantal, Ruynes-en-Margeride. Licensed under CC BY-SA 2.0 via Wikimedia Commons -
18 Sporeforming bacteria in processing environments Post-processing contamination Inadequate cleaning and sanitization leading to biofilm formation Indicators of PPC in fluid milk include Gram-negative bacteria (e.g., Pseudomonas) Sporeformers can also be introduced into processed dairy products through biofilm formation "Biofilm" by D. Davis - From: Looking for Chinks in the Armor of Bacterial Biofilms Monroe D PLoS Biology Vol. 5, No. 11, e307 doi: /journal.pbio Licensed under CC BY 2.5 via Wikimedia Commons -
19 Sporeforming bacteria in processing environments Scott et al., 2007
20 Testing methods matter Spore Pasteurization 80 C (176 F)/12m Highly Heat Resistant Spore Treatment 100 C (212 F)/30m Specially Heat Resistant Spore Treatment 106 C (222.8 F)/30m Butyric Acid Bacteria (BAB) 75 C (167 F)/15m PSC 6 C (42.8 F)/10d incubation MSC 32 C (89.6 F)/48h incubation TSC 55 C (131 F)/48h incubation HHR-MSC 32 C/48h incubation HHR-TSC 55 C/48h incubation STSC 55 C/48h incubation BAB Most Probable Number (MPN) 37 C (98.6 F)/7d incubation
21
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23 Spore populations in raw milk vs. dairy powders
24 Spore testing challenges Low level contamination, stochastic variation Population diversity Germination and recovery variability on different media Countability Swarmers, spreaders and microaerophiles, oh my! What is the right method? Must consider implementation
25 Spore enumeration
26 Milk powder A, SP TSC on BHI, Pour Plate Milk powder A, SP TSC on BHI, Spread plate Milk powder A, SP TSC on BHI, Pour Plate with overlay
27 Milk powder A, SP TSC on BHI, Pour Plate Plating type is a source of Milk powder A, SP TSC on BHI, Spread plate variation Milk powder A, SP TSC on BHI, Pour Plate with overlay
28
29 Summary and moving forward Spores are ubiquitous and must be controlled at the farm and processing level Sample type (raw milk, powder, etc), Media type, plating type, heat treatment and incubation parameters all play an important role in spore method variation Populations in different dairy products can vary widely contributing to variation seen between spore testing methods Identify a set of methods and parameters that provide standards for spore testing
30 Acknowledgments NYFVI NYSDPAB NDC Staff and students of the Milk Quality Improvement Program and Food Safety Laboratory Participating producers, cooperatives and processors
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