N.H. Martin

Identification and Characterization of Psychrotolerant Sporeformers Associated with Fluid Milk Production and Processing

ABSTRACT Psychrotolerant spore-forming bacteria represent a major challenge to the goal of extending the shelf life of pasteurized dairy products. The objective of this study was to identify prominent phylogenetic groups of dairy-associated aerobic sporeformers and to characterize representative isolates for phenotypes relevant to growth in milk. Analysis of sequence data for a 632-nucleotide fragment of rpoB showed that 1,288 dairy-associated isolates (obtained from raw and pasteurized milk and from dairy farm environments) clustered into two major divisions representing (i) the genus Paenibacillus (737 isolates, including the species Paenibacillus odorifer, Paenibacillus graminis, and Paenibacillus amylolyticus sensu lato) and (ii) Bacillus (n = 467) (e.g., Bacillus licheniformis sensu lato, Bacillus pumilus, Bacillus weihenstephanensis) and genera formerly classified as Bacillus (n = 84) (e.g., Viridibacillus spp.). When isolates representing the most common rpoB allelic types (ATs) were tested for growth in skim milk broth at 6°C, 6/9 Paenibacillus isolates, but only 2/8 isolates representing Bacillus subtypes, grew >5 log CFU/ml over 21 days. In addition, 38/40 Paenibacillus isolates but only 3/47 Bacillus isolates tested were positive for β-galactosidase activity (including some isolates representing Bacillus licheniformis sensu lato, a common dairy-associated clade). Our study confirms that Paenibacillus spp. are the predominant psychrotolerant sporeformers in fluid milk and provides 16S rRNA gene and rpoB subtype data and phenotypic characteristics facilitating the identification of aerobic spore-forming spoilage organisms of concern. These data will be critical for the development of detection methods and control strategies that will reduce the introduction of psychrotolerant sporeformers and extend the shelf life of dairy products.

When cheese gets the blues: Pseudomonas fluorescens as the causative agent of cheese spoilage

A bacterial contamination of fresh, low-acid cheese that resulted in production of a blue fluorescent pigment on the surface of the cheese was determined to be caused by Pseudomonas fluorescens biovar IV, a gram-negative bacteria that produces a blue, nondiffusible pigment as well as the soluble pigment pyoverdin, which fluoresces under UV light. Ten isolates collected from contaminated cheese and environmental samples were initially identified as P. fluorescens using 16S rDNA sequencing, but only 8 of the isolates produced blue pigment and fluoresced under UV light when re-inoculated onto fresh, low-acid cheese. The Biolog Metabolic Fingerprint system (Biolog Inc., Hayward, CA) and the Analytical Profile Index (BioMerieux Vitek Inc., Hazelwood, MO) for nonenteric gram-negative species as well as EcoRI ribotyping did not differentiate between the isolates that produced blue color and those that did not. Pulsed field gel electrophoresis with the enzyme XbaI was able to distinguish between the isolates that produced pigment and those that did not and allowed for identification of a specific environmental site (i.e., an overhead cheese vat agitator system) as the likely source of product contamination.

Evaluation of dairy powder products implicates thermophilic sporeformers as the primary organisms of interest

Dairy powder products (e.g., sweet whey, nonfat dry milk, acid whey, and whey protein concentrate-80) are of economic interest to the dairy industry. According to the US Dairy Export Council, customers have set strict tolerances (<500 to <1,000/g) for thermophilic and mesophilic spores in dairy powders; therefore, understanding proliferation and survival of sporeforming organisms within dairy powder processing plants is necessary to control and reduce sporeformer counts. Raw, work-in-process, and finished product samples were collected from 4 dairy powder processing facilities in the northeastern United States over a 1-yr period. Two separate spore treatments: (1) 80°C for 12min (to detect sporeformers) and (2) 100°C for 30min (to detect highly heat resistant sporeformers) were applied to samples before microbiological analyses. Raw material, work-in-process, and finished product samples were analyzed for thermophilic, mesophilic, and psychrotolerant sporeformers, with 77.5, 71.0, and 4.6% of samples being positive for those organisms, respectively. Work-in-process and finished product samples were also analyzed for highly heat resistant thermophilic and mesophilic sporeformers, with 63.7 and 42.6% of samples being positive, respectively. Sporeformer prevalence and counts varied considerably by product and plant; sweet whey and nonfat dry milk showed a higher prevalence of thermophilic and mesophilic sporeformers compared with acid whey and whey protein concentrate-80. Unlike previous reports, we found limited evidence for increased spore counts toward the end of processing runs. Our data provide important insight into spore contamination patterns associated with production of different types of dairy powders and support that thermophilic sporeformers are the primary organism of concern in dairy powders.

Results from raw milk microbiological tests do not predict the shelf-life performance of commercially pasteurized fluid milk.

Analytical tools that accurately predict the performance of raw milk following its manufacture into commercial food products are of economic interest to the dairy industry. To evaluate the ability of currently applied raw milk microbiological tests to predict the quality of commercially pasteurized fluid milk products, samples of raw milk and 2% fat pasteurized milk were obtained from 4 New York State fluid milk processors for a 1-yr period. Raw milk samples were examined using a variety of tests commonly applied to raw milk, including somatic cell count, standard plate count, psychrotrophic bacteria count, ropy milk test, coliform count, preliminary incubation count, laboratory pasteurization count, and spore pasteurization count. Differential and selective media were used to identify groups of bacteria present in raw milk. Pasteurized milk samples were held at 6°C for 21 d and evaluated for standard plate count, coliform count, and sensory quality throughout shelf-life. Bacterial isolates from select raw and pasteurized milk tests were identified using 16S ribosomal DNA sequencing. Linear regression analysis of raw milk test results versus results reflecting pasteurized milk quality consistently showed low R(2) values (<0.45); the majority of R(2) values were <0.25, indicating small relationship between the results from the raw milk tests and results from tests used to evaluate pasteurized milk quality. Our findings suggest the need for new raw milk tests that measure the specific biological barriers that limit shelf-life and quality of fluid milk products.

Spore populations among bulk tank raw milk and dairy powders are significantly different.

To accommodate stringent spore limits mandated for the export of dairy powders, a more thorough understanding of the spore species present will be necessary to develop prospective strategies to identify and reduce sources (i.e., raw materials or in-plant) of contamination. We characterized 1,523 spore isolates obtained from bulk tank raw milk (n=33 farms) and samples collected from 4 different dairy powder-processing plants producing acid whey, nonfat dry milk, sweet whey, or whey protein concentrate 80. The spores isolated comprised 12 genera, at least 44 species, and 216 rpoB allelic types. Bacillus and Geobacillus represented the most commonly isolated spore genera (approximately 68.9 and 12.1%, respectively, of all spore isolates). Whereas Bacillus licheniformis was isolated from samples collected from all plants and farms, Geobacillus spp. were isolated from samples from 3 out of 4 plants and just 1 out of 33 farms. We found significant differences between the spore population isolated from bulk tank raw milk and those isolated from dairy powder plant samples, except samples from the plant producing acid whey. A comparison of spore species isolated from raw materials and finished powders showed that although certain species, such as B. licheniformis, were found in both raw and finished product samples, other species, such as Geobacillus spp. and Anoxybacillus spp., were more frequently isolated from finished powders. Importantly, we found that 8 out of 12 genera were isolated from at least 2 different spore count methods, suggesting that some spore count methods may provide redundant information if used in parallel. Together, our results suggest that (1) Bacillus and Geobacillus are the predominant spore contaminants in a variety of dairy powders, implying that future research efforts targeted at elucidating approaches to reduce levels of spores in dairy powders should focus on controlling levels of spore isolates from these genera; and (2) the spore populations isolated from bulk tank raw milk and some dairy powder products are significantly different, suggesting that targeting in-plant sources of contamination may be important for achieving low spore counts in the finished product. These data provide important insight regarding the diversity of spore populations isolated from dairy powders and bulk tank raw milk, and demonstrate that several spore genera are detected by multiple spore count methods.

Identification of dairy farm management practices associated with the presence of psychrotolerant sporeformers in bulk tank milk

Some strains of sporeforming bacteria (e.g., Bacillus spp. and Paenibacillus spp.) can survive pasteurization and subsequently grow at refrigeration temperatures, causing pasteurized fluid milk spoilage. To identify farm management practices associated with different levels of sporeformers in raw milk, a bulk tank sample was obtained from and a management and herd health questionnaire was administered to 99 New York State dairy farms. Milk samples were spore pasteurized [80°C (176°F) for 12 min] and subsequently analyzed for most-probable number and for sporeformer counts on the initial day of spore pasteurization (SP), and after refrigerated storage (6°C) at 7, 14, and 21 d after SP. Management practices were analyzed for association with sporeformer counts and bulk tank somatic cell counts. Sixty-two farms had high sporeformer growth (≥3 log cfu/mL at any day after SP), with an average sporeformer count of 5.20 ± 1.41 mean log10 cfu/mL at 21 d after SP. Thirty-seven farms had low sporeformer numbers (<3 log cfu/mL for all days after SP), with an average sporeformer count of 0.75 ± 0.94 mean log10 cfu/mL at 21 d after SP. Farms with >25% of cows with dirty udders in the milking parlor were 3.15 times more likely to be in the high category than farms with ≤10% of milking cows with dirty udders. Farms with <200 cows were 3.61 times more likely to be in the high category than farms with ≥200 cows. Management practices significantly associated with increased bulk tank somatic cell count were a lack of use of the California mastitis test at freshening and >25% of cows with dirty udders observed in the milking parlor. Changes in management practices associated with cow cleanliness may directly ensure longer shelf life and higher quality of pasteurized fluid milk.

A decade of improvement: New York State fluid milk quality

The microbiological and sensory qualities of New York State (NYS) fluid milk products were assessed as part of an ongoing fluid milk quality program. Commercially packaged pasteurized fluid milk samples were collected twice a year over the 10-yr period from 2001 to 2010 from 14 NYS dairy processing facilities and analyzed at the Milk Quality Improvement Program (MQIP) laboratory. Each sample was tested throughout refrigerated storage (6°C) on day initial, 7, 10, and 14 for standard plate count (SPC), coliform count (CC), and sensory quality. Over the 10-yr period, the percentage of samples with bacterial numbers below the Pasteurized Milk Ordinance (PMO) limit of 20,000 cfu/mL at d 14 postprocessing ranged from a low of 21.1% in 2002 to a high of 48.6% in 2010. Percent samples positive for coliforms during that same period ranged from a high of 26.6% in 2002 to a low of 7.5% in 2007. Mean d 14 sensory scores ranged from a low of 6.0 in 2002 to a high of 7.3 in 2007. Samples contaminated with coliforms after pasteurization have significantly higher SPC counts and significantly lower sensory scores on d 14 of shelf-life than those not contaminated with coliforms. Product factors such as fat level were not significantly associated with SPC, CC, or sensory quality of the product, whereas the factor processing plant significantly affected overall product quality. This study demonstrates that overall fluid milk quality in NYS, as determined by microbiological and sensory analyses, has improved over the last decade, and identifies some challenges that remain.

Different management practices are associated with mesophilic and thermophilic spore levels in bulk tank raw milk

Bacterial endospores (also referred to as spores) present in raw milk are capable of surviving pasteurization and other adverse conditions encountered during dairy powder production. Therefore, requiring low spore levels in raw ingredients (e.g., raw milk) may be necessary for producing dairy powders with low spore counts. To identify potential associations between management practices and spore levels in raw milk, we sampled bulk tank raw milk from 33 farms throughout New York State every other month for 1yr. Following spore pasteurization (80°C for 12min), samples were incubated at 3 different temperatures to enumerate psychrotolerant (6°C for 10 d), mesophilic (32°C for 48h), and thermophilic (55°C for 48h) spores. An additional enrichment procedure was used to detect spores present at low levels (<10 spores/mL). Overall, psychrotolerant, mesophilic, and thermophilic spores were detected (at levels ≥10 spores/mL) in 1, 74, and 58% of bulk tank raw milk samples, respectively. Although thermophilic spore levels could not be quantified (due to bacterial swarming), mesophilic spore levels ranged from below detection (<10 spores/mL) to 680 spores/mL. Data collected through surveys were used to identify management practices associated with either mesophilic or thermophilic spore levels. We found that different management practices are associated with mesophilic and thermophilic spore levels. Low mesophilic spore levels in bulk tank raw milk samples were associated with (1) large herd size, (2) use of sawdust or sand bedding, and (3) not fore stripping during the premilking routine. Management practices that were associated with lower odds of having a thermophilic spore level ≥10 spores/mL are (1) large herd size, (2) spray-based application of the postmilking disinfectant, (3) dry massaging the udder during the premilking routine, and (4) the use of straw bedding. Collectively, these results suggest that different management practices may influence mesophilic and thermophilic spore levels in raw milk.

Influence of raw milk quality on processed dairy products: How do raw milk quality test results relate to product quality and yield?

This article provides an overview of the influence of raw milk quality on the quality of processed dairy products and offers a perspective on the merits of investing in quality. Dairy farmers are frequently offered monetary premium incentives to provide high-quality milk to processors. These incentives are most often based on raw milk somatic cell and bacteria count levels well below the regulatory public health-based limits. Justification for these incentive payments can be based on improved processed product quality and manufacturing efficiencies that provide the processor with a return on their investment for high-quality raw milk. In some cases, this return on investment is difficult to measure. Raw milks with high levels of somatic cells and bacteria are associated with increased enzyme activity that can result in product defects. Use of raw milk with somatic cell counts >100,000cells/mL has been shown to reduce cheese yields, and higher levels, generally >400,000 cells/mL, have been associated with textural and flavor defects in cheese and other products. Although most research indicates that fairly high total bacteria counts (>1,000,000 cfu/mL) in raw milk are needed to cause defects in most processed dairy products, receiving high-quality milk from the farm allows some flexibility for handling raw milk, which can increase efficiencies and reduce the risk of raw milk reaching bacterial levels of concern. Monitoring total bacterial numbers in regard to raw milk quality is imperative, but determining levels of specific types of bacteria present has gained increasing importance. For example, spores of certain spore-forming bacteria present in raw milk at very low levels (e.g., <1/mL) can survive pasteurization and grow in milk and cheese products to levels that result in defects. With the exception of meeting product specifications often required for milk powders, testing for specific spore-forming groups is currently not used in quality incentive programs in the United States but is used in other countries (e.g., the Netherlands).

A standard bacterial isolate set for research on contemporary dairy spoilage

Food spoilage is an ongoing issue that could be dealt with more efficiently if some standardization and unification was introduced in this field of research. For example, research and development efforts to understand and reduce food spoilage can greatly be enhanced through availability and use of standardized isolate sets. To address this critical issue, we have assembled a standard isolate set of dairy spoilers and other selected nonpathogenic organisms frequently associated with dairy products. This publicly available bacterial set consists of (1) 35 gram-positive isolates including 9 Bacillus and 15 Paenibacillus isolates and (2) 16 gram-negative isolates including 4 Pseudomonas and 8 coliform isolates. The set includes isolates obtained from samples of pasteurized milk (n=43), pasteurized chocolate milk (n=1), raw milk (n=1), cheese (n=2), as well as isolates obtained from samples obtained from dairy-powder production (n=4). Analysis of growth characteristics in skim milk broth identified 16 gram-positive and 13 gram-negative isolates as psychrotolerant. Additional phenotypic characterization of isolates included testing for activity of β-galactosidase and lipolytic and proteolytic enzymes. All groups of isolates included in the isolate set exhibited diversity in growth and enzyme activity. Source data for all isolates in this isolate set are publicly available in the FoodMicrobeTracker database (http://www.foodmicrobetracker.com), which allows for continuous updating of information and advancement of knowledge on dairy-spoilage representatives included in this isolate set. This isolate set along with publicly available isolate data provide a unique resource that will help advance knowledge of dairy-spoilage organisms as well as aid industry in development and validation of new control strategies.