Sources, transmission, and tracking of sporeforming bacterial contaminants in dairy systems.

Bacterial endospores, or simply spores, are formed by a diverse group of members within the phylum Bacillota and include notable genera such as Bacillus, Paenibacillus, and Clostridium. Spores are distributed ubiquitously in natural environments, with soil being an important primary reservoir for these microbes. As such, spores are present throughout the dairy farm environment, and transmission into raw milk occurs through several pathways that coalesce at the point of milk harvest. Despite the very low spore concentrations typically found in bulk tank raw milk, the impact of spores on dairy product quality, safety, and product conformance is widely documented. Processed dairy products affected by the presence of sporeforming bacteria include milk, cheese, dairy powders, ice cream mix, and more. Although raw milk is a major source of spores leading to quality, safety, and conformance issues in dairy products, the impact of other sources should not be discounted and may include ingredients (e.g., cocoa powder), contamination originating from biofilms in processing equipment, and even cross-contamination from the processing environment itself. Addressing spore contamination in the dairy system is complicated by this widespread distribution and by the diversity of these organisms, and successful source tracking often requires discriminatory molecular subtyping tools. Here, we review the key sources of sporeforming bacteria in the dairy system, the factors leading to the transmission of this diverse group of microbes into processed dairy products, and methods employed to enumerate and track spore contaminants.

Gram-negative postpasteurization contamination patterns of single-serve fluid milk produced in 4 different processing facilities.

An analysis of historic data on high temperature, short time (HTST) fluid milk quality showed higher total bacterial counts and lower sensory defect judging scores at d 14 postprocessing for milk packaged in single-serve containers as compared with milk packaged in half-gallon containers from the same processing facilities. As postpasteurization contamination with gram-negative bacteria is likely a major contributor to an increased spoilage risk associated with milk packaged in single-serve containers, we performed a comprehensive assessment of the microbial quality and shelf life of 265 commingled single-serve HTST fluid milk samples (including white [unflavored] skim, white [unflavored] 1%, chocolate skim, and chocolate 1%) collected over 2 visits to 4 commercial fluid milk processing facilities. Over 2 initial sampling visits, the frequency of gram-negative spoilage ranged from 14 to 79% of the product collected from the 4 facilities, with significant differences of gram-negative spoilage frequency between sampling visits, facilities (sampling visit 1, sampling visit 2, and both sampling visits combined), milk types (sampling visit 2), and filler lanes (sampling visit 2). We found no significant differences in the frequency of gram-negative spoilage between sampling time points (e.g., beginning, middle, and end of production run). Across facilities, single-serve containers of milk with gram-negative contamination showed significantly higher bacterial counts on d 7 and 14 and significantly lower sensory scores as compared with those without gram-negative contamination. Follow-up investigations, based on in-facility surveys that identified carton forming mandrels as filler components that frequently failed quality assurance ATP swab checks, found that bacterial genera, including Pseudomonas and Bacillus, isolated from single-serve milk samples were also frequently isolated from mandrels. Although interventions aimed at improving cleaning and sanitation of mandrels did not lead to significant reduction of gram-negative spoilage frequency in a comparison of 398 control and 400 intervention samples, our data still suggest that the unhygienic design of single-serve fillers is likely a root cause of gram-negative contamination of single-serve milk.

Microbacterium represents an emerging microorganism of concern in microfiltered extended shelf-life milk products.

Growing interest in the manufacture of extended shelf-life (ESL) milk, which is typically achieved by a high-temperature treatment called ultra-pasteurization (UP), is driven by distribution challenges, efforts to reduce food waste, and more. Even though high-temperature, short-time (HTST) pasteurized milk has a substantially shorter shelf life than UP milk, HTST milk is preferred in the United States because consumers tend to perceive UP milk as less desirable due to the "cooked" flavor associated with high-temperature processing. While ESL beyond 21 d may be possible for HTST, the survival and outgrowth of psychrotolerant aerobic spore-forming bacteria can still be a limitation to extending shelf life of HTST milk. Microfiltration (MF) is effective for reducing vegetative microorganisms and spores in raw milk, but it is unclear what the effects of membrane pore size, storage temperature, and milk type (i.e., skim vs. whole) are on the microbial shelf life of milk processed by both MF and HTST pasteurization. To investigate these factors, raw skim milk was MF using different pore sizes (0.8 or 1.2 µm), and then MF skim milk and standardized whole milk (MF skim with heat-treated [85°C for 20 s] cream) were HTST pasteurized at 75°C for 20 s. Subsequently, milk was stored at 3°C, 6.5°C, or 10°C and total bacteria counts were measured for up to 63 d. An ANOVA indicated that mean bacterial concentrations between storage temperatures were significantly different from each other, with mean maximum observed concentrations of 3.67, 5.33, and 8.08 log10 cfu/mL for storage temperatures 3°C, 6.5°C, and 10°C, respectively. Additionally, a smaller difference in mean maximum bacterial concentrations throughout shelf life was identified between pore sizes (<1 log cfu/mL), but no significant difference was attributed to milk type. An unexpected outcome of this study was the identification of Microbacterium as a major contributor to the bacterial population in MF ESL milk. Microbacterium is a psychrotolerant, thermoduric gram-positive, non-spore-forming rod with a small cell size (∼0.9 µm length and ∼0.3 µm width), which our data suggest was able to permeate the membranes used in this study, survive HTST pasteurization, and then grow at refrigeration temperatures. While spores continue to be a key concern for the manufacture of MF, ESL milk, our study demonstrates the importance of other psychrotolerant, thermoduric bacteria such as Microbacterium to these products.

Development and deployment of a supply-chain digital tool to predict fluid-milk spoilage due to psychrotolerant sporeformers.

Psychrotolerant sporeformers pose a challenge to maintaining fluid milk quality. Dynamic temperature changes along the supply chain can favor the germination and growth of these bacteria and lead to fluid milk spoilage. In this study, we aim to expand on our previous work on predicting milk spoilage due to psychrotolerant sporeformers. The key model innovations include (1) the ability to account for changing temperatures along the supply chain, and (2) a deployed user-friendly interface to allow easy access to the model. Using the frequencies and concentrations of 8 Bacillales subtypes specific to fluid milk collected in New York, the model simulated sporeformer growth in half-gallons of high-temperature, short-time (HTST) pasteurized fluid milk transported from processing facility to retail store and then to consumer. The Monte Carlo simulations predicted that 44.3% of half-gallons of milk were spoiled (defined as having a bacterial concentration >20,000 cfu/mL, a conservative estimate that represents the Pasteurized Milk Ordinance regulatory limit) after 21 d of refrigerated storage at consumer's home. Model validations showed that the model was the most accurate in predicting the mean sporeformer concentration at low temperatures (i.e., at 3°C and 4°C; compared with higher temperatures at 6°C and 10°C) within the first 21 d of consumer storage, with a root mean square error of 0.29 and 0.34 log10 cfu/mL, respectively. Global sensitivity analyses indicated that home storage temperature, facility-to-retail transportation temperature, and initial spore concentration were the 3 most influential factors for predicting milk spoilage on d 21 of shelf life. What-if scenarios indicated that microfiltration was predicted to be the most effective strategy to reduce spoilage. The implementation of this strategy (assumed to reduce initial spore concentration by 2.2 log10 cfu/mL) was predicted to reduce the percentage of spoiled milk by 17.0 percentage points on d 21 of storage and could delay the date by which 50% of half-gallons of milk were spoiled, from d 25 to 35. Overall, the model is readily deployed as a digital tool for assessing fluid milk spoilage along the supply chain and evaluating the effectiveness of intervention strategies, including those that target storage temperatures at different supply chain stages.

Shelf-life storage temperature has a considerably larger effect than high-temperature, short-time pasteurization temperature on the growth of spore-forming bacteria in fluid milk.

In the absence of postpasteurization contamination, psychrotolerant, aerobic spore-forming bacteria that survive high-temperature, short-time (HTST) pasteurization, limit the ability to achieve HTST extended shelf-life milk. Therefore, the goal of the current study was to evaluate bacterial outgrowth in milk pasteurized at different temperatures (75, 85, or 90°C, each for 20 s) and subsequently stored at 3, 6.5, or 10°C. An initial ANOVA of bacterial concentrations over 14 d of storage revealed a highly significant effect of storage temperatures, but no significant effect of HTST. At d 14, average bacterial counts for milk stored at 3, 6.5, and 10°C were 1.82, 3.55, and 6.86 log10 cfu/mL, respectively. Time to reach 1,000,000 cfu/mL (a bacterial concentration where consumers begin to notice microbially induced sensory defects in fluid milk) was estimated to be 68, 27, and 10 d for milk stored at 3, 6.5, and 10°C, respectively. Out of 95 isolates characterized with rpoB allelic typing, 6 unique genera, 15 unique species, and 44 unique rpoB allelic types were represented. The most common genera identified were Paenibacillus, Bacillus, and Lysinibacillus. Nonmetric multidimensional scaling identified that Bacillus was significantly associated with 3 and 10°C, whereas Paenibacillus was consistently found across all storage temperatures. Overall, our data show that storage temperature has a substantially larger effect on fluid milk shelf life than HTST and suggests that abuse temperatures (e.g., storage at 10°C) allow for growth of Bacillus species (including Bacillus cereus genomospecies) that do not grow at lower temperatures. This indicates that stringent control of storage and distribution temperatures is critical for producing extended shelf-life HTST milk, particularly concerning new distribution pathways for HTST pasteurized milk (e.g., electronic commerce), and when enhanced control of spores in raw milk is not feasible.

Butyric acid-producing bacterial spore levels in conventional raw milk vary by farm.

Anaerobic butyric acid-producing sporeforming bacteria (BAB) are important microbial contaminants in raw milk that may lead to premature spoilage of certain cheeses during aging. A study was conducted to determine the baseline levels of these spores in raw milk from 7 conventional Northeast United States dairy farms over a 1-yr period. The overall mean BAB concentration was 1.79 log10 most probable number per liter with spore levels differing significantly by farm. A post-hoc farm management practices survey was conducted to determine if there was an association between farm practices on BAB levels in raw milk from these farms. Survey questions included variables related to bedding, milking preparation procedures, teat and udder cleanliness scoring, holding area cleaning procedures, and udder clipping or flaming frequency. Each variable was fitted with a linear mixed-effects model, which revealed no significant association between farm-level factors and the initial BAB concentrations in raw milk; this finding was likely due to the small sample size in this study. To demonstrate the usefulness of our data beyond the initial baseline levels of BAB spores in raw milk, we used this data set to calculate minimum number of individual samples that would be needed to be collected in future studies, which was determined to be 96 to 126 samples, to evaluate the association between farm-level factors and BAB spore concentrations in raw milk. Overall, this study provides dairy industry stakeholders with baseline data on BAB spore levels in raw milk, along with a demonstration on how these data could be used in future studies to calculate sample sizes needed to assess the effect of farm management practices on BAB levels in raw milk.

Invited review: Redefining raw milk quality-Evaluation of raw milk microbiological parameters to ensure high-quality processed dairy products.

Raw milk typically has little bacterial contamination as it leaves the udder of the animal; however, through a variety of pathways, it can become contaminated with bacteria originating from environmental sources, the cow herself, and contact with contaminated equipment. Although the types of bacteria found in raw milk are very diverse, select groups are particularly important from the perspective of finished product quality. In particular, psychrophilic and psychrotolerant bacteria that grow quickly at low temperatures (e.g., species in the genus Pseudomonas and the family Enterobacteriaceae) and produce heat-stable enzymes, and sporeforming bacteria that survive processing hurdles in spore form, are the 2 primary groups of bacteria related to effects on processed dairy products. Understanding factors leading to the presence of these important bacterial groups in raw milk is key to reducing their influence on processed dairy product quality. Here we examine the raw milk microbiological parameters used in the contemporary dairy industry for their utility in identifying raw milk supplies that will perform well in processed dairy products. We further recommend the use of a single microbiological indicator of raw milk quality, namely the total bacteria count, and call for the development of a whole-farm approach to raw milk quality that will use data-driven, risk-based tools integrated across the continuum from production to processing and shelf-life to ensure continuous improvement in dairy product quality.

Development of a risk assessment model to predict the occurrence of late blowing defect in Gouda cheese and evaluate potential intervention strategies.

Late blowing defect (LBD) is an important spoilage issue in semi-hard cheese, with the outgrowth of Clostridium tyrobutyricum spores during cheese aging considered to be the primary cause. Although previous studies have explored the microbial and physicochemical factors influencing the defect, a risk assessment tool that allows for improved and rational management of LBD is lacking. The purpose of this study was to develop a predictive model to estimate the probability of LBD in Gouda cheese and evaluate different intervention strategies. The spore concentration distribution of butyric acid bacteria (BAB) in bulk tank milk was obtained from 8 dairy farms over 12 mo. The concentration of C. tyrobutyricum from raw milk to the end of aging was simulated based on Gouda brined for 2 d in saturated brine at 8°C and aged at 13°C. Predicted C. tyrobutyricum concentrations during aging and estimated concentration thresholds in cheese at onset of LBD were used to predict product loss due to LBD during a simulated 1-yr production. With the estimated concentration thresholds in cheese ranging from 4.36 to 4.46 log most probable number (MPN)/kg of cheese, the model predicted that 9.2% (±1.7%) of Gouda cheese showed LBD by d 60; cheeses predicted to show LBD at d 60 showed a mean pH of 5.39 and were produced with raw milk with a mean BAB spore count of 143 MPN/L. By d 90, 36.1% (±3.4%) of cheeses were predicted to show LBD, indicating that LBD typically manifests between d 60 and 90, which is consistent with observations from the literature and the cheese industry. Sensitivity analysis indicated that C. tyrobutyricum maximum growth rate as well as concentration threshold in cheese at onset of LBD are the most important variables, identifying key data needs for development of more accurate models. The implementation of microfiltration or bactofugation of raw milk (assumed to show 98% efficiency of spore removal) in our model prevented occurrence of LBD during the first 60 d of aging. Overall, our findings provide a framework for predicting the occurrence of LBD in Gouda as well as other cheeses and illustrate the value of developing digital tools for managing dairy product quality.

Development of a Monte Carlo simulation model to predict pasteurized fluid milk spoilage due to post-pasteurization contamination with gram-negative bacteria.

Psychrotolerant gram-negative bacteria introduced as post-pasteurization contamination (PPC) are a major cause of spoilage and reduced shelf life of high-temperature, short-time pasteurized fluid milk. To provide improved tools to (1) predict pasteurized fluid milk shelf life as influenced by PPC and (2) assess the effectiveness of different potential interventions that could reduce spoilage due to PPC, we developed a Monte Carlo simulation model that predicts fluid milk spoilage due to psychrotolerant gram-negative bacteria introduced as PPC. As a first step, 17 gram-negative bacterial isolates frequently associated with fluid milk spoilage were selected and used to generate growth data in skim milk broth at 6°C. The resulting growth parameters, frequency of isolation for the 17 different isolates, and initial concentration of bacteria in milk with PPC, were used to develop a Monte Carlo model to predict bacterial number at different days of shelf life based on storage temperature of milk. This model was then validated with data from d 7 and 10 of shelf life, collected from commercial operations. The validated model predicted that the parameters (1) maximum growth rate and (2) storage temperature had the greatest influence on the percentage of containers exceeding 20,000 cfu/mL standard plate count on d 7 and 10 (i.e., spoiling due to PPC), which indicates that accurate data on maximum growth rate and storage temperature are important for accurate predictions. In addition to allowing for prediction of fluid milk shelf life, the model allows for simulation of "what-if" scenarios, which allowed us to predict the effectiveness of different interventions to reduce overall fluid milk spoilage due to PPC through a set of proof-of-concept scenario (e.g., frequency of PPC in containers reduced from 100% to 10%; limiting distribution temperature to a maximum of 6°C). Combined with other models, such as previous models on fluid milk spoilage due to psychrotolerant spore-forming bacteria, the data and tools developed here will allow for rational, digitally enabled, fluid milk shelf life prediction and quality enhancement.

Identification, subtyping, and tracking of dairy spoilage-associated Pseudomonas by sequencing the ileS gene.

Pseudomonas spp. are important spoilage bacteria that negatively affect the quality of refrigerated fluid milk and uncultured cheese by generating unwanted odors, flavors, and pigments. They are frequently found in dairy plant environments and enter dairy products predominantly as postpasteurization contaminants. Current subtyping and characterization methods for dairy-associated Pseudomonas are often labor-intensive and expensive or provide limited and possibly unreliable classification information (e.g., to the species level). Our goal was to identify a single-copy gene that could be analyzed in dairy spoilage-associated Pseudomonas for preliminary species-level identification, subtyping, and phenotype prediction. We tested 7 genes previously targeted in a Pseudomonas fluorescens multilocus sequence typing scheme for their individual suitability in this application using a set of 113 Pseudomonas spp. isolates representing the diversity of typical pasteurized milk contamination. For each of the 7 candidate genes, we determined the success rate of PCR and sequencing for these 113 isolates as well as the level of discrimination for species identification and subtyping that the sequence data provided. Using these metrics, we selected a single gene, isoleucyl tRNA synthetase (ileS), which had the most suitable traits for simple and affordable single-gene Pseudomonas characterization. This was based on the number of isolates successfully sequenced for ileS (113/113), the number of unique allelic types assigned (83, compared with 50 for 16S rDNA), nucleotide and sequence diversity measures (e.g., number of unique SNP and Simpson index), and tests for genetic recombination. The discriminatory ability of ileS sequencing was confirmed by separation of 99 additional dairy Pseudomonas spp. isolates, which were indistinguishable by 16S rDNA sequencing, into 28 different ileS allelic types. Further, we used whole-genome sequencing data to demonstrate the similarities in ileS-based phylogenetic clustering to whole-genome-based clustering for 27 closely related dairy-associated Pseudomonas spp. isolates and for 178 Pseudomonas type strains. We also found that dairy-associated Pseudomonas within an ileS cluster typically shared the same proteolytic and lipolytic activities. Use of ileS sequencing provides a promising strategy for affordable initial characterization of Pseudomonas isolates, which will help the dairy industry identify, characterize, and track Pseudomonas in their facilities and products.

A standard set of testing methods reliably enumerates spores across commercial milk powders.

Contamination of dairy powders with sporeforming bacteria is a concern for dairy processors who wish to penetrate markets with stringent spore count specifications (e.g., infant powders). Despite instituted specifications, no standard methodology is used for spore testing across the dairy industry. Instead, a variety of spore enumeration methods are in use, varying primarily by heat-shock treatments, plating method, recovery medium, and incubation temperature. Importantly, testing the same product using different methodologies leads to differences in spore count outcomes, which is a major issue for those required to meet specifications. As such, we set out to identify method(s) to recommend for standardized milk powder spore testing. To this end, 10 commercial milk powders were evaluated using methods varying by (1) heat treatment (e.g., 80°C/12 min), (2) plating method (e.g., spread plating), (3) medium type (e.g., plate count milk agar), and (4) incubation time and temperature combinations (e.g., 32°C for 48 h). The resulting data set included a total of 48 methods. With this data set, we used a stepwise process to identify optimal method(s) that would explain a high proportion of variance in spore count outcomes and would be practical to implement across the dairy industry. Ultimately, spore pasteurized mesophilic spore count (80°C/12 min, incubated at 32°C for 48 h), highly heat resistant thermophilic spore count (100°C/30 min, incubated at 55°C for 48 h), and specially thermoresistant spore enumeration (106°C/30 min, incubated at 55°C for 48 h) spread plating on plate count milk agar were identified as the optimal method set for reliable enumeration of spores in milk powders. Subsequently, we assessed different powder sampling strategies as a way to reduce variation in powder spore testing outcomes using our recommended method set. Results indicated that 33-g composite sampling may reduce variation in spore testing outcomes for highly heat resistant thermophilic spore count over 11-g and 33-g discrete sampling, whereas there was no significant difference across sampling strategies for specially thermoresistant spore enumeration or spore pasteurized mesophilic spore count. Finally, an interlaboratory study using our recommended method set and a modified method set (using tryptic soy agar with 1% starch) among both university and industry laboratories showed increased variation in spore count outcomes within milk powders, which not only was due to natural variation in powders but also was hypothesized to be due to technical errors, highlighting the need for specialized training for technicians who perform spore testing on milk powders. Overall, this study addresses challenges to milk powder spore testing and recommends a method set for standardized spore testing for implementation across the dairy industry.

Invited review: Controlling dairy product spoilage to reduce food loss and waste.

Food loss and waste is a major concern in the United States and globally, with dairy foods representing one of the top categories of food lost and wasted. Estimates indicate that in the United States, approximately a quarter of dairy products are lost at the production level or wasted at the retail or consumer level annually. Premature microbial spoilage of dairy products, including fluid milk, cheese, and cultured products, is a primary contributor to dairy food waste. Microbial contamination may occur at various points throughout the production and processing continuum and includes organisms such as gram-negative bacteria (e.g., Pseudomonas), gram-positive bacteria (e.g., Paenibacillus), and a wide range of fungal organisms. These organisms grow at refrigerated storage temperatures, often rapidly, and create various degradative enzymes that result in off-odors, flavors, and body defects (e.g., coagulation), rendering them inedible. Reducing premature dairy food spoilage will in turn reduce waste throughout the dairy continuum. Strategies to reduce premature spoilage include reducing raw material contamination on-farm, physically removing microbial contaminants, employing biocontrol agents to reduce outgrowth of microbial contaminants, tracking and eliminating microbial contaminants using advanced molecular microbiological techniques, and others. This review will address the primary microbial causes of premature dairy product spoilage and methods of controlling this spoilage to reduce loss and waste in dairy products.

Interventions designed to control postpasteurization contamination in high-temperature, short-time-pasteurized fluid milk processing facilities: A case study on the effect of employee training, clean-in-place chemical modification, and preventive maintenance programs.

Postpasteurization contamination (PPC) with gram-negative bacteria adversely affects the quality and shelf-life of milk through the development of flavor, odor, texture, and visual defects. Through evaluation of milk quality at 4 large fluid milk processing facilities in the northeast United States, we examined the efficacy of 3 strategies designed to reduce the occurrence of PPC in fluid milk: (1) employee training (focusing on good manufacturing practices) alone and (2) with concurrent implementation of modified clean-in-place chemistry and (3) preventive maintenance (PM) focused on replacement of wearable rubber components. Despite increases in employee knowledge and self-reported behavior change, microbiological evaluation of fluid milk before and after interventions indicated that neither training alone nor training combined with modified clean-in-place interventions significantly decreased PPC. Furthermore, characterization of gram-negative bacterial isolates from milk suggested that specific bacterial taxonomic groups (notably, Pseudomonas sequence types) continued to contribute to PPC even after interventions and that no major changes in the composition of the spoilage-associated microbial populations occurred as a consequence of the interventions. More specifically, in 3 of 4 facilities, gram-negative bacteria with identical 16S rDNA sequence types were isolated on multiple occasions. Evaluation of a PM intervention showed that used rubber goods harbored PPC-associated bacteria and that PPC may have been less frequent following a PM intervention in which wearable rubber goods were replaced (reduction from 3/3 samples with PPC before to 1/3 samples after). Overall, our findings suggest that commonly used "broad stroke interventions" may have a limited effect on reducing PPC. Our case study also demonstrates the inherent complexities of identifying and successfully addressing sanitation problems in large and complex fluid milk processing facilities. For example, broad changes to sanitation practices without improvements in PM and sanitary equipment design may not always lead to reduced PPC. Our data also indicate that although short-term evaluations, such as pre- and post-tests for employee training, may suggest improvements after corrective and preventive actions, extensive microbial testing, ideally in combination with isolate characterization, may be necessary to evaluate return on investment of different interventions.

Short communication: Coliform Petrifilm as an alternative method for detecting total gram-negative bacteria in fluid milk.

Postpasteurization contamination (PPC) of fluid milk remains a challenge for some dairy processors. Pseudomonas is the most common contaminant of fluid milk after pasteurization, and therefore methods to detect PPC should be inclusive of Pseudomonas and other gram-negative contaminants (e.g., coliform bacteria). Our objective was to compare the ability of 3M (St. Paul, MN) coliform and Enterobacteriaceae (EB) Petrifilm to detect total gram-negative bacteria with that of the standard method, crystal violet tetrazolium agar. To that end, we evaluated coliform Petrifilm, EB Petrifilm, and crystal violet tetrazolium agar to detect gram-negative bacteria in naturally contaminated samples of fluid milk. A total of 92 observations derived from shelf-life testing of 33 milk samples from 5 different processing facilities were evaluated for (1) presence of coliforms on coliform Petrifilm at both 24 and 48 h of incubation; (2) presence of any growth, regardless of gas production, on coliform Petrifilm at both 24 and 48 h of incubation; (3) presence of EB on EB Petrifilm at both 24 and 48 h of incubation; (4) presence of any growth, regardless of gas or acid production, on EB Petrifilm at both 24 and 48 h of incubation; and (5) presence of gram-negative bacteria on crystal violet tetrazolium agar after 48 h of incubation. Sensitivity and specificity analysis of results indicated that compared with the standard method (i.e., crystal violet tetrazolium agar), the method that performed the best, based on balanced accuracy (i.e., the average of sensitivity and specificity), was coliform Petrifilm evaluated for the presence of any growth after 48 h of incubation (sensitivity = 0.787; specificity = 0.839). This method can be easily adopted by the dairy industry as many processing facilities already test for coliforms using coliform Petrifilm. Improving the ability of processors to detect PPC will improve the quality of the fluid milk supply.

Bedding and bedding management practices are associated with mesophilic and thermophilic spore levels in bulk tank raw milk.

Mesophilic and thermophilic spore-forming bacteria represent a challenge to the dairy industry, as these bacteria are capable of surviving adverse conditions associated with processing and sanitation and eventually spoil dairy products. The dairy farm environment, including soil, manure, silage, and bedding, has been implicated as a source for spores in raw milk. High levels of spores have previously been isolated from bedding, and different bedding materials have been associated with spore levels in bulk tank (BT) raw milk; however, the effect of different bedding types, bedding management practices, and bedding spore levels on the variance of spore levels in BT raw milk has not been investigated. To this end, farm and bedding management surveys were administered and unused bedding, used bedding, and BT raw milk samples were collected from dairy farms (1 or 2 times per farm) across the United States over 1 yr; the final data set included 182 dairy farms in 18 states. Bedding suspensions and BT raw milk were spore pasteurized (80°C for 12 min), and mesophilic and thermophilic spores were enumerated. Piecewise structural equation modeling analysis was used to determine direct and indirect pathways of association among farm and bedding practices, levels of spores in unused and used bedding, and levels of spores in BT raw milk. Separate models were constructed for mesophilic and thermophilic spore levels. The analyses showed that bedding material had a direct influence on levels of spores in unused and used bedding as well as an indirect association with spore levels in BT raw milk through used bedding spore levels. Specific bedding and farm management practices as well as cow hygiene in the housing area were associated with mesophilic and thermophilic spore levels in unused bedding, used bedding, and BT raw milk. Notably, levels of spores in used bedding were positively related to those in unused bedding, and used bedding spore levels were positively related to those in BT raw milk. The results of this study increase the understanding of the levels and ecology of mesophilic and thermophilic spores in raw milk, emphasize the possible role of bedding as a source of spores on-farm, and present opportunities for dairy producers to reduce spore levels in BT raw milk.

Internal transcribed spacer (ITS) sequence-based characterization of fungal isolates from multiple yogurt facilities-A case study.

Fungal spoilage remains a significant issue in dairy product quality, especially for cultured dairy products such as yogurt formulated without preservatives such as potassium sorbate. Fungal contamination can occur throughout the processing continuum, from the dairy farm environment to the finished product processing environment. As molecular characterization of fungal isolates is used more frequently, we obtained fungal isolates obtained in 2 yogurt processing facilities as part of routine fungal testing of raw materials (e.g., fruit preparations, added ingredients), in-process product samples, environmental samples (e.g., air plates, equipment surfaces such as valves, face plates, air nozzles), and finished product samples, to determine whether internal transcribed spacer (ITS) barcoding data would be helpful to support source tracking of fungal contamination issues. Internal transcribed spacer PCR amplification and sequencing allowed us to classify the 852 isolates from these 2 facilities into 200 unique ITS allelic types (AT), representing the phyla Ascomycota (743 isolates), Basidiomycota (97 isolates), and Mucoromycota (12 isolates). Thirty ITS AT were isolated from both facilities; 62 and 108 ITS AT were isolated from only facility A or only facility B, respectively. Nine ITS AT were each represented by more than 20 isolates; these AT comprised 53% of the 852 isolates. The considerable diversity of fungal isolates even within a single facility illustrates the challenge associated with controlling fungal contamination of dairy products. The ITS barcoding technique, however, did show promise for facilitating the source tracking of fungal contamination, particularly for ITS AT over-represented in a given facility. For example, we found evidence for equipment-specific reservoirs for 2 AT (14 and 219) in facility B. Our data suggest that despite its limited discriminatory power, ITS sequencing can provide initial information that can help trace fungal contamination along the processing continuum. However, development and implementation of discriminatory subtyping methods will be needed to further improve the ability to identify sources of fungal contamination in dairy facilities. Developing and implementing sampling plans that comprehensively capture yeast and mold diversity in a given processing facility remain a considerable challenge.

Evaluation of biopreservatives in Greek yogurt to inhibit yeast and mold spoilage and development of a yogurt spoilage predictive model.

Dairy products, including cultured dairy products such as cheese and yogurt, are susceptible to fungal spoilage. Traditionally, additives such as potassium sorbate have been used to control fungal spoilage; however, with consumer demand for clean-label products, other strategies to control fungal spoilage (e.g., biopreservatives) are increasingly being used in dairy formulations. In order to help the dairy industry better evaluate biopreservatives for control of fungal spoilage, we developed a challenge study protocol, which was applied to evaluate 2 commercially available protective cultures for their ability to control yeast and mold spoilage of Greek yogurt. Greek yogurt formulated with and without protective cultures was inoculated with a cocktail consisting of 5 yeasts and 1 mold to yield inoculum levels of 101 and 103 cfu/g of yogurt. The inoculated yogurts were stored at 7°C and fungal counts as well as time to visible growth, on the yogurt surface, of mycelium mold colonies or yeast were determined over shelf-life. Whereas fungal concentrations increased to spoilage levels (≥105 cfu/g) in all yogurt formulations at both inoculum levels by d 23 of storage at 7°C, no surface mold was observed over 76 d in any of the products formulated with protective cultures. Control yogurts without biopreservatives all showed surface mold by d 23. In order to allow industry to better evaluate the business effects of improved control of surface mold growth that can be achieved with protective cultures, we developed a Monte Carlo simulation model to estimate consumer exposure to visible mold growth in yogurt formulated without fungal inhibitors. Our model showed that initial mold contamination rate has the largest effect on the model outcome, indicating that accurate data on contamination rates are important for use of these models. When air plates were used, in a proof-of-concept approach, to estimate initial contamination rates in a small yogurt manufacturing operation, our model predicted that 550 ± 25.2 consumers (±standard deviation) would be exposed to visible mold growth for every 1 million cups of yogurt produced. With initial contamination rate data for individual facilities, this model could be used by industry to estimate the number of consumers exposed to visible mold spoilage and could allow industry to better assess the value of mold-control strategies.

Pseudomonas fluorescens group bacterial strains are responsible for repeat and sporadic postpasteurization contamination and reduced fluid milk shelf life.

Postpasteurization contamination (PPC) of high temperature, short time-pasteurized fluid milk by gram-negative (GN) bacteria continues to be an issue for processors. To improve PPC control, a better understanding of PPC patterns in dairy processing facilities over time and across equipment is needed. We thus collected samples from 10 fluid milk processing facilities to (1) detect and characterize PPC patterns over time, (2) determine the efficacy of different media to detect PPC, and (3) characterize sensory defects associated with PPC. Specifically, we collected 280 samples of high temperature, short time-pasteurized milk representing different products (2%, skim, and chocolate) and different fillers over 4 samplings performed over 11 mo at each of the 10 facilities. Standard plate count (SPC) as well as total GN, coliform, and Enterobacteriaceae (EB) counts were performed upon receipt and after 7, 10, 14, 17, and 21 d of storage at 6°C. We used 16S rDNA sequencing to characterize representative bacterial isolates from (1) test days with SPC >20,000 cfu/mL and (2) all samples with presumptive GN, coliforms, or EB. Day-21 samples were also evaluated by a trained defect judging panel. By d 21, 226 samples had SPC >20,000 cfu/mL on at least 1 d of shelf life; GN bacteria were found in 132 of these 226 samples, indicating PPC. Crystal violet tetrazolium agar detected PPC with the greatest sensitivity. Spoilage due to PPC was predominantly associated with Pseudomonas (isolated from 101 of the 132 samples with PPC); coliforms and EB were found in 27 and 37 samples with spoilage due to PPC, respectively. Detection of Pseudomonas and Acinetobacter was associated with lower flavor scores; coagulated, fruity fermented, and unclean defects were more prevalent in d-21 samples with PPC. Repeat isolation of Pseudomonas fluorescens group strains with identical partial 16S rDNA sequence types was observed in 8 facilities. In several facilities, specific lines, products, or processing days were linked to repeat product contamination with Pseudomonas with identical sequence types. Our data show that PPC due to Pseudomonas remains a major challenge for fluid milk processors; the inability of coliform and EB tests to detect Pseudomonas may contribute to this. Our data also provide important initial insights into PPC patterns (e.g., line-specific contamination), supporting the importance of molecular subtyping methods for identification of PPC sources.

Psychrotolerant spore-former growth characterization for the development of a dairy spoilage predictive model.

Psychrotolerant spore-forming bacteria represent a major challenge regarding microbial spoilage of fluid milk. These organisms can survive most conventional pasteurization regimens and subsequently germinate and grow to spoilage levels during refrigerated storage. To improve predictions of fluid milk shelf life and assess different approaches to control psychrotolerant spore-forming bacteria in the fluid milk production and processing continuum, we developed a predictive model of spoilage of fluid milk due to germination and growth of psychrotolerant spore-forming bacteria. We characterized 14 psychrotolerant spore-formers, representing the most common Bacillales subtypes isolated from raw and pasteurized milk, for ability to germinate from spores and grow in skim milk broth at 6°C. Complete growth curves were obtained by determining total bacterial count and spore count every 24 h for 30 d. Based on growth curves at 6°C, probability distributions of initial spore counts in bulk tank raw milk, and subtype frequency in bulk tank raw milk, a Monte Carlo simulation model was created to predict spoilage patterns in high temperature, short time-pasteurized fluid milk. Monte Carlo simulations predicted that 66% of half-gallons (1,900 mL) of high temperature, short time fluid milk would reach a cell density greater than 20,000 cfu/mL after 21 d of storage at 6°C, consistent with current spoilage patterns observed in commercial products. Our model also predicted that an intervention that reduces initial spore loads by 2.2 Log10 most probable number/mL (e.g., microfiltration) can extend fluid milk shelf life by 4 d (end of shelf life was defined here as the first day when the mean total bacterial count exceeded 20,000 cfu/mL). This study not only provides a baseline understanding of the growth rates of psychrotolerant spore-formers in fluid milk, it also provides a stochastic model of spoilage by these organisms over the shelf life of fluid milk, which will ultimately allow for the assessment of different approaches to reduce fluid milk spoilage.

Longitudinal assessment of dairy farm management practices associated with the presence of psychrotolerant Bacillales spores in bulk tank milk on 10 New York State dairy farms.

The ability of certain spore-forming bacteria in the order Bacillales (e.g., Bacillus spp., Paenibacillus spp.) to survive pasteurization in spore form and grow at refrigeration temperatures results in product spoilage and limits the shelf life of high temperature, short time (HTST)-pasteurized fluid milk. To facilitate development of strategies to minimize contamination of raw milk with psychrotolerant Bacillales spores, we conducted a longitudinal study of 10 New York State dairy farms, which included yearlong monthly assessments of the frequency and levels of bulk tank raw milk psychrotolerant spore contamination, along with administration of questionnaires to identify farm management practices associated with psychrotolerant spore presence over time. Milk samples were first spore pasteurized (80°C for 12 min) and then analyzed for sporeformer counts on the initial day of spore pasteurization (SP), and after refrigerated storage (6°C) for 7, 14, and 21 d after SP. Overall, 41% of samples showed sporeformer counts of >20,000 cfu/mL at d 21, with Bacillus and Paenibacillus spp. being predominant causes of high sporeformer counts. Statistical analyses identified 3 management factors (more frequent cleaning of the bulk tank area, the use of a skid steer to scrape the housing area, and segregating problem cows during milking) that were all associated with lower probabilities of d-21 Bacillales spore detection in SP-treated bulk tank raw milk. Our data emphasize that appropriate on-farm measures to improve overall cleanliness and cow hygiene will reduce the probability of psychrotolerant Bacillales spore contamination of bulk tank raw milk, allowing for consistent production of raw milk with reduced psychrotolerant spore counts, which will facilitate production of HTST-pasteurized milk with extended refrigerated shelf life.