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.     

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.     

Low-cost,on-farm intervention to reduce spores in bulk tank raw milk benefits producers,processors, and consumers

Bacterial spores, which are found in raw milk, can survive harsh processing conditions encountered in dairy manufacturing, including pasteurization and drying. Low-spore raw milk is desirable for dairy industry stakeholders, especially those who want to extend the shelf life of their product, expand their distribution channels, or reduce product spoilage. A recent previous study showed that an on-farm intervention that included washing towels with chlorine bleach and drying them completely, as well as training milking parlor employees to focus on teat end cleaning, significantly reduced spore levels in bulk tank raw milk. As a follow up to that previous study, here we calculate the costs associated with that previously described intervention as ranging from $9.49 to $13.35 per cow per year, depending on farm size. A Monte Carlo model was used to predict the shelf life of high temperature, short time fluid milk processed from raw milk before and after this low-cost intervention was applied, based on experimental data collected in a previous study. The model predicted that 18.24% of half-gallon containers of fluid milk processed from raw milk receiving no spore intervention would exceed the pasteurized milk ordinance limit of 20,000 cfu/mL by 17 d after pasteurization, while only 16.99% of containers processed from raw milk receiving the spore intervention would reach this level 17 d after pasteurization (a reduction of 1.25 percentage points and a 6.85% reduction). Finally, a survey of consumer milk use was conducted to determine how many consumers regularly consume fluid milk near or past the date printed on the package (i.e., code date), which revealed that over 50% of fluid milk consumers surveyed continue to consume fluid milk after this date, indicating that a considerable proportion of consumers are exposed to fluid milk that is likely to have high levels spore-forming bacterial growth and possibly associated quality defects (e.g., flavor or odor defects). This further highlights the importance of reducing spore levels in raw milk to extend pasteurized fluid milk shelf life and thereby reducing the risk of adverse consumer experiences. Processors who are interested in extending fluid milk shelf life by controlling the levels of spores in the raw milk supply should consider incentivizing low-spore raw milk through premium payments to producers.     

Monte Carlo simulation model predicts bactofugation can extend shelf-life of pasteurized fluid milk,even when raw milk with low spore counts is used as the incoming ingredient

Bacterial spores from raw milk that survive the pasteurization process are responsible for half of all the spoilage of fluid milk. Bactofugation has received more attention as a nonthermal method that can reduce the presence of bacterial spores in milk and with it the spoilage of fluid milk. The objective of this work was to determine the effectiveness of bactofugation in removing spores from raw milk and estimate the effect the spore removal could have on shelf-life of fluid milk. The study was conducted in a commercial fluid milk processing facility where warm spore removal was performed using one-phase bactofuge followed by warm cream separation and high temperature, short time pasteurization. Samples from different stages of fluid milk processing with and without the use of bactofuge were tested for total plate count, mesophilic spore count, psychrotolerant spore count (PSC), and somatic cell count. Results were evaluated to determine the count reductions during different stages of fluid milk processing and compare counts in fluid milk processed with and without bactofugation. Bactofugation on average reduced the total plate count by 1.81 ± 0.72 log cfu/mL, mesophilic spore count by 1.08 ± 0.71 log cfu/mL, PSC by 0.86 ± 0.59 log cfu/mL, and somatic cell count by 135,881 ± 43,942 cells/mL. Psychrotolerant spore count in final pasteurized skim milk processed with and without bactofugation was used to predict the shelf-life of the pasteurized skim milk using the Monte Carlo simulation model. Although PSC in the initial raw milk was already low (?0.63 ± 0.47 log cfu/mL), the predicted values from the simulation model showed that bactofugation would extend the shelf-life of pasteurized skim milk by approximately 2 d. The results of this study will directly help fluid milk processors evaluate the benefits of using bactofugation as an intervention in their plants, and also demonstrate the benefits of using mathematical modeling in decision making.     

Rapid detection and characterization of postpasteurization contaminants in pasteurized fluid milk

Microbial spoilage of pasteurized fluid milk is typically due to either (1) postpasteurization contamination (PPC) with psychrotolerant gram-negative bacteria (predominantly Pseudomonas) or (2) growth of psychrotolerant sporeformers (e.g., Paenibacillus) that have the ability to survive pasteurization when present as spores in raw milk, and to subsequently grow at refrigeration temperatures. While fluid milk quality has improved over the last several decades, continued reduction of PPC is hampered by the lack of rapid, sensitive, and specific methods that allow for detection of PPC in fluid milk, with fluid milk processors still often using time-consuming methods (e.g., Moseley keeping quality test). The goal of this project was to utilize a set of commercial fluid milk samples that are characterized by a mixture of samples with PPC due to psychrotolerant gram-negative bacteria and samples with presence and growth of psychrotolerant sporeforming bacteria to evaluate different approaches for rapid detection of PPC. Comprehensive microbiological shelf-life characterization of 105 pasteurized fluid milk samples obtained from 20 dairy processing plants showed that 60/105 samples reached bacterial counts >20,000 cfu/mL over the shelf-life due to PPC with gram-negative bacteria. Among these 60 samples with evidence of gram-negative PPC spoilage over the shelf-life, 100% (60/60) showed evidence of contamination with noncoliform, non-Enterobacteriaceae (EB) gram-negative bacteria (e.g., Pseudomonas), 20% (12/60) showed evidence of contamination with coliforms, and 7% (4/60) showed evidence of contamination with noncoliform EB. Among the remaining 45 samples, 28 showed levels of gram-positive bacteria above 20,000 cfu/mL and the remaining 17 samples did not exceed 20,000 cfu/mL over the shelf-life. Evaluation of the same set of 105 samples using 6 different approaches {all possible combinations of 2 different enrichment protocols (13°C or 21°C for 18 h) and 3 different plating media [crystal violet tetrazolium agar, EB Petrifilm (3M, St. Paul, MN), and Coliform Petrifilm]} showed that enrichment at 21°C for 18 h, followed by plating on crystal violet tetrazolium agar provided for the most sensitive, accelerated detection of samples that reached >20,000 cfu/mL due to PPC with psychrotolerant gram-negatives (70% sensitivity). These results show that tests still required and traditionally used in the dairy industry (e.g., coliform testing) are not suitable for monitoring for PPC. Rather, approaches that allow for detection of all gram-negative bacteria are essential for improved detection of PPC in fluid milk.     

Antimicrobial effects of a bioactive glycolipid on spore-forming spoilage bacteria in milk

The growth of psychrotolerant aerobic spore-forming bacteria during refrigerated storage often results in the spoilage of fluid milk, leading to off-flavors and curdling. Because of their low toxicity, biodegradability, selectivity, and antimicrobial activity over a range of conditions, glycolipids are a novel and promising intervention to control undesirable microbes. The objective of this study was to determine the efficacy of a commercial glycolipid product to inhibit spore germination, spore outgrowth, and the growth of vegetative cells of Paenibacillus odorifer, Bacillus weihenstephanensis, and Viridibacillus arenosi, which are the predominant spore-forming spoilage bacteria in milk. For spore germination and outgrowth assays, varying concentrations (25–400 mg/L) of the glycolipid product were added to commercial UHT whole and skim milk inoculated with ～4 log₁₀ spores/mL of each bacteria and incubated at 30°C for 5 d. Inhibition of spore germination in inoculated UHT whole milk was only observed for V. arenosi, and only when glycolipid was added at 400 mg/L. However, concentrations of 400 and 200 mg/L markedly inhibited the outgrowth of vegetative cells from spores of P. odorifer and B. weihenstephanensis, respectively. No inhibition of spore germination or outgrowth was observed in inoculated UHT skim milk for any strain at the concentrations tested (25 and 50 mg/L). The effect of glycolipid addition on vegetative cell growth in UHT whole and skim milk when inoculated with ～4 log₁₀ cfu/mL of each bacteria was also determined over 21 d of storage at 7°C. Glycolipid addition at 50 mg/L was bactericidal against P. odorifer and B. weihenstephanensis in inoculated UHT skim milk through 21 d of storage, whereas 100 mg/L was needed for similar control of V. arenosi. Concentrations of 100 and 200 mg/L inhibited the growth of vegetative cells of B. weihenstephanensis and P. odorifer, respectively, in inoculated UHT whole milk, whereas 200 mg/L was also bactericidal to B. weihenstephanensis. Additional studies are necessary to identify effective concentrations for the inhibition of Viridibacillus spp. growth in whole milk beyond 7 d. Findings from this study demonstrate that natural glycolipids have the potential to inhibit the growth of dairy-spoilage bacteria and extend the shelf life of milk.     

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.     

Use of microfiltration to improve fluid milk quality

The objectives of the research were to determine the growth characteristics of bacteria in commercially pasteurized skim milk as a function of storage temperature; to determine the efficacy of a microfiltration and pasteurization process in reducing the number of total bacteria, spores, and coliforms in skim milk; and to estimate the shelf life of pasteurized microfiltered skim milk as a function of storage temperature. For the first objective, commercially pasteurized skim milk was stored at 0.1, 2.0, 4.2, and 6.1 degrees C. A total bacterial count >20,000 cfu/mL was considered the end of shelf life. Shelf life ranged from 16 d at 6.1 degrees C to 66 d at 0.1 degrees C. Decreasing storage temperature increased lag time and reduced logarithmic growth rate of a mixed microbial population. The increased lag time for the mixed microbial population at a lower storage temperature was the biggest contributor to longer shelf life. For the second objective, raw skim milk was microfiltered at 50 degrees C using a Tetra Alcross M7 Pilot Plant equipped with a ceramic Membralox membrane (pore diameter of 1.4 microm). The 50 degrees C permeate was pasteurized at 72 degrees C for 15 s, and cooled to 6 degrees C. Bacterial counts of raw skim milk were determined by standard plate count. Bacterial counts of microfiltered and pasteurized microfiltered skim milk were determined using a most probable number method. Across 3 trials, bacterial counts of the raw milk were reduced from 2,400, 3,600, and 1,475 cfu/mL to 0.240, 0.918, and 0.240 cfu/mL, respectively, by microfiltration. Bacterial counts in the pasteurized microfiltered skim milk for the 3 trials were 0.005, 0.008, and 0.005 cfu/mL, respectively, demonstrating an average 5.6 log reduction from the raw count due to the combination of microfiltration and pasteurization. For the third objective, pasteurized microfiltered skim milk was stored at each of 4 temperatures (0.1, 2.0, 4.2, and 6.1 degrees C) and the total bacterial count was determined weekly over a 92-d period. At 6 time points in the study, samples were also analyzed for noncasein nitrogen and the decrease in casein as a percentage of true protein was calculated. After 92 d, 50% of samples stored at 6.1 degrees C and 12% of samples stored at 4.2 degrees C exceeded a total bacterial count of 20,000 cfu/mL. No samples stored at 0.1 or 2.0 degrees C reached a detectable bacterial level during the study. When the bacterial count was <1,000 cfu/mL, shelf life was limited because sufficient proteolysis had occurred at 32 d at 6.1 degrees C, 46 d at 4.2 degrees C, 78 d at 2.0 degrees C, and >92 d at 0.1 degrees C to produce a detectable off-flavor in skim milk produced from a raw milk with a 240,000 somatic cell count.     

Tracking spore-forming bacterial contaminants in fluid milk-processing systems

The presence of psychrotolerant Bacillus species and related spore formers (e.g., Paenibacillus spp.) in milk has emerged as a key biological obstacle in extending the shelf life of high-temperature, short-time pasteurized fluid milk beyond 14 d. A recently developed rpoB DNA sequence-based subtyping method was applied to characterize spoilage bacteria present in raw milk supplies for 2 processing plants, and to assess transmission of these organisms into pasteurized products. Thirty-nine raw milk samples and 11 pasteurized product samples were collected to represent the processing continuum from incoming truck loads of raw milk to packaged products. Milk samples were held at 6°C for up to 16 d and plated for bacterial enumeration at various times throughout storage. Among the 88 bacterial isolates characterized, a total of 31 rpoB allelic types representing Bacillus and Paenibacillus spp. were identified, including 5 allelic types found in both raw milk and finished product samples. The presence of the same bacterial subtypes in raw and commercially pasteurized milk samples suggests that the raw milk supply represents an important source of these spoilage bacteria. Extension of the shelf life of high-temperature, short-time pasteurized fluid milk products will require elimination of these organisms from milk-processing systems.     

Influence of raw milk quality on fluid milk shelf life

Pasteurized fluid milk shelf life is influenced by raw milk quality. The microbial count and somatic cell count (SCC) determine the load of heat-resistant enzymes in milk. Generally, high levels of psychrotrophic bacteria in raw milk are required to contribute sufficient quantities of heat-stable proteases and lipases to cause breakdown of protein and fat after pasteurization. Sanitation, refrigeration, and the addition of CO2 to milk are used to control both total and psychrotrophic bacteria count. It is not uncommon for total bacterial counts of raw milk to be < 10,000 cfu/mL. In the past, fluid milk processors have not focused much attention on milk SCC. Increased SCC is correlated with increased amounts of heat-stable protease (plasmin) and lipase (lipoprotein lipase) in milk. When starting with raw milk that has a low bacterial count, and in the absence of microbial growth in pasteurized milk, enzymes associated with high SCC will cause protein and fat degradation during refrigerated storage, and produce off-flavors. As the ability to kill, remove, or control microbial growth in pasteurized refrigerated milk continues to improve, the original milk SCC will be the factor limiting the time of refrigerated storage before development of an off-flavor in milk. Most healthy cows in a dairy herd have a milk SCC < 50,000 cell/mL. Bulk tank SCC > 200,000 cell/mL are usually due to the contribution of high SCC milk from a small number of cows in the herd. Technology to identify these cows and keep their milk out of the bulk tank could substantially increase the value of the remaining milk for use in fluid milk processing. To achieve a 60- to 90-d shelf life of refrigerated fluid milk, fluid processors and dairy farmers need to work together to structure economic incentives that allow farmers to produce milk with the SCC needed for extended refrigerated shelf life.     

Bacterial spore levels in bulk tank raw milk are influenced by environmental and cow hygiene factors

Sporeforming bacteria are responsible for the spoilage of several dairy products including fluid milk, cheese, and products manufactured using dried dairy powders as ingredients. Sporeforming bacteria represent a considerable challenge for the dairy industry because they primarily enter the dairy product continuum at the farm, survive processing hurdles, and subsequently grow in finished products. As such, strategies to reduce spoilage due to this group of bacterial contaminants have focused on understanding the effect of farm level factors on the presence of spores in bulk tank raw milk with the goal of reducing spore levels in raw milk, as well as understanding processing contributions to spore levels and outgrowth in finished products. The goal of the current study was to investigate sources of spores in the farm environment and survey farm management practices to identify variables using multimodel inference, a model averaging approach that eliminates the uncertainty of traditional model selection approaches, that affect the presence and levels of spores in bulk tank raw milk. To this end, environmental samples including feed, bedding, manure, soil, water, and so on, and bulk tank raw milk were collected twice from 17 upstate New York dairy farms over a 19-mo period and the presence and levels of various spore types (e.g., psychrotolerant, mesophilic, thermophilic, highly heat resistant thermophilic, specially thermoresistant thermophilic, and anaerobic butyric acid bacteria) were assessed. Manure had the highest level of spores for 4 out of 5 aerobic spore types with mean counts of 5.87, 5.22, 4.35, and 3.68 log cfu/g of mesophilic, thermophilic, highly heat resistant thermophilic, and specially thermoresistant thermophilic spores, respectively. In contrast, bulk tank raw milk had mean spore levels below 1 log cfu/mL across spore types. Multimodel inference was used to determine variables (i.e., management factors, environmental spore levels, and meteorological data from each sampling) that were important for presence or levels of each spore type in bulk tank raw milk. Analyses indicated that variables of importance for more than one spore type included the residual level of spores in milk from individual cows after thorough teat cleaning and forestripping, udder hygiene, clipping or flaming of udders, spore level in feed commodities, spore level in parlor air, how often bedding was topped up or changed, the use of recycled manure bedding, and the use of sawdust bedding. These results improve our understanding of how spores transfer from environmental sources into bulk tank raw milk and provide information that can be used to design intervention trials aimed at reducing spore levels in raw milk.     

Strategies for raw sheep milk storage in smallholdings: Effect of freezing or long-term refrigerated storage on microbial growth

We assessed the effects of freezing and refrigeration over long periods on the microbiological quality of sheep milk. The raw milk was frozen in 1-L plastic bags or 5-L milk buckets and, after 1 mo, thawed at 7 or 25°C. We evaluated these samples immediately after thawing (d 0) and after 1 d of storage at 7°C. Furthermore, we stored fresh raw milk at 7°C for 10 d in the same packages and in a bulk milk cooler at 4°C (adding 10% of fresh raw milk daily). The total bacterial, total psychrotolerant, and proteolytic psychrotolerant counts were evaluated before and after thawing (for previously frozen milk) and daily (for refrigerated milk). The frozen-thawed milks showed no significant increase in bacterial counts immediately after thawing for all samples (<0.7 log cfu/mL), but only the samples packaged in 1-L bags and thawed at 7°C remained microbiologically adequate after 1 d of storage. Findings of the refrigerated samples were modeled using a modified Gompertz equation, obtaining a lag phase of around 0.5 (5-L bucket), 2.6 (1-L bag), and 7.0 (bulk milk cooler) d for total bacterial and total psychrotolerant counts. Maximum growth rates (µ_max) were 1.0 and 1.0 (5-L bucket), 1.2 and 1.3 (1-L bag) and 3.0 and 1.5 (bulk milk cooler) ln(cfu/mL) per day for total bacteria and total psychrotolerant counts, respectively. Compared with total bacteria and total psychrotolerant bacteria, psychrotolerant proteolytic bacteria grew slowly, reaching unacceptable counts only after 9 to 10 d of storage. The studied methods are interesting alternatives for preserving raw sheep milk on smallholdings.     

Shelf life of pasteurized microfiltered milk containing 2% fat

The goal of this research was to produce homogenized milk containing 2% fat with a refrigerated shelf life of 60 to 90 d using minimum high temperature, short time (HTST) pasteurization in combination with other nonthermal processes. Raw skim milk was microfiltered (MF) using a Tetra Alcross MFS-7 pilot plant (Tetra Pak International SA, Pully, Switzerland) equipped with Membralox ceramic membranes (1.4 μm and surface area of 2.31 m²; Pall Corp., East Hills, NY). The unpasteurized MF skim permeate and each of 3 different cream sources were blended together to achieve three 2% fat milks. Each milk was homogenized (first stage: 17 MPa, second stage: 3 MPa) and HTST pasteurized (73.8°C for 15 s). The pasteurized MF skim permeate and the 3 pasteurized homogenized 2% fat milks (made from different fat sources) were stored at 1.7 and 5.7°C and the standard plate count for each milk was determined weekly over 90 d. When the standard plate count was >20,000 cfu/mL, it was considered the end of shelf life for the purpose of this study. Across 4 replicates, a 4.13 log reduction in bacteria was achieved by MF, and a further 0.53 log reduction was achieved by the combination of MF with HTST pasteurization (73.8°C for 15 s), resulting in a 4.66 log reduction in bacteria for the combined process. No containers of MF skim milk that was pasteurized after MF exceeded 20,000 cfu/mL bacteria count during 90 d of storage at 5.7°C. The 3 different approaches used to reduce the initial bacteria and spore count of each cream source used to make the 2% fat milks did not produce any shelf-life advantage over using cold separated raw cream when starting with excellent quality raw whole milk (i.e., low bacteria count). The combination of MF with HTST pasteurization (73.8°C for 15 s), combined with filling and packaging that was protected from microbial contamination, achieved a refrigerated shelf life of 60 to 90 d at both 1.7 and 5.7°C for 2% fat milks.     

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.     

Isolation and genetic identification of spore-forming bacteria associated with concentrated-milk processing in Nebraska

Spore-forming bacteria are heat-resistant microorganisms capable of surviving and germinating in milk after pasteurization. They have been reported to affect the quality of dairy products by the production of enzymes (lipolytic and proteolytic) under low-temperature conditions in fluid milk, and have become a limiting factor for milk powder in reaching some selective markets. The objective of this research was to isolate and identify the population of spore-forming bacteria (psychrotrophic and thermophilic strains) associated with concentrated milk processing in Nebraska. During 2 seasons, in-process milk samples from a commercial plant (raw, pasteurized, and concentrated) were collected and heat-treated (80°C/12 min) to recover only spore-formers. Samples were spread-plated using standard methods agar and incubated at 32°C to enumerate mesophilic spore counts. Heat-treated samples were also stored at 7°C and 55°C to recover spore-formers that had the ability to grow under those temperature conditions. Isolates obtained from incubation or storage conditions were identified using molecular techniques (16S or rpoB sequencing). Based on the identification of the isolates and their relatedness, strains found in raw, pasteurized, and concentrated milk were determined to be similar. Paenibacillus spp. were associated with both raw and concentrated milk. Due to their known ability to cause spoilage under refrigeration, this shows the potential risk associated with the transferring of these problematic organisms into other dairy products. Other Bacillus species found in concentrated milk included Bacillus clausii, Bacillus subtilis, Lysinibacillus sp., Bacillus safensis, Bacillus licheniformis, Bacillus sonorensis, and Brevibacillus sp., with the last 3 organisms being capable of growing at thermophilic temperatures. These strains can also be translocated to other dairy products, such as milk powder, representing a quality problem. The results of this research highlight the importance of understanding spore-formers associated with the processing of condensed milk, which then may allow for specific interventions to be applied to control these microorganisms in this processing chain. To our knowledge, this is the first study evaluating spore-formers associated with concentrated milk in the United States.     

Symposium review: Effect of post-pasteurization contamination on fluid milk quality

Fluid milk quality in the United States has improved steadily over the last 2 decades, in large part due to the reduction in post-pasteurization contamination (PPC). Despite these improvements, some studies suggest that almost 50% of fluid milk still shows evidence of PPC with organisms that are able to grow at 6°C, even though PPC may be much less frequent in some facilities. Several gram-negative bacteria, when introduced as PPC, can grow rapidly at refrigeration temperatures around 6°C and can lead to bacterial levels above 20,000 cfu/mL (the regulatory limit for bacterial numbers in fluid milk in the United States) and spoilage that can be detected sensorially within 7 to 10 d of processing. Importantly, however, storage temperature can have a considerable effect on microbial growth, and fluid milk stored at 4°C and below may show considerably delayed onset of microbial growth and spoilage compared with samples stored at what may be considered mild abuse (6°C and above). Notable organisms that cause PPC and grow at refrigeration temperatures include psychrotolerant Enterobacteriaceae and coliforms, as well as Pseudomonas. These organisms are known to produce a variety of enzymes that lead to flavor, odor, and body defects that can ultimately affect consumer perception and willingness to buy. Detecting PPC in high temperature, short time, freshly pasteurized fluid milk can be challenging because PPC often occurs sporadically and at low levels. Additionally, indicator organisms typically used in fluid milk (i.e., coliforms) have been shown to represent only a fraction of the total PPC. Recent studies indicate that coliforms account for less than 20% of the total gram-negative organisms introduced into fluid milk after pasteurization. In contrast, Pseudomonas, which is not a coliform and therefore is not detected using coliform media, is the most commonly isolated genus in PPC fluid milk. To reduce PPC, processors must (1) use testing methods that can detect both coliforms and non-coliform gram-negatives (i.e., Pseudomonas) to understand true contamination rates and patterns, and (2) establish cleaning and sanitation protocols and employee and management behaviors that target persistent and transient PPC organisms.     

Bacillus and Paenibacillus species associated with extended shelf life milk during processing and storage

Characterisation of spore formers associated with extended shelf life milk was performed by analysing the bacteriological quality of milk samples collected at various processing stages and during storage. Isolates were identified with MALDI‐TOF‐MS. Milk had spore counts <2 log₁₀ cfu/mL and 4 log₁₀ cfu/mL during processing and storage, respectively. Bacillus pumilus dominated the bacterial population. Bacterial species were inoculated into sterile milk for a shelf life study, and the population change was observed over 42 days at 7 °C. Although the extended shelf life milk process was effective in reducing bacterial counts and species diversity, the presence of Bacillus cereus shows a potential safety problem in extended shelf life milk.     

Assessment of the application of an automated electronic milk analyzer for the enumeration of total bacteria in raw goat milk

Automated electronic milk analyzers for rapid enumeration of total bacteria counts (TBC) are widely used for raw milk testing by many analytical laboratories worldwide. In Ontario, Canada, Bactoscan flow cytometry (BsnFC; Foss Electric, Hillerød, Denmark) is the official anchor method for TBC in raw cow milk. Penalties are levied at the BsnFC equivalent level of 50,000 cfu/mL, the standard plate count (SPC) regulatory limit. This study was conducted to assess the BsnFC for TBC in raw goat milk, to determine the mathematical relationship between the SPC and BsnFC methods, and to identify probable reasons for the difference in the SPC:BsnFC equivalents for goat and cow milks. Test procedures were conducted according to International Dairy Federation Bulletin guidelines. Approximately 115 farm bulk tank milk samples per month were tested for inhibitor residues, SPC, BsnFC, psychrotrophic bacteria count, composition (fat, protein, lactose, lactose and other solids, and freezing point), and somatic cell count from March 2009 to February 2010. Data analysis of the results for the samples tested indicated that the BsnFC method would be a good alternative to the SPC method, providing accurate and more precise results with a faster turnaround time. Although a linear regression model showed good correlation and prediction, tests for linearity indicated that the relationship was linear only beyond log 4.1 SPC. The logistic growth curve best modeled the relationship between the SPC and BsnFC for the entire sample population. The BsnFC equivalent to the SPC 50,000 cfu/mL regulatory limit was estimated to be 321,000 individual bacteria count (ibc)/mL. This estimate differs considerably from the BsnFC equivalent for cow milk (121,000 ibc/mL). Because of the low frequency of bulk tank milk pickups at goat farms, 78.5% of the samples had their oldest milking in the tank to be 6.5 to 9.0 d old when tested, compared with the cow milk samples, which had their oldest milking at 4 d old when tested. This may be one of the major factors contributing to the larger goat milk BsnFC equivalence. Correlations and interactions between various test results were also discussed to further understand differences between the 2 methods for goat and cow milks.     

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.