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.     

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.     

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.     

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.     

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.     

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.     

Molecular subtyping and characterization of psychrotolerant endospore-forming bacteria in two New York State fluid milk processing systems

Psychrotolerant endospore-forming bacteria Bacillus and Paenibacillus spp. are important spoilage organisms in fluid milk. A recently developed rpoB subtyping method was applied to characterize the diversity and phylogenetic relationships among Bacillus and related sporeformers associated with milk processing systems. Milk samples representing the processing continuum from raw milk to pasteurized products were collected from two fluid milk processing plants, held at 6 degrees C up to the code date that had been established by each processing plant (i.e., either 18 or 21 days), and plated for bacterial enumeration throughout storage. Bacterial colonies selected to represent the visible diversity in colony morphology on enumeration plates were examined further. Among 385 bacterial isolates characterized, 35% were Bacillus spp., and 65% were Paenibacillus spp. A total of 92 rpoB allelic types were identified among these isolates, indicating considerable diversity among endospore-forming spoilage organisms present in fluid milk systems. Of the 92 allelic types identified, 19 were isolated from samples collected from both processing plants. The same rpoB allelic types were frequently identified in paired raw milk and packaged product samples, indicating that Bacillus and Paenibacillus spp. can enter dairy processing systems through raw milk. Certain subtypes were found exclusively in pasteurized samples, including those that were temporally independent, suggesting the possibility of in-plant sources for these spoilage organisms, including through the persistence of selected subtypes in processing plants. Development of effective control strategies for the diverse array of psychrotolerant endospore-forming organisms that currently limit the shelf lives of high-temperature short-time fluid milk products will require comprehensive, integrated efforts along the entire milk processing continuum.     

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.     

Modelling the relation between bacterial growth and storage temperature in pasteurized milks of varying hygienic quality

The shelf-life of pasteurized milk was mainly determined by the level of contamination with Gram-negative psychrotrophic bacteria. The length of lag phase of the bacteria was also important, although the generation times of the naturally contaminating flora seemed to be of little relevance except for milks where the shelf-life exceeded 10 days at 6°C. The effect of temperature on growth of the contaminants could be accurately determined by the 'square root' plot but the conceptual minimum temperature for growth (T_o) varied. The variation was related to the quality of the pasteurized milk. Effects of temperature on generation time, length of lag phase and shelf-life were most marked at temperatures below 15°C.
The microflora of pasteurized milk varied significantly with storage temperature. At refrigeration temperatures, spoilage was mainly due to the growth of Pseudomonas spp. Enterobacteriaceae and Gram-positive bacteria assumed greater importance in the spoilage of milks stored at temperatures above 10°C. Milks of good quality also contained Bacillus spp and this group of bacteria were not detectable in milks with short shelf-lives.     

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.     

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.     

EFFECT OF POTASSIUM SORBATE ON THE MICROBIOLOGY OF VACUUM-PACKED POULTRY

Treatment of vacuum-packed poultry pieces and muscle fillets with potassium sorbate decreased the rate of microbial development and increased shelf life when the substrates were stored at chill temperatures. Microbial growth on sorbate treated breast fillets was totally inhibited for up to 35 days at 2°C. The effect of sorbate was temperature dependent and completely negated by severe temperature abuse (storage at 12°C). Increased storage temperature also resulted in a greater proportion of gram-negative, facultatively anaerobic bacteria in the spoilage association. At each storage temperature where significant growth occurred, the effect of sorbate was to reduce the proportion of enteric organisms and increase the proportion of lactic acid bacteria in the spoilage association.     

Fluid dairy product quality and safety: looking to the future

The fiercely competitive nature of the US beverage industry will drive the fluid milk sector of the dairy industry to improve product quality and shelf life to enable dairy beverages to compete with innovative new introductions as well as with currently popular shelf-stable products. The recent substantial growth in the volume of flavored milk sales specifically suggests that attention is needed to improve these products. Further, increasing public awareness and regulatory attention directed toward food safety issues highlight the need for the dairy industry to proactively address and eliminate emerging food safety issues that may negatively impact the image of dairy products. Shelf life and sensory profiles of high temperature short time pasteurized fluid milk products are presented, illustrating the need for greater attention to controlling contaminating microorganisms in processed fluid milk products. Bacterial spoilage patterns of flavored versus unflavored milks are compared, and suggestions are presented for extending flavored product shelf lives. Strategies currently applied to extend shelf life are reviewed. Food safety issues facing the dairy industry are presented within the context of an overview of foodborne illnesses in the United States. The pressing need to determine thermal resistance characteristics of Mycobacterium paratuberculosis is described.     

SUBJECT: BITTY CREAM AND RELATED PROBLEMS:PROBLEMS ASSOCIATED WITH BACTERIAL SPORES IN HEAT-TREATED MILK AND DAIRY PRODUCTS

Spore spoilage in pasteurized milk and UHT and sterilized milk is reviewed with particular reference to cream. The problem of the incidence of bitty cream and the effectiveness of refrigeration in checking this is discussed. Results of studies on the bacterial quality of bulk raw milk and tanker reload milk using the thermoduric count are given in detail. Current methods for pasteurizing cream are described and studies on the effect of storage temperature on the shelf life of pasteurized cream are referred to in detail with particular reference to the lag in bacterial multiplication and the use of the Methylene Blue Test.     

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.     

Effect of heat treatment of milk on activation of Bacillus spores

The quality and shelf life of fluid milk products are dependent on the amount and type of microorganisms present following pasteurization. This study evaluated the effects of different pasteurization processes on the microbial populations in fluid milk. The objective was to determine whether certain pasteurization processes lead to an increase in the amount of bacteria present in pasteurized milk by activating Bacillus spores. Samples of raw milk were collected on the day of arrival at the dairy plant. The samples were pasteurized at 63 degrees C for 30 min (low temperature, long time), 72 degrees C for 15 s (high temperature, short time), 76 degrees C for 15 s, and 82 degrees C for 30 min. The pasteurized samples were then stored at 6 and 10 degrees C for 14 days. The samples were analyzed for standard plate count and Bacillus count immediately after pasteurization and after 14 days of storage. Pasteurization of milk at 72 and 76 degrees C significantly (P < 0.05) increased the amount of Bacillus spore activation over that of 63 degrees C. There was no detection of Bacillus in initial samples pasteurized at 82 degrees C for 30 min, but Bacillus was present in samples after storage for 14 days, indicating that injury and recovery time preceded growth. The majority of isolates were characterized as Bacillus mycoides and not Bacillus cereus, suggesting that this organism might be more a cause of sweet curdling of fluid milk than previously reported.     

Short communication: Bacterial ecology of high-temperature, short-time pasteurized milk processed in the United States

To determine the microbial ecology of pasteurized milk within the United States, 2% fat pasteurized fluid milk samples were obtained from 18 dairy plants from 5 geographical areas representing the Northeast, Southeast, South, Midwest, and West. Of the 589 bacterial isolates identified using DNA sequence-based subtyping methods, 346 belonged to genera characterized as gram-positive endospore-forming bacteria (i.e., Bacillus and Paenibacillus). Of the 346 gram-positive endospore-forming bacteria isolated in the present study, 240 were classified into 45 allelic types identical to those previously identified from samples obtained in New York State, indicating the widespread presence of these microbes in fluid milk production and processing systems in the United States. More than 84% of the gram-positive spore-forming isolates characterized at d 1, 7, and 10 were of the genus Bacillus, whereas more than 92% of isolates characterized at d 17 of shelf life were of the genus Paenibacillus, indicating that the predominant gram-positive spoilage genera shifts from Bacillus spp. to Paenibacillus spp. during refrigerated storage.     

不同储藏温度对超巴氏奶货架期理化指标及风味的影响

本研究将生鲜乳均质后进行120 ℃、15 s的热处理,随后进行无菌灌装。分别在4、25、37 ℃三个储藏温度下进行45 d的保藏实验,分析产品在储藏期间理化及风味变化规律。结果表明,4 ℃保藏条件下超巴氏奶细菌蛋白酶酶活以及脂肪酶酶活变化不明显,蛋白酶活在37 ℃保藏条件下第25 d后急剧上升,第45 d达到最大值3.2 U/mL,25和37 ℃保藏温度下的脂肪酶活性均在储藏第10 d后急剧上升,并分别维持在6.25和6.75 U/mL左右。此外,在储藏期间,pH降低,粒径增大,Zeta电位先降低后增高,并随储藏温度增加,变化速度加快,不同储藏温度样品间差异明显。对于色泽,4 ℃保藏下超巴氏奶只在L*值上有明显变化,另外两个温度保藏下L*、a*以及b*值均有明显变化。电子鼻以及电子舌结果显示,4 ℃保藏条件下,保藏期间超巴氏奶气味和滋味无较大差异,而25和37 ℃气味及滋味均差异明显。研究结果表明,超巴氏奶在4 ℃保藏温度下货架期可达45 d,而储藏在25和37 ℃温度下的超巴氏奶货架期无法达到45 d。     

Use of time-temperature integrators and predictive modelling for shelf life control of chilled fish under dynamic storage conditions

A systematic approach for fish shelf life modelling and Time Temperature Integrator (TTI) selection in order to plan and apply an effective quality monitoring scheme for the fish chill chain was developed. The temperature behaviour of the natural microflora of the Mediterranean fish boque (Boops boops) was studied and growth of the specific spoilage bacteria Pseudomonas spp. and Shewanella putrefaciens was modelled and correlated to organoleptic shelf life. Arrhenius and square root functions were used to model temperature dependence of maximum growth rates. Bacterial growth and shelf life models were validated under dynamic storage conditions with independent variable temperature experiments. The response of several TTIs from similar storage experiments was also modelled. The reliability of the TTI monitoring was cumulatively expressed by the error in the TTI derived effective temperature (Teff) for different variable temperature distributions. Teff was directly translated to shelf life of the fish.     

Tracking heat-resistant, cold-thriving fluid milk spoilage bacteria from farm to packaged product

Control of psychrotolerant endospore-forming spoilage bacteria, particularly Bacillus and Paenibacillus spp., is economically important to the dairy industry. These microbes form endospores that can survive high-temperature, short-time pasteurization; hence, their presence in raw milk represents a major potential cause of milk spoilage. A previously developed culture-dependent selection strategy and an rpoB sequence-based subtyping method were applied to bacterial isolates obtained from environmental samples collected on a New York State dairy farm. A total of 54 different rpoB allelic types putatively identified as Bacillus (75% of isolates), Paenibacillus (24%), and Sporosarcina spp. (1%) were identified among 93 isolates. Assembly of a broader data set, including 93 dairy farm isolates, 57 raw milk tank truck isolates, 138 dairy plant storage silo isolates, and 336 pasteurized milk isolates, identified a total of 154 rpoB allelic types, representing an extensive diversity of Bacillus and Paenibacillus spp. Our molecular subtype data clearly showed that certain endospore-forming bacterial subtypes are present in the dairy farm environment as well as in the processing plant. The potential for entry of these ubiquitous heat-resistant spoilage organisms into milk production and processing systems, from the dairy farm to the processing plant, represents a considerable challenge that will require a comprehensive farm-to-table approach to fluid milk quality.