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.     

Reduction of pasteurization temperature leads to lower bacterial outgrowth in pasteurized fluid milk during refrigerated storage: a case study

Bacterial numbers over refrigerated shelf-life were enumerated in high-temperature, short-time (HTST) commercially pasteurized fluid milk for 15 mo before and 15 mo after reducing pasteurization temperature from 79.4°C (175°F) [corrected] to 76.1°C (169°F). Total bacterial counts were measured in whole fat, 2% fat, and fat-free milk products on the day of processing as well as throughout refrigerated storage (6°C) at 7, 14, and 21 d postprocessing. Mean total bacterial counts were significantly lower immediately after processing as well as at 21 d postprocessing in samples pasteurized at 76.1°C versus samples pasteurized at 79.4°C. In addition to mean total bacterial counts, changes in bacterial numbers over time (i.e., bacterial growth) were analyzed and were lower during refrigerated storage of products pasteurized at the lower temperature. Lowering the pasteurization temperature for unflavored fluid milk processed in a commercial processing facility significantly reduced bacterial growth during refrigerated storage.     

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.     

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.     

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.     

A decade of improvement: New York State fluid milk quality

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

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.     

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.     

Short communication: Nα-Lauroyl-l-arginine ethylester monohydrochloride reduces bacterial growth in pasteurized milk

Effective strategies for extending fluid milk product shelf-life by controlling bacterial growth are of economic interest to the dairy industry. To that end, the effects of addition of l-arginine, Nα-lauroyl ethylester monohydrochloride (LAE) on bacterial numbers in fluid milk products were measured. Specifically, LAE was added (125, 170, or 200 mg/L) to conventionally homogenized and pasteurized 3.25% fat chocolate or unflavored milk products. The treated milks and corresponding untreated controls were held at 6°C and plated on standard plate count agar within 24 h of processing and again at 7, 14, 17, and 21 d of storage. Bacterial counts in all unflavored milk samples treated with LAE remained below the Pasteurized Milk Ordinance limit for grade A pasteurized fluid milk of 4.3 log cfu/mL for the entire 21 d. Bacterial counts in unflavored samples containing 170 and 200 mg/L of LAE were significantly lower than those in the untreated unflavored milk at d 17 and 21 postprocessing. Specifically, bacterial counts in the milk treated with 200 mg/L of LAE were 5.77 log cfu/mL lower than in untreated milk at 21 d postprocessing. Bacterial counts in chocolate milk treated with 200 mg/L of LAE were significantly lower than those in the untreated chocolate milk at d 14, 17, and 21. In chocolate milk treated with 200 mg/L of LAE, bacterial counts were 0.9 log cfu/mL lower than in the untreated milk at 21 d postprocessing. Our results show that addition of LAE to milk can reduce bacterial growth. Addition of LAE is more effective at controlling bacterial growth in unflavored milk than in chocolate milk.     

Thermal inactivation of foot-and-mouth disease virus in milk using high-temperature, short-time pasteurization

Previous studies of laboratory simulation of high temperature, short time pasteurization (HTST) to eliminate foot-and-mouth disease virus (FMDV) in milk have shown that the virus is not completely inactivated at the legal pasteurization minimum (71.7°C/15 s) but is inactivated in a flow apparatus at 148°C with holding times of 2 to 3 s. It was the intent of this study to determine whether HTST pasteurization conducted in a continuous-flow pasteurizer that simulates commercial operation would enhance FMDV inactivation in milk. Cows were inoculated in the mammary gland with the field strain of FMDV (01/UK). Infected raw whole milk and 2% milk were then pasteurized using an Arm-field pilot-scale, continuous-flow HTST pasteurizer equipped with a plate-and-frame heat exchanger and a holding tube. The milk samples, containing FMDV at levels of up to 10⁴ plaque-forming units/mL, were pasteurized at temperatures ranging from 72 to 95°C at holding times of either 18.6 or 36 s. Pasteurization decreased virus infectivity by 4 log₁₀ to undetectable levels in tissue culture. However, residual infectivity was still detectable for selected pasteurized milk samples, as shown by intramuscular and intradermal inoculation of milk into naïve steers. Although HTST pasteurization did not completely inactivate viral infectivity in whole and 2% milk, possibly because a fraction of the virus was protected by the milk fat and the casein proteins, it greatly reduced the risk of natural transmission of FMDV by milk.     

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.     

Short communication: Effect of homogenization on heat inactivation of Mycobacterium avium subspecies paratuberculosis in milk

Mycobacterium avium ssp. paratuberculosis (MAP) can be present in cow milk and low numbers may survive high-temperature, short-time (HTST) pasteurization. Although HTST treatment leads to inactivation of at least 5 log₁₀ cycles, it might become necessary to enhance the efficacy of HTST by additional treatments such as homogenization if the debate about the role of MAP in Crohn’s disease of humans concludes that MAP is a zoonotic agent. This study aimed to determine whether disrupting the clumps of MAP in milk by homogenization during the heat treatment process would enhance the inactivation of MAP. We used HTST pasteurization in a continuous-flow pilot-plant pasteurizer and evaluated the effect of upstream, downstream, and in-hold homogenization on inactivation of MAP. Reduction of MAP at 72°C with a holding time of 28 s was between 3.7 and 6.9 log₁₀ cycles, with an overall mean of 5.5 log₁₀ cycles. None of the 3 homogenization modes applied showed a statistically significant additional effect on the inactivation of MAP during HTST treatment.     

Flow characteristics of a pilot-scale high temperature, short time pasteurizer

In this study, we present a method for determining the fastest moving particle (FMP) and residence time distribution (RTD) in a pilot-scale high temperature, short time (HTST) pasteurizer to ensure that laboratory or pilot-scale HTST apparatus meets the Pasteurized Milk Ordinance standards for pasteurization of milk and can be used for obtaining thermal inactivation data. The overall dimensions of the plate in the pasteurizer were 75 x 115 mm, with a thickness of 0.5 mm and effective diameter of 3.0 mm. The pasteurizer was equipped with nominal 21.5- and 52.2-s hold tubes, and flow capacity was variable from 0 to 20 L/h.Tracer studies were used to determine FMP times and RTD data to establish flow characteristics. Using brine milk as tracer, the FMP time for the short holding section was 18.6 s and for the long holding section was 36 s at 72 degrees C, compared with the nominal times of 21.5 and 52.2 s, respectively. The RTD study indicates that the short hold section was 45% back mixed and 55% plug flow for whole milk at 72 degrees C. The long hold section was 91% plug and 9% back mixed for whole milk at 72 degrees C. This study demonstrates that continuous laboratory and pilot-scale pasteurizers may be used to study inactivation of microorganisms only if the flow conditions in the holding tube are established for comparison with commercial HTST systems.     

Maintenance of breast milk immunoglobulin A after high-pressure processing

Human milk is considered the optimal nutritional source for infants. Banked human milk is processed using low-temperature, long-time pasteurization, which assures microbial safety but involves heat denaturation of some desirable milk components such as IgA. High-pressure processing technology, the subject of the current research, has shown minimal destruction of food macromolecules. The objective of this study was to investigate the influence of pressure treatments on IgA content. Moreover, bacterial load was evaluated after pressure treatments. The effects of high-pressure processing on milk IgA content were compared with those of low-temperature, long-time pasteurization. Mature human milk samples were heat treated at 62.5°C for 30 min or pressure processed at 400, 500, or 600 MPa for 5 min at 12°C. An indirect ELISA was used to measure IgA in human milk whey obtained after centrifugation at 800 × g for 10 min at 4°C. All 3 high-pressure treatments were as effective as low-temperature, long-time pasteurization in reducing the bacterial population of the human milk samples studied. After human milk pressure processing at 400 MPa, 100% of IgA content was preserved in milk whey, whereas only 72% was retained in pasteurized milk whey. The higher pressure conditions of 500 and 600 MPa produced IgA retention of 87.9 and 69.3%, respectively. These results indicate that high-pressure processing at 400 MPa for 5 min at 12°C maintains the immunological protective capacity associated with IgA antibodies. This preliminary study suggests that high-pressure processing may be a promising alternative to pasteurization in human milk banking.     

Thermal resistance of Listeria monocytogenes in inoculated and naturally contaminated raw milk

Raw whole milk inoculated with 10(5) CFU/ml of Listeria monocytogenes was thermally processed at 60-72 degrees C for a minimum holding time of 16.2 s with survival being observed at temperatures up to 67.5 degrees C. In addition, milk naturally contaminated with L. monocytogenes serotype 1 (around 10(4) CFU/ml) was pooled for 2 to 2.5 days and then run through an HTST pasteurizer at temperatures ranging from 60-78 degrees C. Viable L. monocytogenes were detected in the temperature range of 60-66 degrees C. No viable Listeria were detected after treatment at temperatures of 69 degrees C and above in any of five trials. Efficacy of pasteurization and widespread use of processing conditions well above the minimum HTST guidelines ensure the absence of Listeria in pasteurized milk products. However, survival of Listeria at sub-pasteurization temperatures (60-67.5 degrees C) is of concern with regard to heat-treated or raw-milk cheeses.     

Characterization of chocolate milk spoilage patterns

Standard plate counts (SPC) and psychrotrophic plate counts (PPC) from chocolate milk samples were compared with those of unflavored milk samples plated within 24 h of processing and at 7, 10, and 14 days of storage at 6 degrees C using matched samples collected over four time periods from four milk-processing plants. Bacterial numbers within 24 h of processing were not significantly different in unflavored and in chocolate milk samples (P > 0.001), with SPC less than 1,000 CFU/ml and PPC below 10 CFU/ml for both types of products. SPC and PPC were higher in chocolate milk samples than in unflavored milk samples collected from all four plants after 14 days of storage (P < 0.001). To examine the effects of chocolate milk components on bacterial numbers, SPC for days 0, 7, 14, and 21 were monitored in samples of experimentally prepared unflavored milk, milk with chocolate powder and sucrose (chocolate milk), milk with sucrose only, and milk containing chocolate powder only. At days 14 and 21, SPC were higher in both chocolate milk and in milk with chocolate powder only, than in either the unflavored milk or milk with sucrose only (P < 0.001). These findings suggest that the addition of chocolate powder to milk can contribute to a greater relative increase in bacterial numbers in pasteurized chocolate milk than in identically processed unflavored milk at 14 days postprocessing.     

Pasteurization of human milk by a benchtop High-Temperature Short-Time device

A new small-scale continuous-flow High-Temperature Short-Time (HTST) pasteurizer has been designed for treating human milk. The efficacy of the new HTST device was assessed on inoculated Listeria monocytogenes, Staphylococcus aureus and Chronobacter sakazakii, as well as on raw human milk bacteria. The milk biochemical quality after HTST pasteurization was assessed in comparison to a standard Holder pasteurization, by determining the secretory IgAs (sIgAs) content, the protein profile, lysozyme and the Bile Salt Stimulated Lipase (BSSL) activities. No pathogen or bacterial growth was detected after HTST pasteurization with the new instrument. Changes in the protein profile were observed in the milk pasteurized according to both processes. The sIgAs content and BSSL activity were significantly higher in the milk pasteurized with the new device than in the same milk treated by the standard Holder pasteurization. In conclusion, the new HTST apparatus: (i) can effectively pasteurize human milk with a better retention of sIgAs content and BSSL activity; (ii) comply to human milk banking safety requirements.Industrial relevanceCurrently, 210 active human milk banks are located in Europe (and 17 more are planned). The majority of the European banks still use Holder-based pasteurizers, which, despite efficacy in ensuring microbiological safety, are known to reduce/disrupt important nutritional and non-nutritional biological factors. Although already widely established in food industry, the advantages of HTST technology were tested only at small laboratory scale for human milk. The device tested in the present research was specifically designed to provide human milk banks with the technology they need to ensure a safe and lower-impact pasteurization process, that is suitable for processing different volumes of donations. The device can pasteurize up to 10 L of milk per hour, with a minimum volume of 100 mL. The system is designed to be cleaned-in-place (CIP) after each pasteurization run and sanitized immediately prior to the next use, being thus more suitable for treating pools of milk from different donors than milk from single donations. Italian and EU patents have been filed for the device, within a partnership between public research institutions, stakeholders (Italian association of donor milk banks), and a private company in the sector of dairy processing equipment. The device has achieved a Technology Readiness Level (TRL) 6 (Prototype demonstration in a relevant environment). The cost of the new device will be comparable to that of a typical human milk Holder pasteurizer.     

Validation of radio-frequency dielectric heating system for destruction of Cronobacter sakazakii and Salmonella species in nonfat dry milk

Cronobacter sakazakii and Salmonella species have been associated with human illnesses from consumption of contaminated nonfat dry milk (NDM), a key ingredient in powdered infant formula and many other foods. Cronobacter sakazakii and Salmonella spp. can survive the spray-drying process if milk is contaminated after pasteurization, and the dried product can be contaminated from environmental sources. Compared with conventional heating, radio-frequency dielectric heating (RFDH) is a faster and more uniform process for heating low-moisture foods. The objective of this study was to design an RFDH process to achieve target destruction (log reductions) of C. sakazakii and Salmonella spp. The thermal destruction (decimal reduction time; D-value) of C. sakazakii and Salmonella spp. in NDM (high-heat, HH; and low-heat, LH) was determined at 75, 80, 85, or 90°C using a thermal-death-time (TDT) disk method, and the z-values (the temperature increase required to obtain a decimal reduction of the D-value) were calculated. Time and temperature requirements to achieve specific destruction of the pathogens were calculated from the thermal destruction parameters, and the efficacy of the RFDH process was validated by heating NDM using RFDH to achieve the target temperatures and holding the product in a convection oven for the required period. Linear regression was used to determine the D-values and z-values. The D-values of C. sakazakii in HH- and LH-NDM were 24.86 and 23.0 min at 75°C, 13.75 and 7.52 min at 80°C, 8.0 and 6.03 min at 85°C, and 5.57 and 5.37 min at 90°C, respectively. The D-values of Salmonella spp. in HH- and LH-NDM were 23.02 and 24.94 min at 75°C, 10.45 and 12.54 min at 80°C, 8.63 and 8.68 min at 85°C, and 5.82 and 4.55 min at 90°C, respectively. The predicted and observed destruction of C. sakazakii and Salmonella spp. were in agreement, indicating that the behavior of the organisms was similar regardless of the heating system (conventional vs. RFDH). Radio-frequency dielectric heating can be used as a faster and more uniform heating method for NDM to achieve target temperatures for a postprocess lethality treatment of NDM before packaging.     

Nonthermal inactivation and sublethal injury of Lactobacillus plantarum in apple cider by a pilot plant scale continuous supercritical carbon dioxide system

The objective of this study was to evaluate the efficacy of supercritical carbon dioxide (SCCO₂) for inactivating Lactobacillus plantarum in apple cider using a continuous system with a gas-liquid metal contactor. Pasteurized apple cider without preservatives was inoculated with L. plantarum and processed using a SCCO₂ system at a CO₂ concentration range of 0-12% (g CO₂/100 g product), outlet temperatures of 34, 38, and 42 °C, a system pressure of 7.6 MPa, and a flow rate of 1 L/min. Processing with SCCO₂ significantly (P < 0.05) enhanced inactivation of L. plantarum in apple cider, resulting in a 5 log reduction with 8% CO₂ at 42 °C. The response surface model indicated that both CO₂ concentration and temperature contributed to the microbial inactivation. The extent of sublethal injury in surviving cells in processed apple cider increased as CO₂ concentration and processing temperature increased, however the percent injury dramatically decreased during SCCO₂ processing at 42 °C. Structural damage in cell membranes after SCCO₂ processing was observed by SEM. Refrigeration (4 °C) after SCCO₂ processing effectively inhibited the re-growth of surviving L. plantarum during storage for 28 days. Thus this study suggests that SCCO₂ processing is effective in eliminating L. plantarum and could be applicable for nonthermal pasteurization of apple cider.     

Pilot-scale crossflow-microfiltration and pasteurization to remove spores of Bacillus anthracis (Sterne) from milk

High-temperature, short-time pasteurization of milk is ineffective against spore-forming bacteria such as Bacillus anthracis (BA), but is lethal to its vegetative cells. Crossflow microfiltration (MF) using ceramic membranes with a pore size of 1.4 μm has been shown to reject most microorganisms from skim milk; and, in combination with pasteurization, has been shown to extend its shelf life. The objectives of this study were to evaluate MF for its efficiency in removing spores of the attenuated Sterne strain of BA from milk; to evaluate the combined efficiency of MF using a 0.8-μm ceramic membrane, followed by pasteurization (72°C, 18.6 s); and to monitor any residual BA in the permeates when stored at temperatures of 4, 10, and 25°C for up to 28 d. In each trial, 95 L of raw skim milk was inoculated with about 6.5 log₁₀ BA spores/mL of milk. It was then microfiltered in total recycle mode at 50°C using ceramic membranes with pore sizes of either 0.8 μm or 1.4 μm, at crossflow velocity of 6.2 m/s and transmembrane pressure of 127.6 kPa, conditions selected to exploit the selectivity of the membrane. Microfiltration using the 0.8-μm membrane removed 5.91 ± 0.05 log₁₀ BA spores/mL of milk and the 1.4-μm membrane removed 4.50 ± 0.35 log₁₀ BA spores/mL of milk. The 0.8-μm membrane showed efficient removal of the native microflora and both membranes showed near complete transmission of the casein proteins. Spore germination was evident in the permeates obtained at 10, 30, and 120 min of MF time (0.8-μm membrane) but when stored at 4 or 10°C, spore levels were decreased to below detection levels (≤0.3 log₁₀ spores/mL) by d 7 or 3 of storage, respectively. Permeates stored at 25°C showed coagulation and were not evaluated further. Pasteurization of the permeate samples immediately after MF resulted in additional spore germination that was related to the length of MF time. Pasteurized permeates obtained at 10 min of MF and stored at 4 or 10°C showed no growth of BA by d 7 and 3, respectively. Pasteurization of permeates obtained at 30 and 120 min of MF resulted in spore germination of up to 2.42 log₁₀ BA spores/mL. Spore levels decreased over the length of the storage period at 4 or 10°C for the samples obtained at 30 min of MF but not for the samples obtained at 120 min of MF. This study confirms that MF using a 0.8-μm membrane before high-temperature, short-time pasteurization may improve the safety and quality of the fluid milk supply; however, the duration of MF should be limited to prevent spore germination following pasteurization.