A review of oleogels applications in dairy foods.

The characteristics of dairy products, such as texture, color, flavor, and nutritional profile, are significantly influenced by the presence of milk fat. However, saturated fatty acids account for 65% of total milk fat. With increased health awareness and regulatory recommendations, consumer preferences have evolved toward low/no saturated fat food products. Reducing the saturated fat content of dairy products to meet market demands is an urgent yet challenging task, as it may compromise product quality and increase production costs. In this regard, oleogels have emerged as a viable milk fat replacement in dairy foods. This review focuses on recent advances in oleogel systems and explores their potential for incorporation into dairy products as a milk fat substitute. Overall, it can be concluded that oleogel can be a potential alternative to replace milk fat fully or partially in the product matrix to improve nutritional profile by mimicking similar rheological and textural product characteristics as milk fat. Furthermore, the impact of consuming oleogel-based dairy foods on digestibility and gut health is also discussed. A thorough comprehension of the application of oleogels in dairy products will provide an opportunity for the dairy sector to develop applications that will appeal to the changing consumer needs.

Environmental persistence of Listeria monocytogenes and its implications in dairy processing plants.

Listeriosis, an invasive illness with a fatality rate between 20% and 30%, is caused by the ubiquitous bacterium Listeria monocytogenes. Human listeriosis has long been associated with foods. This is because the ubiquitous nature of the bacteria renders it a common food contaminant, posing a significant risk to the food processing sector. Although several sophisticated stress coping mechanisms have been identified as significant contributing factors toward the pathogen's persistence, a complete understanding of the mechanisms underlying persistence across various strains remains limited. Moreover, aside from genetic aspects that promote the ability to cope with stress, various environmental factors that exist in food manufacturing plants could also contribute to the persistence of the pathogen. The objective of this review is to provide insight into the challenges faced by the dairy industry because of the pathogens' environmental persistence. Additionally, it also aims to emphasize the diverse adaptation and response mechanisms utilized by L. monocytogenes in food manufacturing plants to evade environmental stressors. The persistence of L. monocytogenes in the food processing environment poses a serious threat to food safety and public health. The emergence of areas with high levels of L. monocytogenes contamination could facilitate Listeria transmission through aerosols, potentially leading to the recontamination of food, particularly from floors and drains, when sanitation is implemented alongside product manufacturing. Hence, to produce safe dairy products and reduce the frequency of outbreaks of listeriosis, it is crucial to understand the factors that contribute to the persistence of this pathogen and to implement efficient control strategies.

Overall Adverse Event Profile of Vadadustat versus Darbepoetin Alfa for the Treatment of Anemia Associated with Chronic Kidney Disease in Phase 3 Trials.

INTRODUCTION: Anemia frequently occurs in chronic kidney disease (CKD), is associated with poor quality of life and cardiovascular outcomes, and its treatment represents a considerable economic burden to the healthcare system. Although effective, the current standard of care for the treatment of anemia in chronic kidney disease patients with erythropoiesis-stimulating agents requires chronic/ongoing injections, making the treatment less accessible or desirable to patients not treated by in-center maintenance hemodialysis. Furthermore, safety concerns, including an increased risk of cardiovascular events and mortality, have emerged from their use in studies targeting hemoglobin concentrations in the normal or near-normal range. The orally active hypoxia-inducible factor prolyl hydroxylase inhibitor vadadustat may offer advantages over erythropoiesis-stimulating agents by correcting anemia via pathways activating endogenous erythropoietin production. METHODS: To comprehensively analyze the safety profile of vadadustat in patients with dialysis-dependent and non-dialysis-dependent CKD-related anemia, we pooled the safety populations from each of the four trials in the phase 3 clinical program (n = 7,373) and compared the risk of treatment-emergent adverse events (TEAEs) for each treatment arm. RESULTS: In patients randomized to vadadustat versus darbepoetin alfa, rates of TEAEs (88.9% vs. 89.3%), treatment-emergent serious adverse events (58.0% vs. 59.3%), and TEAEs leading to death (16.1% vs. 16.2%) were similar, as were rates of adverse events of special interest, including cardiovascular-, hepatic-, and neoplasm-related adverse events. DISCUSSION/CONCLUSION: Among patients with CKD-related anemia treated with vadadustat, we observed similar rates of adverse events relative to those treated with darbepoetin alfa.

Efficacy of a typical clean-in-place protocol against in vitro membrane biofilms.

This study evaluates the effectiveness of a typical clean-in-place (CIP) protocol against in vitro biofilms on whey reverse osmosis (RO) membranes developed under static condition. Bacterial isolates obtained from RO membrane biofilms were used to develop single and multispecies biofilms under laboratory conditions. A typical commercial CIP protocol was tested against the 24-h-old biofilms, and included 6 sequential treatment steps based on alkali, surfactant, acid, enzyme, a second surfactant, and a sanitizer treatment step. Experiments were conducted in 4 replicates and the data were statistically analyzed. The results revealed a variation in the resistance of mixed-species biofilms against the individual steps in the sequential CIP protocol. The overall 6 steps protocol, although resulted in a greater reduction, also resulted in the detection of survivors even after the final sanitizer step, reflect the ineffectiveness of the CIP protocol for complete removal of biofilms. Posttreatment counts of 0.71 log after the sequential CIP of mixed-species biofilm revealed the resistance of biofilm constitutive microbiota. Mixed-species biofilms, constituting different genera including Bacillus, Staphylococcus, and Streptococcus, were observed to be more resistant than most of the single-species biofilms. However, among the single-species biofilms, significantly different resistance pattern was observed for Bacillus isolates compared with the other bacterial isolates. All 5 isolates of Bacillus were found resistant with survivor counts of more than 1.0 log against the sequential CIP protocol tested. Thus, it can be concluded that the tested CIP protocol had a limited effectiveness to clean membrane biofilms formed on the whey RO membranes.

Development of a spray-dried conjugated whey protein hydrolysate powder with entrapped probiotics.

Bifidobacterium animalis ssp. lactis ATCC27536 and Lactobacillus acidophilus ATCC4356 were encapsulated in a conjugated whey protein hydrolysate (WPH10) through spray drying. Probiotic cultures were added at the ratio of 1:1 into the conjugated WPH10 solution at a spiking level of about 10 log10 cfu/mL. The mixture was spray dried in a Niro drier with inlet and outlet temperatures of 200°C and 90°C, respectively. The final dried product was determined for cell viability and further stored for 16 wk at 25°, 4°, and -18°C to monitor viability and functionality. Micro images showed the presence of link bridges in non-conjugated WPH10, whereas, in the case of conjugated WPH10, round particles with pores were observed. The mean probiotic counts before and after spray drying were 10.59 log10 cfu/mL and 8.98 log10 cfu/g, respectively, indicating good retention of viability after spray drying. The solubility and wetting time of the WPH10-maltodextrin (MD) encapsulated probiotic powder were 91.03% and 47 min, whereas for WPH10, the solubility and wetting time were 82.03% and 53 min, respectively. At the end of storage period, the counts were 7.18 log10 cfu/g at 4°C and 7.87 log10 cfu/g at -18°C, whereas at 25°C the counts were significantly reduced, to 3.97 log10 cfu/g. The solubility of WPH-MD powder was 82.36%, 83.1%, and 81.19% at -18°C, 4°C, and 25°C, respectively, and wetting times were 61 min, 60 min, and 63 min at -18°C, 4°C, and 25°C, respectively. By contrast, for WPH10 powder, the solubility significantly reduced to 69.41%, 69.97%, and 68.99% at -18°C, 4°C, and 25°C, and wetting times increased to 71 min, 70 min, and 72 min at -18°C, 4°C, and 25°C, respectively. The conjugated WPH10 is thus demonstrated as a promising carrier for probiotics and can be further used as an ingredient for developing functional foods, to harness their enhanced functionality and health benefits derived from both WPH and probiotics.

Effect of midday pasteurizer washing on thermoduric organisms and their progression through Cheddar cheese manufacturing and ripening.

Thermoduric bacteria are known to affect the quality of Cheddar cheese, with manifested defects including slits, weak body, and blowing. Thermoduric bacteria are likely to increase in numbers during cheese-making, as in-process conditions are conducive to proliferation. The present study was conducted to track thermoduric bacterial progression during an 18- to 20-h Cheddar cheese production run and during ripening when the pasteurizer was washed at midway through the production day. This study also correlated a broad range of chemical changes to the growth of thermoduric bacteria during ripening. Three independent cheese trials were performed at 3.5- ± 0.5-mo intervals. Samples were drawn in duplicates at 4 different times of the day: at the start of the run (vat 1), prior to a midday wash of the pasteurizer (vat 20), after the midday wash of the pasteurizer (vat 21), and at the end of the run (vat 42) for raw milk, pasteurized milk, and cheese. Cheeses were also tested during ripening for 6 mo. Results showed that raw milk total bacterial counts comprised 0.24% thermoduric mesophiles (TM) and 0.12% thermoduric thermophiles (TT). The thermoduric thermophilic bacterial counts increased by log10 1.23 during the pasteurizer run of 9 to 10 h, indicating a buildup of thermoduric thermophilic bacteria during the pasteurization process itself. Midday washing reduced thermophilic counts by log10 1.36, as evident by pre- and post-midday wash counts. However, a thermophilic buildup during post-midday wash was again noticed near the end of the 20-h run. We found that TT bacteria decreased in the first 60 d of ripening, whereas TM bacteria increased during the same period. However, TT bacteria increased later during 60 to 180 d of ripening. Bacillus licheniformis was the most frequently isolated bacteria in this study and was recovered at all production stages sampled during the cheese-making and ripening. We observed a significant increase in the level of orotic and uric acids in the vat made at the end of the day. No significant difference in the overall chemical composition, proteolysis, sugar, or other organic acids was observed in cheese made at the start versus the end of the production run.

Short communication: Evaluating the recovery potential of injured cells of Listeria innocua under product temperature-abuse conditions and passage through simulated gastrointestinal fluids.

Ice cream handling and serving conditions on the consumer side may result in temperature abuse before consumption. Under some extreme conditions, even the sporadic presence of injured bacterial cells might pose a health risk due to the possibility of recovery of those cells. We conducted this investigation to evaluate the potential of injured cells of Listeria innocua to recover under ice cream temperature abuse conditions and on exposure to simulated gastrointestinal (GI) fluids. Ice cream mix samples (42% total solids), spiked with 4 log10 cfu/g of Listeria innocua, were thermally treated at 69°C for 30 min. Potential heat-injured cells were recovered in buffered Listeria broth (BLEB), followed by isolation on Listeria-specific modified Oxford agar (MOX). The ice cream mix samples, containing potentially injured cells of Listeria innocua, were followed through overnight aging (7°C), freezing (-3.3°C), and overnight hardening (-40°C) steps to obtain the final ice cream samples. To simulate temperature abuse conditions, the samples were held for 12 h at 4.4°C, followed by 30 min at room temperature (22°C); this treatment was considered the first cycle of temperature abuse. To generate a worst-case scenario, the samples were exposed to 3 such consecutive temperature abuse cycles. At the end of each cycle, direct plating was done on MOX to recover viable cells, and BLEB enrichment verified the presence of potential injured cells. In addition, the ice cream samples, containing potential injured cells, were passed through simulated GI fluids. As a first step, samples were mixed (1:1) with simulated gastric fluids (pH 1.0 and 2.0 before mixing) and held at 37°C in a shaker incubator. Samples drawn at 15, 30, and 60 min were analyzed for viable and potential injured cells. To study the effect of sequential transit through simulated intestinal fluid, a mixture of ice cream and gastric fluid (1:1) from the gastric fluid experiment above was added to simulated intestinal fluid (pH 6.8) and held at 37°C. Samples were analyzed at 30 and 360 min for viable and potential injured cells. Three trials were conducted and the samples collected in duplicates. The temperature abuse or GI fluid exposure studies did not result in the recovery of potential injured cells of Listeria innocua in the ice cream samples under the conditions tested. Exposure to gastric fluids, however, did not eliminate the potential injured cells. Further studies are necessary to understand the exact risk implications of these findings.

Short communication: A competitive exclusion study reveals the emergence of Bacillus subtilis as a predominant constitutive microorganism of a whey reverse osmosis membrane biofilm matrix.

Microbial attachment and colonization on separation membranes lead to biofilm formation. Some isolates within the biofilm microflora acquire greater resistance to the chemical cleaning protocols on prolonged use of membranes. It is thus likely that the constitutive microflora might compete with each other and result in certain species emerging as predominant, especially within older biofilms. To understand the microbial interactions within biofilms, the emergence of predominance was studied in the current investigation. An 18-mo-old reverse osmosis membrane was procured from a whey processing plant. The membrane pieces (2.54 × 2.54 cm2) were neutralized by dipping in Letheen broth. The resuscitation step was done in tryptic soy broth (TSB) at 37°C, followed by plating on tryptic soy agar (TSA) to recover the constitutive microflora. Distinct colonies of isolates were further identified using MALDI-TOF as Bacillus licheniformis, Exiguobacterium aurantiacum, Acinetobacter radioresistens, Bacillus subtilis (rpoB sequencing), and 1 unidentified species each of Exiguobacterium and Bacillus. Further, the competitive exclusion study helped establish the emergence of predominance using a co-culturing technique. Fifteen combinations (of 2 isolates each) were prepared from the isolates. Pure cultures of the respective isolates were spiked in a ratio of 1:1 in TSB and incubated at 37°C for 24 h, followed by plating on TSA. The enumerated colonies were distinguished based on colony morphology, Gram staining, and MALDI-TOF to identify the type of the isolate. Plate counts of B. subtilis emerged as predominant with mean log counts of 7.22 ± 0.22 cfu/mL. The predominance of B. subtilis was also validated using the process of natural selection in a multispecies growth environment. In this instance, the TSB culture with overnight-incubated membrane piece (with mixed-species biofilm) at 37°C for 12 h was inoculated in fresh TSB and incubated for the second cycle. Overall, 5 such sequential broth-culture incubation cycles were carried out, followed by pour plating on TSA plates, at the end of each cycle. The isolates obtained were identified using a similar methodology as mentioned above. The fifth subsequent transfer depicted the presence of only 1 B. subtilis isolate on plating, thereby validating its predominance under the conditions of the experiment.

Antimicrobial activity as a potential factor influencing the predominance of Bacillus subtilis within the constitutive microflora of a whey reverse osmosis membrane biofilm.

Current cleaning and sanitation protocols may not be adequately effective in cleaning separation membranes and can result in the formation of resilient multispecies biofilms. The matured biofilms may result in a bacterial predominance with resilient strains on membranes with a prolonged use. In our previous study, we isolated organisms such as Bacillus subtilis, Bacillus licheniformis, Exiguobacterium aurantiacum, and Acinetobacter radioresistens from an 18-mo-old reverse osmosis membrane. The competitive exclusion studies revealed the predominance of B. subtilis within the membrane biofilm microflora. This study investigated the antimicrobial activity of the B. subtilis isolate as a potential cause of its predominance. The culture isolate was propagated in tryptic soy broth at 37°C, and microfiltered to prepare cell-free extracts (CFE) at 8-, 10-, 12-, 14-, 16-, and 18-h intervals. The CFE were freeze-dried and suspended in minimum quantities of HPLC-grade water to prepare concentrated solutions. The antimicrobial activities of CFE were tested using the agar-well assay against the biofilm constitutive microflora. The experiments were conducted in triplicates and means were compared for significant differences using a general linear mixed model procedure. The results indicated the highest antimicrobial activity of 12-h CFE of B. subtilis against other constitutive microflora such as Exiguobacterium sp., E. auranticum, and A. radioresistens, with average inhibition zone sizes of 16.5 ± 0.00, 16.25 ± 0.66, and 20.6 ± 0.00 mm, respectively. Upon treatment with proteinase K, the CFE completely lost its antimicrobial activity, establishing it to be a proteinaceous compound. The AA profiling revealed the total crude protein in CFE to be 51% (wt/wt), with its major constituent as glutamic acid (11.30% wt/wt). The freeze-dried CFE was thermally stable on exposure to the common temperature used for sanitizer applications (23.8°C for 5 and 10 min) and over a pH range of 3.0 to 6.3. The study helped us understand the role of the antimicrobial compound produced by B. subtilis as a potential cause of its predominance within the biofilm constitutive microflora.

Short communication: Entrapment of Listeria cells within air pockets of ice cream mix matrix may lead to potentially heat-injured cells.

In our previous study, we observed the sporadic presence of potentially heat-injured cells of Listeria innocua in ice cream mix following a selective enrichment protocol. Although injured cells have not yet been reported to cause any disease outbreaks, it is important to understand their presence in heat-treated food matrices. In this study, we propose a possible protective role of air pockets that may help explain the sporadic presence of potentially heat-injured cells following heat treatment. Challenge studies were conducted by inoculating ice cream mix samples (42% total solids, 16.3% fat, 22.2% total sugar, and 3.4% protein) with Listeria innocua (an established surrogate) at a mean spiking level of 4.0 log cfu/g. The inoculated samples were heat-treated at 69°C for 30 min and potentially heat-injured cells were detected using buffered Listeria enrichment broth, followed by plating on modified Oxford and Rapid'LMono agars. Scanning electron microscopy and atomic force microscopy were conducted on the air-dried, spiked ice cream mix samples, before and after the thermal treatment stages. Although direct plating did not reveal any intact cells in the heat-treated ice cream mix, a more sensitive enrichment protocol was able to identify cells that were potentially heat-injured. The scanning electron micrographs showed air pockets of different sizes in the ice cream mix samples. The spiked mix samples before heat treatment showed some Listeria cells unevenly distributed in the mix matrix and some entrapped within the larger air pockets. After heat treatment, scanning electron and atomic force micrographs showed cells entrapped only within the larger air pockets. The mix matrix, however, did not show any Listeria cells. Confirmation of Listeria at all stages of analysis was done using MALDI-TOF. These observations suggest that the Listeria cells could be entrapped within the larger air pockets and thus may undergo inadequate thermal effect. This could have resulted in their detection as potentially heat-injured cells, as evident under the conditions of the experiment. These results are preliminary observations and further studies are necessary to draw conclusions and understand the true implications of these findings.

Sporulating behavior of Bacillus licheniformis strains influences their population dynamics during raw milk holding.

To understand the role of strain variability, population dynamics of 2 strains of Bacillus licheniformis, ATCC 6634 and ATCC 14580, were modeled as a function of temperature (4.0-12.0°C) and duration (0-72 h) using regression analysis. Based on the initial spiking of vegetative cells (approximately 4.0 log cfu/mL) and spores (approximately 2.0 log cfu/mL), regression equations, elucidating B. licheniformis growth behavior during raw milk holding at low temperature, were obtained. Contour plots were developed to determine the time-temperature combinations, keeping the population changes to less than 1.0 log. In vegetative cell spiking study of B. licheniformis ATCC 6634 (S1), cell population changes remained below 1.0 log up to 72 h at 8°C. For B. licheniformis ATCC 14580 (S2), 1.0 log shift was not observed only after 80 h at 8°C, indicating higher multiplication potential of S1 as compared with S2. As S2 was a readily sporulating strain, the vegetative spiking study showed spore formation at different storage temperatures. Evidence of some parallel germination was observed for this strain at 8°C or higher, when raw milk samples were spiked with spores. The experimental values obtained for sporeformers and spore counts were validated with contour plot-generated values. Overall, for raw milk samples predominated by the low sporulating strain, the contour plots suggested holding at 8°C or below for up to 72 h. In the case of the readily sporulating strain (S2), raw milk could be held at 8°C for 80 h, where little or no sporulation is observed. Sporulation behavior, germination and multiplication ability, strain variability, and temperature and duration of holding raw milk influenced the population dynamics of Bacillus species. However, in the presence of equivalent numbers of both types of sporulating strains in raw milk, despite strain variability, holding milk at 8°C for not more than 72 h would keep any cell population changes below 1.0 log. In addition, under these storage conditions, the population would remain as vegetative cells that are likely to be inactivated by pasteurization. The contour plots, so generated, would help predict the population shifts and define optimum holding conditions for raw milk before further processing.

Feed substrates influence biofilm formation on reverse osmosis membranes and their cleaning efficiency.

The dairy industry is increasingly using reverse osmosis (RO) membranes for concentration of various fluid feed materials such as whey and ultrafiltration (UF) permeate. This study compared the effect of UF permeate and whey on membrane biofilm formation. A Bacillus sp., previously isolated in our laboratory from a cleaning-resistant membrane biofilm, was used to develop 48-h-old static biofilms on RO membrane pieces, using the different feed substrates (UF permeate, whey, and an alternating whey/UF feed). Biofilms were analyzed for viable counts by the swab technique, and we used scanning electron and atomic force microscopy for microstructure imaging. The membrane cleaning process included 6 sequential steps. We observed differences in the resistance pattern of the 3 types of biofilms to the typical cleaning process. The mean pretreatment counts of the 48-h UF permeate biofilms were 5.39 log cfu/cm2, much higher than the whey biofilm counts of 3.44 log, and alternating whey/UF biofilm counts of 4.54 log. After a 6-step cleaning cycle, we found 2.54 log survivors of the Bacillus isolate on UF biofilms, whereas only 1.82 log survivors were found in whey biofilm, and 2.14 log survivors on whey/UF permeate biofilms. In conclusion, the UF permeate biofilms was more resistant to the biofilm cleaning process compared with the whey or whey/UF permeate biofilms. Scanning electron micrographs showed different microstructures of biofilms based on the type of feed. For UF permeate and whey/UF permeate biofilms, bacilli were present in multilayers of cells in aggregates or irregular clusters with foulant layers. In contrast, those in whey biofilms were in monolayers, with a smoother, flatter appearance. Atomic force microscopy analysis indicated that UF permeate biofilms had the greatest surface roughness among the biofilms, reflecting intensified bacterial colonization. The biofilm micro- and nanostructure variations for the 2 feed substrates and their combination may have resulted in differences in their resistance to the cleaning process.

Comparison of adhesion characteristics of common dairy sporeformers and their spores on unmodified and modified stainless steel contact surfaces.

The attachment of aerobic spore-forming bacteria and their spores to the surfaces of dairy processing equipment leads to biofilm formation. Although sporeformers may differ in the degree of attachment, various surface modifications are being studied in order to develop a surface that is least vulnerable to attachment. This study was conducted to compare the extent of adhesion of spores and vegetative cells of the thermotolerant sporeformer Bacillus licheniformis and the high-heat-resistant sporeformers Geobacillus stearothermophilus and Bacillus sporothermodurans on both native and modified stainless steel surfaces. We studied the effect of contact surface and cell surface properties (including surface energy, surface hydrophobicity, cell surface hydrophobicity, and zeta potential) on the adhesion tendency of both types of sporeformers and their spores. Attachment to native and modified (Ni-P-polytetrafluoroethylene, Ni-P-PTFE) stainless steel surfaces was determined by allowing interaction between the respective contact surface and vegetative cells or spores for 1 h at ambient temperature. The hydrophobicity of vegetative cells and spores of aerobic spore-forming bacteria was determined using the hexadecane assay, and zeta potential was determined using the Zeta sizer Nano series instrument (Malvern Panalytical, Malvern, UK). The results indicated a higher adhesion tendency of spores over vegetative cells for both thermotolerant and high-heat-resistant sporeformers. On comparing the sporeformers, B. sporothermodurans demonstrated the highest adhesion tendency followed by G. stearothermophilus; B. licheniformis exhibited minimal attachment on both surfaces. The tendency to adhere varied with cell surface properties, decreasing with lower cell surface hydrophobicity and higher cell surface charge. On the other hand, modifying contact surface properties for higher surface hydrophobicity and lower surface energy decreased attachment.

Listeria cross contamination levels in raw ice cream mix can serve as a predictor of their potential presence as heat-injured cells.

Listeriosis is a life-threatening infection caused by foods contaminated with Listeria monocytogenes. Some of the major ice cream recalls in recent years reaffirm the ability of this food-borne pathogen to survive in diverse dairy processing environments and cause cross contamination. Inspection reports revealed certain lapses in implementing adequate hygienic practices for Listeria persistence in the processing environment, leading to cross contamination of ice cream. The higher levels of cross contamination of raw ice cream mix might result in random heat-injured cells when exposed to minimum heat treatment (69°C for 30 min). These heat-injured cells could later recover under abusive storage and handling conditions and pose a health risk. Evidence about the presence of injured cells in ice cream mix may thus prove useful to establish the overall Listeria risk, which was the aim of this study. Challenge studies were conducted to evaluate the dose-dependent presence of heat-injured cells of Listeria. Ice cream mix formulations of 4 different types (36, 40, 42, and 45% total solids) were inoculated at 2.0, 3.0, and 4.0 log cfu/g levels of Listeria innocua (an established surrogate). The dose levels were selected based on a likely cross contamination on the raw side from environmental Listeria, especially due to their resident nature and growth in harborage sites. The samples were exposed to minimum heat treatment (69°C for 30 min) and the survivors, including heat-injured cells, were enumerated using standard protocols. A binary logistic regression model was fitted for evaluating the severity of risk. The influence of total solids, water activity, and pH variability were also studied on Listeria survival. The enrichment protocol, using buffered Listeria enrichment broth, followed by plating on modified oxford agar and Rapid L'mono medium, revealed the random presence of heat-injured cells in buffered Listeria enrichment broth, only at the highest dose level of 4+ logs. Any potential risk from heat-injured cells was thus limited only to the highest levels of cross contamination, irrespective of the type of the mix. Significantly, none of the pasteurized ice cream mix samples supported the recovery of any heat-injured cells of Listeria during 72 h holding at 7°C, even at the highest dose level of 4+ logs, under the conditions of experimentation. The level of cross contamination (dose) emerged as a predictor of the potential presence of heat-injured cells of Listeria exposed to minimum pasteurization treatment.

Short communication: A comparison of biofilm development on stainless steel and modified-surface plate heat exchangers during a 17-h milk pasteurization run.

Flow of milk through the plate heat exchanger (PHE) results in denaturation of proteins, resulting in fouling. This also accelerates bacterial adhesion on the PHE surface, eventually leading to the development of biofilms. During prolonged processing, these biofilms result in shedding of bacteria and cross-contaminate the milk being processed, thereby limiting the duration of production runs. Altering the surface properties of PHE, such as surface energy and hydrophobicity, could be an effective approach to reduce biofouling. This study was conducted to compare the extent of biofouling on native stainless steel (SS) and modified-surface [Ni-P-polytetrafluoroethylene (PTFE)] PHE during the pasteurization of raw milk for an uninterrupted processing run of 17 h. For microbial studies, raw and pasteurized milk samples were aseptically collected from inlets and outlets of both PHE at various time intervals to examine shedding of bacteria in the milk. At the end of the run, 3M quick swabs (3M, St. Paul, MN) and ATP swabs (Charm Sciences Inc., Lawrence, MA) were used to sample plates from different sections of the pasteurizers (regeneration, heating, and cooling) for biofilm screening and to estimate the efficiency of cleaning in place, respectively. The data were tested for ANOVA, and means were compared. Modified PHE experienced lower mesophilic and thermophilic bacterial attachment and biofilm formation (average log 1.0 and 0.99 cfu/cm2, respectively) in the regenerative section of the pasteurizer compared with SS PHE (average log 1.49 and 1.47, respectively). Similarly, higher relative light units were observed for SS PHE compared with the modified PHE, illustrating the presence of more organic matter on the surface of SS PHE at the end of the run. In addition, at h 17, milk collected from the outlet of SS PHE showed plate counts of 5.44 cfu/cm2, which were significantly higher than those for pasteurized milk collected from modified PHE (4.12 log cfu/cm2). This provided further evidence in favor of the modified PHE achieving better microbial quality of pasteurized milk in long process runs. Moreover, because cleaning SS PHE involves an acid treatment step, whereas an alkali treatment step is sufficient for the modified-surface PHE, use of the latter is both cost and time effective, making it a better surface for thermal processing of milk and other fluid dairy products.

Short communication: Evaluation of a sol-gel-based stainless steel surface modification to reduce fouling and biofilm formation during pasteurization of milk.

Milk fouling and biofilms are common problems in the dairy industry across many types of processing equipment. One way to reduce milk fouling and biofilms is to modify the characteristics of milk contact surfaces. This study examines the viability of using Thermolon (Porcelain Industries Inc., Dickson, TN), a sol-gel-based surface modification of stainless steel, during thermal processing of milk. We used stainless steel 316L (control) and sol-gel-modified coupons in this study to evaluate fouling behavior and bacterial adhesion. The surface roughness as measured by an optical profiler indicated that the control coupons had a slightly smoother finish. Contact angle measurements showed that the modified surface led to a higher water contact angle, suggesting a more hydrophobic surface. The modified surface also had a lower surface energy (32.4 ± 1.4 mN/m) than the control surface (41.36 ± 2.7 mN/m). We evaluated the susceptibility of control and modified stainless steel coupons to fouling in a benchtop plate heat exchanger. We observed a significant reduction in the amount of fouled layer on modified surfaces. We found an average fouling weight of 19.21 mg/cm2 and 0.37 mg/cm2 on the control and modified stainless steel coupons, respectively. We also examined the adhesion of Bacillus and biofilm formation, and observed that the modified stainless steel surface offered greater resistance to biofilm formation. Overall, the Thermolon-modified surface showed potential in the thermal processing of milk, offering significantly lower fouling and bacterial attachment than the control surface.

Evaluation of modified stainless steel surfaces targeted to reduce biofilm formation by common milk sporeformers.

The development of bacterial biofilms on stainless steel (SS) surfaces poses a great threat to the quality of milk and other dairy products as the biofilm-embedded bacteria can survive thermal processing. Established biofilms offer cleaning challenges because they are resistant to most of the regular cleaning protocols. Sporeforming thermoduric organisms entrapped within biofilm matrix can also form heat-resistant spores, and may result in a long-term persistent contamination. The main objective of this study was to evaluate the efficacy of different nonfouling coatings [AMC 18 (Advanced Materials Components Express, Lemont, PA), Dursan (SilcoTek Corporation, Bellefonte, PA), Ni-P-polytetrafluoroethylene (PTFE, Avtec Finishing Systems, New Hope, MN), and Lectrofluor 641 (General Magnaplate Corporation, Linden, NJ)] on SS plate heat exchanger surfaces, to resist the formation of bacterial biofilms. It was hypothesized that modified SS surfaces would promote a lesser amount of deposit buildup and bacterial adhesion as compared with the native SS surface. Vegetative cells of aerobic sporeformers, Geobacillus stearothermophilus (ATCC 15952), Bacillus licheniformis (ATCC 6634), and Bacillus sporothermodurans (DSM 10599), were used to study biofilm development on the modified and native SS surfaces. The adherence of these organisms, though influenced by surface energy and hydrophobicity, exhibited no apparent relation with surface roughness. The Ni-P-PTFE coating exhibited the least bacterial attachment and milk solid deposition, and hence, was the most resistant to biofilm formation. Scanning electron microscopy, which was used to visualize the extent of biofilm formation on modified and native SS surfaces, also revealed lower bacterial attachment on the Ni-P-PTFE as compared with the native SS surface. This study thus provides evidence of reduced biofilm formation on the modified SS surfaces.

Prevalence of thermoduric bacteria and spores in nonfat dry milk powders of Midwest origin.

Samples of nonfat dry milk powder were analyzed for the presence of heat-resistant bacteria. The samples were collected from Midwest manufacturing companies and were evaluated for the presence of spores, thermoduric bacteria, and the total bacterial count. Three companies were included in this study, and results showed differences between each of the companies in the heat-resistant microbial groups tested. Company 3 had the highest levels of total spores and thermoduric bacteria: 3.6±0.14 and 3.5±0.13 log cfu/g, respectively. Interestingly, this company did not have the highest total bacterial count but rather the second lowest total bacterial count for the group, perhaps because of the higher proportion of thermophiles present in the powders from this company. The average level of total bacterial counts was 2.57±0.07 log cfu/g. Isolates obtained from the samples were identified by mass spectrometry, and all of the companies showed Bacillus licheniformis as the most prevalent bacterial species identified.

Bovine adenovirus 3 core protein precursor pVII localizes to mitochondria, and modulates ATP synthesis, mitochondrial Ca2+ and mitochondrial membrane potential.

Viruses modulate the functions of mitochondria by translocating viral proteins to the mitochondria. Subcellular fractionation and sensitivity to proteinase K/Triton X-100 treatment of mitochondrial fractions of bovine adenovirus (BAdV)-3-infected/transfected cells suggested that core protein pVII localizes to the mitochondria and contains a functional mitochondrial localization signal. Moreover, mitochondrial localization of BAdV-3 pVII appears to help in the retention of mitochondrial Ca(2+), inducing a significant increase in the levels of ATP and maintaining the mitochondrial membrane potential (MMP) in transfected cells. In contrast, mitochondrial localization of BAdV-3 pVII has no significant effect on the levels of cytoplasmic Ca(2+) and reactive oxygen species production in the transfected cells. Consistent with these results, expression of pVII in transfected cells treated with staurosporine decreased significantly the activation of caspase-3. Our results suggested that BAdV-3 pVII localizes to mitochondria, and interferes with apoptosis by inhibiting loss of the MMP and by increasing mitochondrial Ca(2+) and ATP production.

Effect of bovine adenovirus 3 on mitochondria.

Viruses alter the structure and the function of mitochondria for survival. Electron microscopy analysis of the cells infected with bovine adenovirus 3 revealed extensive damage to the inner mitochondrial membrane characterized by dissolution of the cristae and amorphous appearance of mitochondrial matrix with little or no damage to the outer mitochondrial membrane. There were fewer cristae with altered morphology. Potential patches of protein synthesis machinary around mitochondria could be observed at 12 hours post infection (hpi). At 24 hpi, the multi vascular bodies were evident throughout the infected cell. ATP production, mitochondrial Ca2+ and mitochondrial membrane potential (MMP) peaked at 18 hpi but decreased significantly at 24 hpi. This decrease coincided with the increased production of superoxide (SO) and reactive oxygen species (ROS), at 24 hpi indicating acute oxidative stress in the cells and suggesting a complete failure of the cellular homeostatic machinary. The results reveal an intericate relationship between Ca2+ homeostasis, the ATP generation ability of cells, SO and ROS production, and regulation of MMP following infection by bovine adenovirus 3.