Modeling the UV-C Inactivation Kinetics and Determination of Fluence Required for Incremental Inactivation of Cronobacter spp.

ABSTRACT: A study was undertaken to model the UV-C inactivation kinetics and determine the fluences required for the incremental inactivation of several strains of Cronobacter spp. suspended in clear phosphate-buffered saline (PBS). In total, 13 strains of Cronobacter spp. were individually suspended in PBS and treated with UV-C doses of 0, 2, 4, 6, 8, and 10 mJ cm-2 with a collimated beam device emitting UV-C at 253.7 nm. The log reduction from each treatment was identified using the plate count method and plotted against the UV-C dose and then curve fitted using several mathematical models. The UV-C dose required for incremental inactivation of each isolate was determined using both linear and nonlinear regression. For the 13 strains tested, a UV-C dose of 10 mJ cm-2 inactivated between 3.66 ± 0.101 and 5.04 ± 0.465 log CFU mL-1. The survival behavior of all strains was best fitted to the Weibull+tail model, with correlation coefficients between 97.17 and 99.71%, and was used to determine the fluences required for incremental inactivation. The UV-C fluences needed to inactivate 1 log (D10-value) of Cronobacter spp. in buffer were between 3.53 and 5.50 mJ cm-2, whereas a fluence greater than 6.57 mJ cm-2 was required to achieve a 4-log inactivation. A clear understanding of the UV-C dose-response of several strains of Cronobacter spp. lays the foundation to design effective UV-based disinfection systems.

Use of 2-hydroxy-4-(methylthio)-butanoic acid to inhibit Salmonella and Listeria in raw meat for feline diets and palatability in domestic cats.

While the raw pet food market continues to grow, the risk of bacterial contamination in these types of diets is a major concern, with Salmonella enterica and Listeria monocytogenes being the most frequently associated pathogens in raw pet food product recalls. dl-Methionine is included in some commercial feline kibble and canned diets to improve protein quality; however, an alternative to this is a liquid methionine supplement, 2-hydroxy-4-(methylthio)-butanoic acid (HMTBa), which is also an organic acid. 2-Hydroxy-4-(methylthio)-butanoic acid has previously demonstrated similar efficacy to formic acid against pathogens in a liquid environment and may be a good candidate to inhibit S. enterica and L. monocytogenes in raw ground meat. First, the minimum inhibitory concentration and minimum bactericidal concentration of HMTBa against these pathogens under laboratory growth conditions were determined by measuring growth of pathogens over 36 h when exposed to 10 concentrations of HMTBa (0.10% to 1.00%) mixed with tryptic soy broth. 2-Hydroxy-4-(methylthio)-butanoic acid included at ≥0.50% was bactericidal to S. enterica and L. monocytogenes (P < 0.05). Next, five levels of HMTBa (0.50% to 1.25%) were included in raw ground meat mixtures inoculated with cocktails of S. enterica or L. monocytogenes, and contamination levels were determined at four timepoints: immediately, and after refrigerated storage (4 °C) at 24, 48, and 72 h after removal from freezer (24 h at -20 °C). 2-Hydroxy-4-(methylthio)-butanoic acid included as 1.25% of the meat mixture reduced S. enterica and L. monocytogenes compared with the control (P < 0.05); however, it did not result in total kill of either of these pathogens. Following this, feeding behaviors of seven domestic cats were assessed when offered a raw chicken diet treated with or without 1.25% HMTBa for 5 d each, after which a 2-d 2-choice preference test was conducted. Cats demonstrated a preference for raw diets without HMTBa, but still readily consumed diets with 1.25% HMTBa, suggesting that such a diet was still palatable to them.

Fatty acids and volatile flavor compounds in commercial plant-based burgers.

Interest in plant-based meat alternatives (PMBAs) has been rapidly growing in both the food research community and the food industry due to higher consumer demands in recent years. However, scientific data regarding the health and aroma aspects of PBMA are rare. In this study, the fatty acids (FAs) and volatile flavor compounds (VFCs) were profiled in four types of plant-based burgers (PBs) and compared to beef burger (BB). Over 40 FAs and 64 VFCs were detected and quantified in the samples. Nonsignificant differences (α = 0.05) were observed in the percentages of most FAs between uncooked and cooked PBs. PBs contained lower percentages of saturated FAs and trans-FAs, higher percentages of unsaturated FAs, and lower ratio of n-6 to n-3 FAs comparing to the BB. The FA profiles in PBs are mainly determined by their ingredients. The VFC profile of cooked PBs was different from that of the uncooked ones. The ingredients, thermally induced Maillard reaction, and lipid oxidation had contributed to the formation of the flavor. For uncooked samples, the VFC profiles of PB 3 and PB 4 were similar to that of BB. While for cooked samples, PB 1 had similar VFC profile as BB. This illustrated the importance of the cooking process for aroma formation; however, ingredients, such as spices, remain an important source of VFCs in these burger samples. Ingredient optimization could be an effective strategy to enhance the flavor of PBs to resemble BB. PRACTICAL APPLICATION: This study provides the knowledge of health and aroma-related components in both raw and cooked PBs, including FA and VFC profiles. It also explains the source of those components. This will not only help consumer's decision making in choosing plant-based meat alternatives, but also help the related industry to choose proper ingredients to optimize the final products.

Impacts of food matrix on bacteriophage and endolysin antimicrobial efficacy and performance.

Food contamination with bacterial pathogens is a persisting threat and challenge for producers, consumers, and health care systems globally. Thus, there is a need for novel and targeted food safety practices. This review discusses the importance of characterizing bacteriophage and endolysins for specific food matrices, as well as characterizing individual properties of food matrices to guide optimized bacteriophage and endolysin usage and engineering. Diverse food parameters and their interactions specific to bacteriophages and endolysins are examined to provide insight into influential factors that affect their efficacy. Food matrix parameters prove to warrant detailed individualistic characterization of bacteriophages and endolysins in order to determine their suitability for specific food systems. Established impacts of food matrix components on bacteriophages and on other antimicrobials are discussed in relation to inferences regarding endolysin performance. Determining food matrix parameters of a food system and understanding how these features impact bacateriophages and endolysins, can also provide a foundation for tailoring their optimized administration. With this knowledge, endolysin enhancements via protein engineering can be introduced in a more tailored fashion, optimizing the innate antimicrobial nature of endolysins for specific applications.

Inactivation of Bacillus and Clostridium Spores in Coconut Water by Ultraviolet Light.

Bacterial spores are generally more resistant than vegetative bacteria to ultraviolet (UV) inactivation. The UV sensitivity of these spores must be known for implementing UV disinfection of low acid liquid foods. UV inactivation kinetics of bacterial spores in coconut water (CW) and distilled sterile water was studied. Populations of Bacillus cereus and Clostridium sporogenes dormant spores were reduced by more than 5.5 log10 at the UV-C photon fluence of 1142 µE·m-2 and 1919 µE·m-2 respectively. C. sporogenes spores showed higher UV-C resistance than B. cereus, with the photon fluence 300 µE·m-2 required for one log inactivation (D10) and 194 µE·m-2, respectively. No significant difference was observed in D10 values of spores suspended in the two fluid types (p > 0.05). The inactivation kinetics of microorganisms were described by log linear models with low root mean square error and high coefficient of determination (R2 > 0.98). This study clearly demonstrated that high levels of inactivation of bacterial spores can be achieved in CW. The baseline data generated from this study will be used to conduct spore inactivation studies in continuous flow UV systems. Further proliferation of the technology will include conducting extensive pilot studies.

Quantitating Phage Efficacy in Ready-To-Eat Meats.

Bacteriophages are being applied in biocontrol of bacterial pathogens in foods and food processing environments. There is need for the development of standardized protocols to quantify the effectiveness of phage preparations in reducing food-borne pathogens on foods. Here, we present a procedure for the verification of the effectiveness of a phage preparation in reducing Listeria monocytogenes on ready-to-eat (RTE) meats. The protocol is designed taking into account real-world scenarios and avoiding common errors reported in previous phage decontamination assays.

Draft Genome Sequences of Two Novel Salmonella enterica subsp. enterica Strains Isolated from Low-Moisture Foods with Applications in Food Safety Research.

The genomes of two strains of Salmonella enterica subsp. enterica serovar Cubana and serovar Muenchen, isolated from dry hazelnuts and chia seeds, respectively, were sequenced using the Illumina MiSeq platform, assembled de novo using the overlap-layout-consensus method, and aligned to their respective most identical sequence genome scaffolds using MUMMER and BLAST searches.

Thermal-Stability and Reconstitution Ability of Listeria Phages P100 and A511.

The study evaluated the thermal-stability of Listeria phages P100 and A511 at temperatures simulating the preparation of ready-to-eat meats. The phage infectivity after heating to 71°C and holding for a minimum of 30 s, before eventually cooling to 4°C were examined. Higher temperatures of 75, 80, and 85°C were also tested to evaluate their effect on phages thermal-stability. This study found that despite minor differences in the amino acid sequences of their structural proteins, the two phages responded differently to high temperatures. P100 activity declined at least 10 log (PFU mL-1) with exposure to 71°C (30 s) and falling below the limit of detection (1 log PFU mL-1) while, A511 dropped from 108 to 105 PFU mL-1. Cooling resulted in partial reconstitution of P100 phage particles to 103 PFU mL-1. Exposure to 75°C (30 s) abolished A511 activity (8 log PFU mL-1) and both phages showed reconstitution during cooling phase after exposure to 75°C. P100 exhibited reconstitution after treatment at 80°C (30 s), conversely A511 showed no reconstitution activity. Heating P100 to 85°C abolished the reconstitution potential. Substantial differences were found in thermal-stability and reconstitution of the examined phages showing A511 to be more thermo-stable than P100, while P100 exhibited reconstitution during cooling after treatment at 80°C which was absent in A511. The differences in predicted melting temperatures of structural proteins of P100 and A511 were consistent with the observed differences in thermal stability and morphological changes observed with transmission electron microscopy.

Use of encapsulated bacteriophages to enhance farm to fork food safety.

Bacteriophages have been successfully applied to control the growth of pathogens in foods and to reduce the colonization and shedding of pathogens by food animals. They are set to play a dominant role in food safety in the future. However, many food-processing operations and the microenvironments in food animals' guts inactivate phages and reduce their infectivity. Encapsulation technologies have been used successfully to protect phages against extreme environments, and have been shown to preserve their activity and enable their release in targeted environments. A number of encapsulation technologies have shown potential for use with bacteriophages. This review discusses the current state of knowledge about the use of encapsulation technologies with bacteriophages to control pathogens in foods and food animals.

Propagation method for persistent high yield of diverse Listeria phages on permissive hosts at refrigeration temperatures.

The efficient production of a high concentration of bacteriophage in large volumes has been a limiting factor in the exploration of the true potential of these organisms for biotechnology, agriculture and medicine. Traditional methods focus on generating small volumes of highly concentrated samples as the end product of extensive mechanical and osmotic processing. To function at an industrial scale mandates extensive investment in infrastructure and input materials not feasible for many smaller facilities. To address this, we developed a novel, scalable, generic method for producing significantly higher titer psychrophilic phage (P < 2.0 × 10(-6)), 2- to 4-fold faster than traditional methods. We generate renewable high yields from single source cultures by propagating phage under refrigeration conditions in which Listeria, Yersinia and their phages grow in equilibrium. Diverse Yersinia and Listeria phages tested yielded averages of 3.49 × 10(8) to 3.36 × 10(12) PFU/ml/day compared to averages of 1.28 × 10(5) to 1.30 × 10(10) PFU/ml/day by traditional methods. Host growth and death kinetics made this method ineffective for extended propagation of mesophilic phages.

Survival of Campylobacter jejuni on beef and pork under vacuum packaged and retail storage conditions: examination of the role of natural meat microflora on C. jejuni survival.

The ability of Campylobacter jejuni ATCC 11168 to survive on beef and pork stored under chilled, vacuum packaged and retail display conditions were examined. In addition, the effect of natural microflora on commercial beef and pork on the survival of C. jejuni under these storage conditions was examined. When sterile cores of beef and pork were inoculated with ∼ 10(5) to 10(6) cfu cm(-2)C. jejuni, and were stored under aerobic or vacuum packaged conditions at -1.5 or 4 °C, its numbers dropped significantly and C. jejuni could not be enumerated by direct plating after 21 d of the 6 wks study. In contrast, survival of C. jejuni on commercial vacuum packaged beef and pork was significantly enhanced, resulting in only 1 log cfu cm(-2) reduction at the end of 6 wks. During 7 d of display in a retail case, numbers of C. jejuni dropped quickly, but could be enumerated by direct plating even after the 7 d. The presence of high numbers of inoculated C. jejuni on beef and pork had no significant effect on the natural microflora numbers compared to uninoculated controls when the meat was stored either in vacuum or in a retail display case. These results show that natural microflora on vacuum packaged meat afford enhanced survival of C. jejuni present on the surfaces of both beef and pork when stored at refrigeration temperatures. Hence, strict hygienic practices or the implementation of decontamination technologies are recommended to ensure safety of meat with respect to this pathogen.

Growth temperature associated protein expression and membrane fatty acid composition profiles of Salmonella enterica serovar Typhimurium.

Total cellular proteins and fatty acid composition profiles of mid-log phase cells of Salmonella enterica serovar Typhimurium grown at 8, 25, 37 or 42 °C were separated by 2D-PAGE and FAME analysis. Growth temperature associated protein expression can be grouped into 3 thermal classes which include proteins whose expression is: I) optimal at 37 °C, meaning their expression peaked at 37 °C; II) up-regulated with an increase in growth temperature; III) down-regulated with increase in growth temperature; meaning their expression peaked at 8 °C. At higher growth temperatures, proteins belonging to the functional groups of amino acid transport and metabolism, nucleotide metabolism, energy metabolism and post-translation modifications (chaperones) are present in substantially higher amounts. This increase in abundance is regulated in a temperature dependent manner. It is important to point out that proteins involved in energy metabolism observed in higher amounts at higher growth temperatures all belong to the glycolysis pathway, while at 8 °C they belonged to the TCA cycle. Increase in growth temperatures results in a decrease in membrane fatty acid unsaturation and an increase in saturated and cyclic fatty acids. These results provide an insight into the dynamic molecular and physiological responses of Salmonella Typhimurium during growth at different temperatures.

Products of glucose and lactate fermentation, and utilization of amino acids by Clostridium estertheticum subspp. laramiense and estertheticum growing in meat juice medium.

The type strains of Clostridium estertheticum subsp. laramiense and C. estertheticum subsp. estertheticum both utilized glucose and glycogen when growing in meat juice medium and fermented lactate, but ceased growth when glucose was exhausted. The fermentation products from glucose were butyrate, acetate, and formate; those from lactate were 1-butanol, ethanol, butyrate, and formate. Both organisms utilized several amino acids (not containing sulfur) during their cultivation in meat juice medium and did not produce H(2)S. The optimum and maximum temperatures for growth of C. estertheticum subsp. laramiense were 10 degrees C, and 20 to 22 degrees C, respectively. Those same optimum and maximum temperatures have previously been determined for C. estertheticum subsp. estertheticum. The pH range for growth of the two organisms, 5.5 to 7.5, was also the same. Both organisms were beta-hemolytic and formed subterminal spores. Thus, the organisms did not display the difference in fermentation products, optimum and maximum temperatures, hemolysis, and spore position that were reported to be the differentiating characteristics of the subspecies. The findings indicate that vacuum-packaged meat should be spoiled similarly by the two type strains.

Effects of temperature and pH on the growth of bacteria isolated from blown packs of vacuum-packaged beef.

Bacteria recovered from the microflora of blown packs of vacuum-packaged beef were identified as Leuconostoc mesenteroides, Lactococcus lactis, Carnobacterium maltaromaticum, and Clostridium estertheticum, with L. mesenteroides predominant. Isolates of these lactic acid bacteria all grew in peptone yeast extract glucose starch broth (PYGSB) at temperatures between -2 and 30 degrees C but generally grew more slowly and over a more restricted temperature range in meat juice medium (MJM). A C. estertheticum isolate and the type strain of C. estertheticum subsp. estertheticum (ATCC 51377) both grew in PYGSB and MJM at similar rates at temperatures between -2 and 17 degrees C and grew at 20 degrees C in MJM but not in PYGSB. Square root models of the variation of the growth rate with temperature indicated that the C. maltaromaticum isolate and the C. estertheticum strains grew at similar rates that were faster than those of the other isolates at temperatures between -2 and 0 degrees C. The L. mesenteroides and L. lactis isolates grew in PYGSB at pH 5.0, but the C. maltaromaticum isolate and both strains of C. estertheticum did not grow in PYGSB at pH

Substrate utilization by Clostridium estertheticum cultivated in meat juice medium.

Blown pack spoilage of vacuum packaged beef, which results in packs being grossly distended with gas, is caused by the psychrophile Clostridium estertheticum. To determine what substrates are utilized by C. estertheticum during growth on beef, C. estertheticum subsp. estertheticum ATCC 51377, the type strain for that organism, and two isolates from blown pack spoiled beef that were identified as C. estertheticum by 16 S rRNA gene sequencing were grown in meat juice medium at 10 degrees C for up to 14 days. Analysis of the growth media showed that all three organisms grew exponentially on glucose with simultaneous hydrolysis of glycogen. Growth ceased when glucose in the media was depleted; but hydrolysis of glycogen continued at a reduced rate, and lactate was consumed rapidly. The pH values of media fell during growth of the organisms, but rose as the concentrations of lactate subsequently decreased. The major products of fermentation during utilization of glucose were butyrate and acetate, with butyrate greatly predominating. During fermentation of lactate the major products were butyrate and butanol, which were produced in similar amounts. The findings suggest that growth of C. estertheticum on vacuum packaged beef may be limited by the availability of glucose, as is the growth of other organisms that usually predominate in the flora of vacuum packaged meat. However, production of gas by fermentation of lactate will likely continue after growth ceases.