Predominance and Distribution of a Persistent Listeria monocytogenes Clone in a Commercial Fresh Mushroom Processing Environment.

A longitudinal study was conducted to determine the prevalence of Listeria spp. in a commercial fresh mushroom slicing and packaging environment. Samples were collected at three different sampling periods within a 13-month time interval. Of the 255 environmental samples collected, 18.8% tested positive for L. monocytogenes, 4.3% for L. innocua, and 2.0% for L. grayi. L. monocytogenes was most often found on wet floors within the washing and slicing and packaging areas. Each of the 171 L. monocytogenes isolates found in the environment could be placed into one of three different serotypes; 1/2c was predominant (93.6%), followed by 1/2b (3.5%) and 1/2a (2.9%). Of 58 isolates subtyped using multi-virulence-locus sequence typing, all 1/2c isolates were identified as virulence type (VT) 11 (VT11), all 1/2b isolates were VT105, and 1/2a isolates were either VT107 or VT56. VT11 was designated as the predominant and persistent clone in the environment because it was isolated repeatedly at numerous locations throughout the study. The overall predominance and persistence of VT11 indicates that it likely colonized the mushroom processing environment. Areas adjacent to the trench drain in the washing and slicing area and a floor crack in the packaging area may represent primary harborage sites (reservoirs) for VT11. Improvements made to sanitation procedures by company management after period 2 coincided with a significant (P ≤ 0.001) reduction in the prevalence of L. monocytogenes from 17.8% in period 1 and 30.7% in period 2 to 8.5% in period 3. This suggests that targeted cleaning and sanitizing procedures can be effective in minimizing the occurrence of L. monocytogenes contamination in processing facilities. Additional research is needed to understand why VT11 was predominant and persistent in the mushroom processing environment.

Modeling development of inhibition zones in an agar diffusion bioassay.

A two-temperature agar diffusion bioassay is commonly used to quantify the concentration of nisin using Micrococcus luteus as the indicator microorganism. A finite element computational model based on Fick's second law of diffusion was used to predict the radius of the inhibition zone in this diffusion bioassay. The model developed was used to calculate nisin concentration profiles as a function of time and position within the agar. The minimum inhibitory concentration (MIC) of nisin against M. luteus was determined experimentally. The critical time (T c) for growth of M. luteus within the agar diffusion bioassay was experimentally determined using incubation studies with nisin. The radius of the inhibition zone was predicted from the computational model as the location where the predicted nisin concentration at T c was equal to MIC. The MIC was experimentally determined to be 0.156 µg mL(-1), and T c was determined to be 7 h. Good agreement (R (2) = 0.984) was obtained between model-predicted and experimentally determined inhibition zone radii.

Bacterial persister cell formation and dormancy.

Bacterial cells may escape the effects of antibiotics without undergoing genetic change; these cells are known as persisters. Unlike resistant cells that grow in the presence of antibiotics, persister cells do not grow in the presence of antibiotics. These persister cells are a small fraction of exponentially growing cells (due to carryover from the inoculum) but become a significant fraction in the stationary phase and in biofilms (up to 1%). Critically, persister cells may be a major cause of chronic infections. The mechanism of persister cell formation is not well understood, and even the metabolic state of these cells is debated. Here, we review studies relevant to the formation of persister cells and their metabolic state and conclude that the best model for persister cells is still dormancy, with the latest mechanistic studies shedding light on how cells reach this dormant state.

Listeria monocytogenes responds to cell density as it transitions to the long-term-survival phase.

Listeria monocytogenes was recently found to enter a long-term-survival (LTS) phase, which may help explain its persistence in natural environments and within food processing plants. The purpose of this study was to investigate the effects of initial cell density, initial pH and type of broth (fresh vs. spent) on the transition of L. monocytogenes to the LTS phase and model the change in viable population density with time. Initial cell density (~10(6)-~10(10)CFU/ml) and initial pH (5.36-6.85) both significantly affected the transition of L. monocytogenes to the LTS phase (P<0.001) with initial cell density being the main determining factor. In contrast, type of broth did not significantly affect cell density change during the transition of stationary-phase cells at high initial density to the LTS phase (P>0.05). After 30-d incubation no significant differences in cell densities were observed between either type of broth or between any of the initial cell density/pH treatment combinations (P>0.05), where the mean viable cell density was 4.3±1.1×10(8)CFU/ml. L. monocytogenes responded to viable cell density in accordance with the logistic equation during transition to the LTS phase. The Agr quorum-sensing system does not appear to play a role in the transition to the LTS phase. Further research is needed to better understand the control mechanisms utilized by L. monocytogenes as it transitions to a coccoid, resistant and stable density state in the LTS phase.

Incidence of Listeria monocytogenes and Listeria spp. in a small-scale mushroom production facility.

Listeria monocytogenes is a foodborne pathogen of significant concern to the agricultural and food processing industry because of its ability to grow and persist in cool and moist environments and its association with listeriosis, a disease with a very high mortality rate. Although there have been no listeriosis outbreaks attributed to fresh mushrooms in the United States, retail surveys and recalls are evidence that L. monocytogenes contamination of mushrooms (Agaricus bisporus) can occur. The objective of this study was to determine the prevalence of Listeria spp., including L. monocytogenes, in a small-scale mushroom production facility on the campus of the Pennsylvania State University in the United States. Of 184 samples taken from five production zones within the facility, 29 (15.8%) samples were positive for Listeria spp. Among the Listeria spp. isolates, L. innocua was most prevalent (10.3%) followed by L. welshimeri (3.3%), L. monocytogenes (1.6%), and L. grayi (0.5%). L. monocytogenes was recovered only from the phase I raw material composting area. Isolates of L. monocytogenes were confirmed and serotyped by multiplex PCR. The epidemiological relatedness of the three L. monocytogenes isolates to those serotypes or lineages frequently encountered in listeriosis infections was determined by multi-virulence-locus sequence typing using six virulence genes, namely, prfA, inlB, inlC, dal, clpP, and lisR. The phylogenetic positions of the three isolates in the dendrogram prepared with data from other isolates of L. monocytogenes showed that all isolates were grouped with serotype 4a, lineage IIIA. To date, this serotype has rarely been reported in foodborne disease outbreaks.

Detection of virulence-associated genes and epidemic clone markers in Listeria monocytogenes isolates from PDO Gorgonzola cheese.

Fifty-three Listeria monocytogenes isolates obtained from Gorgonzola cheese and previously characterized with biochemical typing, serotyping and pulsed field gel electrophoresis (PFGE), were analyzed in this study. Seven virulence-associated genes were selected (actA, inlC, inlJ, plcA, prfA, hlyA and iap) and their presence was investigated using PCR. All virulence-associated genes were detected in 51 isolates. One isolate did not show amplification of the inlC gene and one other isolate, previously mis-identified as L. monocytogenes probably due to atypical phenotypes, resulted negative by PCR for all virulence genes and was identified as Listeria innocua by 16S rRNA gene sequencing analysis. A multiplex PCR assay was used to evaluate the presence of markers specific for epidemic clones (ECs) of L. monocytogenes. The marker specific for the recently identified epidemic clone V (ECV) was detected in 38 of 43 (88%) of serotype 1/2a isolates. These findings suggest that Gorgonzola cheese can represent a significant source of L. monocytogenes and potential health risk for listeriosis as almost all isolates (94%) could be potentially virulent and that 38 (~72%) were presumptive positive ECV. PCR screening for both virulence-associated genes and EC markers may be useful for rapidly evaluating the epidemic potential and public health risk posed by L. monocytogenes in PDO Gorgonzola cheese and other dairy products.

A combined multi-virulence-locus sequence typing and Staphylococcal Cassette Chromosome mec typing scheme possesses enhanced discriminatory power for genotyping MRSA.

Methicillin-resistant Staphylococcus aureus (MRSA) remains a major threat to human populations worldwide. Knowing the extent of MRSA genetic diversity within a healthcare facility may provide important insights into the epidemiology of this important pathogen. MRSA isolates recovered from nasal swabs of patients entering the Intensive Care Unit of the Penn State Milton S. Hershey Medical Center, USA, from 2008 to 2009 were genotyped using Staphylococcal Cassette Chromosome mec (SCCmec), multilocus sequence typing (MLST) and a newly developed multi-virulence-locus sequence typing (MVLST) scheme. Sequence data for seven housekeeping genes (arcC, aroE, glpF, gmk, pta, tpi and yqiL) and six virulence genes (alt, essC, geh, hlgA, htrA and sdrC) were used for MLST and MVLST analyses, respectively. MLST identified 12 sequence types (STs) within the hospital isolates. One ST designated ST5 was the most common subtype (38.8%) followed by ST105 (22.4%) and ST8 (16.4%). In contrast, MVLST identified 29 STs (Virulence Types, VTs) from the same set of isolates, with VT6 (32.8%) being the predominant subtype followed by VT9 (8.9%) and VT2 (8.9%). Subsequent analysis of 25 MRSA isolates associated with an outbreak at a Pennsylvania state prison revealed all isolates were VT2 and SCCmec type IVa. These results suggest that a combination of MVLST and SCCmec typing may clarify the epidemiology of MRSA. Additional research with a more diverse set of strains and correlation with conventional epidemiologic data are needed to validate this new subtyping strategy.

Sequence typing confirms that a predominant Listeria monocytogenes clone caused human listeriosis cases and outbreaks in Canada from 1988 to 2010.

Human listeriosis outbreaks in Canada have been predominantly caused by serotype 1/2a isolates with highly similar pulsed-field gel electrophoresis (PFGE) patterns. Multilocus sequence typing (MLST) and multi-virulence-locus sequence typing (MVLST) each identified a diverse population of Listeria monocytogenes isolates, and within that, both methods had congruent subtypes that substantiated a predominant clone (clonal complex 8; virulence type 59; proposed epidemic clone 5 [ECV]) that has been causing human illness across Canada for more than 2 decades.

Novel multiplex single nucleotide polymorphism-based method for identifying epidemic clones of Listeria monocytogenes.

A novel primer extension-based, multiplex minisequencing assay targeting six highly informative single nucleotide polymorphisms (SNPs) in four virulence genes correctly identified and differentiated all four epidemic clones (ECs) of Listeria monocytogenes and 9 other strains initially misclassified as non-ECs. This assay allows rapid, accurate, and high-throughput screening for all known ECs of L. monocytogenes.

Transcriptomic response of Listeria monocytogenes during the transition to the long-term-survival phase.

Listeria monocytogenes can change its cellular morphology from bacilli to cocci during the transition to the long-term-survival (LTS) phase. The LTS cells demonstrated increased baro- and thermotolerance compared to their vegetative counterparts. So far, the underlying mechanisms that trigger this morphological and physiological transition remain largely unknown. In this study, we compared the transcriptomic profiles of L. monocytogenes serotype 4b strain F2365 at different growth stages in tryptic soy broth with yeast extract (TSBYE) using a whole-genome DNA chip approach. We identified a total of 225 differentially expressed genes (≥4-fold; P < 0.05) during the transition to the LTS phase in TSBYE. Genes related to cell envelope structure, energy metabolism, and transport were most significantly upregulated in the LTS phase. The upregulation of compatible solute transporters may lead to the accumulation of cellular solutes, lowering intracellular water activity and thus increasing bacterial stress resistance during the transition to the LTS phase. The downregulation of genes associated with protein synthesis may indicate a status of metabolic dormancy of the LTS cells. The transcriptomic profiles of resuscitated LTS cells in fresh TSBYE resembled those of log-phase cells (r=0.94), as the LTS cells rapidly resume metabolic activities and transit back to log phase with decreased baro- and thermotolerance.

A novel multiplex PCR method for detecting the major clonal complexes of MRSA in nasal isolates from a Pennsylvania hospital.

A novel multiplex PCR was developed which targeted virulence genes associated with the major clonal complexes (CCs) of healthcare- and community-associated methicillin-resistant Staphylococcus aureus (MRSA) in the USA. Most isolates (40/66) were identified as CC 5, while remaining isolates represented CCs 1, 8, 30, 45, 59, 133, and five isolates were not S. aureus.

Subtyping Salmonella enterica serovar enteritidis isolates from different sources by using sequence typing based on virulence genes and clustered regularly interspaced short palindromic repeats (CRISPRs).

Salmonella enterica subsp. enterica serovar Enteritidis is a major cause of food-borne salmonellosis in the United States. Two major food vehicles for S. Enteritidis are contaminated eggs and chicken meat. Improved subtyping methods are needed to accurately track specific strains of S. Enteritidis related to human salmonellosis throughout the chicken and egg food system. A sequence typing scheme based on virulence genes (fimH and sseL) and clustered regularly interspaced short palindromic repeats (CRISPRs)-CRISPR-including multi-virulence-locus sequence typing (designated CRISPR-MVLST)-was used to characterize 35 human clinical isolates, 46 chicken isolates, 24 egg isolates, and 63 hen house environment isolates of S. Enteritidis. A total of 27 sequence types (STs) were identified among the 167 isolates. CRISPR-MVLST identified three persistent and predominate STs circulating among U.S. human clinical isolates and chicken, egg, and hen house environmental isolates in Pennsylvania, and an ST that was found only in eggs and humans. It also identified a potential environment-specific sequence type. Moreover, cluster analysis based on fimH and sseL identified a number of clusters, of which several were found in more than one outbreak, as well as 11 singletons. Further research is needed to determine if CRISPR-MVLST might help identify the ecological origins of S. Enteritidis strains that contaminate chickens and eggs.

Novel virulence gene and clustered regularly interspaced short palindromic repeat (CRISPR) multilocus sequence typing scheme for subtyping of the major serovars of Salmonella enterica subsp. enterica.

Salmonella enterica subsp. enterica is the leading cause of bacterial food-borne disease in the United States. Molecular subtyping methods are powerful tools for tracking the farm-to-fork spread of food-borne pathogens during outbreaks. In order to develop a novel multilocus sequence typing (MLST) scheme for subtyping the major serovars of S. enterica subsp. enterica, the virulence genes sseL and fimH and clustered regularly interspaced short palindromic repeat (CRISPR) loci were sequenced from 171 clinical isolates from nine Salmonella serovars, Salmonella serovars Typhimurium, Enteritidis, Newport, Heidelberg, Javiana, I 4,[5],12:i:-, Montevideo, Muenchen, and Saintpaul. The MLST scheme using only virulence genes was congruent with serotyping and identified epidemic clones but could not differentiate outbreaks. The addition of CRISPR sequences dramatically improved discriminatory power by differentiating individual outbreak strains/clones. Of particular note, the present MLST scheme provided better discrimination of Salmonella serovar Enteritidis strains than pulsed-field gel electrophoresis (PFGE). This method showed high epidemiologic concordance for all serovars screened except for Salmonella serovar Muenchen. In conclusion, the novel MLST scheme described in the present study accurately differentiated outbreak strains/clones of the major serovars of Salmonella, and therefore, it shows promise for subtyping this important food-borne pathogen during investigations of outbreaks.

Feeding the World Today and Tomorrow: The Importance of Food Science and Technology: An IFT Scientific Review.

by Philip E. Nelson, 2007 World Food Prize Laureate; Professor Emeritus, Food Science Dept., Purdue Univ. Just as society has evolved over time, our food system has also evolved over centuries into a global system of immense size and complexity. The commitment of food science and technology professionals to advancing the science of food, ensuring a safe and abundant food supply, and contributing to healthier people everywhere is integral to that evolution. Food scientists and technologists are versatile, interdisciplinary, and collaborative practitioners in a profession at the crossroads of scientific and technological developments. As the food system has drastically changed, from one centered around family food production on individual farms and home food preservation to the modern system of today, most people are not connected to their food nor are they familiar with agricultural production and food manufacturing designed for better food safety and quality. The Institute of Food Technologists-a nonprofit scientific society of individual members engaged in food science, food technology, and related professions in industry, academia, and government-has the mission to advance the science of food and the long-range vision to ensure a safe and abundant food supply contributing to healthier people everywhere. IFT convened a task force and called on contributing authors to develop this scientific review to inform the general public about the importance and benefits of food science and technology in IFT's efforts to feed a growing world. The main objective of this review is to serve as a foundational resource for public outreach and education and to address misperceptions and misinformation about processed foods. The intended audience includes those who desire to know more about the application of science and technology to meet society's food needs and those involved in public education and outreach. It is IFT's hope that the reader will gain a better understanding of the goals or purposes for various applications of science and technology in the food system, and an appreciation for the complexity of the modern food supply. Abstract: This Institute of Food Technologists scientific review describes the scientific and technological achievements that made possible the modern production-to-consumption food system capable of feeding nearly 7 billion people, and it also discusses the promising potential of ongoing technological advancements to enhance the food supply even further and to increase the health and wellness of the growing global population. This review begins with a historical perspective that summarizes the parallel developments of agriculture and food technology, from the beginnings of modern society to the present. A section on food manufacturing explains why food is processed and details various food processing methods that ensure food safety and preserve the quality of products. A section about potential solutions to future challenges briefly discusses ways in which scientists, the food industry, and policy makers are striving to improve the food supply for a healthier population and feed the future. Applications of science and technology within the food system have allowed production of foods in adequate quantities to meet the needs of society, as it has evolved. Today, our production-to-consumption food system is complex, and our food is largely safe, tasty, nutritious, abundant, diverse, convenient, and less costly and more readily accessible than ever before. Scientific and technological advancements must be accelerated and applied in developed and developing nations alike, if we are to feed a growing world population.

Changes in barotolerance, thermotolerance, and cellular morphology throughout the life cycle of Listeria monocytogenes.

Changes in barotolerance, thermotolerance, and cellular morphology throughout the life cycle of Listeria monocytogenes were investigated. For part 1 of this analysis, L. monocytogenes ATCC 19115 was grown to log, stationary, death, and long-term-survival phases at 35 degrees C in tryptic soy broth with yeast extract (TSBYE). Cells were diluted in whole milk that had been subjected to ultrahigh temperatures (UHT whole milk) and then high-pressure processed (HPP) at 400 MPa for 180 s or thermally processed at 62.8 degrees C for 30 s. As cells transitioned from the log to the long-term-survival phase, the D(400 MPa) and D(62.8 degrees C) values increased 10- and 19-fold, respectively. Cells decreased in size as they transitioned from the log to the long-term-survival phase. Rod-shaped cells transitioned to cocci as they entered the late-death and long-term-survival phases. L. monocytogenes strains F5069 and Scott A showed similar results. For part 2 of the analysis, cells in long-term-survival phase were centrifuged, suspended in fresh TSBYE, and incubated at 35 degrees C. As cells transitioned from the long-term-survival phase to log and the stationary phase, they increased in size and log reductions increased following HPP or heat treatment. In part 3 of this analysis, cells in long-term-survival phase were centrifuged, suspended in UHT whole milk, and incubated at 4 degrees C. After HPP or heat treatment, similar results were observed as for part 2. We hypothesize that cells of L. monocytogenes enter a dormant, long-term-survival phase and become more barotolerant and thermotolerant due to cytoplasmic condensation when they transition from rods to cocci. Further research is needed to test this hypothesis and to determine the practical significance of these findings.

Effect of water activity on inactivation of Listeria monocytogenes and lactate dehydrogenase during high pressure processing.

The aim of this study was to investigate the effect of water activity (aw) on the inactivation of Listeria monocytogenes and lactate dehydrogenase (LDH) during high pressure processing (HPP). For microbial inactivation lyophilized cells of L. monocytogenes 19,115 were left dry or were suspended in 10 ml of 0.1% peptone water, 10 ml of glycerol, or mixtures of glycerol and peptone water. All samples of various aws were high pressure (HP) processed at ambient temperature at 600 MPa for 300 s. Following HPP, samples were serially diluted in 0.1% peptone and spread-plated on Tryptic Soy agar supplemented with Yeast Extract. For enzyme inactivation, 4.2 mg of lyophilized LDH was suspended in 2 ml of 100 mM phosphate buffer (pH 7.4), 2 ml of peptone water or glycerol, or in 2 ml mixtures of glycerol and peptone water. A lyophilized sample with no added liquid was also included. All enzyme samples were subjected to HPP as described above. After HPP, LDH was diluted to 0.28 microg/ml in 100 mM phosphate buffer (pH 7.4). LDH activity was assessed by measuring the change in concentration of beta-NADH as a function of time. Dynamic light scattering analysis (DLS) was performed to examine the size distribution, polydispersity, and hydrodynamic radius of LDH before and after HPP. No significant difference in CFU/g was observed between lyophilized cells not subjected to HPP and lyophilized cells subjected to 600 MPa for 300 s (P<0.05). However, lyophilized cells that were suspended in 100% to 60% peptone water showed a approximately 7.5-log(10) reduction when subjected to HPP. Survival of L. monocytogenes following HPP significantly increased (P<0.05) when the peptone water concentration was decreased below 60% (aw approximately 0.8). DLS results revealed that LDH suspended in buffer underwent aggregation following HPP (600 MPa, 300 s). Inactivation rate constants obtained using a first-order kinetic model indicated that untreated and HP processed lyophilized LDH had similar activities. When LDH was subject to HPP in solutions containing glycerol, enzyme activity decreased as the water content increased (r2=0.95). Lyophilization completely protected L. monocytogenes and LDH from inactivation by high pressure. Furthermore, enzyme activity and cell survival increased as water activity was decreased. We postulate low aw results in protein stabilization, which prevents protein denaturation and cell death during HPP.

Heat shock induces barotolerance in Listeria monocytogenes.

The aim of this study was to investigate the effect of heat shock on the resistance of Listeria monocytogenes to high pressure processing (HPP). L. monocytogenes ATCC 19115 was grown to stationary phase at 15 degrees C and inoculated into whole ultrahigh-temperature milk at approximately 10(7) CFU/ml. Milk samples (5 ml) were placed into plastic transfer pipettes, which were heat sealed and then heated in a water bath at 48 degrees C for 10 min. Immediately after heat shock, the milk was cooled in water (20 degrees C) for 25 min and then placed on ice. The samples were high pressure processed at ambient temperature (approximately 23 degrees C) at 400 MPa for various times up to 150 s. Following HPP, the samples were spread plated on tryptic soy agar supplemented with yeast extract. Heat shock significantly increased the D400 MPa-value of L. monocytogenes from 35 s in non-heat-shocked cells to 127 s in heat-shocked cells (P < 0.05). Addition of chloramphenicol before heat shock eliminated the protective effect of heat shock (P < 0.05). Heat shock for 5, 10, 15, or 30 min at 48 degrees C resulted in maximal barotolerance (P < 0.05); increasing the time to 60 min significantly decreased survival compared with that at 5, 10, 15, or 30 min (P < 0.05). These results indicate that prior heat shock significantly increases the barotolerance of L. monocytogenes and that de novo protein synthesis during heat shock is required for this enhanced barotolerance.

Prophages in Listeria monocytogenes contain single-nucleotide polymorphisms that differentiate outbreak clones within epidemic clones.

A fragment end ligation-mediated PCR strategy was used to analyze the AscI pulsed-field gel electrophoresis patterns of Listeria monocytogenes epidemic clone II (ECII), which led to the identification of single-nucleotide polymorphisms (SNPs) in prophage regions that differentiated the two ECII outbreak clones. SNPs in prophages that differentiated the outbreak clones of ECIII and -IV were also identified.

Analysis of additional virulence genes and virulence gene regions in Listeria monocytogenes confirms the epidemiologic relevance of multi-virulence-locus sequence typing.

Previous molecular subtyping studies have defined four epidemic clones (ECs) of Listeria monocytogenes (ECI, ECII, ECIII, and ECIV). Partial sequences of eight virulence genes were previously shown to be identical within individual ECs of L. monocytogenes. The present study was conducted to determine if the sequences of other virulence genes and virulence gene regions are also conserved within these ECs. Six additional virulence genes--bsh, hly, inlJ, IplA1, pgdA, and srtA--and three additional virulence gene regions of actA, inlA, and inlB were selected based on their role in L. monocytogenes virulence, and intragenic regions of each gene were sequenced. Sequencing was performed on a diverse set of 44 to 48 L. monocytogenes strains. Results demonstrated that the sequenced regions of the nine virulence genes were identical within each of the ECs, and 257 new single nucleotide polymorphism (SNPs) were identified. ECIII (lineage II) was easily distinguishable from the other ECs, as 238 SNPs were observed in ECIII due to its significant evolutionary divergence from lineage I. With regard to the other ECs, there were 5 SNPs that represented an informative set, since these SNPs were able to differentiate specific ECs from all other unrelated strains used in this study. This study confirms our previous finding that virulence gene sequences are highly conserved within individual ECs and contain stable SNPs that can be used to very accurately differentiate ECs of L. monocytogenes from each other and from other diverse strains.

Effect of high-pressure processing on activity and structure of alkaline phosphatase and lactate dehydrogenase in buffer and milk.

Changes in the activity and structure of alkaline phosphatase (ALP) and L-lactate dehydrogenase (LDH) were investigated after high pressure processing (HPP). HPP treatments (206-620 MPa for 6 and 12 min) were applied to ALP and LDH prepared in buffer, fat-free milk, and 2% fat milk. Enzyme activities were measured using enzymatic assays, and changes in structure were investigated using far-ultraviolet circular dichroism (CD) spectroscopy and dynamic light scattetering (DLS). Kinetic data indicated that the activity of ALP was not affected after 6 min of pressure treatments (206-620 MPa), regardless of the medium in which the enzyme was prepared. Increasing the processing time to 12 min did significantly reduce the activity of ALP at 620 MPa (P < 0.001). However, even the lowest HPP treatment of 206 MPa induced a reduction in LDH activity, and the course of reduction increased with HPP treatment until complete inactivation at 482, 515, and 620 MPa. CD data demonstrated a partial change in the secondary structure of ALP at 620 MPa, whereas the structure of LDH showed gradual denaturation after exposure at 206 MPa for 6 min, leading to a random coil structure at both 515 and 620 MPa. DLS results indicated aggregation of ALP only at HPP treatment of 206 MPa and not above and enzyme precipitation as well as aggregation at 345, 415, 482, and 515 MPa. The loss of LDH activity with increasing pressure and time treatment was due to the combined effects of denaturation and aggregation.