Stereoselective Construction of ß-, γ-, and δ-Lactam Rings via Enzymatic C-H Amidation.

Lactam rings are found in many biologically active natural products and pharmaceuticals, including important classes of antibiotics. Methods for the asymmetric synthesis of these molecules are therefore highly desirable, particularly through the selective functionalization of unreactive aliphatic C-H bonds. Here we show the development of a strategy for the asymmetric synthesis of ß-, γ-, and δ-lactams via hemoprotein-catalysed intramolecular C-H amidation reaction with readily available dioxazolone reagents. Engineered myoglobin variants serve as excellent biocatalysts for this transformation yielding the desired lactam products in high yields, high enantioselectivity, and on preparative scale. Mechanistic and computational studies elucidate the nature of the C-H amination and enantiodetermining steps and provide insights into protein-mediated control of regioselectivity and stereoselectivity. Additionally, an alkaloid natural product and a drug molecule were synthesized chemoenzymatically in much fewer steps (7-8 vs. 11-12) than previously reported, further demonstrating the power of biosynthetic strategy for the preparation of complex bioactive molecules.

Biocatalytic strategy for the construction of sp3-rich polycyclic compounds from directed evolution and computational modelling.

Catalysis with engineered enzymes has provided more efficient routes for the production of active pharmaceutical agents. However, the potential of biocatalysis to assist in early-stage drug discovery campaigns remains largely untapped. In this study, we have developed a biocatalytic strategy for the construction of sp3-rich polycyclic compounds via the intramolecular cyclopropanation of benzothiophenes and related heterocycles. Two carbene transferases with complementary regioisomer selectivity were evolved to catalyse the stereoselective cyclization of benzothiophene substrates bearing diazo ester groups at the C2 or C3 position of the heterocycle. The detailed mechanisms of these reactions were elucidated by a combination of crystallographic and computational analyses. Leveraging these insights, the substrate scope of one of the biocatalysts could be expanded to include previously unreactive substrates, highlighting the value of integrating evolutionary and rational strategies to develop enzymes for new-to-nature transformations. The molecular scaffolds accessed here feature a combination of three-dimensional and stereochemical complexity with 'rule-of-three' properties, which should make them highly valuable for fragment-based drug discovery campaigns.

Bio-Mapping Salmonella and Campylobacter Loads in Three Commercial Broiler Processing Facilities in the United States to Identify Strategic Intervention Points.

The poultry industry in the United States is one of the largest in the world. Poultry consumption has significantly increase since the COVID-19 pandemic and is predicted to increase over 16% between 2021 and 2030. Two of the most significant causes of hospitalizations and death in the United States are highly related to poultry consumption. The FSIS regulates poultry processing, enforcing microbial performance standards based on Salmonella and Campylobacter prevalence in poultry processing establishments. This prevalence approach by itself is not a good indicator of food safety. More studies have shown that it is important to evaluate quantification along with prevalence, but there is not much information about poultry mapping using quantification and prevalence. In this study, enumeration and prevalence of Salmonella and Campylobacter were evaluated throughout the process at three different plants in the United States. Important locations were selected in this study to evaluate the effect of differences interventions. Even though there were high differences between the prevalences in the processes, some of the counts were not significantly different, and they were effective in maintaining pathogens at safe levels. Some of the results showed that the intervention and/or process were not well controlled, and they were not effective in controlling pathogens. This study shows that every plant environment is different, and every plant should be encouraged to implement a bio-mapping study. Quantification of pathogens leads to appropriate risk assessment, where physical and chemical interventions can be aimed at specific processing points with higher pathogen concentrations using different concentrations of overall process improvement.

Bio-Mapping of Microbial Indicators and Pathogen Quantitative Loads in Commercial Broiler Processing Facilities in South America.

A bio-mapping study was conducted with the aim of creating a microbiological baseline on indicator organisms and pathogens in commercial broiler processing facilities located in a country in South America. Whole chicken carcass and wing rinses were collected from five stages of the poultry processing line: live receiving (LR), rehanger (R), post-evisceration (PE), post-chilling (PC), and wings (W). Rinses (n = 150) were enumerated using the MicroSnap™ system for total viable counts (TVC) and Enterobacteriaceae (EB), while the BAX®-System-SalQuant® and BAX®-System-CampyQuant™ were used for Salmonella and Campylobacter, respectively. TVC and EB were significantly different between stages at the processing line (p < 0.01). There was a significant reduction from LR to PC for both microbial indicators. TVC and EB counts increased significantly from PC to W. Salmonella counts at PC were significantly different from the other stages at the processing line (p = 0.03). Campylobacter counts were significantly higher than the other stages at PC (p < 0.01). The development of bio-mapping baselines with microbial indicators showed consistent reduction up to the post-chilling stage, followed by an increase at the wings sampling location. The quantification of pathogens demonstrates that prevalence analysis as a sole measurement of food safety is not sufficient to evaluate the performance of processing operations and sanitary dressing procedures in commercial processing facilities.

Mitigation of Salmonella in Ground Pork Products through Gland Removal in Pork Trimmings.

Bio-mapping studies conducted in pork harvest and fabrication facilities have indicated that Salmonella is prevalent and mitigations are needed to reduce the pathogen in trim and ground products. Salmonella can be isolated from the lymph nodes and can cause contamination in comminuted pork products. The objective of this study was to determine if physically removing topical and internal lymph nodes in pork products prior to grinding would result in the mitigation of Salmonella and a reduction in indicators in the final ground/comminuted products. In total, three treatment groups were assigned in a commercial pork processing facility as follows: (1) untreated control, (2) topical (surface) glands removed before grinding, and (3) topical, jowl, and internal lymph nodes and glands removed before grinding. Indicator microorganisms were determined using the BioMérieux TEMPO® system and the quantification of Salmonella was performed using the BAX® System Real-Time Salmonella SalQuant® methodology. The removal of lymph nodes located on the topical and internal surfaces and in the jowl significantly (p < 0.05) reduced the presence of Salmonella and also reduced the presence of indicator organisms according to this study. Briefly, 2.5-Log CFU/sample of Salmonella was initially observed in the trim samples, and the ground samples contained 3.8-Log CFU/sample of Salmonella. The total numbers were reduced to less than 1-Log CFU/sample in both trim and ground products. This study indicates a need for lymph node mitigation strategies beginning prior to harvest, in order to prevent contamination in further-processed pork products.

Cellular Validation of a Chemically Improved Inhibitor Identifies Monoubiquitination on OTUB2.

Ubiquitin thioesterase OTUB2, a cysteine protease from the ovarian tumor (OTU) deubiquitinase superfamily, is often overexpressed during tumor progression and metastasis. Development of OTUB2 inhibitors is therefore believed to be therapeutically important, yet potent and selective small-molecule inhibitors targeting OTUB2 are scarce. Here, we describe the development of an improved OTUB2 inhibitor, LN5P45, comprising a chloroacethydrazide moiety that covalently reacts to the active-site cysteine residue. LN5P45 shows outstanding target engagement and proteome-wide selectivity in living cells. Importantly, LN5P45 as well as other OTUB2 inhibitors strongly induce monoubiquitination of OTUB2 on lysine 31. We present a route to future OTUB2-related therapeutics and have shown that the OTUB2 inhibitor developed in this study can help to uncover new aspects of the related biology and open new questions regarding the understanding of OTUB2 regulation at the post-translational modification level.

Rapid Quantitative Method Development for Beef and Pork Lymph Nodes Using BAX® System Real Time Salmonella Assay.

The goal of this study was to develop a rapid RT-PCR enumeration method for Salmonella in pork and beef lymph nodes (LNs) utilizing BAX®-System-SalQuant® as well as to assess the performance of the methodology in comparison with existing ones. For study one: PCR curve development, pork, and beef LNs (n = 64) were trimmed, sterilized, pulverized, spiked with 0.00 to 5.00 Log CFU/LN using Salmonella Typhimurium, and then homogenized with BAX-MP media. Samples were incubated at 42 °C and tested at several time points using the BAX®-System-RT-PCR Assay for Salmonella. Cycle-Threshold values from the BAX®-System, for each Salmonella concentration were recorded and utilized for statistical analysis. For study two: Method comparison; additional pork and beef LNs (n = 52) were spiked and enumerated by (1) 3M™EB-Petrifilm™ + XLD-replica plate, (2) BAX®-System-SalQuant®, and (3) MPN. Linear-fit equations for LNs were estimated with recovery times of 6 h and a limit of quantification (LOQ) of 10 CFU/LN. Slopes and intercepts for LNs using BAX®-System-SalQuant® when compared with MPN were not significantly different (p < 0.05), while the same parameters for 3M™EB-Petrifilm™ + XLD-replica plate were significantly different (p > 0.05). The results support the capability of BAX®-System-SalQuant® to enumerate Salmonella in pork and beef LNs. This development adds support to the use of PCR-based quantification methodologies for pathogen loads in meat products.

Efficacy of Common Antimicrobial Interventions at and above Regulatory Allowable Pick-Up Levels on Pathogen Reduction.

The objective of this study was to evaluate the food safety efficacy of common antimicrobial interventions at and above required uptake levels for processing aids on the reduction of Shiga-toxin producing E. coli (STEC) and Salmonella spp. through spray and dip applications. Beef trim was inoculated with specific isolates of STEC or Salmonella strains. Trim was intervened with peracetic or lactic acid through spray or dip application. Meat rinses were serially diluted and plated following the drop dilution method; an enumerable range of 2-30 colonies was used to report results before log transformation. The combination of all treatments exhibits an average reduction rate of 0.16 LogCFU/g for STEC and Salmonella spp., suggesting that for every 1% increase in uptake there is an increase of 0.16 LogCFU/g of reduction rate. There is a statistical significance in the reduction rate of Shiga-toxin producing Escherichia coli in relation to the uptake percentage (p < 0.01). The addition of explanatory variables increases the R2 of the regression for STEC, where all the additional explanatory variables are statistically significant for reduction (p < 0.01). The addition of explanatory variables increases the R2 of the regression for Salmonella spp., but only trim type is statistically significant for reduction rate (p < 0.01). An increase in uptake percentages showed a significant increase in reduction rate of pathogens on beef trimmings.

Data-Mining Poultry Processing Bio-Mapping Counts of Pathogens and Indicator Organisms for Food Safety Management Decision Making.

Bio-mapping studies play an important role, as the data collected can be managed and analyzed in multiple ways to look at process trends, find explanations about the effect of process changes, activate a root cause analysis for events, and even compile performance data to demonstrate to inspection authorities or auditors the effect of certain decisions made on a daily basis and their effects over time in commercial settings not only from the food safety perspective but also from the production side. This study presents an alternative analysis of bio-mapping data collected throughout several months in a commercial poultry processing operation as described in the article "Bio-Mapping Indicators and Pathogen Loads in a Commercial Broiler Processing Facility Operating with High and Low Antimicrobial Interventions". The conducted analysis identifies the processing shift effect on microbial loads, attempts to find correlation between microbial indicators data and pathogens loads, and identifies novel visualization approaches and conducts distribution analysis for microbial indicators and pathogens in a commercial poultry processing facility. From the data analyzed, a greater number of locations were statistically different between shifts under reduced levels of chemical interventions with higher means at the second shift for both indicators and pathogens levels. Minimal to negligible correlation was found when comparing aerobic counts and Enterobacteriaceae counts with Salmonella levels, with significant variability between sampling locations. Distribution analysis and visualization as a bio-map of the process resulted in a clear bimodality in reduced chemical conditions for multiple locations mostly explained by shift effect. The development and use of bio-mapping data, including proper data visualization, improves the tools needed for ongoing decision making in food safety systems.

Stereoselective Construction of ß-, γ-, and δ-Lactam Rings via Enzymatic C-H Amidation.

Lactam rings are found in many biologically active natural products and pharmaceuticals, including important classes of antibiotics. Given their widespread presence in bioactive molecules, methods for the asymmetric synthesis of these molecules, in particular through the selective functionalization of ubiquitous yet unreactive aliphatic C-H bonds, are highly desirable. In this study, we report the development of a novel strategy for the asymmetric synthesis of 4-, 5-, and 6-membered lactams via an unprecedented hemoprotein-catalyzed intramolecular C-H amidation reaction with readily available dioxazolone reagents. Engineered myoglobin variants serve as excellent biocatalysts for this transformation producing an array of ß-, γ-, and δ-lactam molecules in high yields, with high enantioselectivity, and on preparative scale. Mechanistic and computational studies elucidate the nature of the C-H amination and enantiodetermining steps in these reactions and provide insights into protein-mediated control of regioselectivity and stereoselectivity. Using this system, it was possible to accomplish the chemoenzymatic total synthesis of an alkaloid natural product and a drug molecule in much fewer steps (7-8 vs. 11-12) than previously possible, which showcases the power of this biosynthetic strategy toward enabling the preparation of complex bioactive molecules.

Dehaloperoxidase Catalyzed Stereoselective Synthesis of Cyclopropanol Esters.

Chiral cyclopropanols are highly desirable building blocks for medicinal chemistry, but the stereoselective synthesis of these molecules remains challenging. Here, a novel strategy is reported for the diastereo- and enantioselective synthesis of cyclopropanol derivatives via the biocatalytic asymmetric cyclopropanation of vinyl esters with ethyl diazoacetate (EDA). A dehaloperoxidase enzyme from Amphitrite ornata was repurposed to catalyze this challenging cyclopropanation reaction, and its activity and stereoselectivity were optimized via protein engineering. Using this system, a broad range of electron-deficient vinyl esters were efficiently converted to the desired cyclopropanation products with up to 99.5:0.5 diastereomeric and enantiomeric ratios. In addition, the engineered dehaloperoxidase-based biocatalyst is able to catalyze a variety of other abiological carbene transfer reactions, including N-H/S-H carbene insertion with EDA as well as cyclopropanation with diazoacetonitrile, thus adding to the multifunctionality of this enzyme and defining it as a valuable new scaffold for the development of novel carbene transferases.

Shelf-Life Evaluation of Pork Loins as Influenced by the Application of Different Antimicrobial Interventions.

The objective of the study was to determine the impact of antimicrobial interventions and refrigerated dark storage on the shelf-life of pork chops. Boneless pork loins (n = 36) were split and stored for 1, 14, 28, and 42 days at 2-4 °C after being treated with the following antimicrobials: water (WAT), Bovibrom 225 ppm (BB225), Bovibrom 500 ppm (BB500), Fit Fresh 3 ppm (FF3), or washing solution 750 ppm (WS750). After the end of dark storage, pork loins were further processed and sliced into chops, overwrapped in trays, and displayed for up to an additional 96 h in a retail case. Instrumental and visual color measurements as well as mesophilic and psychrotrophic aerobic bacteria, and lactic acid bacteria were measured. BB500 and FF3 performed better in inhibiting the growth of indicator bacteria under 6 logs; however, FF3 presented the best stability for color during storage. Principal component analysis clustered initial dark storage days with a* and chroma while % discoloration, hue, b* and microorganisms where clustered with longer dark storage times. In general, treatment FF3 presented the best performance, both in inhibiting microbial growth and maintaining the stability of color, thus increasing the shelf-life of pork loins.

Quantitative Bio-Mapping of Salmonella and Indicator Organisms at Different Stages in a Commercial Pork Processing Facility.

The purpose of this study was to develop a quantitative baseline of indicator organisms and Salmonella by bio-mapping throughout the processing chain from harvest to final product stages within a commercial conventional design pork processing establishment. Swab samples were taken on the harvest floor at different processing steps, gambrel table, after polisher, before final rinse, after the final rinse, post snap chill, and after peroxyacetic acid (PAA) application, while 2-pound product samples were collected for trim and ground samples. The samples were subjected to analysis for indicator microorganism enumeration, Aerobic Count (AC), Enterobacteriaceae (EB), and generic Escherichia coli (EC), with the BioMérieux TEMPO®. Salmonella prevalence and enumeration was evaluated using the BAX® System Real-Time Salmonella and the SalQuant™ methodology. Microbial counts were converted to Log Colony-forming units (CFU) on a per mL, per g or per sample basis, presented as LogCFU/mL, LogCFU/g and LogCFU/sample, prior to statistical analysis. All indicator microorganisms were significantly reduced at the harvest floor (p-value < 0.001), from gambrel table to after PAA cabinet location. The reduction at harvest was 2.27, 2.46 and 2.24 LogCFU/mL for AC, EB and EC, respectively. Trim sample values fluctuated based on cut, with the highest average AC count found at neck trim (2.83 LogCFU/g). Further process samples showed the highest AC count in sausage with a mean of 5.28 LogCFU/g. EB counts in sausage (3.19 LogCFU/g) showed an evident increase, compared to the reduction observed at the end of harvest and throughout trim processing. EC counts showed a similar trend to EB counts with the highest value found in sausage links (1.60 LogCFU/g). Statistical microbial process control (SPC) parameters were also developed for each of the indicator microorganisms, using the overall mean count (X=), the Lower control limit (LCL) and Upper control limit (UCL) at each sampling location. For Salmonella prevalence, a total of 125/650 samples were found positive (19%). From those positive samples, 47 samples (38%) were suitable for enumeration using the BAX® System SalQuant™, the majority detected at the gambrel table location. From those enumerable samples, 60% were estimated to be between 0.97 and 1.97 LogCFU/sample, while the rest (40%) were higher within the 2.00−4.02 LogCFU/sample range. This study provides evidence for the application of indicator and pathogen quantification methodologies for food safety management in commercial pork processing operations.

Tuning Enzyme Thermostability via Computationally Guided Covalent Stapling and Structural Basis of Enhanced Stabilization.

Enhancing the thermostability of enzymes without impacting their catalytic function represents an important yet challenging goal in protein engineering and biocatalysis. We recently introduced a novel method for enzyme thermostabilization that relies on the computationally guided installation of genetically encoded thioether "staples" into a protein via cysteine alkylation with the noncanonical amino acid O-2-bromoethyl tyrosine (O2beY). Here, we demonstrate the functionality of an expanded set of electrophilic amino acids featuring chloroacetamido, acrylamido, and vinylsulfonamido side-chain groups for protein stapling using this strategy. Using a myoglobin-based cyclopropanase as a model enzyme, our studies show that covalent stapling with p-chloroacetamido-phenylalanine (pCaaF) provides higher stapling efficiency and enhanced stability (thermodynamic and kinetic) compared to the other stapled variants and the parent protein. Interestingly, molecular simulations of conformational flexibility of the cross-links show that the pCaaF staple allows fewer energetically feasible conformers than the other staples, and this property may be a broader indicator of stability enhancement. Using this strategy, pCaaF-stapled variants with significantly enhanced stability against thermal denaturation (ΔTm' = +27 °C) and temperature-induced heme loss (ΔT50 = +30 °C) were obtained while maintaining high levels of catalytic activity and stereoselectivity. Crystallographic analyses of singly and doubly stapled variants provide key insights into the structural basis for stabilization, which includes both direct interactions of the staples with protein residues and indirect interactions through adjacent residues involved in heme binding. This work expands the toolbox of protein stapling strategies available for protein stabilization.

Bio-Mapping of Microbial Indicators to Establish Statistical Process Control Parameters in a Commercial Beef Processing Facility.

The objective was to conduct a bio-mapping of microbial indicators to determine statistical process control (SPC) parameters at a beef processing plant to establish microbiological baselines and process control parameters to support food safety management decisions. EZ-ReachTM swabs were used to collect 100 cm2 area samples at seven different locations throughout the beef processing line at four different regions on the carcass. Each of the eight sampling days evaluated included three samples collected per sampling location/carcass region for a total of 84 samples per day. Enumeration of total aerobic bacteria, Enterobacteriaceae, and Escherichia coli was performed on each sample. Microbial SPC parameters were estimated for each sampling point. Statistical differences between sampling points for all carcass locations (p < 0.001) followed an overall trend with higher values at pre- and post-evisceration with a continuous decrease until final interventions with a slight increase in counts during the chilling process and a final increase after fabrication. Variability at sampling points is the result of the nature of the process and highlights open opportunities for improvement of the food safety system. Microbial baselines and SPC parameters will help support decision making for continuous process improvement, validation of intervention schemes, and corrective action implementation for food safety management.

Bio-Mapping Indicators and Pathogen Loads in a Commercial Broiler Processing Facility Operating with High and Low Antimicrobial Intervention Levels.

The poultry industry in the United States has traditionally implemented non-chemical and chemical interventions against Salmonella spp. and Campylobacter spp. on the basis of experience and word-of-mouth information shared among poultry processors. The effects of individual interventions have been assessed with microbiological testing methods for Salmonella spp. and Campylobacter spp. prevalence as well as quantification of indicator organisms, such as aerobic plate counts (APC), to demonstrate efficacy. The current study evaluated the loads of both indicators and pathogens in a commercial chicken processing facility, comparing the "normal chemical", with all chemical interventions turned-on, at typical chemical concentrations set by the processing plant versus low-chemical process ("reduced chemical"), where all interventions were turned off or reduced to the minimum concentrations considered in the facility's HACCP system. Enumeration and prevalence of Salmonella spp. and Campylobacter spp. as well as indicator organisms (APC and Enterobacteriaceae-EB) enumeration were evaluated to compare both treatments throughout a 25-month sampling period. Ten locations were selected in the current bio-mapping study, including live receiving, rehanger, post eviscerator, post cropper, post neck breaker, post IOBW #1, post IOBW #2, prechilling, post chilling, and parts (wings). Statistical process control parameters for each location and processing schemes were developed for each pathogen and indicator evaluated. Despite demonstrating significant statistical differences between the normal and naked processes in Salmonella spp. counts ("normal" significantly lower counts than the "reduced" at each location except for post-eviscerator and post-cropper locations), the prevalence of Salmonella spp. after chilling is comparable on both treatments (~10%), whereas for Campylobacter spp. counts, only at the parts' location was there significant statistical difference between the "normal chemical" and the "reduced chemical". Therefore, not all chemical intervention locations show an overall impact on Salmonella spp. or Campylobacter spp., and certain interventions can be turned off to achieve the same or better microbial performance if strategic intervention locations are enhanced.

In-Plant Intervention Validation of a Novel Ozone Generation Technology (Bio-Safe) Compared to Lactic Acid in Variety Meats.

The objective of this experiment was to compare the antimicrobial efficacy of an aqueous ozone intervention and a lactic acid solution on natural microbiota of variety meats in a commercial beef processing plant. EZ-Reach™ swabs were used to collect 100 cm2 area samples before and after ozone and lactic acid intervention application for three different offals (head, heart, and liver). Each repetition included 54 samples per variety meat and antimicrobial for a total of 162 samples per repetition. Enumeration of total aerobic bacteria (APC) and Escherichia coli (EC) was performed on each sample. Microbial counts for both microorganisms evaluated were significantly reduced (p < 0.001) after lactic acid immersion (2-5%) and ozone intervention for all variety meats, with the exception of ozone intervention in EC counts of the heart samples. APC after lactic acid intervention was reduced on average by 1.73, 1.66, and 1.50 Log CFU/sample in the head, heart, and liver, respectively, while after ozone intervention, counts were reduced on average by 1.66, 0.52, and 1.20 Log CFU/sample. EC counts after lactic acid intervention were reduced on average by 0.96, 0.79, and 1.00 Log CFU/sample in the head, heart, and liver, respectively, while after ozone intervention, counts were reduced on average by 0.75, 0.62, and 1.25 Log CFU/sample. The aqueous ozone antimicrobial scheme proved to be a promising intervention for the in-plant reduction of indicator levels in variety meats, specifically heads, hearts, and livers.

In-Plant Validation of Novel On-Site Ozone Generation Technology (Bio-Safe) Compared to Lactic Acid Beef Carcasses and Trim Using Natural Microbiota and Salmonella and E. coli O157:H7 Surrogate Enumeration.

The purpose of this study was to evaluate the antimicrobial efficacy of an aqueous ozone (Bio-Safe) treatment and lactic acid solutions on natural microbiota and E. coli O157:H7 and Salmonella surrogates on beef carcasses and trim in a commercial beef processing plant. For every repetition, 40 carcass and 40 trim swabs (500 cm2) were collected. Samples were taken using EZ-ReachTM swabs, and plated into aerobic plate count (APC), coliform, and E. coli PetrifilmTM for enumeration. In addition, a five-strain cocktail (MP-26) of E. coli surrogates was inoculated onto trim. For every trim surrogate repetition, 30 trim pieces were sampled after attachment and after ozone intervention. Samples were diluted and counts were determined using the TEMPO® system for E. coli enumeration. Ozone and lactic acid interventions significantly reduced (p < 0.003) bacterial counts in carcasses and trim samples. Moreover, lactic acid further reduced APC and coliforms in trim samples compared to ozone intervention (p < 0.009). In the surrogate trials, ozone significantly reduced (p < 0.001) surrogate concentration. Historical data from the plant revealed a reduction (p < 0.001) of presumptive E. coli O157:H7 in trim after a full year of ozone intervention implementation. The novel technology for ozone generation and application as an antimicrobial can become an alternative option that may also act synergistically with existing interventions, minimizing the risk of pathogens such as Salmonella and E. coli O157:H7.

Microbial Growth Study on Pork Loins as Influenced by the Application of Different Antimicrobials.

The use of antimicrobials in the pork industry is critical in order to ensure food safety and, at the same time, extend shelf life. The objective of the study was to determine the impact of antimicrobials on indicator bacteria on pork loins under long, dark, refrigerated storage conditions. Fresh boneless pork loins (n = 36) were split in five sections and treated with antimicrobials: Water (WAT), Bovibrom 225 ppm (BB225), Bovibrom 500 ppm (BB500), Fit Fresh 3 ppm (FF3), or Washing Solution 750 ppm (WS750). Sections were stored for 1, 14, 28, and 42 days at 2-4 °C. Mesophilic and psychrotrophic aerobic bacteria (APC-M, APC-P), lactic acid bacteria (LAB-M), coliforms, and Escherichia coli were enumerated before intervention, after intervention, and at each storage time. All bacterial enumeration data were converted into log10 for statistical analysis, and the Kruskal-Wallis test was used to find statistical differences (p < 0.05). Initial counts did not differ between treatments, while, after treatment interventions, treatment WS750 did not effectively reduce counts for APC-M, APC-P, and coliforms (p < 0.01). BB500, FF3, and WS750 performed better at inhibiting the growth of indicator bacteria when compared with water until 14 days of dark storage.

Organic solvent stability and long-term storage of myoglobin-based carbene transfer biocatalysts.

Recent years have witnessed a rapid increase in the application of enzymes for chemical synthesis and manufacturing, including the industrial-scale synthesis of pharmaceuticals using multienzyme processes. From an operational standpoint, these bioprocesses often require robust biocatalysts capable of tolerating high concentrations of organic solvents and possessing long shelflife stability. In this work, we investigated the activity and stability of myoglobin (Mb)-based carbene transfer biocatalysts in the presence of organic solvents and after lyophilization. Our studies demonstrate that Mb-based cyclopropanases possess remarkable organic solvent stability, maintaining high levels of activity and stereoselectivity in the presence of up to 30%-50% (v/v) concentrations of various organic solvents, including ethanol, methanol, N,N-dimethylformamide, acetonitrile, and dimethyl sulfoxide. Furthermore, they tolerate long-term storage in lyophilized form, both as purified protein and as whole cells, without significant loss in activity and stereoselectivity. These stability properties are shared by Mb-based carbene transferases optimized for other type of asymmetric carbene transfer reactions. Finally, we report on simple protocols for catalyst recycling as whole-cell system and for obviating the need for strictly anaerobic conditions to perform these transformations. These findings demonstrate the robustness of Mb-based carbene transferases under operationally relevant conditions and should help guide the application of these biocatalysts for synthetic applications.