Use of Doehlert Matrix as a Tool for High-Throughput Screening of Organic Acids and Essential Oils on Miniaturized Pork Loins, Followed by Lab-Scale Validation That Confirmed Tested Compounds Do Not Show Synergistic Effects against Salmonella Typhimurium.

The many possible treatments and continuously changing consumer trends present a challenge when selecting antimicrobial interventions during pork processing. Thirty-five potential antimicrobials were screened at commercial working concentrations by individually adding them to miniaturized (69 cm3) disks of pork loin ends, followed by inoculation with Salmonella Typhimurium ATCC 19585. Two organic acids and nine essential oils significantly inhibited Salmonella counts on pork (p < 0.05). However, six compounds that represent different levels of significance (p < 0.05-p < 0.0001) were selected as independent variables to build a Response Surface Methodology model based on a Doehlert matrix (Doehlert Matrix-RSM): lactic acid 1.25%, formic acid 0.25%, cumin 0.25%, clove 0.25%, peppermint 0.5%, and spearmint 0.5%. The goal of the Doehlert Matrix-RSM was to study single and paired effects of these antimicrobials on the change in Salmonella over 24 h. The Doehlert Matrix-RSM model predicted that lactic acid, formic acid, cumin, peppermint, and spearmint significantly reduced Salmonella when added alone, while no significant interactions between these antimicrobials were found. A laboratory-scale validation was carried out on pork loin end slices, which confirmed the results predicted by the model. While this screening did not identify novel synergistic combinations, our approach to screening a variety of chemical compounds by implementing a miniaturized pork loin disk model allowed us to identify the most promising antimicrobial candidates to then formally design experiments to study potential interactions with other antimicrobials.

Enabling Cost-Effective Screening for Antimicrobials against Listeria monocytogenes in Ham.

ABSTRACT: Ready-to-eat meat products, such as deli ham, can support the growth of Listeria monocytogenes (LM), which can cause severe illness in immunocompromised individuals. The objectives of this study were to validate a miniature ham model (MHM) against the ham slice method and to screen antimicrobial combinations to control LM on ham by using response surface methology (RSM) as a time- and cost-effective high-throughput screening tool. The effect of nisin (Ni), potassium lactate and sodium diacetate, lauric arginate (LAG), lytic bacteriophage (P100), and ε-polylysine (EPL) added alone, or in combination, were determined on the MHM over 12 days of storage. Results showed the MHM accurately mimics the ham slice method because no statistical differences were found (P = 0.526) in the change of LM cell counts in MHM and slice counts after 12 days of storage at 4°C for treated and untreated hams. The MHM was then used to screen antimicrobial combinations by using an on-face design and three center points in a central composite design. The RSM was tested by using a cocktail of five LM strains isolated from foodborne disease outbreaks. Three levels of the previously mentioned antimicrobials were used in combination for a total of 28 runs performed in triplicate. The change of LM cell counts were determined after 12 days of storage at 4°C. All tested antimicrobials were effective on reducing LM cell counts on ham when added alone. A significant antagonistic interaction (P = 0.002) was identified by the RSM between LAG and P100, where this antimicrobial combination caused a 2.2-log CFU/g change of LM cell counts after 12 days of storage. Two interactions, between Ni and EPL (P = 0.058), and Ni and P100 (P = 0.068), showed possible synergistic effects against LM on the MHM. Other interactions were clearly nonsignificant, suggesting additive effects. In future work, the developed MHM in combination with RSM can be used as a high-throughput method to analyze novel antimicrobial treatments against LM.

Non-Destructive Luminescence-Based Screening Tool for Listeria monocytogenes Growth on Ham.

Listeria monocytogenes is a food-borne pathogen often associated with ready-to-eat (RTE) food products. Many antimicrobial compounds have been evaluated in RTE meats. However, the search for optimum antimicrobial treatments is ongoing. The present study developed a rapid, non-destructive preliminary screening tool for large-scale evaluation of antimicrobials utilizing a bioluminescent L. monocytogenes with a model meat system. Miniature hams were produced, surface treated with antimicrobials nisin (at 0-100 ppm) and potassium lactate sodium diacetate (at 0-3.5%) and inoculated with bioluminescent L. monocytogenes. A strong correlation (r = 0.91) was found between log scale relative light units (log RLU, ranging from 0.00 to 3.35) read directly from the ham surface and endpoint enumeration on selective agar (log colony forming units (CFU)/g, ranging from 4.7 to 8.3) when the hams were inoculated with 6 log CFU/g, treated with antimicrobials, and L. monocytogenes were allowed to grow over a 12 d refrigerated shelf life at 4 °C. Then, a threshold of 1 log RLU emitted from a ham surface was determined to separate antimicrobial treatments that allowed more than 2 log CFU/g growth of L. monocytogenes (from 6 log CFU/g inoculation to 8 log CFU/g after 12 d). The proposed threshold was utilized in a luminescent screening of antimicrobials with days-to-detect growth monitoring of luminescent L. monocytogenes. Significantly different (p < 0.05) plate counts were found in antimicrobial treated hams that had reached a 1 log RLU increase (8.1-8.5 log(CFU/g)) and the hams that did not reach the proposed light threshold (5.3-7.5 log(CFU/g)). This confirms the potential use of the proposed light threshold as a qualitative tool to screen antimicrobials with less than or greater than a 2 log CFU/g increase. This screening tool can be used to prioritize novel antimicrobials targeting L. monocytogenes, alone or in combination, for future validation.