Sensitivity of Planktonic Cells of Staphylococcus Aureus to Elevated Hydrostatic Pressure As Aﬀected by Mild Heat, Carvacrol, Nisin, and Caprylic Acid

International Journal of Environmental Research and Public Health

Article Sensitivity of Planktonic Cells of Staphylococcus aureus to Elevated Hydrostatic Pressure as Aﬀected by Mild Heat, Carvacrol, Nisin, and Caprylic Acid

Jyothi George 1,2, Sadiye Aras 1,2, Md Niamul Kabir 1, Sabrina Wadood 1, Shahid Chowdhury 1 and Aliyar Cyrus Fouladkhah 1,3,* 1 Public Health Microbiology Laboratory, Tennessee State University, Nashville, TN 37209, USA; [email protected] (J.G.); [email protected] (S.A.); [email protected] (M.N.K.); [email protected] (S.W.); [email protected] (S.C.) 2 Department of Biological Sciences, Tennessee State University, Nashville, TN 37209, USA 3 Cooperative Extension Program, Tennessee State University, Nashville, TN 37209, USA * Correspondence: [email protected]; Tel.: +1-970-690-7392

Received: 29 July 2020; Accepted: 23 September 2020; Published: 25 September 2020

Abstract: Current study investigated effects of elevated hydrostatic pressure exposure in the presence of mild heat and natural antimicrobials against Staphylococcus aureus. Hydrostatic pressure of 350 to 550 MPa with nisin (5000 IU/mL), carvacrol, or caprylic acid (0.5% v/v) were applied for the reduction in four-strain mixture of S. aureus in HEPES buffer at 4 and 40 ◦C for up to 7 min. Results were statistically analyzed by ANOVA and D-values were additionally calculated using best-fitted linear model. Prior to exposure to treatments at 4 C, counts of the pathogen were 7.95 0.4 log CFU/mL ◦ ± and were reduced (p < 0.05) to 6.44 0.3 log CFU/mL after 7 min of treatment at 450 MPa. D-value ± associated with this treatment was 5.34 min (R2 = 0.72). At 40 C, counts were 8.21 0.7 and 5.77 ◦ ± ± 0.3 log CFU/mL before and after the 7-min treatments, respectively. D-value associated with 40 ◦C treatment was 3.30 min (R2 = 0.62). Application of the antimicrobials provided additional pathogen reduction augmentation for treatments < 5 min. The results of the current study could be incorporated for meeting regulatory requirements such as Food Code, HACCP, and Preventive Control for Human Food of Food Safety Modernization Act for assuring microbiological safety of products against this prevalent pathogen of public health concern.

Keywords: Staphylococcus aureus; high-pressure processing; carvacrol; nisin; caprylic acid

1. Introduction Infections caused by foodborne pathogens of public health concern are persisting challenges in healthcare settings, for the food industry, and for the consumers of raw and processed commodities. The symptoms of foodborne diseases range from mild and self-limiting manifestations such as nausea, vomiting, and diarrhea to severe complications such as kidney and liver failure, and brain and neural disorders that could result in absence from work, potential life-long health complications, and premature death [1]. At least, 31 known foodborne pathogens are estimated to cause 9.4 million illnesses, 56,961 hospitalizations, and 1351 death episodes in a typical year in the United States [2]. Foodborne diseases are an important cause of morbidity and mortality around the world as well. Around 600 million foodborne disease episodes and 420,000 deaths are associated with foodborne diseases globally every year with 30% of deaths occurring among the children under the age of 5 [1]. These challenges are expected to be augmented in the landscape of climate change as increases in environmental temperature could augment the proliferation and persistence of an array of microbial pathogens in food and water supplies [3–6].

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Among these foodborne pathogens, Staphylococcus aureus is a commensal bacterium and can potentially be isolated from the mucosa or skin of both humans and animals [7]. Some studies indicate that S. aureus could be isolated from >20% of food processing employees [8,9]. As such, food handlers and food products are considered one of the main reservoirs of dissemination of this pathogen in the community [10]. Epidemiological data obtained from the U.S. Centers for Disease Control and Prevention indicate that from 1998 to 2017 there were more than 600 outbreaks (single or multi-state events) in the United States leading to around 10,000 cases of illness episodes associated with S. aureus [11]. Among the various methods of food preservation, the application of high-pressure processing is gaining increasing importance and momentum in food commerce. The technology exposes the final packaged products to elevated levels of pressure, typically from 100 to 1000 MPa (most commonly around 650 MPa) for treatments commonly lasting less than 10 min [12–14]. The National Advisory Committee on Microbiological Criteria for Foods (NACMCF) has defined high-pressure pasteurization as a “pasteurization equivalence,” thus an array of products is currently available in the market relying on this emerging technology for microbiological safety [15]. One of the main challenges associated with this technology is the slightly higher operation costs of pressure-treated products relative to traditional heat-treated commodities [16]. The application of treatment at elevated pressures of 650 MPa (currently the most common intensity in food commerce) and higher intensity levels is typically associated with the increased operation and maintenance cost of pressure vessels. Thus, the application of mild pressure in combination with natural antimicrobials could be of importance for the stakeholders of this technology. The application of mild levels coupled with natural antimicrobials, if proven to be microbiologically efficacious, could have additional co-benefits such as the potential for better retention of nutrients, increased shelf-life due to residual effects of antimicrobials, and better organoleptic properties [12,16]. Various antimicrobials in the United States are regulated by the generally recognized as safe (GRAS) list of the U.S. Food and Drug Administration [17]. Although the efficacy of antimicrobials from GRAS list has been extensively studied in the literature, limited studies are available exploring the efficacy of these antimicrobials for augmenting the performance of emerging and non-thermal processes, particularly their antimicrobial effectiveness under elevated hydrostatic pressure [14]. Nisin is a peptide with antimicrobial properties that is produced by bacterium Lactococcus lactis and is the first bacteriocin approved for commercial utilization in food industry. Various concentrations of this chemical, including 1000 to > 5000 IU, have been studied as an antimicrobial agent against an array of microorganisms, particularly Gram-positive bacteria [18–23]. Caprylic acid (C8H16O2), found primarily in bovine milk, extracted palm and coconut oil, and carvacrol (C10H14O), found primarily in oregano, both belong to the GRAS list as well. These bioactive compounds with antimicrobial properties have been utilized in the food industry to assure the microbiological safety and extend the shelf-life of various products [24–28]. Considering the public health importance of S. aureus and its prevalence in food products and food manufacturing facilities, the current study investigates the effects of elevated hydrostatic pressure for decontamination of planktonic cells of the pathogen. This study further investigates the effects of natural antimicrobials (nisin, caprylic acid, and carvacrol) for augmenting the decontamination efficacy of pressure-based pasteurization.

2. Materials and Methods

2.1. Propagation of Bacterial Cell and Inoculation Mixture This study utilized a four-strain mixture of S. aureus from the strain library of the Public Health Microbiology laboratory in Nashville, TN. These strains were selected based on their epidemiological and food industry signiﬁcance and were originally obtained from American Type Culture Collection (ATCC®, Manassas, VA, USA) with strain designation numbers of ATCC® 51740TM, ATCC® 6538TM, Int. J. Environ. Res. Public Health 2020, 17, 7033 3 of 14

ATCC® 25923TM, and ATCC® 9144TM. The glycerol stock of these strains were kept at 80 C and − ◦ prior to the experiment, 100 µL of each strain was aseptically pipetted into 10 mL of Tryptic Soy Broth (Difco, Becton Dickinson, Franklin Lakes, NJ, USA), supplemented with 0.6% yeast extract (TSB + YE), to activate each strain. The addition of yeast extract was designed to minimize the acid-stress of the pathogen during culturing [12]. The inoculated TSB + YE was then aerobically incubated at 37 ◦C for 22–24 h, and after homogenizing the overnight bacterial suspension by a high-speed vortex (Scientific Industries, Model Vortex-2 Genie, Bohemia, NY, USA), a 100 µL aliquot of the homogenized bacterial suspension, for each strain separately, was transferred to new, sterile, 10-mL TSB + YE. In order to prepare the sub-cultured bacterial suspension, the inoculated TSB + YE was again aerobically incubated at 37 ◦C for 22–24 h. The bacterial cells from the sub-cultured suspensions were then harvested, for each strain individually, as elaborated in our recent studies [12–14,16]. In short, bacterial cells (2 mL for each strain) after homogenizing the overnight suspension by vortexing, were harvested by centrifugation (Eppendorf North America, Hauppauge, NY, USA) for 15 min at 6000 RPM (equivalent to approximately 3540 g, for the centrifuge’s 88 mm rotor [Model 5424, Rotor FA-45-24-11]). The cells, for each strain separately, were then resuspended in 2 mL of HEPES buffer (VWR International Inc., Suwanee, GA, USA). This purification step was repeated twice using the same intensity and time and the pellets were resuspended in HEPES buffer. Individually activated, cultured, sub-cultured, and purified strains were then composited before each experiment to assure equal representation of four strains prior to treatments.

2.2. High-Pressure Processing and Antimicrobial Treatments The current study utilized the application of three antimicrobials with concentrations relevant to the food industry. The concentrations and the pressure intensity levels were selected based on preliminary trials and our recent studies [13,14]. A powder form of nisin (Sigma-Aldrich, St. Louis, MO, USA) was used for preparation of the antimicrobial. This antimicrobial, generally considered as safe by the U.S. Food and Drug Administration for application in food products [29], was used at a concentration of 5000 IU per mL (w/v). The International Unit (IU) is the quantity of nisin required to inhibit a single cell of Streptococcus agalactiae in 1000 µL broth medium [30], and 1000 IU of nisin is equivalent to approximately 0.025 mg nisin [14]. The powered nisin was mixed with 2 mL of HEPES buffer and then centrifuged for one minute at 3000 RPM (equivalent to approximately 886 g) using the above-mentioned instrument and rotor to remove the insoluble impurities before filter sterilization [14,31,32]. For carvacrol (Fisher Scientific Company LLC Corp, Hanover Park, IL, USA) and caprylic acid (Fisher Scientific Company LLC Corp.), 0.5% (v/v) of the antimicrobial (i.e., 7.5 µL antibacterial in 1.5 mL of HEPES buffer) were used in the current study. High-pressure treatments were conducted in PULSE tubes (Pressure BioScience Inc., South Easton, MA, USA) with a total volume of 1.5 mL. The pressure transmission fluid was distilled water (total soluble solids < 30 ppm), and pressure treatments were applied using Hub880 Barocycler unit (Pressure BioScience Inc.). Temperature was precisely regulated by a refrigerated circulating water bath (Model1160s, VWR International, Radnor, PA, USA) connected to stainless steel water jacket surrounding the reaction chamber. The temperature was monitored by a T-type thermocouple (Omega Engineering Inc., Norwalk, CT, USA) inserted inside the wall of chamber and secured using thermal paste (Model 5 AS5-3.5G, Arctic Silver, Visalia, CA, USA), connected to Hub880 Barocycler software (HUB PBI 2.3.11 Software, Pressure BioScience Inc., South Easton, MA, USA) that automatically recorded the temperature and pressure intensity of treatments every three seconds. Trials of the study was conducted under various hydrostatic pressure levels of 350 MPa (with presence of caprylic acid and carvacrol), 450 MPa (at two temperatures of 4 and 40 ◦C), and at 550 MPa (with the presence of nisin) for up to 7 min. Treatment times exclude the come-up time (<30 s for Hub880 Barocycler) and come-down time (<3 s for Hub880 Barocycler). Our previous study had illustrated that come-up and come-down times of less than one minute have negligible effects (p 0.05) on the microbial reduction capability of pressure-based treatments [33]. ≥ Int. J. Environ. Res. Public Health 2020, 17, 7033 4 of 14

As such, reductions obtained in the current study are due to eﬀects of antimicrobials, mild heat, and elevated hydrostatic pressure rather than come-up and come-down times.

2.3. Microbiological Enumeration, pH Measurement, and Control of Exposure Time To assure precise exposure time, each antimicrobial compound was added immediately before the pressure treatment to the PULSE tubes and after homogenization by vortexing, the sample was immediately pressure-treated (<5 s). Additionally, immediately after treatment, samples were neutralized with D/E neutralizing (Difco, Becton Dickinson, Franklin Lakes, NJ, USA) broth (1 mL of treated sample to 3 mL of sterilized D/E broth) to assure neutralization of the antimicrobial compound and assure that antimicrobial residue is not transferred onto surface of the enumeration medium. Thus, the detection limit of the current study is 0.60 log CFU/mL. Samples were additionally placed into ice-water slurry immediately after neutralization to assure temperature exposure times were controlled [12]. After neutralizing the eﬀects of antimicrobials and heat, samples were then 10-fold serially diluted by single strength Maximum Recovery Diluent (MRD, Difco, Becton Dickinson, Franklin Lakes, NJ, USA). To assure further recovery of injured cells, 0.6% of yeast extract was added to Tryptic Soy Agar (TSA, Difco, Becton Dickinson, Franklin Lakes, NJ, USA), the medium used for enumeration of the pathogen. The use of tryptic soy agar with yeast extract (TSA + YE) additionally enhances the recovery of pressure-, heat- and antimicrobial-injured microbial cells and improves external validity of the pressure-based microbiological challenge study [12,34]. The 10-fold diluted samples were spread-plated onto TSA + YE, aerobically incubated at 37 ◦C for 48 h, and the colony-forming units (CFU) were calculated based on the Bacteriological Analytical Methods (BAM) of the U.S. Food and Drug Administration [35] using a Quebec colony counter. The pH values of the samples were measured using a calibrated digital pH meter prior and after the neutralization (Mettler Toledo AG, Grelfensee, Switzerland).

2.4. Experimental Design and Statistical Analyses This study is summary of four independent experiments: (i) Application of elevated hydrostatic pressure at 450 MPa at 4 and 40 ◦C; (ii) Application of elevated hydrostatic pressure at 550 MPa and 5000 IU/mL (w/v) of nisin at 4 ◦C; (iii) Application of elevated hydrostatic pressure at 350 MPa and 0.5% (v/v) carvacrol and/or caprylic acid at 25 ◦C; (iv) Comparison of treatments in the first three experiments with untreated control and the current common food industry standard (treated control). Target inoculation level for experiments 1 and 2 were 7 to 8 and for experiments 3 and 4 were 6 to 7 log CFU/mL. The target inoculations, pressure intensity levels, treatment times and temperatures, and concentrations of antimicrobials were selected based on preliminary trials (data not shown) to assure the microbiological efficacy of the treatments and relevance to stakeholders. Each of the four experiments were conducted in two biologically independent repetitions considered as blocking factor of a randomized complete block design. Each block was additionally consisted of three replications and each replication was obtained by averaging the values of two repetitions as microbiological (instrumental) repetitions. Thus, each represented value is the average of 12 independent observations (two blocks, three replications, two instrumental repetitions). Our previous study indicates that at least five and nine repetitions (for a statistical power of 80% and type I error level of 5%) are needed to observe a 0.15 and 0.10 log difference between two pressure-treated log transformed means as statistically significant, respectively [36]. Data for each trial were separately collected and log-transformed using Microsoft Excel. The inferential statistics were conducted using SAS9.4 (SAS Institute Inc., Cary, NC, USA) for conducting analysis of variance (ANOVA) for comparison (Tukey- and/or Dunnett’s-adjusted) of treatments and the controls at type I error level of 5% using the GLM procedures. D-value inactivation index was additionally calculated using reciprocal of slope of the best-fitted linear model by plotting the log-transformed microbial counts as function of treatment time (minutes). The non-linear Int. J. Environ. Res. Public Health 2020, 17, 7033 5 of 14

Int. J. Environ. Res. Public Health 2020, 17, x FOR PEER REVIEW 5 of 13 Int.inactivation J. Environ. Res. index Public k Healthmax was 2020 calculated, 17, x FOR PEER using REVIEW GInaFiT software (Katholieke Universiteit, version 5 of1.7 13 3.Leuven, Results Belgium). 3. Results 3.1.3. Results Inactivation of S. aureus at 450 MPa of Elevated Hydrostatic Pressure as Affected by Mild Heat 3.1. Inactivation of S. aureus at 450 MPa of Elevated Hydrostatic Pressure as Affected by Mild Heat 3.1. InactivationFor samples of treated S. aureus at at 4 450°C, MPacontrol of Elevated counts Hydrostaticof S. aureusPressure was 7.95 as ± A 0.4ffected log by CFU/mL Mild Heat (Figure 1). For samples treated at 4 °C, control counts of S. aureus was 7.95 ± 0.4 log CFU/mL (Figure 1). These counts after treatment at 450 MPa were reduced (p < 0.05) by 0.54, 0.88, 0.98, and 1.51 log TheseFor counts samples after treated treatment at 4 ◦ C,at control450 MPa counts were of reducedS. aureus (wasp < 7.950.05) by0.4 0.54, log CFU 0.88,/mL 0.98, (Figure and1 1.51). These log CFU/mL after 1, 3, 5, and 7 min treatments, respectively (Figure 1).± The application of mild heat at 40 CFU/mLcounts after after treatment 1, 3, 5, and at 4507 min MPa treatments, were reduced respectively (p < 0.05) (Figure by 0.54, 1). 0.88, The 0.98, application and 1.51 of log mild CFU heat/mL at after 40 °C, to a great extent, augmented the decontamination efficacy of the treatment. Control counts and °C,1, 3, to 5, a and great 7 minextent, treatments, augmented respectively the decontamination (Figure1). The efficacy application of the oftreatment. mild heat Control at 40 ◦ C,counts to agreat and those obtained after 1, 3, 5, and 7 min of treatment were 8.21 ± 0.7, 7.02 ± 0.2, 5.93 ± 0.4, 6.13 ± 0.1, and thoseextent, obtained augmented after the 1, 3, decontamination 5, and 7 min of treatment efficacy of we there treatment. 8.21 ± 0.7, 7.02 Control ± 0.2, counts 5.93 ± and 0.4, those6.13 ± obtained0.1, and 5.77 ± 0.3 log CFU/mL, respectively (Figure 1). These reductions (p < 0.05) were appreciably higher 5.77after ± 1,0.3 3, log 5, and CFU/mL, 7 min ofrespectively treatment (Figure were 8.21 1). These0.7, 7.02reductions0.2, 5.93 (p < 0.05)0.4, 6.13were appreciably0.1, and 5.77 higher0.3 than those obtained at 4 °C. As an example, ±while 1.51± log CFU/mL± reductions± (p < 0.05) were± thanlog CFU those/mL, obtained respectively at 4 °C. (Figure As 1an). Theseexample, reductions while 1.51 ( p < log0.05) CFU/mL were appreciably reductions higher (p < 0.05) than were those obtained after 7 min of treatment at 450 MPa and at 4 °C, the same treatment at 40 °C resulted in 2.44 obtainedobtained after at 4 ◦7C. min As of an treatment example, at while 450 MPa 1.51 and log at CFU 4 °C,/mL the reductions same treatment (p < 0.05) at 40 were °C resulted obtained in after2.44 log CFU/mL reductions (p < 0.05) of S. aureus (Figure 1). Linear and non-linear inactivation indices log7 min CFU/mL of treatment reductions at 450 (p MPa < 0.05) and of at S. 4 aureus◦C, the (Figure same treatment 1). Linear at and 40 ◦non-linearC resulted inactivation in 2.44 log CFU indices/mL exhibited similar trends (Figure 2). While 5.34 min was needed for a 90% reduction in S. aureus at 4 exhibitedreductions similar (p < 0.05) trends of S. (Figure aureus (Figure2). While1). 5.34 Linear min and was non-linear needed inactivationfor a 90% reduction indices exhibited in S. aureus similar at 4 °C during a 450-MPa treatment (i.e., D-value = 5.34), the same treatment at 40 °C required 3.30 min °Ctrends during (Figure a 450-MPa2). While treatment 5.34 min (i.e., was neededD-value for = 5.34), a 90% the reduction same treatment in S. aureus at at40 4°C◦C required during a 3.30 450-MPa min for decontamination of the pathogen (Figure 2). The non-linearly calculated Kmax additionally fortreatment decontamination (i.e., D-value of= the5.34), pathogen the same (F treatmentigure 2).at The 40 ◦Cnon-linearly required 3.30 calculated min for decontaminationKmax additionally of illustrated the effects of mild heat for enhancing the decontamination efficacy of the treatment. Kmax illustratedthe pathogen the (Figureeffects 2of). mild The non-linearlyheat for enhancing calculated the decontamination K max additionally efficacy illustrated of the the treatment. effects of mildKmax values for samples treated at 4 and 40 °C at 450 MPa were 0.50 and 2.81 1/min, respectively (Figure valuesheat for for enhancing samples thetreated decontamination at 4 and 40 °C effi atcacy 450 ofMPa the were treatment. 0.50 and Kmax 2.81values 1/min, for respectively samples treated (Figure at 4 2). These findings are in harmony with the previous literature, where S. aureus was reduced by 1.61 2).and These 40 ◦C findings at 450 MPa are werein harmony 0.50 and wi 2.81th the 1/min, previous respectively literature, (Figure where2). TheseS. aureus findings was reduced are in harmony by 1.61 to 2.73 log CFU/mL after 9 min of hydrostatic pressure treatment at 450 MPa [37]. Similarly, 2.69 log towith 2.73 the log previous CFU/mL literature, after 9 min where of hydrostaticS. aureus was pressure reduced treatment by 1.61 at to 450 2.73 MPa log [37]. CFU Similarly,/mL after 92.69 min log of reductions in S. aureus were reported after pressure treatments of samples at 40 °C and at 400 MPa reductionshydrostatic in pressure S. aureus treatment were reported at 450 MPa after [37 pressure]. Similarly, treatments 2.69 log of reductions samples inat S.40aureus °C andwere at 400 reported MPa [38]. [38].after pressure treatments of samples at 40 ◦C and at 400 MPa [38].

FigureFigure 1. 1. InactivationInactivation of of S.S. aureus aureus byby elevated elevated hydrostatic hydrostatic pressure pressure at at 4 4 and and 40 40 °C.C. Values Values followed followed by by Figure 1. Inactivation of S. aureus by elevated hydrostatic pressure at 4 and 40 °C.◦ Values followed by differentdiﬀerent upper-case letters are statistically ( p < 0.05)0.05) different diﬀerent than than each each other other (pair-wise (pair-wise comparisons, comparisons, different upper-case letters are statistically (p < 0.05) different than each other (pair-wise comparisons, Tukey-adjusted ANOVA). Tukey-adjusted ANOVA).

FigureFigure 2. LinearLinear and and non-linear inactivation inactivation indices indices for for inactivation inactivation of of S.S. aureus aureus treatedtreated by by elevated Figure 2. Linear and non-linear inactivation indices for inactivation of S. aureus treated by elevated hydrostatichydrostatic pressurepressure at at 450 450 MPa MPa at 4at and 4 40and◦C 40 with °C no with bacteriocin no bacteriocin or bactericidal or bactericidal compound. compound. Measured hydrostatic pressure at 450 MPa at 4 and 40 °C with no bacteriocin or bactericidal compound. Measured= Actual observations; = Actual observations; Identiﬁed Identified= Best-ﬁtted = Best-fitted non-linear non-linear model. model. Measured = Actual observations; Identified = Best-fitted non-linear model.

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3.2. Inactivation of S. aureus at 550 MPa of Elevated Hydrostatic Pressure as Affected by Nisin 3.2. Inactivation of S. aureus at 550 MPa of Elevated Hydrostatic Pressure as Affected by Nisin Prior to the treatment at 500 MPa, counts of S. aureus were 7.96 ± 0.2 log CFU/mL (mean ± SE). A one-minutePrior to the treatment treatment at at550 500 MPa MPa, was counts not (p of ≥S. 0.05) aureus effectivewere7.96 at reducing0.2 log the CFU pathogen/mL (mean and countsSE). A ± ± ofone-minute S. aureus treatmentwere 7.05 at± 5500.1 MPalog CFU/mL was not (afterp 0.05) the e1ff minective treatment at reducing (Figure the pathogen 3). The 3, and 5, countsand 7 ofminS. ≥ treatmentsaureus were were 7.05 similar0.1 log to CFU each/mL other after (p the≥ 0.05) 1 min and treatment were effective (Figure (p3 <). 0.05) The 3,in 5,reducing and 7 min the treatments pathogen. ± wereAfter similarthe treatments, to each other0.99, (1.36,p 0.05)and and1.61 werelog CFU/mL effective reductions (p < 0.05) in(p reducing< 0.05) of the the pathogen. pathogen Afterwere ≥ observed,the treatments, respectively 0.99, 1.36, (Figure and 1.61 3). Samples log CFU /treatedmL reductions at 550 MPa (p < of0.05) elevated of the hydrostatic pathogen were pressure observed, at 4 °Crespectively and, in the (Figure presence3). of Samples 5000 IU/mL treated of nisin, at 550 exhi MPabited of similar elevated trends. hydrostatic All trea pressuretments of at 1 to 4 7◦ Cmin(s) and, in the presence of 5000nisin IUwere/mL effective of nisin, (p exhibited < 0.05) at similar reducing trends. the pa Allthogen, treatments and log of 1reductions to 7 min(s) (p in< 0.05)the presence for 1, 3, of 5, nisin and were7 min e fftreatedective (samplesp < 0.05) were at reducing 0.85, 0.74, the pathogen,1.25, and and1.59 loglog reductions CFU/mL, (respectivelyp < 0.05) for 1,(Figure 3, 5, and 3). 7 min treated samples were 0.85, 0.74, 1.25, and 1.59 log CFU/mL, respectively (Figure3).

Figure 3. Inactivation of S. aureus by elevated hydrostatic pressure (550 MPa at 4 C) in the presence of Figure 3. Inactivation of S. aureus by elevated hydrostatic pressure (550 MPa at ◦4 °C) in the presence 5000 IU of nisin. Values followed by different upper-case letters are statistically (p < 0.05) different than of 5000 IU of nisin. Values followed by different upper-case letters are statistically (p < 0.05) different each other (pair-wise comparisons, Tukey-adjusted ANOVA). For samples treated without antimicrobial than each other (pair-wise comparisons, Tukey-adjusted ANOVA). For samples treated without (solid bars) and those treated in the presence of nisin (patterned bars) statistical analyses are presented antimicrobial (solid bars) and those treated in the presence of nisin (patterned bars) statistical analyses separately by upper-case letters with different colors. are presented separately by upper-case letters with different colors. The calculation of linear and non-linear inactivation indices exhibits a similar trend. D-value, The calculation of linear and non-linear inactivation indices exhibits a similar trend. D-value, amount of time needed for one log (e.g., 90%) reduction in the pathogen, was similar for elevated amount of time needed for one log (e.g., 90%) reduction in the pathogen, was similar for elevated hydrostatic pressure treatments with and without added nisin (Figure4). These values were 5.15 and hydrostatic pressure treatments with and without added nisin (Figure 4). These values were 5.15 and 5.22 min for samples treated without and with nisin at 4 ◦C. The inactivation index Kmax has the unit of 5.22 min for samples treated without and with nisin at 4 °C. The inactivation index Kmax has the unit 1/min, thus lower values correspond to a higher inactivation rate and vice versa [39]. The Kmax values of 1/min, thus lower values correspond to a higher inactivation rate and vice versa [39]. The Kmax for samples treated with and without nisin at 550 MPa and at 4 ◦C were similar and were 26.70 and values for samples treated with and without nisin at 550 MPa and at 4 °C were similar and were 26.70 24.83, respectively (Figure4). and 24.83, respectively (Figure 4).

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550 MPa at 4°C, No Antimicrobial 550 MPa at 4°C with Nisin 10 10 9 9 8 8 7 7 6 6 5 5 4 Linear Model 4 Linear Model LogCFU/mL D-value= 5.15 min LogCFU/mL D-value= 5.22 min 3 (R2= 0.68) 3 (R2= 0.58) 2 Non-Linear Model 2 Non-Linear Model Kmax= 26.70 1 1 Kmax= 24.83 (R2= 0.81) Measured Identified (R2= 0.61) Measured Identified 0 0 0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8 Time (minute s ) Time (minute s )

Figure 4. Linear and non-linear inactivation indices for S. aureus exposed to elevated hydrostatic Figurepressure 4. Linear (550 MPa and atnon-linear 4 °C) in inactivationthe presence indices of 5000 for IU S. of aureus nisin. exposedMeasure tod =elevated Actual observations;hydrostatic Figure 4. Linear and non-linear inactivation indices for S. aureus exposed to elevated hydrostatic pressureIdentified (550 = BestMPa-fitted at 4 non°C) -inlinear the model.presence of 5000 IU of nisin. Measured = Actual observations; pressure (550 MPa at 4 C) in the presence of 5000 IU of nisin. Measured = Actual observations; Identified = Best-fitted non-linear◦ model. Identified = Best-fitted non-linear model. 3.3. Inactivation of S. aureus at 350 MPa of Elevated Hydrostatic Pressure as Affected by Caprylic Acid and 3.3.Carvacrol Inactivation Inactivation of S. aureus at 350 MPa of Elevated Hydrostatic Pressure as AAffectedffected byby CaprylicCaprylic AcidAcid andand Carvacrol This section summarizes the results of samples treated at 25 °C in the presence of 0.5% (v/v) carvacrolThis section and 0.5% summarizes (v/v) caprylic the results resultsacid. It of is samples noteworthy tr treatedeated that, at atas 25 25 previously °C◦C in in the the elaborated,presence presence of of immediately 0.5% 0.5% ( (vv//vv)) carvacrolafter treatments, and 0.5% samples (v/v) capryliccaprylic were acid.neutralizedacid. It is noteworthy using D/E that,broth as, thus previouslypreviously exposure elaborated,elaborated, times to immediatelyantimicrobials afterwere treatments, controlled samples and residu were al amountneutralized s of antimicrobialusing D/ D/E broth,s on thussurface exposure of the medi timesum to antimicrobialswere mitigated. wereFor controlledcontrolledsamples treated andand residual residual at 350 amountsMPa amounts (without of of antimicrobials antimicrob antimicrobial)ials on on surfacethe surface control of theof counts the medium medium and were those were mitigated. obtained mitigated. Forafter Forsamples1, samples3, 5, treatedand treated 7 min at 350 ofat MPa350treatments MPa (without (without were antimicrobial) 6.81 antimicrob ± 0.1, 6.78 theial) control± the 0.0, control 6.52 counts ± counts0.2, and 6.31 those and ± 0.0, obtainedthose and obtained 3.80 after ± 1, 0.2after 3, log 5, 1,andCFU/mL, 3, 75, min and of respectively7 treatmentsmin of treatments (Figure were 6.81 5). were Th0.1,ese 6.81 6.78values ± 0.1,0.0, remain 6.78 6.52 ± ed0.0, 0.2,statistically 6.52 6.31 ± 0.2,0.0, unchanged 6.31 and ± 3.800.0, andduring0.2 3.80 log the CFU± 0.21, 3,/ mL,log and ± ± ± ± ± CFU/mL,respectively5 min of respectivelytreatment (Figure 5().p (Figure≥ These 0.05) and values5). These only remained a values 7-min rematreatment statisticallyined statisticallyw unchangedas able to unchangedreduce during (p the< 0.05)during 1, 3, S. and aureusthe 51, min 3,(Figure and of 5treatment min5). of treatment (p 0.05) (p and ≥ 0.05) only and a 7-min only a treatment 7-min trea wastment able was toreduce able to (reducep < 0.05) (p S.< 0.05) aureus S.(Figure aureus 5(Figure). ≥ 5). 350 MPa Treatements (25 °C) in HEPES Buffer 350 MPa Treatements (25 °C) in HEPES Buffer B 7 B B B 7 B B B B 5 B A 5 B B A 3 B B A 3 B B A 1 B B A 1 B Treatment Time (minutes) Time Treatment A A 0 A Treatment Time (minutes) Time Treatment A A 0 A A 0 1 2 3 4 5 6 7 01234567S. aureus Log CFU/mL (Mean ± Standard Error) S. aureus Log CFU/mL (Mean ± Standard Error) Caprylic Acid Carvacrol No Antimicrobial Caprylic Acid Carvacrol No Antimicrobial

FigureFigure 5. 5.Inactivation Inactivation of ofS. S. aureus aureusby by elevated elevated hydrostatic hydrostatic pressure pressure in in presence presence of of 0.5% 0.5% (v (/v/)v caprylic) caprylic Figureacidacid and and 5. carvacrol.Inactivation carvacrol. Values Values of S. followedaureus followed byby elevated by di differentﬀerent hydrostatic upper-case upper-case pressure letters letters arein are presence statistically statistically of 0.5% (p (

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Int. J. Environ. Res. Public Health 2020, 17, x FOR PEER REVIEW 8 of 13 for the same treatment without any antimicrobials were 0.03, 0.28, and 0.49, respectively, indicating thatsame the treatment addition without of carvacrol any antimicrobials could, to a great were extent, 0.03, 0.28, augment and 0.49, the respectively, efficacy of pressureindicating pasteurization, that the particularlyaddition of forcarvacrol short-term could, treatments to a great of extent,5 min. augment Caprylic the acid efficacy at 0.5% of pressure was also pasteurization, efficacious (p < 0.05) ≤ forparticularly enhancing for the short-term decontamination treatments e ffiofcacy ≤ 5 min. of the Caprylic treatments. acid at Counts 0.5% was of treatedalso efficacious samples (p at < 3500.05) MPa in thefor presenceenhancing of the caprylic decontamination acid were reducedefficacy of(p the< 0.05) treatments.by 2.77, Counts 3.40, 3.39, of treated and 3.58 samples log CFU at 350/mL MPa after 1, 3, 5,in and the presence 7 min of of treatment caprylic acid (Figure were5 ).reduced It is important (p < 0.05) by to note2.77, 3.40, that, 3.39, for aand long-term 3.58 log CFU/mL treatment after of more 1, 3, 5, and 7 min of treatment (Figure 5). It is important to note that, for a long-term treatment of than 5 min, the efficacy of both caprylic acid and carvacrol faded and log reductions were similar to more than 5 min, the efficacy of both caprylic acid and carvacrol faded and log reductions were the samples treated without any antimicrobial. Thus, our results illustrate that the tested antimicrobials similar to the samples treated without any antimicrobial. Thus, our results illustrate that the tested are efficacious for short-term treatments, such as those that are currently the common processing antimicrobials are efficacious for short-term treatments, such as those that are currently the common parametersprocessing parameters in the private in the food private industry. food industry. Inactivation Inactivation indices illustrate indices illustrate similar similar trends, trends, with the with D-value, and the K of all three treatments of up to 7 min remain nearly identical (Figure6). the D-value,max and the Kmax of all three treatments of up to 7 min remain nearly identical (Figure 6).

FigureFigure 6. LinearLinear and and non-linear non-linear in inactivationactivation indices indices for S. for aureusS. aureus treatedtreated by hydrostatic by hydrostatic pressure pressure (350 (350 MPaMPa at 25 °C)◦C) in in the the presence presence of of 0.5% 0.5% (v/ (vv)/ vcaprylic) caprylic acid acid and and 0.5% 0.5% (v/v) ( vcarvacrol./v) carvacrol. Measured Measured = Actual= Actual observations;observations; Identified Identiﬁed == Best-fittedBest-ﬁtted non-linear non-linear model. model.

3.4.3.4. SummarySummary of of Inactivati Inactivationon Efficacy Efficacy of ofTreatments Treatments Currently,Currently, the food food industry industry relies relies on on treatments treatments of 650 of 650MPa, MPa, commonly commonly lasting lasting for 3 min, for 3 for min, for pressure-basedpressure-based pasteurization of of various various products products [12–14]. [12–14 As]. previously As previously discussed, discussed, one of one the ofmain the main challengeschallenges for the the production production of of pressure-treated pressure-treated commodities commodities is slightly is slightly higher higher operation operation costs costs relativerelative toto traditionaltraditional heat-treated heat-treated products. products. Thus, Thus, this this study study investigated investigated the application the application of lower of lower intensitiesintensities of pressure augmented augmented with with GRAS-listed GRAS-listed bacteriocin bacteriocin andand bactericidal bactericidal compounds. compounds. This This synergismsynergism between elevated elevated hydrostatic hydrostatic pressure pressure and and antimicrobials antimicrobials could could reduce reduce the operation the operation cost cost and thus enhance the competitiveness of pressure-treated commodities. Additionally, it could and thus enhance the competitiveness of pressure-treated commodities. Additionally, it could provide provide protection for products during shelf-life due to the residual effects of antimicrobials [12–14]. protection for products during shelf-life due to the residual effects of antimicrobials [12–14]. This This experiment, summarized in Figure 7, was conducted to compare the treatments from experiment, summarized in Figure7, was conducted to compare the treatments from experiments 1 experiments 1 to 3 to the common industry standard (Trt. A) and to an untreated control (Control). toThis 3 to common the common treatment industry from the standard food industry (Trt. A) (650 and MPa to an at untreated 4 °C, for 3 control min) resulted (Control). in a This0.85 log common treatmentCFU/mL reduction from the foodof the industry pathogen (650 (Figure MPa 7). at The 4 ◦C, 3-min for 3 treatments min) resulted at 550 in aMPa 0.85 (at log 4 CFU°C) and/mL with reduction of5000 the (w pathogen/v) IU nisin (Figure (Trt. B),7). 450 The MPa 3-min at 40 treatments °C (Trt. C), atand 550 350 MPa MPa (at (at 425◦ °C)C) andand 0.5% with ( 5000v/v) carvacrol (w/v) IU nisin (Trt.(Trt. B),D), 450 were MPa more at effective 40 ◦C (Trt. (p < C), 0.05) and for 350 the MPa inactivation (at 25 ◦C) of andS. aureus 0.5%, compared (v/v) carvacrol to Trt. (Trt. A (Figure D), were 7). more eLogffective reductions (p < 0.05) (p < 0.05) for the for inactivationthese three treatments of S. aureus were, compared1.91, 1.54, and to Trt.2.70 log A (FigureCFU/mL,7). respectively Log reductions (p(Figure< 0.05) 7). for these three treatments were 1.91, 1.54, and 2.70 log CFU/mL, respectively (Figure7).

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FigureFigure 7. 7.InactivationInactivation of S. of aureusS. aureus by byelevated elevated hydrostatic hydrostatic pressure pressure in inpresence presence of ofbacteriocin bacteriocin and and

bactericidalbactericidal compounds compounds and and mild mild heat. heat. Control: Control: Untr Untreatedeated control control (0 MPa (0 MPa at 4 at °C 4 ◦forC for3 min); 3 min); Trt. Trt. A: A: TreatedTreated control control (650 (650 MPa MPa at at4 °C 4 ◦ Cfor for 3 min). 3 min). This This treatment treatment is currently is currently the the industry industry standard standard for for pressure-basedpressure-based pasteurization pasteurization [12–14]. [12–14 ].Trt. Trt. B: B: Treatm Treatmentent of of 550 550 MPa MPa at at 4 4 °C◦C for for 3 3 min min with 5000 (w(w/v)/v) IU IU nisin.nisin. This This treatment treatment is further is further investigated investigated under theunder second the experimentsecond experiment of this study, of summarizedthis study, in

summarizedFigures3 and in 4Figures. Trt. C: 3 Treatment and 4. Trt. of C: 450 Treatment MPa at 40 of◦ C450 for MPa 3 min. at 40 This °C treatment for 3 min. is This further treatment investigated is furtherunder investigated the ﬁrst experiment under the of thefirst study, experiment summarized of the instudy, Figures summarized1 and2. Trt. in D: Fi Treatmentgures 1 and of 3502. Trt. MPa D: at

Treatment25 ◦C for of 3 min350 MPa with at 0.5% 25 (v°C/ v)for carvacrol. 3 min with Trt. 0.5% E: Treatment (v/v) carvacrol. of 350 MPaTrt. E: at Treatment 25 ◦C for 3 of min 350 with MPa 0.5% at 25 (v /v) °C caprylicfor 3 min acid. with The 0.5% last (v/v) two caprylic treatments acid. are The further last two investigated treatments under are further the third investigated experiment under of the the study, thirdsummarized experiment in of Figures the study,5 and6 summarized. Values followed in Figu byres di ff5erent and 6. upper-case Values foll lettersowed are by statistically different upper- (p < 0.05) casedi ffletterserent are than statistically each other (p (pair-wise < 0.05) different comparisons, than each Tukey-adjusted other (pair-wise ANOVA). comparisons, Values Tukey-adjusted followed by * are ANOVA).statistically Values (p < followed0.05) diff byerent * are than statistically untreated (p control < 0.05)(Dunnett’s-adjusted different than untreated ANOVA). control Values (Dunnett’s- followed adjustedby ˆ are ANOVA). statistically Values (p < 0.05) follow diedfferent by ^ thanare statistically Trt. A., e.g., ( treatedp < 0.05) control different (Dunnett’s-adjusted than Trt. A., e.g., ANOVA).treated control (Dunnett’s-adjusted ANOVA). 4. Discussion 4. DiscussionIt is important to note that, although the pathogen was reduced by up to > 90% due to treatment withIt is 550 important MPa of elevatedto note that, hydrostatic although pressure the pathog withen and was without reduced 5000 by up IU to/mL > 90% of nisin, due to treatments treatment left withbehind 550 MPa as much of elevated as >5 loghydrostatic CFU/mL pressure of the pathogen, with and indicatingwithout 5000 that IU/mL pressure of nisin, at this treatments intensity left with behindor without as much nisin as >5 is log not CFU/mL solely su offfi cientthe pathogen, to assure in thedicating safety that of apressure product, at and this requiresintensity additionalwith or withoutdecontamination nisin is not hurdle(s). solely sufficient Thus, this to level assure of nisin the andsafety pressure of a product, could be incorporatedand requires onlyadditional as one of decontaminationthe hurdles in a hurdle(s). multiple hurdleThus, this technology level of [nisi40],n not and as pressure the sole could antimicrobial be incorporated treatment. only as one of the hurdlesIt is also in noteworthya multiple hurdle that the technology utilization [40], of thenot HEPESas the sole buff antimicrobialer is common treatment. in the high-pressure processingIt is also literature,noteworthy since that the the medium utilization could of the maintain HEPES a bubufferffered is environmentcommon in the under high-pressure the elevated processinghydrostatic literature, pressure since [34 ].the Thus, medium the reductionscould maintain obtained a buffered in the environment current study under could the be elevated attributed hydrostaticto the effects pressure of elevated [34]. Thus, hydrostatic the reductions pressure, obtained heat, and inthe theantimicrobials current study rathercould be than attributed any intrinsic to thefactor effects of of the elevated medium hydrostatic [12]. The pressure, application heat, of and nisin the was antimicrobials reported to rather be effi thancacious any to intrinsic augment factorthe pressure-basedof the medium pasteurization[12]. The application of bacterial of nisi pathogensn was reported in the past.to be Nisin efficacious was able to augment to significantly the pressure-basedincrease the decontaminationpasteurization of e ffbacterialectiveness pathogens of a pressure-based in the past. treatmentNisin was against able to a Gram-positivesignificantly increasebacterium, the decontaminationListeria monocytogenes effectiveness[14]. Other of a studiespressure-based indicate antreatment even lower against concentration a Gram-positive of nisin, bacterium,such as 100 Listeria IU/mL, monocytogenes could enhance [14]. the Other decontamination studies indicate efficacy an of even high-pressure lower concentration pasteurization of nisin, against suchpathogenic as 100 IU/mL, organisms could such enhance as Escherichia the decontami coli, SalmonellanationEnteritidis, efficacy ofSalmonella high-pressureTyphimurium, pasteurizationShigella againstsonnei pathogenic, Shigella flexneri organisms, Pseudomonas such as Escherichia fluorescens, coliand, SalmonellaS. aureus Enteritidis,[41]. Similarly, Salmonella nisin was Typhimurium, efficacious to Shigellaenhance sonnei the,decontamination Shigella flexneri, ePseudomonasfficacy of other fluorescens, non-thermal and treatments, S. aureus such[41]. asSimilarly, high-intensity nisin pulsedwas efficaciouselectric fields to enhance for the the elimination decontami ofnationS. aureus efficacy[42]. ofStaphylococcus other non-th carnosusermal treatments,was similarly suchreduced as high- by intensitysynergism pulsed of 500 electric MPa fields of pressure for the and elimination nisin, indicating of S. aureus that other [42]. speciesStaphylococcus of Staphylococcus carnosus wascould similarlyadditionally reduced be decontaminatedby synergism ofusing 500 MPa a combination of pressure of and elevated nisin,hydrostatic indicating pressurethat other and species nisin of [43 ]. StaphylococcusThese synergistic could e ffadditionallyects from the be literature decontaminated were typically using obtained a combination for treatments of elevated of as longhydrostatic as 30 min. pressureIn food and processing, nisin [43]. treatments These synergistic longer than effects 5 min from are notthe commonliterature and were the typically vast majority obtained of current for treatmentscommercial of as treatments long as 30 are min. limited In food to 3 minproce [12ssing,–14,16 treatments]. Our results longer indicate than while5 min thisare antimicrobialnot common is and the vast majority of current commercial treatments are limited to 3 min [12–14,16]. Our results

Int. J. Environ. Res. Public Health 2020, 17, 7033 10 of 14 very effective against other Gram-positive pathogens such as Listeria monocytogenes [15], the application of nisin for time intervals of less than 10 min and at 4 ◦C only has mild antibacterial benefits against S. aureus (Figure3). To assimilate the effects of mild heat for augmenting the decontamination efficacy of pressure-based pasteurization, the experiment was conducted at two controlled temperatures of 4 and 40 ◦C. It is noteworthy that a pressure of 450 MPa is more than four times higher than the pressure in Mariana Trench, the earth’s deepest oceanic trench [44]. However, this pressure is still considered as a medium level of pressure in food manufacturing, as vast majority of pressure-treated products in commerce are treated at 650 MPa [12–14,16]. Currently, pressure-treated products typically have slightly higher operation cost relative to traditional thermally treated products, thus the application of lower levels of pressure coupled with mild heat and/or bacteriocin and bactericidal compounds could be of great importance for stakeholders to optimize their cost. The application of pressure at higher levels is typically associated with a higher cost of operation and maintenance of the pressure vessels [12]. This finding is in harmony with the previous literature, where it was shown that antimicrobials known to be effective against Gram-positive organisms such as Listeria monocytogenes are capable of enhancing the decontamination efficacy of elevated hydrostatic pressure for short-term treatments, and the efficacy of the antimicrobial faded as treatment time increased [14]. Studies investigating the synergism of elevated hydrostatic pressure, carvacrol, and caprylic acid for the inactivation of S. aureus is very limited in the literature. However, the positive effects of carvacrol and caprylic acid for enhancing pressure-based inactivation of Gram-positive organisms, such as Listeria monocytogenes, and Gram-negative bacteria, such as Shiga toxin-producing Escherichia coli, were reported in the literature [13, 45]. It is noteworthy that although infections with S. aureus are not as fatal as the vast majority of the main foodborne diseases [2], this pathogen is highly prevalent among food workers, and thus additional studies on the decontamination of this microorganism could be of great importance to the private food industry. One study conducted in the fish-processing factory showed that as many as 62% of the workers in one operation tested positive for S. aureus [8]. Other studies indicate that > 26% of food handlers could be a carrier of this pathogen [9]. As previously articulated, this study was conducted in the HEPES buffer, capable of maintaining a steady pH under the elevated hydrostatic pressure. Thus, the reductions could be solely attributed to the effects of temperature, the antimicrobials and/or elevated hydrostatic pressure. Future studies could further explore the effects of various intrinsic and extrinsic factors, particularly pH, in various food vehicles to identify and optimize the use of various bacteriocin and bactericidal compounds for improving the microbiological safety and cost-effectiveness of pressure-based treatments. This study investigated the effects of three bacteriocin and bactericidal compounds (carvacrol, caprylic acid, and nisin), that are part of the generally recognized as safe (GRAS) list of the U.S. Food and Drug Administration as food additives [17]. Antibiotics efficacious for the treatment of S. aureus infections such as oxacillin, methicillin, or cefoxitin could also be used to augment the decontamination efficacy of elevated hydrostatic pressure to reduce this pathogen in the food chain in future experiments. These antibiotics, if granted GRAS status, could additionally become part of food formulations for the control of this pathogen. In addition, S. aureus is among the 11 pathogens that are considered “serious threats” by the U.S. Centers for Disease Control and Prevention for development of antibiotic resistance [46]. Thus, future studies comparing the susceptible and drug-resistant (such as methicillin-resistant S. aureus) phenotypes of this pathogen could be projects of great importance for continuation of the experiments articulated in our study. Finally, it is noteworthy that S. aureus is a versatile and dangerous pathogen that, in addition to causing systematic infection, could secrete exotoxins that could be relatively stable in the food environment [47]. The current study investigated the effects of the proposed treatments against the planktonic cells of the pathogen to prevent infections and encoding of secondary metabolites such as exotoxins. Studies exploring the pressure sensitivity of S. aureus exotoxins could be novel and very important follow-up experiments for future researchers. Int. J. Environ. Res. Public Health 2020, 17, 7033 11 of 14

5. Conclusions Under the condition of our experiments, we observed that nisin had only minor effects in augmenting the decontamination efficacy of the pressure-based (550 MPa) pasteurization of S. aureus. Carvacrol and caprylic acid, however, were able to greatly enhance the decontamination efficacy of elevated hydrostatic pressure at 350 MPa and at 25 ◦C for treatments shorter than 5 min. Mild heat (40 ◦C) was similarly effective to increase the decontamination efficacy of pressure-based treatments for inactivation of S. aureus. These findings indicate that practitioners of this technology could benefit from the synergism of mild heat, and bacteriocin and/or bactericidal compounds to assure the microbial safety of products while utilizing moderate levels of hydrostatic pressure. This approach optimizes the cost of operation and the competitiveness of pressure-treated products, since extreme levels of pressure are typically associated with higher operation and maintenance cost. Additionally, this could lead to co-benefits such as the potential for the better retention of nutrients and bioactive compounds and better product shelf-life due to the residual effects of antimicrobials in product formulation. Our results additionally illustrate that the product, pathogen, level of elevated hydrostatic pressure, antimicrobial, and temperature would need to be carefully considered in a hurdle microbiological validation study to ensure that selected intrinsic and extrinsic factors are capable of assuring the microbiological safety of the product and its economic feasibility.

Author Contributions: Conceptualization, A.C.F. and J.G.; methodology, A.C.F., J.G., S.A., M.N.K., S.W., and S.C.; software, A.C.F. and J.G.; validation, A.C.F. and J.G.; formal analysis, A.C.F. and J.G.; investigation, A.C.F., J.G., S.A., M.N.K., S.W., and S.C.; resources, A.C.F.; data curation, A.C.F. and J.G.; writing—original draft preparation, A.C.F. and J.G.; writing—review and editing, A.C.F.; visualization, J.G. and A.C.F.; supervision, A.C.F.; project administration, A.C.F.; funding acquisition, A.C.F. All authors have read and agreed to the published version of the manuscript. Funding: This project was funded in part by United States Department of Agriculture [grant numbers: 2017-07534; 2017-07975; 2016-07430] and Pressure BioScience Inc. Acknowledgments: Technical contribution and assistance of students and staff of the Public Health Microbiology Laboratory is greatly appreciated by the corresponding author. Conflicts of Interest: The authors declare no conflict of interest.

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