inactivation of bacillus atrophaeus spores with surface-active peracids and characterization of...

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JFS M: Food Microbiology and Safety

Inactivation of Bacillus atrophaeusSpores with Surface-Active Peracids andCharacterization of Formed Free RadicalsUsing Electron Spin Resonance SpectroscopyANAND MOHAN, JOSEPH DUNN, MELVIN C. HUNT, AND CHARLES E. SIZER

ABSTRACT: This study investigated microbial inactivation via surface-active peracids and used electron spin res-onance spectroscopy to characterize the active components and free radical formation. Bacillus atrophaeus sporeswere injected directly into 3 different concentrations of the peracid disinfectant (1.1%, 1.3%, or 1.5%) for varioustimes (5, 10, 15, or 20 s) at 3 different temperatures (50, 60, or 70 ◦C) to evaluate the sporicidal activity of the dis-infectant mixture. Spectroscopy revealed that the combination of hydrogen peroxide, peracetic acid, and octanoicacid were highly effective at forming a complex mixture of sporicidal, free radical intermediates including hydroxyland superoxide radicals. Individual components of this mixture alone were not as effective as the final combination.This information has practical applications in the food industry for design of effective sanitation and disinfectionagents and suggests that kinetic models could be developed to account for both the physical removal and localizedinactivation of spores on food-contact surfaces.

Keywords: bacillus spores, disinfection, electron spin resonance, hydrogen peroxide, octanoic acid, peraceticacid, surfactant

Introduction

Antimicrobials play an important role in control of foodbornepathogens and spoilage microorganisms. A variety of disinfec-

tants, sanitizers, and antimicrobials are used for disinfecting food-contact surfaces, processing equipment, and machinery. Althoughsome of these disinfectants are generally effective at killing vegeta-tive microorganisms, bacterial spores are known for being relativelyresistant to many disinfection treatments (Russell 1990). Dormantspores produced by some bacteria can survive most environmentalchallenges in either dry or hydrated form (Setlow 1992; Driks 2003).

A variety of chemicals effectively eliminate foodborne pathogensand bacterial spores during cleaning and sanitation of food-contactsurfaces and processing facilities; but there are concerns regard-ing toxicity, expense, stability, and corrosiveness of these chem-icals. This has prompted a search for alternative disinfectantformulations that are effective against a variety of pathogens andalso less corrosive, nontoxic, and safer to handle and use. Peroxygenmixtures (primarily hydrogen peroxide and peroxyacetic acid) havea long history in the food industry as antimicrobial rinses becauseof their broad effectiveness and because they degrade quickly, leav-ing harmless residues (Baldry 1983; Leaper 1984). Using these sub-stances in combination with surface-active alkyl acids like octanoicacid increased the potency of peroxyacetic acid against bacterialspores (Russell 1990).

MS 20090120 Submitted 2/9/2009, Accepted 6/17/2009. Authors Mohan andHunt are with Dept. of Animal Sciences and Industry, Weber Hall, KansasState Univ., Manhattan, KS 66506, U.S.A. Authors Dunn and Sizer are withPilot Aseptic, 204 Dearborn Court, Suite 113, Geneva, IL 60134, U.S.A. Di-rect inquiries to author Hunt (E-mail: [email protected]).

Contribution nr. 09-227-J from the Kansas Agricultural Experiment Sta-tion, Manhattan, Kans. 66506, U.S.A.

Numerous reports (Tennen and others 2000; Setlow and others2002; Cross and others 2003) have suggested various mechanismsduring the killing of bacterial spores under various conditions oftreatment. The presence of a thick proteinaceous coat, reducedpermeability, and the protection of DNA from the active agentplay a significant role in protecting bacterial spores from injuryand death (Setlow 2007). The importance of these resistance fac-tors varies with the type of antimicrobial agent used because thereis a large variation in spore inactivation mechanisms for differentagents (Loshon and others 2001). Spores exhibit elevated resistancetoward oxidizing agents, particularly hydrogen peroxide (Setlowand Setlow 1993; Nicholson and others 2000), because of their pro-teinaceous spore coats (Riesenman and Nicholson 2000), the lowwater content of the spore core (Gilmore and others 2004), rela-tive impermeability of antimicrobials to the inner spore membrane(Nicholson and others 2000), and the α/β-type small acid-solublespore proteins that block DNA damage caused by hydrogen per-oxide (Riesenman and Nicholson 2000). Radiation, formaldehyde,and other alkylating agents are known to cause bacterial spore in-activation via DNA damage (Tennen and others 2000), whereas heatand some other oxidizing agents may inactivate one or more key re-covery mechanisms (Setlow and Setlow 1998).

Sanitizers and antimicrobials commonly used in food indus-tries are often strong oxidizing agents that mediate inactivationthrough free radical formation (Russell 1990; Setlow 1992, 1994;Marquis and others 1995). Spore DNA damage by hydroxyl radi-cals is considered to be a major mechanism for spore inactivationby oxidizing agents. Keszler and others (2003) suggested that thehydroxyl radical (·OH), the quintessential reactive oxygen species,possesses high electrophilicity and high thermo kinetic reactiv-ity that helps in the mediation of the DNA damage. The hydroxyl

C© 2009 Institute of Food Technologists R© Vol. 74, Nr. 7, 2009—JOURNAL OF FOOD SCIENCE M411doi: 10.1111/j.1750-3841.2009.01293.xFurther reproduction without permission is prohibited

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Inactivation of Bacillus atrophaeus spores . . .

radical is an extremely aggressive radical generated by peroxygensanitizers, ionizing radiation, and other antimicrobial compounds;reacting with almost all molecules in living cells including DNA,lipids, proteins, and carbohydrates (Baldry 1983; Setlow and oth-ers 1997). The hydroxyl radical is so reactive that it normally dif-fuses only a few bond lengths before reacting, so hydroxyl radicaldamage is very site specific (Riesenman and Nicholson 2000; Imlay2003). Therefore, microbial inactivation kinetics could be depen-dent upon the rate at which free radicals are produced, theirconcentration, and the immediate location where the radical isgenerated. To our knowledge, there are no reports of bacterial sporeinactivation via localized free radical formation. We hypothesizethat bacterial spore inactivation could be enhanced by the produc-tion of a localized, site-specific high concentration of free radicals.

This study aimed to determine the kinetics of sporicidal prop-erties of peracids and explore the release of hydroxyl, superox-ide, and alkyl radicals from individual and mixtures of the ac-tive components of the sporocide. More specifically, we measuredthe bacterial spore inactivation kinetics of Bacillus atrophaeus andcharacterized the localized formation of free radicals by using elec-tron spin resonance (ESR) spectroscopy.

Materials and Methods

PeracidsA commercial preparation of peracid (Matrixx R© ) was obtained

from Ecolab Inc. (Mendota Heights, Minn., U.S.A.). This prepara-tion contained hydrogen peroxide (10%), peracetic acid (5%), andoctanoic acid (5%) as active ingredients and 80% inactive ingredi-ents. The stock solution was stored at 2 to 5 ◦C. All peracid prepara-tions had a pH of 6.4.

Sodium thiosulfateA stock solution of 1% sodium thiosulfate (pH 7.1) was prepared

in deionized water, autoclaved and stored at 2 to 5 ◦C. Sodiumthiosulfate was used as a neutralizing agent, which leaves nontoxicresidues after reaction with peracid.

Neutralizing brothA 1:3 dilution of the neutralizing broth (NC9399018; Fisher Sci-

entific, Inc., Pittsburg, Pa., U.S.A.) stock solution (pH 7.6) was pre-pared in deionized water, autoclaved, and stored at 5 ◦C.

Spore suspensionA stock spore suspension (>99.9% viable spores) of a 107 colony

forming units (CFU) per milliliter of B. atrophaeus ATCC 9273 (for-merly B. atrophaeus var. niger strain globigii ATCC 9273) in 100%deionized water were purchased from Raven Biological Inc. (RavenLabs, 8607 Park Drive; Omaha, Nebr., U.S.A.). A working stock sus-pension of 105 CFU/mL of spores in triple deionized water was pre-pared from the original stock culture. A 2 mL portion from the work-ing stock culture was subjected to heat–shock treatment at 80 ◦C for5 min to kill any vegetative cells, if present, and plated on trypticasesoy agar (TSA; DifcoTM tryptic soy agar, BD Diagnostics, Sparks,Md., U.S.A.) plates.

Spore inactivation by direct injectionFor direct injection tests, a working stock solution of peracid was

prepared by adding 1.25, 1.5, or 1.75 mL of Matrixx to 114 mL ofwater to achieve 1.1%, 1.3%, and 1.5%, respectively. A total of 3 mLfrom the working stock solution was pipetted into a sterile test tubeand capped. Tubes with 3 mL of sterile deionized water served asa control. These test tubes were then placed in a hot water bath at

50 ◦C. Two additional test tube sets containing 3 mL of 1% sodiumthiosulfate and 1 mL of neutralizing broth as a 1:3 dilution of theoriginal stock concentration were used to stop the reaction.

Tubes containing peracid at 50 ◦C and 10 μL B. atrophaeusspores were vortexed, and incubated for the prescribed processtimes of 5, 10, 15, or 20 s. For longer process reaction times, thetest tube containing the spores and peracid reaction mixture wascapped and then placed into a hot water bath. After the process-ing time had elapsed, 1 mL of the peracid-spore reaction mixturewas pipetted into sodium thiosulfate solution, vortexed for approx-imately 10 s, and then neutralizing broth was added and vortexed.The mixture was then diluted and plated.

EnumerationA total of 0.5 mL of the recovery solution was diluted into 4.5 mL

of phosphate-buffered saline (140 mM NaCl, 2.7 mM KCl, 10 mMNa2HPO4, 1.8 mM KH2PO4; pH 7.2) and then further diluted to 10−4

for the processed samples, and to 10−5 for controls. Two-tenths ofa milliliter from the recovery medium and its serial dilutions wereindividually spread plated in duplicate onto TSA. TSA plates wereplaced in an incubator at 37 ◦C for 24 h and counted. Incubationfor longer periods gave no further increase in survivors.

Processing time and temperatureIn this set of tests, process time, temperature, and concentration

were changed to examine how these variations affected inactiva-tion kinetics. Each test included duplicate samples, and a total of 2independent replicates were tested for each set of conditions. Eachtest was performed at 1.1%, 1.3%, and 1.5% peracid using 4 expo-sure times (5, 10, 15, and 20 s), and 3 temperatures (50, 60, and70 ◦C). Control experiments showed that the treatment conditionsin the absence of peracid, but including thiosulphate and neutral-izing broth treatment, produced no appreciable (<5%) inactivationof spores or vegetative cells.

Detection of free radicals in peracidESR spectrometry was performed using a Varian E 12 (Varian

Associates; Palo Alto, Calif., U.S.A.) spectrometer operated at9400 MHz frequency. This unit provided superior control for sim-ple solution work due to its high sensitivity using small volumes ofexperimental material. ESR assays were performed in a flat cell andthe cavity was tuned using a standard of 0.3 mM of 3-carbamoyl-2,2, 5, 5-tetramethyl-3-pyrrolin-1-yloxy (CTPO) solution to assess thesensitivity of the instrument. A 1.5% stock solution of peracid wasprepared for capturing the spectrum with 5-tert-butoxycarbonyl5-methyl-1-pyrroline N-oxide (BMPO) as the spin trap. A 40-mMstock solution of BMPO was prepared in triple distilled water in ad-vance and stored in liquid nitrogen to prevent auto-degradation atroom temperature.

ESR assays were performed in the flat cell containing 300 μLof experimental sample prepared by adding 11 μL of BMPO and289 μL of the peracid. Assays were conducted at 22 ± 1 ◦C. The re-action solution was exposed to light from a 150 W halogen lampabout 18 cm from the cell for 2 min. The cell was placed in theESR cavity and the spectrum was captured 30 s after the exposureto light. Samples were not exposed to any other illumination be-fore or during the recording of the ESR spectrum. The ESR cavitywas tuned by adjusting the receiver gain and the frequency. ESRspectral recordings were obtained for a minimum of 30 scans. Allscans were stored in the database using a data logger attached tothe spectrometer. Spectra were recorded, analyzed, and plotted us-ing the MATLAB software (version 6, The MathWorks, Inc., Natick,Mass., U.S.A.). The ESR parameters used were: microwave power

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8 mW, modulation frequency 100 Hz, modulation amplitude0.1 mT, response time 0.1 s, gain 3358, sweep time 10 mT/min. TheESR spectra were recorded at different time intervals and sampleswere freshly prepared prior to ESR assay without incubation of theexperimental samples for any extended period of time.

United States Pharmacopeia grade hydrogen peroxide (pH 7.4)was purchased locally. Peracetic acid (catalogue nr 269336,32 weight percent in dilute acetic acid; pH 2.5) and octanoic acid(catalogue nr C 3907, purity 98%; pH 6.8) were purchased fromSigma-Aldrich (St. Louis, Mo., U.S.A.). Working peracid stock so-lutions were prepared by mixing octanoic acid, hydrogen perox-ide, and peracetic acid prior to starting the ESR spectrum captur-ing; stock solutions were prepared on the basis of the concentra-tion in commercial Matrixx preparations (pH 6.5) as follows: hydro-gen peroxide: 52 mM, peracetic acid: 14.5 mM, and octanoic acid:58 mM.

Statistical analysisThe experimental design was a randomized complete block with

2 replications (representing different spore batches) with a 4 × 3 × 3factorial design consisting of 4 different processing times (5, 10, 15,and 20 s), 3 concentrations of peracid (1.1%, 1.3%, and 1.5%), and 3temperatures (50, 60, 70 ◦C). Data were analyzed separately for eachof the 3 variables (time, temperature, and concentration). Thus,for each variable, the analysis consisted of 72 total observations(2 observations × 4 time × 3 concentrations × 3 temperatures).The Proc Mixed procedure of SAS (2003; version 9.1.3) was used toperform type-3 tests of fixed effects. Least squares means for pro-tected F-tests (P < 0.05) were separated by using least significantdifferences (LSD; P < 0.05).

Results and Discussion

Experiment 1—inactivation of sporesB. atrophaeus ATCC 9372 spores were used for testing because

of their known relatively high resistance to chemical disinfectants,peroxide-based sanitizers, and their wide environmental distribu-tion (McDonnell and Russell 1999; Driks 2003). The results of thedirect injection inactivation studies were as expected for a potentsporicidal agent: peracid had a deleterious effect on survival ofB. atrophaeus spores, and this effect was synergistically enhancedby increasing exposure time, temperature, or concentration of dis-infectant (Figure 1). Treatment of B. atrophaeus spores with peracidreduced survival (P < 0.05) and, under some conditions, reducedspore survival below the limits of detection. The highest concentra-tion of peracid tested (1.5%) yielded no survivors with 20 s of expo-sure. After 20 s of exposure at 50 ◦C, more than 2-logarithms of thespores were inactivated (P < 0.05) with 1.1% peracid (Figure 1A).Increasing disinfectant concentration to 1.3% yielded more than a3 logarithm reduction of B. atrophaeus spores, and 1.5% peracid re-duced spore survival below the limits of detection for the experi-ment (P < 0.05).

At higher temperatures and concentrations, B. atrophaeusspores showed a higher rate of inactivation. Spore death at 60 ◦Cwas more rapid than at 50 ◦C (P < 0.05) and 1.3% or 1.5% of peracid.No survivors remained after 15 s at 1.5% peracid and after 20 s ofexposure at 1.3% or 1.5% (Figure 1B). The initial phase of spore in-activation was faster at 60 ◦C than at 50 ◦C; and at 70 ◦C, spore inac-tivation was rapid, and no survivors were detected after 5 s at 1.5%and after 15 s at 1.1% and 1.3% disinfectant (Figure 1C). At highertemperatures and concentrations, B. atrophaeus spores showed ahigher rate of inactivation.

Surface-active effects, localized concentration of free radicals inthe vicinity of spore surfaces, or localized foaming properties of

the peroxide with the surfactant octanoic acid could have con-tributed to the rapid inactivation of the spores (Figure 1). TheB. atrophaeus inner spore coat is composed of multiple mem-brane layers and consists largely of protein (Driks 2003). Interac-tion of peroctanoic produced from the combination of peraceticand octanoic acid could have created easy access through the outerspore coat to the inner membrane, thus degrading the spore cor-tex and inner membrane proteins (Genest and others 2002; Rajuand others 2006). It is possible that the peracid ingredients actedsynergistically at elevated temperature and inactivated most of themembrane proteins because of either direct interaction of organicacid and peracetic acid or a high concentration of lethal free rad-icals generated from the peracid. Spore membrane damage couldhave permitted deeper penetration of hydroxyl and superoxide rad-icals from peracid, causing damage to spore DNA, enzymes, and vi-tal cellular organelles. Leaper (1984) suggested that peracetic acidpromotes oxidation of cellular material by acting on the cytoplas-mic membrane, disrupting its physiological role. He reported thatthe spores of B. atrophaeus might have been destroyed by hydroxylradicals formed by the decomposition of hydrogen peroxide. Palopand others (1998) reported that a combination of peracetic acidand hydrogen peroxide inactivated enzymes within intact sporesof Bacillus megaterium ATCC 19213. These researchers suggestedthat sensitivity of different enzymes to inactivation varied and eachagent inactivated enzymes at different rates due to differences inthe type of radical generated by each peroxide agent.

Setlow and others (1997) used organic hydroperoxide for the in-activation of B. atrophaeus spores and speculated that free radicalsemanating from hydroperoxide radicals were involved in the sporeinactivation. However, these researchers did not provide any evi-dence that rapid spore death occurred due to free radicals gener-ated by the hydroperoxides. Our study suggests the involvement offree radicals generated from a mixture of surface-active peracidsand prompted us to investigate using ESR spectroscopy to deter-mine whether the rapid inactivation of B. atrophaeus spores mightbe due to surface localized concentration of free radicals.

Experiment 2—ESR spectroscopy of peracidResults from ESR spectroscopy of peracid with BMPO pro-

duced a characteristic hydroxyl radical spectrum mixed with loweramounts of superoxide and alkyl radicals attributed to the pres-ence of both hydrogen peroxide and peracetic acid in the mix-ture (Figure 2A). Besides the combination of hydrogen peroxideand peracetic, other active ingredients of the disinfectant such asoctanoic acid could have contributed to the improved biocidalaction of the disinfectant mix by releasing superoxide and alkylradicals adjacent to the spore coat (Figure 2B). McDonnell andRussell (1999) reported that a mixture of peracetic acid and oc-tanoic formed peroctanoic acid, which acted as a stronger biocidethan hydrogen peroxide alone.

ESR spectroscopy of a mixture containing octanoic acid andhydrogen peroxide did not yield as relatively intense peak as thefull mixture (Figure 2A), and the combination of peracetic andoctanoic acid also did not yield as relatively an intense a peak(Figure 2B), suggesting that these solutions apparently did not pro-duce as high a concentration of free radicals as the combinationmixture containing all 3 reagents. When peracetic acid was mixedwith hydrogen peroxide and octanoic acid, the combination of the3 resulted in several orders of magnitude enhancement of peak in-tensity, indicating the presence of a high concentration of free rad-icals (Figure 2C). ESR spectroscopy of 1.5% peracid with the BMPOspin trap allowed the spectral characterization of the most pre-dominant radical present as hydroxyl radical (Figure 3). The ESR

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spectrum of 1.5% peracid shows the presence of hydroxyl radicalmixed with lower amounts of superoxide radicals and alkyl radicalsattributed to the presence of hydrogen peroxide and peracetic acidin the peracid mixture (Figure 3). Insert A of Figure 3 shows a typicalhydroxyl radical spectrum generated using pure hydrogen peroxideand BMPO.

The captured ESR spectral patterns in samples in which 10 μLof B. atrophaeus spores (107 CFU/mL) were added to the peracidmix, show a reduction in some of the peaks and a slightly al-tered pattern of peaks (Figure 4). The efficient generation of highconcentrations of localized radicals and other oxidizing speciesappears to be the primary cause for the inactivation of the

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Figure 1 --- Inactivation of spores of B.atrophaeus after 5, 10, 15, or 20 s ofexposure to various concentrations ofperacid at temperatures 50, 60, and70 ◦C.


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B. atrophaeus spores and the participation of produced localizedreactive intermediates at the interface between the spore surfaceand its aqueous peracid environment may have caused a reduc-tion and alteration of ESR peaks. Yu and Cheng (2008) suggestedthat changes in membrane structural integrity could alter the prop-erties of released radicals due to molecular collisions betweenthe formed radical species and the spin label compound, which

may effectively alter the line width and peak pattern of the ESRspectrum.

The octanoic acid, peracetic, and hydrogen peroxide mixtureseems to have a unique and potent antimicrobial effect on spores.One of the most striking characteristics of the peracid mixture wasthe high level of inactivation achieved at relatively short exposuretimes using elevated temperatures. The efficacy of octanoic acid

Figure 2 --- ESR spectrum from 2A) different combinations of peracid mix with spin adduct BMPO (a) hydrogen peroxideand octanoic acid, (b) peracetic acid with octanoic acid, and (c) hydrogen peroxide, peracetic acid, and octanoicacid; 2B) individual components (BMPO and hydrogen peroxide or octanoic acid or peracetic acid) and the spin trap(BMPO only).


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as an additive to this antimicrobial mixture should perhaps be at-tributed to its surface-active properties. We speculate that the pres-ence of octanoic acid in the peracid mixture might improve theefficacy and consequently biocidal action of the disinfectant mix-ture by contributing surface active effects that elevate the concen-tration of hydroxyl radicals adjacent to the spore surface (Clark2003). The present study suggests that localized or site-specific freeradical generation could increase lethality of antimicrobial peracidmixtures. We believe that it might be possible to elevate the bioci-dal effects of a particular sporicidal agent by selecting componentsof the mixture, which provide a localized enhancement of surfaceactivity.

Hydrogen peroxide and peracetic acid are potent biocides,which have a long history of usage for disinfection and sterilization.A recent trend in disinfection is to combine the 2 biocides in a singletreatment, both agents mediating their effects through the genera-tion of hydroxyl radicals. Surfactants are a class of molecules that

Figure 3 --- ESR spectrum of 1.5% Matrixx with spinadduct BMPO (5-tert-butoxycarbonyl 5-methyl-1-pyrrolineN-oxide) showing presence of hydroxyl radical with someother radicals. The insert (Box A) shows a typical hy-droxyl radical spectrum generated using pure hydrogenperoxide and BMPO.

Figure 4 --- ESR spectrum of the peracid mixture with spinadduct BMPO (bold lines) and the same mixture with abacterial spore suspension added (dotted line) showingthe change in peak and ESR spectral pattern after theaddition of bacterial spores into peracid.

because of their amphiphilic or bipolar nature find it energeticallyfavorable to form oriented layers at aqueous interfaces. For exam-ple, at the interface of a microorganism (or spore) and its wateryenvironment, octanoic acid will coat the microbial surface with itshydrophobic, nonpolar 8-unit alkyl carbon chain positioned in-wards (toward the microbe), and its hydrophilic, enolic, po-lar carboxyl-group outwards (toward the aqueous menstruum).Figure 5 shows an idealized conceptualization of the surfactantproperties of octanoic acid and their affects on molecular config-uration in the near-field region of the cell surface/aqueous phaseinterface. The model depicts a region extending less than a few tensof Angstroms beyond the cell surface. The model does not depict allthe hydrophobic and attractive forces favoring the molecular reori-entation and concentration of octanoic at the cell surface.

In the high hydroxyl radical environment provided by the rel-atively high concentrations of hydrogen peroxide and peraceticacid present in peracid, the enolic carboxyl-group of octanoic acidwould freely participate in hydroxyl radical transfer and exchangethrough the formation of its peracid (Figure 5; Insert A). Indeed,McDonnell and Russell (1999) report that a mixture of peraceticacid and octanoic forms peroctanoic acid, which acts as a strongerbiocide than hydrogen peroxide alone. The rapid and efficientspore inactivation demonstrated in this study support this observa-tion, and the ESR spectroscopic patterns showing a concentrationdependent production of free radicals (Figure 6) suggest a hydroxylradical rich environment populated by highly energetic superoxideand alkyl peroxide molecules in a seething equilibrium stew.

Peroctanoic would be expected to stabilize and enhance this sys-tem. The hydroxyl radical itself is notoriously reactive and shortlived; for example, on an electronegativity scale, the hydroxyl rad-ical is bested only by the fluorine radical. Diffusion being slowerthan the half-life of the hydroxyl radical, its reactive distance isestimated at less than about 10 Angstroms (or the distance of7 to 8 carbon bonds). Peroctanoic in contrast, among the ener-getic oxidizing molecules within the peracid mixture, would be ex-pected to be relatively more stable in terms of half-life and therefore

= Octanoic Acid

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capable of concentrating (and localizing) the oxidative reactivecapacity of the medium, due to its own concentration and local-ization at aqueous interfaces and the surface of cells. Peroctanoicwould also act to preserve the system due to its insensitivity to cata-lase and peroxidases.

Conclusions

This study examined the effects of treatment temperature andaddition of a surfactant (octanoic acid) to a disinfectant so-

lution of hydrogen peroxide and peracetic acid. As expected,increasing temperature increased the sporicidal effects of thismixture. We speculate that the presence of octanoic acid in theperacid mixture improves the biocidal action of the disinfectantmixture by contributing surface-active effects that lead to an en-hanced concentration of hydroxyl radicals adjacent to the spore

Figure 6 --- ESR spectrum of 1.1%, 1.3%, and 1.5% Matrixxsolution with spin adduct BMPO.

surface. This localized or site-specific concentration of free radicalswould be expected to increase the antimicrobial lethality of peracidmixtures. We believe that it is possible to elevate the biocidal effectsof a particular agent by tailoring components of the mixture so asto provide an enhancement of activity through surfactant or othereffects. Results from this study could find practical application forthe inactivation of highly resistant pathogenic spores by conven-tional sanitation methods. The use of wet heat with peroctanoicacid and hydrogen peroxide could be useful in sanitation for foodservice establishments, sterilization of medical equipment, and in-activation of bacterial spores present in food or on food-contactsurfaces.

AcknowledgmentsThe authors would like to acknowledge Dr. Howard J. Halpern andMr. Gene Barth, Center for EPR Imaging for in vivo Physiology, Univ.of Chicago, and Dr. Darsh T. Wasan, Illinois Inst. of Technology,Chicago, for their collaboration and help throughout this project.

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