Journal of Food Engineering 110 (2012) 493–496

Contents lists available at SciVerse ScienceDirect

Journal of Food Engineering

journal homepage: www.elsevier .com/ locate / j foodeng

Inactivation of Clostridium sporogenes spores on stainless-steel using heat andan organic acidic chemical agent

Jaesung Lee, Melvin A. Pascall ⇑Department of Food Science and Technology, Ohio State University, 2015 Fyffe Road, Columbus, OH 43210, USA

a r t i c l e i n f o

Article history:Received 29 July 2011Received in revised form 16 November 2011Accepted 11 December 2011Available online 28 December 2011

Keywords:Acidic sanitizerSterilizing agentsClostridium sporogenes sporesFood contact surface

0260-8774/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.jfoodeng.2011.12.012

⇑ Corresponding author. Tel.: +1 614 292 0287; faxE-mail address: pascall.1@osu.edu (M.A. Pascall).

a b s t r a c t

The efficacy of a food grade acidic chemical agent for the reduction of Clostridium sporogenes spores on astainless steel surface was investigated. The chemical agent was a combination of selected fatty acids andlactate esters. Distilled deionized water and 35% hydrogen peroxide were used as negative and positivecontrols, respectively. Approximately 3log cfu reductions in viable spore numbers were detected on thesteel surfaces for all treatments at room temperature, except the controls. Reductions in the viable sporenumbers significantly increased with increasing exposure times and concentrations of the acidic agent.Five log reduction of viable spore number was achieved after 10 min treatment with the 10% agent solu-tion at 68 �C. No viable spores were observed on the 10% agent treated sample after a 60 min exposuretime at 75 �C. This research showed that the acidic sanitizer tested in this study could be used to reducethe number of C. sporogenes spores on stainless steel surfaces.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Food spoilage and poisoning due to spore forming bacteria is achallenging problem for food processors because of the extremeresistance of spores to heat and disinfecting chemicals. Spores con-tain only the minimal life sustaining necessities such as DNA andan enzymatic machinery (Setlow, 1994). These are necessary forreproduction and give the spore the ability to withstand environ-mental stresses of heat, chemicals and dehydration. The ability toresist chemical penetration is mainly achieved by the hydrophobicnature of the spore’s external surface and the acid soluble proteinsin its central core region (Driks, 2002; Setlow, 2006).

Clostridium perfringens, Clostridium botulinum and Clostridiumdifficile are spore-forming anaerobic bacteria of public health con-cern that are capable of producing potent toxins. These organismsare commonly found in soil and can be sporadically isolated fromthe food supply (Genest et al., 2002). Although the vegetative cellsof Clostridium spp. can be killed during heat processing and sanitiz-ing treatment in food processing facilities, spores can survive andgrow rapidly in prepared food stored at ambient temperature.The safety of canned foods can be compromised if anaerobic sporeformers are present in under-processed cans or if post-processingcontamination occurs. These organisms are capable of producingdeadly neurotoxins in food which may deceptively appear to beunspoiled (Franciosa et al., 1999).

ll rights reserved.

: +1 614 292 0218.

Studies have shown that the elimination of spores is more diffi-cult when they are attached to food contact surfaces such as stain-less steel, glass and plastic materials (Blatchley et al., 2005). To beeffective against spores, disinfecting agents should be sufficientlyconcentrated and be given enough time to kill the organisms.Although spores of Clostridium spp. are strongly resistant to mostchemicals, they are still susceptible to inactivation by disinfectingagents with sporicidal activity. These chemicals include quaternaryammonium compounds, iodophors, phenols, glutaraldehyde, andoxidizing agents such as chlorine dioxide, peroxyacetic acid, hypo-chlorous acid and hydrogen peroxide (Russell and Loosemore,1964; Cupkova et al., 1981; Tennen et al., 2000; Loshon et al.,2001; Granum, 2002; Melly et al., 2002; Young and Setlow,2003). However, many of these chemicals are corrosive to equip-ment and toxic to humans if over exposure occurs. Thus, there isa need for food grade chemicals, without corrosive or toxic poten-tial, for use as sporicidal agents.

In an attempt to provide the food processing industry with suchchemicals, this study investigated esters of fatty acids acting asboth solvents and detergents that are expected to show microbici-dal activity against a broad spectrum of microorganisms. Initialstudies have shown that these chemicals have spore inactivationpotential (Lopes, 2007). These are non-toxic food grade chemicalsthat can significantly impact the food processing industry. Initialstudies have been undertaken to evaluate the efficacy of theseagents against Clostridium sporogenes spores on food contact sur-faces. Since that time, we have enhanced the composition of thesechemicals by adding detergents in order to produce spore inacti-vating capabilities but without toxic or corrosive potential. Thus,

494 J. Lee, M.A. Pascall / Journal of Food Engineering 110 (2012) 493–496

the objective of this study was to investigate the effectiveness ofthe acidic agent and heat on the viability of C. sporogenes sporeson stainless steel material.

2. Methods and materials

2.1. Spore preparation

C. sporogenes ATCC 7955 (PA 3679), obtained from The OhioState University Culture Collection Center, was used as the testorganism. When needed for testing, a loopful of the organismwas revived in 25 ml reinforced clostridial medium broth (RCB;Difco laboratories, Detroit, MI) and incubated for 48 h at 35 �C. Aportion (100 ll) of the 48 h culture was spread plated on rein-forced clostridial medium containing 1.5% agar (RCA, Difco). Thecells on the inoculated plates were grown for 9 days at 35 �C untilmore than 90% sporulation was observed by light microscopy. Thespores were harvested with sterile buffered peptone water (BPW,pH 7.2) by pipetting 10 ml onto the surface of each plate. To per-form this, the surface of each plate was scrapped using a sterileglass spreader, then aspirated by sterile pipetting with the BPW.The spores removed by the BPW were then washed three timesby centrifugation (Sorvall� RC5C Plus, Newtown, CT) at 10,000gfor 10 min at 4 �C then heated at 75 �C for 10 min in a water bathto eliminate any remaining vegetative cells. After washing by cen-trifugation, the spore pellets were resuspended in sterile deionizedwater to obtain �108 cfu/ml spores. They were then stored at 4 �Cuntil needed.

2.2. Spore inoculation on the stainless steel surface

Stainless steel can materials were supplied by Ball Corporation(Columbus, OH). The materials were cut into 4 � 4 cm squares. Thesteel sheets were autoclaved at 121 �C for 15 min then air dried. Aportion (100 ll, �108 cfu/surface) of the spore suspension wasevenly inoculated onto the surface of the steel sheets using a sterilepipette tip. The inoculated surfaces were dried in a laminar flowhood for �1.5 h at 24 �C before treatment.

2.3. Chemical agents

The acidic chemical agent was provided by Microcide Inc.(Detroit, MI). It was a liquid concentrate of esters fortified withfood grade detergents and food grade solvents and a FDA approvedsurface-active agent (sodium dodecylbenzene sulfonate). The 35%H2O2 was obtained from FMC Corporation (Philadelphia, PA).

2.4. Treatments

The sanitizers and control treatments included in this studywere: (1) autoclaved distilled deionized water (negative control);(2) 35% H2O2 (positive control); and (3) 1% (pH 3.2), 5% (pH 2.9)and 10% (pH 2.8) concentrations of the acidic chemical agent atroom temperature (23 �C), 68, and 75 �C. In treating the sporesinoculated onto the surface of the steel material they were im-mersed in the chemical treatments for 10, 30 and 60 min, thenthe surviving organisms enumerated. This immersion was donein sterile glass beakers. All treatments were done in duplicateand each observation tested twice. After each treatment, one setof steel sheet was placed in separate Petri dishes and directly over-laid with RCA containing selected neutralizers [0.07% lechthin, 0.5%Tween 80 and 0.1% sodium thiosulfate; Fisher Scientific (Fair Lawn,NJ)]. A second set of the steel sheet was plated using a dilutionmethod. This was done because of the difficulty in counting highmicrobial numbers in some of the samples. In this dilution method,

the treated samples were transferred to sterile tubes containing30 ml of the neutralizing buffer solution. This was then vigorouslyvortexed to resuspend the contents in the tube, after which thespores were serially diluted and pour plated using the RCA contain-ing the neutralizers. After 48 h anaerobic incubation at 35 �C, via-ble counts from the first and second sets of materials wereobtained. The detection limit for the spores was 1 cfu/stainlesssteel material.

The treatment method on the spores in the sanitizing solutionwas also performed to determine the effect of the chemical agenton planktonic spores. This was done by adjusting the spore num-bers to �106 cfu/ml spores in 20 ml of each sanitizing solution in50 ml sterile centrifuge tube for 10, 30 and 60 min at 23, 68 and75 �C. At the expiration of these times, the spores suspension ineach tube was serially diluted and pour plated using the RCA con-taining the neutralizers mentioned earlier. The detection limit forthe spores in each sanitizing solution was 2log10/20ml.

2.5. Statistical analysis

The data were statistically analyzed by equal-variance t-testusing a Microsoft Excel data analysis program (Ontario, Canada).The level of significance was set for P < 0.05. The statistical analy-ses were performed to determine the effect of concentration of thechemical treatments, exposure time and temperature on inactiva-tion of the spores.

3. Results and discussion

3.1. Survival of spores on the stainless steel surface prior to treatment

After drying at 24 �C on the material surfaces for �1.5 h, viablespore numbers were found to be �6.7log cfu/surface. Although weassumed many spores were removed from the surfaces by the lam-inar flow of air, the reduction in the number of viable spores duringthe drying step was about 0.6log cfu. Compared to the result ofprevious bacterial studies (Lee et al., 2007) where the reductionranged between 1 and 2log units for test Gram negative and Grampositive vegetative bacteria, the spores in this study showed great-er desiccation stability. Similar results were reported by Rönneret al. (1990) and Husmark and Rönner (1992), when they theorizedthat the relatively higher hydrophobicity of spores could be consid-ered an important factor which influences their attachment to foodcontact surfaces and survival in dried conditions.

3.2. Effect of the chemical treatments at room temperature

The effectiveness of water, H2O2, and chemical treatments forreduction of the viable spores on the steel sheets at room temper-ature is shown in Fig. 1. The materials immersed in de-ionizedwater (without acidic agent) had approximately 2.3log cfu/surfacereduction in numbers for all conditions. This reduction could havebeen attributed to a washing away of the un-attached or loosely at-tached spores. After 60 min immersion in water for the inoculatedmaterial surfaces, there were no significant (P > 0.05) differencesbetween the counts obtained for the spores. On the other hand,there were no viable spores on any of the material samples afterexposed to the 35% H2O2 for all exposure times. In the case ofthe pro-oxidant H2O2, the United States Food and Drug Administra-tion (FDA, 2011) regulations set a minimum concentration limit of35% that must be used as a sterilization agent in aseptic packaging.At the same time, however, its residual toxicity and corrosivenesshave been issues of concern.

In the preliminary part of this study, viability of the spores onthe test surfaces exposed to a 0.1% concentration of the acidic

Treatment at RT

0

1

2

3

4

5

6

7

8

0 10 30 60Time (min)

log

of c

fu/s

urfa

ce

water

1% acidic agent

5% acidic agent

10% acidic agent

35% hydrogen peroxide

Fig. 1. Viable counts (log10 cfu) of C. sporogenes spore on the stainless steel surfaceafter treatments at room temperature (RT).

Treatment at 68oC

0

1

2

3

4

5

6

7

8

0 10 30 60Time (min)

log

of c

fu/s

urfa

ce

water

1% acidic agent

5% acidic agent

10% acidic agent

35% hydrogen peroxide

Fig. 2. Viable counts (log10 cfu) of C. sporogenes spore on the stainless steel surfaceafter treatments at 68 �C.

Treatment at 75oC

0

1

2

3

4

5

6

7

8

0 10 30 60Time (min)

log

of c

fu/s

urfa

ce

water

1% acidic agent

5% acidic agent

10% acidic agent

35% hydrogen peroxide

Fig. 3. Viable counts (log10 cfu) of C. sporogenes spore on the stainless steel surfaceafter treatments at 75 �C.

J. Lee, M.A. Pascall / Journal of Food Engineering 110 (2012) 493–496 495

chemical agent were not significantly different (P > 0.05) fromthose exposed to the water (data not shown). Furthermore, a 10%concentration of the agent was shown to be more effective inreducing the viable spore numbers when compared with a treat-ment containing 100% concentration of the agent (data not shown).As a result, we decided to use concentrations of 1%, 5%, and 10% inthis present study. After 10 min exposure time, reductions in theviable numbers of spores on the steels sheets were 2.7 log cfu forthe 1% agent, and 3.8log for 5% and the 10% agents (Fig. 1). Whenthe effect of exposure times was considered, the results showedthat there were no significant differences (P > 0.05) between theexposure times (10, 30, 60 min) for the 1%, 5%, and 10% treatments.Similar results were obtained during our previous study using thesame acidic agent for Bacillus cereus spores on various food packag-ing materials (Lee et al., 2008), where we observed that there wereno significant reductions (P > 0.05) in the viability of the spores onthe steel surfaces at room temperature for all exposure times.

3.3. Effect of the acidic treatments at 68 and 75 �C

Figs. 2 and 3 show the effects of higher temperatures and thetreatments on the viable spores inoculated onto the steel materials.In comparison with the results for the room temperature treat-ments, the reductions in viable spore numbers after the de-ionizedwater treatment were 0.2log cfu greater at 68 �C and 0.4log cfugreater at the 75 �C conditions. Similar to the result for the roomtemperature treatments, there were no significant (P > 0.05) de-creases in the microbial numbers after the samples were treatedwith the water for all exposure times at each temperature.

When the acidic treatments were performed at the higher tem-peratures, they produced a noticeable decrease in the viabilities ofspores on the steel surface. At 68 �C for the 10 min condition,approximately 3.5 log cfu viable spores were eliminated after treat-ment with a 1% concentration treatment while a 4.5log cfu reduc-tion was obtained after the treatment with a 5% chemical agent.Approximately 5.0log cfu reductions were achieved for the 10%concentration treatment (Fig. 2). Except for the spores treated withthe 5% agent, reduction in the viability of the spores after the60 min treatment was significantly greater (P < 0.05) than that ofthe treated spores at 10 and 30 min. In addition to this, the treat-ment with 10% agent for 60 min reduced the viable spores by6log cfu. The application of a higher temperature (75 �C) to thetreatments (Fig. 3) thus markedly reduced the viable spores treatedwith the 1% and 5% agents for 60 min and the 10% agent for all

exposure times when compared with that of the 68 �C condition.After a 30 min exposure time, a 5log cfu reduction was achievedafter treatment with the 5% agent and no viable spores were ob-served on the 10% agent treated sample. These results supportthe ‘‘hurdle technology’’ concept, in which mild heating in con-junction with acidic chemical agents have been utilized to reducethe treatment requirements. The concept was previously appliedto inactivate bacterial spore in foods by combining heating withthe use of acidulants (Silla et al., 1992; Silla and Torres, 1995).

3.4. Viable spore numbers in sanitizing solutions after treatment

To confirm the inactivation ability of the sanitizers on the bac-terial spores, the presence of planktonic spores in dilutions of thesanitizers were enumerated. Table 1 shows the number of sporessurviving in each concentration of the sanitizing solution at differ-ent temperatures and exposure times. When compared with theviable counts for the untreated spores, less than 1log reduction oc-curred in all sanitizing solutions stored for 60 min at room temper-ature (Table 1). As was the case for the result of the spores on thesteel sheets, a significant increase in the rate of spore inactivationwas observed for the samples treated with the sanitizing solution

Table 1Viable counts (log10 cfu) of C. sporogenes spores in acidic sanitizing solutions after treatment at varying temperatures and times.

Time (min) Room temperature 68 �C 75 �C

1% 5% 10% 1% 5% 10% 1% 5% 10%

0 7.7 ± 0.1 7.7 ± 0.1 7.7 ± 0.1 7.7 ± 0.1 7.7 ± 0.1 7.7 ± 0.1 7.7 ± 0.1 7.7 ± 0.1 7.7 ± 0.110 7.5 ± 0.1 7.5 ± 0.1 7.2 ± 0.3 5.2 ± 0.2 4.7 ± 0.1 4.4 ± 0.3 2.9 ± 0.7 3.0 ± 0.7 ND30 7.5 ± 0.2 7.2 ± 0.2 7.1 ± 0.2 5.1 ± 0.2 4.7 ± 0.2 4.1 ± 0.2 2.4 ± 0.4 ND ND60 7.3 ± 0.2 7.3 ± 0.3 7.1 ± 0.4 4.0 ± 0.3 3.6 ± 0.3 2.3 ± 0.3 NDa ND ND

Values of the counts ate the mean ± SD of at least duplicated trials.a No colonies detected on the plate – a detection limit of 2log10 20ml�1.

496 J. Lee, M.A. Pascall / Journal of Food Engineering 110 (2012) 493–496

at high temperatures. Greater than 2.5log cfu reductions of viablespore numbers were detected in all treatments at 68 �C. Exceptfor the spores treated in the 1% and 5% sanitizing solutions for10 min, all treatments at 75 �C produced at least 5log cfu sporereduction (Table 1). These results suggest that most spores onthe steel sheets were eliminated by the inactivation ability of thesanitizer. This suggests that it did not occur by merely detachingthe spores from the treated surfaces.

4. Conclusions

Although further studies are needed to more fully explain themode of action of the chemical agent, this study demonstrated thata 5log (99.999%) reductions in viability of spores can be achievedafter the acidic chemical treatments when they are combined withmoderate heat. Our results can be used to help design systems withpotential to create microbiologically safe food contact surfaceswithout the need to use corrosive or toxic chemical agents.

Acknowledgments

The authors wish to thank the Center for Innovative Food Tech-nology for their financial support for this project and Yoon-Hee Leefor her help with the heat treatment part of this study. We alsothank Dr. John Lopes from Microcide Inc. for providing the acidicsanitizer used in this study.

References

Blatchley, E.R., Meeusen, A., Aronson, A.I., Brewster, L., 2005. Inactivation of Bacillusspores by ultraviolet or gamma radiation. Journal of Environmental Engineering131, 1245–1252.

Cupkova, V., Mlynarcik, D., Devinsky, F., Lacko, I., 1981. The effect of quaternaryammonium compounds and amine oxides on spores of Bacillus cereus.Microbiology (Praha) 26, 189–195.

Driks, A., 2002. Proteins of the spore core and coat. In: Sonenshein, A.L., Hoch, J.A.,Losick, R. (Eds.), Bacillus subtilis and its Closest Relatives: from Genes to Cells.American Society for Microbiology, Washington, DC, pp. 527–535.

Food and Drug Administration, 2011. Code of Federal Regulations (21 CFR178.1005). Indirect Food Additives: Adjuvants, Production Aids and Sanitizers.

<http://www.fda.gov/Food/GuidanceComplianceRegulatoryInformation/GuidanceDocuments/Juice/ucm072557.htm>. Accessed October 31 2011.

Franciosa, G., Pourshaban, M., Ferrini, A.M., Mannoni, V., de Luca, G., Aureli, P., 1999.Clostridium botulinum spore and toxin in mascarpone cheese and other milkproducts. Journal of Food Protection 62, 867–871.

Genest, P.C., Setlow, B., Melly, E., Setlow, P., 2002. Killing of spores of Bacillus subtilisby peroxynitrite appears to be caused by membrane damage. Microbiology 148,307–314.

Granum, P.E., 2002. Bacillus cereus. In: Doyle, M., Beuchat, L., Montville, T. (Eds.),Food Microbiology. Fundamentals and Frontiers, 2nd ed. American Society forMicrobiology, Washington, DC, pp. 373–381.

Husmark, U., Rönner, U., 1992. The influence of hydrophobic, electrostatic andmorphological properties on the adhesion of Bacillus spores. Biofouling 5, 335–344.

Lee, J., Lopes, J., Pascall, M.A., 2007. Development of a sanitizing fabric wipe for useon food contact surfaces. Journal of Food Science 72, M375–M381.

Lee, J., Pascall, M.A., Lopes, J., 2008. Efficacy of an acidic sporicidal agent for Bacilluscereus spore reduction on food packaging materials. In: IFT Annual Meeting,New Orleans.

Lopes, J.A., 2007. The sporicidal properties of PRO-SAN, a non-oxidizing organicagent. Unpublished data.

Loshon, C.A., Melly, E., Setlow, B., Setlow, P., 2001. Analysis of killing of spores ofBacillus subtilis by a new disinfectant, Sterilox�. Journal of Applied Microbiology91, 1051–1058.

Melly, E., Cowan, A.E., Setlow, P., 2002. Studies on the mechanism of killing ofBacillus subtilis spores by hydrogen peroxide. Journal of Applied Microbiology93, 316–325.

Rönner, U., Husmark, U., Henriksson, A., 1990. Adhesion of Bacillus spores in relationto hydrophobicity. Journal of Applied Bacteriology 69, 550–556.

Russell, A.D., Loosemore, M., 1964. Effect of phenol on Bacillus subtilis spores atelevated temperatures. Applied Microbiology 12, 403–406.

Setlow, P., 1994. Mechanisms which contribute to the long-term survival of sporesof Bacillus species. Journal of Applied Bacteriology 176, 49S–60S.

Setlow, P., 2006. Spores of Bacillus subtilis: their resistance to and killing byradiation, heat and chemicals. Journal of Applied Microbiology 101,514–525.

Silla, M.H., Núñez, H., Casado, A., Rodrigo, M., 1992. The effect of pH on the heatresistance of Clostridium sporogenes (PA 3679) in asparagus puree acidified withcitric acid and glucono-d-lactone. International Journal of Food Microbiology 16,275–281.

Silla, M.H., Torres, J., 1995. Glucono-d-lactone and citric acid as acidulants forlowering the heat resistance of Clostridium sporogenes PA 3679 in HTST workingconditions. International Journal of Food Microbiology 25, 191–197.

Tennen, R., Setlow, B., Davis, K.L., Loshon, C.A., Setlow, P., 2000. Mechanisms ofkilling of spores of Bacillus subtilis by iodine, glutaraldehyde and nitrous acid.Journal of Applied Microbiology 89, 330–338.

Young, S.B., Setlow, P., 2003. Mechanisms of killing of Bacillus subtilis spores byhypochlorite and chlorine dioxide. Journal of Applied Microbiology 95,54–67.