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Food Microbiology 38 (2014) 128e136

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Food Microbiology
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Microbial decontamination of red pepper powder by cold plasma

Jung Eun Kim a, Dong-Un Lee b, Sea C. Min a, *
a b

Department of Food Science and Technology, Seoul Womens University, 621 Hwarangro, Nowon-gu, Seoul 139-774, Republic of Korea Department of Food Science and Technology, Chung-Ang University, 72-1 Nae-ri, Daedeok-myeon, Ansung-si, Gyunggi-do 456-756, Republic of Korea

a r t i c l e i n f o
Article history: Received 15 May 2013 Received in revised form 5 August 2013 Accepted 29 August 2013 Available online 11 September 2013 Keywords: Cold plasma Nonthermal process Red pepper Bacillus cereus Fermis model Weibull model

a b s t r a c t
Effects of the microwave-powered cold plasma treatments (CPTs) on the inhibition of microorganisms in red pepper powder, including Aspergillus avus and Bacillus cereus spores, were investigated. Combinations of heat treatment with CPT were investigated for the inhibition of B. cereus spores on the powder. The number of A. avus was reduced by 2.5 0.3 log spores/g by the CPT with nitrogen at 900 W and 667 Pa for 20 min. CPT at 900 W and 667 Pa for 20 min inhibited naturally occurring total aerobic bacteria in the red pepper powder by approximately 1 log CFU/g. B. cereus spores were inhibited (3.4 0.7 log spores/g reduction) only when heat treatment at 90  C for 30 min was integrated with the CPT using a helium-oxygen gas mixture at 900 W. Fermis model and Weibull model adequately described the inhibition of A. avus on the red pepper powder by the CPT. The changes in treatment temperature and water activity were less than 5.0  C (initial temperature: 23.8  C) and 0.22, respectively, and were affected by both treatment power and time (P < 0.05). The CPTs have demonstrated the potential to reduce the microbial counts of red pepper powder and other powder products. 2013 Elsevier Ltd. All rights reserved.

1. Introduction Red pepper (Capsicum annuum L.) is cultivated worldwide and consumed fresh or in dried powdered form as a food ingredient (Rico et al., 2010). Red pepper powder is primarily used to impart a bright red color with a pungent taste and enhance the avor of many processed food products (Akbas and Ozdemir, 2008; Rico et al., 2010). Red pepper powder is of agricultural origin and therefore is generally highly contaminated by microorganisms before any decontamination processes (Buckenhuskes and Rendlen, 2004; Oularbi and Mansouri, 1996). The use of contaminated red pepper powder can result in rapid spoilage of the foods to which the powder was applied. Pathogenic microorganisms, including Aspergillus avus, Bacillus cereus, Clostridium perfringens, and Staphylococcus aureus are often present in red pepper powder (Aydin et al., 2007; Buckenhuskes and Rendlen, 2004). Fumigation with ethylene oxide, irradiation, steam heat sterilization, and ultraviolet (UV) treatments are used to decontaminate undesirable microorganisms in red pepper powder (Schweiggert et al., 2007). Fumigation with ethylene oxide, the technique used for the longest period, effectively inhibits microbes. However, its use is prohibited in many countries due to carcinogenicity (Fowles et al.,

* Corresponding author. Tel.: 82 2 970 5635; fax: 82 2 970 5977. E-mail addresses:, (S.C. Min). 0740-0020/$ e see front matter 2013 Elsevier Ltd. All rights reserved.

2001; Schweiggert et al., 2007). Gamma irradiation at 2e7 kGy has been shown to effectively decontaminate various spices (Farkas, 1998). However, owing to public fear and the legal regulation for the products treated by irradiation or those containing any irradiated ingredients, irradiated red pepper powder is unpopular in some countries. An alternative method is thermal treatment using superheated steam. However, this method is expensive and causes the powder to undergo undesirable sensory and nutritional changes (Moisan et al., 2001a). Although steaming is effective for decontamination, the treatment is usually applied before grinding of dried pepper. Thus, the product can be re-contaminated during grinding (Schweiggert et al., 2007) and requires an additional decontamination step prior to packaging. UV lamps have been installed in many powder production lines but do not inhibit the growth of microbes effectively during production (Fine and Gervais, 2004; Sharma and Demirci, 2003). The ineffectiveness of the UV radiation could be due to the lack of penetration, the strong dependence on the distance from the UV source, and/or signicant absorption of irradiation by glass and plastics, which can result in nonhomogeneous microbial decontamination (Song et al., 2010). CPT, which generates plasma by gas excitation with electron discharge without a marked temperature increase, has been investigated as a non-thermal food preservation method (Niemira, 2012; Perni et al., 2008). Plasma consists of highly energetic species that break covalent bonds and initiate various chemical reactions. These species include UV photons, electrons, positive and negative

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ions, free radicals, and excited or non-excited molecules and atoms, which in combination can inhibit microorganisms, more effectively (Fernandez et al., 2011; Song et al., 2010). Diffusion of the reactive species through cell membranes can cause severe damage by reacting with macromolecules, such as membrane lipids, proteins, and nucleic acids. Electrons, ions, and free radicals can also cause surface erosion and localized lesions in the cell membrane; these result in inhibition of microorganisms. The formation of surface erosion and localized lesions may also facilitate penetration of the reactive species into cells, which can enhance microbial inhibition (Gallagher et al., 2007). The UV also can cause DNA modications and consequent improper cell replication (Bolshakov et al., 2004). Oxidation of cell membranes and amino acids due to reactive oxygen and nitrogen species, including atomic oxygen, ozone, hydroxyl, nitric oxide, and nitrogen dioxide, is also an important mechanism of microbial inhibition mediated by cold plasma (Laroussi and Leipold, 2004). Although several studies have demonstrated the microbial inhibition effects of CPTs, little is known about their effect on microorganisms in foodstuffs, particularly powder food products. Thus, the objectives of this study were to (1) investigate the microbial inhibition effects of CPT on the inhibition of naturallyoccurring aerobic microorganisms in red pepper powder and A. avus and B. cereus spores inoculated on the powder using a microwave-powered CPT system; (2) optimize treatment conditions for the inhibition of A. avus; (3) evaluate deterministic models of A. avus inhibition by CPT; and (4) assess the effect of CPT integrated with heat treatment against B. cereus spores. 2. Materials and methods

inoculum concentration. The method of Finley and Fields (1962) was adopted to prepare B. cereus spores. Cell suspensions grown for 24 h at 37  C in TSB (0.1 mL) were spread onto tryptic soy agar (TSA, Difco) and incubated for 7 days at 37  C until at least 80% of the cells sporulated, as determined by microscopic examination. Spores were harvested by depositing 2 mL of 0.1% (w/v) sterile peptone water onto the surface of TSA culture plates. Spores were dislodged by gently rubbing with a sterile loop. Pooled suspensions of ve plates of each strain were transferred to a 15-mL tube and heated in an 80  C water bath for 10 min (including a 1-min warm-up time). Heat-treated suspensions were centrifuged at 3600 g at 4  C for 20 min and washed three times with 0.1% peptone water. The pellet of each strain was resuspended in 0.1% peptone water. A microbial cocktail was prepared by combining each strain in equal proportions to produce an inoculum of approximately 108 spores/mL. The cocktail was diluted in 0.1% (w/v) sterile peptone water to produce the desired inoculum concentration. 2.3. Inoculation and sample preparation Red pepper powder (200 g) in a 250-mL glass bottle was autoclaved for 15 min at 121  C. The powder (5.0 g) was spread evenly over a 16 cm 16 cm area of the surface of a Teon plate (25 cm 25 cm). A 1.0-g suspension of A. avus or B. cereus spores was inoculated onto the powder using a sterile glass sprayer (BT1270S-100, Joylab Co., Seoul, Korea) and then dried in a laminar ow biohazard hood for 1 h at 22 2  C. Non-autoclaved and noninoculated powder (5.0 g) was also prepared to evaluate the effect of CPT on naturally occurring aerobic microorganisms. 2.4. Cold plasma treatment system

2.1. Red pepper powder Red pepper powder (C. annuum L.) was purchased from a local store. The powder was prepared using the following steps: red pepper harvested in 2012 in Goesan (Korea) was washed in water and cut in half after removing the stalk. The half-cut red pepper was dried for 2 h using a far-infrared dryer at 83  C and ground into ne powder (>1.7 mm). The powder was exposed to UV (320 W, 10 s) prior to packaging. 2.2. Microbial strains and preparation of inoculum subculture A. avus (ATCC 200026, American Type Culture Collection, Manassas, VA, USA) was cultured for 5 days at 20  C on potato dextrose agar (PDA, Difco, Sparks, MD, USA) acidied with 10% tartaric acid (SigmaeAldrich, St. Louis, MO, USA). Sterile distilled water (10 mL) containing 0.1% Tween 80 was added to the PDA for growth of A. avus. The PDA surface was gently scraped with an inoculation loop and the content was transferred to a sterile 15-mL tube (SPL Life Science Co., Pocheon, Korea). The tube was shaken vigorously to liberate spores. The shaken suspension was ltered through sterile cloth and spores ltered were enumerated using a hemocytometer (Paul Marienfeld GmbH & Co. KG, LaudaKonigshofen, Germany). Spore density was adjusted by dilution with 0.1% peptone water (Difco). B. cereus ATCC 10876, ATCC 13061, and W-1 were obtained from the Agricultural Biotechnology Culture Collection at Seoul National University (Seoul, Korea). B. cereus vegetative cells were cultured for 24 h at 37  C in tryptic soy broth (TSB, Difco), harvested by centrifugation at 10,000 rpm for 2 min, and washed twice with 0.1% (w/v) sterile peptone water. The pellets of each strain were resuspended in 0.1% peptone water, corresponding to approximately 108 CFU/mL. The cocktail was prepared by combining equal amounts of each strain, and diluted with 0.1% peptone water to produce the desired The SWU-2 CPT system, illustrated in Fig. 1, consists of a microwave generator, a cooling system, a treatment chamber, a gas mass ow rate controller, a vacuum pump, and a parameter controller (Fig. 1). The magnetron (Magnetron 2M246, LG electronics Inc., Seoul, Korea) in the microwave generator produces a 2.45-GHz wave discharge operated at the 50e1000 W power levels. The treatment chamber is of stainless steel and has dimensions of 43 cm (width) 37 cm (height) 40 cm (length) with a fused silica (quartz) observation window. Cooling water ows at 0.8 m3/min. The plasma-forming gas ows at a maximum of 20 slm (standard liter/min), which is controlled by a gas mass ow-rate controller (2 channels, Model 3660, Kojima Instrument Inc., Osaka, Japan). The pressure in the chamber ranges from 500 to 30,000 Pa, adjusted by a vacuum valve (Model 2-way electric ball valve, DongjooAP, Incheon, Korea). The parameter controller monitors and regulates supplied power (treatment power), gas ow rates, and pressure in the treatment chamber (treatment pressure). 2.5. Cold plasma treatments (CPTs) 2.5.1. Determination of the conditions for forming stable plasma The conditions used for formation of stable plasma for the treatment of red pepper powder were determined for each plasmaforming gas treatment power and pressure. The feed gases for plasma emission were nitrogen (N2), an N2-oxygen (O2) mixture (N2:O2 99.3:0.7), helium (He), and a HeeO2 mixture (He:O2 99.8:0.2). The formulations for the mixtures of N2eO2 and HeeO2 were selected based on the studies of Pintassilgo et al. (2007) and Hong et al. (2009), respectively. The gases were dried and ltered. The power and pressure ranges were 300e900 W and 267e26,680 Pa, respectively. Powder (5.0 g) was evenly spread on the Teon plate and treated with cold plasma. Plasma was observed through the observation window (Fig. 1).


J.E. Kim et al. / Food Microbiology 38 (2014) 128e136

2.6. Combined treatments of heat and cold plasma for B. cereus spore inhibition Heat treatments and CPTs were combined and their effects on B. cereus spores determined. The heat treatment was conducted in a water bath by immersing 5.0 g of red pepper powder in a stomacher bag (19 30 cm, Nasco WHIRL-PAK, Fort Atkinson, WI.). The treatment temperatures and times were 70, 80, and 90  C and 10, 30, and 60 min, respectively. The warm-up-times to reach 70, 80, and 90  C were 2.3, 2.7, and 3.0 min, respectively. The heat-treated powder was then subjected to cold plasma formed using N2, the N2eO2 mixture, He, or the HeeO2 mixture at 1 L/min. The treatment power, pressure, and time were 900 W, 667 Pa, and 20 min, respectively.

2.7. Temperature and water activity measurements of red pepper powder Immediately before and after CPT, the temperature of the plasma-treated surface of the red pepper powder was measured using an infrared thermometer (DT 44L, DIAS Infrared GmbH, Dresden, Germany), calibrated with a thermocouple (Type K, 1.6mm diameter, Fisher Scientic) (Fig. 1). The water activity values of red pepper powder were measured using a water activity meter (Pawkit water activity meter, Decagon Devices Inc., Pullman, WA, USA) right before and after the treatment.

2.8. Microbiological analyses

Fig. 1. Schematic diagram of the cold plasma treatment system.

2.5.2. Microbial inhibition The gases of N2, N2eO2 mixture, He, and HeeO2 mixture were used to form plasma. The pressure in the chamber and the ow rate of the gas were maintained at 667 Pa and 1 L/min, respectively. The treatment powers and times used to evaluate the microbial inhibition effect of CPT against A. avus were determined using the response surface method (Table 1). The explanatory variables were treatment power (X1, 400, 474, 650, 828, 900 W) and treatment time (X2, 4.0, 6.4, 12.5, 18.6, 20 min). The response variable was the level of microbial reduction.

Red pepper powder samples were diluted 10-fold with 0.1% peptone water in stomacher bags (19 30 cm, Nasco WHIRL-PAK) and pummeled for 3 min by a stomacher blender (Stomacher Lab Blender Model 400, Seward Medical, London U.K.) at 230 5% rpm. The homogenate was serially diluted and plated (100 mL or 1 mL) on PDA, TSA, or mannitol-egg yolk-polymyxin (Difco) supplemented with polymyxin B (Antimicrobic Vial P, Difco) (MYPP), and/or plate count agar (PCA, Difco). PDA, MYPP, and PCA were used to enumerate A. avus, B. cereus, and total aerobic microorganisms, respectively. TSA was used to enumerate B. cereus inoculated on autoclaved red pepper powder. PDA plates were incubated for 5 days at 20  C. TSA and MYPP plates were incubated for 48 h at 37  C and PCA plates were incubated for 48 h at 35  C.

Table 1 Experimental variables and their values for the determination of optimum cold plasma treatmenta conditions for inhibiting A. avus on red pepper powder. Experiment number Explanatory variables e treatment power (W): X1, C1; treatment time (min): X2, C2 Coded value X1 1 2 3 4 5 6 7 8 9 10 11 12 13

Response variable Reduction (log spores/g) C2 12.5 18.6 6.4 12.5 12.5 12.5 12.5 12.5 20 4 6.4 18.6 12.5 0.5 0.6 0.6 0.7 0.7 0.7 0.7 0.7 0.7 0.5 1.1 1.4 1.5 0.1 0.0 0.1 0.0 0.1 0.0 0.0 0.0 0.1 0.0 0.2 0.1 0.1

Real value X2 0 1 1 0 0 0 0 0 1.4 - 1.4 1 1 0 C1 400 474 474 650 650 650 650 650 650 650 828 828 900

1.4 1 1 0 0 0 0 0 0 0 1 1 1.4

Nitrogen was used as the plasma-forming gas.

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2.9. Scanning electron microscopy (SEM) Red pepper powder, inoculated with B. cereus spores (approximately 6 log spores/g), was treated with heat (90  C, 30 min) and cold plasma (HeeO2 gas, 900 W, 20 min) in sequence. The microscopy specimens were prepared from the suspensions of B. cereus spores following the method of Cheng and Huber (1996). The specimens were sputter-coated with platinum and viewed by scanning electron microscopy (Field emission-SEM, FE-SEM, S4700, Hitachi, Tokyo, Japan). 2.10. Models of A. avus inhibition by cold plasma The residual microbial concentration (RM) after CPT was dened as:

treatment power and time on the inhibition of A. avus was designed using RSM by Minitab (ver. 15, Minitab, Inc., State College, PA). A two variable second-order center composite RSM design was used to show the interactions of the variables on the inhibition of A. avus in 13 runs, of which 5 were for the center point (Table 1) (Nath et al., 2007). The effect of individual linear, quadratic, and interaction terms was determined using SAS.
2 2 Y b0 b1 X1 b2 X2 b12 X1 X2 b11 X1 b22 X2




where bn are constant regression coefcients, Y is the A. avus reduction (log spores/g), X1 and X2 are the treatment power (W) and time (min), respectively. The optimum conditions for inhibiting A. avus were determined using the Response optimizer function from Minitab. ANOVA and regression analyses were conducted to estimate the model parameters (k1, k2, Ph, and a). 3. Results and discussion 3.1. CPT conditions for stable plasma formation The treatment time and pressure values that resulted in stable plasma formation using each gas are presented in Table 2. Stable plasma was dened if no corona was formed or if plasma was formed without ickering in the treatment chamber. Formation of stable plasma was affected by power and pressure, regardless of the gas used. The power and pressure zones depended upon the type of gas. No matter which gas was used, the combination of high power with low pressure resulted in stable plasma formation. This is in agreement with a previous report that broader and stable plasma was formed by a microwave-powered system with higher power and lower pressure when a mixed gas comprising hydrogen, methane, and nitrogen, and 600e1600-W power and 9000e 21,000-Pa pressure were applied (Yamada et al., 2006). Helium was excited into the plasma state within the narrowest power and pressure ranges under these experimental conditions. Electron discharges for plasma formation are commonly produced by radiofrequency source of power. Microwave can replace the role of radiofrequency. Microwave-generated plasma can result in a higher microbial reduction and a faster processing time than radiofrequency-generated plasma due to a higher energy density of microwave (Abidi and Hequet, 2004). 3.2. A. avus inhibition CPT at 900 W for 20 min with N2, He, and the N2eO2 and Hee O2 mixtures inhibited A. avus inoculated onto red pepper powder by 2.5 0.3, 2.0 0.3, 0.4 0.1, and 0.3 0.1 log spores/ g, respectively. Treatment with N2 resulted in the greatest reduction (P < 0.05). This might be due to potential formation of reactive nitrogen species (RNS) in addition to other reactive species, including UV photons, electrons, and those formed with water molecules evaporated from the powder, such as H atom and oxygen reactive species (e.g., hydroxyl radicals). These reactive species likely oxidized glycoproteins in the fungal cells, resulting in growth inhibition (Gander, 1974). Plasma formed using He and the HeeO2 mixture also yielded signicant reductions in the number of cells (P < 0.05). This could be attributable to the activities of He- and O2-driven reactive species, such as electrons, He, He2, O2, O 2 , O, O3, and metastable state O2 (Lu et al., 2008). Incorporation of oxygen in plasma-forming gases is thought to enhance microbial inhibition by inducing oxygen radicals; these attack the cell membrane via various mechanisms, including etching and oxidizing amino acids,

where A is the microbial concentration after CPT and A0 is the initial microbial concentration before the treatment. Experimental data were t to the rst-order models in Equations (2) and (4), and Fermis kinetic model (Peleg, 1995) in Equation (3) by Minitab (ver. 15, Minitab, Inc., State College, PA, USA), and the Weibull model in Equation (5) by GInaFiT (Geeraerd and Van Impe Inactivation Model Fitting Tool).

lnRM k1 P


where P is the power (W) and k1 is the rst-order kinetic constant.


1   Ph 1 exp P a


where Ph is the treatment power (W), RM is 0.5, and a is the parameter indicating the slope of the curve around Ph.

lnRM k2 t


where t is the treatment time (min) and k2 is the rst-order kinetic constant.

logN logN0 t =d


where d is the scale parameter and s is the shape parameter. 2.11. Surface and extractable color evaluation A colorimeter (Minolta Chroma Meter CR-400, Minolta Camera Co., Osaka, Japan) was used to measure L*, a*, and b* values by CIELab coordinates for the surface of the red pepper powder. The colorimeter was calibrated using white and black standard tiles, Illuminate D 65, and a 10 standard observer. The extractable color of the red pepper powder sample was evaluated using the American Spice Trade Association (ASTA, 1968) method. 2.12. Experimental design and statistical analyses All experiments were performed in triplicate. Each observation within each replicate was determined in duplicate. Analysis of variance (ANOVA) was used to evaluate differences between means and if signicant differences were observed, Duncans multiple range tests were used to evaluate the means to estimate the signicant difference (a 0.05) (SAS version 9.2, SAS Institute Inc., Raleigh, NC). Pearsons correlation coefcients among the A. avus population, treatment power, and treatment time were determined using the SAS software. The experiment studying the effects of

132 Table 2

J.E. Kim et al. / Food Microbiology 38 (2014) 128e136

Formation of cold plasma at various power and pressure levels with different gasesa
Stable plasma formationb Unstable plasma formationc No plasma formation


Power (W) 900 800 700 600 500 400 300 900 800 700 600 500 400 300 900 800 700 600 500 400 300 900 800 700 600 500 400 300

Pressure (Pa) 267 667 800 1,067 1,334 2,668 4,002 6,670 13,340 26,680


Unstable plasma

Nitrogenoxygen (99.3:0.7)

Unstable plasma


Unstable plasma

Heliumoxygen (99.8:0.2)

Unstable plasma

Flow rate: 1 L/min Plasma without flickering and changing in size. No corona formation. c Plasma not steady and changing in size.

nucleic acids, and lipids (Lee et al., 2006; Pintassilgo et al., 2007; Song et al., 2010). However, use of oxygen-containing gas did not enhance microbial inhibition in the current study. Oxygen radicals could have been produced from O2 already present in the chamber or from water evaporated from the red pepper powder. This might have been sufcient to maximally inhibit A. avus in combination with other plasma reactive species under the given conditions. Correlations among treatment power, treatment time, and reduction in A. avus numbers were evaluated with plasma generated using N2, which yielded the greatest inhibition of A. avus. Pearsons correlation coefcient indicated that each treatment variable (treatment power or time) and the A. avus reduction had positive correlations (power: 0.840 (P < 0.0001); time: 0.744 (P < 0.001)). Increases in treatment power and time led to more efcient inhibition of A. avus. Although greater inhibition could be achieved by increasing treatment power and time, in practice, technological challenges and limitations, cost, and negative impact on quality must be considered (Deng et al., 2007). The response surface model for A. avus reduction (Y) was as follows: Y (3.875160) (0.010226)X1 (0.113244) 2 X2 (0.000162)X1X2 (0.000007879)X2 1 (0.001207)X2. The var2 iables were well explained by the model (R 0.89, P < 0.001). The

negative coefcients of the rst-order terms of X1 and X2 indicate that A. avus inhibition was enhanced with increasing treatment power and time. The P value for the interaction term (X1X2) was <0.0001, indicating the interaction of the power and the time is valid for the A. avus reduction. The reduction could increase by the interaction. The variation of the A. avus reduction by the CPT with respect to the treatment power and time is presented using a surface plot (Fig. 2). Microbial log reduction increased with increased input power. The reduction would be less than 1 log spores/g at <700 W. Treatment with 800 W or greater could result in signicant reductions compared to the untreated control, with higher power being more effective (Fig. 2). The inhibition appeared to depend on the treatment time: the longest time (20 min) resulted in the highest reduction. The curves for treatments with a power >800 W were biphasic (Fig. 2). CPT survival curves are characterized by the presence of two or three linear segments (Philip et al., 2002). Biphasic survival curves were observed for S. aureus and Escherichia coli on plasma-treated fabrics. Wintenberg-Kelly et al. (1999) hypothesized that during the rst inhibition segment, active species diffuse through the cell surface and generate alterations, followed by irreversible damage and cell lysis during the second inhibition segment due to the high concentrations of active species. The rst phase, demonstrated by the steeper slope during the early period, may be dominated by UV damage whereas the

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evaporation of moisture from the powder, facilitated by the formation of low pressure (667 Pa). The moisture evaporated could result in formation of oxygen reactive species in the plasma. The lowered aw may benet the powder product by enhancing its microbiological stability. The temperature of the red pepper powder increased from 23.8 to 28.5  C (DT 4.7  C) after CPT at 900 W for 20 min, which resulted in the greatest reduction in spore number (2.5 0.3 log spores/g) (Fig. 4). Microbial inhibition by heat can be disregarded because the temperature of the surface remained below 30  C at the highest power level. This indicates that A. avus in the powder was inhibited non-thermally by cold plasma.

3.3. Inhibition of total aerobic microorganisms CPT at 900 W for 20 min with N2, the N2eO2 mixture, He, and the HeeO2 mixture inhibited naturally occurring aerobic bacteria in red pepper powder (5.9 0.1 CFU/g) by approximately 1 log CFU/g. No signicant differences in reduction were observed among the different treatments (P > 0.05). Kim et al. (2011) reported a 4.5 log CFU/g reduction in total aerobic bacteria on bacon by cold plasma treatment using a helium-oxygen mixture at 125 W and atmospheric pressure for 1.5 min. A greater inhibition than that we report here was achieved using a lower energy level. The differences in the CPT systems and the type of bacteria, as well as the aw of the samples, may have resulted in the variation in microbial inhibition. Wintenberg-Kelly et al. (1998) reported that plasma was more effective against wet-mounted cultures on surfaces, and that longer-duration exposure was required to achieve the same inhibition levels for dry-mounted cultures. The red pepper that was used as the raw material for the powder was supplied from the same company from which the powder was purchased. The aw of the pepper was 0.98 and the initial total aerobic plate count was 7.4 0.1 log CFU/g. CPT with N2 at 900 W for 20 min reduced the count by 2.7 0.1 log CFU/g. Assuming that the naturally occurring microora in both red pepper and dried red pepper powder are identical, it can be concluded that aw had a marked effect on the inhibition of total aerobic bacteria on the red pepper powder. The number of B. cereus spores in the red pepper powder was examined to elucidate the possibility that the resistance of the bacteria against the CPT might be induced by the presence of the spores. The 3.6 0.2 log spores of B. cereus were detected from 1 g of the red pepper powder. The inhibition of the spores is vital if CPT

Fig. 2. Response surface plots generated with treatment power, time, and the reduction of A. avus by the cold plasma treatments using nitrogen.

second phase may be dominated by erosion mediated by reactive species (Moisan et al., 2001b). The goodness-of-t of the inhibition models was compared using R2. The R2 of the rst-order model with respect to the power (Equation (2)), Fermis model (Equation (3)), the rst-order kinetic model (Equation (4)), and Weibull model (Equation (5)) were 0.69, 0.88, 0.61, and 0.91, respectively. Linear models (Equations (2) and (4)) did not t the inhibition data as well as the non-linear models (Equations (3) and (5)). This could be due to the biphasic nature of the survival curves (Perni et al., 2006). Fermis model with Ph and a (Ph 1,092, a 218) and the Weibull model with parameter values (d 63.96, s 0.24) adequately described A. avus inhibition on red pepper powder by CPT. The predicted optimum treatment power and time were 900 W and 20 min, which were the highest values used in this study. The predicted reduction (2.5 log spores/g) agreed with the empirical value (2.5 0.3 log spores/g). The D value, the exposure time required to inhibit 90% of the population, of plasma treatment at 650 W was 26.7 min. The aw of the red pepper powder decreased as the treatment time increased at 650 and 826 W (Fig. 3). This might be due to

Fig. 3. Effect of the treatment time and power on the water activity of red pepper powder with cold plasma treatment using nitrogen.

Fig. 4. Effect of treatment time and power on the surface temperature of red pepper powder with cold plasma treatment using nitrogen.


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is to be considered an effective means of decontaminating red pepper powder. 3.4. Inhibition of B. cereus spores CPTs at 900 W for 20 min with N2, the N2eO2 mixture, He, and the HeeO2 mixture did not inhibit B. cereus spores inoculated onto red pepper powder. Philip et al. (2002) reported effective inhibition of Bacillus subtilis spores on polystyrene by CPTs at 100 W using N2eO2 mixtures. Hong et al. (2009) sterilized B. subtilis spores on a cover glass by CPT at 75 W with HeeO2 mixtures. Roth et al. (2010) also reported the reduction of B. subtilis spores on glass slides (2.3 log spores/g) by the CPT at 4000 W using a mixture of N2 and O2. Although the composition of the mixture gases used in our study were taken from reports of successful reduction of B. subtilis spores (Hong et al., 2009; Roth et al., 2010), these treatments were not effective. This implies that more parameters than simply the gas and energy applied should be considered. For example, equipment parameters, including the geometry of treatment chamber (related to the density of plasma) and the energy source, as well as medium parameters, including aw, pH, ionic strength, shape, and surface should be considered. Compared with the inhibition curves for Salmonella inoculated onto the surface of mangos, those from melon surfaces required longer exposure times to obtain the same levels of reduction (Perni et al., 2008). The authors suggested that the intricate webbing on the surface of melon rinds protects the Salmonella cells (Perni et al., 2008). The red pepper powder surface is rougher than the surfaces of plastics and agar media. The intricate surface of the powder could provide numerous sites for the microorganism to attach and potentially evade the effect of plasma

treatment. In addition, stacking of the red pepper powder may not result in a uniform treatment on inoculated powder. Many powders were stacked on top of one another, resulting in areas of overlap. Reactive species may not effectively penetrate the powder and cannot reach spores in the overlap areas. Thus, uniform plasma treatment of red pepper powder is challenging. This stacking and the inherent geometry of the powder may have resulted in ineffective spore inhibition by CPT. This hypothesis is supported by our experiment result that spores (5.9 0.2 log spores/cm2) inoculated on a smooth polystyrene plate were inhibited by 3.9 0.1 log spores/cm2 by CPT with the HeeO2 mixture at the same operating conditions used for the powder treatment. The resistance of the Bacillus spores to microbial inhibition treatments can be induced by a number of factors, including (i) the structure of the spores, formed with cortex, coat, and exosporangium of the spores; (ii) the low water content in the central region of core of the spore; (iii) the saturation of spore DNA by a group of acid-soluble proteins; and (iv) the low permeability of the inner spore membrane to hydrophilic molecules (Akbas and Ozdemir, 2008; Young and Setlow, 2003). A higher resistance of B. cereus spores than A. avus spores to CPT could be induced by one or more of those factors. Combination treatments are advantageous, principally because many individual treatments do not ensure food safety or stability (Raso and Barbosa-Canovas, 2003). Combining lethal heat treatments with novel nonthermal processes might facilitate eradication of microorganisms resistant to nonthermal processes (Patterson et al., 1995; Rademacher et al., 1998). The heat treatments inhibited the spores (6.3 0.1 log spores/g) as follows: 0.2 0.1, 0.2 0.1, and 0.2 0.2 log spores/g reductions at 70  C

Fig. 5. Scanning electron micrographs of B. cereus. Left, untreated B. cereus vegetative cells and spores; right, B. cereus vegetative cell and spores imposed to the integrated treatments of heat (90  C, 30 min) and cold plasma (helium-oxygen mixture, 900 W, 20 min).

J.E. Kim et al. / Food Microbiology 38 (2014) 128e136


with the treatment times of 10, 30, and 60 min, respectively; 0.1 0.0, 0.7 0.2, and 1.6 0.1 log spores/g reductions at 80  C with the treatment times of 10, 30, and 60 min, respectively; and 0.1 0.2 and 1.4 0.2 log spores/g reductions at 90  C with the treatment times of 10 and 30 min, respectively. No colonies were detected after treatment for 60 min at 90  C. The 10-min treatment was not effective at any of the three temperatures. B. cereus spores on the red pepper powder treated thermally were exposed for 20 min to cold plasma formed at 900 W using N2, an N2eO2 mixture, He, and a HeeO2 mixture. The powder treated at 90  C for 60 min was not exposed to cold plasma because the heat treatment alone resulted in inhibition of most of the cells. Among the CPTs, the treatment exhibited an additional inhibition of the spores was the one with the HeeO2 mixture, which was conducted following heat treatment at 90  C for 30 min. The combination of heat (90  C, 30 min) and cold plasma (HeeO2 mixture, 900 W, 20 min) inhibited B. cereus spores by 3.4 0.7 log spores/g. The combination treatment with the HeeO2 mixture was more effective than the treatment with He alone. Compared to A. avus, more oxygen reactive species might be required for inhibition of B. cereus spores. A synergistic effect on the inhibition of B. cereus spores was observed with this combination. The reduction (3.4 0.7 log spores/g) resulting from the combination treatment was higher than the sum of the reductions (1.4 0.2 log spores/g) due to heat treatment and CPT (no reduction). The heat treatment could damage the material covering the cores of spores and inactivate enzymes in spores (Setlow, 2006; Warth, 1980), which could facilitate the activities of the reactive species in the plasma. The temperature of the powder after CPT (HeeO2 mixture, 900 W, 20 min) was 28.8  C and the DT was 3.8  C, indicating the CPT was conducted with a minimal heat inux to the powder. The SEM images of B. cereus vegetative cells and spores either with or without the combination treatment that resulted in a 3.4 log spores/g reduction are presented in Fig. 5. Damage to the structures of both vegetative cells and spores was evident in treated samples (Fig. 5). Erosion of untreated spores is almost unnoticeable, but is considerable in treated cells. Treated B. cereus spores were shrunken and fractured. Similar shrunken forms were observed after treatment with electron beams or electric elds (Hury et al., 1998; Lara et al., 2002; Park et al., 2004).

values (P > 0.05). The temperature increase during CPT, which corresponds to heat induction, might be too small to affect the color of the powder. However, the combination treatment reduced L* (lightness), a* (redness), and b* (yellowness) signicantly (P < 0.05). The change was likely caused by the heat treatment, as the values of the heat treatment sample were not signicantly different from those of the combination treatment sample (P > 0.05). Red color is the main factor determining the commercial quality of red pepper (Lee et al., 2004). The red color of red pepper is attributed to the presence of capsanthin, a major carotenoid pigment (Lee et al., 2004). The extractable color in red pepper powder is usually expressed as ASTA values, which correspond to the total carotenoid content (Mnguez-Mosquera and Prez-Galvez, 1998). CPT I, II, and the combination treatment did not signicantly change the ASTA values of the red pepper powder (P > 0.05). The change in redness indicated by the CIELab coordinates was not reected by the ASTA measurement. The results from the color measurements demonstrate the potential for application of CPT and the combination treatments for decontamination of red pepper powder without altering color properties noticeably. 4. Conclusions The microwave-powered cold plasma system effectively inhibited A. avus on red pepper powder. With biphasic shapes, the treatment power and time enhanced A. avus inhibition. The aw of the powder was lowered by CPT, which might be due to evaporation under the vacuum conditions of the cold plasma treatment. The moisture evaporated might have resulted in formation of oxygen reactive species in the plasma. The use of CPT to decontaminate powder foods could be evaluated more fully if the effects of stacking of the samples and the smoothness of the surface of the samples on microbial inhibition are further examined. The combination treatment with heat (90  C, 30 min) and cold plasma (900 W, 20 min) using the HeeO2 mixture inhibited B. cereus spores in a synergistic manner. CPT at 900 W for 20 min did not markedly increase the temperature of the powder (max DT 4.7  C), which showed the potential of CPT in nonthermal decontamination of red pepper powder. This potential is supported by the result that the color properties of the red pepper powder were not signicantly altered by CPT. CPT could thus be applied to red pepper powder and other powder food products to improve their microbial safety and extend their microbial shelf life. Acknowledgment This research was supported by iPET (Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries), Ministry of Agriculture, Food and Rural Affairs.

3.5. Surface and extractable color Color change assessment of red pepper powder after treatment with CPT and combination treatments are necessary to determine the extent to which the treatments affect the color of the powder. The effects of CPT and combination treatments on red pepper powder color are summarized in Table 3. Neither CPT I, which effectively inhibited A. avus, nor CPT II, which was used for the combination treatment, signicantly altered the L*, a*, b*, and ASTA

Table 3 Effects of heat treatments, cold plasma treatments (CPTs), and integrated treatments on the color properties of red pepper powder. Color Treatments No treatment L* a* b* ASTA
a,b,c,d a b c

Heat treatmenta 37.19 9.49 8.45 43.24 0.50b 0.50b 0.38b 0.82b

CPT Ib 38.47 12.10 10.32 48.65 0.49a 0.50a 0.38a 0.09a

CPT IIc 38.54 12.38 10.84 50.73 0.53a 0.49a 0.65a 0.47a

Integrated treatment (heat treatment CPT II) 37.49 9.32 8.23 47.37 0.28b 0.41b 0.58b 3.95ab

38.27 11.99 9.89 46.62

0.34a 0.53a 0.66a 1.91ab

Different letters within the same column differ signicantly (P < 0.05). Temperature: 90  C, time: 30 min. Gas: nitrogen, power: 900 W, time: 20 min. Gas: helium-oxygen mixture, power: 900 W, time: 20 min.


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