Inactivation of food by pulsed xenon flash light
Transcription
Inactivation of food by pulsed xenon flash light
Food Control 33 (2013) 15e19 Contents lists available at SciVerse ScienceDirect Food Control journal homepage: www.elsevier.com/locate/foodcont Inactivation of food-related microorganisms in liquid environment by pulsed xenon flash light treatment system Hirokazu Ogihara a, *, Kenji Morimura a, Hikaru Uruga a, Takuya Miyamae b, Masayuki Kogure b, Soichi Furukawa a a b Department of Food Bioscience and Biotechnology, College of Bioresource Sciences, Nihon University, Fujisawa-shi, Kanagawa 252-0880, Japan Iwasaki Electric, Co., Ltd. Ichiriyama 1-1, Gyoda, Saitama-shi 361-8505, Japan a r t i c l e i n f o a b s t r a c t Article history: Received 19 September 2012 Received in revised form 29 January 2013 Accepted 4 February 2013 Effect of pulsed xenon flash light (PLS) treatment on the inactivation of 13 food-related microorganisms including food-poisoning bacteria in liquid environment was investigated. Inactivation ratio was proportional to the irradiation energy and irradiation flush number. Higher inactivation was achieved in transparent environment such as water, phosphate buffer and physiological saline. On the other hand, pulsed xenon flash light treatment could not inactivate microorganisms in Tryptic Soy Broth. Irradiation energy 500 J and 1 time pulse treatment brought about above 5 order inactivation in all used strains, and it was indicated that PLS treatment was effective for inactivating food-related microorganisms including food-poisoning bacteria. Ó 2013 Elsevier Ltd. All rights reserved. Keywords: Pulsed light Inactivation Food-related microorganisms 1. Introduction Prevention of the outbreak induced by food-poisoning bacteria is a most important point in food hygiene. In food industry, heat sterilization is one of the most important process, however, heat treatment sometimes brings about degeneration of the fragrance and the functional ingredient components of foods. Therefore, development of some non-thermal sterilization processes has been studied and developed. Representative non-thermal sterilization processes were high hydrostatic pressure treatment (Hoover, Metrick, Papineau, Farkas, & Knorr, 1989; Meyer, Cooper, Knorr, & Lelieveld, 2000; Zhang & Mittal, 2008), pulsed high electric field treatment (Evrendilek, Zhang, & Richter, 1999; Knorr, Geulen, Grahl, & Sitzman, 1994; Qin et al., 1995; Rodrigo, Martinez, Harte, Barbosa-Canovas, & Rodrigo, 2001), high pressure carbon dioxide treatment (Yuk, Geveke, & Zhang, 2010; Zhang et al., 2006), and light treatment usually using UV light (Bank, John, Schmehl, & Dratch, 1990; Chang et al., 1985; Kuo, Carey, & Ricke, 1997) etc. Out of above treatments, UV light treatment is most effective treatment for surface sterilization, and it has been already used in food and medical fields (Bank et al., 1990; Chang et al., 1985; Kuo, Carey & Ricke; 1997). In general, light treatment needs long period for inactivating microorganisms, on the other hand, high energy short period light * Corresponding author. Tel.: þ81 0466 84 3972. E-mail address: ogihara@brs.nihon-u.ac.jp (H. Ogihara). 0956-7135/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodcont.2013.02.012 treatment has been developed. Usually xenon lamp is used in this type of high energy short period light treatment, named pulsed xenon flash light treatment (PLS), and it use wide range wave length light (200e 1100 nm) including far UV (200e300 nm), near UV (300e380 nm) and infrared (780e1100 nm) for inactivating microorganisms. There were some studies on the effect of PLS on the inactivation of some food related or food-poisoning bacteria on the various surfaces (mainly agar media surface) (Anderson, Rowan, MacGregor, Fouracre, & Farish, 2000; Gomez-Lopez, Devlieghere, Bonduelle, & Debevere, 2005a,b; MacGregor et al., 1998; Rowan et al., 1999; Takeshita et al., 2002). On the other hand, there were a few reports on the inactivation of microorganisms in liquid environment, such as water, apple juice, orange juice and milk (Haffman, Slifko, Salisbury, & Rose, 2000; Palgan et al., 2011; Sauer & Moraru, 2009). PLS treatment is a promising method for inactivating food-poisoning bacteria, however there was no enough studies on the effect of PLS on the food related or food-poisoning bacteria in liquid environment and its inactivation mechanism. Therefore, effect of PLS treatment on some food-related and food-poisoning bacteria in liquid environment was investigated. 2. Materials and methods 2.1. Pulsed xenon flash lamp light (PLS) system PLS system was used (Iwasaki Electric, Co. Saitama, Japan). This system is comprised of xenon lamp (tube type), irradiation vessel, 16 H. Ogihara et al. / Food Control 33 (2013) 15e19 cooling water system and power source (Fig. 1). FLASH UV MONITOR (Iwasaki Electric, Co. Saitama, Japan) was used for measuring the irradiation intensity of 4e16 cm points from lamp. 2.2. Used bacteria and culture The following bacteria, Listeria monocytogenes ATCC 49594, Staphylococcus aureus ATCC 25923, Streptococcus faecalis ATCC 29212, Enterobacter cloacae ATCC 23355, Enterobacter aerogenes ATCC 13047, Escherichia coli ATCC 25922, Providencia alcalifaciens ATCC 51902, Pseudomonas aeruginosa ATCC 27853, Serratia marcescens ATCC 8100, Salmonella Enteritidis IFO 3313, Aeromonas hydrophila subsp. hydrophila IFO 13286, Yersinia enterocolitica JCM 1677, and Salmonella Typhimurium IID 1000 were obtained from the American Type Culture Collection (ATCC) (Rockville, MD, USA), Institute for Fermentation Osaka (IFO) (Osaka, Japan), Japan collection of Microorganisms (JCM) (Saitama, Japan) and Institute of Medical Science, University of Tokyo (IID) (Tokyo, Japan), respectively. These bacteria were cultured in Tryptic Soy Broth (TSB) (Difco, Detroit, MI, USA) at 37 C for 24 h. Second successive full-growth cultures (around 1089 CFU/ml) were washed two times by phosphate buffer (1/15 M), and these cell suspensions were used for PLS treatment. After PLS treatment, inactivation ratios were measured by culturing at 37 C for 48 h on Tryptic Soy Agar (TSA) (Difco, Detroit, MI, USA). Inactivation ratios were directly described by cell numbers. 2.3. Inactivation of food-related bacteria by PLS Applicable irradiation widths at 7e16 cm irradiation distance (from lamp to liquid surface) points by PLS treatment (1 flush, 500 J) were measured by using E. coli (100 ml) on TSA in polystyrene rectangle Petri dish (235 mm 85 mm 15 mm). Effects of solutions of cells on the inactivation of bacteria by PLS treatment (1, 5 and 10 flushes, 500 J, 10 cm distance) were investigated by using E. coli (5 ml) in polystyrene Petri dish (4 48 mm) without cover. E. coli cultures were centrifuged at 8000 rpm for 10 min, and those were dissolved in 0.1% peptone added physiological saline (PSS), TSB, 1/15 M phosphate buffer (PPB) and refined water (RFW). Optical density at 660 nm of each suspensions were measured by BACTOMNITOR$BACT-550 (JIKCO LTD., Tokyo). Effects of container on the inactivation of bacteria by PLS treatment (1 flush, 500 J, 10 cm distance) were investigated by using E. coli (4 ml) in crystal boat (73 mm 17 mm 9 mm; boat like shape), crystal beaker (4 40 mm 60 mm) and polystyrene Petri dish (4 48 mm) without cover. E. coli cells were dissolved in PPB. Irradiation area, suspension depth and thickness of containers were described in Table 1. Table 1 Irradiation area, suspension depth and thickness of containers. Treatment container Irradiation area (mm2) Suspension depth (mm) Thickness (mm) Crystal board Crystal beaker Polystyrene Petri dishes 1.24 102 1.25 102 1.81 102 5.5 3.2 2.2 1.4 1.4 1.2 Effects of PLS treatment (1 flush, 200, 300, 400 and 500 J, 10 cm distance) on the 13 food-related bacteria including 8 pathogenic bacteria, L. monocytogenes, S. aureus, S. faecalis, A. hydrophila, E. cloacae, E. aerogenes, E. coli, P. alcalifaciens, P. aeruginosa, S. Enteritidis, S. Typhimurium, S. marcescens, and Y. enterocolitica (each 4 ml), in crystal boat without cover were investigated. Cells were dissolved in PPB. 2.4. Statistical analysis All experiments were performed in three or more replications. The data presented are the means of at least three replicate experiments. The error bar shows the standard deviation. Significant differences were determined by Student’s t-test (P < 0.05). 3. Results 3.1. Studies on the properties of PLS treatment on the inactivation of bacteria Relationships among irradiation distance, irradiation energy and irradiation intensity in PLS treatment were described (Table 2), and it was indicated that irradiation intensities increased in proportion to the increase of irradiation energy. However, there was no inverse proportional relationship between irradiation intensities and irradiation distance, and 9e12 cm irradiation distances were best for irradiation intensity. Effect of the irradiation distances (7e16 cm) on the applicable irradiation width by PLS treatment of E. coli on the agar plate was investigated (Fig. 2), and irradiation distances (10e12 cm) showed the narrowest applicable irradiation width (4e5 cm). Effects of solutions (TSB, PSS, PPB and RFW) of the cells on the inactivation of E. coli by PLS treatment was investigated (Fig. 3), and inactivation ratios increased in order of TSB, PSS, PPB and RFW. Especially in TSB, increase of flush number brought about no increase in inactivation ratio, on the other hand, 10 flushes PLS treatment brought about above 7 orders in activation ratios in other three solutions. Transmission ratios of the PLS in TSB, PSS, PPB and Fig. 1. Schematic model of pulsed xenon flash lamp system. H. Ogihara et al. / Food Control 33 (2013) 15e19 Table 2 Relationships among irradiation distance, irradiation energy and irradiation intensity in PLS treatment in PLS treatment. 4 cm 5 cm 6 cm 7 cm 8 cm 9 cm 10 cm 11 cm 12 cm 13 cm 14 cm 15 cm 16 cm 8.28 8.07 8.16 8.88 11.60 13.78 15.54 15.32 14.26 11.90 10.16 7.71 7.62 200 J 300 J 0.30 0.77 0.22 0.25 0.27 0.58 0.66 0.56 0.12 0.89 0.55 0.35 1.04 13.49 13.61 13.41 14.94 19.09 22.91 26.09 25.92 22.21 17.63 14.24 13.04 12.74 400 J 0.32 0.98 1.44 0.82 0.46 0.63 1.15 1.76 0.99 3.30 1.26 1.19 1.39 18.56 17.32 17.38 23.22 25.29 28.39 33.06 32.87 30.32 23.95 21.66 19.89 16.43 500 J 1.12 0.48 1.44 1.67 0.77 1.90 0.67 0.56 0.98 1.65 0.95 1.70 1.45 22.9 20.51 21.04 26.71 30.05 37.25 41.48 41.26 35.71 33.21 30.64 25.04 21.85 1.21 0.93 1.03 1.56 1.84 2.93 2.15 2.57 2.00 2.94 3.30 3.09 2.56 9 log 10 CFU/ml [mJ/cm2]/(n ¼ 10) (Mean Standard deviation) 10 8 7 6 Number of cell survivors Irradiation distance (cm) 5 4 3 2 1 0 TSB RFW were described in Table 3, and it was indicated that there were inverse proportional relationships between inactivation ratios and light transmission ratios. Effects of container on the inactivation of E. coli by PLS treatment (1 flush, 500 J, 10 cm distance) were investigated in polystyrene Petri dish, crystal boat and crystal beaker without cover, and inactivation ratios increased in order of polystyrene Petri dish, crystal beaker and crystal boat (Table 4). These results indicated that crystal containers were better for its high light permeability, and difference between crystal boat and crystal beaker was considered to be dependent on their shapes. 3.2. Inactivation of food-related bacteria by PLS Effects of PLS treatment (1 flush, 200, 300, 400 and 500 J, 10 cm distance) on the 13 food-related bacteria including 8 pathogen, L. monocytogenes, S. aureus, S. faecalis, A. hydrophila, E. cloacae, E. aerogenes, E. coli, P. alcalifaciens, P. aeruginosa, S. Enteritidis, S. Typhimurium, S. marcescens, and Y. enterocolitica in crystal boat were investigated (Figs. 4 and 5). Inactivation ratio was proportional to the irradiation energy. It was indicated that except for E. aerogenes and S. Typhimurium, all strains were inactivated more than 6-log-orders by 500 J, 1 flush PLS treatment, and all strains were inactivated completely with the increase of the flush number (data not shown). In many strains, treatment above 400 J showed significantly higher inactivation. Interestingly, gram-positive bacteria showed higher sensitivity to PLS treatment. Non n tretment PSS 1flashes 1fl f ashes PPB RFW 5 flashes f ashes fl 500J 10 flashes Fig. 3. Effects of solutions of the cells on the inactivation of E. coli by PLS treatment. Used solutions were tryptic soy broth (TSB), peptone added physiological saline (PSS), Potassium phosphate buffer (PPB) and refined water (RFW). 4. Discussion There were some reports on the effect of PLS treatment on the inactivation of microorganisms including pathogens on surface of food stuff, such as fish, vegetables, fruit etc (Dunn, 1997; Dunn, Ott, & Clark., 1995; Gomez-Lopez et al., 2005a,b; Kaack & Lyager, 2007; Keklik, Demirci, Patterson, & Puri, 2010; Lagunas-solar, Pina, MacDonald, & Bolkan, 2006; Ozer & Demirci, 2006), and it was indicated that PLS was effective for inactivating microorganisms on food surface and also for extending their shelf-life. On the other hand, there were a few reports on the inactivation of microorganisms in liquid environment (Huffman et al., 2000; Palgan et al., 2011; Sauer & Moraru, 2009). Therefore, effect of PLS treatment on some food-related and food-poisoning bacteria in liquid environment should be investigated, and its inactivation mechanism is also necessary to be discussed. Relationships among irradiation distance, irradiation energy, irradiation intensity and applicable irradiation width in PLS treatment was investigated (Table 2 and Fig. 2), and it was indicated that irradiation intensities increased in independent on the increase of irradiation energy, and it was also indicated that there was Table 3 Transmission ratios of the PLS in TSB, PSS, PPB and RFW. 12 applicable irradiation width [cm] 17 10 8 Replacement solution Transmission rate (%) Tryptic soy broth (TSB) 0.1% peptone added physiological saline (PSS) 1/15 M phosphate buffer (PPB) Refined water (RFW) 0.57 20.00 98.77 98.93 0.05 0.41 0.54 0.54 6 Table 4 Effects of container on the inactivation of E. coli by PLS treatment (1 flush, 500 J, 10 cm distance). 4 2 Treatment container Pulsed light treatment CFU/ml 0 7cm 8cm 9cm 10cm 11cm 12cm 13cm Irradiation distance [cm] 14cm 15cm 16cm (n=3) Fig. 2. Effect of the irradiation distances on the applicable irradiation width by PLS treatment (1 flash, 500 J) of E. coli. Non treatment Crystal board Crystal beaker Polystyrene Petri dishes 9.08 5.21 7.23 8.05 0.05 0.19 0.12 0.05 18 H. Ogihara et al. / Food Control 33 (2013) 15e19 10 Number of cell survivors log 10 CFU/ml 9 8 7 6 5 4 3 2 1 0 Non tretment ment 200J 300J 400J 500J Fig. 4. Effects of PLS treatment (1 flush, 200, 300, 400 and 500 J, 10 cm distance) on the 5 food-related bacteria, S. faecalis, E. cloacae, E. aerogenes, E. coli, and S. marcescens, in crystal boat. optimum irradiation distance at around 10e11 cm for inactivating microorganisms. And then, effects of solutions (TSB, PSS, PPB and RFW) of the cells on the inactivation of E. coli by PLS treatment (Fig. 3), and it was indicated that there were inverse proportional relationships between inactivation ratios and light transmission ratios (Table 3). In TSB, there were some components that can absorb light including UV, and those components would inhibit the transmission of light necessary for inactivating bacteria. PLS treatment also could not inactivate microorganisms enough in milk and juice environment for their high UV absorbancy. Inactivation ratios increased in order of polystyrene Petri dish, crystal beaker and crystal boat (Table 4). These results indicated that crystal containers were better for its high light permeability. Distance was measured between lamp and surface of cell suspensions and suspension depths were inversely proportional to the inactivation ratios. We considered that reflected light from the bottom of the crystal boat also would significantly affect the viability of the cells. In addition boat like shape of the crystal boat would also contribute to the low depth of cell suspensions. From above results, it was indicated that direct irradiation of light including UV to microbial cells would be most important point in inactivating bacteria. This hypothesis was also supported by the results that using crystal boat was most effective for inactivating bacteria by PLS treatment. Mechanisms of the inactivation of microorganisms by PLS treatment would mainly depend on the UV, and inactivation was mainly induced by formation of thymine dimer in DNA (Mitchell, Jen, & Cleaver, 1992). However, PLS treatment brought about higher inactivation ratio more than expected from UV treatment, and it was indicated that higher inactivation ratios would be induced by broad wave length and momentary high-energy flush. Thermal energy would support the inactivation. However the temperatures after 500 J and 10 time pulse have just increased (0.9 C) crystal boat, (1.2 C) crystal beaker and (0.7 C) polystyrene Petri dish (4 48 mm), respectively. From these results, it was concluded that thermal energy did not support the inactivation. Effects of PLS treatment on the 13 food-related bacteria including 8 pathogen in crystal boat were investigated (Figs. 4 and 5), and PLS treatment (500 J, 1 flush) inactivated most used bacteria more than 6-log-orders except for E. aerogenes and S. Typhimurium. All strains were inactivated completely with the increase of the flush number. E. aerogenes aggregated in its preparation, and that would be one of the reasons of their resistance. There were many studies on the biofilm formation of S. Typhimurium, and S. Typhimurium also would form slight aggregates. Used E. aerogenes and S. Typhimurium would have higher UV resistance than other used strains. Their genes related to UV resistance would have higher activity or they would produce some extracellular substances that can absorb UV. From these results, PLS treatment was promising method to inactivate food-related bacteria including pathogen in high light permeable liquid environment. The precise mechanism of the inactivation of microorganisms by PLS treatment should be investigated. Number of cell survivors log 10 CFU/ml 10 9 8 7 6 5 4 3 2 1 0 Non tretment 200J 300J 400J 500J Fig. 5. Effects of PLS treatment (1 flush, 200, 300, 400 and 500 J, 10 cm distance) on the 8 food-related pathogenic bacteria, L. monocytogenes, S. aureus, A. hydrophila, P. alcalifaciens, P. aeruginosa, S. Enteritidis, S. Typhimurium, and Y. enterocolitica in crystal boat were investigated. H. Ogihara et al. / Food Control 33 (2013) 15e19 References Anderson, J. G., Rowan, N. J., MacGregor, S. J., Fouracre, R. A., & Farish, O. (2000). Inactivation of food-borne enteropathogenic bacteria and spoilage fungi using pulsed-light. IEEE Transactions on Plasma Science, 28, 83e88. Bank, H. L., John, J., Schmehl, M. K., & Dratch, R. J. (1990). 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