(high-intensity pulsed light) 정명수이화여자대학교환경ㆍ식품공학부 1. 1.1 연구목적및필요성 21,,.,,.,.,..,,,,,,. 3,000 2005 1,500,, 597
. 2004,.,,..,., Bacillus, Pseudomonas 10 2 ~10 5 cfu/g, Bacillus cereus,,,. E. coli, Listeria.. ( ),..,,,,,,,,, oxidizer, alcohol,,,,. 598
.,.,,,. 1.2 광펄스 (Intense pulsed light) 기술 Intensed light pulse, pulsed white light(whl), broad-spectrum white light. 170~2,600nm (UV) (NIR) UV. (PEF) ( 1) UV. High voltage pulse generator A.C 220V TRANSFORMER RECTIFIER OSCILLOSCOPE D.C HIGH VOLTAGE CAPACITOR INDUCTOR THYRATRON TRIGGER CIRCUIT Water Bath PULSE GENERATOR Peristaltic pump Sample reservoir GROUND 1 Intense Pulsed Light Magnetic Stirrer (high-intensity pulsed light) 599
0.01~50J/cm 2 (energy density) pulse 1µs~0.1s 1~20 flash, 450torr Xenon clear fused quartz tube. sterilizing,.,, pulse,,, Lamp, pulse. UV DNA. UV DNA cell repair system, UV UV. 1990 (1,2).,. Anderson (3) ( 2),. Gomez-Lopez (4),. Dunn (1),,, sample 5~7 mold colonies 11 sample 11 mold colonies. 7 600
Cell/Spore Numbers (log10 CFU/ml) 11 10 9 8 7 6 5 4 3 2 E. coli S. enteritidis B. cereus F. culm orum A. niger 0 200 400 600 800 1,000 Number of Pulses 2 Pulsed-light inactivation of surface-inoculated pathogenic bacteria and spoilage fungi. 9 Cell Number (log10 CFU/plate) 8 7 6 5 4 3 High UV Low UV 2 0 50 100 150 200 250 300 Number of Pulses 3 Pulsed light inactivation of surface-inoculated E. coli using two light sources which contained either a low or high UV content., 2log. Rowan (5) high UV low UV high UV ( 3), Huffman (6). (high-intensity pulsed light) 601
4 Photos of glass Petri dishes containing colonies of A. cinnamomeus generated by the ILP treatment surviving spores after 20 10-3 s duration of treatment at different energetic densities: (a) control sample; (b) 0.497 J/cm 2 (c) 0.716 J/cm 2 and (d) 0.977 J/cm 2. 5 Photos of glass Petri dishes containing colonies of C. herbarum generated by the ILP treatment surviving spores when treated at 0.497 J/cm 2 energetic density for different times: (a) 10 10-3 s; (b) 20 10-3 s; (c) 30 10-3 s. Marquenie (7,8) UV-C, heat strawberry 2. Turtoi (9) paper-polyethylene packaging material, packaging material ( 4, 5). ( 1). data, data. 602
1 The effects of PL treatments on microbial inactivation in foods Microoorganisms Liquids Serratia marcescens Staphlococcus aureus Plant foods Aspergillus niger spores Botrytis cinerea Escherichia coli O157:H7 Mesophilic aerobic microorganisms Monilia fructigena Salmonella enteric Salmonella spp. Other foods Clostridium sporogenes Eschrichia coli O157:H7 Listeria monocytogenes Packaging material Cladosporium herbaruml Food product and food contact materials Milk Milk Corn meal Strawberries Blueberries Raspberries Strawberries Alfalfa seeds Shredded spinach Grated celeriac Chopped green pa prika Soybean sprouts Shredded radicchio Great carrot Shredded iceberg lettuce Shredded white cabbage Strawberries Blueberries Raspberries Strawberries Honey Salmon fillets Salmon fillets Pulse Energy (J) 12.6 1.27 5.6 7 32.4 72.0 64.8 5.6 7 7 7 7 7 7 7 32.4 59.4 64.8 5.6 5.6 5.6 Number pulses - 16 300 3,750 180 180 180 135 2,700 675 2,700 675 2,700 675 2,700 2,700 3,750 180 180 180 135 135 Pulse width 20 ns - - 30 - - - - 30 30 30 30 30 30 30 30 30 - - - - - Log reduction > 2.0 7.2 4.93 3 4.9 3.9 3.3 0.94~1.82 0.9 0.21 0.56 0.65 0.56 1.67 2.0 0.8 4 3.8 3.4 4.3 0.89~5.46 0.24~0.91 0.72~0.8 Reference Smith et al. (2002) Krishnamurthy et al. (2007) Jun et al. (2003) Marquenie et al. (2003a) Bialka and Demirci (2007) Bialka and Demirci (2007) Bialka and Demirci (2007) Sharma and Demirci (2003) Gomez-Lopez et al. (2005a) Gomez-Lopez et al. (2005a) Gomez-Lopez et al. (2005a) Gomez-Lopez et al. (2005a) Gomez-Lopez et al. (2005a) Gomez-Lopez et al. (2005a) Gomez-Lopez et al. (2005a) Gomez-Lopez et al. (2005a) Marquenie et al. (2003a) Bialka and Demirci (2007) Bialka and Demirci (2007) Bialka and Demirci (2007) Hillegas and Demici (2003) Ozer and Dermici (2006) Ozer and Dermici (2006) Paper-polyethylene Packaging material 0.977 30-2.7 Turtoi and Nicolau (2007) Food contact surfaces Listeria innocua Stainless steel surfaces 1.27 3-1.93~2.77 Woedling and Moraru (2005) (high-intensity pulsed light) 603
,. Cho (10) UV-C heat Botritis cinerea Monilia Fructigena, 3~4log UV-C heat 2~3log. Cho (10),,,, ( 6~8).,., le-00 le-01 le-02 Survival fraction (N/No) le-03 le-04 le-05 le-06 le-07 0 500 1,000 1,500 2,000 2,500 Treatment time ( s) 6 Effet of the distance between the lamp and the surface of sample on inactivation of L. plantarum by intense light pulse (1 pulse width) treatment at room temperature. ; 15kV, ; 20kV, ; 25kV. 604
le-00 le-01 le-02 Survival fraction (N/No) le-03 le-04 le-05 le-06 le-07 0 500 1,000 1,500 2,000 2,500 Treatment time ( s) 7 Susceptibility of Lactobacillus, Leuconostoc and Pediococcus expore to intense light pulse treatments carried out at 15 kv and room temperature. ; Leuconostoc mesenteroides, ; Lactobacillus plantarum, ; Lactobacillus brevis, ; Pediococcus pentosaceus.,. (,, ),, (,, ), (critical process factors) (12,13).. 254nm (UV), (mechanisms of microbial inactivation). (high-intensity pulsed light) 605
le-00 le-01 le-02 Survival fraction (N/No) le-03 le-04 le-05 le-06 le-07 0 500 1,000 1,500 2,000 2,500 Treatment time ( s) 8 Effect of the distance between the lamp and the surface of sample on inactivation of L. plantarum by intense light pulse carried out at 25kV and room temperature. ; 60 mm, ; 80 mm, ; 110 mm, ; 135 mm. 2. 2.1 광펄스기술의위해세균에대한저감화효과검토. - E. coli O157:H7 - Listeria monocytogenes - Bacillus cereus - Pseudomonas aeruginosa E. coli O157:H7, Listeria monocytogenes, Bacillus cereus, Pseudomonas aeruginosa 5mL TSB(Tryptic 606
soy broth), 37, 200rpm shaking incubator overnight. 1mL 100mL TSB E. coli O157:H7 37, 200rpm 10, Listeria monocytogenes 37, 200rpm 8, Bacillus cereus 37, 200rpm 10, Pseudomonas aeruginosa 37, 200rpm 10. (0.85% NaCl solution) 2 4. 0.1mL Tryptic soy agar(tsa) spreading. Treatment time, kv, Hz High-intensity pulsed light. plate 37 48 incubator. E. coli O157:H7 Listeria monocytogenes KCCM 40307 Bacillus cereus MEDIA dilution MEDIA culture Tryptic soy broth (Difco) 37, 200 rpm shaking incubation, 10 h 37, 200 rpm shaking incubation, 8 h 37, 200 rpm shaking incubation, 10 h 0.85% NaCl solution Tryptic soy agar (Difco) 37 36 h 37 48 h 37 24 h Pseudomonas aeruginosa 37, 200 rpm shaking incubation, 10 h 37 24 h,. 25kV(triggering voltage), DC,. 220V, AC, 25A 30kV(triggering voltage), DC, 25kW. capacitor. (lamp) 59kPa Xenon quartz Heraeus Noblight Series, lamp 147mm 7.14mm. (high-intensity pulsed light) 607
Lamp oscilloscope(lecroy Digital Oscilloscope, Model 9300 AM, Dual 400 MHz Switzerland). 2.2 광펄스에의한분말, 신선식품속일반세균의살균 10. -.. - Plate Count Agar(PCA, difco Co.). - 1g 0.88% NaCl 10-3 ~10-5 0.1 ml PCA spreading. - 25kV triggering voltage, 5Hz pulse frequency, plate 12cm. 500V 700V. - plate 35 1 24~48, plate colony CFU/mL. - PCA colony colony 16S rrna. - sequencing. (http://dna.macrogen.com/kor/) - DNA homology BLASTN online program. (http://www.ncbi. nlm.nih.gov/blast/) 2.3 Transmission electron microscope(tem) 측정 Transmission electron microscopy(tem). 25kV, 1,500 Bacillus cereus, Listeria monocytogenes, E. coli O157:H7, Pseudomonas aeruginosa (2% glutaraldehyde and 2% paraformaldegyde in 0.05M sodium cacodylate buffer) overnight 0.05M sodium cacodylate buffer. 1% osmium tetroxide, 2, 0.5% uranyl acetate en bloc staining 1. 608
ethanol 50, 70, 80, 90% 10, 100% ethanol 10, 10, 10, 100% propylene oxide 15 2. spurr s resin propylene oxide 1:1 2 (embeding). Ultramicron(MT-X, RMC, Tucson, Az, USA) sample semi-section, thin section, section 2% uranyl acetate 7, reynolds lead citrate 7. washing TEM(Libra 120, Carl zeiss, Germany). 3. 3.1 광펄스기술의위해세균에대한저감화효과검토 E. coli O157:H7 5Hz 10, 15, 20, 25kV triggering voltage E. coli le+00 le-01 Survival fraction (N/No) le-02 le-03 le-04 le-05 le-06 0 100 200 300 400 500 600 700 800 900 Treatment time ( s) 9 Inactivation of E. coli O157:H7 as triggering voltage by intense pulsed light. ( ; 10 KV, ; 15 KV, ; 20 KV, ; 25 KV) (high-intensity pulsed light) 609
O157:H7 triggering voltage, ( 9).. 15kV, 5Hz, 450 3log, 25kV, 5Hz, 750 6log.. Listeria monocytogenes 5Hz 10, 15, 20, 25kV triggering voltage Listeria monocytogenes triggering voltage, ( 10). 15kV, 5Hz, 450 1log, 25kV, 5Hz, 900 6log. 10, 15kV. le+00 le-01 Survival fraction (N/No) le-02 le-03 le-04 le-05 le-06 0 200 400 600 800 1,000 1,200 1,400 Treatment time ( s) 10 Inactivation of Listeria monocytogenes as triggering voltage by intense pulsed light. ( ; 10 kv, ; 15 kv, ; 20 kv, ; 25 kv) 610
Bacillus cereus 5Hz 10, 15, 20, 25kV Bacillus cereus, ( 11). 15kV, 5Hz, 450 1log, 25kV, 5 Hz, 750 6 log. 10, 15kV. l.e+00 l.e-01 Survival fraction (N/No) l.e-02 l.e-03 l.e-04 l.e-05 l.e-06 0 150 300 450 600 750 Treatment time ( s) 11 Inactivation of Bacillus cereus as triggering voltage by intense pulsed light. ( ; 10 kv, ; 15 kv, ; 20 kv, ; 25 kv ) Pseudomonas aeruginosa 15Hz 10, 15kV triggering voltage Pseudomonas aeruginosa triggering voltage, ( 12). 15kV, 5Hz, 600 3log, 25kV, 5Hz, 800 6log.. (high-intensity pulsed light) 611
le+00 le-01 Survival fraction (N/No) le-02 le-03 le-04 le-05 le-06 0 200 400 600 800 1,000 Treatment time ( s) 12 Inactivation of Pseudomonas aeruginosa as triggering voltage by intense pulsed light. ( ; 10 kv, ; 15 kv, ; 20 kv, ; 25 kv) 13 A photo of petri dishes containing colonies of E. coli O157:H7 generated by the IPL treatment at 25 kv of triggering voltage. 612
3.2 광펄스기술을이용한분말신선식품에존재하는위해미생물의저감효과확인 sequencing Bacillus (96%) ( 14). 10 5. 25kV triggering voltage, 5Hz pulse frequency, plate 12cm, 500, 600, 700V maintaining voltage maintaining voltage, ( 15). Maintaining voltage 700V, 5Hz 2 3log. ACTCAGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATG TTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCT AATACCGGATGGTTGTTTGAACCGCATGGTTCAAACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCC GCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATC GGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGA AAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAA GTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCG CGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTG ATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGG AATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTA ACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATG AGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACG GTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGC AACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAG TGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCT TGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATG ACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAAC CGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGG AATCGCTAGTATCGCGGATCAGCATGCGCGGTTGAATACGTTCCCGGGCCTGTACACACCGCCCGTCACACCAC GAGAGTTTGTACACCGAGTCGTGAGTACNTTAGAGCAGCGCGANTGGANGATGANGGTGACNNNNNCCCCCCCN CNNANNNNC 14 Nucleotide sequence of 16S rrna gene from isolate. (high-intensity pulsed light) 613
l.e+00 Survival fraction (N/No) l.e-01 l.e-02 l.e-03 0 5 10 15 20 25 Treatment time (min) 15 Survival fraction of microorganisms in Sengsick as function of treatment time of IPL. (maintaining voltage: ; 500 V, ; 600 V, ; 700 V) 5Hz, plate 12cm 500V, 600V maintaining voltage maintaining voltage, ( 16~19). 10 4. Maintaining voltage. A 600V, 5Hz 30s, B 600V, 5Hz 120s, C 600V, 5Hz 60s D 500V, 5Hz 30s 3log. 614
l.e+00 Survival fraction (N/No) l.e-01 l.e-02 l.e-03 0 20 40 60 80 100 Treatment time (sec) 16 Survival fraction of microorganisms in ramen soup powder A as function of treatment time of IPL.(maintaining voltage: ; 500 V, ; 600 V) l.e+00 Survival fraction (N/No) l.e-01 l.e-02 l.e-03 0 20 40 60 80 100 120 140 160 Treatment time (sec) 17 Survival fraction of microorganisms in ramen soup powder B as function of treatment time of IPL.(maintaining voltage: ; 500 V, ; 600 V) (high-intensity pulsed light) 615
l.e+00 Survival fraction (N/No) l.e-01 l.e-02 l.e-03 0 20 40 60 80 100 Treatment time (sec) 18 Survival fraction of microorganisms in ramen soup powder C as function of treatment time of IPL.(maintaining voltage: ; 500 V, ; 600 V) l.e+00 Survival fraction (N/No) l.e-01 l.e-02 l.e-03 0 20 40 60 80 100 Treatment time (sec) 19 Survival fraction of microorganisms in ramen soup powder D as function of treatment time of IPL.(maintaining voltage: ; 500 V) 3.3 Transmission electron microscope(tem) 측정 Bacillus cereus, Listeria monocytogenes, E. coli O157:H7, Pseudomonas aeruginosa 25KV 1500 Transmission electron microscopy(tem). 20 intense pulsed light control,, intense pulsed light 616
(a) (b) 1. 2. 3. 4. 그림 20 Morphology of IPL damaged microorganisms observed by TEM. (a) before IPL treatment (b) after IPL treatment; 1. Bacillus cereus, 2. Listeria monocytogenes, 3. E. coli O157:H7, 4. Pseudomonas aeruginosa. 광펄스(high-intensity pulsed light) 기술을 이용한 분말 신선식품에 존재하는 위해세균 저감화 연구 617
,.. intense pulsed light,. (Bacillus cereus, Listeria monocytogenes) (E. coli O157:H7, Pseudomonas aeruginosa), Bacillus cereus, Listeria monocytogenes. E. coli O157:H7, Pseudomonas aeruginosa. intense pulsed light. 4. ( ),,,,,,.... 10. E. coli O157:H7, Listeria monocytogenes, Pseudomonas aeruginosa, Bacillus cereus,. E. coli O157:H7 25kV triggering voltage 750 6log, Listeria monocytogenes 25kV triggering voltage 618
900 6log. Pseudomonas aeruginosa 25kV triggering voltage 800 5log, Bacillus cereus 25kV triggering voltage, 750 6log.. triggering voltage 25kV, frequency 5Hz, plate 12cm, (s) maintaining voltage(v). 10 5 maintaining voltage 700V, 5Hz, 2min 3log. 10 4 3log A maintaining voltage 600V 30s, B 600V 120s, C 600V 60s D 500V 30s. 25kV (TEM).,, (cytoplasm) (outer membrane),....,,. (E. sakazakii),. data. data. (high-intensity pulsed light) 619
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5423.2003.00752.x. 17. Marquenie D, Geeraerd AH, Lammertyn J, soontjens C, Van impe JF, Michiels CW et al. 2003a. Combinations of pulsed light and UV-C or mild heat treatment to inactivate conidia of Botrytis cinerea and Monilia fructigena. International Journal of Food Microbiolog y, 85, 185-196. doi:10.1016/s0168-1605(02)00538-x. 18. Bialka KL, & Demirci A. 2007. Decontamination of Escherichia coli O157:H7 and Salmonella enterica on blueberries using ozone and pulsed UV-light. Journal of Food Science, 72(9), 391-396. doi:10.1111/j. 1750-3841.2007.00517.x. 19. Bialka KI, & Demirci A. 2008. Efficacy of pulsed UV-light on the decontamination of Escherichia coli O157:H7 and Salmonella spp on raspberries and strawberries. Journal of Food Science, 73(5), M201-7. 20. Sharma RR, & Demirci A. 2003. Inactivation of Escherichia coli O157:H7 on inoculated alfalfa seeds with pulsed ultraviolet light and reponse surface modelling. Journal of Food Science, 68, 1448-1453. doi:10.1111/ j.1365-2621.2003. tb09665.x. 21. Gomez-Lopez VM, Devlieghere F, Bonduelle V, & Debevere, J. 2005a. Factors affecting the inactivation of microorganisms by intense light pulses. Journal of Applied Microbiolog y, 99, 460-470. doi:10.1111/j.1365-2672. 2005. 02641.x. 22. Hillegas SL, & Demirci A. 2003. Inactivation of Clostridium sporogenes in clover honey by pulsed UV-light treatment. Agricultural Engineering international, V. Manuscritp FP 03 009. 23. Ozer NP, & Demirci A. 2006. Inactivation of Escherichia coli O157:H7 and Listeria monocytogenes inoculated on raw salmon fillets, by pulsed UV-light treatment. International Journal of Food Science & Technolog y, 41, 354-360. doi:10.1111/j.1365-2621. 2005. 01071.x. 24. Turtoi M, & Nicolau A. 2007. Intense light pulse treatment as alternative method for mould spores destruction on paper polyethylene packaging material. Journal of Food Engineering, 83, 47-53. doi :10.1016/ j.jfoodeng.2006.11.017. 25. Woodling SE, & Moraru CI. 2005. Influence of surface topography on the effectiveness of pulsed light treatment for the inactivation of Listeria innocua on stainless-steel surfaces. Journal of Food Science, 70(7), M345-M351. doi:10.1111/j.1365-2621. 2005. tb11478.x. (high-intensity pulsed light) 621