Alaire)
`of
`mtyiys
`MCHA
`
`www.foodprotection.org
`
`“The mission of the International Peoria Cele neler cee
`is to provide food safety MUCOUS forumto
`exchange informationonprotecting the food supply.”
`
`NTUArte
`ATL Oo
`Pages 1465-1624
`CODEN: JFPRDR 63(11)1465-1624(2000)
`ISSN:0362-028X
`
`Published Monthly by
`International Associationfor
`ANIMANCHAOLUL
`6200 Aurora Avenue, Suite 200W
`Des Moines, lowa 50322-2863 USA
`
`EXHIBIT 1008
`
`1
`
`EXHIBIT 1008
`
`

`

`Journal of Food Protection
`
`ISSN: 0362-028X
`Official Publication
`
`1465
`
`International Association for
`
`FoodProtection
`
`Reg.U.S. Pat. Off.
`
`Vol. 63
`
`November 2000
`
`No. 11
`
`The Effect of Different Grain Diets on Fecal Shedding of Escherichia coli 0157:H7 by Steers S. J: Buchko, R. A. Holley, W.
`©. Cleon Vor. Jd Gannon, and De Mi VOUas ccccccsccaxcssnnwccevessenisecawcies cuasaciwesdvedasdesvavwacacepnceiierewdvapdasencieeseravaeeieies. 1467
`Comparison of Aqueous Chemical Treatments To Eliminate Sa/monelia on Alfalfa Seeds W.R. Weissinger and L. R.
`BOUGHOR ciiae orcas eiccoetscslcsneeisdsuaed caves secevausboosatsivusretuaburessdushedsbedeutecsnbeleecinedusvaadskeserasscscusvéceuebensesseucsebueGe 1475
`Enrichment Procedures and Plating Media for Isolation of Yersinia enterocolitica G.C. Jiang, Dong-Hyun Kang,* and Daniel
`MNS PONG: ceccectatietinia catunsaventeb-ddatien te cciendcnnavennnediadd acenncecenascensheatateacepahelensrauavaphiddiad ceadelasasdsseey tae tadiadseeiece 1483
`Genotypes and Enterotoxicity of Staphylococcus aureus Isolated from the Hands and Nasal Cavities of Flight-Catering
`:
`Employees M. Hatakka,* K. J. Bjérkroth, K. Asplund, N. Maki-Petdys, and H. J. Korkeala.............seccsecessseeenereeeceerersneeeenens 1487
`Stimulation of Starter Culture for Further Reduction of Foodborne Pathogens during Salami Fermentation Dong-Hyun
`Heaaragy? eared: heart Ve Pin asics oie is acaccasnie dn ag ete Go 0d abe ape vadbv a RU AE gs RR en es ee MS 1492
`Bacillus cereus Group Strains, Their Hemotysin BL Activity, and Their Detection in Foods Using a 16S RNA and Hemolysin
`BL Gene-Targeted Multiplex Polymerase Chain Reaction System Hau-Yang Tsen,* Ming-Lun Chen, You-Miin Hsieh, Sen-Je
`SHG, ANG VEN-LIGN CHO E iisiiccciin candan dntenercecubinaciaecccccGacwuweitacedeasiuedscusdiaadvevantevarhuaars suacnadcunaxsdsdvecagecucdsswebadervance 1496
`Bacterial Spore Inhibition and Inactivation in Foods by Pressure, Chemical Preservatives, and Mild Heat Adrienne E. H.
`Shearer, C. Patrick Dunne, Anthony Sikes, and Dallas G. HOOVOr ........:-ccecersecaccesceteetscteeeceteateesaeqesenseenecateneenseneeesnes 1503
`Nonproteolytic Clostridium botulinum Toxigenesis in Cooked Turkey Stored under Modified Atmospheres Kathleen A.
`Lawlor,” Merle D. Pierson, Cameron R. Hackney, James R. Claus, and Joseph E. Marcy .........0.cccceceenereeeeeeetenecennereeeeeeeunes 1511
`Detection of Gualacol Produced by Alicyclobacillus acidoterrestris in Apple Juice by Sensory and Chromatographic
`Analyses, and Comparison with Spore and Vegetative Cell Populations Rachel V. Orr, Robert L. Shewfelt, C. J. Huang,
`Sebdhat Tefera, and Larry A. Beuchat........ccecccccscssercoassenedenneeeceteceuecsecnusesneeteasecneceusenseetsecneseaenrestnesnecuaeseaseneeness 1517
`Aeroblology of a High-Line Speed Cattle Abattoir K.W. F. Jericho," J. Ho, and G. C. Kozub..................ce cece cence eeeeeneeeeee 1523
`The Synergistic Effect of Excimer and Low-Pressure Mercury Lamps onthe Disinfection of Flowing Water
`lan A. Ramsay,
`Jean-Christophe Niedziela, and lain D. Ogden* ..........cscccssceuseeecsscusceusteeeseeeucnsseeacuecarcuseneeseceuaneedsucaensustrencuecesecusions 1529
`Ultraviolet Spectrophotometric Characterization and Bactericidal Properties of Electrolyzed Oxidizing Water as Influenced
`by Amperage and pH Soo-Voon Len, Yen-Con Hung,” Marilyn Erickson, and Chyer Kim ..............::.c00sceeeeesseeeeeeeeeeeaeeeneees 1534
`Prevalence of High-Risk Food Consumption and Food-Handling Practices among Adults: A Multistate Survey, 1996 to 1997
`Beletshachew Shiferaw,” Samantha Yang, Paul Cieslak, Duc Vugia, Ruthanne Marcus, Jane Koehler, Valerie Deneen, Frederick
`Angulo, and the FoodNet: Wording Group. ciisserscrecessaceusesiuessssuanesuenensdstesueuasedsaiens vans sadayavesadseacerteusieseeneaetenedieenaiens 1538
`Influence of Hygienic Quality of Raw Materials on Biogenic Amine Production during Ripening and Storage of Dry
`Fermented Sausages Sara Bover-Cid, Maria Izquierdo-Pulido, and M, Carmen Vidal-Carou" .............0cseecseeersernecevecnesurenenes 1544
`Formation of Biogenic Amines in Raw Milk Hispanico Cheese Manufactured with Proteinases and Different Levels of
`Starter Culture E. Ferndndez-Garcia,* J. Tomillo, and M. Nufie ...........cccceceeeeeseueeesenseeeeeeneceneeeeeeeeeueeeeseenecsesneesuneeeans 1551
`Mixed Starter Cultures To Control Biogenic Amine Production in Dry Fermented Sausages Sara Bover-Cid, Maria Izquierdo-
`Pulido, and M. Carmen Vidal-Carou" ......0cccccccessccsessevsedsaccecescteseuecsuetacteustsusaceacecsanseesenevavsdadenaepenecsucnasensensareseenes 1556
`Detection of Radiation-Iinduced Hydrocarbons and 2-Alkyicyclobutanonesin Irradiated Perilla Seeds Hae-Jung Lee, Myung-
`Woo Byun, and KyGngeSil RUT cies secciesccsncianscanssvadeninvasanys consineyeauassets sannesieenseie tebe dua vaanar vue ieebewewiwaliaciaaeieea icasedees 1563
`A Differential Medium for the Enumeration of the Spoilage Yeast Z2ygosaccharomyces bailii in Wine D. Schuller, M. Cérte-
`Real,” and C. Ledo........ dU adusawei aw edavbavineddsaa ves eintael Nl wbssuwadinnedasbbedsdivacoiidul ed aweediv de sada dcdku Wee dunsLubiadwe haa 1570
`Norwalk-like Virus Sequences Detected by Reverse Transcription-Polymerase Chain Reaction In Mineral Waters Imported
`into or Bottled in Switzerland Christian Beuret,* Dorothe Kohler, and Thomas LOthi ............cccsseceeeeeesnesseetesueenecenseeeeanens 1576
`Research Notes
`Prevalence and Characteristics of Shiga Toxin-Producing Escherichia coli in Beef Cattle Slaughtered on Prince Edward
`Island R. Douglas Schurman, Harry Hariharan,” Susan B. Heaney, and Kris Raln.............cccccceeseseeeeeseeeteeueeeeseuneeeeereeneen 1583
`Analysis and Modeling of the Variability Associated with UV Inactivation of Escherichia coli in Apple Cider Siobain Duffy,
`John Churey, Randy W. Worobo, and Donald W. Schaffner’ ..................cccceee cence eeeeeeeeeeeeeseea ee ceaeeeeueeeneeeaeaeeeeseeaneeaeeeeee 1587
`corava enterocolitica Biogroup 1A, Serotype 0:5 in Chicken Carcasses Mirtha E. Floccari, Maria M. Carranza, and Jose L.
`TEA” ose eee eee e een eet eneneeanenanceeneeenceeeseneseenseensenesaneeenenseeneeenensanasecesenesaneneateensnesueessniseneeansesenrensaeesaneas 1591
`Reduction of Campylobacterjejuni in a Simulated Chicken Digestive Tract by Lactobacilli Cultures M.H. Chang and T. C.
`CARI ice ura nie ree OER AER nae Ina REIN EHR ink in ONT neni ns cE arEN ar eR rece AMIE RL RIG NW cea eee ERA RRCOee 1594
`ThermalStability of Moniliformin at Varying Temperature, pH, and Time in an Aqueous Environment Graciela Pineda-
`Valdes and Lloyd B. Bullerman®..........ccccccscessscsctecesececersecetseaeeeneceeeuteaunen PEO E ET CETTE CL CTR TROT ER ET eT Pere TELLS Tee 1598
`Review
`Detection and Analysis of Animal Materials in Food and Feed Momcilovic Dragan and Avraham Rasooly* ...............0.ccc000e 1602
`
`* Asterisk indicates author for correspondence.
`
`The publishers do not warrant, either expressly or by implication, the factual accuracy of the articles or descriptions herein, nor do they so warrant any views or
`opinions offered by the authors of said articles and descriptions.
`
`2
`
`

`

`1466
`
`Scientific Editors
`
`DR. LARRY R. BEUCHAT, Center for Food Safety and Quality
`Enhancement, University of Georgia, Griffin, Georgia 30223-1797, USA
`DR. JOHN N. SOFOS, Department of Animal Sciences, Colorado State
`University, Fort Collins, Colorado 80523-1171, USA
`
`:
`Journal Editorial Staff
`DAVID W. THARP, CAE, Executive Director
`LISA K. HOVEY, Managing Editor
`
`BEV CORRON,Administrative Editor
`DIDI LOYNACHAN, Administrative Assistant
`
`J. Food Prot., Vol. 63, No. 11
`
`Journal Management Committee Chairperson
`
`DR. DONALD E, CONNER, Auburn University, Department of Poultry
`Science, 236 Ann Upchurch Hall, Auburn, Alabama 36849-5416, USA
`
`Journal Editorial Office
`
`International Association‘for Food Protection, 6200 Aurora Avenue,
`Suite 200W, Des Moines, Iowa 50322-2863, USA; Phone 515.276.3344;
`Fax 515.276.8655; E-mail: bcorron@foodprotection.org
`
`Executive Board
`
`President, JENNY SCOTT, National Food Processors Association,
`1350 I Street N.W., Suite 300, Washington, D.C. 20005-3305, USA;
`Phone 202.639.5985; Fax 202.639.5991; E-mail: jscott@nfpa-food.org
`President-Elect, JAMES S. DICKSON, Iowa State University,
`Department of Microbiology, 207 Science I, Ames, Iowa 50011-0001,
`USA; Phone 515.294.4733; Fax 515.294.6019; E-mail:
`jdickson@iastate.edu
`Vice President, ANNA M. LAMMERDING,Health Canada, Health
`Protection Branch, 110 Stone Road W., Guelph, Ontario, Canada NIG
`3W4, Phone 519.822.3300; Fax 519.822.2280;
`E-mail: annalammerding@hc-sc.ge.ca
`Secretary, PAUL A. HALL, Kraft Foods, Inc., 801 Waukegan Road,
`Glenview, Illinois 60025-4312, USA; Phone 847.646.3678;
`646.
`H
`il:
`.
`Fax 847.646.4820; E-mail: phall@kraft.com
`
`Past-President, JACK GUZEWICH, Food and Drug Administration,
`Division of Enforcement and Programs, HFS-605, 200 C Street S.W.,
`Washington, D.C. 20204-0001, USA; Phone 202.260.3847;
`Fax 202.260.0133; E-mail: john.guzewich@cfsan.fda.gov
`
`Affiliate Council Chairperson, FRED WEBER, WeberScientific,
`2732 Kuser Road, Hamilton, New Jersey 08691-9430, USA;
`Phone 609.584.7677, Fax 609.584.8388; E-mail: fredweber@earthlink.net
`
`Executive Director, DAVID W. THARP. CAE, International Association
`for Food Protection, 6200 Aurora Avenue, Suite 200W,
`Des Moines, Iowa 50322-2863, USA; Phone 800.369.6337;
`Fax 515.276.8655; E-mail: dtharp@foodprotection.org
`
`E. J. RHODEHAMEL,SC (01)
`P. E. KOEHLER, GA (00)
`. R. ACUFE TX (00)
`. A. GLATZ, IA (01)
`S. C. RICKE, TX (01)
`R. G. LABBE, MA (01)
`. H. ANDREWS, DC (01)
`. A. GOLDEN,TN (02)
`. G. M. GORRIS, NETH (02)
`E. T. RYSER,MI (01)
`R. V. LACHICA, MA (02)
`. §. BAILEY, GA (02)
`D. W. SCHAFFNER,NJ (01)
`R. E. LEVIN, MA (00)
`. J, BARRETT, GA (01)
`. W. GRIFFITHS, CAN (02)
`. D, HANCOCK, WA (01)
`B. W. SHELDON, NC(00)
`D. L. MARSHALL, MS (01)
`. E. BEATTIE, CA (01)
`WA.
`L. A. SHELER MI (00)
`R. T. MARSHALL, MO(00)
`. E. BRACKETT, DC (02)
`HARRISON,GA (00)
`LL.
`M. EFSLAVIK, AR (01)
`S. E. MARTIN,IL (01)
`BUCHANAN,DC (02)
`HERWIG, WA(00)
`.P
`LA.
`. B.
`HILL, WA (00)
`D. M. SMITH,MI (02)
`S. A. MeCARTHY, AL (02)
`BUSHWAY, ME (02)
`. AL
`HIMELBLOOM,AK(01)
`J. L. SMITH, PA (62)
`R. E McFEETERS,NC (02)
`. CASTILLO, MEX (02)
`.D.
`
`
`. G. CERVENY, WI (00) HITCHINS, DC (02) T. A. McMEEKIN, AUSTRAL (01)_S. S. SUMNER, VA (01)
`. D.
`HOCKING, AUSTRAL (01)
`2 §. CHU, WI (02)
`A. M. McNAMARA,VA(00)
`M. L. TAMPLIN,FL (01)
`. G. HOOVER,DE(00)
`. O. CLIVER, CA (02)
`J. MENG, MD (00)
`D. W. THAYER, PA (00)
`. G.
`. A. COUSIN,IN (00)
`HOTCHKISS, NY (00)
`C. MICHIELS, BELG (02)
`E. C. D. TODD, CAN (00)
`. W.
`L. J. MOBERG,OH (99)
`S. B. TURNIPSEED, CO (02)
`-Y. D’AOUST, CAN (00)
`HUTKINS,NE (00)
`.C,
`2» M. DAVIDSON,TN (01)
`INGHAM, WI (00)
`P. V. NIELSEN, DEN (00)
`K. S. VENKITANARAYANAN,
`A.
`. DEMIRCI,PA (02)
`JAYKUS, NC (02)
`G.-I. E. NYCHAS, GRE (02)
`CT (02)
`A,
`.D
`ePAOLA, AL (02)
`JOHNSON,WI (00)
`S. A. PALUMBO,PA (01)
`A. VON HOLY, SAFR (01)
`.G,
`.S.
`JOHNSON,AR (00)
`DICKSON,IA (02)
`M.W. PECK, UK (02)
`M. R. WACHTEL, CA (1)
`ORDANO, SPA (01)
`. EF ESCARTIN, MEX (01)
`J. M. PEINADO,SPA (00)
`LT WALLS, DC (02)
`me
`. JUNEJA,PA (02)
`. M. FARBER, CAN (00)
`J. J. PESTKA, MI (01)
`M. M. WEKELL, NY (00)
`2, FENG, DC (02)
`M. E. POTTER,DC (00)
`R. C. WHITING,DC (01)
` W. KASPAR, WI (02)
`. KATHARIOU,HI (00)
`_ E FRANK,GA (02)
`D. A. POWELL, CAN (02)
`C. E. WOLF-HALL, ND (01)
`. KATZ, NJ (01)
`. FRATAMICO, PA (02)
`K. J. RAJKOWSKI, PA (01)
`R. W. WOROBO, NY (02)
`AE.
`. KNABEL, PA (00)
`. GILL, CAN (00)
`B. RAY, WY (01)
`YOUSEEOH (00)
`
`OagEnOameong
`
`of?
`
`Editorial Board
`
`
`
`CEDSPRRORverearRvareemM
`
`Journal of Food Protection (SSN-0362-028X) is published monthly beginning with the January issue by the International Association for Food
`Protection. Executive offices are located at 6200 Aurora Avenue, Suite 200W, Des Moines, Iowa 50322-2863, USA. Each volumeconsists of 12
`issues. Periodical postage paid at Des Moines, Jowa 50318, and additional entry offices.
`Postmaster: Send address changes to Journal of Food Protection, LAFP, 6200 Aurora Avenue, Suite 200W, Des Moines, Iowa 50322-2863, USA.
`Microfilm of Journal of Food Protection is available from Xerox University Microfilms, 300 N. Zeeb Road, Ann Arbor, Michigan 48106-1346, USA.
`Permission to reprint any portion of Journal of Food Protection must be obtained from International Association for Food Protection. Claims for
`missing issues must be submitted to the Association within 30 days (US, Canada, and Mexico). International claims must be submitted within 60
`days. Address changes and membership dues should be directed to the editorial office. Instructions for Authors are available at
`www.foodprotcction.org or from the Journal of Food Protection Administrative Assistant.
`Membership in the association is available to individuals. Dues including the Journal of Food Protection and Dairy, Food and Environmental
`Sanitation are $150.00 US, $175.00 Canada/Mexico, and $220.00 International. Studentrates are $45.00 US, $60.00 Canada/Mexico, and $90.00
`International for Journal of Food Protection; $45.00 US, $55.00 Canada/Mexico, and $70.00 International for Dairy, Feod and Environmental
`Sanitation; and $75.00 US, $100.00 Canada/Mexico, and $145.00 for International for Journal of Food Protection and Dairy, Food and
`Environmental Sanitation. All membership dues include shipping and handling. Journal of Food Protection is available by subscription for $284.00
`US, $299.00 Canada/Mexico, and $329.00 International. Single copies are available for $36.00 US and $45,00 other countries. All rates include
`shipping and handling. No cancellations accepted.
`
`3
`
`

`

`Journal of Food Protection, Vol. 63, No. 11, 2000, Pages 1467-1474
`
`1467
`
`LINDA NgZC
` 2000
`
`Institute, Lethbridge,
`
`yee iq: 4He
`&xtts Kawetbops,
`Animal Diseases Research Institute, Lethbridge, Alberta, Canada; and “Agriculture and Agri-Food Canada, Range Restwal§i
`British Columbia, Canada V2B 8A9
`
`MS00-58: Received 24 February 2000/Accepted 22 May 2000
`
`ABSTRACT
`
`Three groupsof six yearling steers (three rumen fistulated plus three nonfistulated) fed one of three different grain diets
`(85% cracked corn, 15% whole cottonseed and 70% barley, or 85% barley) were inoculated with 10'° CFU of Escherichia
`coli 0157:H7 strain 3081, and the presence of the inoculated strain was followed in the rumen fluid and feces for a 10-week
`period. FE. coli 0157:H7 was rapidly eliminated from the rumen ofthe animals on all three diets but persisted in the feces of
`some animals up to 67 days after inoculation, suggesting that the bovine hindgut is the site of E. coli O157:H7 persistence.
`A significant difference existed in the levels of E. coli 0157:H7 shed by the animals among diets on days 5, 7, 49, and 63
`after inoculation (P < 0.05). No significant difference was found between the levels shed among diets on days 9 through 42
`and on day 67 (P > 0.05). The number of animals that were culture positive for E. coli 0157:H7 strain 3081 during the 10-
`week period was significantly higher for the barley fed group (72 of 114 samplings) as opposed to the corn fed group (44 of
`114 samplings) (P < 0.005) and the cottonseed and barley fed group (57 of 114 samplings) (P < 0.05). The fecal pH of the
`animals fed the corn diet was significantly lower (P < 0.05) than the fecal pH of the animals fed the cottonseed and barley
`* and barley diets, likely resulting in a less suitable environment for E. coli 0157:H7 in the hindgut of the corn fed animals.
`E. coli 0157:H7 strain 3081 was presentin 3 of 30 (corn, 1 of 10; cottonseed, 1 of 10; barley, 1 of 10) animal drinking water
`samples, 3 of 30 (corn, 1 of 10; cottonseed, 0 of 10; barley, 2 of 10) water trough biofilm swabs, 5 of 30 (corn, 0 of 10;
`cottonseed, 2 of 10; barley, 3 of 10) feed samples, and 30 of 30 manure samples taken from the pens during the entire
`experimental period. Mouth swabs of the steers were also culture positive for E. coli O157:H7 strain 3081 in 30 of 180
`samples (corn, 7 of 60; cottonseed, 4 of 60; barley, 19 of 60) taken during the 10-week period. Minimizing environmental
`dissemination of E. coli 0157:H7 in conjunction with diet modification may reduce numbers of £. coli 0157:H7-positive
`cattle.
`
`Since 1982, when it was first identified as a human
`H7 during the spring and summer months (/8, 26, 38). It
`has also been reported that weaned dairy calves and year-
`pathogen, enterohemorthagic Escherichia coliO157:H7 has
`ling beef cattle at slaughter are more likely to shed the
`been implicated in numerous outbreaks of hemorrhagic co-
`organism in their feces than adult cattle (14, 19, 36). In
`litis and life-threatening hemolytic uremic syndrome (31,
`addition to these factors, a numberof recent studies suggest
`32). Epidemiological investigations demonstrate that cattle,
`that diet also influences the fecal shedding of E. coli 0157:
`both beef and dairy, are a principal reservoir of E. coli
`H7bycattle (8, 14, 17, 20).
`O157:H7 (17, 38). This association is further supported by
`Grain feeding is amongthe dietary factors that are con-
`numerousfield surveys and trace-back studies that link E.
`sidered to be importantin respect to E. coli 0157:H7 fecal
`coli 0157:H7 directly and indirectly with bovine sources
`shedding. Beef cattle are commonly fed energy-rich grain
`(J2, 17, 38). Although contaminated and improperly
`diets during the finishing periods of beef production before
`cooked ground beef has been implicated as the primary
`slaughter, and fecal shedding of E. coli O0157:H7 at slaugh-
`vehicle of transmission (15, 17),
`foods such as radish
`ter is considered to beasignificant source of contamination
`sprouts (23), apple cider (4), unpasteurized milk (6, 26),
`for beef (36). Rapid fermentation of grains lowers the ru-
`mayonnaise (30), yogurt (27), venison jerky (24), and water
`minal and intestinal pH of cattle, favoring acid-resistant E.
`(1) have also been linked to E. coli 0157:H7 outbreaks.
`coli such as E. coli O157:H7. Althoughit has been reported
`Fecal shedding of E. coli O157:H7 in cattle herds is
`that grain feeding as opposed to hay feeding favors acid-
`widespread and intermittent in nature (17, 38). It is well
`resistant E. coli, a more recent study has indicated that the
`known that the season and age of the animal have a sig-
`acid sensitivity of E. coli O0157:H7 is not affected by the
`nificant effect on the level and duration of fecal shedding
`diet of cattle (9, 21). Much debate still exists concerning
`of E. coli 0157:H7 by cattle and other ruminants. Several
`these findings, and it is not clear if the cattle feeding in-
`studies have reported peak fecal shedding of E. coli 0157:
`dustry will advocate management changes until the issue
`resolves.
`Information provided by numerous farm surveys sug-
`
`* Author for correspondence: Tel: 250-554-5205; Fax: 250-554-5229,
`E-mail: veirad@em.agr.ca.
`
`4
`
`

`

`1529
`
`Journal of Food Protection, Vol. 63, No. 11, 2000, Pages 1529-1533
`‘Copyright ©, Intemational Association for Food Protection
`
`The Synergistic Effect of Excimer and Low-Pressure Mercury
`Lamps onthe Disinfection of Flowing Water
`
`IAN A. RAMSAY,' JEAN-CHRISTOPHE NIEDZIELA,? anp IAIN D. OGDEN?*
`
`‘Laser Installations Limited, Arbirlot, by Arbroath, DD11 2PY, UK; and ?Applied Food Microbiology Group, Department of Medical Microbiology,
`University ofAberdeen, Foresterhill, Aberdeen, AB25 2ZD, UK
`
`MS 00-60: Received 24 February 2000/Accepted 22 May 2000
`
`ABSTRACT
`
`Microorganisms in flowing water were disinfected by UV radiation from two excimer (excited dimer) lamps (emitting at
`172 and 222 nm) in combination with two low-pressure mercury lamps (emitting at 254 nm). Synergies were investigated
`among the three types of radiation in the treatment of water spiked in turn with Escherichia coli, Listeria innocua, Shewanella
`putrefaciens, and spores of Bacillus subtilis and Bacillus cereus. Synergy was demonstrated between radiations at 222 and
`254 nm in the treatment of E. coli, L. innocua, and S. putrefaciens, butlittle or no synergy was observed in the treatment of
`B. subtilis and B. cereus. At maximum flow rates (60 liters/min), 5-log reductions in E. coli were achieved at 254 nm,although
`at 222 nm, less than 1-log reductions were observed. No bacterial kill was observed with 172-nm radiation alone, despite
`increasing exposure time by reducing flow rates to less than 3 liters/min.
`
`The availability of safe drinking water is essential for
`human health and well-being. Water contamination and re-
`lated food poisoning incidents cause considerable economic
`costs throughout the world (2). The social disruption and
`financial consequences are great, and the illness suffered
`can be debilitating and sometimes life threatening, e.g.,
`Escherichia coli 0157, which has been linked to water-
`borne incidents in the UK (J2). The requirement for mi-
`crobiologically pure water is further necessitated since
`some ready-to-eat foods, such as vegetables, are disinfected
`by aqueous treatments, The motivation for evolving new
`processes or new combinations of processes to disinfect and
`clean foods comes also from changing legislation. There
`are moves under way to eliminate the use of chlorine in the
`microbiological treatment of some foods, and processes that
`involve UV (4), ozone (6, 8), gammairradiation (J, 5), and
`pulsed-electric fields (7, 13) are receiving increasing atten-
`tion.
`
`Surface water is often contaminated with waste from
`animal and human activity. Underground water supplies can
`also be contaminated, particularly so in the case of porous
`soils in periods of high rainfall. The microbiological safety
`limit for drinking water in the UK is less than 1 coliform
`per 100 ml. The use of mercury UV light at 254 nm is now
`widespread both in the production of potable water and,to
`a lesser extent, in the treatment of waste water. The maxi-
`mum absorption of DNA occurs at 257 nm. Higher and
`lower wavelengths, e.g., 222 nm, are absorbed to a lesser
`extent. The UV absorption causes photocleavage (severing),
`resulting in bacterial death (5) at relatively small doses (typ-
`ically <10 mJ/cm~?). Higher doses are required to elimi-
`
`* Author for correspondence. Tel: +44 1224 551132; Fax: +44 1224
`685604; E-mail: iogden@abdn.ac.uk.
`
`nate some viruses and still higher doses (330 mJ/cm~*) for
`Cryptosporidium parvum (10).
`Oppenlinder etal. (1/1) suggested that bactericidal ac-
`tion at 254-nm radiation could be improved by supplemen-
`tary radiation from excimer lamps. An excimer is an ex-
`cited dimer that is a combination of two adjacent atoms or
`molecules that behave as a unit. Excimer lamps, developed
`by Kogelschatz (9), make use of the excited dimer fluores-
`cence from pairs of atoms that are excited by an electrical
`discharge. There are many excimer combinations, some
`with pairs of like atoms and others with pairs of different
`atoms. At the appropriate gas pressure for a chosen com-
`bination, the lamp gives an outputof fluorescence centered
`on a specific wavelength in a narrow waveband, usually
`+5 nm.
`
`Two lamps that exhibit a high, essentially monochro-
`matic output are the Xe, at 172 nm and the KrCl at 222
`nm. At the lower wavelength of 172 nm, high quantum
`energy is producedthatis sufficient to hydrolyze water into
`hydrogen atoms and hydroxyl radicals (JJ). The hydroxyl
`radical is very short lived however (<10 ws), and the pen-
`etration depth of 172-nm radiation in water is very short
`(approximately 30 jzm). For radiation at this wavelength, a
`vacuum is required between lamp and flow tube, and the
`envelope materials must be Spectrosil (or an equivalent)
`and not Vitreosil (fused silica). Photons at the higher wave-
`length, 222 nm, do not require a vacuum or the Spectrosil
`material, and they have a much deeper penetration into
`clear water, approximately 3 cm. In the theory of Oppen-
`lander etal. (11), radiation at 222 nm catalyses the reaction
`between hydroxyl radicals (which are created by the 172-
`nm radiation) and wateritself to produce hydrogen perox-
`ide (HO) and ozone. These products are weaker oxidizing
`agents than the hydroxylradical but exist for longer periods
`
`5
`
`

`

`1530
`
`RAMSAYETAL.
`
`J. Food Prot., Vol. 63, No. 11
`
`Excimer chamber
`
`Right angle bends
`to facilitate mixing
`
`Mercury lamp
`chamber
`
`
`
`
`
`
`Reservoir
`
`An elevated reservoir (4 liters) was placed 1 m above the cham-
`bers sited on the laboratory bench. The reservoir was linked to a
`30-mm-diameter Spectrosil tube in the excimer chamberwith sil-
`icone rubber tubing. The connections to the 30-mm-diameter Vi-
`treosil tube in the mercury chamber via three right-angled glass
`(Pyrex) bends were also madeusingsilicone rubbertubing.A final
`length of silicone rubber tubing conducted the water under gravity
`from the mercury chamber into a waste receptacle on the floor.
`This tubing was clamped and released by handtoinitiate flow.
`
`Excimer lamps and the excimer chamber. The two lamp
`sets (KrCl Excimer and Xe* Excivac laboratory systems) were
`purchased from Heraeus Noblelight Ltd, Cambridge, UK. The
`steel excimer chamberheld a Spectrosil tube (32 mm diameter by
`60 cm length) that was submitted to UV light emitted by the two
`lamps. Two mirrors of polished stainless steel, curved to focus
`the irradiation onto the sample tube, were included. A vacuum
`pump created a vacuum to 0.3 mbar in the chamber. Before test-
`ing, it was important to exclude air bubbles manually from the
`sample tube that would otherwise prevent penetration by the 172-
`nm radiation.
`
`Mercury lamp chamber. Two mercury-indium lamps (NNI,
`120/80 from Heraeus, each having a discharge length of 80 cm)
`were housed parallel to a central Vitreosil sample tube within a
`doubleelliptical reflector (aluminum) to focus radiation from both
`sides onto the sample. Such a system prevents shadowing oftarget
`bacteria.
`
`Bacterial strains undertest. Isolates from the Applied Food
`Group’s laboratory collection included six strains of E. coli (50,
`60, 65, 67, 888, and D), three strains of Listeria innocua (GS1,
`MMB, and SC1), five strains of Shewanella putrefaciens (Ga2,
`Ga3, H1, Nn9, and Pp1), and onestrain each of Bacillus subtilis
`and Bacillus cereus. E. coli and L. innocua were grown in 10-ml
`volumes of tryptone soya broth (Oxoid CM129) at 37°C for 18 h
`before use, and S. putrefaciens was grown in 10-ml volumesnu-
`trient broth (Oxoid CM1) at 20°C for 24 h. B. subtilis and B.
`cereus spores were prepared by inoculation of vegetative cells
`onto nutrient agar plates incubated for 3 days at 20°C. The re-
`sulting growths were aseptically transferred to 100-ml sterile glass
`containers, heated in a water bath to 82°C for 10 min to kill veg-
`etative cells, and the spore crops were quickly cooled and stored
`at 4°C before use. Different volumes (5 to 15 mil) of microorgan-
`isms under test were inoculated separately into the system reser-
`voir and aliquots (10 ml) removed to estimate bacterial numbers
`by a modified method of Miles and Misra (3).
`
`Disinfection experiments. The mercury lamps were warmed
`up for 10 min to reach full power before use. The excimer lamps
`warmed up instantaneously. The system was filled with 4 liters of
`bacterial suspension, and the flow rate on releasing the clamp at
`the output end was measured at 60 liters/min. Aliquots (10 ml)
`for bacterial analysis were aseptically collected after 2 liters had
`flowed to waste, which was the volumeheld in the system down-
`stream of the second chamber and ensured that the test sample
`had been subjected to irradiation.
`
`Syuergy experiments. Preliminary tests showed that bacte-
`rial Kill from the mercury lamps was significantly greater than
`with excimer radiation. To observe synergy amongall lamps,it
`wastherefore necessary to reduce the output power from the mer-
`cury Jamps, set the 172- and 222-nm lamps at the highest power
`setting, and increase the contact time between sample and radia-
`tion source. The latter was achieved by the application of a con-
`strictor on the output tube end of the system. Flow rates of 8
`
`Clamp/constrictor
`
`P| Waste
`FIGURE 1. Schematic diagram offlow through UV disinfection
`system.
`
`in water under normal conditions and have some bacteri-
`cidal action.
`For 172-nm radiation alone, only a marginal outer lay-
`er of water would receive treatment, and the hydroxyl rad-
`ical would quickly decay as soon as the water flowed out
`of the region of excitation. For this reason, the logic of the
`theory requires that the 172- and 222-nm lamps should ir-
`radiate the Spectrosil flow tube simultaneously, although
`the diagram illustrating the theory (JJ) shows successive
`rather than simultaneous excitation by excimer lamps.
`By causing mixing of the water with a series of right-
`angled bendsas the water flows between the excimer cham-
`ber (where simultaneous excitation is assumed to have oc-
`curred) and the mercury lamp chamber, a dilute, uniform
`distribution of HO, is achieved before radiation by 254
`nm. Then, according to Oppenlinder et al. (11), this radi-
`ation dissociates the H,0, into pairs of hydroxyl radicals
`that are now distributed over the entire cross section. So,
`in addition to the killing of bacteria by photocleavage by
`the radiations at 222 and 254 nm, there is the additional
`antimicrobial action of the hydroxyl radicals. This is the
`major source of synergy that is predicted (/7) between the
`excimer lamps on one hand and the mercury Jamps on the
`other. This theory also predicts a minor synergy between
`the two types of excimer lamp through killing by the less
`potent H,0,.
`The objective of this study was to validate the synergy
`theory and to develop an improved UV method for the
`disinfection of flowing liquids. The effect of excimer and
`mercury radiation on gram-positive and gram-negative mi-
`croorganisms and bacillus spores was investigated.
`MATERIALS AND METHODS
`
`The flow-through system. A flow-through system was built
`to connect two UV chambers for dynamic experiments (Fig. 1).
`
`6
`
`

`

`J. Food Prot., Vol. 63, No. 11
`
`UV DISINFECTION OF WATER
`
`1531
`
`TABLE 1. Effect of different UV lamp combinations on E. coli
`65 in water*
`
`
`
`Lamp combinations Number of survivors
`
`adjusted to 4.1 (with HCl) and 10.0 (with NaOH) and compared
`with neutral water (pH 7.4) when irradiated with the mercury-
`indium lamp alone.
`
`4X 10°/ml
`Control (no lamps)
`4 x 105/ml
`172 nm
`1 x 105/ml
`222 nm
`<1 X 10'/ml
`254 nm
`1 X 10/ml
`172 + 222 nm
`<1 10'/ml
`222 + 254 nm
`
`172 + 222+ 254 nm <1 < 10m!
`
`“Flow rate was 60 liters/min.
`
`liters/min were used for E. coli, L. innocua, and S. putrefaciens
`and 2.9 liters/min for bacillus spores.
`
`Cleaning the system. Between replicate tests on the same
`microorganism, the system was rinsed thoroughly with tap water
`to ensure no residual contamination (confirmed by analysis of the
`water after such rinsing). At the end of each working day, the
`system was soaked overnight with proprietary hyperchlorite at the
`recommended strength.
`
`Testing for hydrogen peroxide. The presence of H,Oin
`water (after passing through both chambers) was determined using
`a test kit (HYP-1, HACH Company, Loveland, Colo.). Ammoni-
`um molybdate (1 ml) was added to a 10-ml aqueous sample fol-
`lowed by a measured quantity of sulfite reagent. The presence of |
`H,0, was indicated by a blue color. After 5 min, a specific number
`of drops of sodium thiosulfate titrant was added until the sample
`became faintly yellow. One drop of thiosulfate was equivalent to
`0.2 mg/liter of H,O, in the test sample.
`
`Role of hydrogen peroxide in the disinfection. To test
`whether the production of HO, contributed to synergy between
`excimer and mercury radiation, tap water was allowed to flow
`through the system at a flow rate of 0.29 liter/min, exactly 10
`times slower than therate of flow used for 222- and 254-nm tests.
`Samples for bacterial analysis were taken after 5 s and 10.5 min
`under various power settings on the lamps (to maximize H,O,
`production). To test whether the production of H,O, by 222-nm
`radiation was responsible for the observed synergy, the 222-nm
`lamp was switched off and H2O(Sigma, Poole, Dorset, UK) add-
`ed separately at 0.22- and 2.2-mg/liter concentrations to a suspen-
`sion of £. coli located within the system.
`
`Effects of temperature and pH. Water under treatment (be-
`fore spiking) was left to equilibrate at 6, 18, and 42°C.After spiking
`with E. coli, yolumes were in turn subjected to 222-, 254-, and
`222- + 254