Journal of Food Protection, Vol. 68, No. 7, 2005, Pages 1393–1398 Efficacy of Ozonated and Electrolyzed Oxidative Waters To
Decontaminate Hides of Cattle before Slaughter
U.S. Department of Agriculture, Agricultural Research Service, Roman L. Hruska U.S. Meat Animal Research Center, Clay Center, MS 04-517: Received 15 November 2004/Accepted 28 February 2005 ABSTRACT
The hides of cattle are the primary source of pathogens such as Escherichia coli O157:H7 that contaminate preevisceration carcasses during commercial beef processing. A number of interventions that reduce hide contamination and subsequent carcasscontamination are currently being developed. The objective of this study was to determine the efficacy of ozonated andelectrolyzed oxidizing (EO) waters to decontaminate beef hides and to compare these treatments with similar washing in waterwithout the active antimicrobial compounds. Cattle hides draped over barrels were used as the model system. Ozonated water(2 ppm) was applied at 4,800 kPa (700 lb in2) and 15ЊC for 10 s. Alkaline EO water and acidic EO water were sequentiallyapplied at 60ЊC for 10 s at 4,800 and 1,700 kPa (250 lb in2), respectively. Treatment using ozonated water reduced hideaerobic plate counts by 2.1 log CFU/100 cm2 and reduced Enterobacteriaceae counts by 3.4 log CFU/100 cm2. EO watertreatment reduced aerobic plate counts by 3.5 log CFU/100 cm2 and reduced Enterobacteriaceae counts by 4.3 log CFU/100cm2. Water controls that matched the wash conditions of the ozonated and EO treatments reduced aerobic plate counts by only0.5 and 1.0 log CFU/100 cm2, respectively, and each reduced Enterobacteriaceae counts by 0.9 log CFU/100 cm2. Theprevalence of E. coli O157 on hides was reduced from 89 to 31% following treatment with ozonated water and from 82 to35% following EO water treatment. Control wash treatments had no significant effect on the prevalence of E. coli O157:H7.
These results demonstrate that ozonated and EO waters can be used to decontaminate hides during processing and may beviable treatments for significantly reducing pathogen loads on beef hides, thereby reducing pathogens on beef carcasses.
The pathogen Escherichia coli O157:H7 has been a beef carcasses during processing originates on the hides of concern to the meat processing industry for the last 20 cattle (4, 6, 21). During the hide removal process, E. coli years. In the early 1980s, cases of hemorrhagic colitis O157:H7 and other pathogens such as Salmonella are trans- caused by E. coli O157:H7 were associated with consump- ferred from the hide, where they are highly prevalent, to tion of undercooked ground beef (25), and a ground beef- the carcass (2, 4, 6, 21). Processes that effectively clean the related E. coli O157:H7 outbreak caused hundreds of ill- hides before hide removal have been effective in lowering nesses and four deaths during 1992 and 1993 (28). In re- carcass microbial concentrations (6, 7, 21). Chemical de- sponse to these events, the U.S. Department of Agriculture hairing of hide-on carcasses was the first intervention to Food Safety and Inspection Service declared E. coli O157: indicate that the prevalence of E. coli O157:H7 on the H7 to be an adulterant in ground beef and required meat preevisceration carcass was almost eliminated when bac- processors to establish hazard analysis and critical control terial contamination of the hide was greatly reduced before point plans (14). Since then, several interventions that focus hide removal (21). Subsequent studies of an on-line hide on preventing carcass contamination and on decontaminat- washing cabinet revealed that hide washes prior to slaughter ing carcasses have been designed, tested, and put into use.
were highly effective in reducing carcass contamination These antimicrobial interventions, combined with strict hy- during hide removal (7). Chemicals and antimicrobial com- giene practices, have significantly improved microbial qual- pounds that have been evaluated for use in hide interven- ity of beef carcasses and reduced the incidence of E. coli tions include cetylpyridinium chloride, NaOH, trisodium O157:H7 in processing plants (2–4, 12). However, cattle phosphate, acidified chlorine, and phosphoric acid (6, 7). occasionally present for slaughter with a higher level of The data reported by Bosilevac et al. (7) formed the basis contamination than can be removed effectively with the for effective hide-washing systems now installed in all Car- gill Meat Solutions beef processing plants (1). Because hide The vast majority of E. coli O157:H7 that contaminates interventions may be the most effective means to reducepathogens on beef, we are attempting to provide as many * Author for correspondence. Tel: 402-762-4225; Fax: 402-762-4149; viable alternatives as possible to increase the rate and ease E-mail:
of implementation of these interventions by all processors.
† Names are necessary to report factually on available data; however, the Ozonated water and electrolyzed oxidizing (EO) water U.S. Department of Agriculture neither guarantees nor warrants the stan- are generally recognized as safe (GRAS) and can be used dard of the product, and the use of the name by the U.S. Department ofAgriculture implies no approval of the product to the exclusion of others inside a processing plant. Ozone has been used since the 1940s to disinfect drinking water at many municipal water treatment plants, and most bottled water has been treatedwith ozone since the 1980s. In 1997, ozone was grantedGRAS status, and its use has been investigated in the pro-cessing of fresh produce and red meat. EO water is pro-duced by passing a current of electricity through a dilutesaltwater solution. One product of the reaction is NaOHand the other is hypochlorous acid, which has a low pH,contains active chlorine, and has a strong oxidation-reduc-tion potential similar to that of ozone. The efficacy of bothozonated water and EO water has been demonstratedagainst E. coli O157:H7 and Salmonella Typhimurium (13,24) and other food-related pathogens such as Listeria mon-ocytogenes (15, 24), the same organisms that are of concernto beef processors. No information exists describing the po- FIGURE 1. Model hide washing system used to evaluate ozonated tential utility of ozonated or EO waters as hide-washing and EO water washes. Whole pulled hides were draped over bar- compounds. The objective of these studies was to evaluate rels to simulate hide-on carcasses. The hides were taken directly the use of ozonated or EO waters in wash steps to reduce from the processing line immediately after having been pulled hide contamination in experiments using a model hide- from the carcasses and before any hide processing steps had oc- curred. The anterior (ant.) and posterior (post.) of one side ofeach hide were used to collect data (treatment area 1 and treat- MATERIALS AND METHODS
ment area 2). Control samples were obtained from each treatment Experimental protocol. At a beef processing plant, hides
area before spraying. Controls (c) were taken from 500-cm2 areas were selected randomly from the processing line. The selected of alternating angles across the hide. After control samples were hides represented a variety of fed cattle breeds (some with long obtained, the wash treatments were applied to the hides, first to hair and some with short hair). The hides were collected imme- one end of the hide and then to the other end, with pauses to diately after removal from the carcasses and before any hide pro- allow sample collection. Treated samples (t) were collected from cessing steps (e.g., trimming, defleshing, or water immersion) oc- 200-cm2 areas adjacent to the control areas. All samples were curred. As a matter of standard operating procedure at the partic- collected using Speci-Sponge Whirl-Pak bags containing 20 ml of ipating processing plant, all animals in holding pens and alley ways had been sprayed using low-pressure tap water to removeas much visible contamination as possible before the animals en-tered the plant. No hide-directed interventions were used during to-side passes of the pressure sprayer. The sprayer nozzle was maintained at a distance of 65 cm from the hide surface during To evaluate hide decontamination treatments, whole pulled spraying. A 30-s dwell time for antimicrobial activity was allowed hides were draped over barrels to simulate hide-on carcasses. One after the final spray before samples were collected for microbial side of each pulled hide was used to collect two sets of data.
Control samples were obtained before treatment from the anterior Ozonated water application. Ozonated water was applied
and posterior of each hide (Fig. 1). Then, wash treatments were using a Mobile Whitewater System (Ozone International, Bain- applied to the anterior end of the hide and a sample was collected.
bridge Island, Wash.). This machine generates ozonated water of The treatment was then applied to the posterior end of the hide 2 Ϯ 0.2 ppm and dispenses it at 240 kPa (35 lb in2) in a stream and a sample was collected. Ninety-four hides (46 for EO water parallel to a high-pressure water stream. Ozone concentration was treatment and 48 for ozonated water treatment) were sampled dur- monitored periodically during use by visual comparison to optical ing two separate sample collection trips. An additional 72 hides standards. Atmospheric ozone was also monitored throughout ap- were sampled at a later time to determine the potential effects of plications to ensure that concentrations did not exceed Occupa- plain water washing at similar temperatures, durations, and pres- tional Safety & Health Administration standards. Ozonated water sures. Thirty-six hides were used for evaluation of EO water treat- was applied to the hide at 4,800 kPa and 15ЊC with 10 to 15 side- ments and for ozonated water treatments.
to-side passes for 10 s, then the high-pressure stream was turnedoff and ozonated water alone was applied at the dispensing pres- EO water application conditions. EO water was generated
sure (240 kPa) for an additional 5 s. Samples were collected 30 with a P-5000 EO water generator (Electric Aquagenics Unlim- s after the final low-pressure ozonated water spray.
ited, Inc., Lindon, Utah). Both the alkaline and acidic forms ofEO water were used. First, alkaline EO water (NaOH) was applied Application of control water washes. To determine the ef-
for 10 s at 4,800 kPa (700 lb in2) with a gasoline-powered pres- fects of pressure and temperature of the wash procedures, control sure sprayer (Briggs & Stratton, Milwaukee, Wis.) that was fitted washes were performed that mimicked the ozonated and EO water with a rotating nozzle and an adjustable pressure valve. The al- wash conditions in pressure, temperature, and duration. A pressure kaline EO water was pH 11.2 and 52ЊC when applied. A 10-s sprayer (model P14030, Precision Industries, Germantown, Wis.) pause occurred before the 10-s application of the acidic EO water was used for the control washes. Ozonated water controls were (hypochlorous acid) using a second power sprayer (Electric Aqua- applied for 10 s at 15ЊC and 4,800 kPa with end-to-end passes of genics) at 1,700 kPa (250 lb in2). Acidic EO water was pH 2.4, the sprayer. A control for the low-pressure 5-s ozonated water 70 ppm chlorine, and 60ЊC. Before each use, the free chlorine treatment was not included because low-pressure washes such as content of the acidic EO water was confirmed by titration. Both this have had no effect on hide microbial status (6, 22). Samples the alkaline and acidic EO waters were applied with 10 to 15 side- were collected after a 30-s dwell time. Control EO water washes HIDE DECONTAMINATION USING OZONATED AND EO WATERS consisted of two sequential 10-s washes at 4,800 kPa and 60ЊC.
TABLE 1. Effects of ozonated water treatment on the microbial Samples were collected after a 30-s dwell time.
Sampling. All hide samples were collected using Speci-
Sponge Whirl-Pak bags (Nasco, Fort Atkinson, Wis.) containing 20 ml of neutralization buffer (Difco, Becton Dickinson, Sparks,Md.). Controls (nontreated samples) were taken from 500-cm2 ar- eas of alternated angles across hide (in an ‘‘M’’ or ‘‘W’’ pattern).
Treated samples were collected from 200-cm2 areas adjacent to the control areas. Samples were collected using 10 bidirectional strokes of the sponge, which was turned over halfway through theprocess. All samples were thoroughly massaged by hand before a Values for APC and EBC are means, n ϭ 96. SEM was 0.06 being placed on ice and transported to the laboratory to be pro- for APC and 0.08 for EBC. Values for E. coli O157 prevalence are percentage of positive samples (fraction of positive samples).
b All samples were analyzed directly with a Bactometer, and treat- Bacterial counts. Aerobic plate counts (APC) and Entero-
ed samples were also analyzed using Petrifilm.
bacteriaceae counts (EBC) were determined using a Bactometer c Prevalence of E. coli O157 was determined by selective enrich- (bioMe´rieux, Hazelwood, Mo.) and Petrifilm count plates (3M Mi- ment and immunomagnetic separation isolation followed by crobiology, St. Paul, Minn.). The APC and EBC of hide samples plating on sorbitol MacConkey agar with cefixime and potassi- were performed by impedance measurements of 1-ml samples in um tellurite on O157 chromogenic media. Positive colonies were the Bactometer. Before use, APC samples were diluted 1,000-fold in General Purpose Medium Plus (bioMe´rieux) supplemented with d Treatment was 10 to 15 end-to-end passes (10 s each) of high- 18 g/liter dextrose (for a final concentration of 2% dextrose), and pressure (4,800 kPa or 700 lb in2) 15ЊC ozonated water, followed EBC samples were diluted 100-fold in Entero Medium (bio- by 5-s application of low-pressure (240 kPa or 35 lb in2) ozon- Me´rieux). The Bactometer incubated samples for 16 h at 37ЊC ated water. Samples were collected after a 30-s dwell time.
while measuring the initial detection time for each sample, which e Values were determined using general linear model procedures was converted to log CFU per milliliter using standard curves of SAS for APC and EBC. The model included the main effect derived for each test. The standard curves were determined by of treatment, and the least squares means separation was accom- performing quadratic regression analysis of initial detection times plished by the probability of difference (PDIFF) option (a pair- and log CFU per milliliter, which had been determined using Pe- wise t test). Pairwise comparisons of frequencies of E. coli O157 trifilm aerobic count plates for APC or Petrifilm Enterobacteria- detection were made using PROC FREQ and Mantel-Haenszel ceae count plates for EBC as the standards. The Bactometer in our experiments was therefore calibrated to Petrifilm. The reliablelower limit of detection using the Bactometer is 100 CFU/ml;therefore, all samples from treated hides were also directly plated main effect of treatment. For significant main effects (P Ͻ 0.05), to Petrifilm to determine APC and EBC. A 1-ml aliquot from each least squares mean separation was accomplished by the probability treated sample was serially diluted to 10Ϫ1 for EBC and 10Ϫ2 for of difference (PDIFF) option (a pairwise t test). Data for APC and APC in buffered peptone water. One milliliter of the appropriate EBC were log transformed before analysis of variance. Pairwise dilution was plated to Petrifilm aerobic count plates or Petrifilm comparisons of frequencies of E. coli O157 detection were made Enterobacteriaceae count plates. Petrifilm plates were incubated using PROC FREQ and Mantel-Haenszel chi-square analysis for 16 h at 37ЊC and colonies were counted manually.
E. coli O157 detection. The procedure for detection of E.
coli O157 consisted of enrichment, immunomagnetic separation,and plating as described previously (5, 7) with minor modifica- Recent studies have confirmed that the hide is one of tions for plating as follows. Bacterial cells bound to the immu- the most significant sites for pathogen intervention in nomagnetic separation beads were plated on sorbitol MacConkey slaughter cattle (6, 7, 21). Not all processors can implement agar (Difco, Becton Dickinson) plates supplemented with 0.05 systems such as chemical dehairing (21) or on-line hide mg/liter cefixime and 2.5 mg/liter potassium tellurite (Dynal, Lake wash cabinets (7). We continue to investigate viable alter- Success, N.Y.) and on E. coli O157 chromogenic agar plates.
natives that can be applied in an economical fashion. The Chromogenic media were either CHROMagar O157 agar latest of these are ozonated water and EO water. Both were (CHROMagar, Paris, France) supplemented with 5 mg/liter no- evaluated using a hide-washing model system (Fig. 1) to vobiocin (Sigma, St. Louis, Mo.) and 1 mg/liter potassium tellur- ite (Sigma) or Rainbow Agar (Biolog, Hayward, Calif.) supple-mented with 10 mg/liter novobiocin and 8 mg/liter potassium tel- Ozonated water washes of hides resulted in significant lurite. The use of different chromogenic media was based solely reductions (P Ͻ 0.05) of APC and EBC (Table 1). Before on availability from the manufacturers. All plates were incubated treatment, hide APC were 8.4 log CFU/100 cm2 and hide at 37ЊC for 16 h, and suspect colonies (i.e., sorbitol negative on EBC were 6.6 log CFU/100 cm2. The ozone wash reduced supplemented sorbitol MacConkey agar, characteristic magenta on APC by 2.1 log CFU/100 cm2 and reduced EBC by 3.4 log supplemented CHROMagar, or characteristic blue on supplement- CFU/100 cm2. The prevalence of E. coli O157 was also ed Rainbow agar) were confirmed as E. coli O157 using DrySpot reduced (P Ͻ 0.05) following ozonated water washing.
O157 latex agglutination tests (Oxoid, Ogdensburg, N.Y.).
Before treatment, E. coli O157 prevalence was 89%, and Statistical analyses. APC and EBC of hides were evaluated
after treatment the prevalence was 31%, a reduction of by analysis of variance using the general linear model procedures of SAS (SAS Institute, Inc., Cary, N.C.). The model included the Cold (15ЊC) water applied at 4,800 kPa for 10 s, in the TABLE 2. Effects of control water treatments on the microbial TABLE 3. Effects of electrolyzed water treatment on the micro- Ozone water control (n ϭ 72)d a Values for APC and EBC are means, n ϭ 92. SEM was 0.07 EO water control (n ϭ 72)f for APC and EBC. Values for E. coli O157 prevalence are per- centage of positive samples (fraction of positive samples).
b All samples were analyzed directly with a Bactometer, and treat- ed samples were also analyzed using Petrifilm.
c Prevalence of E. coli O157 was determined by selective enrich- ment and immunomagnetic separation isolation followed by a Values for APC and EBC are means, SEM ranged from 0.04 to plating on sorbitol MacConkey agar with cefixime and potassi- 0.05 for APC and EBC. Values for E. coli O157 prevalence are um tellurite and on O157 chromogenic media. Positive colonies percentage of positive samples (fraction of positive samples).
were confirmed by latex agglutination.
b All samples were analyzed directly with a Bactometer.
d Treatment was two 10-s applications of EO water: alkaline EO c Prevalence of E. coli O157 was determined by selective enrich- water applied at 52ЊC and 4,800 kPa (700 lb in2) and acidic EO ment and immunomagnetic separation isolation followed by water applied at 1,700 kPa (250 lb in2) and 60ЊC. Samples were plating on sorbitol MacConkey agar with cefixime and potassi- um tellurite and on O157 chromogenic media. Positive colonies e Values were determined using general linear model procedures were confirmed by latex agglutination.
of SAS for APC and EBC. The model included the main effect d Treatment was 10 to 15 end-to-end passes (10 s each) of water of treatment, and the least squares means separation was accom- at 4,800 kPa (700 lb in2) and 15ЊC. Samples were collected after plished by the probability of difference (PDIFF) option (a pair- wise t test). Pairwise comparisons of frequencies of E. coli O157 e Values were determined using general linear model procedures detection were made using PROC FREQ and Mantel-Haenszel of SAS for APC and EBC. The model included the main effect of treatment, and the least squares means separation was accom-plished by the probability of difference (PDIFF) option (a pair-wise t test). Pairwise comparisons of frequencies of E. coli O157 s steps (in the same manner as the EO water treatment) detection were made using PROC FREQ and Mantel-Haenszel reduced APC by 1.0 log CFU/100 cm2 and reduced EBC by 0.9 log CFU/100 cm2 (Table 2). The conditions of the f Treatment was 10 to 15 end-to-end passes (repeated twice, 10 s EO water washes were demonstrably more effective than each) of water at 4,800 kPa (700 lb in2) and 60ЊC. Samples werecollected after a 30-s dwell time.
those of the ozonated water washes, possibly because ofthe length of time and/or the application pressure. The liq-uid pressure at which a hide is washed affects the efficacy same fashion as the ozone treatment, reduced APC by 0.5 of the washing treatment (8). The difference of 0.5 log log CFU/100 cm2 and reduced EBC by 0.9 log CFU/100 CFU/100 cm2 in APC between wash conditions was sig- cm2 (Table 2). We concluded that the effect of ozonated nificant (P Ͻ 0.05), but there was no difference in the EBC water was therefore at least a reduction of 1.5 log CFU/100 or the prevalence of E. coli O157. In a previous study in cm2 for APC and 2.5 log CFU/100 cm2 for EBC. This which water was applied to hides at 8,300 kPa (1,200 lb water wash control had no effect on the prevalence of E. in2) for two 20-s periods, APC were reduced by 0.4 log coli O157, so the 65% reduction of E. coli O157 following CFU/100 cm2 and EBC were reduced by 0.6 log CFU/100 ozonated water washing can be directly attributed to the cm2 (8). Neither of these reductions was significantly dif- antimicrobial activity of the ozone.
ferent (P Ͼ 0.05) from those for the controls. In another The results of EO water washing also demonstrated study, a similar hide-washing treatment using water signif- significant effects (P Ͻ 0.05) on APC, EBC, and the prev- icantly reduced EBC by 1.6 log CFU/100 cm2 (7). The alence of E. coli O157 (Table 3). The hides used for the findings of the current study and those of the previous stud- EO washing experiments were not different (P Ͼ 0.05) ies suggest that water washes have variable effects on the from those washed with ozonated water. Reductions of microbial status of hides, possibly because of differences APC and EBC were 3.5 and 4.3 log CFU/100 cm2, respec- tively. The reduction of E. coli O157 was also significant The observed differences between the EO and ozon- and similar to that observed with ozonated water; 82% of ated water washes, however, were not solely due to the the hide samples contained detectable E. coli O157 before differences in wash conditions, because when the effect of EO water treatment, but only 35% of the hides were posi- the hot water washes was taken into account, treatments tive after treatment, a reduction of 57%.
with EO water still had a greater effect on hide cleanliness Hot (60ЊC) water applied for a total of 20 s in two 10- than did treatments with ozonated water. Adjusting results HIDE DECONTAMINATION USING OZONATED AND EO WATERS of treatments using EO water to take into account the hot on hides, which may be due to the difference in antimicro- water effects resulted in APC reductions of 2.5 log CFU/ bial action rather than the differences in application time 100 cm2 and EBC reductions of 3.4 log CFU/100 cm2. The and temperature. EO water and ozone both have a strong hot water wash also had a small (8%) but significant (P Ͻ oxidation-reduction potential, but only EO water contains 0.05) effect on the prevalence of E. coli O157. These find- free chlorine, an additional bactericide. EO water treatments ings suggest that the temperature and/or duration of the EO expose organisms to two extremes of pH (alkaline at pH water treatments also contributed to some extent to the an- 11.2 and acid at pH 2.4) that can stress and damage cells to a greater extent than can ozonated water, which is only We previously examined other hide decontamination methods using the same model system described here (7). The presence of foodborne pathogens on cattle hides Using a similar two-stage application treatment, we evalu- has been examined, and the hide is considered a likely ated combinations of phosphoric acid, trisodium phosphate, source of cross-contamination of beef during the hide re- and NaOH as wash and water or acidified chlorine as rinse moval process because the brisket area of the hide is fre- to reduce hide EBC. The greatest reduction in EBC was quently contaminated with pathogens (23). Improved hy- observed with the combination of phosphoric acid and acid- gienic practices during removal of hides from dirty animals ified chlorine (7), and that reduction was similar to that resulted in significant reductions of bacteria on the carcass obtained in the present study for EBC following ozonated (19), and a direct relationship between the prevalence of E. coli O157 on hides and the prevalence of this pathogen on Ozonated water can instantly reduce populations of E. preevisceration carcasses has been established (1). Several coli, Salmonella Typhimurium, and other food-related path- hide interventions have been used with various degrees of ogens by 5 to 6 log CFU/ml (24). However, direct appli- success. Washing the hides of cattle with water did not re- cations of ozonated water to various food products have not sult in significant reductions in hide or carcass contamina- resulted in such dramatic reductions. When alfalfa seeds tion (9, 20). The inclusion of an antimicrobial (cetylpyri- were treated with 4 ppm ozone for 2 min to inactivate E. dinium chloride) or a decontamination step (chemical de- coli O157:H7 (inoculated at 105 CFU/g), no reduction in hairing) was required to obtain improvements in cleanliness E. coli O157:H7 numbers was observed (27). Further ex- of hides and subsequent preevisceration carcasses, but use periments revealed that reductions of 0.51 to 1.75 log CFU/ of antimicrobials (6, 20) or chemical dehairing (26) did not g required 4 to 21 ppm ozone and exposure times of up to have a significant effect. Later use of chemical dehairing 64 min (27). Treatment of beef carcass surfaces with ozone on a large sample in an industry setting did clearly dem- and water to reduce populations of inoculated E. coli O157: onstrate the efficacy of hide decontamination and improved H7 and Salmonella Typhimurium resulted in no significant carcass cleanliness (21). Other successful hide interventions differences between the two treatments (10). In our system, that followed chemical dehairing included treatment with ozonated water was much more effective than water con- cetylpyridinium chloride (6) and treatment with NaOH and trols, and concentrations and exposure times were much water in an on-line hide wash cabinet (7). These treatments lower. The efficacy of ozone treatment is increased when have resulted in effective reduction of hide contamination, competing organic particles are removed and when a me- and the hide intervention resulted in less contamination of chanical means is used to dislodge bacteria (16). We hy- the corresponding carcasses compared with carcasses that pothesize that our results are due in part to the high-pres- were processed conventionally without hide intervention (6, sure application, which satisfies both of these requirements.
7, 21). Ozonated and EO waters are not currently validated In previous studies, acidic EO water reduced E. coli for use in hide interventions. These experiments were per- O157 and L. monocytogenes by 8 log CFU/ml when ex- formed in anticipation of potential on-line applications us- posed for 30 and 60 s, respectively (15). Salmonella Ty- ing either a hide wash cabinet or some alternative equip- phimurium was reduced by 7 log CFU/ml when treated in ment. The results of the model system reported here suggest a similar manner (13). In studies similar to those with ozon- that on-line implementation of processes using either ozon- ated water, direct application to food did not result in re- ated or EO water will be just as successful in controlling ductions of such magnitude. EO water applied to tomatoes pathogens on carcasses as the currently used hide interven- reduced populations of E. coli O157:H7, L. monocytogenes, and Salmonella Typhimurium by 4 log CFU/cm2 each after ACKNOWLEDGMENTS
exposures of 30 and 60 s (11). Our EO water wash lasteda total of 20 s (10 s for each phase) and was as effective We are grateful to the participating processing plant and its personnel for cooperation in these experiments. We thank the representatives from as treatments used on produce products, but not nearly as Ozone International and Electric Aquagenics Unlimited, Inc., for provid- effective as treatments of pathogens suspended in EO water.
ing the necessary equipment for use in these studies. We also thank Dr.
The efficacies of ozonated water, EO water, and other Xiangwu Nou and Dr. Terrance Arthur for their scientific assistance; Bruce solutions to disinfect lettuce, cucumbers, and strawberries Jasch, Ann Goding, and Gregory Smith for their technical support; and have been compared (17, 18). In each case, electrolyzed Carol Grummert for her secretarial skills.
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Pii: s0378-5122(01)00275-4

Effect of continuous combined therapy with vitamin K andvitamin D on bone mineral density and coagulofibrinolysisTakahisa Ushiroyama *, Atushi Ikeda, Minoru Ueki Department of Obstetrics and Gynecology , Osaka Medical College , 2-7 Daigaku - machi , Takatsuki , Osaka 569-8686, Japan Received 28 July 2000; received in revised form 7 February 2001; accepted 14 September 2001 Abstract


W E E K L Y • W E E K L Y • W E E K L Y • W E E K L Y • W E E K L Y Future of GA Forestry Committee Welcomes SPAN to Meeting As mentioned in a previous Management Update Weekly newsletter, a joint Senate and House committee was formed by GA Governor Sonny Perdue to explore ways to revive the state’s forest products industry. The committee held it’s first meeting earlier this week

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