1275

Journal of Food Protection, Vol. 61, No. 10, 1998, Pages 1275-1280 Copyright ©, International Association of Milk, Food and Environmental Sanitarians

The Effect of Reuterin on Listeria monocytogenes and Escherichia coli 0157:H7 in Milk and Cottage Cheese

M. G. EL-ZINEyl,2 AND J. M. DEBEVEREh

lDepartment of Food Technology and Nutrition, Faculty of Agricultural and Applied Biological Sciences, University of Ghent, B-9000 Ghent, Belgium; and 2Department of Dairy Science and Technology, Alexandria University, Alexandria, Egypt

MS 97-128: Received 17 July 1997/Accepted 2 February 1998 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/61/10/1275/1670818/0362-028x-61_10_1275.pdf by guest on 26 September 2021 ABSTRACT

A broad-spectrum reuterin produced during anaerobic fermentation of glycerol by Lactobacillus reuteri strain 12002 was found to be inhibitory and bactericidal for Listeria monocytogenes and Escherichia coli OI57:H7. Lyophilized reuterin was prepared by a two-step fermentation process. A batch fermentation in a IS-liter fermentor was applied to produce a maximum biomass of L. reuteri using a modified MRS broth at pH 4.3. Further, harvested cells were used to ferment glycerol (250 mM) under anaerobic conditions, The sensitivity to reuterin of 10strains of Listeria spp., including 6 strains ofL. monocytogenes, and 6 strains of E. coli, including one enterotoxigenic E. coli strain and two enterohemorrhagic E. coli strains, was estimated. Strains of L. monocytogenes were more resistant to reuterin than E. coli strains. In cottage cheese, pH 5.4, L. monocytogenes increased by 0.4 log while E. coli 0157:H7 decreased by 0.5 log in 21 days at 7°C; addition of reuterin (50 to 250 units per g) to the cottage cheese reduced the viability of both organisms. The inactivation rate was more pronounced (P :=; 0.05) with E. coli 0157:H7 than L. monocytogenes and it was dependent on reuterin concentration. The rate of E. coli 0157:H7 population reduction reached to 2, 3, and 6 log cycles by day 7 for reuterin concentrations of 50, 100, and 150 units per g of creamed cottage cheese, respectively. While, 100, 150, and 250 units of reuterin per g caused reductions in L. monocytogenes counts by 2, 5, and greater than 5 log cycles, respectively. In UHT skim milk with 150 units of reuterin per ml, stored at rc, the decline in the numbers of L. monocytogenes cells was higher than that in cottage cheese. Milk fat in the range of 0.5 to 3% did not affect the reuterin activity (P :=; 0.05). Addition of 3% salt enhanced the lethal effect of reuterin and diminished the initial population of L. monocytogenes by 4.5 log cycles in three days at 7°C.

Reuterin is a potent broad-spectrum antimicrobial sub- tions of 50 and 100 units per g in ground beef kept at 4°C for stance produced by Lactobacillus reuteri during anaerobic 6 days reduced the viability of coliforms by 0.5 and 2 log fermentation of glycerol (3, 10). Reuterin exhibits an cycles respectively, while the viability increased by 4 log inhibitory activity against a wide range of microorganisms cycles in the control (8). including gram-positive and gram-negative bacteria, yeast, In recent years Listeria monocytogenes and Escherichia and fungi (5, 19). Reuterin has been isolated and identified coli 0157:H7 have become the most recognizable pathogens as an equilibrium mixture of monomeric, hydrated mono- of concern to public health and food agencies and the food meric, and cyclic dimeric forms of a metabolic intermediate, industry because of several outbreaks and cases associated l3-hydroxypropionaldehyde (29). with consumption of foods including milk and dairy prod- Reuterin is a neutral water-soluble product, effective in ucts (12, 21). It has been suggested that dairy cattle are a a wide range of pH (2 to 8) and resistant to proteolytic and natural reservoir for E, coli 0157:H7 (20). Further, it is lipolytic enzymes (5, 28). These advantages raise the known that L. monocytogenes can cause mastitis and possibility of applying reuterin successfully in a variety of abortion leading to excretion of the organism in milk from foodstuffs to increase their safety and extend their shelf life. the infected animal (15,16). Recently, L. reuteri has been applied to foods as a It has been reported that in cottage cheese L. monocyto- probiotic and/or starter culture. In bio-milk, named "BRA" genes survived fermentation and manufacturing processes in Sweden, the organism is used as an additional culture with (17, 25) whereas, E. coli 0157:H7 was able to multiply (1). Lactobacillus acidophilus and Bifidobacter spp. (26). It is also included in the production of Emmental-type cheese in Further, these organisms remained viable during storage at France (9). However, there is limited information concern- refrigeration temperatures (1, 17, 23, 25). Hence, the ing the application of reuterin in different food systems. In potential risk of consumption of milk or contaminated soft primary study, it is reported that reuterin added at concentra- cheese such as cottage cheese with L. monocytogenes or E. coli 0157:H7 prompted us to investigate the efficacy of reuterin in reducing the viability of these pathogens in * Author for correspondence. Mailing address: Department of Food Tech- cottage cheese at refrigeration temperature (70C) and to nology and Nutrition, University of Ghent, Coupure Links 653, B-9000 Ghent, Belgium. Tel: +32/92646177: Fax: +32/92255510; E-mail: johan. determine the effect of fat and salt content on reuterin [email protected]. activity in milk. 1276 EL-ZINEY AND DEBEVERE J. Food Prot., Vol. 61, No. 10

MATERIALS AND METHODS flow rate of 0.6 ml/min (10 mM H2S04 eluant); 20 111sample, loop injected, at 30°C. Reuterin was detected by refractive index Microorganisms. The strains of Listeria spp. included six measurement with a model RI 1047 detector (Hewlett-Packard). strains of L. monocytogenes, LMG 10470 (serotype I), LMG The retention time of reuterin under these conditions was 15 min; 13305 (serotype 4b), Ohio (OH, serotype 4b, isolated from reuterin eluted between glycerol (14 min) and trimethylene glycol Liederkranz cheese manufactured in Ohio), Scott A (clinical (18 min). Reuterin produced by the known producer strain DSM isolate, serotype 4b), V7 (milk isolate, serotype Ib, provided from 20016 (3) was used as a reference for reuterin. The presence of M. Bergdoll, The Food Research Institute, University of Wiscon- reuterin in the collected fractions was confirmed by the method of sin), and 121 (serotype 4b, associated with the outbreak of Circle et al. (6) and by the biological activity assay (5). listeriosis in Demark in 1989 and obtained from the Danish Research Meat Institute). Other Listeria strains used were Listeria Quantification of reuterin. Reuterin activity was quantified innocua strain LMG 11387, Listeria ivanovii strain LMG 11388, by the MIC method as described by Chung et al. (5). In general, the Listeria seeligeri strain LMG 11383, and Listeria welshimeri strain culture of E. coli LMG 8223 (indicator strain) grown overnight was LMG 11389. The strains of E. coli used through this study included harvested, washed twice with phosphate buffer (pH 7.2, 50 mM), Downloaded from http://meridian.allenpress.com/jfp/article-pdf/61/10/1275/1670818/0362-028x-61_10_1275.pdf by guest on 26 September 2021 verotoxigenic E. coli (VTEC) strain LMG 8223, Kl2 strains LMG suspended in the same buffer, and diluted to an A420 of 0.2 2579 and LMG 2578, enterotoxigenic E. coli (ETEC) strain CIP measured with a Shimadzu spectrophotometer (Shimadzu, Tokyo). 81.86 (Louis Pasteur Institute, Paris), and E. coli 0157:H7 strains This suspension was diluted III 0, which corresponds to about 6 932 (obtained from K. Glass, The Food Research Institute, 10glO CFU/ml. The diluted suspension (0.1 ml) was used to University of Wisconsin) and MRK 1542 (provided by Robert inoculate 1 ml of serial dilutions of reuterin diluted in basal Clark, University of Guelph, Ontario). Other strains woce from the medium (BM) containing (per liter in distilled water) 3 g casein Laboratory of Microbiology, University of Ghent, Belgium. L. hydrolysate, vitamin-free (Difco); 1.9 g ammonium citrate; 0.63 g monocytogenes strain OH and E. coli 0157:H7 strain 932 were citric acid; 12.6 g KH2P04; and 0.2 g MgS04·7H20; the pH was used in cottage cheese experiments. Survival experiments in the adjusted to 7.0 prior to autoclaving and 20 mM of filter-sterilized reuterin-treated UHT milk were performed using L. monocyto- glucose was added after sterilization. The tubes were incubated at genes OH. Cultures were maintained in tryptone soy broth (TSB, 3rC for 48 h. Growth was examined after 24 and 48 h. Reuterin Oxoid, Basingstoke, U.K.) containing 50% glycerol at -70°C and concentration was defined as the reciprocal of the highest dilution were propagated in TSB supplemented with 0.6% yeast extract that did not permit visible growth of the indicator strain. (TSYB) at 3rC. The cultures were transferred to individual tubes Determination of MIC values of reuterin against Listeria of the same broth and incubated at 3rC for 18 hprior to use. spp. and E. coli. The previously described method of Chung et al. Reuterin production. A reuterin producer strain L. reuteri (5) was followed with some modification. The MIC assay was strain 12002 was isolated from pig intestine and identified (11) performed in microtiter plates (Flow Laboratories, Rockville, Md.). using computerized comparisons of total soluble protein patterns Quantified reuterin was serially diluted in Mueller-Hinton medium (SDS-PAGE) as described by Pot et al. (24). Concentrated reuterin (Difco) supplemented with 20 mM glucose. The tested strains were used in the milk and cheese experiments was prepared by a grown in TSYB for two successive cultures at 3rC for 24 h. two-step fermentation process. For production of L. reuteri bio- Inocula of the cultures were then prepared (ca. 6 log to CFU/ml) as mass, batch fermentations were performed in a 15-liter fermentor mentioned above and used to inoculate aliquots of the diluted equipped with an Applikon AD! 1020 control unit (Applikon, reuterin (200 fll). The plates were incubated in a humid atmosphere Schidon, Netherlands). Modified MRS broth contained (per liter in in a plastic box at 37°C, and the growth inhibition of the tested distilled water) 10 g peptone (Merck, Darmstad, Germany), 8 g organisms was determined visually or measured as Asso in a Model meat extract (Merck), 4 g yeast extract (Difco Laboratories, 210 automated microplate reader (Microwell system, Organon

Detroit, Mich.), 1.5 g sodium acetate, 2 g K2HP04, 1.2 g Tween 80 Teknika, Boxtel, Netherlands) after 24 and 48 h. The MIC was (Sigma Chemical Co., St. Louis, Mo.), 2 g ammonium citrate, 0.2 g recorded as the lowest concentration that did not permit visible

MgS04·7H20, 0.02 g MnS04' and 0.25 g antifoam agent at pH 4.3. growth or kept the absorbance (550 nm) at a level below 0.06. The After autoclaving the medium at 121°C for 15 min, filter-sterilized MIC for each organism was evaluated twice. glucose (60 mM; Sigma) was added. The effective fermentor Cottage cheese. Creamed-style cottage cheese commercially volume was 14 liters, the stirring rate was 250 rpm, the temperature available in plastic containers (500 g per package) were obtained was 37°C, the pH was monitored with an installed Ingold pH directly from a local market on the day of delivery from the electrode and maintained by the addition of 6 N NaOH and 3 N manufacturer and transported to the laboratory in chilled insulated H3P04 and the fermentor was flushed with filter-sterilized nitrogen boxes and used immediately in the experiments. Fresh cottage at 60 ml/min to maintain anaerobic conditions. Cells were har- cheese was analyzed for moisture by oven drying (5 g of cheese at vested after 20 h by centrifugation (7000 X g, 10 min, 4°C) and 125°C for 2 h), fat content by the Mojonnier method (2), total washed twice with phosphate buffer (pH 7.2, 50 mM). Washed cells protein by the Kjeldahl method using a Kjeltec (Hoganas, Sweden) were incubated in water-glycerol solution (250 mM) under anaero- system 1002, NaCI content according to AOAC methodology (7), bic conditions (flushed with nitrogen). The resulting supernatant minerals by ashing (in a muffle furnace at 550°C for 16 h), and 11m; was sterilized by filtration (pore size 0.22 Millipore, Bedford, lactic acid by enzymatic assay with D- and L-lactate dehydroge- Mass.), then lyophilized to a reduced volume in a Heto Holten nases as described by the supplier (Boehringer GmbH, Mannheim, lyophilizer (model FD3, Allerod, Denmark). Lyophilized reuterin Germany). The pH of the cheese samples was determined by was stored at 4°C for more than a year without any loss in activity. homogenizing 5 g with 10 mI of deionized water and then The reuterin production was monitored by HPLC (28). The measuring the pH with a pH meter (model 763, Knick, Germany) in Hewlett-Packard LC system 1100 (Hewlett-Packard Co., Palo Alto, conjunction with an Ingold electrode (MGDX K57, Mettler- Calif.) in conjunction with HP Chemstation software for LC Toledo, Urdorf, Switzerland). (version 11.04.01) was used. Separation took place on an HPX-87H column (300 by 7.8 mm, Bio-Rad, Richmond, Calif.) protected by The inhibitory effect of reuterin against L. monocytogenes a cation-H+ Micro-Guard column (30 by 4.6 mm, Bio-Rad) at a and E. coli 0157:H7 in creamed cottage cheese. Respective J. Food Prot., Vol. 61, No. 10 INHIBITION OF L. MONOCYTOGENES AND E. COLI 0l57:H7 BY REUTERIN 1277 overnight cultures of L. monocytogenes strain Ohio and E. coli The chemical analysis of the cottage cheese used in this 0157:H7 strain 932 grown in TSYB were harvested, washed, and study revealed that it was composed of 15.5% protein, 5.5% suspended in phosphate buffer. Cottage cheese samples were then fat, 0.5% sodium chloride, 0.28% lactic acid, and 74.5% inoculated with the bacterial suspensions to give ca. 5 to 6 10gIO moisture. In the absence of reuterin the inoculated number of CFU/g. A 500-g portion of cheese was used for each treatment. viable listeria (L. monocytogenes strain Ohio) in creamed Reuterin stock solution was diluted in sterile distilled water (20 ml) cottage cheese, with 5.5% fat and a pH of 5.4, increased by and then added to achieve final concentrations of 50, 100, and 150 0.4 log (Figure 1a), whereas E. coli 0157:H7 strain 932 units per g or 50, 100, 150, and 250 units per g in cheese contaminated with E. coli 0157:H7 or L. monocytogenes, respec- decreased by 0.5 log (Figure 2a) within 21 days at 7°C. The tively. The addition of reuterin did not affect the initial pH of the addition of reuterin led to a reduced viability of both cottage cheese. The samples were placed, 25 g per pack, in organisms. The reduction in the E. coli cultures was more duplicate sterile plastic bags (Sidamil UCB, Ghent, Belgium; pronounced (P::5 0.05) compared to the cultures of L. permeability values: 6 ml of Oz, 15 ml of COz, and 2 ml of Nz per monocytogenes. At a reuterin concentration of 50 units per g Z m in 24 h at 25°C and 100% RH). The bags were sealed under 99% the population of L. monocytogenes decreased by 1.5 log Downloaded from http://meridian.allenpress.com/jfp/article-pdf/61/10/1275/1670818/0362-028x-61_10_1275.pdf by guest on 26 September 2021 vacuum in a Multivac apparatus (Haggemu11er KG, Wolfertschwe- den, Germany). Samples were incubated at rc for 21 days. a The inhibitory effect of reuterin against L. monocytogenes 7 in milk at different levels of fat and salt. UHT skim (0.5% fat), half-fat (1.5% fat), and whole-fat (3% fat) milks were obtained 6 from a local market. Salt (NaCl 10%, wtlvol) was added to UHT skim milk to give final concentrations of I and 3%. Duplicate milk 5 C> portions (20 ml) Were aseptically added to sterilized Scott bottles :::> (100 ml) and inoculated with L. monocytogenes Ohio to give ca. 5 - 4 LL 10gIOCFU/ml. Stock solution of reuterin was added to give a final () concentration of 150 units per ml. Samples were incubated at rc, C> 3 0 and survival of L. monocytogenes was monitored in time. .-J 2 Enumeration of microorganisms. In cheese experiments duplicate samples (25 g) were taken from each treatment at time 1 intervals, transferred to sterile Stomacher bags (Seward, London, U.K.), and pummeled for 2 min with 100 ml of buffered peptone 0 saline (BPS, 0.1 % peptone, Difco; 0.85% NaCl) in a Stomacher 400 (Seward). Homogenized samples were serially diluted, and 0 3 6 9 12 15 18 21 viable cultures were surface plated onto MacConkey sorbitol agar (Difco) for E. coli 0157:H7 and onto listeria selective agar base, Time (Days) Oxford formulation (CM 856, Oxoid), with its supplement (SR 140E, Oxoid) for L. monocytogenes. Colonies were counted after incubation for 24 h at 3rc. Total plate counts (TPCs) of mesophilic aerobic microorganisms were enumerated with plate b count agar (PCA, Oxoid) incubated aerobically at 22°C for 3 days. 8 In milk experiments viable cultures of L. monocytogenes Ohio were determined by surface plating on tryptone soy yeast extract 7 agar (TSYA, Oxoid) supplemented with 50 mg nalidixic acid 6 (Sigma) per liter. Representative presumptive colonies of listeria C> were confirmed by a r3-hemolysis test and by observation of typical 5 growth in listeria motility test medium. Two trials were performed :3 for each treatment, and duplicate samples were taken from each ~ 4 trial at every sampling time. 8>3 .-J Statistical analysis. Data were analyzed by SPSS software 2 (version 6, SPSS Inc., Chicago) for the analysis of variance (ANOVA), with significance (P < 0.05) between the treatments 1 determined using Duncan's multiple range test and least square means (LSMs), as appropriate. D values were determined as the o inverse of the slope of the survivor regression curve. Data were o 3 6 9 12 15 18 21 reported as the mean of two replicates. Time (Days) RESULTS FIGURE 1. Effect of reuterin on the growth of (a) L. monocyto- Strains of E. coli including the enterohemorrhagic genes OH and (b) mesophilic aerobic microorganisms in creamed strains did not differ in their sensitivity to reuterin, and a cottage cheese at rc. Symbols: 0, without added listeria and concentration of 4 units per ml was shown to be sufficient to reuterin; +, listeria control (no reuterin); ., 50 units of reuterin stop the growth of all the tested strains. Reuterin at a per g; _, 100 units of reuterin per g; +, 150 units of reuterin per g; concentration of 8 units per m1 was capable of inhibiting all 0, 250 units of reuterin per g; , detection limit. Bars represent the Listeria spp. including the strains of L. monocytogenes. the standard deviations. 1278 EL-ZINEY AND DEBEVERE J. Food Prot., Vol. 61, No. 10

a with L. monocytogenes and E. coli 0157:H7, in the presence 7 of reuterin, gave results similar to those obtained on the selective media, but the survival rates were higher (Figures 6 Ib and 2b). The pH of reuterin-free cottage cheese decreased from 5.4 to 5.2, 5.14, and 5.0 after 7, 14, and 21 days, 5 respectively. C> In the reuterin-treated cheese the pH did not drop below :J u.- 4 5.24 at a concentration of 50 units of reuterin per g while it () was 5.3 at higher reuterin concentrations. Recovery of C> 3 reuterin from the cottage cheese did not fully succeed. 0 ....J Difficulties were experienced in regard to the extraction 2 method. Variable results were obtained which did not allow

detection of the actual presence of residual active reuterin. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/61/10/1275/1670818/0362-028x-61_10_1275.pdf by guest on 26 September 2021 1 In UHT skim milk (0.5% fat) without reuterin addition the numbers of viable L. monocytogenes increased approxi- 0 mately 100-fold within 5 days at 7°C (Figure 3). The addition of 150 units of reuterin per ml killed nearly all of 0 3 6 9 12 15 18 21 the initial L. monocytogenes population (ca. 4.8 10glO CFU/ml) after 3 days (Figure 3). The content of milk fat, up Time (Days) to 3%, in the UHT milk did not significantly affect the reuterin activity (P::5 0.05). The decimal reduction value (D) for listeria during the first 3 days of incubation was 17.14 h in skim milk (0.5% fat) and was higher at 20.2 hand b 21.6 h in half-fat (1.5% fat) and whole-fat (3% fat) milk, 8 respectively. However, after 5 days, the L. monocytogenes 7 was not detected in any of the three types of milk. Increasing the sodium chloride concentration in reuterin-free milk 6 retarded the growth of listeria and enhanced the reuterin activity in skim UHT milk (Figure 4). The synergistic effect :35 between reuterin and salt was found to be concentration ~ 4 dependent. The population of listeria after first day of storage decreased by 80% (0.7 log unit) in the presence of ....J8'3 reuterin compared to 97.5% (1.6 log units) when reuterin was combined with 3% salt. However, salt at a concentration 2 of 1% did not enhance the killing effect of reuterin at 7°C. 1 o 7 o 3 6 9 12 15 18 21 6 Time (Days) E 5 ::>-- 4 FIGURE 2. Effectofreuterin on the survival of (a) E. coli 0157:H7 LL () 932 and (b) the growth of mesophilic aerobic microorganisms in Ol 3 creamed cottage cheese at rc. Symbols: 0, without added 0 hemorrhagic E. coli and reuterin; +, hemorrhagic E. coli control -J 2 (no reuterin); .A., 50 units ofreuterin per g; _,100 units ofreuterin per g; +, 150 units of reuterin per g; , detection limit. Bars 1 represent the standard deviations. 0 cycles after three weeks, whereas hemorrhagic E. coli 0 1 2 3 4 5 decreased by 3.5 log cycles during the same time. Increasing reuterin concentration to 150 units per g diminished hemor- Time (Days) rhagic E. coli to non detectable limit within 7 days whereas, FIGURE 3. Effect of reuterin at 150 units of reuterin per ml on the 250 units per g of reuterin was required with L. monocyto- growth ofL. monocytogenes OH in UHT milks with different levels genes to obtain a complete die-off. The natural microflora in of milk fat at rc. Symbols: 0, UHT skim milk (0.5% milk fat) the cottage cheese, without added reuterin, increased by 1 without added reuterin; _, UHT skim milk (0.5% milk fat) with log cycle during the 3 weeks of storage at 7°C. reuterin; .A., UHT half-fat milk (1.5% milk fat) with reuterin; e, The total viable counts (TPCs) of mesophilic aerobic UHTwholejat milk (3% milk fat) with reuterin. Bars represent the microorganisms on the PCA plates for cheese inoculated standard deviations. J. Food Prot., Vol. 61, No. 10 INHIBITION OF L. MONOCYTOGENES AND E. COll 0157:H7 BY REUTERIN 1279

7 positive and gram-negative bacteria, yeasts, fungi, viruses, and protozoa have been reported (3,5). Reuterin is thought 6 to be an analog of D-ribose capable of inhibiting DNA E 5 synthesis by inhibiting ribonucleotide reductases, the first -::) 4 step in de novo synthesis of deoxyribonucleotides for DNA u. synthesis (29). () 3 It is of interest to reiterate that milk fat in UHT milk in a C') range of 0.5 to 3% did not significantly alter the activity of ..9 2 reuterin. This could be attributed to the high solubility of 1 reuterin in the aqueous phase and negligible binding to the milk fat. Further, salt enhanced the reuterin activity and o increased the rate of decline of listeria. o 1 2 3 4 5 The results of these studies indicate that reuterin shows Downloaded from http://meridian.allenpress.com/jfp/article-pdf/61/10/1275/1670818/0362-028x-61_10_1275.pdf by guest on 26 September 2021 a bactericidal effect against L. monocytogenes and E. coli Time (Days) 0157:H7 in synthetic media, in milk, and in cottage cheese. Reuterin could be used as a biopreservative agent against FIGURE 4. Effect of reuterin at 150 units of reuterin per ml in pathogens such as L. monocytogenes and E. coli 0157:H7 in combination with salt on the growth ofL. monocytogenes OH in milk and soft cheeses like cottage cheese stored under UHT skim mi~k (0.5% milk fat) at rc. Symbols: 0, UHT skim milk refrigeration. Reuterin was not affected by milk fat in a without added reuterin with 1% salt; 1:::., UHT skim milk without range of 0.5 to 3%. The addition of 3% salt in combination added reuterin with 3% salt; e, UHT skim milk with reuterin with reuterin could be useful in order to enhance the reuterin without added salt; UHT skim milk with reuterin and 1% salt; +, effect. A, UHT skim milk with reuterin and 3% salt. Bars represent the standard deviations. ACKNOWLEDGMENTS

The production of reuterin was performed in the Lab of Fermentation, DISCUSSION Department of Food and Dairy Science, The Royal University of Veterinary and Agricultural Science, Copenhagen, in collaboration with Mogens In the present study reuterin at a concentration of 4 units Jakobsen. We wish to thank Mieke Uyttendaele (NFWO, National Fund for of reuterin per m1 was able to inhibit all the tested strains of Scientific Research) for valuable comments on the manuscript. E. coli including the hemorrhagic strains. The same MIC REFERENCES value was reported by Chung et al. (5) for E. coli K12 and 1. Aeocha, M. M., M. Mcvey, S. D. Loder, J. H. Rupnow, and L. six ETEC strains. There is no available information concern- Bullerman. 1992. Behavior of hemorrhagic Escherichia coli 0157:H7 ing the MIC value of reuterin against Listeria spp. during the manufacture of cottage cheese. J. Food Prot. 55:379-381. In cottage cheese at pH 5.4 listeria grew and increased 2. Atherton, H. V., and J. A. Newlander. 1977. Chemistry and testing of by 0.4 log after 21 days of storage at 7°e. In results dairy products, 4th ed. AVI Publishing Co., Westport, Conn. 3. Axelsson, L., T. C. Chung, W. J. Dobrogosz, and S. Lindgren. 1989. consistent with the present study, Genigeorgis et al. (14) Production of a broad spectrum antimicrobial substance by Lactoba- reported multiplication of L. monocytogenes in three of five cillus reuteri. Microb. Ecol. Health Dis. 2: 131-136. types of cottage cheese, with pH values of 5.1 to 4.9, by a 4. Benkerroum, N., and W. E. Sandine. 1988. Inhibitory action of nisin factor of 10 within 15 to 24 days at g0e. However, a rapid against Listeria monocytogenes. J. Dairy Res 71:3237-3245. 5. Chung, T. C., L.Axelsson, S. E. Lindgren, and W. J. Dobrogosz. 1989. growth of this bacterium in sterilized cottage cheese had In vitro studies on reuterin synthesis by Lactobacillus reuteri. Microb. previously been observed at pH 2: 5 both at 5 and 37°C (4). Beol. Health Dis. 2:137-144. It was suggested that the changes in the cheese induced by 6. Circle, S. J., L. Stone, and C. S. Boruff. 1945. Acrolein determination sterilization in the latter study initiated the growth of listeria by means of tryptophane. A colorimetric micromethod. Ind. Eng. Chern. Ed. 2:259-262. (23). However, in long-life cottage cheese at low pH (4.6 to 7. Cuniff, P. (ed.). 1995. Official methods of analysis, 16th ed., p. 10-12, 4.7), Ferreira and Lund (13) reported that L. monocytogenes 6l.Association of Official Analytical Chemists, Arlington, Va. decreased lO-fold within 7 days at 20°e. 8. Daeschel, M. A. 1989. Antimicrobial substances from lactic acid bacteria. Food Technol. 43:164-167. In current study, enterohemorrhagic E. coli did not grow 9. Delespaul, G., G. Leclerc, M. Lepeltier, and M. H. Chassagne. 1994. in cottage cheese and declined by 0.5 log unit within 21 days Cheesemaking process and the cheese obtained. French patent 2,696, at 7°C. 620. Mesophilic pathogens such as E. coli 0157:H7 gener- 10. Dobrogosz, W. J., I. A. Casas, G. A. Pangano, T. L. Talarico, B. M. Sorberg, and M. Karlson. 1989. Lactobacillus reuteri and the enteric ally grow only at temperatures higher than about lO°C (22, microbiota, p. 283-292. In R. Grubb, T. Midvedt, and E. Norin (eds.), 27). Kasrazadeh and Genigeorgis (18) studied the behavior The regulatory and protective role of the normal microflora. Macmil- of E. coli 0157:H7 in soft hispanic-type cheese at pH 6.6 lan Ltd., London. using the same strain (E. coli 0157:H7 strain 932) in this 11. EI-Ziney, M., T. van den Tempel, J. Debevere, and M. Jakobsen. 1996. Reuterin as an effective tool for meat decontamination, p. 123. Abstr. study and reported a decline of 2 log units after 2 months of 16th International Symposium, International Committee on Food storage at goC. Microbiology and Hygiene, Budapest, Hungary. In this study the addition of reuterin to cottage cheese 12. Farber, M., and P. I. Peterkin. 1991. Listeria monocytogenes, a produced a bactericidal effect against L. monocytogenes, food-borne pathogen. Microbiol. Rev. 55:476-511. 13. Ferreira, M. A. S. S., and B. M. Lund. 1996. The effect of nisin on hemorrhagic E. coli, and the inherent microflora. Listeria monocytogenes in culture medium and long-life cottage The microbicidal effects of reuterin against gram- cheese. Lett. Appl. Microbiol. 22:433-438. 1280 EL-ZINEY AND DEBEVERE J. Food Prot., Vol. 61, No. 10

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