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Application of Reuterin Produced by <I>Lactobacillus Reuteri</I> 12002

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Application of Reuterin Produced by <I>Lactobacillus Reuteri</I> 12002 257 Journal of Food Protection, Vol. 62, No. 3, 1999, Pages 257±261 Copyright Q, International Association of Milk, Food and Environmental Sanitarians Application of Reuterin Produced by Lactobacillus reuteri 12002 for Meat Decontamination and Preservation M. G. EL-ZINEY,1 T. VAN DEN TEMPEL,2 J. DEBEVERE,1 AND M. JAKOBSEN2* 1Department of Food Technology and Nutrition, Faculty of Agricultural and Applied Biological Sciences, Ghent University, Coupure-Links 653, B-9000 Ghent, Belgium, and 2Department of Dairy and Food Science, The Royal Veterinary and Agricultural University, Rolighedsvej 30, 1958 Frederiksberg C, Denmark MS 98-59: Received 3 March 1998/Accepted 23 October 1998 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/62/3/257/1671360/0362-028x-62_3_257.pdf by guest on 27 September 2021 ABSTRACT Lactobacillus reuteri strain 12002 was used for reuterin production in the two-step fermentation process. A batch culture fermentation was used to produce a maximum biomass of L. reuteri. Then cells were harvested, resuspended in a glycerol- water solution, and anaerobically incubated to produce reuterin. The lyophilized supernatants (approximately 4000 activity units (AU) of reuterin per ml) were diluted in distilled water for decontamination and preservation trials. The MIC values of reuterin for Escherichia coli O157:H7 and Listeria monocytogenes were 4 and 8 AU/ml, respectively. In meat decontamination experiments, the surface of cooked pork was inoculated with either L. monocytogenes or E. coli O157:H7 at a level of 2 8 approximately log10 5 CFU/cm , incubated for 30 min at 7 C, and decontaminated by exposure to reuterin (500 AU/ml). The bactericidal effect of reuterin was analyzed 15 s and 24 h after exposure at 78C. After 15 s of exposure to reuterin, viable 2 numbers decreased by 0.45 and 0.3 log10 CFU/cm for E. coli O157:H7 and L. monocytogenes, respectively. After 24 h the 2 2 numbers decreased by 2.7 log10 CFU/cm for E. coli O157:H7 and by 0.63 log10 CFU/cm for L. monocytogenes. In the same experiment, the combined effect of reuterin and lactic acid was also investigated. Adding lactic acid (5%, vol/vol) to reuterin signi®cantly enhanced (P # 0.05) the ef®cacy of reuterin. No additional effect (P # 0.05) was found when ethanol (40%) was added to the mixture of reuterin and lactic acid. To evaluate the preservative effect of reuterin during meat storage, reuterin was added to raw ground pork contaminated with E. coli O157:H7 or L. monocytogenes. Reuterin at a concentration of 100 8 AU/g resulted in a 5.0-log10 reduction of the viability of E. coli O157:H7 after 1 day of storage at 7 C. Reuterin at a concentration of 250 AU/g reduced the number of the viable cells of L. monocytogenes by log10 3.0 cycles after 1 week of storage at 78C. A variety of methods have been reported for the de- terin is a broad-spectrum antimicrobial substance, and its contamination of meat surfaces, including thermal treat- inhibitory effect is related to its action on DNA synthesis ment using hot water (22, 23), infrared radiation (25), gam- (27). It is water soluble, effective over a wide pH range ma radiation (1), and spray chilling system (12) with var- (3), and resistant to proteolytic and lipolytic enzymes (3, ious chemicals added such as chlorine (26), trisodium phos- 15). These advantages raise the possibility of applying reu- phate (4), hydrogen peroxide and ozonated water (17), and terin successfully as a preservative in different foods, in- ethanol (11). Furthermore, a wide range of organic acids, cluding meat and meat products. It has previously been re- such as acetic acid (7, 12, 16, 18, 20), citric acid (7), pro- ported that L. reuteri strain 12002 has the ability to produce pionic acid (20, 22), and lactic acid (18, 24, 28±30), have reuterin, which shows a potential inhibitory effect against been used. a wide range of gram-positive bacteria, e.g., Bacillus ce- Studies have also been carried out investigating the ef- reus. Staphylococcus aureus, and Listeria monocytogenes, ®cacy of bacteriocins for meat decontamination and pres- and gram-negative bacteria, e.g., Escherichia coli, Yersinia ervation (9, 19). Among the bacteriocins, nisin has been enterocolitica, and Pseudomonas ¯uorescens, in synthetic extensively studied as a meat-decontaminating agent (5, 8, media (15). Recently (14), it was demonstrated that reuterin 21). However, the effects observed are unsatisfactory, was able to reduce the viability of L. monocytogenes and which may be explained by binding of nisin to the meat E. coli O157:H7 in milk and cottage cheese at refrigeration particles and surfaces, uneven distribution, poor solubility, temperature (78C). and sensitivity to meat enzymes (10). The main objective of the present work is to investigate the Reuterin is a neutral broad-spectrum antimicrobial sub- bactericidal effect of reuterin produced by L. reuteri 12002 stance (3) formed during anaerobic growth of Lactobacillus alone and in combination with lactic acid and ethanol reuteri in the presence of glycerol. Reuterin is an equilib- against L. monocytogenes and E. coli O157:H7 on pork rium mixture of monomeric, hydrated monomeric, and cy- surfaces and in raw ground ham at 78C. clic dimeric forms of b-hydroxypropionaldehyde (27). Reu- MATERIALS AND METHODS * Author for correspondence. Tel: 145 35 28 32 16; Fax: 145 35 28 32 Microorganisms. L. reuteri 12002 was isolated from small 14; E-mail: [email protected]. pig intestine and identi®ed as previously described (14). E. coli 258 EL-ZINEY ET AL. J. Food Prot., Vol. 62, No. 3 strains included enteroinvasive E. coli strain LMG 8223, the in- bags, and homogenized with peptone saline (0.1 g/liter of peptone dicator strain (Laboratory of Microbiology, Gent University), and and 8.5 g/liter of NaCl) in a Seward Stomacher (Seward 400, enterohemorrhagic E. coli serotype O157:H7 strain MRK 1542 London, England) for 2 min. Homogenized samples were serially provided by Robert C. Clark, Institute of Agriculture, Guelph, diluted in phosphate-buffered saline and surface plated onto blood Ontario, Canada. L. monocytogenes 121 (serotype 4b) was a clin- agar base (Merck) supplemented with 5% (wt/vol) de®brinated ical isolate obtained from the culture collection of the Department horse blood (Difco) for L. monocytogenes or onto tryptone glu- of Veterinary Medicine, The Royal Veterinary and Agricultural cose beef extract agar (Difco) for E. coli O157:H7. Colonies were University, Copenhagen, Denmark. Cultures of E. coli and L. mon- counted after incubation of the plates for 48 h at 378C and veri®ed ocytogenes were maintained in brain-heart infusion broth (BHI, Dif- by haemolysis, colony morphology, and microscopy. co Laboratories, Detroit, Mich.) that contained 25% (wt/vol) glyc- Four independent replications were performed for each treat- erol at 2408C and propagated in BHI at 378C for 18 h (late sta- ment. For each analysis duplicate samples were examined and the 2 tionary phase) prior to use. L. reuteri 12002 was maintained in reduction of viable cells expressed as log10 N0 log10 Nt, where MRS broth (Merck, Darmsted, Germany) containing 25% (wt/vol) N0 and Nt are the counts of before and after treatment, respec- glycerol at 2408C and propagated in the same broth at 378C for tively. Average values of the four replicates are given. 18 h under anaerobic conditions (Anaerocult A, Merck). Downloaded from http://meridian.allenpress.com/jfp/article-pdf/62/3/257/1671360/0362-028x-62_3_257.pdf by guest on 27 September 2021 Inactivation of L. monocytogenes and E. coli O157:H7 in Production of reuterin. A two-step fermentation process raw ground pork at 78C. Lean pork from freshly slaughtered was used for reuterin production as previously described (14). The pigs was obtained from a slaughterhouse and kept in sterile plastic bioassay method and high-pressure liquid chromatography were bags at 48C. The outer layers were removed aseptically, and the used to quantify the reuterin activity units (AU) (6, 14, 15) with remaining parts of the meat minced in a sterilized food processor. 1 AU of reuterin being de®ned as the reciprocal of the highest For each treatment, the minced meat, in portions of 450 g in dilution that did not permit visible growth of E. coli LMG 8223 sterile Stomacher bags, was inoculated with E. coli O157:H7 (the indicator strain). Freeze-dried reuterin was stored at 48C for strain MRK 1542 or L. monocytogenes strain 121 at a level of more than a year without any loss in activity (unpublished results). log10 7 CFU/g. Samples in the bags were mixed by hand for 10 To determine MIC values, the bioassay method of Chung et min. Freeze-dried reuterin was diluted in sterile distilled water (20 al. (6) with slight modi®cations (14) was followed. ml) and used at ®nal concentrations of 50, 100, and 150 AU/g or 50, 100, 150, and 250 AU/g for samples inoculated with E. coli In vitro inactivation of L. monocytogenes and E. coli O157:H7 MRK 1542 and L. monocytogenes 121, respectively. Af- O157:H7 by reuterin. L. monocytogenes strain 121 and E. coli ter mixing in the bags, samples (25 g) were transferred in dupli- O157:H7 strain MRK 1542 were grown, harvested, washed as cate to sterile plastic bags (Sidamil UCB, Belgium; permeability: 2 described above, and then suspended in Butter®eld phosphate 6mlofO2,15mlofCO2,and2mlofN2 per m and 24 h at buffer (2) at room temperature. The bacterial suspension (approx- 258C and 100% relative humidity). The bags were vacuum-packed imately log10 8 CFU/ml determined by the pour plate method as (Komet gas packaging, type X200, Stuttgart, Germany) and stored described below) was used to inoculate 10 ml of reuterin solution at 78C for 14 days.
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