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Journal of Food Protection, Vol. 62, No. 3, 1999, Pages 257±261 Copyright ᮊ, International Association of Milk, Food and Environmental Sanitarians

Application of Reuterin Produced by 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 - 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 Њ 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 7ЊC. 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 Յ 0.05) the ef®cacy of reuterin. No additional effect (P Յ 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 Њ 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 7ЊC.

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 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 (7ЊC). 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 7ЊC. clic dimeric forms of ␤-hydroxypropionaldehyde (27). Reu- MATERIALS AND METHODS

* Author for correspondence. Tel: ϩ45 35 28 32 16; Fax: ϩ45 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 37ЊC 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 Ϫ40ЊC and propagated in BHI at 37ЊC for 18 h (late sta- ment. For each analysis duplicate samples were examined and the Ϫ 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 Ϫ40ЊC and propagated in the same broth at 37ЊC 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 7؇C. 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 4ЊC. 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 4ЊC 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- 25ЊC 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 7ЊC for 14 days. At speci®c intervals samples (10 g) were taken (500 AU/ml). After the de®ned exposure times at room temper- in duplicate for microbiological analysis and pH measurements. ature, 1 ml was transferred to a solution containing 0.5% (wt/vol) The samples were homogenized and diluted as previously de- peptone (Merck), 0.8% (wt/vol) NaCl, and about 0.1 ml of 15% scribed. MacConkey sorbitol agar (Difco) plates incubated for (wt/vol) K3PO4, which gives a pH of 9.0 to neutralize the anti- 37ЊC for 24 h were used for enumeration of E. coli O157:H7 and microbial effect of reuterin (3). The exposure times were 10, 20, modi®ed Oxford agar (Oxoid) for enumeration of L. monocyto- and 30 s and 1, 5, 10, 15, 20, and 30 min. Viable cells were genes. Mesophilic aerobic plate counts were performed in PCA determined by the pour plate method in plate count agar (PCA, incubated aerobically at 30ЊC for 48 h. Representative colonies Difco) incubated at 37ЊC for 48 h. from all the plates were examined by morphology and microscopy to verify the presence of the test organisms. Two separate trials Inactivation of L. monocytogenes and E. coli O157:H7 on were performed for each organism. -a meat surface by reuterin at 7؇C. Blocks of cooked pork, ob tained from a slaughterhouse immediately after cooking, were pH measurements. Samples were added to nine parts deion- aseptically sliced into pieces (5 ϫ 5 ϫ 0.5 cm) and stored at 7ЊC ized water and blended for 1 min. A pH meter (model 763, Knick, for 1 h before use. The samples contained less than 10 CFU/g as Germany) in conjunction with an Ingold electrode (MGDX K57, determined by a pour plate count in PCA incubated at 30ЊC for Urdorf, Switzerland) was used. 48 h. The meat surfaces were inoculated with E. coli O157:H7 Sensory evaluations. Pieces of fresh pork were dipped in strain MRK 1542 or L. monocytogenes strain 121 by spreading solutions of reuterin, lactic acid, and ethanol as described above. ␮ 75 l of 18-h cultures in BHI using a sterile, L-shaped, glass rod The treated samples were evaluated for color, texture, and overall 2 to a level of approximately log10 5 CFU/cm . Attachment of cells quality after 24-h storage at 7ЊC by 17 to 21 untrained judges Њ Њ was allowed to proceed for 30 min at 7 C. Treatment at 7 C was requested to score the coded samples on a nine-point descriptive performed by dipping the meat pieces for 15 s in 25 ml of solu- scale for color (1 ϭ bright, very undesirable color, 9 ϭ very tions containing (i) reuterin, (ii) reuterin and lactic acid, (iii) reu- desirable color), texture (1 ϭ extremely dry, 9 ϭ juicy), and over- terin, lactic acid, and ethanol, (iv) lactic acid and ethanol. The all quality (from 1 to 9; dislike extremely, dislike very much, concentrations used were 500 AU/ml of reuterin, 5% (vol/vol) dislike moderately, dislike slightly, neither like nor dislike, like ϩ lactic acid (L[ ]-lactic acid 80% [wt/wt], Purac SP800, Nordisk slightly, like moderately, like very much, like extremely). Droge Handels A/S, Glostrup, Denmark), and 40% (vol/vol) eth- anol (Merck). Treated samples and controls (untreated) were kept Statistical analysis. Data were analyzed by SPSS software in tightly closed sterile Petri dishes at 7ЊC. Samples were exam- (Version 6, SPSS Inc., Chicago, Ill.) for the analysis of variance ined for the survival of E. coli O157:H7 and L. monogytogenes (ANOVA) and determination of signi®cance (P Ͻ 0.05) between immediately (15 s) after the treatment and again after 24 h. Each the treatments using Duncan's multiple range test and least- slice was cut into two equal parts, transferred to sterile Stomacher squares means as appropriate. J. Food Prot., Vol. 62, No. 3 REUTERIN AS A MEAT DECONTAMINANT 259 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 FIGURE 1. Survival curves of E. coli O157:H7 MRK 1452 and L. monocytogenes 121 exposed to reuterin, 500 AU/ml, in But- ter®eld phosphate buffer at room temperature. Treated (ⅷ) and nontreated (⅜) E. coli O157:H7; treated (Ⅵ) and nontreated (Ⅺ) L. monocytogenes. Bars indicate standard deviation.

FIGURE 2. Mean reduction CFU/cm2 of E. coli O157:H7 strain RESULTS AND DISCUSSION MRK 1452 inoculated onto the surface of cooked pork and ex- posed to reuterin, 500 AU/ml; lactic acid, 5% (vol/vol), and eth- The reuterin concentration required to inhibit the anol, 40% (vol/vol) for 15 s as indicated at 7ЊC. Determination growth of E. coli O157:H7 MRK 1452 was 4 AU/ml, while of viable cells was carried out immediately (15 s) and after 24 h Њ it was found to be 8 AU/ml for L. monocytogenes 121. of storage at 7 C. The initial inoculum level of E. coli was ap- These results agree with our previous report of investigating proximately log10 5. Results with different letters are signi®cantly different (P Յ 0.05). the MIC values of several strains of E. coli and L. mono- cytogenes (14). Survival curves for E. coli O157:H7 MRK 1452 and with time, i.e., 24 h, at 7ЊC. After 24 h no signi®cant dif- L. monocytogenes 121 in Butter®eld phosphate buffer with ferences (P Յ 0.05) between treatments were observed. 500 AU/ml of reuterin added are shown in Figure 1. No In ground ham, reuterin caused a pronounced reduction signi®cant reduction (P Յ 0.05) in counts was observed in in the viability of E. coli O157:H7 (Fig. 4). After 1 day at the control experiments, whereas during the same 30-min 7ЊC, reuterin at a level of 100 AU/g reduced the bacterial period reuterin resulted in an immediate effect, leading to population by 5 log10. For L. monocytogenes, the effect of a 10-fold reduction of both organisms followed by a re- reuterin was signi®cant but less pronounced (Fig. 5). At a duction of 3 and 5 log10 in viable cells of E. coli O157:H7 and L. monocytogenes, respectively. In the inactivation studies on the surface of cooked pork (Figs. 2 and 3), a three-way ANOVA analysis showed that the three parameters of treatment, time, and type of organism had signi®cant effects (P Յ 0.05). The results indicate that both organisms showed a signi®cant reduction in viability immediately, i.e., after 15 s, of treatment. For E. coli O157:H7 (Fig. 2), the reduction of viable cells 2 Њ amounted to a log10 reduction of 0.45 CFU/cm at 7 C. The effect of reuterin was comparable to the effect of the lactic acid±ethanol mixture. However, addition of lactic acid to reuterin signi®cantly enhanced the effect of reuterin (P Յ 0.05). Addition of ethanol to this mixture did not improve the bactericidal effect (Fig. 2). Only treatments including reuterin showed increasing effect (P Յ 0.05) with time, i.e., after 24 h, at 7ЊC. In trials with L. monocytogenes, the immediate effect of reuterin alone amounted to a log10 reduction of 0.3 CFU/ 2 Њ cm at 7 C (Fig. 3). The effect of reuterin for L. monocy- 2 FIGURE 3. Mean log10 reduction CFU/cm of L. monocytogenes togenes was similar to the effect of the lactic acid±ethanol strain 121 inoculated onto the surface of cooked pork and exposed mixture (Fig. 3). Again, addition of lactic acid to reuterin to reuterin, 500 AU/ml; lactic acid, 5% (vol/vol); and ethanol, improved the immediate decontamination effect (Fig. 3). 40% (vol/vol) for 15 s at 7ЊC. For further details see legend of The effect of reuterin increased signi®cantly (P Յ 0.05) Figure 2. 260 EL-ZINEY ET AL. J. Food Prot., Vol. 62, No. 3

FIGURE 4. Bactericidal effect of reuterin against E. coli O157: FIGURE 5. Bactericidal effect of reuterin against L. monocyto- H7 strain MRK 1542 in ground pork stored under vacuum at 7ЊC genes strain 121 in ground pork stored under vacuum at 7ЊC (ⅷ, 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 (ⅷ, control; ᭡, with 50 AU/g; Ⅵ, 100 AU/g; , 150 AU/g). control; ᭡, with 50 AU/g; Ⅵ, 100 AU/g; , 150 AU/g; ϫ, 250 For clarity, coinciding symbols are not indicated. Bars indicate AU/g). For clarity, coinciding symbols are not indicated. Bars standard deviation. indicate standard deviation. reuterin concentration of 150 AU/g, the number of viable ferences between these results are likely to be explained by cells decreased by 1.3 log10 after 7 days. At a concentration variations in the experimental conditions, e.g., exposure of 250 AU/g the reduction was 3.2 log10 for the same pe- time, temperature, and contamination level. For dairy prod- Њ Њ riod at 7 C. During the 14 days of storage at 7 C, no growth ucts (14) a bactericidal effect against E. coli O157:H7 and was observed for surviving cells of L. monocytogenes. For L. monocytogenes comparable to the present study was ob- both organisms, comparative studies with recovery on PCA served. indicated that the selectivity of the media used, i.e., Reuterin alone did not alter the pH, color, texture, or MacConkey sorbitol agar and modi®ed Oxford agar, did not quality of the meat. Addition of lactic acid was responsible affect these data (results not shown). for unacceptable quality changes in the meat even for the Measurements of the pH on meat surface demonstrated low-temperature (7ЊC) and short-time (15 s) treatments. We (Table 1) that reuterin alone did not alter the surface, where- noticed that the quality defect caused by lactic acid is nor- Յ as this was the case for lactic acid (P 0.05). The addition mally diminished with storage time but cannot be avoided of ethanol seemed to reduce the acidifying effect of lactic unless an antioxidant, such as ascorbic acid, is added to the acid. lactic acid (29). Furthermore, the results obtained have in- Concerning the effect of various treatments on the col- dicated the advantage of using a combination of reuterin or, texture, and overall quality, reuterin alone did not cause and lactic acid for inactivation of E. coli O157:H7 and L. Յ any signi®cant (P 0.05) changes (results not shown). monocytogenes. It may even be possible to obtain a signif- Lactic acid (5%, vol/vol) alone or in combination with reu- icant bactericidal effect by combining reuterin with lower terin was responsible for meat discoloration and unaccept- concentrations of lactic acid not leading to undesired chang- Յ able texture and quality (P 0.05), but the use of ethanol es of the meat surfaces. (40%, vol/vol) in combination with lactic acid seemed to The results of the current study suggest that reuterin diminish the damaging effect of the acid (results not alone could be applied through a meat-chilling system to shown). produce an immediate decontamination effect against path- The current study shows the bactericidal effect of reu- ogens without alteration of meat quality. Further, it has been terin applied to (at 7ЊC for 15 s) a cooked pork surface that was arti®cially inoculated with E. coli O157:H7 or L. mon- ocytogenes. As seen in the culture medium, reuterin was TABLE 1. Effect of different treatments on the surface pH of found to be more effective in decontaminating meat in- cooked pork at 7ЊCa fected with E. coli O157:H7 than L. monocytogenes. Time A synergistic effect was obtained when reuterin was combined with lactic acid. This combination improved the Treatment 15 s 24 h immediate inactivation effect, which to a degree was more Control 6.18 Ϯ 0.02 Cb 6.19 Ϯ 0.01 C noticeable with E. coli O157:H7 than L. monocytogenes. Reuterin (500 AU/ml) 6.1 Ϯ 0.012 C 6.19 Ϯ 0.02 C Meanwhile, addition of ethanol to a reuterin±lactic acid Reuterin (500 AU/ml) and lac- mixture did not increase the rate of reduction. tic acid (5%) 5.14 Ϯ 0.02 A 5.35 Ϯ 0.02 B Decontamination properties of lactic acid against E. Reuterin (500 AU/ml), lactic coli O157:H7 and L. monocytogenes have been reported (7, acid (5%), and ethanol (40%) 5.36 Ϯ 0.04 B 5.41 Ϯ 0.03 B 13). 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