Journal of Medical and Biological Engineering, 21(4): 205-212

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A Natural Sterilant (Reuterin) Fermented from Glycerol Using Lactobacillus Reuteri: Fermentation Conditions
Chin-Nan Chen
1

Hsiang-Fa Liang1

Min-Huang Lin1

Hsing-Wen Sung*,1

Department of Chemical Engineering, National Central University, Chung-Li, Taiwan, 320 ROC Department of Chemical Engineering, National Tsing Hua University, Hsinchu, Taiwan, 300 ROC Received 1 May 2001; Accepted 8 October 2001

Abstract
Cultures of Lactobacillus reuteri have been shown to accumulate a large quantity of reuterin during anaerobic growth in the presence of glycerol. It is known that reuterin has broad-spectrum antibacterial, antimycotic, and antiprotozoal activities. Therefore, it may be feasible using reuterin as a sterilant for the sterilization of biological tissues. The objective of the study was to investigate the optimal conditions (cell mass, fermentation time, fermentation temperature, and initial glycerol concentration) to produce reuterin fermented form glycerol using Lactobacillus reuteri. Additionally, the antimicrobial activity of reuterin was examined. Glutaraldehyde, the most commonly used sterilant for the sterilization of biological materials, was employed as a control. In the study, reuterin was successfully isolated from the fermented mixtures by a high-performance liquid chromatographer. The results revealed that the optimal fermentation condition was: 100 mg Lactobacillus reuteri cells/ml suspended in 300 mM glycerol in distilled water incubated under anaerobic condition at 37C for 1 h. Additionally, as per the minimal inhibitory and bactericidal concentrations, the antimicrobial activity of reuterin is significantly superior to glutaraldehyde. Keywords: Lactobacillus reuteri, Reuterin, Biological tissue, Sterilant

Introduction
Bioprostheses derived from biological tissues require sterilization to prevent the risk of transmitting infections to patients [1]. Sterilization is destruction of all forms of life on the implanted materials. It can be achieved via either physical or chemical means. Presently, available means for sterilization of biomaterials include dry heat, steam under pressure, irradiation, filtration, and chemical solution [2-6]. Sterilization of biological tissues is generally accomplished by exposing tissues to chemical solutions such as aqueous glutaraldehyde or formaldehyde. However, these chemical solutions are all highly cytotoxic, which may impair the biocompatibility of the sterilized tissues [7-9]. It is therefore desirable to provide a sterilant or antimicrobial reagent that is of low cytotoxicity and that may form germless and biocompatible products. Axelsson and co-workers reported discovery of a broadspectrum antimicrobial reagent, reuterin (3hydroxypropinoaldehyde). Reuterin is a fermented product of Lactobacillus reuteri [10]. Lactobacillus reuteri resides in
* Corresponding author: Hsing-Wen Sung Tel:+886-3-5719036; Fax: +886-3-572-6832 E-mail: hwsung@che.nthu.edu.tw

the gastrointestinal tract of humans and animals. Cultures of Lactobacillus reuteri have been shown to accumulate a large quantity of reuterin during anaerobic growth in the presence of glycerol [11,12]. It was reported in the literature that reuterin is a low-molecule-weight, pH-neutral, and watersoluble substance, which has antibacterial, antimycotic, and antiprotozoal activities [11]. Enteric Lactobacilli have been implicated as contributors to healthy gastrointestinal functions in humans and animals [13]. Evidence continues to prove that dietary Lactobacillus therapy affords protection from colon cancer for human populations on Western diets [14,15]. Additionally, using Lactobacillus reuteri as food supplements have been acknowledged to be safe and healthy by the Food and Drug Administration (FDA, USA, 1989) [16]. El-Ziney et al. investigated the efficacy of reuterin for meat decontamination and preservation [17]. The results demonstrated that reuterin is able to reduce the viability of Listeria monocytogenes and Escherichia coli O157:H7 on pork surfaces and in raw ground ham at refrigeration temperature (7C). The aforementioned results prompted us to evaluate the feasibility of using reuterin as a natural sterilant for the sterilization of biological materials.

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The objective of the study was to investigate the optimal conditions (cell mass, fermentation time, fermentation temperature, and initial glycerol concentration) to produce reuterin fermented form glycerol using Lactobacillus reuteri. Additionally, the antimicrobial activity of reuterin was examined. Glutaraldehyde, the most commonly used sterilant for the sterilization of biological materials, was employed as a control.

Figure 1.

Chemical structures of reuterin and glutaraldehyde

Materials and Methods

Growth of Lactobacillus reuteri Lactobacillus reuteri obtained from a local company (Challenge Bioproducts, Taichung, Taiwan) were cultured at 37C overnight in lactobacilli MRS broth medium (#288130, DIFCO, Sparks, Maryland, USA). A 1% inoculum from an overnight culture broth medium was added to fresh MRS medium and grown for 24 h at 37C under anaerobic conditions with nitrogen gas. Lactobacillus reuteri were harvested by centrifugation at 4000 g for 10 min and washed twice with phosphate buffer saline (PBS, pH 7.4). These Lactobacillus reuteri were used for the production of reuterin. Production of reuterin Reuterin (Figure 1) was produced as per the method reported by Axelsson et al. and Chung et al. [10,11] with a slight modification. The harvested Lactobacillus reuteri (250 mg wet weight) were suspended in 5 ml of 250 mM glycerol in distilled water and incubated for 2 h under anaerobic conditions (nitrogen sparging). After fermentation, the cells were collected by centrifugation at 4000 g for 10 min. The supernatant fraction was filtered using a filter menbrane (0.45 m pore size, nylon, Millipore) and stored at 4C in a container for purification subseqently. Purification and analysis of reuterin Reuterin was isolated out of other compounds obtained in the fermentation reaction using a high-performance liquid chromatographer (HPLC) equipped with a preparative glass column (30025 mm, OMNI, Cambridge, England) packed with ion-exchange resin (50W-X8, 200-400 mesh, Bio-Rad, Hercules, California, USA). A solvent composed of 60% acetonitrile and 40% distilled-deionized water containing 1.1 g of trifluoroacetic acid (Merck, Rahway, New Jersey, USA) was delivered by an HPLC pump (TSP, P-100, Riviera Beach, FL33419, USA). The solvent flow rate was 3 ml/min. A refractive index detector (RI2000-F, SFD, Torrance, California, USA) was employed to identify reuterin. Fractions were collected with a fraction collector (SF2100W, ADVANTEC, Dublin, California, USA) and then evaporated to remove acetonitrile solvent. Quantification of reuterin was performed by passing the fractions through an analytical column (87H3, Transgenomic, Omaha, Nevada, USA). The HPLC equipment used was the same as described above. The solvent consisted of a 65:35 mixture of deionized water and acetonitrile containing 0.01 N H2SO4. The flow rate was adjusted to be 0.7 ml/min at an ambient temperature.

Fermentation conditions In an attempt to allow Lactobacillus reuteri to synthesize a relatively large quantity of reuterin, the effects of fermentation conditions (cell mass, fermentation time, fermentation temperature, and initial glycerol concentration) were examined. The effects of cell mass on the production of reuterin were investigated by suspending 10, 25, 50, 100, or 150 mg/ml (wet weight) of washed Lactobacillus reuteri in 250 mM glycerol solution and incubated at 37C for 2 h. The effects of fermentation time were evaluated at 0.5, 1, 2, 3, 4, and 5 h. To elucidate the effects of fermentation temperature, distinct temperatures (4, 25, 37, and 45C) were used. Additionally, the effects of initial glycerol concentration (100, 200, 250, 300, 400 and 500 mM) on the production of reuterin were studied. After incubation, Lactobacillus reuteri were removed and the supernatant fractions were subjected to chromatography. Antimicrobial activity Dilution tests were used to determine the minimal concentrations of reuterin required to inhibit or kill microorganisms [18]. Serial dilutions of reuterin were inoculated with the test microorganisms and then incubated. The minimal inhibitory concentration (MIC) of reuterin was defined as the lowest concentration used without a visible growth of the test microorganism. On the other hand, the minimal bactericidal concentration (MBC) of reuterin was the lowest concentration employed without the formation of colony on the medium-based agar plate. In the study, glutaraldehyde (Figure 1) was used as a control. The microorganisms tested in the study were Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Staphylococcus aureus (ATCC 25923), and Bacillus subtillis (ATCC 6633). A series of tubes containing decreasing concentrations of reuterin were prepared. Generally, twofold dilutions can be prepared in the tubes for different concentrations of reuterin. To the first tube was added 2 ml solution of reuterin. Each remaining tube was added 1 ml of nutrient broth. With a sterile pipette, 1 ml stock solution was transferred from the first tube to the second tube. After entirely vortex of the second tube, 1 ml mixed solution was transferred from second tube to third tube. The process was continued to the last tube. The tube without adding reuterin was used as the growth control.

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-hydroxypropionic acid

reuterin glycerol

Figure 2. Example of the chromatogram of the fermented compounds obtained with 50 mg cells/ml of Lactobacillus reuteri and 250 mM of glycerol

reuterin

Figure 3. Chromatogram of reuterin after fraction collection

The inoculum was prepared to contain 106 colonyformatted units (CFU) of selected microorganism in 1 ml for each tube. Each test tube contained 1 ml of the inoculum solution and 1 ml of reuterin solution. The final concentrations of reuterin in tubes were half of the initial concentration because of the addition of an equal volume of inoculum solution. The tubes were incubated at 37C for 24 h. After incubation, the lowest concentration of reuterin resulting in complete inhibition of visible growth represents MIC. The tubes containing reuterin that show no visible growth of the microorganism can be subcultured onto medium-based agar plate and incubated at 37C for 24 h to see whether living microorganisms remain. The lowest concentration of reuterin caused no colony formation on the plate represents MBC.

Results
Production of reuterin Figure 2 shows an example of the chromatogram of the fermented compounds obtained with 50 mg cells/ml of Lactobacillus reuteri and 250 mM of glycerol. Separation of the compounds obtained in this fermentation condition was accomplished with an analytical ion-exchange column. As shown in the figure, three distinct compounds were identified: -hydroxypropionic acid (-HPA), reuterin, and glycerol. An analytical HPLC with an ion-exchange column was used to quantify these three compounds. A linear relationship was established between the concentrations of the products (-HPA, reuterin and glycerol) and the peak

areas in the chromatogram. Subsequently, reuterin was isolated out of the mixed compounds with a preparative column. Figure 3 presents the chromatogram of reuterin after fraction collection. As indicated in the figure, there was only one peak observed in the chromatogram. Effects of cell mass The cell mass played an important role in affecting the percentage of glycerol converted into reuterin after fermentation. Figure 4 presents the concentrations of reuterin produced with distinct amounts of cell mass. As shown in the figure, the concentrations of reuterin produced at 100 and 150 mg cells/ml were significantly greater than those produced at 10, 25, and 50 mg cells/ml. In addition, the degree of increasing the production of reuterin with 10 to 100 mg cells/ml appeared to be higher than those with 100 to 150 mg cells/ml. Effects of fermentation time In this study, Lactobacillus reuteri (100 mg of cells/ml) were suspended in 250 mM glycerol and incubated at 37C. Samples were retrieved at distinct duration and analyzed by an HPLC. Figure 5 presents the reuterin concentrations produced at various fermentation durations. As shown in the figure, the production of reuterin was maximal at 1 h after incubation. At this condition, cells were found dead after incubation. This observation indicated that reuterin could inhibit the growth of Lactobacillus reuteri. Effects of fermentation temperature The production of reuterin was significantly influenced by the fermentation temperature. The effects of fermentation temperature on the amount of reuterin produced are presented in Figure 6. As indicated in the figure, the concentration of reuterin produced at 37C was maximal. In contrast, the concentrations of reuterin produced at 4 and 50C were significantly lower than those produced at 25 and 37C. Effects of initial glycerol concentration The effects of initial glycerol concentration on the production of reuterin were remarkable (Figure 7). The production of reuterin increased significantly with increasing the initial glycerol concentration. However, as the initial concentration of glycerol was greater than 300 mM, the production of reuterin stayed approximately equivalent. Antimicrobial activity The results of MIC and MBC obtained in the study for each test microorganism are presented in Figures 8 and 9, respectively. As shown in the figures, all test microorganisms, including both the gram-positive bacteria (Staphylococcus aureus and Bacillus subtillis) and the gramnegative bacteria (Escherichia coli and Pseudomonas aeruginosa) were sensitive to reuterin. Generally, a 20 to 35 ppm concentration of reuterin can effectively prevent the growth of all test microorganisms, while a 40 to 50 ppm concentration of reuterin resulted in the death of all tested microorganisms. However, the values of MIC and MBC for glutaraldehyde were significantly greater than those for reuterin (approximately 2~3 times higher). These results indicated that the antimicrobial activity of reuterin is significantly superior to that of glutaraldehyde.

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Reuterin Concentration (ppm)

7000 6000 5000 4000 3000 2000 1000 0 0 20 40 60 80 100 120 140 160

Reuterin Concentration (ppm)

8000

(n = 3) 8000 7500 7000 6500 6000 5500 5000 4500 4000 3500 0 1 2 3 4

(n = 3)

Cell Mass (mg/ml in 250mM glycerol) Figure 4. Effects of cell mass on the production of reuterin

Fermentation Time (hr) Figure 5. Effects of fermentation time on the production of reuterin

Reuterin Concentration (ppm)

Reuterin Concentration (ppm)

7000 6000 5000 4000 3000 2000 1000 0 10 20 30 40

(n = 3)

6500 6000 5500 5000 4500 4000 3500 3000 2500 100 200 300 400

(n = 3)

50

500

Fermentation Temperature () Figure 6. Effects of fermentation temperature on the production of reuterin

Glycerol Concentration (mM) Figure 7. Effects of initial glycerol concentration on the production of reuterin

Reuterin Concentration (ppm)

140 130.0 8.2 120 35.0 0.0 60.0 0.0 33.0 2.9 100 80 60 40 20 0 E P

Reuterin Concentration (ppm)

Reuterin Glutaraldehyde (n = 3) 23.0 2.9 60.0 0.0 35.0 0.0

Reuterin Glutaraldeh (n = 3) 43.0 2.9 95.0 5.8 110.0 0.0 S 50.0 0.0 200 150 100 50 0 103.0 17.1 B

85.0 10.0

Figure 8. Results of minimal inhibitory concentrations (MIC). E: Escherichia coli; P: Pseudomonas aeruginosa; S: Staphylococcus aureus; B: Bacillus subtillis.

Figure 9. Results of minimal bactericidal concentrations (MBC). E: Escherichia coli; P: Pseudomonas aeruginosa; S: Staphylococcus aureus; B: Bacillus subtillis.

180.0 18.3

41.0 2.5

30.0 0.0

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H H C O H

H C O H

H C H O H

H2O

O H H H C C C O H H H
Reuterin

O H H H C C C O H OH H
-hydroxypropionic acid

+ H H H H C C C H O H O H H
1,3-proanediol

Glycerol

Figure 10. Fermentation pathway for the production of reuterin in Lactobacillus reuteri.

OH HC O O OH H2C H2C OH H2C H2C OH O HC OH

OH

Reuterin dimer

Reuterin monomer

Reuterin hydrated monomer

Figure 11. Chemical structures of monomeric, hydrated monomeric, and cyclic dimeric forms of -hydroxypropioaldehyde.

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Discussion
Sterilization is clinically performed using steam, ethylene oxide, dry heat, and chemical solution [2-6]. Reuterin and Lactobacillus reuteri have been applied in the preservation of foods [14, 15]. Therefore, it is expected that reuterin may be used as a sterilant for the sterilization of biological tissues. Lactobacillus species have been used to convert glycerol into 1,3-propanediol, -hydroxypropionic acid ( -HPA), and reuterin via a fermentation process [19]. This fermentation process occurs in two steps (Figure 10): the first step is catalyzed by glycerol dehydrase, which converts glycerol to reuterin (-hydroxypropioaldehyde); the second step involves a conversion of -hydroxypropioaldehyde into 1,3-propanediol and -HPA [12]. However, the results obtained in our study (Figure 2) showed the presence of only reuterin and-HPA. The absence of 1,3-propanediol in our study may be due to the different strains of Lactobacillus reuteri used, that may have a different fermentation pathway. Earnshaw et al. failed to show any antimicrobial activity with two different strains of Lactobacillus reuteri under controlled fermentation conditions [20]. It is therefore probable that the ability to produce reuterin is restricted to certain strains of Lactobacillus reuteri. It was reported that reuterin is an intermediate in the fermentation process by Lactobacillus reuteri (Figure 10). The determination of the optimal fermentation condition can help one to produce a large quantity of reuterin. The result of fermentation time (Figure 5) showed that the production of reuterin was maximal at approximately 1 h after incubation. Subsequently, the concentration of reuterin started to decrease due to the conversion of reuterin into -HPA (Figure 10). The results shown in Figure 6 indicated that the optimal temperature for Lactobacillus reuteri to produce reuterin was 37C (i.e., the gastrointestinal tract temperature). Other temperatures higher or lower than 37C may inhibit the activity of Lactobacillus reuteri to synthesize reuterin. In addition, increasing the cell mass and initial concentration of glycerol can increase the production of reuterin (Figures 4, 7). Based on the aforementioned results, the optimal fermentation condition was: 100 mg cells/ml Lactobacillus reuteri and 300 mM glycerol incubated at 37C for 1 h. It was reported that reuterin is an equilibrium mixture of monomeric, hydrated monomeric, and cyclic dimeric forms of -hydroxypropioaldehyde [12]. The chemical structures of monomeric, hydrated monomeric, and cyclic dimeric forms of -hydroxypropioaldehyde are illustrated in Figure 11. In the acidic condition, the hydroxyl group on the first carbon of -hydroxypropioaldehyde reacted reversibly with the aldehyde group in another-hydroxypropioaldehyde to form a cyclic dimeric molecule. In contrast, in the alkaline environment, the aldehyde group of hydroxypropioaldehyde undertook the aldol-condensation with the aldehyde group in another hydroxypropioaldehyde. The aldol-condensation might stop -hydroxypropioaldehyde to form a cyclic dimeric

molecule. Therefore, the production of -hydroxypropionic acid can result in the acidification of the fermentation system and may increase the production of reuterin dimeric molecule. In the antimicrobial activity study, the values of MIC and MBC for glutaraldehyde were significantly greater than those for reuterin (approximately 2~3 times higher). These results indicated that reuterin is more efficient than glutaraldehyde in sterilization. It was reported that reuterin could effectively inhibit the activity of ribonucleotide reductase of Escherichia coli in vitro [12]. Because of the activity of ribonucleotide reductase dominates the DNA synthesis, the broad-spectrum activity of reuterin may be explained on this basis.

Conclusions
The results of the study demonstrated that the optimal fermentation condition to produce reuterin was: 100 mg Lactobacillus reuteri cells/ml suspended in 300 mM glycerol in distilled water incubated under anaerobic condition at 37C for 1 h. Additionally, as per the results obtained in the MIC and MBC studies, the antimicrobial activity of reuterin is significantly superior to that of glutaraldehyde.

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