Appl Environ Microbiol 66(10): 4325-4333

Characterization of Reutericyclin Produced by <em>Lactobacillus reuteri</em> LTH2584

Institut für Lebensmitteltechnologie, Universität Hohenheim, D-70599 Stuttgart,^{and Institut für Organische Chemie, Universität Tübingen, D-72076 Tübingen,}^Germany

^{Corresponding author. Present address: Lehrstuhl für Technische Mikrobiologie, TU Müchen, Weihenstephaner Steig 16, D-85350 Freising, Germany. Phone: 49 8161 713959. Fax: 49 8161 713327. E-mail:}ed.nehcneum-ut.mlb@elzneag.leahcim.

Received 2000 Feb 28; Accepted 2000 Jul 6.

Abstract

Lactobacillus reuteri LTH2584 exhibits antimicrobial activity that can be attributed neither to bacteriocins nor to the production of reuterin or organic acids. We have purified the active compound, named reutericyclin, to homogeneity and characterized its antimicrobial activity. Reutericyclin exhibited a broad inhibitory spectrum including Lactobacillus spp., Bacillus subtilis, B. cereus, Enterococcus faecalis, Staphylococcus aureus, and Listeria innocua. It did not affect the growth of gram-negative bacteria; however, the growth of lipopolysaccharide mutant strains of Escherichia coli was inhibited. Reutericyclin exhibited a bactericidal mode of action against Lactobacillus sanfranciscensis, Staphylococcus aureus, and B. subtilis and triggered the lysis of cells of L. sanfranciscensis in a dose-dependent manner. Germination of spores of B. subtilis was inhibited, but the spores remained unaffected under conditions that do not permit germination. The fatty acid supply of the growth media had a strong effect on reutericyclin production and its distribution between producer cells and the culture supernatant. Reutericyclin was purified from cell extracts and culture supernatant of L. reuteri LTH2584 cultures grown in mMRS by solvent extraction, gel filtration, RP-C₈ chromatography, and anion-exchange chromatography, followed by rechromatography by reversed-phase high-pressure liquid chromatography. Reutericyclin was characterized as a negatively charged, highly hydrophobic molecule with a molecular mass of 349 Da. Structural characterization (A. Höltzel, M. G. Gänzle, G. J. Nicholson, W. P. Hammes, and G. Jung, Angew. Chem. Int. Ed. 39:2766–2768, 2000) revealed that reutericyclin is a novel tetramic acid derivative. The inhibitory activity of culture supernatant of L. reuteri LTH2584 corresponded to that of purified as well as synthetic reutericyclin.

Abstract

Lactic acid bacteria (LAB) are the biological basis for the production of a great multitude of fermented foods. Their metabolic activity during these fermentative processes determines and maintains food quality. Food preservation by lactic fermentations relies mainly on the accumulation of organic acids and the acidification of the substrate. Metabolites such as acetaldehyde, diacetyl, hydrogen peroxide, and carbon dioxide contribute to this preservative effect (15). Niku-Paavola et al. (40) have identified low-molecular-weight compounds from cultures of Lactobacillus plantarum that contribute to the inhibitory effect of lactic acid. Certain strains of Lactobacillus reuteri produce a unique antagonistic activity, reuterin (1). This antimicrobial activity against a broad range of microorganisms was attributed to monomers, hydrated monomers, and cyclic dimers of β-hydroxypropionic aldehyde formed during anaerobic catabolism of glycerol. Furthermore, a great number of strains of LAB produce bacteriocins, ribosomally synthesized peptides that exhibit antagonistic activity against closely related species (32, 54). These compounds have received increasing attention since they have the potential to inhibit food pathogens (24, 51). Furthermore, lactobacilli of intestinal origin exhibit antimicrobial activity that could not be attributed to either bacteriocins or organic acids (10, 49). However, to date, no nonbacteriocin antibiotic of lactobacilli has been purified and characterized on the molecular level.

The applications of antagonistic compounds produced by lactobacilli are not limited to food preservation. Antimicrobials of LAB have been employed successfully to prevent the formation of biogenic amines (30), to inhibit pathogens causing mastitis (46), and to inhibit enteropathogens in the small intestines of animals (3). Furthermore, bacteriocin formation by meat starter cultures contributes to the competitiveness of the producer strain during sausage fermentation (59).

The majority of bacteriocins and antagonistic compounds characterized to date are produced by lactobacilli originating from meat or milk fermentations. Few data are available on antimicrobials produced by the lactobacilli employed in cereal fermentations. The metabolism and the physiological properties of lactobacilli from sourdoughs are highly adapted to their natural substrate (19, 26), and several studies suggest that the production of antagonists may further account for their dominance in the dough environment (11, 35, 41). Gänzle et al. (21) screened 65 strains of lactobacilli previously isolated from wheat and rye sourdoughs. Two of these 65 strains, L. mucosae LTH3566 and L. reuteri LTH2854, produced inhibitory activity against L. sanfranciscensis ATCC 27651. This study was undertaken to characterize the active compound produced by L. reuteri LTH2584, reutericyclin, on the molecular level and to determine a possible role for this antagonistic compound in the microecology of sourdough.

ACKNOWLEDGMENTS

We thank G. Reuter (Berlin), H. Maidhof (Berlin), W. Röcken (Detmold), and W. Brabetz (Borstel) for providing bacterial strains; Dagmar Glenewinkel for excellent technical assistance; and Christian Hertel and Gudrun Wolf for helpful discussions during the work. We are further indebted to Udo Marquardt, EMC microcollections GmbH, Tübingen, Germany, for kindly providing synthetic reutericyclin.

ACKNOWLEDGMENTS

REFERENCES

References

1. Axelsson L T, Chung T C, Dobrogosz W J, Lindgren S EProduction of a broad spectrum antimicrobial substance by Lactobacillus reuteri. Microb Ecol Health Dis. 1989;2:131–136.[PubMed][Google Scholar]
2. Bennik M H J, Verheul A, Abee T, Naaktgeboren-Stoffels G, Gorris L G M, Smid E JInteractions of nisin and pediocin PA-1 with closely related lactic acid bacteria that manifest over 100-fold differences in bacteriocin sensitivity. Appl Environ Microbiol. 1997;63:3628–3639.[Google Scholar]
3. Bernet-Camard M-F, Lievin V, Brassart D, Neeser J-R, Servin A L, Hudault SThe human Lactobacillus acidophilus strain LA1 secretes a nonbacteriocin antibacterial substance(s) active in vitro and in vivo. Appl Environ Microbiol. 1997;63:2747–2753.[Google Scholar]
4. Blom H, Katla T, Hagen B F, Axelsson LA model assay to demonstrate how intrinsic factors affect diffusion of bacteriocins. Int J Food Microbiol. 1997;38:103–109.[PubMed][Google Scholar]
5. Böcker G, Stolz P, Hammes W PNeue Erkenntnisse zum Ökosystem Sauerteig und zur Physiologie der sauerteig-typischen Stämme Lactobacillus sanfrancisco und Lactobacillus pontis. Getreide Mehl Brot. 1995;49:370–374.[PubMed][Google Scholar]
6. Böcker G, Vogel R F, Hammes W P. Lactobacillus sanfrancisco als stabiles Element in einem Reinzucht-Sauerteig Präparat. Getreide Mehl Brot. 1990;44:269–271.[PubMed]
7. Brabetz W, Müller-Loennies S, Holst O, Brade HDeletion of the heptosyltransferase genes rfaC and rfaF in Escherichia coli K-12 results in an Re-type lipopolysaccharide with a high degree of 2-aminoethanol phosphate substitution. Eur J Biochem. 1997;247:716–724.[PubMed][Google Scholar]
8. Casas I A, Dobrogosz W J. Lactobacillus reuteri: overview of a new probiotic for humans and animals. Microecol Ther. 1997;26:221–231.[PubMed]
9. Coconnier M-H, Lievin V, Hemery E, Servin A LAntagonistic activity against Helicobacter infection in vitro and in vivo by the human Lactobacillus acidophilus strain LB. Appl Environ Microbiol. 1998;64:4573–4580.[Google Scholar]
10. Coconnier M-H, Lievin V, Bernet-Camard M-F, Hudault S, Servin A LAntibacterial effect of the adhering human Lactobacillus acidophilus strain LB. Antimicrob Agents Chemother. 1997;41:1046–1052.[Google Scholar]
11. Corsetti A, Gobbetti M, Smacchi EAntibacterial activity of sourdough lactic acid bacteria: isolation of a bacteriocin-like inhibitory substance from Lactobacillus sanfrancisco C57. Food Microbiol. 1996;13:447–456.[PubMed][Google Scholar]
12. Daba H, Lacroix C, Huang J, Simard R E, Lemieux LSimple method of purification and sequencing of a bacteriocin produced by Pediococcus acidilactici UL5. J Appl Bacteriol. 1994;77:682–688.[PubMed][Google Scholar]
13. De Angelis M, Pollacci P, Gobbetti MAutolysis of Lactobacillus sanfranciscensis. Eur Food Res Technol. 1999;210:57–61.[PubMed][Google Scholar]
14. De Vuyst L, Callewaert R, Crabbé KPrimary metabolite kinetics of bacteriocin biosynthesis by Lactobacillus amylovorus and evidence for stimulation of bacteriocin production under unfavourable growth conditions. Microbiology. 1996;142:817–827.[PubMed][Google Scholar]
15. De Vuyst L, Vandamme E J. Antimicrobial potential of lactic acid bacteria. In: De Vuyst L, Vandamme E J, editors. Bacteriocins of lactic acid bacteria. London, United Kingdom: Chapman & Hall; 1994. pp. 91–142. [PubMed]
16. Eijsink V G, Skeie M, Middelhoven P H, Brurberg M-B, Nes I FComparative studies of class IIa bacteriocins of lactic acid bacteria. Appl Environ Microbiol. 1998;64:3275–3281.[Google Scholar]
17. Gänzle M G, Hertel C, Hammes W PResistance of Escherichia coli and Salmonella against nisin and curvacin A. Int J Food Microbiol. 1999;48:37–50.[PubMed][Google Scholar]
18. Gänzle M G, Weber S, Hammes W PEffect of ecological factors on the inhibitory spectrum and activity of bacteriocins. Int J Food Microbiol. 1999;48:207–217.[PubMed][Google Scholar]
19. Gänzle M G, Ehmann M, Hammes W PModeling of growth of Lactobacillus sanfranciscensis and Candida milleri in response to process parameters of the sourdough fermentation. Appl Environ Microbiol. 1998;64:2616–2623.[Google Scholar]
20. Gänzle M G, Hertel C, Hammes W P. Antimicrobial activity of bacteriocin-producing cultures in meat products. Modelling of the effect of pH, NaCl, and nitrite concentrations on the antimicrobial activity of sakacin P against Listeria ivanovii DSM20750. Fleischwirtschaft. 1996;76:409–412.[PubMed]
21. Gänzle M G, Hertel C, Hammes W P. Antimicrobial activity in lactobacilli from sourdough. In: Scheffers H W A, van Dijken J P, editors. Beijerinck Centennial. Microbial physiology and gene regulation: emerging principles and applications. Delft, The Netherlands: Delft University Press; 1995. pp. 380–381. [PubMed]
22. Gao Y, van Belkum M J, Stiles M EThe outer membrane of gram-negative bacteria inhibits antibacterial activity of brochocin-C. Appl Environ Microbiol. 1999;65:4329–4333.[Google Scholar]
23. Gittermann C OAntitumor, cytotoxic, and antibacterial activities of tenuazonic acid and congeneric tetramic acids. J Med Chem. 1965;8:483–486.[PubMed][Google Scholar]
24. Hammes W P, Hertel CNew developments in meat starter cultures. Meat Sci. 1998;49:S125–S138.[PubMed][Google Scholar]
25. Hammes W P, Haller D, Brassart D, Bode CTraditional starter cultures as probiotics. Microecol Ther. 1997;26:97–114.[PubMed][Google Scholar]
26. Hammes W P, Stolz P, Gänzle M GMetabolism of lactobacilli in traditional sourdoughs. Adv Food Sci. 1996;18:176–184.[PubMed][Google Scholar]
27. Höltzel A, Gänzle M G, Nicholson G J, Hammes W P, Jung GThe first low-molecular-weight antibiotic from lactic acid bacteria: reutericyclin, a new tetramic acid. Angew Chem Int Ed. 2000;39:2766–2768.[PubMed][Google Scholar]
28. Huis in't Veld J H J, Havenaar RSelection criteria and applications of probiotic microorganisms in man and animal. Microecol Ther. 1997;26:43–57.[PubMed][Google Scholar]
29. Huot E, Barrena-Bonzalez C, Petitdemange HTween 80 effect on bacteriocin synthesis by Lactococcus lactic subsp. cremoris J46. Lett Appl Microbiol. 1996;22:307–310.[PubMed][Google Scholar]
30. Joosten H M L J, Nunez MPrevention of histamine formation in cheese by bacteriocin-producing lactic acid bacteria. Appl Environ Microbiol. 1996;62:1178–1181.[Google Scholar]
31. Kabuchi T, Saito T, Kawai Y, Uemura J, Itoh TProduction, purification and characterization of reutericin 6, a bacteriocin with lytic activity produced by Lactobacillus reuteri LA6. Int J Food Microbiol. 1997;34:145–156.[PubMed][Google Scholar]
32. Klaenhammer T RGenetics of bacteriocins produced by lactic acid bacteria. FEMS Microbiol Rev. 1993;12:39–86.[PubMed][Google Scholar]
33. Klein G, Eichberg J, Grund S, Reuter GCharacterisation of a potential probiotic strain of Lactobacillus reuteri isolated from pigeon crop. Microecol Ther. 1997;26:233–241.[PubMed][Google Scholar]
34. Kline L, Sugihara T F. Microorganisms of the San Francisco sour dough bread process. II. Isolation and characterization of undescribed bacterial species responsible for the souring activity. Appl Microbiol. 1971;21:456–465.
35. Larsen A G, Vogensen F K, Josephsen JAntimicrobial activity of lactic acid bacteria isolated from sour doughs: purification and characterization of bavaricin A, a bacteriocin produced by Lactobacillus bavaricus MI401. J Appl Bacteriol. 1993;75:113–122.[PubMed][Google Scholar]
36. Marquardt U, Schmid D, Jung GRacemic synthesis of the new antibiotic tetramic acid reutericyclin. Synlett. 2000;2000:1131–1132.[PubMed][Google Scholar]
37. Morgan S, Ross R P, Hill CIncreasing starter cell lysis in cheddar cheese using a bacteriocin-producing adjunct. J Dairy Sci. 1997;80:1–10.[PubMed][Google Scholar]
38. Morgan S, Ross R P, Hill CBacteriolytic activity caused by the presence of a novel lactococcal plasmid encoding lactococcins A, B, and M. Appl Environ Microbiol. 1995;61:2995–3001.[Google Scholar]
39. Nikaido H. Outer membrane. In: Neidhardt F C, et al., editors. Escherichia coli and Salmonella: cellular and molecular biology. 2nd ed. Washington, D.C.: ASM Press; 1996. pp. 29–47. [PubMed]
40. Niku-Paavola M-L, Laitila A, Mattila-Sandholm T, Haikara ANew types of antimicrobial compounds produced by Lactobacillus plantarum. J Appl Microbiol. 1999;86:29–36.[PubMed][Google Scholar]
41. Olsen A, Halm M, Jakobsen MThe antimicrobial activity of lactic acid bacteria from fermented maize (kenkey) and their interactions during fermentation. J Appl Bacteriol. 1995;79:506–512.[PubMed][Google Scholar]
42. Peleg MA model of microbial growth and decay in a closed habitat based on combined Fermi's and the logistic equations. J Sci Food Agric. 1996;71:225–231.[PubMed][Google Scholar]
43. Röcken W, Spicher GFadenziehende Bakterien—Vorkommen, Bedeutung, Gegenmaßnahmen. Getreide Mehl Brot. 1993;47:30–35.[PubMed][Google Scholar]
44. Rosenquist H, Hansen AThe antimicrobial effect of organic acids, sour dough and nisin against Bacillus subtilis and B. licheniformis isolated from wheat bread. J Appl Microbiol. 1998;85:621–631.[PubMed][Google Scholar]
45. Rosenkvist H, Hansen AContamination profiles and characterisation of Bacillus species in wheat bread and raw materials for bread production. Int J Food Microbiol. 1995;26:353–363.[PubMed][Google Scholar]
46. Ryan M P, Meaney W J, Ross R P, Hill CEvaluation of lacticin 3147 and a teat seal containing this bacteriocin for inhibition of mastitis pathogens. Appl Environ Microbiol. 1998;64:2287–2290.[Google Scholar]
47. Schmidt G, Jann B, Jann KImmunochemistry of R lipopolysaccharides of Escherichia coli. Eur J Biochem. 1970;16:382–392.[PubMed][Google Scholar]
48. Shefet S M, Sheldon B W, Klaenhammer T REfficacy of optimized nisin-based treatments to inhibit Salmonella typhimurium and extend shelf life of broiler carcasses. J Food Prot. 1995;58:1077–1082.[PubMed][Google Scholar]
49. Silva M, Jacobus N V, Deneke C, Gorbach S LAntimicrobial substance from a human Lactobacillus strain. Antimicrob Agents Chemother. 1987;31:1231–1233.[Google Scholar]
50. Stevens K A, Sheldon B W, Klapes N A, Klaenhammer T RNisin treatment for inactivation of Salmonella species and other gram-negative bacteria. Appl Environ Microbiol. 1991;57:3613–3615.[Google Scholar]
51. Stiles M EBiopreservation by lactic acid bacteria. Antonie Leeuwenhoek. 1996;70:331–345.[PubMed][Google Scholar]
52. Stolz P, Böcker G, Vogel R F, Hammes W PUtilisation of maltose and glucose by lactobacilli isolated from sourdough. FEMS Microbiol Lett. 1993;109:237–242.[PubMed][Google Scholar]
53. Sugihara T F, Kline LFurther studies on a growth medium for Lactobacillus sanfrancisco. J Milk Food Technol. 1975;38:667–672.[PubMed][Google Scholar]
54. Tagg J R, Dajani A S, Wannamaker L WBacteriocins of gram-positive bacteria. Microbiol Rev. 1976;40:722–756.[Google Scholar]
55. Tichaczek P S, Nissen-Meyer J, Nes I F, Vogel R F, Hammes W PCharacterization of the bacteriocins curvacin A from Lactobacillus curvatus LTH1174 and sakacin P from L. sake LTH673. Syst Appl Microbiol. 1992;15:460–468.[PubMed][Google Scholar]
56. Vaara MAntibiotic supersusceptible mutants of Escherichia coli and Salmonella typhimurium. Antimicrob Agents Chemother. 1993;37:2255–2260.[Google Scholar]
57. Vogel R F, Knorr R, Müller M R A, Steudel U, Gänzle M G, Ehrmann M ANon-dairy lactic fermentations: the cereal world. Antonie Leeuwenhoek. 1999;76:403–411.[PubMed][Google Scholar]
58. Vogel R F, Böcker G, Stolz P, Ehrmann M, Fanta D, Ludwig W, Pot B, Kersters K, Schleifer K H, Hammes W PIdentification of lactobacilli from sourdough and description of Lactobacillus pontis sp. nov. Int J Syst Bacteriol. 1994;44:223–229.[PubMed][Google Scholar]
59. Vogel R F, Pohle B S, Tichaczek P S, Hammes W PThe competitive advantage of Lactobacillus curvatus LTH1174 in sausage fermentations is caused by formation of curvacin A. Syst Appl Microbiol. 1993;16:457–462.[PubMed][Google Scholar]
60. Yang R, Johnson M C, Ray BNovel method to extract large amounts of bacteriocins from lactic acid bacteria. Appl Environ Microbiol. 1992;58:3355–3359.[Google Scholar]