WO2000039152A1 - Subtiline stable et ses procedes de preparation - Google Patents

Subtiline stable et ses procedes de preparation Download PDF

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Publication number
WO2000039152A1
WO2000039152A1 PCT/US1999/030938 US9930938W WO0039152A1 WO 2000039152 A1 WO2000039152 A1 WO 2000039152A1 US 9930938 W US9930938 W US 9930938W WO 0039152 A1 WO0039152 A1 WO 0039152A1
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WIPO (PCT)
Prior art keywords
subtilin
stable
composition
column
effective
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Application number
PCT/US1999/030938
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English (en)
Inventor
Norman J. Hansen
Original Assignee
University Of Maryland
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
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Priority to CA002356955A priority Critical patent/CA2356955A1/fr
Priority to AU23888/00A priority patent/AU2388800A/en
Priority to EP99967636A priority patent/EP1140975A4/fr
Publication of WO2000039152A1 publication Critical patent/WO2000039152A1/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
    • A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
    • A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
    • A23L3/3463—Organic compounds; Microorganisms; Enzymes
    • A23L3/34635—Antibiotics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to compositions and methods of making a stable lantibiotic, such as nisin or subtilin, preferably subtilin.
  • Nisin is a 34-residue long peptide produced by Lactococcus lactis.
  • Subtilin is a 32-residue long peptide produced by Bacillus subtilis.
  • Subtilin sequences are disclosed in, e.g., U.S. Pat. Nos. 5,914,250, 5,885,811, 5,861,275, 5,576,420, 5,516,682, and 5,218,101.
  • a stable subtilin in accordance with the present invention is a subtilin which possesses improved subtilin stability in comparison to a subtilin in the prior art, where the stable subtilin and the prior art subtilin have the same amino acid sequence, preferably a naturally occurring amino acid sequence as disclosed in, e.g., U.S. Pat Nos. 5,576,420 and 5,861,275.
  • Improved stability refers to subtilin's resistance to losing activity when subjected to certain conditions.
  • subtilin prepared according to Jensen and Hirschman, Arch. Biochem., 4:197-309, 1994 is light-sensitive and stores poorly.
  • the subtilin of Jensen and Hirschman loses activity rapidly when stored at 4°C or 20° C.
  • a subtilin prepared according to Jensen and Hirschman shows only a minimal amount of activity, e.g., less than 5%.
  • a stable subtilin in accordance with the present invention when stored for 5 days at the same temperature retains substantially all its activity, e.g., greater than 50%, more preferably, 75% or higher, or 90% or higher.
  • stable subtilin refers to this improved stability in accordance with the present invention.
  • a stable subtilin can mean a subtilin which has been purified by hydrophobic interaction chromatography (HIC) and thus can also be referred to as a HIC- purified stable subtilin.
  • HIC-purified stable subtilin refers to a composition that which has been subjected to fractionation, e.g., HlC-purified stable subtilin is, e.g., about 10-fold, preferably about 20-fold, more preferably about 50-fold more concentrated than prior to its application to an HIC column.
  • a stable subtilin can comprise subtilins which have been post-translationally-modified, e.g., having a succinyl residue at its N-terminus.
  • the improved stability of subtilin can be measured in variety of ways, including, by looking directly at its structure, e.g., by NMR, or by assaying one or more of its biological activities. For instance, subtilin inhibits spore outgrowth and inhibits the growth of proliferating bacterial cells, especially gram-negative bacteria. These assays can be performed as described in, e.g., Liu and Hansen, Appl. Environ. Microbiol., 59:648-651, 1993; Morris et al, J. Biol. Chem., 259, 13590-13594 (1984); Appl. Environ.
  • a stable subtilin possesses more biological activity than a prior art subtilin, e.g., about 5-fold, about 10-fold, about 20-fold, about 50-fold, about 60-fold, or more, when measured after storage, e.g., after storage at 4°C or 20°C for 2, 3, 4, 5, or 7 days, 1 week or more, 2 weeks or more, 1 month or more, etc., i.e., it has an improved storage or shelf-life.
  • Biological activity in inhibiting spore outgrowth can be carried out by adding heat-shocked B. cereus spores, or other target spores, to a final concentration of 75 ⁇ g/ml in 1% tryptone-100 mM Tris-phosphate buffer, pH 6.8, with a total volume of 2 ml and incubating the mixture in 15 ml. polypropylene tubes in a rotating drum shaker at 37 C at 15 rpm for 3 hours. A series of tubes, each containing a different concentration can be assayed. After incubation, the amount of inhibition can be assessed by phase contrast microscopy, which allows spores at different states of germination and outgrowth to be distinguished. For example, in the absence of inhibitor, spores were vegetative in about an hour. Spores were judged to be inhibited if more than 50% were still in early outgrowth stage after the three hour incubation period.
  • vegetative cells can be obtained by incubating heat-shocked B. cereus T spores under the aforementioned conditions in the absence of inhibitor for about 2 hours at 37 °C, when the spores were in the vegetative state.
  • Various amounts of the antimicrobial peptides were added to tubes containing the vegetative cell culture and incubated for one additional hour.
  • the turbidity of the cell suspension was measured (in Klett units) before and after the final incubation by using a Klett-Summerson colorimeter with a green (500- to 570-nm-range) filter, and the integrity of the cells was determined by phase-contrast microscopy.
  • the present invention relates to methods of isolating a composition comprising a stable subtilin, comprising one or more of the following steps: culturing a Bacillus subtilis under effective conditions to express subtilin; applying a composition, comprising a subtilin having a naturally-occurring amino acid sequence, to a hydrophobic interaction column under conditions effective for the subtilin to bind to said column; eluting said subtilin from the column with an effective elution reagent to form a composition comprising stable subtilin; performing reverse phase HPLC on said stable subtilin.
  • a starting composition comprising a subtilin can be obtained from any suitable source.
  • a starting composition is obtained by culturing a cell comprising a subtilin gene under conditions effective for expression of subtilin.
  • expression it is meant that the cell carries out the cellular processes which result in the production of a subtilin polypeptide.
  • Effective conditions for expression include any culture conditions which are suitable for achieving production of the subtilin polypeptide by the cell, including effective temperatures, pH, medias, cell densities, culture dishes, flasks, or other receptacles for growing cells, additives (e.g., cycloheximide, protease inhibitors, etc.).
  • subtilin gene Any cell comprising a subtilin gene can be used in accordance with the present invention, including naturally-occurring and genetically-engineered cells.
  • a preferred cell which expresses subtilin is Bacillus subtilis ATCC No. 6633, or derivatives thereof.
  • the present invention also relates to the production of subtilin by organisms which have been genetically-engineered to express subtilin.
  • B. subtilis strain 168, or substrain B. subtilis strain BR151 (ATCC No. 33677) is transformed with an expressible subtilin gene, e.g., as described in Liu and Hansen, J. Bacteriol., 173:7387-7390, 1991, to form strain LH45 which was deposited under the terms of the Budapest Treaty and assigned ATCC No.
  • subtilin polypeptide i.e., transcription and translation of the subtilin gene.
  • subtilin coding sequence is operably linked to an expression control .
  • expression control sequence means a nucleic acid sequence which regulates expression of a polypeptide coded for by a nucleic acid to which it is operably linked. Expression can be regulated at the level of the mRNA or polypeptide.
  • the expression control sequence includes mRNA-related elements and protein-related elements.
  • Such elements include promoters, enhancers (viral or cellular), ribosome binding sequences, transcriptional terminators, etc.
  • An expression control sequence is operably linked to a nucleotide coding sequence when the expression control sequence is positioned in such a manner to effect or achieve expression of the coding sequence. For example, when a promoter is operably linked 5' to a coding sequence, expression of the coding sequence is driven by the promoter.
  • Expression control sequences can be heterologous or endogenous to the normal gene.
  • Any suitable growth media can be used to culture the subtilin-expressing cells, e.g., media comprising, nitrogen sources, such as yeast extracts, soy bean tripticase, peptone, salts, metal ions, citric acid, buffers, carbohydrates, such as glucose, glycerol, lactose, sucrose, molasses, chalk, phosphates, ammonium sulfate, oil. See, examples for specific embodiments.
  • nitrogen sources such as yeast extracts, soy bean tripticase, peptone, salts, metal ions, citric acid, buffers, carbohydrates, such as glucose, glycerol, lactose, sucrose, molasses, chalk, phosphates, ammonium sulfate, oil. See, examples for specific embodiments.
  • a composition comprising a subtilin can be pre-treated prior to its application to a hydrophobic interaction column in any desired manner.
  • the composition can be diluted, concentrated, precipitated, filtered, filtered through a porous membrane to separate insoluble debris, acidified, centrifuged, heat-treated, etc. It can be supplemented with additives, such as protease inhibitors, anti-oxidants, detergents, salts, chaotropic agents, chelating agents such as EDTA or EGTA, metal ions, cofactors, etc.
  • the culture media containing subtilin prior to applying it the column, is acidified to pH 2.5 with 85% phosphoric acid (14.6 M) to form an acidified composition. Acidification can be performed with any suitable acid, e.g., acetic acid, carboxylic acids, glycine HC1, etc.
  • the subtilin is secreted into the culture medium.
  • the cells can be separated from the culture medium by low-speed centrifugation.
  • the resulting supernatant comprising the subtilin can then be harvested for further manipulations. Centrifugation to remove cells can be performed prior to acidification or after it.
  • the cells can be homogenized to form a homogeneously composition comprising subtilin.
  • a stable subtilin is obtained by reversed phase chromatography, preferably hydrophobic interaction chromatography (HIC)
  • Hydrophobic interaction chromatography uses a nonpolar stationary phase and a polar mobile phase to separate compounds according to their hydrophobicity, using a chromatographic support material having a hydrophobic surface.
  • the binding of a polypeptide to a hydrophobic interaction column can be induced by the addition of high salt concentrations to the sample comprising the polypeptide.
  • the polypeptides in the sample effectively "salt out" on to the hydrophobic surface of the chromatography support.
  • Binding strength can be modulated by manipulating the salt concentration and choosing supports with different hydrophobicities.
  • the binding interaction between the hydrophobic column surface and the polypeptide is reversible. Reducing the salt concentration, changing ionic strength, or adding agents which disrupt the hydrophobic binding can be used to elute the polypeptide from the hydrophobic support surface.
  • chromatographic material Any suitable chromatographic material can be used.
  • a variety of different chromatographic materials supports are commercially available which have hydrophobic ligands attached to a chromatographic support, e.g., having an alkyl chain ranging from about two to twenty- four or more carbons in length, linear or branched, and can contain or terminate in other hydrophobic groups such as a phenyl ring. Increasing chain length results in media with greater hydrophobic character.
  • Commonly used ligands are phenyl-, butyl-, and octyl- residues.
  • hydrophobic interaction chromatographic materials includes, e.g., POROS HP2 (surface covered with phenyl groups), POROS PE (surface coated with phenyl ether groups), POROS ET (surface coated with ether groups), Bio-Rad Macro-Prep HIC Supports, Bio-Rad Methyl
  • HIC support Bio-Rad-t-butyl HIC support, Bio-rad Econo column butyl-650m, TosoHaas TSK-GEL® HIC Columns, TosoHaas TOKYOPEARL® HIC, Fractogel® EMD Propyl (S), Fractogel® EMD Phenyl I (S), IEC PH-814, PolyMETHYL ATM, PolyETHYL ATM, PolyPROPYL ATM, PolyBUTYL ATM, HiPrepTM 16/10 Phenyl, HiPrepTM 16/1- Butyl, HiPrepTM 16/10 Octyl , etc. See, also, U.S. Pat. No. 5,641,403.
  • high salt is added to a composition comprising subtilin in order to increase its salt concentration and facitiate the subtilin 's binding to the HIC column.
  • Any suitable salt can be used, including, e.g, NaCl, such as 0.75 M, 1 M, 1.2 M, 1.5 M, 1.75 M, 2 M, 4 M, etc., ammonium sulfate, such as 0.75 M, 1 M, 1.2 M, 1.5 M, 1.75 M, 2 M, 4 M, etc., sodium acetate, sodium phosphate, etc.
  • the strength of the hydrophobic interactions is also influenced by the ionic strength, pH and polarity of the solvent.
  • a high concentration (i.e., 4 molar) of ammonium sulphate in the solvent would promote hydrophobic interaction and, hence, binding, between the resin and the hydrophobic domains of the solute proteins.
  • subtilin Once the subtilin is bound or adsorbed to the HIC column, and the other material in the composition which do not bind to the column are washed out, the subtilin can be removed from the column by interfering its interactions with the hydrophobic ligands present on the column support. The subtilin can thus be eluted from the column with an effective elution reagent.
  • effective elution reagent it is meant any agent which can be used to remove the subtilin from the column, including, compounds, compositions, mixtures, etc.
  • any effective reagent and/or procedure can be used to elute a subtilin from the resin, including, but not limited to, a buffer with lower ionic strength, chaotropic ions, polarity-lowering additives, such as ethylene glycol, detergents, methanol, acetonitrile, THF, and/or dichlormethane.
  • the elution reagent is acetonitrile, e.g., 30% acetonitrile.
  • the stable subtilin eluted from the HIC column can be further purified according to any suitable methods, including, chromatography, affinity chromatography, thin-layer chromatography, HPLC, SDS-PAGE electrophoresis.
  • a stable subtilin can also be isolated according to prior art procedures in combination with hydrophobic interaction chromatography, e.g. by a modification of the previously published procedures, Jensen and Hirschman, Arch. Biochem., 4, 197-309 (1944). In this procedure, cells are cultured in a medium A (Bannerjee and Hansen, J Biol Chem., 263, 9508-9514, 1988) containing 10% sucrose and incubated with good aeration for 30-35 hours at 35 °C.
  • the culture is then acidified to pH 2.8 with phosphoric acid and heated in an autoclave at 121 °C for 3 min to inactivate proteases, and cooled to room temperature.
  • One-half volume of n-butanol is added, stirred at 4°C, for 2 hours, and centrifuged.
  • 2.5 volume of acetone is added to the supernatant, allowed to stand at -20°C. for at least 2 hours, and then centrifuged.
  • Most of the pellet is subtilin which is washed with 95% ethanol, briefly lyophilized, and dissolved in 0.1% trifluoroacetic acid. This is immediately purified by RP-HPLC as described previously for nisin (Liu, W. and J. N.
  • a subtilin in accordance with the present invention has bactericidal and bacteriostatic activity, e.g., preventing spore outgrowth and inhibiting bacterial cell growth proliferation, especially gram-negative bacteria.
  • subtilin is useful in treating microbial infections, as a food preservative, and in other applications where antibiotic activity is useful.
  • subtilin can be used analogously to other antimicrobial drugs, especially antibiotics, as described in, e.g., Remington 's Pharmaceutical Sciences, Eighteenth Edition, 1990, Chapter 62.
  • Any effective route of administration can be used, e.g., topical or oral.
  • Subtilin can be used alone, or in combination with other agents, such as other bactericides, in combination with nisin or any effective antibiotic.
  • a stable subtilin can also be used as a food preservative to keep food safe for consumption, by inhibiting or prevents nutrient deterioration and organoleptic changes produced by bacterial activity.
  • the subtilin can be directly incorporated into the food, such as a wine, fish, cheese, dairy product, or processed meat, or it can be used in food packaging materials. See, e.g., U.S. Pat. Nos. 4,584,199, 4,597,972, 4,980, 163, and 5,573,800. Subtilin can also be used in mouthwash.
  • a composition comprising a stable subtilin can contain buffers, carriers, etc.
  • the present invention relates to methods of treating a bacterial infection in a host, comprising administering an effective amount of a stable subtilin to a host in need thereof, and methods of preserving food, comprising adding an effective amount of a stable subtilin to a food in need thereof.
  • effective amount it is meant any amount of subtilin which is useful in achieving the claimed purpose, e.g., treating an infection or preserving food.
  • the effective amounts can be determined routinely, and/or in analogy to nisin.
  • Prior art subtilin e.g., having a naturally-occurring sequence, preferably not modified by genetic engineering, was so unstable that it was not useful.
  • Subtilin was purified as follows: One liter of medium A containing 2% sucrose was inoculated with 5-10 ml of overnight culture grown in PAB for 24-28 hr at 37°C with vigorous shaking. The subtilin production was monitored by the pink-brown color, fruity odor, and pH (5.9-6.9) of the medium. All of the purification steps were performed at room temperature (RT). The culture was acidified to pH 2.5 with 85% phosphoric acid(14.6 M), centrifuged at 10,000 x g for 20 min to remove cells, and the subtilin in supernatant fraction purified to homogeneity in two steps.
  • Medium A was prepared by combining 780 ml of solution I (20g of sucrose in 780 ml of distilled water), 200 ml of solution II (53.5 g of citric acid, 20.0 g of Na ⁇ O ⁇ 25.0 g of yeast extract, and 21.0 g of (NH 4 ) 2 HPO 4 in 1 1 of distilled water, with the pH adjusted to 6.9 with 14.8 N NH 4 OH), 10 ml of solution III (7.62 g KC1, 41.8 g
  • subtilin lantibiotic can be used alone as an antimicrobial compound or in combination with other lantibiotics and antibiotics for treating or preventing microbial growth.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
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  • Gastroenterology & Hepatology (AREA)
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Abstract

La présente invention concerne de la subtiline stable et ses procédés de préparation. Un procédé préféré de réalisation consiste à suivre une ou plusieurs de ces opérations: déposer d'abord une composition acidifiée, contenant de la subtiline à séquence d'acide aminé d'origine naturelle, dans une colonne d'interaction hydrophobe dans des conditions permettant à la subtiline de se fixer à ladite colonne; et procéder ensuite à l'élution de la subtiline de la colonne avec un réactif d'élution efficace de manière à former une composition à base de subtiline stable.
PCT/US1999/030938 1998-12-24 1999-12-23 Subtiline stable et ses procedes de preparation WO2000039152A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
CA002356955A CA2356955A1 (fr) 1998-12-24 1999-12-23 Subtiline stable et ses procedes de preparation
AU23888/00A AU2388800A (en) 1998-12-24 1999-12-23 Stable subtilin and methods of manufacture
EP99967636A EP1140975A4 (fr) 1998-12-24 1999-12-23 Subtiline stable et ses procedes de preparation

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US11384398P 1998-12-24 1998-12-24
US60/113,843 1998-12-24

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6541607B1 (en) 1997-07-18 2003-04-01 University Of Maryland Sublancin lantibiotic produced by Bacillus subtilis 168
US6759205B2 (en) 2000-06-29 2004-07-06 University Of Maryland Construction of a strain of Bacillus subtilis 168 that displays the sublancin lantibiotic on the surface of the cell
US6846804B2 (en) 2000-06-29 2005-01-25 University Of Maryland, College Park Office Of Technology Commercialization Construction of a structural variant of sublancin to facilitate its isolation and use in bioremediation of environmental contamination by gram-positive spore formers such as bacillus anthrasis
US6953683B2 (en) 1997-07-18 2005-10-11 University Of Maryland Compositions and methods of inhibiting bacterial spore germination
US7033766B2 (en) 2000-06-29 2006-04-25 University Of Maryland Office Of Technology Commercialization Construction and screening of lantibody display libraries
WO2020185562A1 (fr) * 2019-03-08 2020-09-17 Fraunhofer Usa, Inc. Nouveau peptide lantibiotique antimicrobien et utilisations associées

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101776665B (zh) * 2009-01-13 2013-09-11 上海医药工业研究院 一种乳酸链球菌素的hplc检测方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5218101A (en) * 1988-07-05 1993-06-08 The University Of Maryland Leader sequence inducing a post-translational modification of polypeptides in bacteria, and gene therefor
US5516682A (en) * 1988-07-05 1996-05-14 University Of Maryland Subtilin variant of enhanced stability and activity
US5861275A (en) * 1995-09-28 1999-01-19 The University Of Maryland Lantibiotic mutants and chimeras of enhanced stability and activity, leader sequences therefor, genes encoding the same, and methods of producing and using the same

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5218101A (en) * 1988-07-05 1993-06-08 The University Of Maryland Leader sequence inducing a post-translational modification of polypeptides in bacteria, and gene therefor
US5516682A (en) * 1988-07-05 1996-05-14 University Of Maryland Subtilin variant of enhanced stability and activity
US5576420A (en) * 1988-07-05 1996-11-19 University Of Maryland Subtilin variant of enhanced stability and activity
US5861275A (en) * 1995-09-28 1999-01-19 The University Of Maryland Lantibiotic mutants and chimeras of enhanced stability and activity, leader sequences therefor, genes encoding the same, and methods of producing and using the same

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP1140975A4 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6541607B1 (en) 1997-07-18 2003-04-01 University Of Maryland Sublancin lantibiotic produced by Bacillus subtilis 168
US6953683B2 (en) 1997-07-18 2005-10-11 University Of Maryland Compositions and methods of inhibiting bacterial spore germination
US7358076B2 (en) 1997-07-18 2008-04-15 The University Of Maryland Sublancin lantibiotic produced by Bacillus subtilis 168
US6759205B2 (en) 2000-06-29 2004-07-06 University Of Maryland Construction of a strain of Bacillus subtilis 168 that displays the sublancin lantibiotic on the surface of the cell
US6846804B2 (en) 2000-06-29 2005-01-25 University Of Maryland, College Park Office Of Technology Commercialization Construction of a structural variant of sublancin to facilitate its isolation and use in bioremediation of environmental contamination by gram-positive spore formers such as bacillus anthrasis
US7033766B2 (en) 2000-06-29 2006-04-25 University Of Maryland Office Of Technology Commercialization Construction and screening of lantibody display libraries
WO2020185562A1 (fr) * 2019-03-08 2020-09-17 Fraunhofer Usa, Inc. Nouveau peptide lantibiotique antimicrobien et utilisations associées

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AU2388800A (en) 2000-07-31
EP1140975A4 (fr) 2003-01-08
EP1140975A1 (fr) 2001-10-10

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