Bacteriocin piscicolin 126

Food or edible material: processes – compositions – and products – Inhibiting chemical or physical change of food by contact... – Biocidal or disinfecting chemical agent

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426 36, 530324, A61K 3816

Patent

active

060541636

DESCRIPTION:

BRIEF SUMMARY
This invention relates to novel bacteriocin molecules having antimicrobial activity. In one particular application, the bacteriocin according to the invention is used as a food preservative.


BACKGROUND OF THE INVENTION

There is a continual need for new food preservatives bearing new and useful properties. Further, there is growing interest in replacing traditional "chemical" food preservatives with effective "natural" preservatives, especially those which are specific for pathogenic microorganisms and do not harm beneficial food-producing strains. In this regard, considerable research has been conducted on bacterial peptides known as bacteriocins which are often heat stable and have antimicrobial activity. Two such bacteriocins which are commercially produced for use as food preservatives are nisin and pediocin PA-1. Nisin has been given the status of Generally Regarded as Safe for human consumption (GRAS) by the United States FDA. Nisin and pediocin PA-1 however, have broad spectrum activity, affecting not only pathogenic, but also beneficial (in the food system) microorganisms.


SUMMARY OF THE INVENTION

Novel bacteriocins having a range of activity that is different to and preferably narrower than those of nisin and pediocin PA-1 would be desirable. The present inventors have now identified and isolated a new bacteriocin which has a limited range of activity in comparison to, for example, nisin and pediocin PA-1.
Thus, in a first aspect, the present invention consists in a substantially pure preparation of a bacteriocin having a molecular mass of about 4.4 kDa and antimicrobial activity substantially according to Table 2.
In a preferred embodiment of the present invention the bacteriocin has an amino acid sequence substantially identical to the sequence shown in FIG. 3.
In a second aspect the present invention consists in a biologically pure culture of bacterial strain JG126.
A sample of bacterial strain JG126 was deposited under the Budapest Treaty with Australian Government Analytical Laboratories (AGAL), 1 Saukin Street, Pymble, New South Wales, Australia, on Dec. 21 1994, and was accorded accession No. N94/61478.
In a third aspect the present invention consists in an isolated DNA molecule encoding the bacteriocin of the first aspect of the present invention.
In a preferred embodiment of this aspect of the present invention the DNA molecule includes a nucleotide sequence as shown in FIG. 6.
In a fourth aspect the invention consists in a method of preserving foods or beverages, the method comprising adding to the food or beverage an effective antimicrobial amount of a bacteriocin of the first aspect of the present invention.
The bacteriocin of the present invention may be isolated from natural sources, or produced by recombinant DNA techniques well known in the art.


BRIEF DESCRIPTION OF THE DRAWINGS

In order that the nature of the present invention may be more clearly understood, preferred forms thereof will now be described with reference to the following non-limiting examples and the accompanying figures.


Figure Legends

FIGS. 1A-1C: Purification of piscicolin 126 by reversed-phase HPLC
(A) C18 reversed-phase HPLC separation of piscicolin 126 on a 4.6.times.250 mm Spherisorb ODS II (80 .ANG. pore size, 5 .mu.m particle size) column developed with the gradient of increasing acetonitrile concentration indicated by the dashed line. The piscicolin 126 had been previously partially purified by ion-exchange chromatography (not shown). The peak indicated by the arrow was collected and shown to have antimicrobial activity. (B) Rechromatography by C18 reversed-phase HPLC of the peak of piscicolin 126 collected as described in panel A. The column (2.1.times.250 mm Spherisorb ODS II [80 .ANG. pore size, 5 .mu.m particle size]) was developed with the gradient of increasing acetonitrile concentration indicated by the dashed line. (C) Electropherogram of purified piscicolin 126 separated by CZE through a 50 .mu.m.times.50 cm uncoated silica capillary in 20 mM sodium citrate (pH 2.5) at 20 kV.
FIG. 2. MAL

REFERENCES:
Worobo et al, "Characteristics and genetic determinant of a . . . ," Microbiology, vol. 140, pp. 517-526 (1994).
Jack et al, "Characterization of the Chemical and Antimicrobial Properties," Applied and Environmental Microbiology, vol. 62, No. 8, pp. 2897-2903 (1996).
Fems Microbiology Letters, vol. 115, 1994, A. Holck et al., Purification and Cloning of Sakacin 674, a Bacteriocin from Lactobacillus sake Lb 674 pp. 143-149.
Journal of General Microbiology, vol. 138, 1992, A. Holck et al., Purification and Amino Acid Sequence of Sakacin A, a Bacteriocin from Lactobacillus sake Lb 706, pp. 2715-2720.
The Journal of Biological Chemistry, vol. 269, No. 16, 1994, L. Quadri et al., Chemical and Genetic Characterization of Bacteriocins Produced by Carnobacterium piscicola LV17B, pp. 12204-12211.
Applied and Environmental Microbiology, vol. 58, No. 8, 1992, J. Marugg et al., Cloning Expression and Nucleotide Sequence of Genes Involved in Production of Pediocin PA-1, a Bacteriocin from Pediococcus acidilactici PAC 1.0,, pp. 2350-2367.
Microbiology, vol. 140, 1994, P. Tichaczek et al., Cloning and Sequencing of Sak P Encoding Sakacin P, the Bacteriocin Produced by Lactobacillus sake LTH 673, pp. 2360-2367.
Waite Library, Department of Food Science, University of Alberta, Edmonton, Alberta, Canada, T6G 2P5, Chapter 18, "Bacteriocins Produced by Carnobacterium Species", Michael E. Stiles, pp. 451-459, 1996.
Applied and Environmental Microbiology, Aug. 1990, p. 2503-2510, vol. 56, No. 8, "Plasmid-Associated Bacteriocin Production by a Strain of Carnobacterium piscicola from meat".

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