Bacteriocins

Details Bacteriocins Bacteriocins

1998-04-13

2001-03-27

Sisson, Bradley (Department: 1653)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S252900, C435S320100, C435S252300, C435S173300, C514S012200, C530S324000, C530S326000, C530S325000, C536S023700

Reexamination Certificate

active

06207411

ABSTRACT:

This application claims priority to PCT/IE96/00022, filed Apr. 12, 1996 and Irish patent application S950269, filed Apr. 12, 1995.
The present invention relates to a novel anti-microbial agent, more particularly, a novel bacteriocin with nisin-like properties.
Recent years have seen major advances in the development of microbial metabolites with antagonistic activities towards spoilage and pathogenic micro-organisms associated with food. Although, there now exists in excess of seven thousand known antibiotic compounds of microbial origin, very few have been evaluated for food use. The danger exists that antibiotic-resistant microorganisms of clinical importance will appear with repeated exposure to antibiotics in food, thus compromising the clinical usefulness of the antibiotics (although, of course, clinically important antibiotics are not used for food applications). Additionally, consumer emphasis is now on minimally processed foods which are natural and preservative free. Because of this considerable and justifiable resistance to the use of chemical additives and antibiotics as food preservatives, other biological inhibitors produced by microorganisms are currently being investigated for use in foods. Of particular interest are those inhibitory substances produced by the Lactic Acid Bacteria (LAB) which include hydrogen peroxide, diacetyl, bacteriocins, as well as secondary reaction products such as hypothiocyanite. Bacteriocins are potentially very attractive natural preservatives as they are produced as normal by-products of microbial metabolism. The LAB are industrially important microorganisms and include the genera Lactococcus, Streptococcus, Pediococcus, Leuconostoc, Lactobacillus and Carnobacterium. They have been used for the production of fermented foods which have been consumed safely for hundreds of years. Given their status as “safe” organisms, they are a particularly suitable source for natural antimicrobials such as bacteriocins for use in foods.
In 1925, the first prototype bacteriocin was discovered by Gratia et. al. from a strain of
Escherchia coli.
Bacteriocins are always proteins which can be broad or narrow spectrum and are not lethal to the cells which produce them. Bacteria protect themselves from the lethal effects of their own bacteriocins by such mechanisms as post-translational modification or the production of an immunity protein(s). In any case, bacteriocins are potent antibacterial substances produced by a large and diverse assortment of species. They form a heterogeneous group with respect to their producer, inhibitory spectrum, mode of action and chemical properties. There are four distinct classes of LAB bacteriocins:
A. Lantibiotics—small peptides of less than 5 kDa which contain unusual amino acids such as lanthionine, &bgr;-methyllanthionine and dehydrated residue, e.g. nisin, lacticin 481, carnocin U149 and lactocin S.
B. Non-Lanthionine containing Peptides—small peptides of 10 kDa or less and can be subdivided as follows: (i) Listeria-active peptides e.g. Pediocin PA-1 and Sakacin A. (ii) Poration complexes consisting of two proteinaceous peptides e.g. Lactacin F. (iii) Thiol-activated peptides requiring reduced cysteine residues for activity e.g. Lactococcin B.
C. Large Heat-Labile Proteins—larger proteins, generally having a molecular weight greater than 31 kDa e.g. Helveticin V-1829.
D. Complex Bacteriocins—composed of a protein with one or more chemical moieties which may be of a lipid or carbohydrate nature e.g. Pediocin SJ-1.
Lactococci are widely used as starter cultures in the dairy industry and several strains of dairy species can produce bacteriocins.
L. lactis
subspecies can produce diplococcin, lactococcin, lactostrepcins or nisin. Diplococcin and lactococcins are small molecular weight proteins, active against other lactococci while nisin is a lantibiotic with a broad spectrum of activity against many Gram positive bacteria.
Nisin is the most extensively characterised bacteriocin of the antimicrobial proteins produced by LAB and has found widespread application in the food industry. Nisin was the first “antibiotic” compound to be used on a commercial scale in the food industry. It is used to prevent spore outgrowth and toxin production by
Clostridium botulinum
in processed cheese and cheese spreads. In some countries, it has been used to extend the shelf-life of dairy products and to prevent the spoilage of canned foods by thermophiles.
Nisin is a pentacyclic, subtype A lantibiotic and it displays an amphiphathic character, with a hydrophobic residue (Ile) at its N-terminus and a hydrophilic residue (Lys) at its C-terminus. It is a peptide of 34 amino acids and is inactivated by proteases including chymotrypsin, pancreatin and subtilopeptidase. It is insensitive to carboxypeptidase A, elastase, erepsin, pepsin and trypsin. It contains one lanthionine residue, four &bgr;-methyllanthionines, a dehydroalanine and a dehydrobutyrine. The thioether amino acids, (lanthionine and &bgr;-methyllanthionine) account for the high sulphur content of nisin. The usual amino acid residues are thought to be responsible for the important functional properties of nisin e.g. the associated acid tolerance and thermostable properties of nisin are attributed to the stable thioether linkages while the specific bactericidal activity is thought to be due to the very reactive double bonds. Nisin has a molecular mass of 3.5 kDa (Gross & Morell, 1971) and often forms dimers and oligomers.
Nisin has a very broad spectrum of activity against Gram positive vegetative bacterial cells. The closely related lactococci are especially sensitive but nisin is also inhibitory to several strains of bacilli and clostridia (particularly their spores), lactobacilli, leuconostocs, micrococci, pediococci, streptococci and actinonycetes. Other sensitive strains include
Mycobacterium tuberculosis, Staphylococcus pyogenes, S. aureus, S. epidermidis
and
Listeria monocytogenes
(de Vuyst & Vandamme, 1994). Nisin does not display activity against Gram negative bacteria, except for three Neisseria strains (Mattick & Hirsch, 1947) and three Flavobacter strains (Ogden & Tubb, 1985), nor does it inhibit yeasts or viruses. However, Salmonella subspecies and other Gram negative bacteria can be made sensitive by using a chelating agent in combination with nisin (Stevens et. al. 1992). The site of action of nisin appears to be the cytoplasmic membrane. The outer membrane of Gram negative bacteria is thought to exclude the bacteriocin making contact with the cytoplasmic membrane. Hence, by incorporating a chelating agent such as EDTA with nisin, the structure of the outer membrane undergoes alteration, resulting in destabilization of the lipopolysaccharide (LPS) layer with a corresponding increase in cell permeability. Binding of the bacteriocin to the membrane leads to the aggregation of similar peptides, thus initiating oligomerization. Such aggregates adopt a transmembrane orientation so that the hydrophobic portion is exposed to the core of the membrane and the hydrophilic part forms the aqueous channel. Membrane insertion, pore formation, (both of which require a transmembrane potential) and subsequent depolarization leads to the efflux of small cellular constituents and destruction of energy metabolism of the cell (Ruhr & Sahl, 1985). This results in a deficiency of metabolic intermediates and hence, inhibition of synthesis of DNA, RNA, proteins and polysaccharides. There appears to be a separate mechanism for the prevention of spore outgrowth. Unlike vegetative cells, bacterial spores never lyse when treated with nisin. The dehydro residues of nisin provide possible covalent attachment sites for membrane sulfhydryl groups. These residues appear to have no such role in membrane pore formation (Morris et. al. 1984).
Nisin is encoded by a conjugative transposon which can insert into either plasmid DNA or chromosomal DNA (Horn et. al., 1991). Analysis of the nisin operon indicates that the nisin structural gene, as well as the genes required for processing and maturation a

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