Title of Invention	A BACTERICIDAL, BACTERIOSTATIC, FUNGICIDAL AND/OR FUNGISTATIC COMPOSITION COMPRISING A BASIC PROTEIN OR PEPTIDE
Abstract	A composition consisting essentially of a basic protein or peptide capable of killing microbial cells, e.g. a protamine or protamine sulphate, in combination with a cell-wall degrading enzyme and/or an oxidoreductase, e.g. an endoglycosidase Type II, a lysozyme, chitinase, peroxidase enzyme system (EC 1.11.1.7) or laccase enzyme (EC 1.10.3.2), has bactericidal, bacteriostatic, fungi¬cidal and/or fungistatic properties and is useful in detergent and hard surface cleaning compositions and in methods for killing microbial cells present on a hard surface, for killing microbial cells or inhibiting grow¬ing microbial cells present on laundry, for killing microbial cells present on human or animal skin, mucous membranes, wounds, bruises or in the eye; and in preser¬vation of food, beverages, cosmetics, contact lens pro¬ducts, food ingredients or enzyme compositions.

Title of Invention

A BACTERICIDAL, BACTERIOSTATIC, FUNGICIDAL AND/OR FUNGISTATIC COMPOSITION COMPRISING A BASIC PROTEIN OR PEPTIDE

Abstract

A composition consisting essentially of a basic protein or peptide capable of killing microbial cells, e.g. a protamine or protamine sulphate, in combination with a cell-wall degrading enzyme and/or an oxidoreductase, e.g. an endoglycosidase Type II, a lysozyme, chitinase, peroxidase enzyme system (EC 1.11.1.7) or laccase enzyme (EC 1.10.3.2), has bactericidal, bacteriostatic, fungi¬cidal and/or fungistatic properties and is useful in detergent and hard surface cleaning compositions and in methods for killing microbial cells present on a hard surface, for killing microbial cells or inhibiting grow¬ing microbial cells present on laundry, for killing microbial cells present on human or animal skin, mucous membranes, wounds, bruises or in the eye; and in preser¬vation of food, beverages, cosmetics, contact lens pro¬ducts, food ingredients or enzyme compositions.

Full Text	The present invention relates to a composition capable of killing microbial cells or inhibiting growing microbial cells, i.e. a bactericidal, bacteriostatic, fungicidal and/or fungistatic composition; a cleaning or detergent composition comprising a substance capable of killing microbial cells or inhibiting growing microbial cells; and methods for killing microbial cells present on a hard surface, on skin or in laundry, and for preserving food products, cosmetics etc. BACKGROUND OF THE INVENTION At this time of increased public interest in reducing the use of chemical additives, it is relevant to consider na¬tural alternatives for antimicrobial agents used e.g. for preserving foods, as disinfectants, and as an antimicro¬bial ingredient of detergent and cleaning compositions. This has increased interest in preservation using live bacterial cultures (Jeppesen & Huss 1993) and enzymes like lactoperoxidase (Farrag & Marth 1992), glucose oxi¬dase (Jeong et al. 1992) and lysozyme (Johansen et al. 1994) . Protamines are basic proteins with a high arginine con¬tent found in association with DNA of spermatozoon nuclei of fish, birds, mammals etc. (Rodman et al. 1984; Kossel 1928) . Protamine is used clinically as an antidote to heparin (Jaques 1973) and as a carrier of insulin, pro¬longing the absorption of subcutaneously administered insulin (Brange 1987). Attention has also been paid to the functional properties of protamine as a stabilizing agent (Phillips et al. 1989). Protamine has been shown to have an antibacterial effect (Hitsch 1958), but this .)( aspect has not been thoroughly studied. The Gram-negative bacteria are often resistant to a large number of harmful agents due to the effective permeabili¬ty barrier function of the outer membrane (Nakae 1985). However, protamine and most other cationic peptides are under certain conditions apparently able to traverse the outer membrane of Gram-negative bacteria (Vaara 1992, Vaara & Vaara 1983), probably as a result of their bin¬ding to the anionic lipopolysaccharide-covered surface of the Gram-negative cell. The mechanism of the antibac¬terial action of basic peptides is not known, but it has been suggested that they form a channel in the cytoplas¬mic membrane, thus uncoupling electron transport and cau¬sing leakage (Christensen et al. 1988; Hugo 1978; Kagan et al. 1990). It has also been proposed that they induce autolysis due to activation of the autolytic enzymes (Bi-erbaum & Sahl 1991). In general, the occurrence of highly-basic peptides such as protamine is relatively rare in nature. However, of those studied, several have been found to demonstrate antibacterial properties: eg. nisin (Sahl 1987), defensin (Lehrer et al. 1993), cecropins (Christensen et al. 1988), Pep5 (Bierbaum & Sahl 1987) and melittins (Vaara 1992) . Antibacterial activity is usually measured as a decrease in colony counts, a decrease in the absorbance of a bac¬terial suspension or as inhibition zones on agar plates (Trevors 1986). However, these methods may not be suit¬able when assaying the antibacterial effect of a basic peptide or protein such as protamine due to the aggluti¬nation of the positively-charged protamine and the nega¬tively-charged bacterial cells as described by Islam et al. (1984). Thus, the object of invention was to provide an antibac¬terial and/or antifungicidal composition comprising a natural active compound or substance, i.e. which is non¬toxic, of biological origin, easily available and rela¬tively inexpensive, optionally in combination with other antimicrobial compounds or substances. SUMMARY OF THE INVENTION It has now surprisingly been found that it is possible to kdJ.1 microbial cells or inhibit growing microbial cells /by means of a basic protein or peptide of biological ori¬gin, e.g. protamine or protamine sulphate. For certain bacteria or fungi, it may be necessary to combine the basic protein with a cell-wall degrading enzyme or an oxidoreductase in order to obtain the desired antimicro¬bial effect. Accordingly, the growth inhibitory effect of protamine and the potential lethal effect of this basic protein on non-growing cells has been investigated. Due to the methodological shortcomings described above, impedimetric measurements were used and compared to traditional plate counts (Firstenberg-Eden & Eden 1984; Connolly et al. 1993) . Thus, based these findings the present invention provides a bactericidal, bacteriostatic, fungicidal and/or fun¬gistatic composition comprising a basic protein or pep¬tide capable of killing microbial cells in combination with a cell-wall degrading enzyme or an oxidoreductase. In another aspect, the present invention provides a detergent or cleaning composition comprising a basic pro¬tein or peptide capable of killing microbial cells and a surfactant. Such compositions have a pH in the alkaline range and it has been found that basic proteins such as protamine and protamine sulphate exhibit their optimum antimicrobial effect at alkaline pH, thus making such proteins very suitable for incorporation in compositions for cleaning purposes. The composition of the invention is useful as antimicro¬bial ingredient wherever such an ingredient is needed, for example for the preservation of food, beverages, cos¬metics, contact lens products, food ingredients or enzyme compositions; as a disinfectant for use e.g. on human or animal skin, mucous membranes, wounds, bruises or in the eye; for killing microbial cells in laundry; and for incorporation in cleaning compositions for hard surface cleaning. THE DRAWINGS The invention is further illustrated by the drawings, in which Figure 1 shows the effect of protamine on growth of Yer¬sinia enterocolitica growing in TSB at 25°C. Growth is measured as % change in conductance; Figure 2 shows calibration curves relating conductance ▲ (Shewanella putrefaciens strain A2) or capacitance ■ (Li¬steria monocytogenes strain 032) detection times in TSB at 25°C to colony counts in the absence of protamine; Figure 3 shows the effect of various concentrations of protamine on the Gram-negative bacteria Pseudomonas aeruginosa in dependence of pH. Figure 4 shows the effect of various concentrations of protamine on the Gram-positive bacteria Listeria monocytogenes in dependence of cell concentration (inoculum, log CFU/ml). Figure 5 shows the effect of various concentrations of protamine on the Gram-negative bacteria Shewanella putre-faciens in dependence of cell concentration (inoculum, log CFU/ml). Figure 6 is a response surface plot showing the synergis¬tic effect of a composition of the invention (various concentrations of protamine and lysozyme) on the Gram-ne¬gative bacteria Shewanella putrefaciens in dependence of cell concentration (inoculum, log CFU/ml). DETAILED DESCRIPTION OF THE INVENTION In the present context, the terra "bactericidal" is to be understood as capable of killing bacterial cells. In the present context, the term "bacteriostatic" is to be understood as capable of inhibiting bacterial growth, i.e. inhibiting growing bacterial cells. In the present context, the term "fungicidal" is to be understood as capable of killing fungal cells. In the present context, the term "fungistatic" is to be understood as capable of inhibiting fungal growth, i.e. inhibiting growing fungal cells. The term "growing cell" is to be understood as a cell having access to a suitable nutrient and thus being capable of reproduction/propagation. By the term "non-growing cell" is meant a living, but dormant, cell, i.e. a cell in the non-growing, non-dividing, non-multiplying and non-energized state with metabolic processes at a minimum. The term "cell-wall degrading enzyme" is to be understood as an enzyme which degrades components of the cell wall, e.g. peptidoglucans such as murein and pseudomurein; chi-tin; and teichoic acid. Examples of cell-wall degrading enzymes which are useful in compositions of the present invention are endoglycosidases Type II, e.g. the endo-glycosidases Type II disclosed in EP-A2-0 425 018 which is hereby incorporated by reference, lysozymes and chitinases. The term "amino acids present in mammalian cells" denotes the 2 0 amino acids constituting the proteins being part of mammals, i.e. alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histi-dine, isoleucine, leucine, lysine, methionine, phenylala¬nine, proline, serine, threonine, tryptophan, tyrosine, valine. Preferably, the basic peptides or proteins of the composition of the invention consist of one or more of the mentioned 20 amino acids, i.e. the basic peptides or proteins may not be recovered from e.g. bacteria such as for example nisin and Pep5. The term "oxidoreductase" means an enzyme classified as EC 1. according to the Enzyme Nomenclature (1992) , i.e. any enzyme classified as EC 1.1 (acting on the CH-OH group of donors), EC 1.2 (acting on the aldehyde or oxo group of donors), EC 1.3 (acting on the CH-CH group of donors), EC 1.4 (acting on the CH-NHj group of donors), EC 1.5 (acting on the CH-NH group of donors), EC 1.6 (acting on NADH or NADPH), EC 1.7 (acting on other nitro¬genous compounds as donors), EC 1.8 (acting on a sulfur group of donors) , EC 1.9 (acting on a heme group of donors), EC 1.10 (acting on diphenols and related sub¬stances as donors), EC 1.11 (acting on a peroxide as acceptor), EC 1.12 (acting on hydrogen as donor), EC 1.13 (acting on single donors with incorporation of molecular oxygen (oxygenases), EC 1.14 (acting on paired donors with incorporation of molecular oxygen), EC 1.15 (acting on superoxide radicals as acceptor), EC 1.16 (oxidizing metal ions), EC 1.17 (acting on -CHz- groups), EC 1.18 (acting on reduced ferredoxin as donor), EC 1.19 (acting on reduced flavodoxin as donor), and EC 1.97 (other oxidoreductases). The term "peroxidase enzyme system" is to be understood as a peroxidase (EC 1.11.1) in combination with a source of hydrogen peroxide which may be hydrogen peroxide or a hydrogen peroxide precursor for in situ production of hydrogen peroxide, e.g. percarbonate or perborate, or a hydrogen peroxide generating enzyme system, e.g. an oxi¬dase and a substrate for the oxidase or an amino acid oxidase and a suitable amino acid, or a peroxycarboxylic acid or a salt thereof. Examples of useful peroxidases are lactoperoxidase, horseradish peroxidase, peroxidases producible by culti¬vation of a peroxidase producing strain Myxococcus vires-cens, DSM 8593, Myxococcus fulvus, DSM 8969, or Myxococcus xanthus, DSM 8970, of a peroxidase producing strain of the genus Corallococcus, preferably belonging to Corallococcus coralloides, DSM 8967, or Corallococcus exiguus, DSM 8969. In case of lactoperoxidase, thiocyanate may be used as a substrate. Laccases are enzymes that catalyze the oxidation of a substrate with oxygen; they are known from microbial, plant and animal origins. More specifically, laccases (EC 1.10.3.2) are oxidoreductases that function with molecu¬lar oxygen as electron acceptor. Molecular oxygen from the atmosphere will usually be present in sufficient quantity, so normally it is not necessary to add extra oxygen to the process medium. Examples of a laccase enzyme useful in the compositions of the present inven¬tion is laccase obtainable from the strain Coprinus cinereus, IFO30116, or from a laccase having immunochemical properties identical to those of a laccase derived from Coprinus cinereus, IFO30116; or obtainable from a strain of Myceliophthora thermophile as disclosed in WO 91/05839. The term "microbial cells" denotes bacterial or fungal c cells. By the term "of biological origin" is to be understood that the substance or compounds is recovered or regener¬ated from biological material such as humans, animals or plants. Similarly, the term "of microbiological origin" denotes that the substance or compounds is recovered or regenerated from microbiological material such as bacteria, fungi, yeast or that a parent or native sub¬stance or compound is producible by a microbiological 0 organism. The term "biological material" denotes living material obtainable from Nature or previously living material obtainable from Nature. The term "synthesized polypeptide" denotes a synthesized assembly, i.e. a chain, built of peptide monomers. Polypeptides which are useful in the present compositions are basic polypeptides, i.e. polylysins and polyarginins and co-polymers thereof. It is preferred that the polypeptides have a chain length of less than about 100 amino acids but it is contemplated that polypeptides of less than about 1000 kD are useful. Preferably, the polypeptides to be used in the composition of the inven¬tion is of almost identical chain length or molecular weight but mixtures of polypeptides having various chain lengths or molecular weights are also useful. It is contemplated that the basic protein of the composi¬tion of the invention may be a recombinant protein. In case of protamine, it is contemplated that the protamine may be a recombinant protamine, i.e. produced by cloning of a DNA sequence encoding the protein and subsequent cell transformed with the DNA sequence and expressed in a host, i.e. a suitable fungal or bacterial host. A recom¬binant protamine/protamine sulphate may be cloned and ex¬pressed according to standard techniques conventional to the skilled person. Preferred basic proteins to be used in the compositions of the present invention are protamines, protamine sul¬phates, defensins, magainins, melittin, cecropins and protegrins; more preferably protamines and protamine sul¬phates . Hitherto it has been known that protamine from salmon has a bactericidal effect on growing Gram-positive bacteria (1000 jug/ml) . Islam et al. (1984) found that it inhibited growth but did not determine whether the effect was bactericidal or bacteriostatic. Other studies have reported that protamine is not effective against Gram-negative bacteria (Islam et al. 1984; Yanagimoto et al. 1992). Contrary to this observation, it has now been found that protamine is effective against Gram-positive bacteria. Gram-negative bacteria and fungi. The same applies for protamine sulphate. It has been suggested the primary antibacterial effect of many of the basic peptides is their ability to penetrate the cytoplasmatic membrane, disrupting the electron transport and induce leakage of intracellular compounds. Without being bound to this theory, this mechanism may explain in part the effect of protamine on some of the strains tested in the experiments described in the Examples below. In another aspect, the present invention relates to a :;leaning or detergent composition comprising a basic pro¬tein or peptide capable of killing microbial cells and a surfactant. rhe detergent or cleaning composition may further com¬prise other enzymes conventionally used in detergent or cleaning compositions. Preferably, the detergent or cleaning composition of the invention comprises at least one enzyme selected from the group consisting of protea¬ses, amylases, cellulases, and lipases. The surfactant of the detergent or cleaning composition is preferably a detergent surfactant, more preferably a detergent surfactant selected from the group consisting of anionic, nonionic, ampholytic, zwitterionic and catio-nic surfactants. In a preferred embodiment, the detergent or cleaning composition comprises as the basic protein a protamine or a protamine sulphate in an amount effective for killing cells or inhibiting growth of cells, preferably in an amount corresponding to between 1 and 4000 /xg per ml cle¬aning liquor or washing liquor, more preferably between 1 and 2 000 fig per ml cleaning liquor or washing liquor, especially between 5 and 1000 jug per ml cleaning liquor or washing liquor. In a further aspect, the present invention relates to the use of the compositions of the invention for various pur¬poses, i.e. the invention also relates to a method for killing microbial cells present on a hard surface which method comprises contacting the surface with a cleaning composition of the invention, preferably a composition comprising a protamine or a protamine sulphate, in an amount effective for killing the cells. Also, in yet another aspect the invention relates to a method for killing microbial cells or inhibiting growing microbial cells present on laundry which method comprises contacting the laundry with a detergent composition of the present invention, preferably a composition compri¬sing a protamine or a protamine sulphate, in an amount effective for killing the cells or for inhibiting growing cells. In yet another aspect the invention relates to a method for preservation of food, beverages, cosmetics such as lotions, creams, gels, soaps, shampoos, conditioners, an-tiperspirants; contact lens products, food ingredients or enzyme compositions which method comprises incorporating into the unpreserved food, beverages, cosmetics, contact lens products, food ingredients or enzyme compositions a basic protein or basic peptide or a composition of the present invention in an amount effective for inhibiting growing microbial cells, preferably a protamine or a pro¬tamine sulphate or a composition comprising a protamine or a protamine sulphate. In yet another aspect the invention relates to a method of killing microbial cells present on human or animal skin, mucous membranes, wounds, bruises or in the eye which method comprises contacting the cells to be killed with a basic protein or peptide in an amount effective for killing the cells, preferably a protamine or prota¬mine sulphate. Thus, the compositions of the invention and/or the basic peptides or proteins used in these com¬positions, especially protamines and protamine sulphates, may by useful as disinfectants, e.g in the treatment of acne, infections in the eye, skin infections; in antiperspirants; for cleaning end disinfection of contact lenses etc. The invention is further illustrated by the following non-limiting examples. EXAMPLE 1 Bactericidal and bacteriostatic effect of protamine Materials and Methods BACTERIA, INOCULUM, MEDIA AND REAGENTS The bacteria used in the study are listed in Table 1 be¬low. Stock cultures were maintained in Tryptone Soy Broth (TSB)(Oxoid CM129) with 0.5% glucose, 2% skimmed milk powder, 4% glycerol and stored at -SCC. Cells from the stock culture were streaked on TSB plates (TSB with 1.2% agar) and incubated 48 h at 25°C. One co¬lony of bacteria was inoculated in 5 ml of TSB and grown 24-36 h at 25°C. This culture was used as inoculum. TSB with 1.2% agar was used for plate counts which were done by surface inoculation and incubation of the plates at 25°C. Ten-fold dilution rates were prepared using sterile peptone saline (0.1% peptone, 0.85% NaCl). Protamine from Salmon (P-4005) was obtained from Sigma Chemical Company (St. Louis, USA), dissolved in distilled water, filter sterilized (0.2 nm) and used immediately after preparation. (d) Department of Biotechnology, Technical Uni¬versity of Denmark. (e) Campden Food and Drink Association, Chipping Campden, United Kingdom. (f) Department of Clinical Microbiology, Statens Seruminstitut, Denmark (g) Knochel, 1989. (h) Gram et al., 1990. (j) Jorgensen, 1986. (k) J0rgensen and Huss, 1989. (m) Ben Embarek and Huss 1993. (n) Chung, Steen and Hansen, 1994. IMPEDANCE DETERMINATIONS Volumes (1.0 ml) of TSB medium were added to Bactometer® wells. Protamine solutions (0.1 ml) were transferred to the wells which were sealed, connected to the Bactometer B123-2 (bioM^rieux UK ltd., UK) and incubated at 25°C until a significant detectable increase in the electrical conductivity of the medium was registered and the detec¬tion time (DT) was recorded (maximum 100 h). Detection usually occurs when the cell concentration reaches 10^-10' cfu/ml. For the Gram-positive strains the capacitance signal was monitored, while change in conductance was used for Gram-negative strains. EFFECT OF PROTAMINE ON GROWING CELLS The antibacterial activity of protamine on growing cells was measured in the Bactometer modules. The wells (con¬taining 1 ml TSB and 0.1 ml protamine solution) were ino¬culated with 0.1 ml from a 10"^ dilution of an inoculum culture, giving a final cell concentration in the well of approximately 10^ cfu/ml. The concentration of protamine varied from 1 to 4000 /xg/ml depending on the sensitivity of the strain investigated. Minimum Inhibition Concentration (MIC) was determined as the lowest concentration of protamine resulting in absen¬ce of a DT. When no DT was measured, the lethal/inhibitory effect on the cells were tested by plating the total well volume. EFFECT OF PROTAMINE ON NON-GROWING CELLS 1 ml of inoculum diluted to 10'^ was inoculated in 250 ml TSB (approximately 10^ cfu/ml) and incubated at 25°C for 24 h. Cells were harvested by centrifugation (2000g for 10 min), washed twice with 0.067 M sterile sodium phosp¬hate buffer pH 7.0 (Weisner 1984) and resuspended in the same buffer. The absorbance at 450 nm (OD450) of the bac¬terial suspension was adjusted to 1.0 (approximately 10* cfu/ml), measured on a Perkin Elmer Lambda 2 spectropho¬tometer (tJberlingen, Germany) . The cell suspension was diluted in sterile phosphate buffer to concentrations of 10* and 10^ cfu/ml. Protamine was added to the cell su¬spensions (10^, 10* and 10* cfu/ml) in concentrations of 0, 50, 100 and 500 jug/ml, and the suspensions were incu¬bated at 25°C for 24 h. CALIBRATION CURVES A series 10-fold dilution rate was prepared from the 10* cfu/ml suspension with no protamine added. A calibration curve relating cfu/ml of the 10-fold dilutions to capaci¬tance or conductance DT in TSB was constructed for each strain using a minimum of 8 dilution steps. From the protamine treated suspensions, 0.1 ml was ino¬culated in TSB in Bactometer wells and the DT determined. This DT was, converted to a colony count using the call- bration curve. Thus, colony counts were not made directly on the protamine treated suspensions as protamine caused significant clumping of the bacterial cells. When no DT was measured, the total well volume was pi¬petted onto agar plates to evaluate whether protamine had a bacteriostatic or bactericidal effect. Results The MIC values determined from capacitance or conductance DT of cells growing in TSB are shown in Table 2. Table 2: Minimum inhibition concentration (MIC) measured impedi- metric as a total inhibition after 100 hours at 25>c. Strain MIC ()Ltg/ml) Aeromonas sobria > 4000 Aeromonas salmonicidae 4000 Escherichia coli 0157:H7 1000 Pseudomonas aeruginosa 4000 Pseudomonas fluorescens 3000 Shewanella putrefaciens (4 strains) 500-1000 Vijbrlo anguillarum 1000 Vibrio paraheamolyticus 500-1000 yersinia enterocolitica >4000 Bacillus subtilis 100 Brochotrix thermosphacta 20 Corynebacterium jeikeium 100 Listeria monocytogenes (2 strains) 1000 Staphylococcus aureus (2 strains) 500-1000 The Gram-positive strains were more sensitive to protami¬ne than the Gram-negative. The MIC values determined for Gram-positive strains varied from 20 to 1000 /xg/ml and varied from 500 /xg/ml to more than 4000 nq/inl for the Gram-negative strains. Brochotrix thermosphacta was the most sensitive strain, and a protamine concentration of 20 )ug/ml TSB caused a total kill of the inoculum (10^ cfu/ml) , measured by pla¬ting the well volume after 100 h of incubation in the Bactometer. A protamine concentration of 1000 /ug/ml re¬sulted in a 100% lethal effect on the two strains of Li¬steria monocytogenes and Staphylococcus aureus (10^ cfu/ml). The MIC for protamine on S. aureus was 500 )Lig/ml, however, this concentration did not have a lethal effect. The DT's for Aeromonas sobria and Yersinia enterocolitica increased with increasing protamine concentration, sug¬gesting a prolonged lagphase. However, the cultures were not totally inhibited and no MIC was determined. The DT for a 10^ cfu/ml suspension of A. sobria was 33 h when incubated with 4000 ixq/ml compared to 12 h when no pro¬tamine was added. For Y. enterocolitica the detection time of 10^ cfu/ml was prolonged from 21 h to 60 h when 4000 jLtg/ml of protamine was added, see Figure 1. Aeromo¬nas salmonicidae and Pseudomonas fluorescens were inhibi¬ted by protamine in concentrations of 4000 and 3000 jug/ml TSB, respectively. Thus, no change in conductance was seen after 100 hours incubation, but live cells were iso¬lated from the well. Shewanella putrefaciens (A2), (All) and (A22) and VlJbrlo anguillarum were inhibited by pro¬tamine in the concentration 1000 jug/ml TSB, and ViJbrio paraheamolyticus and S. putrefaciens (A6) were inhibited by 500 jug/ml TSB. S. putrefaciens was the only Gram-nega¬tive bacteria which was killed by protamine, thus, 2000 /ig/ml of protamine killed 100% of the inoculum (lo' cfu/ml) of all the four tested strains. The bactericidal effect of protamine on non-growing cells was tested on four strains of S. putrefaciens and two strains of L. monocytogenes. After protamine treatment, detection times were measured and converted to a cell count using the calibration curve generated for the particular strain (Fig. 2). The sensitivity of S. putre¬faciens varied from strain to strain, see Table 3 below (see also table 1 for code and reference for each strain). Strain A2 and All were similar in sensitivity and more resistant than A6 and A22. Thus, 100 ng protamine/ml killed S. putrefaciens strain A6 and A22 when suspended at low cell concentrations (10 and 10^ cfu/ml). The same level was not 100% lethal on S. putrefaciens strain A2 and All, however the cell number was reduced by 90-99.9%. The initial cell number was estimated by absorbance measurements. k: No surviving cells (determined by spread plating the medium from the well, c): Long detection time (DT) corresponding to a very low cell number (approximately 1 surviving cell). Protamine at 100 and 500 jug/ml had no effect on non-growing L. monocytogenes cells, and increasing the pro¬tamine concentration to 1000 /xg/ml did not cause any let¬hal effect of protamine on non-growing L. monocytogenes. The results show that salmine (salmon protamine) in con¬centrations of 100-4000 /ig/ml prolonged the lag phase of several Gram-negative bacteria significantly. Protamine was more effective on actively growing L. monocytogenes cells as compared to cells suspended in buffer. The bac¬tericidal effect of protamine on Shewanella putrefaciens was seen on both growing and non-growing cells. A pro¬tamine concentration of 2000 /ig/ml was required to kill growing S. putrefaciens (10^ cfu/ml), whereas non-growing cells were killed by only 50 /xg/ml. When the cell con¬centration was raised the bactericidal effect of protami¬ne was decreased, probably as a result of the higher cell/protamine ratio. The impedimetric method used in this study proved useful for the measurement of the antibacterial activity of a cationic protein which caused cellular agglutination. Excellent correlations exit between detection time and cfu of untreated cells, see Figure 2. The correlation between protamine treated cells and cfu was statistically similar to the correlation for untreated cells (data not shown), however, plating the protamine treated cells cau¬sed a great degree of variation on the cells count. It is demonstrated that protamine inhibits growth of all the tested strains, determined as a prolonged lag phase for the most resistant bacteria or a lethal effect on a few of the tested strains, the Gram-positive bacteria in particular. The fact that protamine is naturally occur¬ring and non-toxic makes it an antibacterial protein that might hold great promise for the control of e.g. spoilage bacteria and food-borne pathogens. EXAMPLE 2 Comparison of minimum inhibition concentrations for basic proteins and enzymes The minimum inhibition concentration (MIC) of various substances was determined as described in Example 1. The following substances were tested: protamine (A), pro¬tamine sulphate (B), a peroxidase enzyme system (i.e. lactoperoxidase/glucoseoxidase (C)), subtilisin A (D), polyarginine (E) having an average molecular weight of about 6 kD and lysozyme (F) (150 000 units/mg; Johansen, C. et al., 1994). The results are shown in Table 4 below. It is demonstrated that protamine and protamine sulphate are very effective substances for inhibiting all the te¬sted strains, whereas polyarginine is effective for in¬hibiting all strains but Pseudomonas spp.. Apart from the effect of lysozyme on Listeria monocytogenes, none of the tested enzyme showed any effect. Table 4: Comparative minimum inhibition concentrations Substance Strain Minimum Inhibitory Concentration (Mg/ml) A B C D E F Listeria monocytogenes Staphylococcus aureus Escherichia coli Pseudomonas aeruginosa Pseudomonas fluorescens Shewanella putrefaciens Vibrio paraheamolyticus 1000 1000 n.e. n.e. 2000 2000 1000 2000 n.e. n.e. 1500 n.e 1000 n.d. n.e. n.e. 1500 n.e 4000 n.d. n.e. n.e. n.e. n.e 3000 4000 n.e. n.e. n.e. n.e • 1000 500 n.e. n.e. 1500 n.e 1000 500 n.e. n.e. 2000 n.e • n.d.: not done n.e.: not effective * The lactoperoxidase system was effective for maximum 70 hours. The definition of MIC require an inhibition of at least 100 hours. EXAMPLE 3 The influence of pH and cell concentration on the anti¬bacterial effect of protamine The influence of pH on the antibacterial effect of pro¬tamine was tested using the materials and methods descri- bed in Example 1. The results are shown in Figure 3 and demonstrate clearly that the antibacterial effect of protamine depend signi¬ficantly on pH. At low pH, protamine (1 mg/ml) has no ef¬fect on the Gram-negative bacteria Pseudomonas aeruginosa, however, at high pH protamine (1 mg/ml) pro¬longed the detection time from 32 to 71 hours. The inter¬action between pH and protamine has been observed for all the tested strains. Further, the influence of cell concentration on the anti¬bacterial effect of protamine was tested using the ma¬terials and methods described in Example 1, i.e.the cor¬relation between cell concentration and protamine concen¬tration has been measured by the impedimetric assay. The results are shown in Figure 4 and Figure 5 and demon¬strate clearly that a significant synergistic effect bet¬ween the cell concentration and the protamine concentra¬tion has been observed. Thus, at low cell concentration, protamine (1 mg/ml) caused a prolongation of the detection time from 12 to above 100 hours for the Gram-negative bacteria Shewanella putrefaciens (4 strains), compared to a prolongation at high cell concentration from 6 to 18 hours. The detection time for the Gram-po¬sitive bacteria Listeria monocytogenes (2 strains) was at low cell concentration prolonged from 18 to 55 hours when treated with protamine (1 mg/ml), at high cell concentra¬tion the same protamine concentration only prolonged the detection time from 4 to 15 hours. EXAMPLE 4 Synergistic antimicrobial effect between a basic protein, a cell-wall degrading enzyme and a peroxidase enzyme system Impedimetric measurements carried out as described in example 1 have shown an synergistic effect between basic peptides as protamine, polyarginine or polylysine and lysozyme and/or glucose oxidase and/or the lactoperoxidase enzyme system depending on pH and NaCl concentration. Growth inhibition experiments were conducted, wherein synergistic and additional effects were determined by mixing compounds in low concentrations not having any activity on their own and using a factorial design. The effects were measured as growth inhibition or a 100% bactericidal effect with a total kill of the inoculum. Protamine (250 jug/ml) or poly lysine (500 /xg/ml) in combi¬nation with lactoperoxidase (2 U/ml) and glucose oxidase (2 U/ml) had a 100% lethal effect on Pseudomonas fluorescens, whereas the same strain was not inhibited when treated with any of these three compounds alone in the concentrations mentioned above. A synergistic effect was as observed against Pseudomonas fluorescens when combining protamine (250 ng/mil) and poly lysine (500 /xg/ml) and lysozyme (50000 U/ml) and lactoperoxidase (2 U/ml) and glucose oxidase (1 U/ml) or lysozyme (50.000 U/ml) and poly lysine (500 jig/ml) and lactoperoxidase (2 U/ml) and glucose oxidase (1 U/ml). Experiments where the antibacterial effect was measured as growth inhibition of Shewanella putrefaciens in TSB at 25 00 and pH 7.2, showed a synergistic effect between protamine and lysozyme; the results are shown in Figure 6 as a response surface plot. Lysozyme alone had no effect on the Gram-negative bacteria Shewanella putrefaciens neither had protamine at concentrations below 500 jug/ml, whereas combinations caused a 100% bactericidal activity at protamine concentrations above 300 /xg/ml and lysozyme concentrations from iC-lO* U/ml (lysozyme activity: 150000 U/mg). EXAMPLE 5 Fungistatic and fungicidal activities This experiment was carried out as described in example 1 using the Bactometer substrate: 0.75 g Yeast extract (Difco) , 3.0 g D(+)Glucose, 1 g KH2PO4, 0.8 g isogel agarose lEF (Pharmacia) and 100 ml distilled water. Protamine was added to the substrate immediately before inoculation with a spore suspension of the test fungi (approximately lO^-lO* cfu/ml) . The minimum inhibitory concentration was determined as the lowest concentration of protamine resulting in a absence of DT during 100 h measurement (see table 5 below). When no DT was determined a fungicidal activity was evaluated by plating from a dilution of the total well volume. Table 5 Strain pH MIC (Mg/ml) Alternaria infectoria Aspergillus niger Botrytis aclada Cladosporium herbarum Eurotium repens Fusarium culmorum Penicillium comcam Penicillium crustosum Penicillium roqueforti Ulocladium atrum 5.2 240 7.1 1000 5.2 120 5.2 120 5.2 240 5.2 240 6.1 1000 6.2 1000 5.2 240 5.2 240 The fungistatic and fungicidal effect of protamine was optimal at high pH and low inoculum size as the effect on bacteria. Increasing the pH caused a significant decrease in the MIC-value. A fungistatic effect was obtained with an 2-5 fold lower protamine concentration than used when a fungicidal effect was determined. The most resistant strains shown in table 5 were not inhibited by protamine for 100 h at low pH, thus a 100 h inhibition was not obtained before increasing the pH to the values given in the table. EXAMPLE 6 Survival and transfer of bacteria during mini-wash Materials: Ariel Color (DF-9412330). Swatches (white cotton, DF-9415585), sterilized by autoclaving. Tryptone Soya Broth (TSB). Sterilized water (12°dH). Strains: Staphylococcus aureus (skin isolate) Pseudomonas aeruginosa (skin isolate) Methods: Inoculum: S. aureus and P. aeruginosa were grown in Tryptone Soya Broth (TSB) at 25'C for 30 hours. For each strain six sterile swatches were inoculated with approximately 10* cfu/swatch and air dried for 30 minutes. Mini-wash: 0.56 g of Ariel Color was dissolved in 80 ml sterile water (12"dH, 35°C) in each wash beaker, giving the final concentration of 7 g/1. Protamine, dissolved in water and filter sterilized, were added to half of the wash beakers giving the final concentration of 500 jLtg/ml. After 70 sec. 1 inoculated swatch and 2 sterile swatches were transferred to each wash beaker, and washed at 35°C for 15 min. In one beaker 3 sterile swatches were washed as control. From each wash beaker 0.1 ml of detergent solution were transferred to Malthus (in-direct cells) con¬taining TSB and incubated. The swatches were rinsed in sterile water for 10 min (stirring). From each beaker 0.1 ml of rinse water were incubated in Malthus (in-direct cells). After wash all swatches were slightly air dried in sterile air for 10 min, and each swatch was trans¬ferred to an in-direct Malthus cell. All materials and instruments except the detergent, were sterilized before use to avoid contamination. In-direct Malthus: Indirect-Malthus measurements were used when estimat¬ing the number of viable cells. 3 ml of TSB were transferred to the outer chamber of the in-direct Malthus cells, and 0.5 ml of sterile KOH (0.1 M) were transferred to the inner chamber. As cells are growing in the outer chamber they produce CO2 (g) which will dissolve in the KOH in the inner chamber and thereby change the conductance of the KOH. When the conductance change is measurable by the Malthus, a detection time (DT) will be recorded. The DT's were converted to colony counts by use of a calibration curve relating cfu/ml to DT (see Figure 1 and 2). A series 10-fold dilution rate was prepared from the 10* cfu/ml suspension of cells. Conductance DT of each dilution step was determined in TSB, and a cali¬bration curve relating cfu/ml of the 10 fold dilu¬tions to DT in TSB was constructed for each strain (see Figure 1 and 2). Results: The number of cells surviving mini-wash, in the detergent solution, attached to the contaminated swatches, trans¬ferred to the rinse water or to the sterile swatches, were determined by in-direct Malthus (table 6). A relatively high number of cells were washed of the swatches and found in the wash water, however, approxima¬tely 10^ cfu were still attached to the swatches after mini-wash and rinsing for 10 min, and during wash cells were transferred from the contaminated swatch to the sterile swatches. The S. aureus cells were found very sensitive to protamine, which can be explained by the increased antibacterial activity of protamine at high pH. A high number of P. aeruginosa were determined in the detergent solution, and protamine was found active against the cells in the detergent solution, however, P. aeruginosa attached to the swatches were not inhibited or killed by protamine, and all the sterile swatches in the washes inoculated with P. aeruginosa were contaminated during wash. The rinse water were almost sterile, thus the cells may adhere actively to the textile. Table 6: Cell number in the detergent solution, the rinse water, attached to the contaminated swatches and transferred to sterile swatches, before and after mini-wash at 35°C for 15 min with Ariel Color. Each wash were done in triplets, A, B and C are the three wash beakers with the same combinations of strain and protamine. staphylococcus aureus (pg/ml) 0 500 Pseudomonas aeruginosa (/jg/ml) 0 500 Before wash (cfu/swatch) contaminated swatch A 1.610 1 610 4.010 4. 010' B 1.610' 1. 610' 4.010' 4. 010' C 1.610' 1 610 4.010' 4. 010' sterile swatch 1 A 0 0 0 0 B 0 0 0 0 C 0 0 0 0 sterile swatch 2 A 0 0 0 0 B 0 0 0 0 C 0 0 0 0 After mini-wash (cfu/swatch) contaminated swatch A 1.710' 0 8.010' 2 510' B 3.010' 0 1.210^ 6 010' C 2.910^ 0 6.010' 1 310' swatch 1 A 1 0 4.010^ 1 B 0 0 5.010' 1 C 2.810^ 0 1 1 swatch 2 A 6.110^ 0 7.010^ 1 B 1 0 1 1 C 0 0 3.010' 1 Detergent solution (cfu/ml) total volume; 80 ml A 2.010^ 1 - (a) 0 B 6.310' 0 5.810^ 0 0 2.010' 0 1.910" 0 Rinse water (cfu/ml) total volume;100 ml A 0 0 0 1 B 0 0 0 1 0 0 0 1 0 (a): No DT determined by Malthus, growth in the Malthus cell was observed by eye. From the results it can be concluded that Ariel Color has no significant bactericidal effect on S. aureus and P. aeruginosa (pathogenic skin isolates). A high number of cells were washed of the swatches, and found in the detergent solution, and when no protamine were added, cells from the inoculated swatch contaminated the sterile swatches in the wash beaker. REFERENCES Ben Embarek, P. K. & Huss, H. H. 1993 Heat resistance of Listeria monocytogenes in vacuum packaged pasteurized fish fillets. International Journal of Food Microbiology 20, 85-95. Bierbaum, G. & Sahl, H. G. 1987 Autolytic system of Staphylococcus simulans 22: Influence of cationic peptides on activity of n-acetylmuramoyl-L-alanine amidase. Journal of Bacteriology 169, 5452-5458. Bierbaum, G. & Sahl, H. G. 1991 Induction of autolysis of Staphylococcus simulans 22 by Pep5 and nisin and influen¬ce of the cationic peptides on the activity of the auto¬lytic enzymes. In Nisin and novel antibiotics ed. Jung, G. & Sahl, H. G, p. 347-358. ESCOM, Leiden. Brange, J. 1987 Galenics of insulin. Ch. 3, pp. 34-36. Berlin: Springer-Verlag. Christensen, B., Fink, J., Merrifield, R. B. & Mauzerall, D. 1988 Channel-forming properties of cecropins and rela¬ted model compounds incorporated into planar lipid mem¬branes. Proceedings of the National Academy of Sciences of the United States of America 85, 5072-5076. Chung, Steen & Hansen, (1994). Subtilisin gene of Bacil¬lus subtilis ATCC 6633 is incoded in an operon that contains homologues of the hemolysin B transport protein, J. Bact.. Vol. 174 (1994), p. 1414-1422. Connolly, P., Bloomfield, S. F. & Denyer, S. P. 1993 A study of the use of rapid methods for preservative effi¬cacy testing of pharmaceuticals and cosmetics. Journal of Applied Bacteriology 75, 456-462. Farrag, S. A. & Marth, E. H. 1992 Escherichia coli 0157:H7, Yersinia enterocolitica and their control in milk by the lactoperoxidase system: a review. Lebensmit-tel-Wissenschaft und Technologie 25, 201-211. Firstenberg-Eden, R. & Eden, G. 1984 Impedance microbio¬logy. Letchworth: John Wiley & Sons Inc. Gram, L., Wedell-Nedergaard, C. & Huss, H. H. 1990 The bacteriology of fresh and spoiling Lake Victorian Nile perch (Lates niloticus). International Journal of Food Microbiology 10, 303-316. Hitsch, J. G. 1958 Bactericidal action of histone. Jour¬nal of experimental medicine 108, 925-944. Borrow, J. C. 1985 Protamine: A review of its toxicity. Anaesthesia Analgesia 64, 51-55. Hugo, W. B. 1978 Membrane-active antimicrobial drugs - a reappraisal of their mode of action in the light of the chemosmotic theory. International Journal of Pharmaceu¬tics 1, 127-131. Islam, N. MD., Itakura, T. & Motohiro, T. 1984 Antibac¬terial spectra and minimum inhibition concentration of clupeine and salmine. Bulletin of the Japanese Society of Scientific Fisheries 50, 1705-1708. Jaques, L. B. 1973 Protamine - antagonist to heparin. Canadian Medical Association Journal 108, 1291-1297. Jeong, D. K., Harrison, M. A., Frank, J. F. & Wicker, L. 1992 Trials on the antibacterial effect of glucose oxida¬se on chicken breast skin and muscle. Journal of Food safety 13, 43-49. Jeppesen, V. F. & Huss, H. H. 1993 Characteristics and antagonistic activity of lactic acid bacteria isolated from chilled fish products. International Journal of Food Microbiology 18, 305-320. Johansen, C., Gram, L. & Meyer, A. S. 1994 The combined inhibitory effect of lysozyme and low pH on the growth of Listeria monocytogenes. Journal of Food Protection, in press. J0rgensen, B. R. 1986 Sorbic acid - effect on fish - in¬fluence of pH on the effect. Masters thesis (in Danish). Technological Laboratory, Danish Ministry of Fisheries, Technological University of Denmark. Jorgensen, B. R. & Huss, H. H. 1989 Growth and activity of Shewanella putrefaciens isolated from spoiling fish. International Journal of Food Microbiology 9, 51-62. Kagan, B. L., Selsted, m. E., Ganz, T. & Lehrer, R. I. 1990 Antimicrobial defensin peptides from voltage-depen¬dent ion-permeable channels in planar lipid bilayer mem¬branes. Proceedings of the National Academy of Sciences of the United States of America 87, 210-214. Kn0chel, S. 1989 Aeromonas spp. - ecology and significan¬ce in food and water hygiene. Ph.D. thesis. Technological Laboratory and the Royal Veterinary and Agricultural Uni¬versity , Freder iksberg, Denmark. Kossel, A. 1928 The protamines and histones, Longmans, Green and Co., London, New York, Toronto, 1-63. Lehrer, R. I., Lichtentstein, A. K. St Ganz, T. 1993 De-fensins: Antimicrobial and cytotoxic peptides of animal cells. Annual Review of Immunology 11, 105-128. Nakae, T. 1985 Outer-membrane permeability of bacteria. In Critical Reviews in Microbiology ed. O'Leary, W. M., pp. 1-62. Boca Raton, Florida: CRC Press, Inc. Phillips, L. G., Yang, S. T., Schulman, W. & Kinsella, J. E. 1989 Effect of lysozyme, clupeine, and sucrose on the foaming properties of whey protein isolate and j8-lactag-lobulin. Journal of Food Science 54, 743-747. Rodman, T. C, Pruslin, F. H. & Allfrey, V. G. 1984 Pro-tamine-DNA association in mammalian Spermatozoa. Experi¬mental Cell Research 150, 269-281. Sahl, H. G. 1987 Influence of the staphylococci-like pep¬tide Pep5 on membrane potential of bacterial cells and cytoplasmic membrane vesicles. Journal of Bacteriology 162, 833-836. Trevors, J. T. 1986 Bacterial growth and activity as in¬dicators of toxicity. In Toxicity testing using microor¬ganisms ed. Bitton, G. & Dutka, B. J. Boca Raton, Flori¬da: CRC Press, Inc. Vaara, M. 1992 Agents that increase the permeability of the outer membrane. Microbiological Reviews 56, 395-411. Vaara, M. & Vaara, T. 1983 Polycations as outer membrane-disorganizing agents. Antimicrobial Agents and Chemother¬apy 24, 114-122. Weisner, B. 1984 Lysozyme (muramidase). In Methods of En¬zymatic Analysis Vol. 4, ed. Bergmeyer, H. U. pp. 189-195. Weinheim: Verlag Chemie. Yanagimoto, T., Tanaka, M. & Nagashima, Y. 1992 Changes in the antibacterial activity of salmine during Its Mail-lard reaction. Bulletin of the Japanese Society of Scien¬tific Fisheries 58, 2153-2158. WE CLAIM: 1. A bactericidal, bacteriostatic, fungicidal and/or fungistatic composition comprising or consisting essentially of a basic proteta^or peptide selected from protamines, protamine sulphates, and polylysins and polyarginins and co-polymers thereof, capable of killing microbial cells, in combination with a chitinase. 2. The composition according to claim 1, which further comprises an oxidoreductase. 3. The composition according to claim 2, wherein the oxidoreductase is selected from the group consisting of oxidases (EC 1.10.3) and peroxidases (EC 1.11.1), preferably from peroxidase enzyme systems (EC 1.11.1.7) and laccase enzymes (EC 1.10.3.2). 4. The composition according to claim 3, wherein the peroxidase enzyme system comprises at least one peroxidase enzyme and a hydrogen peroxide generating enzyme system such as an oxidase and a substrate for the oxidase or an amino acid oxidase and a suitable amino acid, or a peroxycarboxylic acid or a salt thereof 5. A cleaning or detergent composition comprising a basic protein or peptide selected from protamines, protamine sulphates, and polylysins and polyarginins and co¬polymers thereof, capable of killing microbial cells, and a chitinase and a surfactant. 6. The composition according to claim 5, which further comprises an oxidoreductase. 7. The composition according to claim 6, wherein the oxidoreductase is selected from peroxidase enzyme systems (EC 1.11.1.7) and laccase enzymes (EC 1.10.3.2). 8. The composition according to claim 7, wherein the peroxidase system comprises at least one peroxidase enzyme and a hydrogen peroxide generating enzyme system such as an oxidase and a substrate for the oxidase or an amino acid oxidase and a suitable amino acid, or a peroxycarboxylic acid or a salt thereof. 9. The composition according to any of the claims 1-8, which further comprises at least one enzyme selected from the group consisting of proteases, amylases, cellulases, and lipases. 10. The composition according to any of the claims 1-9, wherein the surfactant is a detergent surfactant, preferably selected from the group consisting of anionic, nonionic, ampholytic, zwitterionic and cationic surfactants. 11. The composition according to any of the claims 14-18, wherein the basic protein is a protamine or a protamine sulphate in an amount corresponding to between 1 and 4000 mg per 1 cleaning liquor or washing liquor. 12. A method for killing microbial cells present on a hard surface comprising contacting the surface with a cleaning composition according to any of the claims 5- 11 or a composition according to any of the claims 1-4. 13. A method for killing microbial cells or inhibiting growing microbial cells present on laundry comprising contacting the laundry with a detergent composition according to any of the claims 5-11 or a composition according to any of the claims 1-4. 14. A method for preservation of food, beverages, cosmetics, contact lens products, food ingredients or enzyme compositions comprising incorporating into the unpreserved food, beverages, cosmetics, contact lens products, food ingredients or enzyme compositions a basic protein or basic peptide or a composition according to any of the claims 1-4 in an amount effective for inhibiting growing microbial cells.

Full Text

The present invention relates to a composition capable of killing microbial cells or inhibiting growing microbial cells, i.e. a bactericidal, bacteriostatic, fungicidal and/or fungistatic composition; a cleaning or detergent composition comprising a substance capable of killing microbial cells or inhibiting growing microbial cells; and methods for killing microbial cells present on a hard surface, on skin or in laundry, and for preserving food products, cosmetics etc.
BACKGROUND OF THE INVENTION
At this time of increased public interest in reducing the use of chemical additives, it is relevant to consider na¬tural alternatives for antimicrobial agents used e.g. for preserving foods, as disinfectants, and as an antimicro¬bial ingredient of detergent and cleaning compositions. This has increased interest in preservation using live bacterial cultures (Jeppesen & Huss 1993) and enzymes like lactoperoxidase (Farrag & Marth 1992), glucose oxi¬dase (Jeong et al. 1992) and lysozyme (Johansen et al. 1994) .
Protamines are basic proteins with a high arginine con¬tent found in association with DNA of spermatozoon nuclei of fish, birds, mammals etc. (Rodman et al. 1984; Kossel 1928) . Protamine is used clinically as an antidote to heparin (Jaques 1973) and as a carrier of insulin, pro¬longing the absorption of subcutaneously administered insulin (Brange 1987). Attention has also been paid to the functional properties of protamine as a stabilizing agent (Phillips et al. 1989). Protamine has been shown to have an antibacterial effect (Hitsch 1958), but this

.)(

aspect has not been thoroughly studied.
The Gram-negative bacteria are often resistant to a large number of harmful agents due to the effective permeabili¬ty barrier function of the outer membrane (Nakae 1985). However, protamine and most other cationic peptides are under certain conditions apparently able to traverse the outer membrane of Gram-negative bacteria (Vaara 1992, Vaara & Vaara 1983), probably as a result of their bin¬ding to the anionic lipopolysaccharide-covered surface of the Gram-negative cell. The mechanism of the antibac¬terial action of basic peptides is not known, but it has been suggested that they form a channel in the cytoplas¬mic membrane, thus uncoupling electron transport and cau¬sing leakage (Christensen et al. 1988; Hugo 1978; Kagan et al. 1990). It has also been proposed that they induce autolysis due to activation of the autolytic enzymes (Bi-erbaum & Sahl 1991).
In general, the occurrence of highly-basic peptides such as protamine is relatively rare in nature. However, of those studied, several have been found to demonstrate antibacterial properties: eg. nisin (Sahl 1987), defensin (Lehrer et al. 1993), cecropins (Christensen et al. 1988), Pep5 (Bierbaum & Sahl 1987) and melittins (Vaara 1992) .
Antibacterial activity is usually measured as a decrease in colony counts, a decrease in the absorbance of a bac¬terial suspension or as inhibition zones on agar plates (Trevors 1986). However, these methods may not be suit¬able when assaying the antibacterial effect of a basic peptide or protein such as protamine due to the aggluti¬nation of the positively-charged protamine and the nega¬tively-charged bacterial cells as described by Islam et al. (1984).

Thus, the object of invention was to provide an antibac¬terial and/or antifungicidal composition comprising a natural active compound or substance, i.e. which is non¬toxic, of biological origin, easily available and rela¬tively inexpensive, optionally in combination with other antimicrobial compounds or substances.
SUMMARY OF THE INVENTION
It has now surprisingly been found that it is possible to kdJ.1 microbial cells or inhibit growing microbial cells /by means of a basic protein or peptide of biological ori¬gin, e.g. protamine or protamine sulphate. For certain bacteria or fungi, it may be necessary to combine the basic protein with a cell-wall degrading enzyme or an oxidoreductase in order to obtain the desired antimicro¬bial effect.
Accordingly, the growth inhibitory effect of protamine and the potential lethal effect of this basic protein on non-growing cells has been investigated. Due to the methodological shortcomings described above, impedimetric measurements were used and compared to traditional plate counts (Firstenberg-Eden & Eden 1984; Connolly et al. 1993) .
Thus, based these findings the present invention provides a bactericidal, bacteriostatic, fungicidal and/or fun¬gistatic composition comprising a basic protein or pep¬tide capable of killing microbial cells in combination with a cell-wall degrading enzyme or an oxidoreductase.
In another aspect, the present invention provides a detergent or cleaning composition comprising a basic pro¬tein or peptide capable of killing microbial cells and a surfactant. Such compositions have a pH in the alkaline

range and it has been found that basic proteins such as protamine and protamine sulphate exhibit their optimum antimicrobial effect at alkaline pH, thus making such proteins very suitable for incorporation in compositions for cleaning purposes.
The composition of the invention is useful as antimicro¬bial ingredient wherever such an ingredient is needed, for example for the preservation of food, beverages, cos¬metics, contact lens products, food ingredients or enzyme compositions; as a disinfectant for use e.g. on human or animal skin, mucous membranes, wounds, bruises or in the eye; for killing microbial cells in laundry; and for incorporation in cleaning compositions for hard surface cleaning.
THE DRAWINGS
The invention is further illustrated by the drawings, in which
Figure 1 shows the effect of protamine on growth of Yer¬sinia enterocolitica growing in TSB at 25°C. Growth is measured as % change in conductance;
Figure 2 shows calibration curves relating conductance ▲ (Shewanella putrefaciens strain A2) or capacitance ■ (Li¬steria monocytogenes strain 032) detection times in TSB at 25°C to colony counts in the absence of protamine;
Figure 3 shows the effect of various concentrations of protamine on the Gram-negative bacteria Pseudomonas aeruginosa in dependence of pH.
Figure 4 shows the effect of various concentrations of protamine on the Gram-positive bacteria Listeria monocytogenes in dependence of cell concentration

(inoculum, log CFU/ml).
Figure 5 shows the effect of various concentrations of protamine on the Gram-negative bacteria Shewanella putre-faciens in dependence of cell concentration (inoculum, log CFU/ml).
Figure 6 is a response surface plot showing the synergis¬tic effect of a composition of the invention (various concentrations of protamine and lysozyme) on the Gram-ne¬gative bacteria Shewanella putrefaciens in dependence of cell concentration (inoculum, log CFU/ml).
DETAILED DESCRIPTION OF THE INVENTION
In the present context, the terra "bactericidal" is to be understood as capable of killing bacterial cells.
In the present context, the term "bacteriostatic" is to be understood as capable of inhibiting bacterial growth, i.e. inhibiting growing bacterial cells.
In the present context, the term "fungicidal" is to be understood as capable of killing fungal cells.
In the present context, the term "fungistatic" is to be understood as capable of inhibiting fungal growth, i.e. inhibiting growing fungal cells.
The term "growing cell" is to be understood as a cell having access to a suitable nutrient and thus being capable of reproduction/propagation. By the term "non-growing cell" is meant a living, but dormant, cell, i.e. a cell in the non-growing, non-dividing, non-multiplying and non-energized state with metabolic processes at a minimum.

The term "cell-wall degrading enzyme" is to be understood as an enzyme which degrades components of the cell wall, e.g. peptidoglucans such as murein and pseudomurein; chi-tin; and teichoic acid. Examples of cell-wall degrading enzymes which are useful in compositions of the present invention are endoglycosidases Type II, e.g. the endo-glycosidases Type II disclosed in EP-A2-0 425 018 which is hereby incorporated by reference, lysozymes and chitinases.
The term "amino acids present in mammalian cells" denotes the 2 0 amino acids constituting the proteins being part of mammals, i.e. alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histi-dine, isoleucine, leucine, lysine, methionine, phenylala¬nine, proline, serine, threonine, tryptophan, tyrosine, valine. Preferably, the basic peptides or proteins of the composition of the invention consist of one or more of the mentioned 20 amino acids, i.e. the basic peptides or proteins may not be recovered from e.g. bacteria such as for example nisin and Pep5.
The term "oxidoreductase" means an enzyme classified as EC 1. according to the Enzyme Nomenclature (1992) , i.e. any enzyme classified as EC 1.1 (acting on the CH-OH group of donors), EC 1.2 (acting on the aldehyde or oxo group of donors), EC 1.3 (acting on the CH-CH group of donors), EC 1.4 (acting on the CH-NHj group of donors), EC 1.5 (acting on the CH-NH group of donors), EC 1.6 (acting on NADH or NADPH), EC 1.7 (acting on other nitro¬genous compounds as donors), EC 1.8 (acting on a sulfur group of donors) , EC 1.9 (acting on a heme group of donors), EC 1.10 (acting on diphenols and related sub¬stances as donors), EC 1.11 (acting on a peroxide as acceptor), EC 1.12 (acting on hydrogen as donor), EC 1.13 (acting on single donors with incorporation of molecular oxygen (oxygenases), EC 1.14 (acting on paired donors

with incorporation of molecular oxygen), EC 1.15 (acting on superoxide radicals as acceptor), EC 1.16 (oxidizing metal ions), EC 1.17 (acting on -CHz- groups), EC 1.18 (acting on reduced ferredoxin as donor), EC 1.19 (acting on reduced flavodoxin as donor), and EC 1.97 (other oxidoreductases).
The term "peroxidase enzyme system" is to be understood as a peroxidase (EC 1.11.1) in combination with a source of hydrogen peroxide which may be hydrogen peroxide or a hydrogen peroxide precursor for in situ production of hydrogen peroxide, e.g. percarbonate or perborate, or a hydrogen peroxide generating enzyme system, e.g. an oxi¬dase and a substrate for the oxidase or an amino acid oxidase and a suitable amino acid, or a peroxycarboxylic acid or a salt thereof.
Examples of useful peroxidases are lactoperoxidase, horseradish peroxidase, peroxidases producible by culti¬vation of a peroxidase producing strain Myxococcus vires-cens, DSM 8593, Myxococcus fulvus, DSM 8969, or Myxococcus xanthus, DSM 8970, of a peroxidase producing strain of the genus Corallococcus, preferably belonging to Corallococcus coralloides, DSM 8967, or Corallococcus exiguus, DSM 8969.
In case of lactoperoxidase, thiocyanate may be used as a substrate.
Laccases are enzymes that catalyze the oxidation of a substrate with oxygen; they are known from microbial, plant and animal origins. More specifically, laccases (EC 1.10.3.2) are oxidoreductases that function with molecu¬lar oxygen as electron acceptor. Molecular oxygen from the atmosphere will usually be present in sufficient quantity, so normally it is not necessary to add extra oxygen to the process medium. Examples of a laccase

enzyme useful in the compositions of the present inven¬tion is laccase obtainable from the strain Coprinus cinereus, IFO30116, or from a laccase having immunochemical properties identical to those of a laccase derived from Coprinus cinereus, IFO30116; or obtainable from a strain of Myceliophthora thermophile as disclosed in WO 91/05839.
The term "microbial cells" denotes bacterial or fungal c cells.
By the term "of biological origin" is to be understood that the substance or compounds is recovered or regener¬ated from biological material such as humans, animals or plants. Similarly, the term "of microbiological origin" denotes that the substance or compounds is recovered or regenerated from microbiological material such as bacteria, fungi, yeast or that a parent or native sub¬stance or compound is producible by a microbiological 0 organism.
The term "biological material" denotes living material obtainable from Nature or previously living material obtainable from Nature.
The term "synthesized polypeptide" denotes a synthesized assembly, i.e. a chain, built of peptide monomers. Polypeptides which are useful in the present compositions are basic polypeptides, i.e. polylysins and polyarginins and co-polymers thereof. It is preferred that the polypeptides have a chain length of less than about 100 amino acids but it is contemplated that polypeptides of less than about 1000 kD are useful. Preferably, the polypeptides to be used in the composition of the inven¬tion is of almost identical chain length or molecular weight but mixtures of polypeptides having various chain lengths or molecular weights are also useful.

It is contemplated that the basic protein of the composi¬tion of the invention may be a recombinant protein. In case of protamine, it is contemplated that the protamine may be a recombinant protamine, i.e. produced by cloning of a DNA sequence encoding the protein and subsequent cell transformed with the DNA sequence and expressed in a host, i.e. a suitable fungal or bacterial host. A recom¬binant protamine/protamine sulphate may be cloned and ex¬pressed according to standard techniques conventional to the skilled person.
Preferred basic proteins to be used in the compositions of the present invention are protamines, protamine sul¬phates, defensins, magainins, melittin, cecropins and protegrins; more preferably protamines and protamine sul¬phates .
Hitherto it has been known that protamine from salmon has a bactericidal effect on growing Gram-positive bacteria (1000 jug/ml) . Islam et al. (1984) found that it inhibited growth but did not determine whether the effect was bactericidal or bacteriostatic. Other studies have reported that protamine is not effective against Gram-negative bacteria (Islam et al. 1984; Yanagimoto et al. 1992). Contrary to this observation, it has now been found that protamine is effective against Gram-positive bacteria. Gram-negative bacteria and fungi. The same applies for protamine sulphate.
It has been suggested the primary antibacterial effect of many of the basic peptides is their ability to penetrate the cytoplasmatic membrane, disrupting the electron transport and induce leakage of intracellular compounds. Without being bound to this theory, this mechanism may explain in part the effect of protamine on some of the strains tested in the experiments described in the Examples below.

In another aspect, the present invention relates to a :;leaning or detergent composition comprising a basic pro¬tein or peptide capable of killing microbial cells and a surfactant.
rhe detergent or cleaning composition may further com¬prise other enzymes conventionally used in detergent or cleaning compositions. Preferably, the detergent or cleaning composition of the invention comprises at least one enzyme selected from the group consisting of protea¬ses, amylases, cellulases, and lipases.
The surfactant of the detergent or cleaning composition is preferably a detergent surfactant, more preferably a detergent surfactant selected from the group consisting of anionic, nonionic, ampholytic, zwitterionic and catio-nic surfactants.
In a preferred embodiment, the detergent or cleaning composition comprises as the basic protein a protamine or a protamine sulphate in an amount effective for killing cells or inhibiting growth of cells, preferably in an amount corresponding to between 1 and 4000 /xg per ml cle¬aning liquor or washing liquor, more preferably between 1 and 2 000 fig per ml cleaning liquor or washing liquor, especially between 5 and 1000 jug per ml cleaning liquor or washing liquor.
In a further aspect, the present invention relates to the use of the compositions of the invention for various pur¬poses, i.e. the invention also relates to a method for killing microbial cells present on a hard surface which method comprises contacting the surface with a cleaning composition of the invention, preferably a composition comprising a protamine or a protamine sulphate, in an amount effective for killing the cells.

Also, in yet another aspect the invention relates to a method for killing microbial cells or inhibiting growing microbial cells present on laundry which method comprises contacting the laundry with a detergent composition of the present invention, preferably a composition compri¬sing a protamine or a protamine sulphate, in an amount effective for killing the cells or for inhibiting growing cells.
In yet another aspect the invention relates to a method for preservation of food, beverages, cosmetics such as lotions, creams, gels, soaps, shampoos, conditioners, an-tiperspirants; contact lens products, food ingredients or enzyme compositions which method comprises incorporating into the unpreserved food, beverages, cosmetics, contact lens products, food ingredients or enzyme compositions a basic protein or basic peptide or a composition of the present invention in an amount effective for inhibiting growing microbial cells, preferably a protamine or a pro¬tamine sulphate or a composition comprising a protamine or a protamine sulphate.
In yet another aspect the invention relates to a method of killing microbial cells present on human or animal skin, mucous membranes, wounds, bruises or in the eye which method comprises contacting the cells to be killed with a basic protein or peptide in an amount effective for killing the cells, preferably a protamine or prota¬mine sulphate. Thus, the compositions of the invention and/or the basic peptides or proteins used in these com¬positions, especially protamines and protamine sulphates, may by useful as disinfectants, e.g in the treatment of acne, infections in the eye, skin infections; in antiperspirants; for cleaning end disinfection of contact lenses etc.

The invention is further illustrated by the following non-limiting examples.
EXAMPLE 1
Bactericidal and bacteriostatic effect of protamine
Materials and Methods
BACTERIA, INOCULUM, MEDIA AND REAGENTS
The bacteria used in the study are listed in Table 1 be¬low. Stock cultures were maintained in Tryptone Soy Broth (TSB)(Oxoid CM129) with 0.5% glucose, 2% skimmed milk powder, 4% glycerol and stored at -SCC.
Cells from the stock culture were streaked on TSB plates (TSB with 1.2% agar) and incubated 48 h at 25°C. One co¬lony of bacteria was inoculated in 5 ml of TSB and grown 24-36 h at 25°C. This culture was used as inoculum.
TSB with 1.2% agar was used for plate counts which were done by surface inoculation and incubation of the plates at 25°C. Ten-fold dilution rates were prepared using sterile peptone saline (0.1% peptone, 0.85% NaCl).
Protamine from Salmon (P-4005) was obtained from Sigma Chemical Company (St. Louis, USA), dissolved in distilled water, filter sterilized (0.2 nm) and used immediately after preparation.

(d) Department of Biotechnology, Technical Uni¬versity of Denmark.
(e) Campden Food and Drink Association, Chipping Campden, United Kingdom.
(f) Department of Clinical Microbiology, Statens Seruminstitut, Denmark
(g) Knochel, 1989.
(h) Gram et al., 1990.
(j) Jorgensen, 1986.
(k) J0rgensen and Huss, 1989.
(m) Ben Embarek and Huss 1993.
(n) Chung, Steen and Hansen, 1994.
IMPEDANCE DETERMINATIONS
Volumes (1.0 ml) of TSB medium were added to Bactometer® wells. Protamine solutions (0.1 ml) were transferred to the wells which were sealed, connected to the Bactometer B123-2 (bioM^rieux UK ltd., UK) and incubated at 25°C until a significant detectable increase in the electrical conductivity of the medium was registered and the detec¬tion time (DT) was recorded (maximum 100 h). Detection usually occurs when the cell concentration reaches 10*^-10' cfu/ml. For the Gram-positive strains the capacitance signal was monitored, while change in conductance was used for Gram-negative strains.
EFFECT OF PROTAMINE ON GROWING CELLS
The antibacterial activity of protamine on growing cells was measured in the Bactometer modules. The wells (con¬taining 1 ml TSB and 0.1 ml protamine solution) were ino¬culated with 0.1 ml from a 10"^ dilution of an inoculum culture, giving a final cell concentration in the well of approximately 10^ cfu/ml. The concentration of protamine varied from 1 to 4000 /xg/ml depending on the sensitivity

of the strain investigated.
Minimum Inhibition Concentration (MIC) was determined as the lowest concentration of protamine resulting in absen¬ce of a DT.
When no DT was measured, the lethal/inhibitory effect on the cells were tested by plating the total well volume.
EFFECT OF PROTAMINE ON NON-GROWING CELLS
1 ml of inoculum diluted to 10'^ was inoculated in 250 ml TSB (approximately 10^ cfu/ml) and incubated at 25°C for 24 h. Cells were harvested by centrifugation (2000*g for 10 min), washed twice with 0.067 M sterile sodium phosp¬hate buffer pH 7.0 (Weisner 1984) and resuspended in the same buffer. The absorbance at 450 nm (OD450) of the bac¬terial suspension was adjusted to 1.0 (approximately 10* cfu/ml), measured on a Perkin Elmer Lambda 2 spectropho¬tometer (tJberlingen, Germany) . The cell suspension was diluted in sterile phosphate buffer to concentrations of 10* and 10^ cfu/ml. Protamine was added to the cell su¬spensions (10^, 10* and 10* cfu/ml) in concentrations of 0, 50, 100 and 500 jug/ml, and the suspensions were incu¬bated at 25°C for 24 h.
CALIBRATION CURVES
A series 10-fold dilution rate was prepared from the 10* cfu/ml suspension with no protamine added. A calibration curve relating cfu/ml of the 10-fold dilutions to capaci¬tance or conductance DT in TSB was constructed for each strain using a minimum of 8 dilution steps.
From the protamine treated suspensions, 0.1 ml was ino¬culated in TSB in Bactometer wells and the DT determined. This DT was, converted to a colony count using the call-

bration curve. Thus, colony counts were not made directly on the protamine treated suspensions as protamine caused significant clumping of the bacterial cells.
When no DT was measured, the total well volume was pi¬petted onto agar plates to evaluate whether protamine had a bacteriostatic or bactericidal effect.
Results
The MIC values determined from capacitance or conductance DT of cells growing in TSB are shown in Table 2.
Table 2:
Minimum inhibition concentration (MIC) measured impedi-
metric as a total inhibition after 100 hours at 25*>c.
Strain MIC ()Ltg/ml)
Aeromonas sobria > 4000
Aeromonas salmonicidae 4000
Escherichia coli 0157:H7 1000
Pseudomonas aeruginosa 4000
Pseudomonas fluorescens 3000
Shewanella putrefaciens (4 strains) 500-1000
Vijbrlo anguillarum 1000
Vibrio paraheamolyticus 500-1000
yersinia enterocolitica >4000
Bacillus subtilis 100
Brochotrix thermosphacta 20
Corynebacterium jeikeium 100
Listeria monocytogenes (2 strains) 1000
Staphylococcus aureus (2 strains) 500-1000
The Gram-positive strains were more sensitive to protami¬ne than the Gram-negative. The MIC values determined for Gram-positive strains varied from 20 to 1000 /xg/ml and varied from 500 /xg/ml to more than 4000 nq/inl for the

Gram-negative strains.
Brochotrix thermosphacta was the most sensitive strain, and a protamine concentration of 20 )ug/ml TSB caused a total kill of the inoculum (10^ cfu/ml) , measured by pla¬ting the well volume after 100 h of incubation in the Bactometer. A protamine concentration of 1000 /ug/ml re¬sulted in a 100% lethal effect on the two strains of Li¬steria monocytogenes and Staphylococcus aureus (10^ cfu/ml). The MIC for protamine on S. aureus was 500 )Lig/ml, however, this concentration did not have a lethal effect.
The DT's for Aeromonas sobria and Yersinia enterocolitica increased with increasing protamine concentration, sug¬gesting a prolonged lagphase. However, the cultures were not totally inhibited and no MIC was determined. The DT for a 10^ cfu/ml suspension of A. sobria was 33 h when incubated with 4000 ixq/ml compared to 12 h when no pro¬tamine was added. For Y. enterocolitica the detection time of 10^ cfu/ml was prolonged from 21 h to 60 h when 4000 jLtg/ml of protamine was added, see Figure 1. Aeromo¬nas salmonicidae and Pseudomonas fluorescens were inhibi¬ted by protamine in concentrations of 4000 and 3000 jug/ml TSB, respectively. Thus, no change in conductance was seen after 100 hours incubation, but live cells were iso¬lated from the well. Shewanella putrefaciens (A2), (All) and (A22) and VlJbrlo anguillarum were inhibited by pro¬tamine in the concentration 1000 jug/ml TSB, and ViJbrio paraheamolyticus and S. putrefaciens (A6) were inhibited by 500 jug/ml TSB. S. putrefaciens was the only Gram-nega¬tive bacteria which was killed by protamine, thus, 2000 /ig/ml of protamine killed 100% of the inoculum (lo' cfu/ml) of all the four tested strains.
The bactericidal effect of protamine on non-growing cells was tested on four strains of S. putrefaciens and two

strains of L. monocytogenes. After protamine treatment, detection times were measured and converted to a cell count using the calibration curve generated for the particular strain (Fig. 2). The sensitivity of S. putre¬faciens varied from strain to strain, see Table 3 below (see also table 1 for code and reference for each strain). Strain A2 and All were similar in sensitivity and more resistant than A6 and A22. Thus, 100 ng protamine/ml killed S. putrefaciens strain A6 and A22 when suspended at low cell concentrations (10* and 10^ cfu/ml). The same level was not 100% lethal on S. putrefaciens strain A2 and All, however the cell number was reduced by 90-99.9%.
The initial cell number was estimated by absorbance measurements.

k: No surviving cells (determined by spread plating
the medium from the well, c): Long detection time (DT) corresponding to a very
low cell number (approximately 1 surviving
cell).
Protamine at 100 and 500 jug/ml had no effect on non-growing L. monocytogenes cells, and increasing the pro¬tamine concentration to 1000 /xg/ml did not cause any let¬hal effect of protamine on non-growing L. monocytogenes.
The results show that salmine (salmon protamine) in con¬centrations of 100-4000 /ig/ml prolonged the lag phase of several Gram-negative bacteria significantly. Protamine was more effective on actively growing L. monocytogenes cells as compared to cells suspended in buffer. The bac¬tericidal effect of protamine on Shewanella putrefaciens was seen on both growing and non-growing cells. A pro¬tamine concentration of 2000 /ig/ml was required to kill growing S. putrefaciens (10^ cfu/ml), whereas non-growing cells were killed by only 50 /xg/ml. When the cell con¬centration was raised the bactericidal effect of protami¬ne was decreased, probably as a result of the higher cell/protamine ratio.
The impedimetric method used in this study proved useful for the measurement of the antibacterial activity of a cationic protein which caused cellular agglutination. Excellent correlations exit between detection time and cfu of untreated cells, see Figure 2. The correlation between protamine treated cells and cfu was statistically similar to the correlation for untreated cells (data not shown), however, plating the protamine treated cells cau¬sed a great degree of variation on the cells count. It is demonstrated that protamine inhibits growth of all the tested strains, determined as a prolonged lag phase

for the most resistant bacteria or a lethal effect on a few of the tested strains, the Gram-positive bacteria in particular. The fact that protamine is naturally occur¬ring and non-toxic makes it an antibacterial protein that might hold great promise for the control of e.g. spoilage bacteria and food-borne pathogens.
EXAMPLE 2
Comparison of minimum inhibition concentrations for basic proteins and enzymes
The minimum inhibition concentration (MIC) of various substances was determined as described in Example 1.
The following substances were tested: protamine (A), pro¬tamine sulphate (B), a peroxidase enzyme system (i.e. lactoperoxidase/glucoseoxidase (C)), subtilisin A (D), polyarginine (E) having an average molecular weight of about 6 kD and lysozyme (F) (150 000 units/mg; Johansen, C. et al., 1994). The results are shown in Table 4 below.
It is demonstrated that protamine and protamine sulphate are very effective substances for inhibiting all the te¬sted strains, whereas polyarginine is effective for in¬hibiting all strains but Pseudomonas spp.. Apart from the effect of lysozyme on Listeria monocytogenes, none of the tested enzyme showed any effect.

Table 4:
Comparative minimum inhibition concentrations

Substance Strain Minimum Inhibitory Concentration (Mg/ml)
A B C D E F
Listeria monocytogenes
Staphylococcus aureus
Escherichia coli
Pseudomonas aeruginosa
Pseudomonas fluorescens
Shewanella putrefaciens
Vibrio paraheamolyticus 1000 1000 n.e. n.e. 2000 2000

1000 2000 n.e. n.e. 1500 n.e

1000 n.d. n.e. n.e. 1500 n.e

4000 n.d. n.e. n.e. n.e. n.e

3000 4000 n.e. n.e. n.e. n.e
•

1000 500 n.e. n.e. 1500 n.e

1000 500 n.e. n.e. 2000 n.e
•
n.d.: not done
n.e.: not effective
* The lactoperoxidase system was effective for maximum 70
hours. The definition of MIC require an inhibition of at
least 100 hours.
EXAMPLE 3
The influence of pH and cell concentration on the anti¬bacterial effect of protamine
The influence of pH on the antibacterial effect of pro¬tamine was tested using the materials and methods descri-

bed in Example 1.
The results are shown in Figure 3 and demonstrate clearly that the antibacterial effect of protamine depend signi¬ficantly on pH. At low pH, protamine (1 mg/ml) has no ef¬fect on the Gram-negative bacteria Pseudomonas aeruginosa, however, at high pH protamine (1 mg/ml) pro¬longed the detection time from 32 to 71 hours. The inter¬action between pH and protamine has been observed for all the tested strains.
Further, the influence of cell concentration on the anti¬bacterial effect of protamine was tested using the ma¬terials and methods described in Example 1, i.e.the cor¬relation between cell concentration and protamine concen¬tration has been measured by the impedimetric assay.
The results are shown in Figure 4 and Figure 5 and demon¬strate clearly that a significant synergistic effect bet¬ween the cell concentration and the protamine concentra¬tion has been observed. Thus, at low cell concentration, protamine (1 mg/ml) caused a prolongation of the detection time from 12 to above 100 hours for the Gram-negative bacteria Shewanella putrefaciens (4 strains), compared to a prolongation at high cell concentration from 6 to 18 hours. The detection time for the Gram-po¬sitive bacteria Listeria monocytogenes (2 strains) was at low cell concentration prolonged from 18 to 55 hours when treated with protamine (1 mg/ml), at high cell concentra¬tion the same protamine concentration only prolonged the detection time from 4 to 15 hours.

EXAMPLE 4
Synergistic antimicrobial effect between a basic protein, a cell-wall degrading enzyme and a peroxidase enzyme system
Impedimetric measurements carried out as described in example 1 have shown an synergistic effect between basic peptides as protamine, polyarginine or polylysine and lysozyme and/or glucose oxidase and/or the lactoperoxidase enzyme system depending on pH and NaCl concentration.
Growth inhibition experiments were conducted, wherein synergistic and additional effects were determined by mixing compounds in low concentrations not having any activity on their own and using a factorial design. The effects were measured as growth inhibition or a 100% bactericidal effect with a total kill of the inoculum.
Protamine (250 jug/ml) or poly lysine (500 /xg/ml) in combi¬nation with lactoperoxidase (2 U/ml) and glucose oxidase (2 U/ml) had a 100% lethal effect on Pseudomonas fluorescens, whereas the same strain was not inhibited when treated with any of these three compounds alone in the concentrations mentioned above.
A synergistic effect was as observed against Pseudomonas fluorescens when combining protamine (250 ng/mil) and poly lysine (500 /xg/ml) and lysozyme (50000 U/ml) and lactoperoxidase (2 U/ml) and glucose oxidase (1 U/ml) or lysozyme (50.000 U/ml) and poly lysine (500 jig/ml) and lactoperoxidase (2 U/ml) and glucose oxidase (1 U/ml).
Experiments where the antibacterial effect was measured as growth inhibition of Shewanella putrefaciens in TSB at

25 00 and pH 7.2, showed a synergistic effect between protamine and lysozyme; the results are shown in Figure 6 as a response surface plot. Lysozyme alone had no effect on the Gram-negative bacteria Shewanella putrefaciens neither had protamine at concentrations below 500 jug/ml, whereas combinations caused a 100% bactericidal activity at protamine concentrations above 300 /xg/ml and lysozyme concentrations from iC-lO* U/ml (lysozyme activity: 150000 U/mg).
EXAMPLE 5
Fungistatic and fungicidal activities
This experiment was carried out as described in example 1 using the Bactometer substrate: 0.75 g Yeast extract (Difco) , 3.0 g D(+)Glucose, 1 g KH2PO4, 0.8 g isogel agarose lEF (Pharmacia) and 100 ml distilled water.
Protamine was added to the substrate immediately before inoculation with a spore suspension of the test fungi (approximately lO^-lO* cfu/ml) .
The minimum inhibitory concentration was determined as the lowest concentration of protamine resulting in a absence of DT during 100 h measurement (see table 5 below). When no DT was determined a fungicidal activity was evaluated by plating from a dilution of the total well volume.

Table 5

Strain

pH

MIC (Mg/ml)

Alternaria infectoria Aspergillus niger Botrytis aclada Cladosporium herbarum Eurotium repens Fusarium culmorum Penicillium comcam Penicillium crustosum Penicillium roqueforti Ulocladium atrum

5.2 240
7.1 1000
5.2 120
5.2 120
5.2 240
5.2 240
6.1 1000
6.2 1000
5.2 240
5.2 240

The fungistatic and fungicidal effect of protamine was optimal at high pH and low inoculum size as the effect on bacteria. Increasing the pH caused a significant decrease in the MIC-value. A fungistatic effect was obtained with an 2-5 fold lower protamine concentration than used when a fungicidal effect was determined. The most resistant strains shown in table 5 were not inhibited by protamine for 100 h at low pH, thus a 100 h inhibition was not obtained before increasing the pH to the values given in the table.
EXAMPLE 6
Survival and transfer of bacteria during mini-wash
Materials:
Ariel Color (DF-9412330).
Swatches (white cotton, DF-9415585), sterilized by

autoclaving.
Tryptone Soya Broth (TSB).
Sterilized water (12°dH).
Strains:
Staphylococcus aureus (skin isolate)
Pseudomonas aeruginosa (skin isolate)
Methods:
Inoculum:
S. aureus and P. aeruginosa were grown in Tryptone Soya Broth (TSB) at 25'C for 30 hours. For each strain six sterile swatches were inoculated with approximately 10* cfu/swatch and air dried for 30 minutes.
Mini-wash:
0.56 g of Ariel Color was dissolved in 80 ml sterile water (12"dH, 35°C) in each wash beaker, giving the final concentration of 7 g/1. Protamine, dissolved in water and filter sterilized, were added to half of the wash beakers giving the final concentration of 500 jLtg/ml.
After 70 sec. 1 inoculated swatch and 2 sterile swatches were transferred to each wash beaker, and washed at 35°C for 15 min. In one beaker 3 sterile swatches were washed as control.
From each wash beaker 0.1 ml of detergent solution were transferred to Malthus (in-direct cells) con¬taining TSB and incubated.
The swatches were rinsed in sterile water for 10 min (stirring). From each beaker 0.1 ml of rinse water

were incubated in Malthus (in-direct cells).
After wash all swatches were slightly air dried in sterile air for 10 min, and each swatch was trans¬ferred to an in-direct Malthus cell.
All materials and instruments except the detergent, were sterilized before use to avoid contamination.
In-direct Malthus:
Indirect-Malthus measurements were used when estimat¬ing the number of viable cells.
3 ml of TSB were transferred to the outer chamber of the in-direct Malthus cells, and 0.5 ml of sterile KOH (0.1 M) were transferred to the inner chamber. As cells are growing in the outer chamber they produce CO2 (g) which will dissolve in the KOH in the inner chamber and thereby change the conductance of the KOH. When the conductance change is measurable by the Malthus, a detection time (DT) will be recorded. The DT's were converted to colony counts by use of a calibration curve relating cfu/ml to DT (see Figure 1 and 2).
A series 10-fold dilution rate was prepared from the 10* cfu/ml suspension of cells. Conductance DT of each dilution step was determined in TSB, and a cali¬bration curve relating cfu/ml of the 10 fold dilu¬tions to DT in TSB was constructed for each strain (see Figure 1 and 2).
Results:
The number of cells surviving mini-wash, in the detergent solution, attached to the contaminated swatches, trans¬ferred to the rinse water or to the sterile swatches, were determined by in-direct Malthus (table 6).

A relatively high number of cells were washed of the swatches and found in the wash water, however, approxima¬tely 10^ cfu were still attached to the swatches after mini-wash and rinsing for 10 min, and during wash cells were transferred from the contaminated swatch to the sterile swatches. The S. aureus cells were found very sensitive to protamine, which can be explained by the increased antibacterial activity of protamine at high pH. A high number of P. aeruginosa were determined in the detergent solution, and protamine was found active against the cells in the detergent solution, however, P. aeruginosa attached to the swatches were not inhibited or killed by protamine, and all the sterile swatches in the washes inoculated with P. aeruginosa were contaminated during wash.
The rinse water were almost sterile, thus the cells may adhere actively to the textile.
Table 6:
Cell number in the detergent solution, the rinse water, attached to the contaminated swatches and transferred to sterile swatches, before and after mini-wash at 35°C for 15 min with Ariel Color. Each wash were done in triplets, A, B and C are the three wash beakers with the same combinations of strain and protamine.

staphylococcus aureus (pg/ml)
0 500

Pseudomonas aeruginosa
(/jg/ml)
0 500

Before wash
(cfu/swatch)
contaminated swatch
A 1.6*10* 1 6*10* 4.0*10* 4. 0*10'
B 1.6*10' 1. 6*10' 4.0*10' 4. 0*10'
C 1.6*10' 1 6*10* 4.0*10' 4. 0*10'
sterile swatch 1
A 0 0 0 0
B 0 0 0 0
C 0 0 0 0
sterile swatch 2
A 0 0 0 0
B 0 0 0 0
C 0 0 0 0
After mini-wash

(cfu/swatch)
contaminated swatch
A 1.7*10' 0 8.0*10' 2 5*10'
B 3.0*10' 0 1.2*10^ 6 0*10'
C 2.9*10^ 0 6.0*10' 1 3*10'
swatch 1
A 1 0 4.0*10^ 1
B 0 0 5.0*10' 1
C 2.8*10^ 0 1 1
swatch 2
A 6.1*10^ 0 7.0*10^ 1
B 1 0 1 1
C 0 0 3.0*10' 1

Detergent solution
(cfu/ml)
total volume; 80 ml
A 2.0*10^ 1 - (a) 0
B 6.3*10' 0 5.8*10^ 0
0 2.0*10' 0 1.9*10" 0
Rinse water (cfu/ml)

total volume;100 ml
A 0 0 0 1
B 0 0 0 1
0 0 0 1 0
(a): No DT determined by Malthus, growth in the Malthus cell was observed by eye.
From the results it can be concluded that Ariel Color has no significant bactericidal effect on S. aureus and P. aeruginosa (pathogenic skin isolates).
A high number of cells were washed of the swatches, and found in the detergent solution, and when no protamine were added, cells from the inoculated swatch contaminated the sterile swatches in the wash beaker.

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WE CLAIM:
1. A bactericidal, bacteriostatic, fungicidal and/or fungistatic composition comprising or consisting essentially of a basic proteta^or peptide selected from protamines, protamine sulphates, and polylysins and polyarginins and co-polymers thereof, capable of killing microbial cells, in combination with a chitinase.
2. The composition according to claim 1, which further comprises an oxidoreductase.
3. The composition according to claim 2, wherein the oxidoreductase is selected from the group consisting of oxidases (EC 1.10.3) and peroxidases (EC 1.11.1), preferably from peroxidase enzyme systems (EC 1.11.1.7) and laccase enzymes (EC 1.10.3.2).
4. The composition according to claim 3, wherein the peroxidase enzyme system comprises at least one peroxidase enzyme and a hydrogen peroxide generating enzyme system such as an oxidase and a substrate for the oxidase or an amino acid oxidase and a suitable amino acid, or a peroxycarboxylic acid or a salt thereof
5. A cleaning or detergent composition comprising a basic protein or peptide selected from protamines, protamine sulphates, and polylysins and polyarginins and co¬polymers thereof, capable of killing microbial cells, and a chitinase and a surfactant.
6. The composition according to claim 5, which further comprises an oxidoreductase.
7. The composition according to claim 6, wherein the oxidoreductase is selected from peroxidase enzyme systems (EC 1.11.1.7) and laccase enzymes (EC 1.10.3.2).

8. The composition according to claim 7, wherein the peroxidase system comprises at least one peroxidase enzyme and a hydrogen peroxide generating enzyme system such as an oxidase and a substrate for the oxidase or an amino acid oxidase and a suitable amino acid, or a peroxycarboxylic acid or a salt thereof.
9. The composition according to any of the claims 1-8, which further comprises at least one enzyme selected from the group consisting of proteases, amylases, cellulases, and lipases.

10. The composition according to any of the claims 1-9, wherein the surfactant is a detergent surfactant, preferably selected from the group consisting of anionic, nonionic, ampholytic, zwitterionic and cationic surfactants.
11. The composition according to any of the claims 14-18, wherein the basic protein is a protamine or a protamine sulphate in an amount corresponding to between 1 and 4000 mg per 1 cleaning liquor or washing liquor.
12. A method for killing microbial cells present on a hard surface comprising
contacting the surface with a cleaning composition according to any of the claims 5-
11 or a composition according to any of the claims 1-4.
13. A method for killing microbial cells or inhibiting growing microbial cells present
on laundry comprising contacting the laundry with a detergent composition according
to any of the claims 5-11 or a composition according to any of the claims 1-4.

14. A method for preservation of food, beverages, cosmetics, contact lens products, food ingredients or enzyme compositions comprising incorporating into the unpreserved food, beverages, cosmetics, contact lens products, food ingredients or enzyme compositions a basic protein or basic peptide or a composition according to any of the claims 1-4 in an amount effective for inhibiting growing microbial cells.

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Patent Number

225571

Indian Patent Application Number

1245/MAS/1995

PG Journal Number

52/2008

Publication Date

26-Dec-2008

Grant Date

19-Nov-2008

Date of Filing

26-Sep-1995

Name of Patentee

NOVOZYMES A/S

Applicant Address

KROGSHOJVEJ 36 DK-2880 BAGSVAERD,

Inventors:

#	Inventor's Name	Inventor's Address
1	CHARLOTTE JOHANSEN	C/O NOVO NORDISK A/S, NOVO ALLE DK-2880 BAGSVEARD,

PCT International Classification Number

AOIN63/00

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA