TITLE

Antimicrobial peptides and bacterial strains that produce them.

FIELD OF THE INVENTION The present invention relates generally to the field of health promoting agents, in particular antimicrobial agents and provides antimicrobial peptides and bacterial strains that provide the antimicrobial peptides.

BACKGROUND TO THE INVENTION Significant health problems can occur in both humans and animals with infections by pathogenic bacteria. In particular, infections by pathogenic strains such as E. coli, E. sakazakii, Streptococcus mutans and Listeria innocua can be extremely debilitating. It has been recognized that there exists a need to provide antimicrobial agents to pathogenic bacteria.

Increasing resistance of pathogenic bacteria to antibiotics is a major problem and the search for novel drugs continues (35). Multi-drug resistant E. coli, Klebsiella spp., as well as strains of E. sakazakii have increased at an alarming rate and are frequently associated with immunocompromised hosts for whom they may have devastating effects (6, 14). Enterobacter species are recognised as increasingly important pathogens in recent years due to their innate resistance to older antimicrobial agents and their increased association with nosocomial infections (41). For over three decades, E. sakazakii has been recognised as the cause of a distinctive syndrome of meningitis in neonates (20). A mortality rate of 40-80% has been reported and there is increased compelling evidence ((20), (33), (34)) that milk based infant formulas serve as a reservoir of infection. Current industry efforts to control E. sakazakii infection have focused on improving manufacturing hygiene practices and end product testing for the organism (34). One possible route to identifying candidate novel antimicrobial peptides is in the investigation of milk proteins.

Milk proteins are a rich source of bioactive peptides, reported to have a range of health- promoting properties including angiotensin-I converting enzyme (ACE) inhibition, opioid,

immunomodulation, antithrombosis, and antimicrobial activity (9). Bioactive peptides are present in milk proteins such as casein and whey in an encrypted form, stored as propeptides or mature C-terminal peptides (13, 19) with proteolysis required for their release (11). The best characterised of these functional peptides are derived from casein and have been shown to have effects in the cardiovascular system, mainly via ACE inhibition and consequently have antihypertensive features (43). Maruyama and Suzuki (28) reported that tryptic hydrolysates of casein inhibited the in vitro activity of ACE. Pihlanto-Leppala et al. (37) studied the ACE-inhibitory activity of peptides from whey and casein proteins fermented with lactic acid bacteria (LAB) and hydrolysed afterwards with digestive enzymes. Peptides identified were <x _sl -casein f(142-147), f(194-199) and β-casein f(108-l 13) as well as two ACE-inhibitory peptides from whey (37). hi addition, a range of antibacterial peptides have been isolated from bovine α _s2 -casein (23). These include: Casocidin-1, f(164-179): KTKLTEEEKN RLNFLKKISQ RYQKFALPQY LKTVYQHQK, f(164-179): KKTKLTEEEKNRLNFL, and f(l 87-207): QKFALPQ YLK TVYQHQKAMK Q.

These three peptides are derived from treating bovine milk with acetic acid and calcium sulphate to form a mixture which is then heated to form a precipitate and a supernatant. The supernatant is removed and treated with a cation-exchange resin. The peptides are eluted using chromatographic procedures and the active peptide casocidin-1 is released using digestive proteases. The cost of production is one possible limitation to the widespread application of these three peptides, hi particular, Casocidin-1 has been shown to be bactericidal against Escherichia coli and Staphylococcus carnosus in vitro (51).

However, no known bacterial strain produces casocidin-I directly, meaning that production of casocidin-I requires the use of enzymes and chemicals, which add to the cost of production. Further information can be found in US 6,579,849 entitled "Antibiotic peptides from bovine milk". No limitations on the activity of these peptides are mentioned in the above patent but Zucht et al, (FEBS Letters 372 (1995) 185-188), describes the spectrum of inhibition of these peptides and it appears that only Gram-negative bacteria are affected by these peptides, meaning that Gram-positive infections and contamination could not be treated with the peptides of US 6,579,849.

Proteinases of microbial origin, including LAB, by their specificity and activities play a primary role in the generation of peptides from casein and potentially the release of antimicrobial peptides (29). Consequently, during fermentation the microorganisms producing these proteinases must be able to degrade milk proteins such as casein and whey in order to grow in milk. Casein degradation and subsequent utilization of the degradation products by LAB requires a complex proteolytic system (9). Due to the highly proteolytic nature of LAB such as Lactococcus lactis (22, 38) and Lactobacillus helveticus (27, 50), and their need to degrade milk proteins for growth in milk, their use as starter cultures for the generation of bioactive peptides has been reported (12, 25, 32, 45). Miniervini et al. (30) used a proteinase from Lactobacillus helveticus PR4 to produce bioactive peptides exhibiting antimicrobial activity from sodium caseinates of the milk from six species of mammals. An antimicrobial peptide corresponding to human β-casein f( 184-210) was produced by hydrolysis of human sodium caseinate with a partially purified proteinase of L. helveticus PR4 and showed antimicrobial activity against several Gram-positive bacteria and also against E. coli (30). Characterization of the peptides produced during casein degradation has been described for L. helveticus (11, 49) and to a lesser extent for L. casei (7). Also, the cell wall bound proteinase of L. delbrueckii subsp. lactis ACA-DC 178 has been characterised and its specificity for β- casein has been documented (45).

Few casein derived antibacterial peptides have been reported and those that have been described result from the action of enzymes on the mature C-terminal peptide, leading to release of the active peptide sequence (43). The first antimicrobial peptides of casein origin were identified by Hill et al. (17) who isolated antibacterial glycopeptides, known as casecidins, following proteolysis of casein with chymosin at pH 6.4. Isracidin, a positively charged antimicrobial peptide, α _sl -casein peptide residue 1-23 with the primary amino acid structure R ₁ PKHPIKHQGLPQEVLNENLLRF ₂₃ (17), was shown to have a broad spectrum of activity against both Gram-positive and Gram-negative bacteria (23) and to prevent mastitic infections in sheep and cows (12). There is also a persistent problem in that pathogenic bacteria over time develop resistance to existing antimicrobial compounds. There is therefore an ongoing need for the development of

new antimicrobial peptides in order to continually meet the challenge presented by resistant strains. In some situations, novel antimicrobial compounds may require more onerous application methodologies, such as increased concentration, increased number of applications, or may only be efficacious against a narrower range of target species. Even in these circumstances, the novel antimicrobial is still advantageous over the existing compounds, due to the problem of increased resistance. Furthermore, it would be of great benefit to be able to provide a stain of bacteria to produce such antimicrobial peptides, as this can firstly offer an economical method of producing the peptide and secondly that can provide a means to produce the peptides in situ situations.

OBJECT OF THE INVENTION

One of the objects of the present invention is to provide a novel stain of bacteria that produces at least one antimicrobial peptide. It is a further object of the invention to provide at least one antimicrobial peptide. A further object is to provide an antimicrobial peptide for use against multiple drug resistant organisms. It is also an object of the invention to provide a foodstuff with improved safety containing antimicrobial agents. It is a further object of the invention to provide a method of ACE inhibition. It is also an object of the invention to provide a milk formula with improved antimicrobial properties.

SUMMARY OF THE INVENTION

In one embodiment, the invention provides an antimicrobial compound comprising one or more peptides selected from the group consisting of SEQ ID NO: 1 (IKHQHPQE) ₅ SEQ ID NO: 2 (SDIPNPIGSENSEK), and SEQ ID NO: 3 (VLNENLLR). In an alternative embodiment, the invention provides a biologically pure culture of Lactobacillus acidophilus , strain DPC6026, a sample of which has been deposited at the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland on 18 November 2005 under the accession number NCIMB 41354, or a derivative or mutant thereof capable of producing from milk or a milk product, peptides having antimicrobial activity.

The invention also provides for proteins produced by DPC 6026. In particular, the invention provides for novel protyolitic enzymatic proteins produced by DPC6026 that are capable of

producing one or more of peptides capable of producing anti-microbial peptides from milk.

These peptides may be those with sequences substantially equal to one or more of the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 from milk or milk products.

The invention also provides for a method of producing an anti-microbial peptide from milk or milk products comprising adding to milk or a milk product, one or more of the group consisting of a biologically pure culture of Lactobacillus acidophilus strain DPC6026, or a derivative or mutant thereof capable of producing a from milk or a milk product, peptides having antimicrobial activity, a cell-free culture supernatant which is obtained from the DPC6026 culture, or a fraction thereof, and a protein produced by DPC6026, wherein the protein is capable of producing one or antimicrobial peptides from milk or milk products.

The invention also provides for a method of producing one or more proteins from DCP6026 that have proteolytic, catalytic and/or enzymatic properties that are capable of producing at least one of SEQ ID NO: 1, SEQ, ID NO: 2 or SEQ ID NO: 3 from milk or milk products.

An antimicrobial composition comprising one or more selected from the group consisting of an antimicrobial compound as claimed in claim 1, a culture as claimed in claim 2, a supernatant as claimed in claim 3 and a protein as claimed in claim 4.

The invention also provides a cell-free culture supernatant, or a fraction thereof, which is obtained from strain DPC6026, or an antimicrobial composition comprising one or more of the peptides, compounds, supernatant or strain as described above, together with a suitable carrier or diluent.

The invention also provides for the use of the compounds, peptides, strain, proteins, supernatant or composition as described above in the control of microbial infections or microbial contamination. Aspects of the invention provide methods of preventing, or treating or limiting microbial infection or microbial contamination comprising administering one or more of the compounds strain, supernatant or composition of the invention to an animal or human. The microbial infection or microbial contamination may be a contamination or

infection caused by one or more of the group consisting of Escherichia coli, E. sakazakii, Streptococcus mutans, Listeria innocua, Klebsiella spp and Staphylococcus carnosus. In particular, the infection or contamination may be mastitis.

In alternative embodiments, the infection or contamination maybe meningitis, and may be meningitis in neonates. In some embodiments the compound, peptides, strain, supernatant or composition may be provided in a milk-based formula. Thus, the peptides of the invention can be used as an in built protection system in the manufacture of infant formula products. One or more of the peptides may be particularly efficacious against Enterobacter Sakazakii Contaiminant.

Due to the diverse nature of bioactive peptides in terms of structure, spectrum of activity and potency (39) these peptides are prime targets for potential new drug design. The possibility of using antimicrobial peptides such as SEQ ID NO: 1. (IKHQGLPQE) and SEQ ID NO: 3 (VLNENLLR) derived from milk proteins as an in-built mechanism of protection against pathogenic strains such as E. sakazakii 5920fATCC12868) may provide a useful approach for enhancing the safety of milk powders, such as those used in infant formula manufacture.

The invention also provides methods of inhibition of angiotensin-I converting enzyme (ACE), opioid modulation, immunomodulation, and antithrombosis, comprising treating the candidate animal or human with one or more of the compositions, compounds, supernatants, peptides, or strains of the invention.

The peptides, strains, compounds, compositions and supematants of the invention can also be used in the treatment of bovine and ovine mastitis. SEQ ID NO: 1 and SEQ ID NO: 3 are similar to cleavage products of the antimicrobial peptide isracidin. SEQ ID NO: 2 may also be used. Isracidin has been documented as having a strong protective effect against S. aureus, Streptococcus pyogenes and Z. monocytogenes when administered at doses as low as 10 μg per mouse and has also been used previously in the treatment of ovine and bovine mastitis.

The peptides, strains, compounds, compositions and supernatants of the invention can be used to contribute bitter flavours in the manufacture of bitter cheese flavours (Pederson et at, 1999, J. Bacteriology 181,4592-4597) as the first 9 residues of α _sl -casein accumulate in cheese making and are responsible for the bitter flavours attributed to some cheeses (Fox et at, 1995, Chemistry of Structure-Function Relationships in Cheese (Malin, E. L., and Tunick, M. H., eds), Plenum Press, New York, pp 59-98).

The peptides, strains, compounds, compositions and supernatants of the invention can be used as preservatives of food or perishable goods against pathogenic Gram-negative and Gram- Positive bacteria due to the broad spectrum of activity of SEQ ID NO; 1 , SEQ ID NO;3 and SEQ ID NO: 2 as described.

L. acidophilus DPC6026 is a novel strain, isolated from the porcine small intestine and stocked in the Dairy Products Research Centre (DPRC), Teagasc Moorepark, Fermoy, Co. Cork. No bacterial strain has been documented showing the production of isracidin or its fragments previously. Isracidin was derived from α _sl -casein treated with chymosin (Hill et al. J of Dairy research 1974; 41 : 147). This is advantageous economically as L. acidophilus DPC6026 in its natural state produces the three peptides IKHQGLPQE, VLNENLLR and SDϊPIGSENSEK without the need for enzymes, avoiding additional expense to the fermentation process at an industrial level. The peptide sequence of Isracidin is more expensive than SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 to chemically synthesis due to its longer chain length SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 each have shorter chain lengths and are therefore cheaper to chemically synthesis.

Isracidin α _sl .casein f(l-23) appears to maintain a relatively stable conformation in solution (Malin et at, Journal of Protein Chemistry, Vol. 20, 391-404). Conformationally stable peptides are known to be characteristic of allergens and this segment of α _s i.casein is known to be allergenic (Spuergin et al., 1996. Allergy 51, 306-312). The region of α _sl -casein surrounding residue 20 has been suggested to contain a significant allergenic epitope recognised by human IgE (Spuergin et at, 1996). In addition, SEQ ID NO: 3 may not present

an allergenic epitope as it lacks residue 20 of α _sl -casein, which is thought to contain a significant allergenic epitope recognised by human IgE (Spuergin et al., 1996).

Penetration in vivo. Isracidin has been shown to display a protective effect against Staphylococcus carnosus in vivo. SEQ ID NO: 1 and SEQ ID NO: 3 display a greater protective effect in vivo against pathogenic mastitis causing strains. It is hypothesised that the reason for this is that the shorter bioactive peptides display a better in vivo effect (Vermeirssen et al., British Journal of Nutrition (2004), 92, 357-366).

ct _s i-casein f(l-9) (RPKHPIKHQ) has been shown to possess antihypertensive effect in the Spontaneously Hypertensive Rat (SHR) (Saito et al, 2000, J. Dairy Science, 83, 1434-1440). SEQ ID NO: 1 shares 100% homology with the last 4 amino acids (IKHQ). It also possesses proline, glutamine and glutamatic acid at the C-terminal end of the peptide. ACE activity is drastically slowed down by the presence of proline in the C-terminal tripeptides region

(Vermeirssen et al., 2004, British Journal of Nutrition, 92, 357-366). This evidence would suggest that SEQ ID NO: 1 could possess ACE inhibitory activity and suggests a possible advantage of cleaving isracidin into two shorter peptides.

It is also hypothesised that under certain circumstances, the mechanism for Isracidin resistance mediated by an 'antigenic' region of the molecule not presented by either SEQ ID NO: l or SEQ ID NO: 3.

Other cationic antimicrobial peptides such as Lactoferricin B are known to have resistant strains such as Escherichia coli and Staphylococcus aureus due to protease production and this has been suggest as the main resistance mechanism against other cationic antimicrobial peptides such as magainins by E. coli and Staphylococcus aureus (Ulvatne et al., Journal of Antimicrobial Chemotherapy (2003) 50, 461-467). Cleaving isracidin into two of the peptides of the invention, SEQ ID NO: 1 and SEQ ID NO: 3, is likely to reduce the possibility of resistant strain development as fewer proteases can cleave these shorter sequences (as demonstrated using the computer program http://ca.expasy.org/cgi- bin/peptidecutter/peptidecutter.pl compared to isracidin.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 : PFGE macrorestriction patterns for the restriction enzyme Apa I. Pulse time was 1 to 15 s for the gel shown. Lane 1 contained low range PFG marker (A mixture of lambda DNA-Hind III fragments and lambda concatemers embedded in 1% LMP agarose), lanes 2 to 6, Lactobacillus acidophilus DPC6026, L. johnsonii DPC6092, L. salivarius DPC6027, L. animalis DPC6028 and L. delbrueckii sp. bulgaricus DPC6104.

Figure 2: Proteolytic activity of GRAS strains. High Performance Liquid Chromatography (HPLC) peptide profile of bovine sodium caseinate (A), and of its sodium caseinate hydrolysates after fermentation with Lactobacillus johnsonii DPC6092 (B), L. salivarius DPC6027 (C) and L. acidophilus DPC6026 (D). HPLC was monitored at 214nm for 24 h. Sample 30 μl was loaded onto a Delta-Pak C-18 (600mm X 7.5 cm), flow rate of lml/min. Proteolysis was carried out at room temperature. L. acidophilus DPC6026 showed almost complete breakdown of sodium caseinate (D).

Figure 3

A. Reverse Phase-High Performance Liquid chromatography (RP-HPLC) chromatogram of sodium caseinate at pH 7 incubated with L. acidophilus DPC6026 for 24 h. Arrows indicate position of peptide fractions Al -45 and A 1-54. RP-HPLC was carried out at room temperature and following the conditions shown in the materials and methods section.

B. Matrix Assisted Laser Desorption Ionisation-Time of Flight (MALDI-TOF) spectrum of fraction A 1-45 (isolated using sodium caseinate as tfie substrate and the strain L. acidophilus DPC6026) recorded in the m/z region where peptides were detected. Peptide fraction Al -45 was found to contain a peptide of mass 1049.43, which was found after sequencing to be IKHQGLPQE, which corresponds to α _sl -CN f (21-29).

1. Inhibition of E. coli DPC6053 by the chemically synthesised peptide IKHQGLPQE identified in fraction A 1-45 of the sodium caseinate hydrolysate with L. acidophilus

DPC6026. A zone 2.5cm in diameter was recorded using the agar well diffusion assay method.

2. Inhibition oϊ Enter obacter sakazakii5920 (ATCC128682) by the chemically synthesised peptide IKHQGLPQE identified in fraction Al-45 of the sodium caseinate hydrolysate with L. acidophilus DPC6026. A zone 2.0cm in diameter was recorded using the agar well diffusion assay method.

C. Matrix Assisted Laser Desorption Ionisation-Time of Flight (MALDI-TOF) spectrum of fraction Al-54 (isolated using sodium caseinate as the substrate and the strain L. acidophilus DPC6026) recorded in the m/z region where peptides were detected. Peptide fraction Al-54 was found to contain a peptide of mass 970.119, which was found after sequencing to be VLNENLLR corresponding to α _sl -CN f (30-37).

1. Inhibition of E. coli DPC6053 by the chemically synthesised peptide VLNENLLR identified in fraction Al-54 of the sodium caseinate hydrolysate with L. acidophilus DPC6026. A zone 1.5cm in diameter was recorded using the agar well diffusion assay method.

2. Inhibition of Enterobacter sakazaMiω.12 (NCTC8155) by the chemically synthesised peptide VLNENLLR identified in fraction Al-54 of the sodium caseinate hydrolysate with L. acidophilus DPC6026. A zone 2.2cm in diameter was recorded using the agar well diffusion assay method.

Figure 4: A. Effect of isracidin at various concentrations on the OD ₆ oo of E. coli DPC6053 (an overnight culture diluted 1:10 with LB broth) using the 96-well plate assay. Cecropin Pl is used as a control peptide.

Symbols: ■, E. coli DPC6053 incubated with Cecropin Pl (0.52mM).

- , E. coli DPC6053 control with no additions. ♦, E. coli DPC6053 incubated with isracidin (1.9mM). x, sterility control (LB broth with no additions).

#, E. coli DPC6053 incubated with isracidin at a concentration of 0.23mM. X ₃ viability of E. coli DPC6053 incubated with isracidin (0.059mM).

B. Effect of peptide IKHQGLPQE at various concentrations on the OD ₆ oo of E. coli DPC6053 (an overnight culture diluted 1:10 with LB broth) using the 96-well plate assay.

Symbols: - , E. coli DPC6053 with no additions. X ₃ E. coli DPC6053 incubated with IKHQGLPQE (0.078mM). m, E. coli DPC6053 incubated with Cecropin Pl (0.52mM). •, E. coli DPC6053 incubated with IKHQGLPQE (0.15mM). •, E. coli DPC6053 incubated with IKHQGLPQE (0.3 ImM). ♦, E. coli DPC6053 incubated with IKHQGLPQE (0.625mM). x, sterility control (LB broth with no additions).

C. Effect of peptide VLNENLLR at various concentrations on OD ₆₀₀ of E. coli DPC6053 ( an overnight culture diluted 1:10 with LB broth) using the 96-well plate assay.

Symbols: X ₃ E. coli DPC6053 with no additions. ■. E. coli DPC6053 incubated with VLNENLLR (0.22mM). - , E. coli DPC6053 incubated with VLNENLLR (0.45mM). ♦ ₃ E. coli DPC6053 incubated with VLNENLLR (1.2mM). X ₃ viability of E. coli DPC6053 incubated with Cecropin Pl (0.52mM). •, E. coli DPC6053 incubated with VLNENLLR (2.5mM). A ₃ Sterility control - LB broth without additions.

MATERIALS AND METHODS Substrate and chemicals

Peptides Indolicidin and Cecropin Pl and the enzyme Proteinase K were purchased from Sigma (Sigma Aldrich Chemie, Steinheim, Germany). Pimaricin was purchased from Merck (Merck, Darmstadt, Germany). The peptide Isracidin was synthesised by Peptide Protein Research Ltd., (Fareham, UK.). Maximum Recovery Diluent (MRD) was purchased from Oxoid (Oxoid Ltd., Basingstoke, England). Bovine sodium caseinate was from Dairygold

(Mitchelstown, Cork, Ireland). Pulsed Field Certified agarose was from Bio-Rad Laboratories

(Hercules, CA94547, USA). Enzyme Apa I and the Low Range PFG Marker (NO350S) were purchased from New England Biolabs (Hertfordshire SG4 OTY, England). Low melting point agarose was purchased from Bio-Rad Laboratories (Richmond, California).

Microorganisms and culture conditions

L. acidophilus DPC6026, L.johnsonii DPC6092 and L. salivarius DPC6027 were isolated from the porcine small intestine (data unpublished) and stocked in the culture collection of Teagasc Dairy Products Research Centre, Cork, Ireland. These strains were propagated in MRS broth (Oxoid Ltd, Basingstoke, United Kingdom) anaerobically for 24 h at 37°C. Standard cultures were prepared by inoculation of 10 ml MRS broth with 10 μl of a frozen stock (-80 ⁰ C) and then incubated at 37°C for 16-24 h. L. innocua DPC3306, E. sakazakii 5920 (ATCC 12868), E. sakazakii 8272 (NCTC8155) purchased from the NCIMB (National Collection of Industrial and Marine Bacteria, Aberdeen AB24 3RY, Scotland), and E. coli DPC6053 (Teagasc Dairy Products Research Centre, Cork, Ireland) were employed as the test strains.

Initial screening for proteolytic activity on skim milk agar (SMA) plates Two hundred and five bacterial isolates of porcine intestinal origin and 55 isolates of human adult and infant faecal origin (data unpublished) were screened for proteolytic activity using SMA plates as previously described (36). Clearing zones surrounding the spotted culture was indicative of protein degradation. Isolates producing zones >4.0 mm in diameter were selected for further study.

High performance liquid chromatography (HPLC) Isolates showing proteolytic activity using SMA plates were inoculated (1% w/v) in a sodium caseinate solution (2.5% w/v) and incubated overnight anaerobically at 37°C. High Performance Liquid Chromatography using Delta -Pak Cl 8 Column, (size; 600mm X 7.5cm, Varian Chromatography Systems, Walnut creek, California, USA) was then performed on the resultant fermentates. The mobile phase was a binary mixture of acetonitrile and HPLC grade water (30% v/v) containing trifluorpacetic acid (0.1 % v/v). The flow rate was 1 ml/min.

Casein breakdown by both porcine intestinal isolates and infant faecal isolates was monitored by measurement of UV absorbance at 214nm using a HPl 100 diode array detector.

Pulse-field gel electrophoresis Preparation of high-molecular weight DNA from each of the 15 isolates from MRS broth cultures was as previously described by Simpson et al. (42) except that 20 U of the restriction enzyme Apa I was used for the restriction digest of the plug slices.

16S rDNA sequencing of isolates to speciate strains Fifteen isolates of pig intestinal and infant human and adult faeces, were shown to exhibit significant proteolytic activity and were subjected to 16s ribosomal DNA (rDNA) sequencing (Lark Technologies Inc., Essex, UK) to speciate the individual strains. Two 16S rDNA primers - COl for the 5' end (5'-AGTTTGATCCTGGCTCAG-S') and CO2 for the 3' end (5'-TACCTTGTTACGACT-S') -were used to generate an approximate 1.5kb 16S rDNA product under Polymerase Chain Reaction (PCR) conditions described previously (42). This PCR product was partially sequenced using the COl primer (Lark). Comparison of the 16S rDNA sequences obtained by using the BLAST program allowed the assignment of a strain to a particular species. Generally, when 16S rDNA sequence similarity values exceed 97% using the BLAST program, the strains are considered to belong to the same species (44).

Large scale fermentation with GRAS strains

Of the fifteen isolates exhibiting proteolytic activity, three genetically distinct strains, Lactobacillus acidophilus DPC6026, L. salivarius DPC6027 and L. johnsonii DPC6092 were used individually in further fermentations on the basis of their proteolytic capabilities and as they were all generally recognised as safe (GRAS). Three separate fermentations were performed in triplicate. The sodium caseinate substrate (2.5% w/v) was inoculated with each strain (1 % w/v) and incubated at 37°C for 24 h with mixing at 100 rpm at constant pH 7 maintained via addition of 0. IM NaOH. The fermentates were then heated to 8O ⁰ C to inactivate the cultures and subsequently filtered through a size-exclusion SlYlO 10 kDa spiral cartridge filter (Millipore Ltd., Hertfordshire, UK), to separate the peptides less than 1OkDa. Fractions containing these peptides were freeze- dried and stored at -20°C until further use.

Reverse-phase HPLC analysis of fermentates

Peptides <10kDa in size were separated from sodium caseinate hydrolysates using an RP- HPLC reverse-phase high performance liquid chromatography system containing a narrow- bore column (Nucleosil C 18, 5mm X 250mm: Varian Chromatography Systems, Walnut creek, California, USA) and an UV detector operating at 214nm. Aliquots of the freeze-dried powders were diluted in distilled HPLC-grade water and filtered through a 0.45 μm filter (Millipore) and 30mg/ml of the fermentate loaded onto the column. The mobile phase was a binary mixture of acetonitrile and HPLC grade water (100% v/v) containing trifluoroacetic acid (0.1% v/v). The content of acetonitrile in the mobile phase was increased linearly from 0 to 100 % for 72 min at a flow rate of lml/min. Peptides were detected using a detector operating at a wavelength of 214nm. Solvents were removed from the collected fractions by evaporation using a centrivap console (Labconco Corporation, Kansas City, USA). The fractions were redissolved in 1 ml of distilled water prior to subsequent assays for antimicrobial activity.

Determination of protein concentration

The protein concentration of the fractions was determined using the Biorad Protein Assay Method (24). Absorbance at 595nm was determined and protein concentration reported as mg/ml.

Antibacterial activity

Preliminary characterisation of the antibacterial activity of peptides and fractions synthesised involved measurement of growth inhibition in a 96- well plate assay (1, 10) and utilisation of an agar diffusion method (16,39). The 96-well plate assay involved the use of controls of each peptide fraction alone (Cecropin Pl and indolicidin), as well as controls without peptide fractions (growth control). A control without bacteria (sterility control) was also included. The plates were incubated for 6 h at 37°C and culture growth monitored hourly. Minimum inhibitory concentrations (MICs) were taken as the lowest concentration without visible growth, measured by recording the OD ₆ O ₀ in a micro-titre plate reader. A well diffusion assay (16, 40) was used to detect antibacterial activity of purified and chemically synthesised

peptides. These assays were performed in either Brain Heart Infusion (BHI), Luria Bertani (LB) or Nutrient agar (NA) seeded with 1 ml of an overnight culture of the indicator strains E. coli DPC6053, L. innocua DPC3306, E. sakazakii 5920 (ATCC12868) or E. sakazakii 8272 (NCTC8155) respectively. Wells, 4.6 mm in diameter, were cut into these agar plates, and 30 μl (0.162 mg/ml) of the peptide fraction placed in each well. Plates were stored at 4°C for 4 h to permit radial diffusion of the peptide, incubated at 30 ⁰ C anaerobically or 37°C aerobically for 24 h, and examined for zones of inhibition. The sensitivity of a strain to the peptides was scored according to the diameter of the zone of inhibition surrounding the well. The experiments were performed in triplicate and mean zone size calculated. Isracidin, Cecropin Pl for E. coli DPC6053, E. sakazakii 5920 (ATCC12868) and E. sakazakii 8272 (NCTC8155) and Indolicidin against L. innocua DPC3306 were used as controls.

Purification, sequencing and synthesis of antimicrobial peptides

Protein fractions exhibiting antibacterial activity were refractionated by RP-HPLC conditions as described above, and those fractions that inhibited growth of the indicator strain E. coli DPC6053 at a similar protein concentration to the commercial peptide Cecropin Pl were collected and the peptide composition analysed by Mass spectrometry (MS) with a matrix assisted laser desorption ionisation time of flight (MALDI-TOF) mass spectrophotometer (PE Biosystems Voyager-DE STR Biospectrometry Workstation, Aberdeen Proteome Facility) with a laser operating at 337 nm and an acceleration voltage of 2OkV. The amino acid sequences of the peptides in each fraction were determined after derivatization and Edman degradation. These steps were performed on a 494A protein sequencer (Applied Biosystems). AU peptides identified (by MS analysis and amino acid sequencing) in each fraction exhibiting antimicrobial activity were subsequently chemically synthesised by Peptide Protein Research Ltd., (Fareham, UK). The purity of the synthesised peptides was greater than 95% as determined by HPLC analysis as certified by the manufacturer.

Treatment of chemically synthesised peptides and crude fractions with proteinase K Chemically synthesised peptides and fractions exhibiting antimicrobial activity against E. coli DPC6053 were tested for susceptibility to proteinase K (Sigma) by incubation of proteinase K (2050 U/ml) with the peptide or fraction (0.554mg/ml) in a 1 : 1 volume ratio at 4°C for 6 h

using the agar well diffusion assay (40). Plates were incubated at 37°C for 24 h. The positive controls used were the peptides isracidin (Peptide Protein Research Ltd.,) and Cecropin Pl (Sigma TM).

EXAMPLES

One of the objectives of this study was to discover novel bioactive peptides from bovine casein released using the proteolytic capabilities of LAB of mammalian intestinal origin. 205 isolates obtained from the porcine small intestine and 55 isolates of human adults and infant faecal origin were used in this study.

Screening for isolates with proteolytic activity

All 260 isolates on LAB agar plates were screened for proteolytic activity using SMA and sodium caseinate as substrates. 5 isolates of porcine intestinal origin and 10 isolates of human infant and adult faecal origin were selected based on their proteolytic capabilities and potential to generate peptides <10 kDa from sodium caseinate. All 15 isolates produced a zone size >4mm in diameter size indicative of proteolytic activity, and HPLC analysis of small-scale casein fermentations of all 15 isolates showed the most complex peptide profiles. Of the fifteen isolates selected, 9 were identified as distinct species of lactobacilli following PFGE and 16S rDNA sequencing (Table 1). 5 isolates, all of human adult faecal origin, showed between 99- 100% homology with Enterococcus faecalis (using the BLAST program) and therefore were considered unsuitable for further use in large-scale fermentations, as enterococci are not generally recognised as safe (GRAS) (21). Isolate 3L7 and 33Ll (human infant faecal isolates) showed 100% homology (BLAST program) with E. hirae and Staphylococcus epidermis, respectively, and these were also eliminated from further study due to non-GRAS status. 3 of the 9 lactobacilli isolates were identified as L. acidophilus

DPC6026, Ljohnsonii DPC6092 and L. salivarius DPC6027 by PFGE (Figure 1) and 16S- rDNA sequencing.

The peptide profiles obtained following incubation of L. acidophilus DPC6026, L. salivarius DPC6027 and L. johnsonii DPC6092 in sodium caseinate (2.5% w/v) after 24 h and subsequent filtration through a 1OkDa membrane is shown in Figure 2. Fermentation of

sodium casemate with these 3 lactobacilli resulted in substantial degradation of the sodium caseinate (Figure 2A) to lower molecular weight peptides (Figures 2B-2D) with L. acidophilus DPC6026 exhibiting the greatest degree of breakdown of sodium caseinate into smaller weight peptides (Table 1). L. acidophilus DPC6026 produced a fermentate where 60.74% of the peptides obtained were less than < 0.5kDa, 17.49% were peptides between 1- 0.5kDa and 11.57% peptides between l-2kDa. This strain produced more peptides between 0.5kDa-2.0kDa than L. johnsonii DPC6092, L salivarius DPC6027 and L. animalis DPC6028 (Table 1). The HPLC peptide profiles produced by L. reuteri DPC6100, L. gasserri DPC6093, L. rhamnosus DPC6095 and L. brevi DPC6102 did not indicate breakdown of sodium caseinate to the same extent (HPLC profiles not shown) as the strain chosen (Table 1) and therefore were not chosen for further study. L. delbrueckii sp. bulgaricus DPC6104 was not chosen as its use for the generation of bioactive peptides has been documented previously (11).

Detection of antimicrobial peptides

Sodium caseinate fermentates produced by Lactobacillus acidophilus DPC6026, L salivarius DPC6027 and L. johnsonii DPC6092 were antibacterial fractions. However, only fractions generated from L. acidophilus DPC6026 were assayed for antibacterial activity, as this strain was characterised by the highest proteolytic activity of the three strains as shown from the chromatogram profiles shown (Figure 2). Antimicrobial activity in the crude fermentates, using either the 96-well plate assay or the well diffusion assay against the strains E. coli DPC6053, Listeria innocua DPC3306, E. sakazaMi 5920 (ATCC12868) and E. sakazakii 8272 (NCTC8155) was not detected. The sodium caseinate fermentate produced by L. acidophilus DPC6026 was filtered through a size-exclusion SlYlO 1OkDa spiral cartridge filter to obtain a permeate containing peptides < 1OkDa. 72 fractions of the filtered sodium caseinate fermentate produced by L. acidophilus DPC6026 were collected by RP-HPLC and assayed for antibacterial activity against E. coli DPC6053, E. sakazakii 5920 (ATCC12868), E. sakazakii 8272 (NCTC8155) and L. innocua DPC3306 by a well diffusion assay. The antimicrobial activity of these fractions against E. coli DPC6053 and L. innocua DPC3306 was also assayed by measuring the OD ₆₀₀ using the 96-well plate assay method. Three fractions, Al-45, Al-49, and Al-54 had the most potent antibacterial activity and had peptide

concentrations of 0.554mg/ml, 0.5mg/ml and 1.24mg/ml respectively. All these fractions exhibited inhibitory activity against the test strain E. coli DPC6053 at these concentrations, respectively.

Purification., sequencing and characterisation of peptides

The peptide mixtures in fractions Al-45, Al-49 and Al-54 were subjected to mass spectrum analysis and Edman degradation. The amino-acid composition was also determined by sequencing (Aberdeen Proteome Facility). Fraction Al-45 was found to contain the peptide sequence DCHQGLPQE (SEQ ID NO: 1) (Table 2). This peptide sequence corresponds to α _sl -casein f(21-29). Fraction Al-49 contained the peptide sequence SDIPNPIGSENSEK (SEQ ID NO: 2) that corresponds to α _sl -casein f(183-207). Al-54 was found to contain the peptide sequence VLNENLLR (SEQ ID NO: 3) that corresponds to α _sl -casein f(30-37). The expected and calculated masses for each peptide are reported (Table 2). As determined by the agar well-diffusion assay, SEQ ID NO: 1 present in fraction 45 inhibited the indicator organism, E. coli DPC6053 at a concentration of 0.05mM (Figure 3 B). This peptide also showed inhibition against potentially pathogenic bacteria of clinical interest such as E. coli 0157: H7 derivatives (E. coli DPC6054 and E. coli DPC6055), E. sakazakii 5920(ATCC 12868) (Figure 3 B) at the same concentration (0.05mM). SεQ ID NO: 3 present in fraction 54 inhibited the indicator organism, E. coli DPC6053 at a concentration of 0.22mM (Figure 3 C) and also against potentially pathogenic strains such as E. sakazakii DPC6091 at the same concentration (Figure 3 C) ₃ while fraction Al-49 containing SεQ ID NO: 2 displayed only minor inhibitory activity against Listeria innocua DPC3306 but no activity against E. coli DPC6053 (Table 3).

Chemical synthesis of peptides

Peptides identified from fractions that showed strong antibacterial activity and had a broad spectrum of activity were chemically synthesised to ensure that the sequence identified by MALDI-TOF analysis was the antibacterial sequence within the fractions. These were α _sl -CN f(21-29) (SεQ ID NO: 1), α _si -CN f(30-37) (SεQ ID NO: 3) and α _s i-CN f(183-207) (SεQ ID NO: 2). Chemically synthesised peptides SεQ ID NO: 1 and SεQ ID NO: 3 inhibited the same microorganisms as the fractions containing the unpurified peptides. Synthesised peptide

SEQ K ⁾ NO: 2 (present in fraction Al -49) displayed some inhibitory activity against Listeria innocua DPC3306 but no activity against E. coli DPC6053 (Table 3).

The minimum inhibitory concentration (MIC) of these peptides was determined using isracidin as a positive control. Isracidin inhibited E. coli DPC6053 at concentrations ranging from 0.05mM to 1.9mM (Figure 4A). The MIC for isracidin was found to be 0.059mM under the experimental conditions described while the MIC for SEQ ID NO: 1 was 0.078mM shown to inhibit growth of E. coli DPC6053 (Figure 4 B) and SEQ ID NO: 3 inhibited this microorganism at concentrations ranging from 0.22mM to 1.2mM. (Figure 4 C). The MIC obtained for SEQ ID NO: 3 was 0.22mM. Thus, the present invention has surprisingly produced three novel peptides all with antimicrobial properties. Isracidin was considered as a gold standard positive control: SEQ ID NO: 1 compares favourably with isracidin, whereas SEQ ID: NO 2 and SEQ ID NO: 3 are a factor less effective at the same concentration. However, notwithstanding the requirement for greater concentration, both SEQ ID NO: 2 and SEQ ID NO: 3 clearly demonstrated antimicrobial properties.

The concentrations require for SEQ TD NO: 2 and SEQ ID NO: 3 are within the normal concentration ranges for antimicrobial compounds. They compare well with, for example, Casocidin-1 and the peptides described in US 6,579,849 "Antibiotic peptides from bovine milk", which lists desired concentrations of 0. lmg-lmg of the composition. Floris et ah,

Current Pharmaceutical Design, 2003, 9, 1257-1275, state that isracidin "was found to inhibit the in vitro growth of lactobacilli and other Gram-positive bacteria, but only at relatively high concentrations (0.1-lmg/mL)."

Under assay conditions, all the chemically synthesised peptides and crude fractions lost their antimicrobial activity when treated with Proteinase K. Using the computer program Expasy Peptide cutter (http://ca.expasy.org/cgi-bin/peptidecutter/peptidecutter.pl ) it was found that peptide sequences IKHQGLPQE (SEQ ID NO: 1), VLNENLLR (SEQ ID NO: 3), SDIPIGSENSEK (SEQ ID NO: 2) are all hydrolysed by trypsin and chymotrypsin.

Discussion

In this study, sodium caseinate was subjected to proteolysis using the proteolytic strains L. acidophilus DPC6026, L. salivarius DPC6027 and L. johnsonii DPC6092. The strain L. acidophilus DPC6026 was chosen for use in fermentations and peptides generated assayed for antimicrobial activity against potentially pathogenic strains such as E. coli JM109 DPC6053 and Enter obacter sakazakii DPC6090 (ATCC 12868).

The peptides sequences IKHQGLPQE (SEQ ID NO: 1), VLNENLLR (SEQ ID NO: 3) and SDIPIGSENSEK (SEQ ID NO: 2) identified in this study have not been reported previously for antimicrobial activity (8, 47).

(Dziuba et ai, 1999 (8)) makes reference to a bioactive database including a range of bioactive peptides derived from a variety of sources. Peptides included in the database are: antimicrobial peptides, angiotensin-I converting enzyme inhibitory peptides (ACE-I inhibitory peptides), opioid peptides and Bradykinin potentiating peptides. The peptide isracidin (Peptide Data ID 3035, name isracidin, sequence RPKHPIKHQGLPQEVLNENLLRP) used as the gold standard positive control against pathogenic bacteria in the experiments carried out is detailed in this database.

(Wang et ah, 2004 (47)) details an Antimicrobial Peptide Database only, and only briefly mentions isracidin.

Conclusion of study

A known antimicrobial peptide from bovine milk protein is isracidin, α _sl -CN fl-23, with the primary amino acid sequence determined as R ₁ PKHPIKHQGLPQEVLNENLLRF ₂₃ (26). This peptide has a broad spectrum of activity (23) and was used as a positive control in the current study because of the high degree of homology between it and SEQ ID NO: 1 and SEQ ID NO: 3. SEQ ID NO: 1 has nine residues in common with isracidin, while SEQ ID NO: 3 has eight residues in common with the C-terminal end of isracidin. Peptide SEQ ID NO: 1 exhibited a MIC of 0.078mM, comparable with isracidin, which exhibited a MIC of 0.05mM, and the more potent commercially available peptides Cecropin Pl and indolicidin (MIC of 0.05mM). SEQ ID NO: 3 exhibited a MIC of 0.22mM against E. coli DPC6053, comparable

with the MIC of isracidin (0.05mM). Peptides of the SEQ ID NO: 1 (IKHQGLPQE) contains a positive charge of +2, has a hydrophobic (isoleucine) end and a hydrophilic (glutamate) domain and displays better activity against Gram-negative bacteria such as E. sakazakii and E. coli than Gram-positive bacteria.

Antimicrobial peptides usually possess between 5 and 60 amino acids, have molecular masses of less than 1OkDa, are usually amphipathic, and usually have a broad spectrum of activity (Floris et al., Current Pharmaceutical Design 2003). SEQ ID NO: 3 (VLNENLLR) shares these traits.

It is known that the dual cationic and hydrophobic nature of peptides is important for the initial interaction between the peptide and the bacterial membrane. Positivity promotes interaction with the bacterial outer and cytoplasmic membranes (Wu et ah, Journal of Biol. Chem., 1999). The strong positive charge of arginine (R) allow ID SEQ: 3 (VLNENLLR) to interact with the lipopoysaccharide region of Gram-negative bacteria and to penetrate the negatively charged cytoplasmic membrane as described by Hancock et al., 1999 Peptide Antibiotics, Antimicrobial agents and chemotherapy: 1317-1323.

SEQ ID NO: 3 also has a hydrophobic region (VL) and a hydrophilic region (R). It also displays more potent activity against the pathogenic Gram-negative bacteria than against

Gram-positive strains such as L. innocua. Also, the chemically synthesised peptides exhibited more potent activity against the Gram-negative strains such as E. coli DPC6053 than against L. innocua DPC3306.

The proteinases of LAB have been shown to hydrolyse more than 40% of the peptide bonds of β-CN and α _sl -CN resulting in the generation of oligopeptides (22). The complex peptidases of LAB then act upon these oligopeptides (31). Minervini et al. reported the generation of ACE inhibitory peptides using a partially purified proteinase from L. helveticus PR4 (30). This group also identified an antimicrobial peptide from human β-CN f(l 84-210) that displayed activity against both Gram-positive and Gram-negative bacteria. Several casokinins derived from β-CN have been liberated by a cell- wall-associated serine-type proteinase of

Lactobacillus helveticus CP790 (49). A cell-wall-bound proteinase from L. delbrueckii subsp. lactis ACA-DC 178 liberated four peptides from β-CN (45), however; biological activity of these peptides were not reported.

L. acidophilus DPC6026 was chosen based on initial assays demonstrating its proteolytic ability against casein and therefore its potential to generate a large number of peptides with bioactivities such as antibacterial activity. Cecropin Pl was used as a control as it is a peptide isolated from the porcine small intestine and its spectrum of activity is chiefly against Gram- negative bacteria (5).

Chemically synthesised peptides of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 and fractions containing these peptides were found to lose their antibacterial activity when treated with proteinase K. Trypsin and chymotrypsin inactivated all of the chemically synthesised peptides identified in the crude antibacterial fractions. Using the program Expasy Peptide cutter (http://ca.expasy.org/cgi-bin/peptidecutter/peptidecutter.pl ) it was found that bovine α _sl - CN could be hydrolysed by a combination of three endopeptidases and proteinases to release the antibacterial peptides SEQ ID NO: 1 and SEQ ID NO: 3. A proline endopeptidase cleaves bovine α _sl -CN on the C-terminal side of PrO ₂₀ and an endopeptidase restricts at the C-terminal side of Glu2 ₉ . This combination of enzymes would release the antibacterial peptide IKHQGLPQE (SEQ ID NO: 1) from bovine α _sl -CN. The presence of proline-specific peptidases is necessary for optimal growth of LAB because of their ability to degrade proline- rich oligopeptides from casein and proline-specific peptidases have previously been isolated from L. delbrueckii subsp bulgaricus (2). A combination of the Arg-C proteinase, which restricts bovine αsl-CN at position Arg ₃₇ , and the endopeptidase that restricts bovine α _sl -CN at position GIu ₂₉ would release the antimicrobial peptide VLNENLLR (SEQ ID NO: 3).

While not wishing to be bound by theory, it is believed that L. acidophilus DPC6026 used in fermentation under the conditions herein described (pH 7, maintained with 0.1 M NaOH, 100 rpm, 37-42"C) produces a novel collection of enzymes (proteinases and peptidases) such as Arg-C proteinase, capable of cleaving α _sl -CN into peptides SEQ ID NO 1 (IKHQGLPQE) and SEQ ID NO 3 (VLNENLLR) and SEQ ID NO 2 (SDIPIGSENSEK). This supposition is

based on cleavage analysis of α _s i-Cn using the computer program http://ca.expasy.org/cgi- bin/peptidecutter/peptidecutter.pl and enzymes from the enzyme list provided that may cleave ccsi-CN to produce SEQ ID NO 1, SEQ ID NO 2 and SEQ ID NO 3.

Most antimicrobial peptides possess between 6 and 50 amino acid residues (48). The smallest antimicrobial peptide known at present is named hexapeptide with the sequence R ₁ RWQWR isolated from bovine lactoferricin (46). Most possess a positive charge of +2 or greater, and fold into amphipathic structures with both hydrophobic and hydrophilic domains (47). These features allow them to interact with the lipopolysaccharide region of Gram-negative bacteria and to penetrate the negatively charged cytoplasmic membranes (15).

From the sequence data alone, it can be difficult to predict either the activity of a peptide or the secondary structure that it may form (4). Peptides lacking disulfide bridges (as is the case with peptides IKHQGLPQE (SEQ ID NO: 1) and VLNENLLR (SEQ ID NO: 3) have random structures in water and only when they bind to a membrane or self-aggregate can they form a structure (10). However, some features of these peptides suggest that the mechanism of action may involve self-promoted uptake across the cytoplasmic membrane followed by interference with the cytoplasmic membrane barrier, a mechanism that has been described previously for cationic and neutral antimicrobial peptides (15, 48).

The features include (1) Positively charged peptides such as arginine contributing to the cationicity of the peptide which is important for the initial interaction with the bacterial cytoplasmic membrane. (2) Hydrophobicity - this is contributed to the peptide SEQ ID NO: 1 by the amino acid isoleucine and to peptide SEQ ID NO: 3 by the amino acids Valine (V) and Leucine (L). The present invention also provides for increasing the hydrophobicity of these peptides increase binding of the peptides to the membrane due to increased hydrophobic interactions between lipid acyl chains and the hydrophobic core.

Conclusion This study shows that the strain L. acidophilus DPC6026 is suitable for the generation of antimicrobial peptides from casein and that sodium caseinate fermentates produced with this

strain of Lactobacillus may be considered as a component of functional foods with antibacterial benefits, as the MIC concentration of the antibacterial peptides are comparable with the commercially synthesised peptides (Cecropin Pl) and the known antimicrobial peptide isracidin (22).

It also shows that bovine α- _sl casein may be considered as a precursor of the broad spectrum antibacterial peptides isolated here and finally that these peptides may have the potential to be used in infant formula products as an in-built protection system against pathogenic bacteria such as E. sakazakii.

Examples of a Method of Preparation of a Sodium casemate powder with anti- Enterobacter sakazakii effects.

(1) Sodium casemate substrate (5% w/v) maybe inoculated with (1% w/v) of L. acidophilus DPC 6026 and incubated at 37°C for 24 hr with mixing at 100 rpm at constant pH 7 maintained via addition of 0.1 M NaOH. This fermentate can then be heated to 80 ⁰ C to inactivate the cultures which are then freeze dried to produce a fine powder.

(2) Sodium caseinate substrate (5% w/v) maybe inoculated with (1% w/v) of L. acidophilus DPC6026 and incubated at 37°C for 24 hr with mixing at 100 rpm at constant pH 7 maintained via addition of 0.1 M NaOH. (3) This fermentate may then be is then heated to 80°C to inactivate the cultures and subsequently filtered through a size exclusion column 1OkDa to make a powder containing peptides less than 1OkDa. This downstream processing increases the activity of the active peptides. The 1OkDa membrane filtrate is freeze dried and stored at -20°C and vacuum packed. (4) Step (2) maybe followed as described above but a 3kDa membrane filter maybe used to further increase the activity of the peptides SEQ ID NO;1 and SEQ ID NO:3.

The above powders could be added at a concentration of 10%- 15% to dried milk based infant formulas to prevent contamination with Enterobacter sakazakii and subsequently enteric colitis and meningitis caused by this pathogen.

The aiάi-Enterobacter sakazakii powder manufactured as above provides an internal and inbuilt protection mechanism against Enterobacter sakazakii and other pathogens, especially if added to casein based milk baby formula products.

In some embodiments, the peptides of the invention can be used separately or together as adjuvants for processes for the preparation of milk-based infant formula to safeguard against Enterobacter sakazakii contamination - a prominent cause of infection in immunocompromised and premature neonates.

The peptides of the invention can be used to contribute to bitter flavours in cheese manufacture as the first 9 residues of α _s i-CN (casein) are resistant to cleavage and have been documented as contributing to bitter flavours in cheese ripening (Pederson et al., 1999).

The production of α _sl -CN derived antimicrobial peptides by L. acidophilus DPC6026 has not been previously reported. The peptide sequences generated have not been found in various literature and database searches and are thought to be novel in this respect.

The words "comprises/comprising" and the words "having/including" when used herein with reference to the present invention are used to specify the presence of stated features, integers, steps or components but does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

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Table 1: Sodium caseinate protein-derived hydrolysate fractions derived using lactobacilli strains that generated peptide sizes of between <l-20kDa.

Lactobacilli Peptide Peptide Peptide Peptide Peptide strains used for in vitro hydrolyses of Fraction Fraction Fraction Fraction Fraction sodium caseinate (2.5% w/v). 20-1OkDa 10-5kDa 5-2kDa 2-IkDa <lkDa

% % % % %

L. acidophilus DPC6026 0.27 0.66 9.72 11.12 78.23 Ljohnsonii DPC6092 0.06 0.19 1.76 4.36 93.11 L. salivarius DPC6027 0.08 0.32 2.19 3.56 93.85 L. murinus DPC6028 0 0.04 2.36 4.71 92.89 L. delbrueckii sp. bulgaricus DPC6105 0.02 0.18 3.98 14.49 81.34 L. brevi OVC6102 0.01 2.62 1.59 2.66 93.12 L. reuteή OVC6100 0.07 0.3 2 3.5 94.13 L. gasserri DPC6093 0.01 0.14 2.58 5.09 92.19 L. rhamnosus DPC6095 0.02 0.32 1.87 3.02 94.78

Table 2: 16S rDNA sequencing of human infant and adult faecal isolates and pig small intestinal isolates.

Isolate Isolate source Strain 16S rDNA sequencing % homology

3L6 Human adult faeces L gasserri DPC6093 100 ^a

3L8 Human adult faeces Efaecalis DPC6094 100 ^a

15L23 Human adult faeces L rhamnosusωPC6095 99 ^b

23L2 Human infant faeces Efaecdis DPC6096 100 ^a

23L3 Human infant faeces Efaecalis DPC6097 100 ^a

23L4 Human infant faeces Efaecdis DPC6098 99-100 ³

23L5 Human infant faeces Efaecalis DPC6099 100 ^a

28Ll Human infant faeces L reuteri DPC6100 98 ^b

33Ll Human infant faeces S. epidermis DPC6101 100 ^c

37Ll Human infant faeces L breviOVOβlQl 98 ^b

Al Pig small intestine L acidophilus DPC6026 99 ^b

BIl Rg small intestine Ljohnsomi DPC6092 98 ^b

B24 Pig small intestine L saHvarius DPC6027 100 ^b

B28 Pig small intestine L murinus DPC6 ^Q 28 98 ^b

55 Kg small intestine L deϊbmecMi sp. bulgaricus DPC6105 98 ^b

DPC; Dairy Products Research Centre, Teagasc Moorepark, Fermoy, Co. Cork, Ireland. ^a ' Percentage homology with Enterococcus species. ^bi Percentage homology with Lactobacillus species. ^c ' Percentage homology with Staphylococcus species.

41

Table 3: Sequences and corresponding casein (CN) fragments of peptides contained in crude fractions from sodium caseinate hydrolysates produced by Z. acidophilus DPC6026.

Sequence and Fraction Overall A. A. ^a Calculated Mass Expected Mass ^b CN-Fragment Charge Chain length (m/z) (m/z)

KHQGLPQE Al-45 Positive 1049.177 1049.43 α _s ,-CN21-29 VLNENLLR Al-54 Positive 8 970.119 970.119 αsl-CNf 30-37

SDIPNPIGSENSEK Al-49 Positive 14 1486.536 1486.7 αsl-CNf 183-207

^a One letter symbols are used for amino acids. ^b Averages masses are reported for the expected mass of each peptide.

Table 4: The inhibitory spectrum of pure peptides synthesized following fermentation of L. acidophilus DPC6026 in sodium caseinate.

Indicator species Strain Peptide Peptide Peptide details IKHQGLPQE VLNENLLR SDIPIGSENSEK 0,1-CN f(21-29) 0, ₁ -0^(30-38) O _5I -CN f(183-207)

Staplψlococcus DPC5246 ³ aureus

Escherichia coli DPC6053 ³ JM109

K coli O157:H7 DPC6054 ^a +++

E coli O157:H7 DPC6055 ³

Enterobacter DPC6090 ^a sakazddi ATCC12868

E sάkazaSdi DPC6091 ³ NCTC8155

Listeria innocua +

Lactobacillus DPC5383 ³ bidgaricus ATCCl 1842

Streptococcus DPC4069 ³ mutans

^a ' Dairy Products Research Centre, Cork, Ireland. +++, Zone of inhibition > 2.0cm in diameter produced. ++, Zone of inhibition > 1.5cm in diameter produced. +, Zone of inhibition > 1.Ocm in diameter produced. No zone detected.