(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date 10 February 2011 (10.02.2011) WO 2011/014925 Al

(51) International Patent Classification: (74) Agent: FB RICE & CO; Level 23, 200 Queen Street, A23J3/34 (2006.01) A61P 29/00 (2006.01) Melbourne, Victoria 3000 (AU). A61P 25/00 (2006.01) A61K 38/01 (2006.01) (81) Designated States (unless otherwise indicated, for every A61P 37/00 (2006.01) A61P 35/00 (2006.01) kind of national protection available): AE, AG, AL, AM, A61K 9/00 (2006.01) AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, (21) International Application Number: CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, PCT/AU20 10/000994 DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, (22) International Filing Date: KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, 6 August 2010 (06.08.2010) ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, (25) Filing Language: English NO, NZ, OM, PE, PG, PH, PL, PT, RO, RS, RU, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, (26) Publication Language: English TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (30) Priority Data: (84) Designated States (unless otherwise indicated, for every 2009903698 7 August 2009 (07.08.2009) AU kind of regional protection available): ARIPO (BW, GH, 2009903699 7 August 2009 (07.08.2009) AU GM, KE, LR, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, (71) Applicant (for all designated States except US): COM¬ ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, MONWEALTH SCIENTIFIC AND INDUSTRIAL TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, RESEARCH ORGANISATION [AU/AU]; Limestone EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, Avenue, Campbell, Australian Capital Territory 2612 LV, MC, MK, MT, NL, NO, PL, PT, RO, SE, SI, SK, (AU). SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG). (72) Inventors; and (75) Inventors/ Applicants (for US only): BENNETT, Louise Published: [AU/AU]; 671 Sneydes Road, Werribee, Victoria 3030 — with international search report (Art. 21(3)) (AU). CRITTENDEN, ROSS [AU/AU]; 671 Sneydes Road, Werribee, Victoria 3030 (AU). MUENCH, Gerald [AU/AU]; 2/17 Clydesdale Drive, Blairmount, New South Wales 2559 (AU).

(54) Title: PROCESS FOR OBTAINING PEPTIDE FRACTIONS (57) Abstract: The present invention relates to a process for isolating one or more peptide fractions from a protein substrate. The invention also relates to peptide fractions obtained by the process and their use in food and drink products and for the treatment and prevention of disease. PROCESS FOR OBTAINING PEPTIDE FRACTIONS

FIELD OF THE INVENTION The present invention relates to a process for isolating one or more peptide fractions from a protein substrate. The invention also relates to isolated peptide fractions and their use in food and drink products and for the treatment or prevention of disease.

BACKGROUND OF THE INVENTION There is increasing interest in the use of protein hydrolysates for both medical and non-medical applications. In both applications, an easily assimilable diet featuring facilitated gastrointestinal uptake of peptides is a factor of prime importance. Peptides from partial enzymatic hydrolysates of food proteins have been shown to provide health benefits such as, for example, antihypertensive activity. These peptides are hidden in a latent state within the sequence of the parent protein and may be released by proteolytic processes during hydrolysis or during food processing. The commercialisation of various protein hydrolysates claiming antihypertensive effects emphasize the increased scope of use of protein hydrolysates containing bioactive peptides in medical and non-medical applications. Examples of these bioactive peptides and protein hydrolysates containing such bioactive peptides include hydrolysates obtained by incubation of protein substrates with probiotic bacteria or enzymes obtained from such bacteria as well as specific tripeptides obtained by fermentation of Lactobacillus which inhibit angiotensin-converting enzyme (ACE). Products containing antihypertensive peptides by fermenting casein with lactic acid bacteria, as well as the use of highly purified or chemically synthesized peptides for reducing blood pressure or treating diabetes, renal impairment or obesity, have also been described. Processes aimed at optimising protein hydrolysate characteristics, for example biological activity useful for therapeutic or nutritional purposes, have used single or mixed endoproteases such as animal-derived endoproteases like trypsin, chymotrypsin and pancreatin. However, there remains a need for processes to derive peptides from hydrolysed food proteins that would be useful in the development of novel food additives and for therapeutic uses. SUMMARY OF THE INVENTION The present inventors have found that food proteins can be hydrolysed by safe and food grade enzymes in a food-compatible process to yield peptide products that are not otherwise released by digestion. By using a strategic combination of enzymes with complementary specificities to normal digestive enzymes, peptide fractions are produced that are stable to intestinal digestion and which have biological activity. Accordingly, in one aspect, the present invention provides a process for obtaining a peptide fraction, the process comprising: i) contacting a protein substrate with a series of enzymes selected from: a) glutaminase, subtilisin, oryzin, leucyl aminopeptidase, and trypsin, b) glutaminase, oryzin, subtilisin, leucyl aminopeptidase, and trypsin, c) glutaminase, subtilisin, leucyl aminopeptidase, oryzin, and trypsin, d) glutaminase, oryzin, leucyl aminopeptidase, subtilisin, and trypsin, e) glutaminase, leucyl aminopeptidase, oryzin, subtilisin, and trypsin, and f) glutaminase, leucyl aminopeptidase, subtilisin, oryzin, and trypsin, ii) selecting peptides obtained from step i) which are less than about 11 kDa. In another aspect, the present invention provides a process for obtaining a peptide fraction, the process comprising: i) contacting a protein substrate with a series of enzymes selected from: a) trypsin, subtilisin, oryzin, leucyl aminopeptidase, and glutaminase, b) trypsin, subtilisin, leucyl aminopeptidase, oryzin, and glutaminase, c) trypsin, oryzin, subtilisin, leucyl aminopeptidase, and glutaminase, d) trypsin, oryzin, leucyl aminopeptidase, subtilisin, and glutaminase, e) trypsin, leucyl aminopeptidase, oryzin, subtilisin, and glutaminase, and f) trypsin, leucyl aminopeptidase, subtilisin, oryzin, and glutaminase, ii) selecting peptides obtained from step i) which are less than about 11 kDa. In one embodiment, step i) comprises contacting the protein substrate with a series of enzymes selected from: a) glutaminase, subtilisin, oryzin, leucyl aminopeptidase, and trypsin, b) glutaminase, oryzin, subtilisin, leucyl aminopeptidase, and trypsin, and c) glutaminase, leucyl aminopeptidase, oryzin, subtilisin, and trypsin. The enrichment for peptide fractions of like physicochemical properties, i.e., neutral, cationic and anionic fractions, reflecting the systematic fractionation of related peptides, permits the concentration of a key physicochemical property required for biological activity. Thus, in one embodiment, the process further comprises: iii) separating the peptides which are less than about 11 kDa into one or more of cation, anion and/or neutral peptide fractions. In an embodiment, the peptides are separated into one or more of cation, anion and/or neutral peptide fractions by ion- exchange chromatography. In one embodiment the ion-exchange chromatography is performed using cation and anion exchange columns in series. Although the ion-exchange chromatography may be performed at any suitable pH, preferably, the ion-exchange chromatography is performed at about pH 6.0 to about pH 8.0. In one particular embodiment, the ion-exchange chromatography is performed at about pH 7.4. In one embodiment, the step ii) is performed by dialysis, membrane filtration or size-exclusion chromatography. The protein substrate may be any suitable substance that contains protein, for example a food or food protein including animal, plant or microbial proteins, or a non- food animal, plant or microbial protein, or a waste product or by-product from a process. In one embodiment, the protein substrate is a protein-rich extract. In one particular embodiment, the protein-rich extract is a milk extract, meat extract, plant extract, yeast extract and/or bacterial extract. In yet another embodiment, the protein-rich extract is a protein hydrolysate. High pressure treatment exposes protein domains to hydrolysis that would not otherwise be exposed. Thus, in one embodiment, the protein substrate may be high pressure treated prior to contacting the protein substrate with the series of enzymes. For example, the protein substrate may be high pressure treated at about 400 MPa to about 800 MPa for about 15 minutes to about 1 hour. In one embodiment, the protein substrate is high pressure treated at about 600 MPa for about 30 minutes. In another embodiment, the protein substrate is contacted with the series of enzymes glutaminase, subtilisin, oryzin, leucyl aminopeptidase, and trypsin, wherein the protein substrate is selected from fish, soy flour, yeast, pork, beef, chicken, lamb, bovine caseinate, spinach, sheep milk, buffalo milk, spirulina, mushroom, bovine lactoferrin, bovine colostrum, millet, sheep wool, barley, sorghum, amaranth, probiotic microorganisms and bovine whey protein. In another embodiment, the protein substrate is contacted with the series of enzymes glutaminase, oryzin, subtilisin, leucyl aminopeptidase, and trypsin, wherein the protein substrate is selected from bovine caseinate, bovine whey protein, fish, soy flour, yeast, pork, beef, chicken, lamb, spinach, sheep milk, buffalo milk, bovine lactoferrin, bovine colostrum, chicken feather, millet, probiotic organisms, and spirulina. In yet another embodiment, the protein substrate is contacted with the series of enzymes glutaminase, leucyl aminopeptidase, oryzin, subtilisin, and trypsin, wherein the protein substrate is selected from bovine caseinate, bovine whey protein, lactoferrin, fish, soy flour, yeast, beef, chicken, lamb, spinach, spirulina, sheep wool, probiotic organisms, and buffalo milk. In another embodiment, the process of the invention further comprises isolating one or more peptides from the peptide fraction. Once a peptide fraction has been obtained by performing the method of the invention, the person skilled in the art may test the peptide fraction and/or isolated peptide for biological activity using any suitable known assay. Thus, in one embodiment, the process further comprises testing the peptide fraction and/or isolated peptide for biological activity. For example, the process may comprise testing the peptide fraction and/or peptide for biological activity selected from angiotensin converting enzyme (ACE) inhibition activity, angiotensin II receptor inhibition activity, anti-inflammatory activity, fibril inhibition activity, beta-secretase I inhibition activity, and inhibition of receptor for advanced glycation endproduct-mediated inflammation activity. In one embodiment of the process of the invention, the protein substrate is selected from bovine caseinate, bovine whey protein, lactoferrin, fish, soy flour, yeast, pork, beef, chicken, and lamb, and the peptide fraction obtained by the process has angiotensin converting enzyme (ACE) inhibition activity. In another embodiment, the protein substrate is selected from bovine caseinate, bovine whey protein, sheep milk, buffalo milk, spinach, and spirulina, and the peptide fraction obtained by the process of the invention has angiotensin II receptor inhibition activity. In yet another embodiment, the protein substrate is selected from bovine whey protein and yeast, and the peptide fraction obtained by the process of the invention has anti-inflammatory activity. In another embodiment, the protein substrate is selected from bovine caseinate, bovine lactoferrin, bovine colostrum, bovine whey protein, sheep milk, buffalo milk, millet, sheep wool, chicken feather, barley, sorghum, amaranth, mushroom, wheat, and wheat gluten, and the peptide fraction obtained by the process of the invention has fibril inhibition activity. In yet another embodiment, the protein substrate is probiotic microorganisms, and the peptide fraction obtained by the process of the invention has beta-secretase I inhibition activity. In another aspect, the present invention provides a peptide fraction obtained by the process of the invention. The peptide fraction obtained by the process of the invention may be a mixture of charged and neutral peptides. Alternatively, the peptide fraction may be a cation or anion peptide fraction. In one embodiment, the peptide fraction of the invention is a cation peptide fraction at about pH 7.4. In one embodiment, the peptide fraction of the invention has biological activity. In one particular embodiment, the biological activity is selected from angiotensin converting enzyme (ACE) inhibition activity, angiotensin II receptor inhibition activity anti-inflammatory activity, fibril inhibition activity, beta-secretase I inhibition activity, and inhibition of receptor for advanced glycation endproduct-mediated inflammation activity. In another aspect, the present invention provides a composition comprising the peptide fraction of the invention. In yet another aspect, the present invention provides use of the peptide fraction of the invention or the composition of the invention in the manufacture of a food or drink product. In one embodiment, the food or drink product is a nutritional supplement. In one aspect, the present invention provides a food or drink product comprising the peptide fraction of the invention or the composition of the invention. In another aspect, the present invention further provides use of the peptide fraction of the invention or the composition of the invention in the manufacture of a medicament for the treatment or prevention of disease in a subject. In yet another aspect, the present invention provides a method for the treatment or prevention of disease in a subject, the method comprising administering to the subject the peptide fraction of the invention or the composition of the invention. In one embodiment of the use or method of the invention, the disease is selected from cardiovascular disease, cancer, inflammatory or autoimmune disease, and/or a neurological disorder. In another embodiment, the disease is selected from cardiovascular disease and cancer. In one embodiment, the cardiovascular disease that is treated or prevented is selected from hypertension, atherosclerosis and arteriosclerosis. In another embodiment, the cancer is selected from carcinoma, lymphoma, blastoma, sarcoma, and leukemia. In yet another embodiment, the cancer is selected from breast cancer, prostate cancer, colon cancer, squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, ovarian cancer, cervical cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, liver cancer, bladder cancer, hepatoma, colorectal cancer, uterine cervical cancer, endometrial carcinoma, salivary gland carcinoma, mesothelioma, kidney cancer, vulval cancer, thyroid cancer, hepatic carcinoma, skin cancer, melanoma, brain cancer, neuroblastoma, myeloma, various types of head and neck cancer, acute lymphoblastic leukemia, acute myeloid leukemia, Ewing sarcoma and peripheral neuroepithelioma. In one embodiment, the inflammatory or autoimmune disease is selected from asthma, rheumatoid arthritis, inflammatory bowel disease, psoriasis and multiple sclerosis. In another embodiment, the neurological disorder is selected from Alzheimer's disease, Parkinson's disease, dementia, Huntington's disease, Shy-Drager syndrome, progressive supranuclear palsy, Lewy body disease and amyotrophic lateral sclerosis. The present invention further provides a peptide fraction of the invention, wherein the peptide fraction is a cation peptide fraction at about pH 7.4, and the peptide fraction was obtained by contacting bovine whey protein isolate with the series of enzymes glutaminase, oryzin, subtilisin, leucyl aminopeptidase, and trypsin. The present invention further provides a peptide fraction of the invention, wherein the peptide fraction was obtained from a protein substrate comprising mushroom, and the protein substrate was contacted with the series of enzymes glutaminase, subtilisin, oryzin, leucyl aminopeptidase, and trypsin. As will be apparent, preferred features and characteristics of one aspect of the invention are applicable to many other aspects of the invention. Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. The invention is hereinafter described by way of the following non-limiting Examples and with reference to the accompanying figures. BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS FIGURE 1. Effects of feeding Spontaneous Hypertensive rats with lead dairy anti-hypertensive product versus commercial hydrolysate DH 17, showing (a) rat body masses and (b) tailcuff systolic blood pressures measured at fortnightly intervals over treatment period, starting with rats at 2 weeks of age. FIGURE 2. Effects of feeding Spontaneous Hypertensive rats with P O147 at 200 mg/kg/day, showing (a) rat body masses and (b) tailcuff systolic blood pressures measured at fortnightly intervals over treatment period, starting with rats at 2 weeks of age. FIGURE 3. Inhibition kinetics by Lineweaver-Burk plot for control and lead products: (a) NaCl, (b) P0148 from bovine whey, (c) P0147 from bovine whey and (d) P0052 from bovine caseinate; (e) P0609 from sheep whey and (f) P0498 from soy protein, after concentration of the active fraction by Cl 8 Solid Phase Extraction (SPE). FIGURE 4. Transmission Electron microscopic imaging of effects of Aβ42 in the absence (a: Aβ42 control incubated for 80 hr at minus 180C; b : Aβ42 control incubated for 80 hr (48 h at 370C, then to 80 hr at 2 O0C)) or presence (c: Aβ42 + BM0263-SPE40 incubated for 80 hr at minus 180C (note scale difference from other images); d : Aβ42 + BM0263-SPE40 incubated for 80 hr (48 h at 370C, then to 80 hr at 2 O0C)) of BM0263-SPE40 showing significant suppression of fibril assembly at either minus 180C or 37/2O0C, following incubation for 80 hr. FIGURE 5. ThT fluorescence monitoring showing effects of selected inhibitors on further fibril growth, in the presence of pre-formed fibrils of Aβ42 (0.043 mg/ml, after 52 hr at 370C) and similar pre-formed fibrils of Aβ42 in the presence of (a) P0263-SPE40 (millet at 0.17 mg/ml) and (b) BM0263-SPE40 (button mushroom at 0.17 mg/ml). FIGURE 6. Reverse Phase HPLC profiles of SPE40 and SPElOO sub-fractions ofP0147. FIGURE 7. Modulation of RCM-kCn fibrils by SPE fractions of P O147, as monitored by RCM-kCn fibril inhibition assay. FIGURE 8. Concentration-dependent modulation of Aβ42 toxicity to yeast by SPE fractions of P O147 after 20 h at 3 O0C. FIGURE 9. CD profiles of human Aβ42 (sample subtracted) in the absence and presence of (a) SPE40 and (b) SPElOO fractions of P O147 after incubation at 3 O C for 2 O h. FIGURE 10. Western Blot analysis of Aβ42+P0147 mixtures after incubation at 3 O C for 20 h. Freshly prepared Aβ42 (10 µM final) was incubated at with increasing concentrations of P0147-SPE40+SPE100 (Lanes 1-4: 0.1, 0.05, 0.01 and 0 mg/ml, respectively), and visualized by 6E10 antibody immunoblotting. FIGURE 11. Transmission Electron microscopy of (a) Aβ42 alone; (b) Aβ42 and P0147-SPE total and (c) P0147-SPE total alone, each incubated at 3 O C for 20 hr. FIGURE 12. Whole blood inflammation assay results showing (a) SPE non- binding and binding fractions eluting in 40% and 100% acetonitrile tested at 200 µg/ml, • Vegemite, I Marmite, AEpiCor, with IL-10 and hydrocortisone included as positive controls, and (b) untreated Vegemite, reference (Epicor) and total SPE bound fractions of BR0263 and BA0263. Each point is an average of data from four donors with results also shown for individual donors (O ∆ VO), error bars indicate one SD. 100% = no change, <100% indicates an anti-inflammatory effect. FIGURE 13. Dose response curve for total SPE bound fractions of: (a) Vegemite, (b) Bakers yeast hydrolysate, BA0263-SPE, (c) Brewers yeast hydrolysate, BR0263-SPE and (d) Epicor-SPE. Results are expressed by representing the average for four donors, error bars indicate 1 SD, and also results for individual donors (O ∆ VO). 100% = no change, <100% indicates an anti-inflammatory effect based on reduction in level of IFNγ relative to stimulated control in the absence of sample.

DETAILED DESCRIPTION OF THE INVENTION General techniques and definitions Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in protein chemistry, biochemistry, food science, cell culture, molecular genetics, microbiology, and immunology). Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in the present invention are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al, Molecular Cloning: A Laboratory Manual, 3rd edn, Cold Spring Harbour Laboratory Press (2001), R. Scopes, Protein Purification - Principals and Practice, 3 edn, Springer (1994), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley- Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J.E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present). As used herein the term "protein substrate" refers to any substance which comprises protein. The protein substrate may be, for example, food or may contain food proteins, for example animal, plant or microbial proteins, or the protein substrate may be a non-food substrate such as, for example, a non-food animal, plant or microbial protein, or a waste product or by-product from a process. By way of non- limiting example, the protein substrate may include proteins from dairy, for example milk or milk protein, or from meat, vegetable, grain, yeast or bacteria. The term "peptide" as used herein is typically used to refer to chains of amino acids which are not large, for instance 100 or less residues in length. As used herein, the term "peptide fraction" will be understood to indicate a fraction of a hydrolysed protein substrate that contains peptides that may be of different lengths. The "peptide fraction" may also include non-peptide components such as, for example, free amino acids, carbohydrates or lipids. As used herein, the term "subject" relates to an animal. Preferably, the subject is a mammal such as a human, or a companion animal such as a dog or cat, or a livestock animal such as horse, cow, pig or sheep. Alternatively, the subject may be avian, for example, poultry such as a chicken, turkey or duck. Most preferably, the subject is a human. "Administering" as used herein is to be construed broadly and includes administering a peptide fraction as described herein to a subject as well as providing a peptide fraction as described herein to a cell. As used herein, the terms "treating", "treat" or "treatment" include administering a therapeutically effective amount of a peptide fraction as described herein sufficient to reduce or delay the onset or progression of specified disease, or to reduce or eliminate at least one symptom of the disease. As used herein, the terms "preventing", "prevent" or "prevention" include administering a therapeutically effective amount of a peptide fraction sufficient to stop or hinder the development of at least one symptom of the specified condition. The term "about" as used herein refers to a range of +1-5% of the specified value. Process for obtaining peptide fractions Protein substrate In some embodiments, it may desirable to first process a substance comprising protein to form a protein-rich extract which is then used as a protein substrate in the process of the invention. For example, it may be desirable to process protein substrates that have relatively low protein content, such as plant materials, to form a protein-rich extract. If desired plant materials may first be homogenized and treated to remove or reduce the amount of non-protein constituents such as cellulose. Seed grains may optionally be homogenized and proteins such as albumins, glutelins and prolamins extracted by known techniques prior to use as a protein substrate. When probiotic microorganisms are used it is preferred that they are lysed prior to use as a protein substrate in the process of the invention. The probiotic microorganisms may be lysed using known techniques, for example, by French pressing. In some instances, a substance such as a food containing protein may first be minced or chopped and then finely ground to form a protein substrate. Other additional or alternative treatments include high pressure treatment, oven drying and/or freeze drying prior to use of the substance as a protein substrate in the process of the invention. The protein-rich extract may also be a protein hydrolysate. Examples of protein hydrolysates suitable for use in the process of the invention include hydrolysates of native food proteins, such as soy proteins, dairy proteins (e.g., whey, casein), wheat proteins, canola proteins, corn proteins, vegetable proteins, grain proteins, animal proteins, yeast proteins, including those available from commercial sources, for example, hydrolysates such as wheat hydrolysate (Gempr HiQ) from Manildra Group, NSW, Australia, hydrolysates such as bovine whey hydrolysate (DH 17) from Myopure, NSW, Australia, and hydrolysates such as rice protein hydrolysate (Hyprol 8115) from Quest International, the Netherlands. Although these protein hydrolysates have been at last partially broken down or digested either by processing or enzymatic cleavage, they still serve as substrates for the enzymes used in the process of the present invention. In the case of protein hydrolysates, the hydrolysates may be desalted using known techniques such as dialysis prior to use as a protein substrate in the process of the invention. If desired, other substances, for example, milk or skim milk, may also be desalted prior to use as a protein substrate. Non-limiting examples of protein substrates suitable for use in the process of the invention include bovine proteins such as bovine caseinate, bovine casein hydrolysate bovine whey protein, wheat, wheat hydrolysate, wheat gluten hydrolysate, soy, soy hydrolysate, soy flour, rice protein hydrolysate, pea protein hydrolysate, milk including cow, buffalo and sheep milk, lactoferrin, fishmeal, fish meat, yeast, yeast extract, pork, beef, chicken meat, chicken feathers, egg or egg proteins, lamb, spinach, spirulina, millet, barley, brewer's spent grain, sheep wool, sorghum, amaranth, mushroom, or probiotic cultures of microorganisms. Although the protein substrate need only contain protein to be useful in the process of the invention, preferably the protein substrate comprises at least 5% protein, or more preferably at least 10%, 20%, 30%, 40%, or 50% or more protein.

Enzymes The present inventors have advantageously identified a combination of food- compatible enzymes that yield peptide fractions that may not otherwise be released by digestion. In the process of the invention, the enzymes the protein substrate is processed with are glutaminase, subtilisin, oryzin, leucyl aminopeptidase and trypsin. Use of these exo and endo proteases and peptidases in different sequential combinations generates peptides fractions with variation of terminal amino acids. These peptide fractions, that are produced by hydrolysis of the protein substrate in series, advantageously have anti-disease, nutritional or other desirable properties. Glutaminase is a hydrolase which acts on carbon nitrogen bonds other than peptide bonds. When reacted with a protein substrate, glutaminase converts glutamine residues to glutamic acid in a deamidation reaction. Enzymes with this activity have been classified as EC 3.5.1.2. Synonyms for glutaminase include glutaminase I, L- glutaminase, and glutamine aminohydrolase. Glutaminase is commercially available and can be obtained, for example, from Daiwa Kasei K.K. Shiga, Japan. Subtilisin is a serine endopeptidase with broad specificity for peptide bonds, but with a preference for cleavage at glutamic acid residues and also hydrolyses peptide amides. Enzymes with this activity have been classified as EC 3.4.21.62. Alternative names for subtilisin include alcalase, alcalase 0.6L, alcalase 2.5L, alcalase 2.4 L FG, ALK-enzyme, bacillopeptidase A, bacillopeptidase B, Bacillus subtilis alkaline proteinase, bioprase AL 15, bioprase APL 30, colistinase, subtilisin J, subtilisin S41, subtilisin Sendai, subtilisin GX, subtilisin E, subtilisin BL, genease I, esperase, maxatase, thermoase PC 10, protease XXVII, superase, subtilisin DY, subtilopeptidase, SP 266, savinase 8.0L, savinase 4.0T, kazusase, protease VIII, opticlean, protin A 3L, savinase 16.0L, savinase 32.0L EX, orientase 1OB and protease S. Subtilisin is commercially available and can be obtained, for example, from Novozymes, Bagsvaerd, Denmark under the tradename Alcalase® 2.4 L FG. Oryzin is a serine endopeptidase which acts on peptide bonds and hydrolyses proteins with broad specificity. Enzymes with this activity have been classified as EC 3.4.21.63. Alternative names for oryzin include Aspergillus alkaline proteinase, aspergillopeptidase B, API 21, aspergillopepsin B, aspergillopepsin F, Aspergillus candidus alkaline proteinase, Aspergillus flavus alkaline proteinase, Aspergillus melleus semi-alkaline proteinase, Aspergillus oryzae alkaline proteinase, Aspergillus serine proteinase, Aspergillus sydowi alkaline proteinase, Aspergillus soya alkaline proteinase, Aspergillus melleus alkaline proteinase, Aspergillus sulphureus alkaline proteinase, prozyme, P 5380, kyorinase, seaprose S, semi-alkaline proteinase, sumizyme MP, prozyme 10, onoprose, onoprose SA, protease P, and promelase. Oryzin is commercially available and can be obtained from, for example, AB Enzymes, Darmstadt, Germany under the tradename Corolase PN-L. Leucyl aminopeptidase is a hydrolase that acts on peptide bonds to release an N- terminal amino acid from XaaYaa in which Xaa is preferably Leu, but may be other amino acids including Pro, although not Arg or Lys, and Yaa may be proline. Enzymes with this activity are classified as EC 3.4.1 1.1. Alternative names for leucyl aminopeptidase include leucine aminopeptidase, leucyl peptidase, peptidase S, cytosol aminopeptidase, cathepsin III, L-Leucine aminopeptidase, leucinamide aminopeptidase, FTBL proteins, proteinates FBTL, aminopeptidase I, aminopeptidase II, aminopeptidase III. Leucyl aminopeptidase is commercially available and can be obtained from, for example, Novozymes, Bagsvaerd, Denmark under the tradename Flavourzyme. Trypsin is a serine endopeptidase which acts on peptide bonds and preferentially cleaves arginine and lysine. Enzymes with this activity are classified as EC 3.4.21.4. Alternative names for trypsin include alpha-trypsin, beta-trypsin, cocoonase, parenzyme, parenzymol, tryptar, trypure, pseudotrypsin, tripcellim and sperm receptor hydrolase. Trypsin is commercially available and can be obtained, for example, from Novozymes. Although the protein substrate may be completely hydrolysed by a given enzyme, the person skilled in the art would understand that in some instances a given enzyme may only at least partially hydrolyse the protein substrate. In one embodiment of the process of the invention, the protein substrate is first contacted with glutaminase to achieve conversion of glutamine residues to glutamic acid, thereby increasing the anionic character of peptides at physiological pH. Contacting a protein substrate with glutaminase also allows for the production of peptides having potentially enhanced biological activity. The protein substrate that has been at least partially hydrolysed by glutaminase is then contacted with the other hydrolytic enzymes in series. In another embodiment, the final enzyme with which the partially hydrolysed protein substrate is contacted is trypsin. The use of trypsin as a final enzyme advantageously achieves digestive stability of the peptide fraction produced by the process of the invention and which contains a heterogeneous mixture of peptides. In the process of the invention, subtilisin, oryzin and leucyl aminopeptidase may be used in any order so as to produce heterogeneity in peptide length, sequence and biological activity. Preferably, however, the protein substrate is contacted with the enzymes in a series selected from: a) glutaminase, subtilisin, oryzin, leucyl aminopeptidase, and trypsin, b) glutaminase, oryzin, subtilisin, leucyl aminopeptidase, and trypsin, and c) glutaminase, leucyl aminopeptidase, oryzin, subtilisin, and trypsin. For hydrolysis with the aforementioned enzymes, protein substrates or processed products thereof are dissolved or suspended in a suitable buffer. Such buffers that are suitable for hydrolysis are well known to those skilled in the art and include Tris, triethanolamine (TEA), sodium borate, PBS, HEPES amongst other well known buffers. Determining a suitable buffer, its concentration and pH for performing hydrolysis is a matter of routine for the person skilled in the art. Typically the pH of the buffer will fall within the range of pH 6.0 to pH 9.0, but preferably will be about pH 6.0 to about pH 8.0, and in one embodiment is about pH 7.4. The buffer may also comprise other components such as cofactors or small quantities of organic solvent to increase the solubility of the protein substrate. One example of a buffer suitable for use in the process of the present invention is 10 mM TEA, 10% ethanol, pH 7.4. The amount of protein in the protein substrate suspension or solution may vary. For example, the solution or suspension may contain about 5% to about 25% protein (w/w), or preferably about 10% to about 20% protein (w/w). Other reaction conditions such as reaction time and incubation temperature can be readily determined by the skilled person. For example, a protein substrate may be reacted with each enzyme for a period from about 5 minutes to about 24 hours, preferably about 30 minutes to 2 hours. In one embodiment, the protein substrate is reacted with each enzyme for about 1 hour. The temperature of the hydrolysis reaction will depend on the particular enzyme that is being reacted with the protein substrate. For example, glutaminase, leucyl aminopeptidase, subtilisin and oryzin may be reacted with the protein substrate at about 45°C to about 55 0C, preferably at about 500C. For trypsin, the temperature of the reaction is preferably about 37°C and the reaction is an hour or more, or alternatively at least 2, 3, 5, 10 or more hours. An exemplary hydrolysis reaction suitable for use in the process of the present invention involves dissolving or suspending a protein substrate or processed product thereof at about 10% total protein (w/w) in 10 mM TEA, 10% EtOH, pH 7.4 and maintained at pH 7.4 throughout the hydrolysis process. After rehydration, the first enzyme, for example, Glutaminase may be added to a final concentration of 0.5% (w/w) and incubated with agitation at 500C for 1 hour. The enzymes subtilisin (Alcalase) (0.5%, w/w), oryzin (Corolase) (0.5%, w/w) and leucyl aminopeptidase (Flavourzyme) (0.5%, w/w) may be subsequently introduced sequentially at 1 hr intervals at 500C, according to one of 3 possible sequences. When used as the final enzyme, trypsin (0.5%, w/w) may be introduced to the mixture and incubated at 37°C for 17 hours (overnight). Following hydrolysis, the enzymes may be inactivated, for example, by heating at 900C for 30 min. In the exemplary hydrolysis reaction, each of the enzymes is added to the reaction mixture sequentially, with each enzyme at least partially hydrolysing the protein substrate prior to the addition of the next enzyme. In this exemplary reaction, the enzymes are not heat inactivated before the addition of the next enzyme in the series. Further, the partially hydrolysed protein substrate is not isolated or purified between each reaction with a different enzyme. The skilled person would understand, however, that following the at least partial hydrolysis of the protein substrate with an enzyme, the enzyme could be heat inactivated prior to the addition of the next enzyme in the series. Alternatively, if desired, the at least partially hydrolysed protein substrate could be isolated or purified prior to contacting the substrate with the next enzyme in the series.

Sizefractionation Following hydrolysis of the protein substrate, peptides of about 11 kDa or less are selected. In one embodiment, the process of the invention involves selecting peptides about 8 kDa or less. There are several techniques known in the art for separating peptides based on size. By way of non-limiting example, peptides of about 11 kDa or less may be selected by dialysis, membrane filtration or size-exclusion chromatography. Recovery of a peptide fraction by dialysis may be achieved by performing dialysis of the hydrolysate with a suitable dialysis membrane such as a regenerated cellulose or cellulose ester dialysis tubing with an appropriate molecular weight cut-off. Membrane filtration is a process for separating small particles and dissolved molecules from fluids and is suited to the selection of peptides about 11 kDa or less. A membrane's pore size rating, typically given as a micron value, indicates that peptides, proteins or other molecules larger than the rating will be retained. Ultrafiltration membranes which are particularly suited to selecting peptides are rated according to the nominal molecular weight limit (NMWL), also sometimes referred to as molecular weight cut-off (MWCO). The NMWL indicates that most dissolved macromolecules with molecular weights higher than the NMWL will be retained. Typically membranes are made of materials such as regenerated cellulose and microporous PVDF. As would be understood by one of skill in the art, the retention and recovery of peptides by membrane filtration may be affected by other factors, including the molecular shape and size of the molecule; electrical characteristics; sample concentration and composition; operating conditions; and device or system configuration. Two membranes may have the same NMWL but will exhibit different retention of molecules within a relatively narrow range of sizes. In addition, slender, linear molecules may find their way through pores that will retain a globular species of the same weight. Retention can also be affected by hydration with counter-ions. The person skilled in the art would routinely be able to determine an appropriate membrane for use in the process of the invention to select peptides about 11 kDa or less.

Chargefractionation The present inventors found that the fractionation of the selected peptides at physiological pH to resolve neutral, cationic and anionic fractions further permits the enrichment of fractions with bioactivity as may be required in a particular physiological setting. Thus in one embodiment, the process of the invention may optionally include the step of separating the peptides into one or more of a cation, anion and/or neutral peptide fraction. Techniques for separating peptides based on charge are known to those skilled in the art. One technique that is suited to the process of the invention is ion-exchange, for example ion-exchange chromatography. As is known to the person skilled in the art, Ion Exchange Chromatography relies on charge-charge interactions between the proteins in a sample and the charges immobilized on a chosen resin. Ion exchange chromatography can be subdivided into cation exchange chromatography, in which positively charged ions bind to a negatively charged resin; and anion exchange chromatography, in which the binding ions are negative, and the immobilized functional group is positive. As used herein, the term "neutral peptide fraction" refers to a peptide fraction which has no net charge and does not bind to either a cation exchange resin or anion exchange resin at a given pH. Any type of ion-exchange material can be used. For example, ion exchange resins can be cationic, anionic, mixtures of cation and anionic, or biologically related. Examples of ion exchange resins useful in this invention include, but are not limited to, those made of cross-linked polyvinylpyrolodone and polystyrene, and those having ion exchange functional groups such as, but not limited to, halogen ions, sulfonic acid, carboxylic acid, iminodiacetic acid, and tertiary and quaternary amines. Examples of biologically related resins that can be used in the process of the invention include, but are not limited to, Sephadex™, CM C-25, CM C-50, DEAE A-25, DEAE A-50, QAE A-25, QAE A-50, SP C-25, and SP C-50. These cationic, anionic, mixed cationic and anionic, and biologically related ion exchange resins are commercially available. A variety of buffers at different pH values (e.g., Tris-HCL, HEPES, and multi- pH phosphate buffers) can be used to tailor charge distribution. A linear or step gradient of pH or salt can be used to elute peptide fractions from ion-exchange media. As would be understood in the art, the charge on a peptide affects its behaviour in ion-exchange chromatography. Peptides may contain ionizable groups on the side chains of their amino acids as well as their amino- and carboxyl-termini. These include basic groups on the side chains of lysine, arginine and histidine and acidic groups on the side chains or glutamate, aspartate, cysteine and tyrosine. The pH of the solution, the pK of the side chain and the side chain's environment influence the charge on each side chain. In one exemplary method of ion-exchange chromatography suitable for use in the process of the invention, cation exchange (SP Sepharose BB) and anion exchange (Q Sepharose BB) columns may be connected in series. Batches of size selected peptides may be loaded onto the columns with non-binding neutral peptide fractions collected. The columns may then be uncoupled and the fraction bound to the cation exchange column eluted with a 1 M NaCl solution for the recovery of a cationic peptide fraction. The fraction bound to the anion exchange column may be eluted with 1 M NaCl to recovery an anionic peptide fraction. Using such an exemplary method, a size selected peptide fraction may be further fractionated into non-binding (neutral), cationic and anionic fractions. Testing the peptide fractions for biological activity The present inventors have found that the peptide fractions of the invention may exhibit biological activity and be useful for the modulation of biological pathways associated with disease. As used herein, a "biologically active" peptide or peptide fraction, or a peptide or peptide fraction having "biological activity" may be one which is capable of regulating or modulating for example, the cardiovascular system such as by modulating blood pressure, the immunohemopoietic system, or which may affect the viability, growth and differentiation of a variety of normal or neoplastic cells in the body, or which modulates immune-mediated inflammatory or autoimmune disorders, or neurological disorders. Alternatively, the biologically active peptide may be one which is capable of affecting immune regulation or which is capable of enhancing or inducing resistance to infection of cells and tissues, or which modulates energy metabolism as relevant to diabetes and metabolic disease. Non-health related biological activity includes anti-fibril chaperone activity useful for the stabilisation of proteins during thermal processing. There are many assays known to those skilled in the art for determining whether a peptide fraction of the invention has a particular biological activity. Such assays include, by way of non-limiting example, assays for determining whether a peptide fraction of the invention has angiotensin converting enzyme (ACE) inhibition activity, angiotensin II receptor inhibition activity, anti-inflammatory activity or fibril inhibition activity. ACE inhibition activity may be determined, for example, by monitoring the decrease in absorbance at 340 nm as a result of ACE-mediated hydrolysis of the substrate 3-(2-furylacryloyl)-L-phenylalanylglyclglycine (FAPGG) (Sigma-Aldrich, Sydney, Australia) to 3-(2-furylacryloylphenylalanine (FAP) and glyclglycine (GG) (Harjanne, 1984) in the presence of a peptide fraction of the invention. Anti-inflammatory activity may be determined, for example, by generating a ThI response in human peripheral blood mononuclear cells culture by the addition of IL-7 and IL- 12. The stimulation of naϊve T-cells to mature into ThI cells causes the production of ThI associated cytokines including IFN-γ (Gutcher and Becher, 2007). A decrease in the level of IFN-γ in a sample comprising a peptide fraction of the invention relative to a control culture is indicative of anti-inflammatory biological activity. One example of an assay for determining fibril inhibition activity of a peptide fraction utilises reduced and carboxymethylated κ-casein (RCM-K-CN) which forms amyloid fibrils under physiological conditions. The RCM-K-CN is incubated in the presence and absence of test peptide fractions and the formation of amyloid fibrils monitored. Other suitable assays for determining a biological activity of a peptide fraction of the invention would be know to those of skill in the art.

Food and drink products and nutritional supplements In one embodiment of the invention, a peptide fraction obtained by the process of the invention is included in a food or drink product. Food or drink products according to the invention can be prepared by the skilled person using known techniques. Non-limiting examples of such food or drink products are baked goods, dairy type foods and drinks, snacks, etc. The amount of the peptide fraction of the invention to be used in a food or drink product can be variable as the peptides themselves are nutrients. In one embodiment, the food or drink product is a nutritional supplement. As used herein, a "nutritional supplement" is an orally ingestible product consumed to improve overall nutrition, health, well-being, or performance of a subject in an activity and/or an orally ingestible product which provides additional perceived nutritional or biological benefit to a subject. As would be understood, the nutritional supplement may be provided in a concentrated form, thus allowing for the addition of the nutritional supplement to a food or drink product to allow for the consumption of a desired quantity of a peptide fraction of the invention in a reasonable serving size. The food or drink product of the present invention can include additional ingredients including an orally ingestible diluent or carrier. Many orally ingestible diluents or carriers are known in the food sciences. These include, but are not limited to, manufactured cereals, fruit or vegetable products, beverages or beverage concentrates, ground meat products or vegetable analogues thereof, and any inert diluent, carrier, or excipient known in the pharmaceutical art. Preferably, the peptide fractions of the current invention constitute from about 0.0001 to about 10.0% by weight of the food or drink product. The nutritional supplement of the current invention can include additional ingredients. In some embodiments, more than one of the peptide fractions of the current invention can be included in the same nutritional supplement formulation. Other additional ingredients include any ingestible product. Preferred additional ingredients include, but are not limited to, other active food supplement ingredients such as vitamins and minerals. The food additive may also include acceptable dispersing and suspending agents, and water. Other conventional nutritional supplements can also be included if desired. The nutritional supplement can take many forms including, but not limited to, powders, tablets, capsules, solutions, concentrates, syrups, suspensions, or dispersions.

Therapeutic methods The peptide fractions obtained by the methods of the invention are of use for the inhibition of biological pathways associated with disease including cardiovascular disease, cancer, inflammatory or autoimmune disease, or neurological disorders. Thus, in one aspect, the present invention provides methods of treating or preventing disease by administering to a subject a peptide fraction of the invention or a composition of the invention. In one embodiment, a peptide fraction of the invention may be used in the treatment or prevention of cardiovascular disease in a subject, such as for example, for lowering blood pressure in a subject. Using the peptide fractions of the invention to lower blood pressure in a subject may decrease the risk of, for example, heart failure, kidney failure, and the harmful effects of diabetes. In addition, the peptide fraction of the invention may be used for the treatment or prevention of cardiovascular diseases including, but not limited to hypertension, atherosclerosis and arteriosclerosis. The peptide fractions of the invention may be useful in the treatment or prevention of cancer in a subject, including, but not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include breast cancer, prostate cancer, colon cancer, squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, ovarian cancer, cervical cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, liver cancer, bladder cancer, hepatoma, colorectal cancer, uterine cervical cancer, endometrial carcinoma, salivary gland carcinoma, mesothelioma, kidney cancer, vulval cancer, thyroid cancer, hepatic carcinoma, skin cancer, melanoma, brain cancer, neuroblastoma, myeloma, various types of head and neck cancer, acute lymphoblastic leukemia, acute myeloid leukemia, Ewing sarcoma and peripheral neuroepithelioma. The peptide fractions obtained by the methods of the invention are of use for the inhibition of biological pathways associated with disease including inflammatory and autoimmune disease and/or neurological disorders. Inflammatory disorders and autoimmune diseases which may be treated with peptide fractions of the invention include asthma, rheumatoid arthritis, inflammatory bowel disease, psoriasis and multiple sclerosis. Autoimmune diseases arise from an overactive immune response of the body against substances and tissues normally present in the body. Examples of autoimmune diseases include Addison's disease, alopecia areata, ankylosing spondylitis, antiphospholipid antibody syndrome (APS), autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, bullous pemphigoid, coeliac disease, chagas disease , chronic obstructive pulmonary disease, Crohns disease (one of two types of idiopathic inflammatory bowel disease "IBD"), dermatomyositis, diabetes mellitus type 1, endometriosis, goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome (GBS), Hashimoto's disease, hidradenitis suppurativa, Kawasaki disease, IgA nephropathy, idiopathic thrombocytopenic purpura, Interstitial cystitis, lupus erythematosus, mixed connective tissue disease, morphea, multiple sclerosis (MS), myasthenia gravis, narcolepsy, neuromyotonia, pemphigus vulgaris, pernicious anaemia, psoriasis, psoriatic arthritis, polymyositis, primary biliary cirrhosis, rheumatoid arthritis, scleroderma, Sjogren's syndrome, ulcerative colitis, vasculitis, vitiligo and Wegener's granulomatosis. The peptide fractions of the present invention may also be used for the treatment or prevention of a neurological disorder, such as, but not limited to Alzheimer's disease, Parkinson's disease, dementia, Huntington's disease, Shy-Drager syndrome, progressive supranuclear palsy, Lewy body disease or amyotrophic lateral sclerosis.

Compositions and administration In certain embodiments, the present invention provides compositions comprising a peptide fraction of the invention and a suitable carrier or excipient. In one embodiment, the composition is a pharmaceutical composition comprising a pharmaceutically acceptable carrier. The peptide fractions are incorporated into pharmaceutical compositions suitable for administration to a mammalian subject, e.g., a human. Such compositions typically comprise the "active" compound (i.e. the peptide fraction) and a "pharmaceutically acceptable carrier". As used hereinafter the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, such media can be used in the compositions of the invention. Supplementary active compounds can also be incorporated into the compositions. A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral (e.g., intravenous, intradermal, subcutaneous, intramuscular, intraperitoneal, intrathecal), mucosal (e.g., oral, rectal, intranasal, buccal, vaginal, respiratory), enteral (e.g., orally, such as by tablets, capsules or drops, rectally) and transdermal (topical, e.g., epicutaneous, inhalational, intranasal, eyedrops, vaginal). Solutions or suspensions used for parenteral, intradermal, enteral or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier is a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity is maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms is achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid and the like. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions is brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions may be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound is incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions are also prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Systemic administration can also be by mucosal or transdermal means. For mucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for mucosal administration, detergents, bile salts, and fusidic acid derivatives. Mucosal administration is accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. A pharmaceutically acceptable vehicle is understood to designate a compound or a combination of compounds entering into a pharmaceutical composition which does not cause side effects and which makes it possible, for example, to facilitate the administration of the active compound, to increase its life and/or its efficacy in the body, to increase its solubility in solution or alternatively to enhance its preservation. These pharmaceutically acceptable vehicles are well known and will be adapted by persons skilled in the art according to the nature and the mode of administration of the active compound chosen.

EXAMPLES Example 1. Protocols for preparation of protein substrates Materials Chlorhexidine gluconate (20%) and Tris-Hydrochloride (Tris-HCl) were from ICN Biomedicals, Aurora, Ohio. Spectra/Por regenerated cellulose membranes with molecular weight cut-off (MWCO) 6000-8000 Daltons were obtained from Spectrum Laboratories, Inc., Dominguez, CA. Ethanol (EtOH) was from CSR Distilleries, Victoria, Australia. Triethanolamine (TEA), Phosphate buffered saline (PBS) tablets, Sodium bicarbonate (NaHCO3), Ethylenediaminetetraacetic acid (EDTA), L-cysteine, polyvinylpyrrolidone (PVP) and Sodium acetate were from Sigma Chemical Co., St. Louis, MO, USA. Sodium Chloride (NaCl) and Magnesium chloride (MgC^) were obtained from Univar, NSW, Australia. Isopropanol (IP) and sodium dodecyl sulphate (SDS) were from BDH, VWR International Ltd., Poole, England. Urea and Acetic acid were from Merk, Darmstadt, Germany. Ammonium Hydroxide (25%) was from BASF, Singapore. SPI extraction buffer: 20 mM Tris-HCl, pH 8.0, 10 mM NaHCO3, 10 mM MgC12, 1 mM EDTA, 1 mM L-Cysteine, 1% insoluble PVP. 5.0 M acetic acid. Water soluble protein extractant: 0.5 M NaCl. Prolamins extractant: 50% IP containing 1% acetic acid and 1% L-Cysteine. Glutelins extractant: 4 M urea, 1% ammonium hydroxide, 1% L-Cysteine dissolved in water. Protocol 1. No further processing Substrates purchased from commercial sources were hydrolysed without further processing.

Protocol 2. Commercial substrates desalted prior to hydrolysis Substrate reconstituted to 10% total solids in milliQ water and placed in regenerated cellulose dialysis tubing (6-8 KDa MWCO). Dialysis was conducted against 2 changes of 0.001% Chlorhexidine gluconate and 3 changes of distilled water over 48 hours. The resulting substrate free of minerals and salts was freeze-dried and stored at -200C pending hydrolysis.

Protocol 3. Desalting of skimmed milk by ultra-filtration 10 litres of fresh milk was skimmed by centrifugation (3000g /30 min, J6-HC, Beckman Coulter, Inc. Fullerton, CA, USA) at 100C. The skimmed milk was processed through a 1OK (SlYlO, Millipore, Billerica, MA, USA) membrane to concentrate to 500 ml and diafiltered with 5 volumes of distilled water. The concentrated purified retentate was freeze dried and stored at -200C pending hydrolysis.

Protocol 4. Substrate denaturation by high pressure processing (HPP) Substrate reconstituted to 1% total protein (w/w) in 10% ethanol and subjected to 600 MPa pressure in HPP unit (QFP 35L, Flow Pressure Systems, S-72166 Vasteras, Sweden) for 15 minutes at 22°C. The resulting denatured substrate was freeze-dried and stored at -200C pending hydrolysis.

Protocol 5. Commercially available probiotic cells lysed by French pressing Materials CHR-HANSEN La-5: (Direct Vat Set (DVS)) Thermophilic Lactic Culture nu- trish. Lot 264795; DOM 28. 10.2005; BBD 29. 10.2007. DSM DELVO Pro LAFTI-L26: Direct Set Lyophilized starter culture (DSL); Lot 129338/10; BBD 14./01.2007; 10 units/bag.

Method 45 g of freeze dried probiotic cell culture was washed 3 times with phosphate buffered saline (PBS) by suspension and centrifugation (14,000rpm/30,100g for 20 mins at 40C (J2-MC centrifuge, JA- 14 rotor, Beckman Coulter, Inc., Fullerton, CA, USA)) in 205 g of PBS. The washed cell pellet was re-suspended in equal mass of hydrolysis buffer (10 mM TEA in 10% ethanol) and lysed by three consecutive passes through the French press (manufactured at Melbourne University, Victoria, Australia as described in Vanderheiden et al. (1970). The cells were cooled on ice between passes to prevent over heating. The lysed cell slurry preparations were stored at -200C pending hydrolysis.

Protocol 6. Fresh fish fillet purchased from local fish-monger/supermarket and freeze- dried 500 g of fish fillet (deboned and skin removed) were cut into 2 cm3 pieces and freeze-dried. The freeze-dried pieces were ground to fine powder in a blender (Semak Australia, Victoria, Australia) and stored at -200C pending hydrolysis.

Protocol 7. Fresh meat portions purchased from local butcher/supermarket and oven- dried 1 kg of fat-free meat portions were cut into 2 cm pieces and minced using a meat grinder (HV6, Moulinex, France). The minced meat was oven dried (Rational combi-Dampfer oven, Comcater, Victoria, Australia) at 1200C for 2 hours. The dried mince was ground to fine powder in a blender (Semak Australia, Victoria, Australia) and stored at -200C pending hydrolysis.

Protocol 8. Preparation of spinach protein isolate (SPI) for hydrolysis 12 kg of spinach (roots discarded) was washed and chopped coarsely. The leaves were blanched in 2 litres of boiling water to inactivate enzymes and soften cellulosic structures and chilled immediately. 5 volumes of SPI extraction buffer was added and leaves homogenised using a blender (Semak Australia, Victoria, Australia). The mixture was centrifuged (4200rpm /30 min, J6-HC, Beckman Coulter, Inc., Fullerton, CA, USA) and supernatant strained through a sieve (50 µm) to remove solids. The clear filtrate was processed through UF membrane (10 K- SlYlO, Millipore, Billerica, MA, USA) to remove salts, using 5 volumes of 10% ethanol, before concentrating to 5 litres. 2.5 litres of retentate was processed as per Protocol 4 to denature the proteins and freeze-dried and stored at -200C pending hydrolysis. Remaining portion of retentate was Freeze dried (native SPI) and stored at-20°C pending hydrolysis. Protocol 9. Preparation of sheep wool and chicken feathers The substrate was washed using distilled water and excess moisture was removed by shaking and blotting in absorbent paper. The clean wool/feathers were chopped into pieces (2 - 6mm) with the help of sharp scissors.

Protocol 10. Preparation of lactoferrin-enriched bovine colostral whey The pH of bovine colostrum was adjusted to 4.6 with 5.0 M acetic acid and the mixture was warmed to 400C for 30 min (to melt fat) in a water bath. Fat was removed by centrifugation (3000g /30 min, J6-HC, Beckman Coulter, Inc.Fullerton, CA, USA) at 100C. After discarding fat, the supernatants (colostral whey) were pooled and diluted by 1/3 using distilled water. The pH was readjusted to 5.2 using 5.0 M sodium acetate and the whey was processed through 5 m2 UF plant (30 K membrane). Retentate was diafiltered 1 fold with distilled water. The retentate was collected; spray dried and stored at -200C pending hydrolysis.

Protocol 11. Milling of seed grains Seeds were cleaned of any extraneous matter and milled using a blender (Semak Australia, Victoria, Australia) or hammer mill (> 2kg, Ernest Fleming Pty Ltd, New South Wales, Australia) to a particle size range of 0.5 mm- 1.2 mm.

Protocol 12. Preparation of albumin extract from seed grains

2 kg of millet was prepared as per Protocol 11. Water-soluble proteins (albumin) were extracted by adding 2 litres of 0.5 M NaCl to the millet sample and mixing vigorously by overhead stirring at room temperature (RT) for 1.0 hr. The soluble proteins were recovered by centrifugation (4200rpm /30 min, J6-HC, Beckman Coulter, Inc., Fullerton, CA, USA) at RT and the process was repeated with 3 litres of 0.5 M NaCl. The supernatants (pellet was saved for protocol 12) were pooled and processed through a IK (Prep scale TFF, Millipore, Billerica, MA, USA) membrane to concentrate to 200 ml and diafiltered with 5 volumes of distilled water. The concentrated desalted retentate was freeze dried and stored at -200C pending hydrolysis.

Protocol 13. Preparation of prolamin extract from panorama millet Alcohol soluble proteins (Prolamins) were extracted by adding 2 litres of prolamin extractant to the millet solid fraction from Protocol 12 and mixing vigorously by overhead stirring at RT for 1.0 hr. The soluble proteins were recovered by centrifugation (4200rpm /30 min, J6-HC, Beckman Coulter, Inc.Fullerton, CA, USA) at RT and the process was repeated with 3 litres of prolamin extractant. The supernatants (pellet was saved for protocol 14) were pooled and 2 volumes of 1.5 M NaCl was added to allow protein fraction to precipitate overnight at 4°C. The precipitate was recovered and placed in regenerated cellulose dialysis tubing (6-8 KDa MWCO). Dialysis was conducted against 2 changes of 0.001% Chlorhexidine gluconate and 3 changes of distilled water over 48 hours. The resulting desalted protein solution was freeze-dried and stored at -200C pending hydrolysis.

Protocol 14. Preparation of glutelin extract from panorama millet Alkali soluble proteins (Glutelins) were extracted by adding 2 litres of glutelin extractant to the millet solid fraction from Protocol 13 and mixing vigorously by overhead stirring at RT for 1.0 hr. The soluble proteins were recovered by centrifugation (4200rpm /30 min, J6-HC, Beckman Coulter, Inc., Fullerton, CA, USA) at RT and the process was repeated with 3 litres of glutelin extractant. The supernatants were pooled and placed in regenerated cellulose dialysis tubing (6-8 KDa MWCO). Dialysis was conducted against 2 changes of 0.001% Chlorhexidine gluconate and 3 changes of distilled water over 48 hours. The resulting desalted protein solution was freeze-dried and stored at -200C pending hydrolysis.

Protocol 15. Preparation of total protein extract from panorama millet

The wet extracts from Protocol 12, 13 and 14 were pooled together and mixed thoroughly before placing in regenerated cellulose dialysis tubing (6-8 KDa MWCO). Dialysis was conducted against 2 changes of 0.001% Chlorhexidine gluconate and 3 changes of distilled water over 48 hours. The resulting desalted protein solution was freeze-dried and stored at -200C pending hydrolysis.

Protocol 16. Preparation offish meal protein isolate (FMPI) A 10% solution of fishmeal in 5% SDS was homogenised using Ultra-turrax (T25, Crown Scientific P/L, NSW, Australia) for 5 minutes at 13500 rpm and heated to 800C for 1 hour. The mixture was cooled to room temperature and centrifuged (RC5C Sorvall Instruments, Minnesota, USA) at 8000 rpm for 20 minutes. The supernatant was filtered and processed through a 3K (S1Y3, Millipore, Billerica, MA, USA) membrane to concentrate to 200 ml and diafiltered with 3 volumes of distilled water. The concentrated purified retentate was freeze dried and stored at -20 0C pending hydrolysis.

Example 2. Preparation of peptide library products Materials All reagents are approved for use in food processing according to Standard 1.3.3 describing the use of processing aids (Food Standards Australia New Zealand). All laboratory-scale processing involved reagent or better grade reagents whereas all materials used in the pilot plant were food grade. Triethanolamine (TEA) was from Sigma Chemical Co., St. Louis, MO, USA. Ethanol (EtOH) was from CSR Distilleries, Victoria, Australia. Sodium Chloride (NaCl) was obtained from Univar, NSW, Australia. Alcalase 2.4L, Flavourzyme IOOOL and Trypsin enzymes were obtained from Novozymes, Bagsvaerd, Denmark. Corolase PN-L was obtained from AB Enzymes GmbH, Darmstadt, Germany. Glutaminase was obtained from Daiwa Kasei K.K., Shiga, Japan. Dialysis membrane (Spectra/Por regenerated cellulose, molecular weight cut-off (MWCO) 6000-8000 Daltons) were from Spectrum Laboratories, Inc., Dominguez, CA. SP and Q Sepharose Big Beads ion exchange resins (BB) were obtained from GE Healthcare, Uppsala, Sweden.

Substrate hydrolysis Food source refers to the original material from which a protein-rich 'substrate' was prepared, where necessary. In some cases, the food source and substrate, e.g., dairy whey protein isolate, were one and the same material. In other cases, e.g., Panorama millet, a protein-rich substrate was prepared from the original food source. If not available in a powder form, all food sources were washed and ground or milled to a particle size range of 0.5 to 1.2 mm before further processing. Methods for preparation of protein-rich substrates from specific food sources are described for specific 'leads' in tables below. Substrates were dissolved or suspended at 10% total protein (w/w) in 10 mM TEA, 10% EtOH, pH 7.4 and maintained at pH 7.4 throughout the hydrolysis process. After rehydration, the first enzyme Glutaminase was added to a final concentration of 0.5% (w/w) and incubated with agitation at 500C for 1 hour. The enzymes Alcalase (0.5%, w/w), Corolase (0.5%, w/w) and Flavourzyme (0.5%, w/w) were subsequently introduced sequentially at 1 hr intervals at 500C, according in one of 3 possible sequences (Table 1). The final enzyme Trypsin (0.5%, w/w) was introduced to the mixture and incubated at 37°C for 17 hours (overnight). The enzymes were inactivated by heating at 900C for 30 min, and the product cooled to 220C for at least 15 mins.

Table 1. Use of enzyme sequence to generate molecular heterogeneity for bioactivity discovery.

Fractionation of substrate hydrolysates For each hydrolysate product, the molecular size fraction <8 KDa was recovered by dialysis using regenerated cellulose membrane (6-8 KDa MWCO). Each dialysate was subjected to ion exchange chromatographic fractionation, as follows. Peptide fractionation was performed using 2 columns (4.6 x 10 cm) connected in series. Column 1 was packed with cation exchange resin (SP Sepharose BB) and Column 2 was packed with anion exchange resin (Q Sepharose BB) The eluant was monitored by UV detection at 280 nm (Shimadzu SPD-10AV, Kyoto, Japan). Batches of dialysate (400 ml) were loaded onto the two columns with 400 ml of dialysate collected containing non-binding species (designated 'IEO' fraction), corresponding to recovery of non-binding species absorbing at 280 nm. The columns were uncoupled and the fraction bound to the cation exchange column was eluted in 200 ml of 1 M NaCl solution (designated 'IE+' ), corresponding to recovery of cationic species absorbing at 280 nm. Finally, the fraction bound to the anion exchange column was eluted in 200 ml of 1 M NaCl solution (designated 'IE-' ), corresponding to recovery of anionic species absorbing at 280 nm. Thus, each dialysate was fractionated into 3 products, yielding a total of 9 distinct products from each protein substrate (Table 2) or 6 different products where the substrates were commercially available hydrolysates (Table 3). All products were freeze dried and stored at -200C pending bioactivity testing and other methods of characterisation.

Table 2. Use of enzyme sequence and chromatographic fractionation to generate molecular heterogeneity for bioactivity discovery, yielding 9 distinct products from each protein substrate, as used in unhydrolysed form. When required, ion-exchange fractionation of the hydrolysate was omitted (NIE).

Table 3. Use of enzyme sequence and chromatographic fractionation to generate molecular heterogeneity for bioactivity discovery, yielding 6 distinct products from each substrate, where substrates were commercially available hydrolysates. Example 3. Angiotensin converting enzyme (ACE) inhibition activity Assay protocol The assay for the determination of pig kidney ACE (EC 3.4.15.1) (Sigma- Aldrich, Sydney, Australia) employed a COBAS BIO autoanalyser (Roche Diagnostics Ltd, Rotkreuz, Switzerland) which monitored the decrease in absorbance at 340 nm as a result of ACE-mediated hydrolysis of the substrate 3-(2-furylacryloyl)-L- phenylalanylglyclglycine (FAPGG) (Sigma-Aldrich, Sydney, Australia) to 3-(2- furylacryloylphenylalanine (FAP) and glyclglycine (GG) (Harjanne, 1984). Initially µ µ 250 L reagent buffer as 100 mM Tris-HCl pH 8.3, 300 mM NaCl, 10 M ZnCl2 containing ACE at 0.032 U/ml was loaded from a buffer boat and 20 µL of test sample (10-50 mg/ml) or known ACE inhibitors such as captopril (1-100 nM) or the tripeptide valinealanineproline (VAP, 0.1-20 µM) (Auspep Pty Ltd, Melbourne, Australia) found in a s1-casein from the sample carousel are transferred into the multiple cuvette and spun in 2 minutes to warm to 37°C. At that time 50 µL of substrate FAPGG at final concentration 0.5 mM is transferred to the plastic multiple cuvette and quickly mixed and spun with the decrease in OD monitored from zero time every two minutes for 16 minutes which was in the linear range of the assay. The final ACE concentration was 0.025U/ml.

Results A broad range of substrates yielded peptide products with significant capacity for inhibition of ACE (Table 4). Substrates included several classes of dairy protein, vegetable, grain, yeast and meat protein sources. In vitro activity was significantly associated with IEO chromatographic fractions and all enzyme sequence codes were represented. In vivo bioactivity appeared to be associated with IE+ fractions.

Table 4. Angiotensin converting enzyme inhibition bioactivity of food protein-derived peptide fractions. Example 4. Angiotensin II receptor inhibition activity Assay protocol Rat livers or pig hearts were snap frozen in liquid nitrogen and coarsely ground in a pestle and mortar and membranes prepared by differential centrifugation (Leifert et al., 2009). To 1 g of frozen tissue was added 10 ml of ice cold isolation medium containing (in mM) 250 sucrose, 50 mM Tris, 1 MgCl2, 1 EDTA, pH 7.4 and homogenised with an Ultra-Tarrax (Jankee & Kundel GmbH, Staufen, Germany) on setting 4 for 3 bursts of 10 seconds with a short rest in between bursts. The solution was filtered through two pieces of nylon mesh and washed through with 5 ml of isolation media and centrifuged at 3,000 x g for rat liver or 6,000 x g for pig heart in a J2-21 centrifuge (Beckman Instrument Co., Palo Alto, CA) for 30 min at 4°C. The supernatant was decanted and retained and the pellet resuspended in isolation medium and centrifuged as before. The supernatants were combined and centrifuged at 46,000 x g for 30 min. The final pellet was suspended (in mM) 50 Tris, 10 MgCl2, 1 EGTA, pH 7.4 assay buffer with 5 stokes of a dounce glass/Teflon homogeniser that contained protease inhibitors diluted into the buffer at 1:1000 (vol/vol). The stock protease inhibitor concentrations were (mg/ml, solvent) phenylmethylsulfonyl fluoride 17.4,

DMSA; aprotinin 1, H2O; iodoacetate 184, H2O; and pepstatin A 1.37, DMSO. The final stock concentration of liver or heart membrane preparations was 1-2 mg/ml and stored in aliquots at -800C.

Assay of 125I-Sa^-IIe 8I angiotensin II receptor binding to rat liver membrane Receptor radioligand binding activity was determined for rat liver membrane fraction (3,000-46,000 x g) with the All antagonist [^IHSar'-Ile^-angiotensin II (2200 Ci/mmol, 10 µCi in 200 µl assay buffer diluted to 1 nM stock) (Perkin Elmer, MA, USA) which is specific for the ATi receptor at sub K concentration of 0.1 nM (Del Carmen Caro et al., 1998; Leifert et al., 2009). The assay final volume of 75 µl in a 4 ml plastic tube contained (in mM) 90 Tris, 10 MgCl2, 1 EGTA, pH with 0.2% BSA with buffer or the antagonists losartan (10-1000 nM), saralasin (1-100 nM), PD12319

(10-10,000 nM, AT2 antagonist) or agonist angiotensin II (1-100 nM) (all Sigma- Aldrich, Sydney, Australia) or the prepared library fractions or products. Peptides or plant products were dissolved in 10% ethanol (v/v) in (mM) 90 Tris, 10 MgCl2, 1 EGTA adjusted to pH 7.4 with 2 N NaOH, centrifuged at 8,000 x g (Sigma 113 Laborzentrifugen, Ostrode am Harz, Germany) and added at 0.01-10 mg/mL final in the assay. The assay contents were pre-incubated with of 7 µl of rat liver membrane preparation containing approximately 7 µg protein for at least 10 min. The assay is commenced by the addition of 7.5 µl 1 nM [125I]-[Sar 1-Ile8]-Angiotensin II (0.1 nM final) and the binding reaction allowed to proceed for 50 min while shaking at 25°C (Del Carmen Caro et al., 1998). Non specific binding was determined in the presence of unlabelled All or losartan and did not exceed 5%. To separate bound radioligand from free unbound radioligand, 3 ml of ice cold was buffer (mM) 90 Tris, 10 MgCl2, 1 EGTA, pH 7.4 was added to the assay tube and the contents rapidly filtered over GF/C filters (Whatman International Ltd, Maidstone, England) that were pre-soaked in wash buffer containing 1% polyethylenimine (Sigma-Aldrich, Sydney, Australia) and washed 3 more times. Total counts on the GF/C filters were measured in a gamma counter (LKB Wallac Multigamma, Stockholm, Sweden).

Results A range of substrates yielded peptide products with significant capacity for inhibition of angiotensin 1 receptor. Substrates included several classes of dairy protein, vegetable and cellular sources. In vitro activity was significantly associated with IE+ chromatographic fractions and only enzyme sequence codes 1-3 were represented indicating that commercial hydrolysates tested were not bioactive in this assay. A selection of substrates, required non-thermal, high pressure protein denaturation to release bioactive peptides.

Table 5. Summary of Angiotensin I receptor inhibition bioactivity of food protein- derived peptide fractions generated using systematic method of hydrolysis and chromatographic fractionation. Example 5. In vivo testing protocol in spontaneous hypertensive rat study 1 The lead anti-hypertensive products were tested in the Spontaneous Hypertensive rat model by dietary intervention over 20 weeks of feeding. The test feed was given as a mixture of leads comprising P0147+P0148+P0052 (1:1:1). Rats were randomised into 4 treatment groups based on the following dietary interventions (control, dairy lead mixture at 20 mg/kg, dairy lead mixture at 200 mg/kg and a commercial whey protein hydrolysate (Myopure) at 200 mg/kg. Tailcuff blood pressures and body weights were monitored every 14 days with peripheral blood flow and activity of plasma ACE measured at completion of study. The results from the SH Rat Study 1 are shown as effects on body weight (Figure Ia) and tailcuff blood pressures (Figure Ib). The experimental feeds did not significantly alter weight gain in intervention compared groups with the control group. However, the P0147/P0148/P0052 mixture included in solid feeds significantly attenuated development of blood pressure in a dose-dependent manner. The reference hydrolysate (unfractionated commercial hydrolysate, Myopure) produced a similar extent of BP lowering at 200 mg/kg/day to the mixture fed at 20 mg/kg/day. This showed that the specific activity of the test mixture was higher than the Myopure although their in vitro bioactivities were similar. Thus, the process used to produce the products in the mixture yielded a product of superior bioavailability to Myopure.

Example 6. In vivo testing protocol in spontaneous hypertensive rat study 2 The component of the mixture responsible for the in vivo bioactivity observed in SH rats Study 1 was investigated in a subsequent SH rat feeding study (Study 2). In an equivalent study protocol to Study 1, P O147 was tested alone in the Spontaneous Hypertensive rat model, i.e., by dietary intervention over 20 weeks of feeding at 200 mg/kg/day. Rats were randomised into 2 treatment groups (control, P O147 at 200 mg/kg. Tailcuff pressures and body weights were monitored every 14 days. The results (Figure 2) suggest that P O147 was not responsible for the anti-hypertensive effect observed in Study 1 even though P O147 exhibited the highest specific ACE inhibitory activity by in vitro assay.

Example 7. Inhibition kinetics of angiotensin converting enzyme Assay for determination of Angiotensin converting enzyme inhibition The HPLC-based method described was adapted from that of Cushman and Cheung (1971). In brief, hippuric acid (HA) is released from a substrate hippuryl-L- histidyl-L-leucine (HHL) in the presence of Angiotensin I-converting enzyme (ACE) and HA is isolated by extraction into ethyl acetate and measured by absorbance at 228 nm. The concentration of the HA released from HHL is directly related to ACE activity or its inhibition. In the adapted method, the concentration of HA and HHL was quantified by HPLC. Hippuryl-L-histidyl-L-leucine (4 mM, St. Louis, MO, USA) and angiotensin I- converting enzyme (25 µL, 0.05 unit/mL, rabbit lung, St. Louis, MO, USA) were dissolved in reaction buffer (0.1 M dipotassium hydrogen phosphate, 0.3 M NaCl, pH 8.3). HHL (50 µL, St. Louis, MO, USA) was prepared in 4 different concentrations (4, 2, 1 and 0.5 mM). Test inhibitor samples were dissolved in MQ water and tested each at 0, 0.1, 0.5, 1.0 and 2.0 mg/mL for each concentration of substrate. The reaction mixture was incubated at 37°C for 30 min using an Eppendorf Thermomixer (Brinkmann Instruments, New York, USA) with continuous agitation (500 rpm) before inactivation of the enzyme by addition of 1 M HCl (50 µL). By omission of enzyme, the HHL concentration corresponding to zero enzyme activity (i.e., 100% inhibition) was determined and used to calculate the 5 inhibition of test samples. Inhibition was expressed as the percentage of inhibition of ACE activity compared with the control. Data were analysed using MS Excel XP and Prism (V4.0, GraphPad Software Inc., CA, USA). HA and HHL analysis was performed by HPLC (ThermoFinnigan HPLC system, with diode-array detection, a MS pump, and Xcalibur Version 1.4 SRl software, ThermoFinnigan USA). Samples (10 µL) were injected onto C18 reverse phase column (Vydac 238 EV52, monomeric column, 2.1 x 250 mm, 5 µm). HA and HHL were detected at 228 nm. Column temperature was 350C. Gradient elution using solvent systems: (A) 0.08% TFA (St. Louis, MO, USA) in acetonitrile (HPLC-grade), and (B) 0.1% TFA in Milli-Q water, at 150 µl/min flow rate (0 to 5 min at 10% A, increase to 18% A at 8 min and to 90% A at 12 min, hold at 90% A for 2 min and then return to 10% A over 1 min and re-equilibrate before next injection). The concentration of HA produced from enzyme reaction was quantified by comparison with a calibration curve using fresh standards of HA in water, over the range 1 to 250 µM.

Sample preparation by solid phase extraction (SPE) Test samples were fractionated using solid phase extraction cartridge media in order to isolate the active peptide fraction and eliminate salts. Samples were dissolved in MQ water 0.1 to 0.5 g total solids loaded per SPE column (Strata-X reversed phase polymeric sorbent, 500 mg/6 ml, particle size 33 µm, pore size 85 A, surface area 800 m /g, Phenomenex Australia). A 12-position SPE vacuum manifold set (AHO-60230) was used for loading, washing and elution cycles (Phenomenex Australia). SPE columns were conditioned with 5 ml methanol and equilibrated with 5 ml MQ water. Sample (5 ml) was loaded onto the column and washed with 5 ml MQ water. Void, wash and samples eluted into 5 ml of 10%, 30%, 50% or 100% acetonitrile were collected after drying under vacuum at 350C, before testing ACE inhibition activity.

Results In order to understand the lack of correlation of in vitro data with the in vivo result, the competitive kinetics of inhibition of components of the mixture were studied (Figure 3). The active fractions of leads tested in SHR Study 1 were evaluated in terms of kinetic mode of enzyme inhibition by in vitro assay. The results show that the active fraction of P O148 (as isolated by Cl 8 Solid Phase Extraction), exhibits competitive inhibition kinetics whereas active fractions of P O147 (IEO) and P0052 (IE-) exhibit either uncompetitive or non-competitive inhibition kinetics respectively. These results support that the P O148 component of the mixture tested in Study 1, which was recovered by cation exchange (IE+) fractionation, was most likely responsible for in vivo bioactivity. Furthermore, active fractions recovered by SPE of the IE+, but not IEO or IE- fractions of sheep whey and soy protein hydrolysates using a similar processing method (i.e., enzyme sequence), also exhibited competitive inhibition kinetics (Figure 3e and 3f). These results suggest that in some instances the recovery of the cationic fraction of peptides, following enzymatic hydrolysis using the enzyme method described in this invention, facilitates the identification of peptide fractions having in vivo bioactivity. Example 8. Anti-T helper Cell type 1-mediated inflammation activity Assay protocol A ThI response in a human Peripheral Blood Mononuclear Cell (PMBC) culture system was generated by the addition of interleukin 7 (IL7), for homeostatic proliferation and survival of naϊve T-cells (Rathmell et al., 2001); and interleukin 12 (IL12) to promote the stimulation of naϊve T- cells to mature into ThI cells. This mimics, in part, the cytokine environment created by antigen presenting cells (APCs). The stimulation of naϊve T- cells to mature into ThI cells causes the production of ThI associated cytokines including IFN-γ (Gutcher and Becher, 2007). Anti-inflammatory effects of a food extract, protein, or peptide were demonstrated by introducing the test substance into a culture containing PBMC, IL- 12 and IL-7 and monitoring levels of IFN-γ. A decrease in the levels of IFN-γ relative to a control culture, where the extract, protein, or peptide was not present, was taken as indicating an anti-inflammatory effect while an increase in the level of IFN-γ relative to control was taken to indicate a pro-inflammatory response.

Results A small selection of substrates yielded peptide products with significant capacity for inhibition of T Helper cell Type 1-mediated inflammation (Table 6). Substrates included bovine whey protein after high pressure denaturation and several sources of yeast protein including commercial hydrolysates (i.e., Vegemite) and both Brewers and Bakers yeast sources. In vitro activity was significantly associated with IE+ chromatographic fractions and enzyme sequence codes 1, 2 or 4 were represented.

Table 6. Summary of inhibition of T Helper Cell Type 1 receptor inhibition bioactivity of food protein-derived peptide fractions generated using systematic method of hydrolysis and chromatographic fractionation. Example 9. Fibril inhibition activity by RCM-kCn Assay protocol For use in these assays, κ-casein was reduced and carboxymethylated as previously described (Bennett et al., 2009). Our model target protein, RCM-K-CN, which forms amyloid fibrils under physiological conditions, was used in a primary high throughput microtitre plate screen for potential inhibitors of amyloid formation (Carver et al., 2010). RCM-K-CN was incubated at 37°C in 50 mM phosphate buffer, pH 7.4 at 0.5 mg/mL. Screened samples were prepared by weighing out appropriate quantities and diluted to double final concentration in the assay using MiIIiQ water as solvent for peptide samples and 10% ethanol for plant samples. All samples in the assay were prepared in duplicate and incubated with 10 µM ThT. The plates were sealed to prevent evaporation and the fluorescence levels measured with a Fluostar Optima plate reader (BMG Labtechnologies, Melbourne, Australia) with a 440/490 nm excitation/emission filter set over 1000 mins. As ThT fluorescence is temperature dependent and RCM-K-CN binds a small amount of ThT in its native state, the first 45 mins of the assay show a slight decrease in ThT fluorescence intensity as the samples equilibrate to 37°C. Following each assay the percentage inhibition after 1000 mins (i.e. the difference in the change in ThT fluorescence between the sample with and without added tested samples after 1000 mins incubation, expressed as a percentage - see equation below) was calculated for each sample and is reported in the results. The change in ThT fluorescence in the absence of RCM-K-CN was negligible for most samples. For those in which there was a small increase in ThT fluorescence in the absence of the RCM-K-CN, this has been taken into account in the calculations of the Inhibition percentage (IP).

% inhibition = 100 * (∆ I - ∆ ) ∆ I

∆ ∆ Where: I and IS represents the change in ThT fluorescence after 1000 mins for RCM-K-CN in the absence and presence of the tested sample respectively. In the concentration-dependent studies, the samples were used at a number of concentrations and the percent inhibition data were used to calculate the IC50 for that tested samples (i.e. the concentration of solids able to inhibit the increase in ThT fluorescence by 50%) using PRISM software. Results A selection of substrates yielded peptide products with significant capacity for inhibition of RCM-kCn fibril assembly (Table 7). Substrates included several bovine and non-bovine dairy sources, sheep wool, chicken feather and millet. The application of the same process for millet P0263 yielded anti-fibril fractions also from Brewers spent grain and sorghum. In vitro activity was significantly associated with IEO chromatographic fractions and several enzyme sequence codes were represented. Activity in millet, sorghum and spent grain was elicited following 1-NIE processing.

Table 7. Summary of inhibition of reduced carboxymethylated kappa casein fibril assembly bioactivity of food protein-derived peptide fractions generated using systematic method of hydrolysis and chromatographic fractionation. Example 10. Fibril inhibition activity by Abl-42 Assay protocol Beta-Amyloid (1-42), Aβl-42, was purchased from Anaspec Company (San µ Jose, CA, USA) and then dissolved with 60 L 1.0% NH4OH. MiIIiQ water was added to make a 250 µM stock solution, which was aliquoted and stored at - 80 0C until use. Amyloid fibril formation by Aβl-42 was monitored using an adapted in situ ThT binding assay similar to that described above for RCM-K-CN. 25 µM Aβl-42 in 50 mM phosphate buffer (pH 7.4) with 100 mM NaCl was incubated at 37°C in black µclear 96-microwell plates (Greiner Bio-One, Stonehouse, UK) either in the presence or absence of tested samples. The percent protection afforded by each sample was calculated from the ThT fluorescence data at 200 mins by the same equation as used for the RCM-K-CN ThT assay (see above).

Results Bioactivity of a selection of leads from Table 7 were tested in the Amyloid beta 1-42 fibril inhibition assay and bioactivity confirmed. A peptide and analogues identified in P0263 exhibited anti-fibril activity (Table 8).

Table 8. Summary of inhibition of Amyloid beta 1-42 peptide fibril assembly bioactivity of food protein-derived peptide fractions generated using systematic method of hydrolysis and chromatographic fractionation.

Example 11. Oligomer inhibition activity by GFP-Ab yeast assay The Saccharomyces cerevisiae assay system employs an Aβ42-MRF fusion with functional MRF being required for growth of yeast (Bagrianstev et al., 2006). The Aβ42-MRF fusion readily aggregates into non-functional high n-oligomers due to Aβ42-induced oligomerisation. However, several compounds that decrease the oligomerisation, allow growth. Growth assays are convenient and can be performed on solid or liquid media. Our system is a hybrid of the above two in that it uses a GFP-Aβ fusion in yeast. We have previously used it to examine how GFP-Aβ affects yeast, finding a growth stress and a heat shock response (Caine et al., 2007). This effect appears relevant to AD, where elevated levels of heat shock proteins are observed in AD brains, suggesting that yeast may be a relevant model for AD pathogenesis.

Assay protocol

In 5". cerevisiae, plasmids pASIN.GFP and pAS IN. GFP-A β direct the constitutive synthesis of GFP and GFP-A β, respectively. Standard yeast strains transformed with these plasmids have been previously described (Bayly et al., 2001). Cells were spread at 107 cells per plate of solidified media and incubated for 16 hours. Plates of 90 mm diameter contained 20 ml solidified media. Cells were re-suspended from the surface of the plate into water immediately before flow cytometry analyses. LCYl [pASlN.GFP-A β] and LCYl [pASIN.GFP] cells were grown in solidified minimal media (with tryptophan but lacking leucine) supplemented with folinic acid at 50, 100, 200, and 300 µg/ml concentrations. Propidium iodide (PI) was used to identify dead cells. GFP fluorescence was assessed using a flow cytometer (FACSCalibur, BD Biosciences). Data were analyzed using CellQuest analysis software. Cell population gated on a forward and side scatter dot plot was analyzed for PI staining. GFP fluorescence was estimated in the population of Pi-negative (live) cells. Fifteen thousand cells per sample were analyzed.

Results Bioactivity of a selection of leads from Table 7 were also tested in the yeast expression system of GDP-tagged amyloid beta 1-42. In this assay fluorescence increases >10% represent significant anti-fibril activity and the assay also confirms cellular uptake of samples associated with positive results. As for previous fibril- specific assays, millet and related grains following 1-NIE processing exhibited significant anti-fibril assay in this cell system. Some additional bioactive peptides were identified from dairy and Spirulina. Bioactivity was associated with IEO fractions indicating that low net charge was required for cell uptake (Table 9). Table 9. Summary of inhibition of Aβ1-42 oligomerisation as indicated by increase in GFP fluorescence, by food protein-derived peptide fractions generated using systematic method of hydrolysis and chromatographic fractionation.

Example 12. Beta secretase 1 inhibition activity Assay protocol Dried plant samples were dispersed in 10% ethanol at approximately 50 mg/mL total solids by vortexing and extracted for 30 mins using gentle agitation, at room temperature. Supernatants containing extractable solids were freeze dried before reconstitution at 20 mg/mL in 10% ethanol. The stock solution was diluted by 1/100 into sodium acetate buffer (0.1 M, pH 4.5) to a final concentration of 200 µg/mLH. A working sample aliquot (25 µl) was pre-incubated (15 mins at room temperature) with BACEl enzyme (25 µL at 4 µg/mL, Calbiochem) before adding the fluorescent substrate (50 µL at 25 µM, Calbiochem) and commencing monitoring of the reaction. The enzyme inhibition assay was adapted to a high-throughput format using 96-well plate format and microplate fluorescence reader (Biotek FL600, Bio-Tek Instruments Inc, VT, USA), monitoring for 2 hours at room temperature, at excitation and emission wavelength settings of 360 and 485 nm, respectively. Enzyme activity was detected as a linear increase in florescence over the 2 hour monitoring timeframe, and inhibition was measured as the percentage reduction in the fluorescence increase, compared to the control reaction. For dose response experiments, the concentration of extracts was the only variable altered, with all other conditions remaining constant.

Results Capacity for inhibition of beta secretase was associated with protein substrates from bovine dairy and egg protein sources. In addition, a peptides product prepared from a probiotic cell biomass was bioactive following high pressure denaturation and hydrolysis (3-IEO) (Table 10).

Table 10. Summary of inhibition of Beta Secretase Type 1 enzyme bioactivity of food protein-derived peptide fractions generated using systematic method of hydrolysis and chromatographic fractionation.

Example 13. Inhibition of receptor for advanced glycation endproduct-mediated inflammation Assay protocol Cell maintenance RAW264.7 cells (obtained from Ian Cassidy, University of Queensland, Australia) and Nil microglia (obtained from the University of Tublingen, Germany) were grown in 175cm2 cell culture flasks on Dulbecco's Modified Eagle's Medium (DMEM) containing 5% fetal calf serum (FCS), supplemented with penicillin (200 U/ml), streptomycin (200 µg/ml) and Fungizone (2.6 µg/ml). Both cell lines were maintained at 37°C in a humid environment containing 5% CO2.

Activation of macrophages and microglia with LPS and Interferon-y After cells had grown to confluence in culture flasks, they were removed using a rubber cell scraper. Use of trypsin was avoided as it can catalyse the removal of membrane -bound receptors. Cells were concentrated by centrifugation for 3 min at 900 rpm, resuspended in a small volume of fresh DMEM (5% FCS), cell densities estimated using a Neubauer counting chamber and cell densities adjusted to 106 cells/ml. Cells (100 µl) were dispensed into the 60 inner wells of 96-well plates. Sterile distilled water was added to the outer row of wells. Plates were incubated at 37°C for 24 hours with 5% FCS DMEM to allow growth to confluence. The media was removed by aspiration and cell were the grown in 0.1% FCS containing media for 18 hours.

Samples to be tested were dissolved in DMSO, 95% ethanol or ddH2O to concentrations of 100 mg/ml. Dilutions were then made in media from these concentrates, so that the maximum solvent content did not exceed 0.05% of the final well volume. All stock solutions were stored at -200C and dilutions in media were stored at 4°C for no longer than one week. After the cells had been in media for 18 hours, the media was removed by aspiration. For assays with extracts, the dilutions in media were added one hour prior to addition of the activation mixture containing 25 µg/ml LPS and 10 U/ml IFN-γ (Shanmugam et al, 2008, Berbaum et al, 2008). Plates were incubated at 37°C (5%

CO2) for 24 hours. Every plate contained three wells that only contained media as a negative control, while 10 µg/ml LPS was used as a positive control. Vehicle controls were initially present, where all solvents used were tested alone at 0.05% of total volume, but did not have a significant effect on cells in terms of activating ability or effect on viability.

Nitric oxide determination in cell culture supernatant the Griess reagent Nitric oxide production was monitored by measuring the concentration of nitrite in the media using the 'Griess reagent' (Fox et al., 1982). Conditioned media (50 µl) from each well was transferred to a fresh 96-well plate and 25 µl of Reagent 1 (1% w/v µ sulfanilamide in ddH2O) and 25 l of Reagent 2 (0.1% w/v naphthyethylene-diamine in 5% HCl) were added and the absorbance at 540nm determined using a plate reader (Multiskan Ascent with Ascent software v2.4, Labsystems).

Determination of TNF in cell culture supernatant by ELISA The concentration of TNF, following 24 hours of incubation of cells with LPS was determined by a Sandwich Enzyme-Linked Immunosorbent Assay (ELISA), according to the manufacturer's manual (Peprotech). Briefly, capture antibody was µ used at a concentration of 1 g/ml in PBS (1.9 mM NaH 2PO4, 8.1 mM Na2HPO4, 154 mM NaCl) (pH 7.4). Serial dilutions of TNF standard from 0 to 10000 pg/ml in diluent (0.05% Tween-20, 0.1% BSA in PBS) were used as internal standard. TNF was detected with a biotinylated second antibody and an Avidin peroxidase conjugate with ABTS as detection reagent. Colour was determined at 405nm in a 96 well plate reader.

Cell viability assays Cell viability was assessed using MTT (3-(4,5-dimethylthiazol-2-yl)2,5- diphenyl tetrazolium bromide) which measures the level of energy production and respiration in a cell (Takahashi et al., 2002). DMEM (50 µl) containing lmg/ml MTT was added to each well and incubated for 1 hour at 37°C (5%CO2). Media was then removed and 100 µl of 95% ethanol added. Following shaking for at least 30 minutes, the absorbance at 595 nm was measured. Cell viability was also assessed using Neutral Red uptake (Borden & Leonhardt, 1977). Following removal of media of 50 µl of DMEM containing 25 µg/ml Neutral Red was added to the wells and incubated for 1 µ hour at 37°C (5% CO2). The media was then removed and 100 l of the cell lysis solution (50% ethanol, 10% acetic acid) added to each well. Plates were then shaken for 30 minutes and the absorbance at 540 nm was measures with a 96 well plate reader.

Results Substrates from fish, soy and probiotic biomass yielded peptide products with significant capacity for inhibition RAW macrophage inflammation (Table 11). The anti-inflammatory activity of P0263 was thought to be related to its anti-fibril activity as demonstrated previously. In vitro activity was significantly associated with IEO chromatographic fractions and enzyme sequence codes 1-3 were represented. Bioactivity associated with IEO fractions may indicate cell uptake but anti- inflammatory effects were also possible by inhibition of extra-cellular processes IE- and IE+ fractions. Table 11. Inhibition of RAW macrophage inflammation in selected food protein- derived peptide fractions.

Example 14. Anti-fibril activity of BM0263 from button mushroom Materials and methods Sample preparation BM0263 was prepared according to the method applied for millet 'P0263' as per Table 8, with the exception that the button mushroom was processed without prior concentration of protein fraction and dispersed initially at a stock concentration of 10% total solids on a dry weight basis, before addition of enzymes. BM0263-spe40 sub- fraction was prepared by recovering the fraction that eluted from C18 Solid Phase Extraction cartridges in 40% acetonitrile after elution of the non-binding fraction with water.

RCM-kCnfibril inhibition assay

Performed as described previously in Example 9.

TEM imaging Sample concentrations were optimised in order to permit visualisation of fibrils without background interference from inhibitor. The reduction of three dimensions of volume to a two-dimensional area rendered changes in extent of fibrillisation difficult to assess, requiring large excess of inhibitor to observe effects. Amyloid beta 1-42 (Ab 1-42, Rpeptides, USA ) was dissolved in DMSO ( 1 mg/ml) and immediately stored in aliquots (100 µl) at -180C. An aliquot of Ab 1-42 was diluted by 1/10 in water producing a stock solution of 0.1 mg/ml in 10% DMSO. The mushroom product stock solution was 10 mg/ml in water. Ab 1-42 and mushrooms stock solutions were mixed 1:1 (v/v) and divided in half with one set of samples (including Ab 1-42 only controls) incubated at -180C and the other set of samples incubated at 370C, each for 48 hr. The final concentrations were 0.05 and 10 mg/ml for Ab 1-42 and mushroom solids, in 5% DMSO. Carbon-coated 300-mesh copper grids were glow-discharged in nitrogen to render the carbon film hydrophilic. A sample aliquot (4 µl) was pipetted onto each grid with excess liquid drawn off using filter paper, after 30 seconds adsorption time, before staining with 2% aqueous potassium phosphotungstate at pH 7.2, for 10 seconds. Grids were air-dried pending analysis. The samples were examined using a Tecnai 12 Transmission Electron Microscope (FEI, Eindhoven, The Netherlands) at an operating voltage of 120 KV. Images were recorded using a Megaview III CCD camera and AnalySIS camera control software (Olympus, Tokyo, Japan). Each grid was systematically examined and imaged to reflect a representative view of the sample.

Results The 'P0263' analogue prepared from button mushroom exhibited significant anti-fibril activity in the RCM-kCn assay and this activity was supported by the suppression of progression of self-assembly of human Ab42 peptide, as monitored by TEM (Table 12; Figure 4).

Table 12. Summary of inhibition of reduced carboxymethylated kappa casein fibril assembly bioactivity of food protein-derived peptide fractions, generated using systematic method of enzyme hydrolysis and chromatographic fractionation.

Example 15. Dissolution of Ab42 fibrils by P0263 and BM0263 Materials and methods Dissolution of fibrillar Aβ42 by SPE40fractions of millet and button mushroom Freshly prepared Aβ42 was dissolved in MQ water and sonicated for 10 min and then placed in 370C oven for 52 hr before being used for dissolution study. P0263- SPE40 (from Panorama millet) and BM0263-SPE40 (from Button mushroom were dissolved in MQ water, respectively. Equal volumes of Aβ42 and P0263-SPE40 or BM0263-SPE40 (100 µl each) and 30 µl 20 µM ThT were added to a 96 microplate (OptiPlate-96F, PerkinElmer Inc., MA, USA), each in triplicate. The final concentrations for fibrillar Aβ42 was 0.043 mg/ml, and for PP0263SPE40 or BM0263SPE40 were 0.17 mg/ml, respectively. They were incubated at 370C for 66 hr with fluorescence intensity readings at 10 min intervals (Varioskan Flash, Thermo Scientific, USA and 442/482 nm excitation and Emission wavelengths, respectively.

Results The extent of preformed fibrillization of Aβ42 was reflected in the starting level of ThT fluorescence with the growth of fluorescence in the Aβ42 controls indicating clear capacity for further fibril growth (Figure 5a). In the presence of either P0263- SPE40 (Figure 5a) or BM0263-SPE40 (Figure 5b), ThT fluorescence was not only suppressed completely but a downward trend in fluorescence was evident over the 66 hr incubation. These trends suggested that the inhibitory factors present in the millet and mushroom products were able to both prevent assembly of new fibrils and also to dissolve pre-existing fibrils. The downward trend was greater for the mushroom compared with the millet-derived product, when compared at the same level of sample solids indicating higher specific activity of the mushroom compared with the millet product.

Example 16. Regulation of toxicity of amyloid beta peptide by SPE fractions of dairy hydrolysate fraction P0147 Materials and methods Preparation of P O147 SPEfractions

P O147 hydrolysate was prepared from bovine whey protein isolate using confidential project methods and contained 77.6% protein (w/w). P0147 was sub- fractionated using Cl 8 solid phase cartridges (Phenomenex, California, USA). The void and unbound species (LW) were eluted with water and bound fractions sequentially eluted with 40% and 100% acetonitrile (Aj ax Fine Chem, NSW, Australia, SPE40, and SPElOO respectively).

Reversephase HPLCprofiling Fractions were analysed by reverse phase HPLC (Jupiter 5µ C18 300A, 25O x 4.6, Phenomenex, California, USA) connected to Waters Alliance HPLC with Photo Diode array Detector (Waters, Massachusetts, USA). A gradient elution was employed with mobile phase A (0.1% TFA in water) and B (0.1% TFA in 95% acetonitrile) at a flow rate of 1.0 ml/min over 60 mins.

Preparation ofA β solutions Synthetic human Aβ42 (Aβ) was purchased from the W. M. Keck Laboratory (Yale University, New Haven, CT). Solutions of Aβ were prepared according to the method of Bharadwaj et al. (2008) with some modifications. Aβ was dissolved in l,l,l,3,3,3-hexafluoro-2-propanol (HFIP) and incubated overnight at RT. The HFIP solution was centrifuged at 14000 rpm for 10 min and the supernatant removed. The HFIP was then evaporated and the resulting peptide film dissolved in sterile double- distilled water. The solution was sonicated on ice for 5 min and centrifuged for 10 min at 14000 rpm. The supernatant contained soluble oligomers and was used for the toxicity experiments. The Aβ peptide concentration was determined by measuring absorbance at 214 nm. The final concentration was calculated using the formula Aβ (M) = Abs214 x DF / 75887. All solvents used for the preparation of Aβ solutions were filtered using 0.2 µm filters and the entire procedure was performed in laminar air flow hoods.

In situfluorescent RCM kappa-casein assay Inhibition of fibril assembly was monitored using reduced and carboxymethylated-kappa casein (RCM-kCn, 0.5 mg/ml) prepared according to Schecter et al, 1973. The RCM-kCn was used as a model for protein fibril self assembly as follows. RCM-kCn (0.5 mg/mL) was incubated with test peptide samples for 24 h at 370C in black Clear 96-microwell plates that were sealed to prevent evaporation. ThT (Thioflavin T) was added to a final concentration of 10 µM. The ThT fluorescence intensity of each sample was recorded every 5 min using a FLUOstar OPTIMA plate reader (BMG Lab technologies) with 440-/490-nm excitation/emission filters set. Percentage fibril inhibition (Fi) was calculated as follows: Fi (%) = 100 *

(dl-c - dl-s )/dI-c , where dl_c and dl_s represent the change in ThT fluorescence in the absence (control) and presence of sample, respectively. Fibril inhibition (Fi) was calculated as the percentage change in ThT fluorescence in the absence and presence of sample, after correction for any background fluorescence of the sample.

Yeast cell viability assay Aβ42 toxicity assay in yeast was done according to the method of Bharadwaj et al. (2008). The prototrophic yeast strain Candida glabrata ATCC 90030 was used in this study. Cultures of yeast cells were grown in YEPD (1% yeast extract; 2% peptone, 2% dextrose), to achieve exponential growth phase. These cultures, containing -10 yeast cells/ml, were diluted to ~5 x 10 yeast cells/ml in sterile, pure water. Cells were then aliquoted into 96-well microtitre plates for peptide treatments. Peptide preparations were added to the diluted cell suspension to required concentrations. The final volume in each well was made up to 125 µl. The microtitre plate was then sealed with gas permeable membrane (Diversified Biotech Inc.) and incubated at 300C constantly shaken at 150 rpm for 20 h. Cell survival was determined by plating aliquots of peptide treated cell suspensions onto YEPD agar plates to measure the number of colony- forming units (cfu). Circular dichroism spectroscopy

Aβ42 peptide solutions pre-incubated with different concentrations of P O147 were analysed by Jasco J-810 CD Spectropolarimeter (JASCO Inc., Easton, MD, USA) with computerised data collection. Ab 1-42 Oligomer solution was prepared as described in the methods. Oligomeric Abl-42 (0.05mg/ml) was incubated with P0147 sub-fractions spe40 and spelOO, at 0.005, 0.05 & 0.25 mg/ml, respectively in MiIIiQ water. The mean residue ellipticities of samples were recorded across the far UV range of 190-260 nm wavelength using a 0.1 cm path-length quartz cuvette, at room temperature, monitoring at 0.1 nm intervals. The acquisition parameters were 100 nm/min with 1 sec response times, 1.0 nm bandwidth, and 0.1 nm data pitch, and data sets were averaged over 3 scans. Spectra of P0147 fractions alone at the appropriate experimental concentration were subtracted from respective profiles of Ab 1-42+P0 147, but were otherwise unsmoothed. The instrument was calibrated with deionised water (MiIIiQ).

SDS-PAGE electrophoresis and Western Blot Analysis

Aβ42 peptide solutions pre-incubated with different concentrations of P O147 fractions. The samples were fractionated on a 4-12% Bis-Tris NuPage gel in a MES buffering system and visualized by 6E10 immunoblotting followed by TMB development.

Results ThT-binding assay (Figure 7) and yeast toxicity results (Figure 8) revealed interesting dose-dependent capacity for modulation of Aβ fibril morphology and toxicity, respectively, by SPE40 and SPElOO (see Reverse Phase HPLC profiles of respective hydrolysate fractions, Figure 6), apparently driving self-assembly progressively from relatively more toxic to less toxic forms of Aβ42. The suppression of ThT binding by dairy peptides correlated with: loss of beta sheet absorbance by CD (Figure 9); suppression of oligomeric Aβ42 by Western blot with 6E10 antibody (Figure 10) and disappearance of macroscopic fibril structures by TEM (Figure 11). The modulation of Aβ42 self-assembly by dairy peptides, eliciting either potentiation or suppression of amyloid self-assembly, can assist in understanding the determinants of toxicity of self-assembled forms of Aβ42. Example 17. Anti-inflammatory activity of Vegemite, Brewers and Bakers yeast products in whole blood Materials and methods Preparation of low molecular weight yeast fractions by hydrolysis Peptide hydrolysates of brewer's and baker's yeasts were prepared as follows. Dried Brewers yeast (Lotus Foods Pty Ltd, Cheltenham, Victoria, Australia) and dried Bakers yeast (Lowan Whole Foods, Glendenning, New South Wales, Australia) were dissolved or suspended at 10% total protein (w/w) in 10 mM tri-ethanolamine, 10% EtOH, pH 7.4 and maintained at pH 7.4 throughout the hydrolysis process. After rehydration, Glutaminase was added to a final concentration of 0.5% (w/w) and incubated with agitation at 500C for 1 hour. The enzymes Alcalase (0.5%, w/w), Corolase (0.5%, w/w) and Flavourzyme (0.5%, w/w) were subsequently introduced sequentially at 1 hr intervals at 500C. The final enzyme Trypsin (0.5%, w/w) was introduced to the mixture and incubated at 37°C for 17 hours (overnight). The enzymes were inactivated heating at 900C for 30 min, and the product cooled to 220C for at least 15 mins. The hydrolysates were dialysed and the permeate (regenerated cellulose membrane, 6-8 KDa MWCO) recovered and freeze dried. Vegemite™ (Kraft Foods, Port Melbourne, Victoria, Australia) and Marmite™ (Australian Health and Nutrition Association Ltd, Berkley Vale, NSW, Australia) were obtained from local retail suppliers and Epicor® was purchased on-line (Vitamin research products, Carson City, USA).

Fractionation by solid phase extraction (SPE) SPE fractions of the hydrolysate products and the yeast derived foods were obtained by dispersing the samples in water at a concentration of 100 mg/mL then centrifuging to remove insoluble matter. Supernatants were fractionated using Strata-X 33 µm polymeric reverse phase cartridges (500 mg/6 ml, Phenomenex). After washing with methanol and re-equilibrating with water, 5.0 ml of dissolved sample (100 mg/mL) was loaded and non-binding material eluted with a further 5.0 ml of water (designated wash sample). Bound fractions were subsequently eluted with two 5.0 ml aliquots of aqueous acetonitrile at increasing proportions of solvent, typically, 40 and 100%, under vacuum and designated 'SPE40, SPElOO for fractions eluted with 40% or 100% acetonitrile, respectively. For some of the work reported here, the 40% and 100% fractions were pooled then dried (designated 'SPE'). Whole blood Inflammation assay An assay system utilising diluted blood was adapted from the protocol of Matalka (2003). Test samples were dispersed in 10% ethanol and filter sterilised by passing through 0.22 µm membranes then diluted in RPMI 1640 supplemented with 2 mM Glutamine, 100 U/mL Penicillin and 100 ug/mL streptomycin. Blood was obtained from healthy volunteers (into heparinised tubes) and diluted 1+9 with supplemented RPMI 1640 as above. An inflammatory state was initiated by the addition of 1 ug/mL Lipopolysaccharide (LPS) and 5 ug/mL phytohemeagglutinin (PHA). Diluted blood, LPS, PHA, and test compounds were incubated together in 24 well plates (total volume per well 2 mL) incubated for 2 or 3 days at 37°C, in a 5 % CO2 atmosphere. Each test compound / concentration point was tested in two culture wells, each well being assayed in triplicate. Interleukin 10 (IL-10, 250 pg/mL) and Hydrocortisone (HC, 200 ng/mL) were used as a positive controls. Supernatants were removed at the conclusion of the culture period and stored at -800C until tested. Supernatants were tested for IFN-γ by ELISA using the Human IFN- γ DuoSet; DY28E (R&D systems, USA) as per instructions with a standard curve from 5000 to 5 pg/ml prepared by serial 1:2 dilutions of the supplied standard. Results for ELISAs were read at absorbance wavelengths 460 nm and 590 nm in an FL600 Fluorescence Microplate Reader (BioTek, USA) and the KC4 software (version 3.1, BioTek, USA).

Results As distinct from a previous method of preparation of Brewers and bakers yeast hydrolysates (P0613, P0713), the revised method did not involve chromatographic fractionation and so the hydrolysate was not diluted in salt required for elution from the ion-exchange resin. Basic compositional data obtained for the two yeast hydrolysate products were: Baker's yeast: BA0263 8.6%N 41.7%C 7.6%H 6.8% Ash. Brewer's yeast: BR0263 8.5%N 41.4%C 7.6%H 8.1% Ash. The assay was also revised from that used to test the P0613 and P0713 products. In this assay, LPS activates the Toll Like Receptor 4 present on macrophages and monocytes and PHA activates phosphorylation of mitogen activated protein kinases (MAPK), stimulating the release of inflammatory cytokines including Tumour necrosis Factor-alpha (TNF-a) and Interferon -gamma (IFN-γ). Using both PHA and LPS activates the proliferation of both T-cells and B-cells respectively in assays used to investigate inflammation and immunomodulatory effects of compounds. When tested without fractionation, neither Vegemite™ nor EpiCor® effected the production of IFN-γ in the assay (Figure 12b). In contrast, the SPE fractions generated a strong effect with considerable suppression of IFN-γ production (Figure 12a and 12b). The level of suppression was similar in magnitude to both positive controls used in the test; hydrocortisone and Interlukin-10. It is noted that there was some donor to donor variation in the response. The EpiCor® SPE fractions showed the strongest and most consistent effect followed by the Brewer's 0263 SPE fraction. Dose response curves were evaluated for the total SPE fractions of each of the four samples (Figure 13). Each of the fractions exhibited a dose response effect with almost complete inhibition of IFN-γ production at 2000 µg/mL. The differences between samples were most pronounced at 20 µg/mL. The SPE fractions of unadulterated Vegemite, and processed forms of Brewers and Bakers yeast exhibited comparable, although slightly inferior specific activity to Epicor suggesting that the processing methods applied to Brewers and Bakers yeast recovered bioactive components of similar activity to those in Epicor.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive. All publications discussed and/or referenced herein are incorporated herein in their entirety. The present application claims priority from AU 2009903698 and AU 2009903699 filed 7 August 2009, the entire contents of which are incorporated herein by reference. Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application. REFERENCES Bagrianstev et al. (2006) Methods Enzymol, 412:33-48. Bayly et al. (2001) FEMS Microbiol Lett, 204:387-390. Bennett et al. (2009) Australian Journal of Dairy Technology, 64:1 17-121. Berbaum et al. (2008) Cytokine, 4 1:198-203. Bharadwaj et al. (2008) J Alzheimers Dis, 13:147-150. Borden and Leonhardt (1977) J Lab Clin Med, 89:1036-1042. Caine et al. (2007) FEMS Yeast Res, 7:1230-1236. Carver et al. (2010) Bioorg Med Chem, 18:222-228. Cushman and Cheung (1971). Biochem. Pharmacol, 20:1637-1648. Del Carmen Caro et al. (1998) Life Sci, 62:51-57. Fox et al. (1982) J Assoc Off Anal Chem, 65:690-695.

Gutcher and Becher (2007) J Clin Invest, 117: 1 1 19-1 127. Harjanne (1984) Clin Chem. 30:901-902. Leifert et al. (2009) Methods MoI Biol, 552:131-141. Matalka (2003) Cytokine, 21:187-194. Rathmell et al. (2001) J Immunol, 167:6869-6876. Shanmugam et al. (2008) MoI Nutr Food Res, 52:427-438. Schechter et al. (1973) Biochem, 12:3407-3413 Takahashi et al. (2002) Neurochem Int, 40:441-448. Vanderheiden et al. (1970) Appl Microbiol, 19:875. CLAIMS;

1. A process for obtaining a peptide fraction, the process comprising: i) contacting a protein substrate with a series of enzymes selected from: a) glutaminase, subtilisin, oryzin, leucyl aminopeptidase, and trypsin, b) glutaminase, oryzin, subtilisin, leucyl aminopeptidase, and trypsin, c) glutaminase, subtilisin, leucyl aminopeptidase, oryzin, and trypsin, d) glutaminase, oryzin, leucyl aminopeptidase, subtilisin, and trypsin, e) glutaminase, leucyl aminopeptidase, oryzin, subtilisin, and trypsin, and f) glutaminase, leucyl aminopeptidase, subtilisin, oryzin, and trypsin, ii) selecting peptides obtained from step i) which are less than about 11 kDa.

2. A process for obtaining a peptide fraction, the process comprising: i) contacting a protein substrate with a series of enzymes selected from: a) trypsin, subtilisin, oryzin, leucyl aminopeptidase, and glutaminase, b) trypsin, subtilisin, leucyl aminopeptidase, oryzin, and glutaminase, c) trypsin, oryzin, subtilisin, leucyl aminopeptidase, and glutaminase, d) trypsin, oryzin, leucyl aminopeptidase, subtilisin, and glutaminase, e) trypsin, leucyl aminopeptidase, oryzin, subtilisin, and glutaminase, and f) trypsin, leucyl aminopeptidase, subtilisin, oryzin, and glutaminase, ii) selecting peptides obtained from step i) which are less than about 11 kDa.

3. The process of claim 1, wherein step i) comprises contacting the protein substrate with a series of enzymes selected from: a) glutaminase, subtilisin, oryzin, leucyl aminopeptidase, and trypsin, b) glutaminase, oryzin, subtilisin, leucyl aminopeptidase, and trypsin, and c) glutaminase, leucyl aminopeptidase, oryzin, subtilisin, and trypsin.

4. The process of any one of claims 1to 3, wherein the process further comprises: iii) separating the peptides which are less than about 11 kDa into one or more of cation, anion and/or neutral peptide fractions.

5. The process of claim 4, wherein the peptides are separated into one or more of cation, anion and/or neutral peptide fractions by ion-exchange chromatography. 6. The process of claim 5, wherein the ion-exchange chromatography is performed using cation and anion exchange columns in series.

7. The process of claim 5 or claim 6, wherein the ion-exchange chromatography is performed at about pH 6.0 to about 8.0.

8. The method of claim 7, wherein the ion-exchange chromatography is performed at about pH 7.4

9. The process of any one of claims 1 to 8, wherein the step ii) is performed by dialysis, membrane filtration or size-exclusion chromatography.

10. The process of any one of claims 1 to 9, wherein the protein substrate is a protein-rich extract.

11. The process of claim 10, wherein the protein-rich extract is a milk extract, meat extract, plant extract, yeast extract and/or bacterial extract.

12. The process of any one of claims 1 to 11, wherein the protein substrate has been high pressure treated prior to contacting the protein substrate with the series of enzymes.

13. The process of any one of claims 1 to 12, wherein the process further comprises isolating one or more peptides from the peptide fraction.

14. The process of any one of claims 1 to 13 further comprising testing the peptide fraction and/or isolated peptide for biological activity.

15. A peptide fraction obtained by the process of any one of claims 1to 14.

16. The peptide fraction of claim 15 which is a cation peptide fraction at about pH 7.4.

17. A composition comprising the peptide fraction of claim 15 or claim 16. 18. Use of the peptide fraction of claim 15 or claim 16 or the composition of claim 17 in the manufacture of a food or drink product.

19. The use of claim 18, wherein the food or drink product is a nutritional supplement.

20. A food or drink product comprising the peptide fraction of claim 15 or claim 16 or the composition of claim 17.

21. Use of the peptide fraction of claim 15 or claim 16 or the composition of claim 17 in the manufacture of a medicament for the treatment or prevention of disease in a subject.

22. A method for the treatment or prevention of disease in a subject, the method comprising administering to the subject the peptide fraction of claim 15 or claim 16 or the composition of claim 17.

23. The use of claim 2 1 or method of claim 22, wherein the disease is selected from cardiovascular disease, cancer, inflammatory or autoimmune disease, and/or a neurological disorder.

24. The use or method of claim 23, wherein the cardiovascular disease is hypertension, atherosclerosis and/or arteriosclerosis.

25. The use or method of claim 23, wherein the cancer is selected from carcinoma, lymphoma, blastoma, sarcoma, and leukemia.

26. The use or method of claim 23, wherein the inflammatory or autoimmune disease is selected from asthma, rheumatoid arthritis, inflammatory bowel disease, psoriasis and multiple sclerosis.

27. The use or method of claim 23, wherein the neurological disorder is selected from Alzheimer's disease, Parkinson's disease, dementia, Huntington's disease, Shy- Drager syndrome, progressive supranuclear palsy, Lewy body disease and amyotrophic lateral sclerosis.

International application No. INTERNATIONAL SEARCH REPORT PCT/AU20 10/000994

A. CLASSIFICATION OF SUBJECT MATTER Int. Cl. /425/5/54 (2006.0I) A61P 25/00 (2006.01) A61P 37/00 (2006.01) A61K 9/00 (2006.01) A61P 29/00 (2006.01) A61K 38/01 (2006.01) A61P 35/00 (2006.01)

According to International Patent Classification (IPC) or to both national classification and IPC B. FIELDS SEARCHED Minimum documentation searched (classification system followed by classification symbols)

Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched

Electronic data base consulted during the international search (name of data base and, where practicable, search terms used) MEDLINE, WPI, EPODOC, WEB BASED: glutaminase & like terms, subtilisin & like terms, oryzin & like terms, leucyl aminopeptidase & like terms, trypsin & like terms, hydrolysis, serial proteolysis

C. DOCUMENTS CONSIDERED TO BE RELEVANT

Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No. RAGHUNATH M . & NARSINGA RAO B.S., "Release of Peptides & Amino Acids from Dietary Proteins during Sequential Enzymatic Digestion in vitro with Pepsin, Pancreatin + Trypsin & Erepsin", Food Chemistry, 1984, VoI 15, pp 285-306 See page 287 1-20

WANG S et al, "Neuroprotective Activities of Enzymaticaljy Hydrolysed Peptides from Porcine Hyde Gelatin". Int J Clin Exp Med, 2008, VoI 1, pp 283-293 X See pages 286, 290 1-27

CLEMENTE, A., "Enzymatic Protein Hydrolysates in Human Nutrition". Trends in Food Science & Technology, 2000, VoI l 1, pp 254-262 X See page 256, table 2 1-27

Further documents are listed in the continuation of Box C See Patent famil y annex

* Special categories of cited documents: "A" document defining the general state 'of the art which is not "T" later document published after the international filing date or priority date and not in considered to be of particular relevance conflict with the application but cited to understand the principle or theory underlying the invention "E" earlier application or patent but published on or after the "X" document of particular relevance; the claimed invention cannot be considered novel international filing date or cannot be considered to involve an inventive step when the document is taken alone "L" document which may throw doubts on priority claim(s) or "Y" document of particular relevance; the claimed invention cannot be considered to which is cited to establish the publication date of another involve an inventive step when the document is combined with one or more other citation or other special reason (as specified) such documents, such combination being obvious to a person skilled in the art "O" document referring to an oral disclosure, use, exhibition "&" document member of the same patent family or other means "P" document published prior to the international filing date but later than the priority date claimed Date of the actual completion of the international search Date of mailing of the international search report 4 November 2010 NOV 2010 Name and mailing address of the ISA/AU Authorized officer NESRIN OZSARAC AUSTRALIAN PATENT OFFICE PO BOX 200, WODEN ACT 2606, AUSTRALIA AUSTRALIAN PATENT OFFICE E-mail address: [email protected] (ISO 9001 Quality Certified Service) Facsimile No. +61 2 6283 7999 Telephone No : +61 2 6283 7958

Form PCT/ISA/210 (second sheet) (July 2009)