AU2003204321B2 - Bioactive Whey Protein Hydrolysate - Google Patents

Description

e: Regulation 3.2

AUSTRALIA

PATENTS ACT, 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

Name of Applicant: Actual Inventors: Address for service in Australia: Invention Title: NEW ZEALAND DAIRY BOARD RALF-CHRISTIAN SCHLOTHAUER; LINDA MAY SCHOLLUM; ANNE MARIA SINGH; JULIAN ROBERT

REID

A J PARK, Level 11, 60 Marcus Clarke Street, Canberra ACT 2601, Australia Bioactive Whey Protein Hydrolysate The following statement is a full description of this invention, including the best method of performing it known to us.

BIOACTIVE WHEY PROTEIN HYDROLYSATE TECHNICAL FIELD This invention relates to bioactive peptides. The peptides are preferably produced from whey proteins. The peptides may be incorporated into functional foods. This is a divisional of Australian patent application 761477.

BACKGROUND ART A number of food ingredients and foodstuffs have been produced from the hydrolysis of a protein source such as the milk proteins, casein and whey proteins.

Hydrolysed protein foodstuffs may have advantages over non-hydrolysed protein foodstuffs in a number of areas of health care. For example, it is known that enzymatically hydrolysed proteins are less allergenic. They are also more rapidly digested and absorbed than whole proteins.

Foodstuffs containing hydrolysed proteins are also useful in the alimentation of hospital patients with digestive diseases for example.

Hydrolysis of whey proteins and caseins is known to release bioactive peptides that can exhibit a number of physiological effects (Maubois et al, 1991; EP 475506). A number of publications describe such bioactive peptides, for example, ACE inhibiting peptides which have antihypertensive properties have been released through an enzymatic treatment of bovine plactoglobulin and whey protein concentrates (Mullally et al, 1997). ACE inhibitorypeptides are also found in sour milk and in hydrolysates of as and 3 casein (JP 4282400; Nakamura et al 1994, Yamamoto et al 1994).

EP 4745506 discloses the hydrolysis of the milk protein lactoferrin in whey to release lactoferricin which acts as an antimicrobial agent useful for treating diarrhoea, athlete's foot, eye infections, mastitis etc in humans and animals.

However, the hydrolysis of most food proteins, especially the hydrolysis of whey and casein containing products, is known to generate bitterness. This causes palatability problems particularly when attempting to formulate orally ingestible products incorporating milk protein hydrolysates as a source of bioactive peptides.

In the field of protein hydrolysis one or both of two approaches are commonly used for controlling or removing bitterness in protein hydrolysates to increase palatability of the products.

The extensive hydrolysis of the protein substrate is known to reduce bitterness in milk protein hydrolysates (EP 065663; EP 117047; US 3970520). Less bitter products are produced relatively easily and cheaply in this way. However, extensive hydrolysis reduces the chain lengths of all peptides, including the bioactive peptides of interest. Extensive hydrolysis of the protein substrate destroys the functional and biological activity of the peptide of interest. In addition soapy and brothy off-flavours often develop, with the consequence that the palatability of the final product remains poor compared to the original bland tasting protein substrate. A final disadvantage is that for some hydrolysates the bitterness is only partially removed (Roy 1992 and 1997).

A second common method for the control of bitterness in protein hydrolysates is to use debittering enzymes, in particular those sourced from Aspergillus oryzae.

"Bitterness" generation in protein hydrolysis is thought to be due to the presence of large hydrophobic 'bitter' peptides. Debittering enzymes selectively hydrolyse bitter peptides present in the protein hydrolysates. A worker skilled in the art can by the judicious selection of debittering enzymes and the conditions of treatment effectively debitter milk protein hydrolysates leaving intact the particular bioactive peptides of interest. However, use of debittering enzymes makes the process more expensive, and preservation of some of the bioactive peptide is not easily or sucessfully achieved. A further disadvantage is that debittering enzymes treatments have a tendency to release free amino acids into the final product and, as a consequence, the hydrolysates develop unpleasant brothy or soapy flavours (Roy 1992 and 1997).

The various methods of debittering the protein hydrolysates result in additional process steps and add costs to the manufacture of the final product. In addition the final product also becomes overbalanced in its supply of free amino acids.

It would be most advantageous if a process for hydrolysing protein could be developed which releases bioactive peptides of interest and which limits the formation of bitter peptides and free amino acids, thereby allowing the original bland taste of the milk proteins substrates to be retained.

Some bioactive peptides in particular the antihypertensive peptides are relatively stable during protein hydrolysis and are released very early during the hydrolysis of the milk protein substrate as shown in Figure 1.

The bitter flavours of milk protein hydrolysates can be improved by adding sugars or by hydrolysing natural sugars, such as lactose, already present in the milk protein substrate (Bemal and Jelen, 1989). For example sour wheys and cheese wheys are made more palatable when they have been sweetened by P-galactosidase and lactase hydrolysis of lactose (FR 2309154; US 4358464; JP 8056568).

In order to achieve a high flavour acceptability for a hydrolysed protein product which contains bioactive peptides, precise control of hydrolysis is required to prevent bitterness occurring.

A common method of termination of hydrolysis is by deactivation of the enzymes, usually by thermal deactivation at high temperatures, typically 90-100 0 C for an extended period of time.

However, this method cannot be used to stop the hydrolysis of whey proteins as any intact unhydrolysed whey proteins remaining in the mixture would denature and precipitate making the final product less soluble and less acceptable for the use as a food ingredient.

It would be advantageous if a process of hydrolysing whey protein could be controlled so that it directly produced a hydrolysate comprising bioactive peptides for incorporation into functional foods which did not taste bitter and where the enzyme inactivation steps did not compromise the integrity of the intact proteins in the final product.

It is an object of the invention to go some way towards achieving these desiderata or at least to offer the.public a useful choice.

SUMMARY OF THE INVENTION Accordingly the invention may be said broadly to consist in a bioactive peptide selected from the group consisting of AFE, LFSH, ILKEKH, LIVTQ, MKG, LDIQK, ALPMH, VTSTAV, LVYPFPGPIPNSLPQNIPP and LFRQ.

In one embodiment the invention is a combination of any two or more of the bioactive peptides as defined in the preceding paragraph.

In another embodiment the invention is the bioactive peptide AFE.

In another embodiment the invention is the bioactive peptide LFSH.

In another embodiment the invention is the bioactive peptide ILKEKH.

In another embodiment the invention is the bioactive peptide LIVTQ.

In another embodiment the invention is the bioactive peptide MKG.

In another embodiment the invention is the bioactive peptide LDIQK.

In another embodiment the invention is the bioactive peptide ALPMH.

In another embodiment the invention is the bioactive peptide VTSTAV.

In another embodiment the invention is the bioactive peptide LVYPFPGPIPNSLPQNIPP.

In another embodiment the invention is the bioactive peptide LFRQ.

In a further embodiment the invention consists in a method of reducing systolic blood pressure in a subject which comprises administering to that subject an effective amount of peptide as claimed in any one of the preceding claims.

The methods of forming an hydrolysate containing peptides according to this invention and the method of isolating the peptides and testing for their bioactivity are claimed in parent patent application 761477.

The invention consists in the foregoing and also envisages constructions of which the following gives examples.

BRIEF DESCRIPTION OF THE DRAWINGS The present invention will now be described with reference to the accompanying drawings in which: Figure 1 is a plot of bitterness and bioactivity on the ordinant against the degree of hydrolysis on the abscissa. The 'opportunity window' of obtaining a product according to the present invention containing bioactive peptides and having acceptable flavours before the hydrolysis reaction produces bitter peptides is between the lines xl and X2.

Figure 2 is a plot of systolic blood pressure of four groups of hypertensive rats fed different diets over a period of eight weeks.

Figure 3 is a plot of a least squares means analysis of rats fed with a control of commercial rat chow against groups of rats fed with hydrolysate at two different concentrations per day.

DETAILED DESCRIPTION OF THE INVENTION As discussed above, the present invention provides a process for producing a hydrolysed whey protein product containing bioactive peptides, whereby the hydrolysis is carried out under a high degree of control to prevent undesirable flavours developing during hydrolysis (eg bitter, soapy and brothy). The hydrolysis is terminated within the "opportunity window", ie before the emergence of substantial bitterness as shown in Figure 1 to provide hydrolysates having good organoleptic properties and maximum bioactive peptides. In Figure 1 the degree ofhydrolysis is represented qualitatively on the x axis. The window of opportunity is between the points xl and

X

2 which will vary depending on the enzyme which is used. The optimum conditions sought are a maximum bioactivity with an acceptable level of bitterness.

In particularly preferred embodiments of the process of the invention, the enzyme which hydrolyses the whey proteins is selected from the group consisting of Protease P6, Protease A, Protease M, Peptidase, Neutrase, Validase and AFP 2000 (all as herein defined) and the hydrolysis of the whey proteins is terminated by heat treatment for a short time at a high temperature (about 85-100 0 C for 1-10 seconds). The applicants have surprisingly found that the above enzymes are able to produce a whey protein hydrolysate containing a good level of bioactive peptides, and can be inactivated by a short time, high temperature treatment which causes only partial denaturation of the whey proteins in the hydrolysate, and surprisingly improves the organoleptic properties of the whey proteins, in terms of providing a product which is creamy in texture (has a relatively small particle size) and substantially white in appearance.

The present invention is now exemplified by the following examples: Example 1 A 10% solution of a sweet whey protein concentrate with 80% protein content (ALACEN T M 392, 2L) was hydrolysed at 50 0 C with the commercially available enzyme Neutrase sourced from Bacillus subtilis (Novo Nordisk, Denmark). A pH of 7.0 and an enzyme substrate ratio of 6 0.3% w/w was used for the hydrolysis. The hydrolysate was adjusted to pH 5.0 and heated at 0 C for 30min to inactivate the enzyme. The hydrolysate (DH of was spray dried and tested for angiotensin-converting enzyme inhibitor (ACE-I) activity and flavour. ACE-I activity in the dried product was determined using FAPGG as a substrate (Product 305-10 ex Sigma Chemical Corporation, St Louis, MO, USA) according of the method ofD W Cushman H S Cheung (1971). ACE-I activities are expressed as the amount of material needed to reduce the activity of the ACE-I enzyme by 50%. IC5o ACE-I activity in the hydrolysate was 0.44g/L and flavour acceptability score, as determined by a taste panel, was very high.

Example 2 A 50% solution ofALACENTM 421 whey protein concentrate (56% protein content, 10L) was treated with commercial lactase sourced from Kluveromyces lactis (Lactozyme 3000L ex Novo Nordisk) at an enzyme to substrate ratio of 0.3% at 50 0 C for 2 hours. The lactase treated solution was hydrolysed with Neutrase (Novo Nordisk, Denmark) for 1 hour at 50C at an enzyme substrate ratio of Active enzymes were inactivated by UHT treatment (5sec at C) after a five fold dilution of the mixture. The hydrolysate was spray dried. The dry powder (DH contained no traces of active enzyme and had an ACE-I activity of 2.18g/L. The flavour score was exceptionally high due to the introduction of a low level of sweetening into the product. ACE-I measurements and flavour acceptability scoring were determined as for Example 1.

Example 3 A 500L hydrolysate, made from ALACENT 392 in a similar way to that in example 1, was cooled to 10 0 C after enzyme inactivation. A sub-sample of the original hydrolysate was dried.

The remaining hydrolysate was subjected to ultrafiltration at 10 0 C with a 10,000 dalton nominal molecular weight cutoff membrane (HFK 131, Koch Membrane Systems, USA). The hydrolysate (at a DH between 3.8% and was concentrated to a VCF 10 and the retentate was dried directly. The permeate was concentrated by evaporation to approx 25% solids and dried. ACE-I measurement and flavour acceptability scoring were determined as for Example 1.

The ACE-I activity was enriched in the permeate powder (IC 50 of the permeate powder was 0.15g/L). ACE-I activity in the sub-sample of the dried hydrolysate before ultrafiltration was 0.43g/L. The flavour acceptability scores on the retentate powder and the spray dried powder of the feed were both high.

Example 4 Three different solutions from ALACEN T 392, ALACEN T 421 and a mixture ofALACEN

T

392 and lactose were made up at 15% solids to yield 150 L. The solution was treated with a commercial protease from Bacillus subtilis Neutrase (Novo, Nordisk Denmark) and a commercial lactase from Klyvermyces lactis (Lactozyme 3000L ex Novo Nordisk). The addition rate of enzyme was 0.3% w/w (on protein basis) for Neutrase and 1.2% w/w (on lactase basis) for Lactozyme. The reaction continued for 2h at 50 0 C at a pH of 7.0. Samples of were taken every 0.5h inactivated at 88 0 C for 3 seconds and subsequently spray dried. The ACE-I activity as specified in example 1 yielded 0.424g/L, 0.336g/L and 0.432g/L for the three mixtures on a protein basis. The bitterness of the samples from ALACEN T 392 was formally evaluated against two standard hydrolysates. The scores for bitterness on a scale of 1 to 10, being most bitter were 1.9 for a sample after 0.5h hydrolysis, 2.3 for the 2h hydrolysis compared to 5 and 7 for the standard hydrolysis samples of greater degrees of hydrolysis.

The samples ofALACENTM 421 and a mixture ofALACEN 392 and lactose taken after 2h had a mean particle size of 3pm or 2p.m respectively. The sample of ALACENTM 392 had a mean particle size of 6mu after 2h hydrolysis and inactivation as specified. Less grittiness and chalkiness was attributed to the smaller particle size samples.

The solubility of the hydrolysed ALACEN T 392/lactose mixture was the highest with approximately 85% across the pH range. The ALACENT 392, ALACEN T 421 samples are soluble to about 70% with a slight drop in solubility to 65% at pH Example Three different solutions from ALACEN T 392, ALACEN T 421 and a mixture ofALACEN

T

392 and lactose were made up of 30% solids to yield 75L. The enzyme treatment was done using the same conditions as example 4. The samples taken at half hourly intervals were diluted to 15% solids. Otherwise the heat treatment was done as in example 4. The ACE-I activity measured as specified in example 1 was 0.560g/L, 0.440g/L and 0.728g/L.

Samples from example 4 and 5 were added in a concentration of 0.1% to the standard growth media ofBifidobacterium lactis and resulted in a faster cell growth and higher final cell density of the strain than the control without any supplement.

The oligosaccharide level (trisaccharides and higher) of those three hydrolysed samples was 2.1% and 7.0% in ALACEN T 392, ALACEN T 421 and the mixture ofALACENTM 392 and lactose, respectively.

Example 6 Hydrolysate powders prepared in example 5 were used as a supplement for yoghurts in addition rates from 2.5% and 5% of the final yoghurt and resulted in an increased creaminess and improved the texture compared to the control.

Example 7 The hydrolysate powders prepared in example 5 were used as the protein source in a muesli bar recipe on a 6% and 12% w/w addition rate. All tasters preferred the hydrolysate bars over the unhydrolysed WPC control. The best results were achieved with hydrolysed ALACEN T 421 and a mixture of ALACEN T 392 and lactose prepared in example Example 8 The hydrolysate powder prepared in example 5 was used as an ingredient in a meal replacer concept sample. ALACENTM 421 hydrolysed in lactose and protein was added at a rate of w/w to whole milk powder, malto dextrin, sucrose and milk calcium (ALAMIN

T

to result in a powder meal replacer prototype. In comparison with a control sample without hydrolysed whey protein, hydrolysed whey protein prepared in example 5 was found to be more acceptable.

Example 9 A nutritional whey protein drink was formulated containing 8% w/w of ALACEN T 392 or

ALACEN

T 421 or a mixture of ALACENTM 392 and lactose hydrolysed as specified in example 5. The drink also contained sucrose, citric acid, flavouring and colouring agents. The pH of the drink was adjusted to 4.3. The drink combined the nutritional and health advantages of whey protein with the refreshing taste of a soft drink. Compared to a drink containing untreated whey protein control the pH stability was improved and the drink had a more milk like appearance than the control.

Example A further nutritional protein drink was formulated containing 12.5% w/w of ALACEN T 421 hydrolysed as in example 5 in water mixed with pasteurised whole milk. Sucrose was added to yield 6% of the final formulation as well as stabiliser. The drink was flavoured when desired with banana, vanilla or similar flavours. To achieve an extended shelf life the drink was ultra high heated to 140 0 C for 3 seconds. The mean particle size remains at 3 microns after the additional UHT heat treatment.

Example 11 The hydrolysis was carried out as specified in example 5 but instead of reconstituting ALACENTM 421 powder a fresh retentate of ALACENTM was concentrated to solids in the solution. The neutrase addition rate was varied to 0.9% w/w (on a protein base), the lactase level as specified. The reaction mixture was inactivated at 15% solids after 2h. The ACE-I activity yielded 0.480g/L. The organoleptic properties, particle size and food application were very similar to example 4 and Example 12 The hydrolysis was carried out as specified in example 4 with ALACEN T 421 powder. The Neutrase addition rate was varied to 0.9% w/w (on a protein basis). The lactose was converted with a lactase from Aspergillus oryzae (Fungal lactase 30,000, Kyowa Enzymes Co. Ltd.

Japan) on an addition rate of 0.4% w/w (on lactose base). The reaction mixture was inactivated after 1.5h with direct steam injection to achieve a temperature of 88 0 C for either 1.5 seconds or 3 seconds.

The particle size was 2.3 microns. Organoleptic properties and food application were very similar to the product of example 4.

Example 13 A 10% w/w solution of ALACEN T 392 was hydrolysed with a commercial protease from Bacillus subtilis Neutrase (Novo, Nordisk Denmark) at an enzyme concentration of 0.9% w/w.

The reaction continued for 6h at 50 0 C. Samples of 200ml were taken every lh, inactivated at 88 0 C for 8 seconds and subsequently freeze dried.

ACE-I activity, degree of hydrolysis, pH of solution and bitterness developed over time as follows. The higher the bitterness score the more bitter is the taste. The smaller the level measured, the higher is the ACE-I activity.

TABLE 1: Hydrolysis of ALACENTM 392 WPC Hydrolysis ACE-I activity Degree of pH of solution Bitterness score time (IC 50 hydrolysis [informal, 0-10] 1 0.420 3.86 7.01 0 2 0.280 3.78 6.96 1 3 0.230 4.53 6.92 1 4 0.220 4.89 6.89 0.210 5.20 6.87 2 6 0.190 5.37 6.87 Example 14 A 10% w/w solution of ALACENTM 392 was hydrolysed with the following commercial proteases at 1% w/w, 50 0 C for lh. The reaction was inactivated at 88 0 C for 8 seconds and subsequently the hydrolysate was freeze dried.

TABLE 2: Hydrolysis with Different Enzymes Enzyme ACE-I pH Degree of activity hydrolysis [g/L](ICso) Protease P6, neutral protease, Aspergillus strains, 0.274 7.0 8.9 Amano Enzymes Protease A, neutral protease, Aspergillus oryzae, 0.443 7.0 9.2 Amano Enzymes Protease M, acid protease, Aspergillus oryzae, Amano 0.450 4.0 7.4 Enzymes Peptidase, neutral peptidase, Aspergillus oryzae, 0.540 7.0 6.9 Amano Enzymes Neutrase, neutral bacterial protease, Bacillus subtillis, 0.510 7.0 4.3 Novo Nordisk DK Validase (Genancor), acid fungal protease, Aspergillus 0.510 4.0 5.6 niger, Enzyme Services Ltd. NZ AFP 2000 (Genancor), acid fungal protease, 0.550 4.0 3.9 Aspergillus niger, Enzyme Services Ltd. NZ Example Identification of ACEI-Peptides and Measuring their Activities 200 mg of permeate from example 3 was dissolved in 0.1% trifluoroacetic acid (TFA) and applied to a Jupiter preparative reverse-phase HPLC column (10 micron, C18, 22 x 250 mm [Phenomenex NZ]) equilibrated with solvent A (0.1%TFA) and connected to an FPLC system (Pharmacia). Peptides were sequentially eluted from the column with a gradient of 0 to 43% solvent B (0.08% TFA in acetonitrile) in 245 min at a flow rate of 10mL/min. Peptides eluting from the column were detected by monitoring the absorbance of the eluate at 214nm. The eluate was collected by an automatic fraction collector set to collect 3 min fractions.

Each fraction was lyophilised and the amount of peptide material present was measured gravimetrically. Fractions were assayed for ACE-I activity using an in vitro assay system (reagents from Sigma product 305-10) consisting of rabbit lung ACE and the colorimetric ACE substrate furylacryloylphenylalanylglycylglycine (FAPGG); ACE hydrolyses FAPGG to give the products FAP and GG which results in a decrease in absorbance at 340mm. If a peptide inhibits ACE, the change in absorbance at 340nm is reduced. Fractions containing the highest ACE inhibitory activity per mg peptide material were re-applied to the preparative reverse-phase HPLC column and eluted using a shallow gradient of solvent B i.e. 0.09% increase in solvent B concentration/min. The eluate was collected using the fraction collector set to collect 0.5 min fractions.

Samples from each fraction were analysed using an analytical reverse-phase HPLC column, and those fractions containing a single, identical peptide were pooled. Each pooled fraction was lyophilised and the weight of the peptide present was determined gravimetrically. The purified peptides were assayed for ACE-I activity as before and the IC 5 0 was calculated for each individual peptide.

The molecular weight of each peptide was determined by Electrospray lonisation Mass Spectrometry (Sciex API 300 triple quadrupole mass spectrometer). Tandem mass spectrometry was also done for each peptide to generate CAD spectra using MSMS experiment scans. Each peptide was also analysed by an automated N-terminal sequencer (ABI model 476A protein sequencer). Data collected from all three techniques was used to deduce the sequence of all of the peptides possessing ACE-I activity. The origin of each of the active peptides was determined by searching a database containing the known sequences of all bovine milk proteins.

The peptides, their origins, activities and known similarities are set out in table 3. Although the last three peptides are of a casein origin they were from a whey protein hydrolysate. The rennet used to precipitate casein did not precipitate these casein fractions and they remained with the whey proteins.

TABLE 3: ACE-I Peptides and their Activities Peptide Sequence'a Origin ActiVityb Similarity to (1C 5 o in mg L- 1 known ACE-I Peptides AFE PP 3(129-131) (Ala-Phe-Glu) LFSH PP3(125-128) (Leu-Phe-Ser-His) ILKEKH PP3(71-76) (Ile-Leu-Lys-Glu-Lys-His) LJVTQ 1-LGe(1-5) 17 (Leu-Ile-Val-Thr-Gln) MKG P-LG(7-9) 24 (Met-Lys-Gly) LDIQKC f3-LG( 10-14) 17 1-LG(9- 14) (Leu-Asp-Ile-Gln-Lys) VF P-LG(81-82) 19 (Val-Phe) ALPMH 1-LG( 142-146) 12 P-LG(1 42-148) (Ala-Leu-Pro-Met-His) VTSTAV GMPT(59-64) (Val-Thr-Ser-Thr-Ala-Val) LHLPLP O3-CN9(133-138) 7 (Leu-His-Leu-Pro-Leu-Pro) LVYPFPGPIPNSLPQNIPP f-CN(58-76) 19 J-CN(74-76) (Leu-Val-Tyr-Pro-Phe-Pro- Gly-Pro-Ile-Pro-Asn-Ser-Leu- Pro-Gln-Asn-Ile-Pro-Pro) LFRQ a, 1 -CN(136-139) 17 h (Leu-Phe-Arg-Glu) a sequence given using the single-letter amino acid code with the corresponding three-letter code in brackets b using the colorimetric substrate FAPGG c most abundant ACE-I in hydrolysate d protease peptone e P-lactoglobulin f glycomacropeptide g p-casein h activity measured with that of another peptide of unknown origin Example 16 The effect of the hydrolysate powder prepared in example 3 (without ultrafiltration) on in vivo blood pressure was tested using spontaneously hypertensive rats (SHR/N). The rat strain has been specifically selected for their development of high blood pressure on maturing, and is used extensively to monitor the effect of blood pressure lowering agents. They were purchased from Animal Resources Centre, P 0 Box 1180 Canning Vale, Western Australia 6155.

Eight week old rats were individually housed in plastic rat cages and kept in temperature controlled facilities throughout the trial. They had unlimited access to water and were fed commercial rat chow ad libitum. The test products were given orally as a single daily dose for 8 weeks during which time changes in blood pressure were monitored. Their blood pressure was measured using a specially designed tail cuff and blood pressure monitoring apparatus (IITC Inc., Life Science Instruments, 23924 Victory Blvd, Woodland Hilld, CA 91367). The experimental design was approved by the Massey University Animal Ethics Committee, protocol number 98/141.

The changes in the systolic blood pressures of each group of animals over the eight weeks are plotted in Figure 2 (as least squares means). The hydrolysate at both 2g/Kg bodyweight/day and 4g/Kg bodyweight/day significantly lowered the systolic blood pressure of SHRs compared to animals fed commercial rat chow only (p<0.004 by least-squares means analysis, see Figure The effect of the hydrolysate was not as great as that of captopril, a known ACE-I inhibitory drug administered at bodyweight/day, but was a significant improvement for animals fed commercial rat chow only.

REFERENCES

Bernal V Jelen P (1989). Effectiveness of lactose hydrolysis in Cottage cheese whey for the development of whey drinks. Milchwissenchqft 44: 222-225 Cushman D W Cheung H S (1971). Spectrophotometric assay and properties of the angiotensin converting enzyme in rabbit lung. Biochem Pharmacol 20: 163 7-1648.

FR 2309154, 30 December 1976 Fromageries Bel La Vache Qui (From), France.

US 3970520, 20 July 1976, General Electric Co, USA.

EP0117047, 29 August 1984, General Foods Corporation, USA.

Maubois J L, L6onil J, Trouv6 R Bouhallab S (1991) Les peptides du lait A activit6 physiologique III. Peptides du lait A effect cardiovasculaire: activit6s antithrombotique et antihypertensive. Lait, 71, 249-255.

JP 4282400, 7 October 1992, Calpis Shokuhin Kogyo KK, Japan.

EP065663, 1 December 1982, Miles Laboratories Incorporated, USA.

JP 8056568, 17 August 1994. Morinaga Milk Co Ltd Japan.

EP4745506, 11 March 1992, Morinaga Milk Co Ltd, Japan.

Mullally M M, Meisel H FitzGerald R J (1997) Identification of a novel angiotensin-I-converting enzyme inhibitory peptide corresponding to a tryptic fragment of bovine 1-lactoglobulin. Federation of European Biochemical Societies Letters, 402, 99-101.

Nakamura Y, Yamamoto N, Sakai K Takano T (1994) Antihypertensive effect of the peptides derived from casein by an extracellular proteinase from Lactobacillus helveticus CP790. Journal of Dairy Science, 77, 917-922.

Roy G (1992). Bitterness: reduction and inhibition. Trends in Food Science and Technology 3 91 Roy G (1997). Modifying bitterness: Mechanism, ingredients and applications. Technomic Publishers, Lancaster, UK.

US 4358464, 9 September 1982, Superior Dairy Company, USA.

Yamamoto N (1997). Antihypertensive peptides derived from food proteins. Biopolymers 43:129- 134.

Claims (11)

1. A bioactive peptide selected from the group consisting of AFE, LFSH, ILKEKH, LIVTQ, MKG, LDIQK, ALPMH, VTSTAV, LVYPFPGPIPNSLPQNIPP and LFRQ.

2. A combination of any two or more of the bioactive peptides as claimed in claim 1.

3. The bioactive peptide AFE.

4. The bioactive peptide LFSH. The bioactive peptide ILKEKH.

6. The bioactive peptide LIVTQ.

7. The bioactive peptide MKG.

8. The bioactive peptide LDIQK.

9. The bioactive peptide ALPMH. The bioactive peptide VTSTAV.

11. The bioactive peptide LVYPFPGPIPNSLPQNIPP.

12. The bioactive peptide LFRQ.

13. A method of reducing systolic blood pressure in a subject which comprises administering to that subject an effective amount of a peptide as claimed in any one of the preceding claims.