Edible emulsions with peptide

Field of the invention

The invention relates to an edible emulsion comprising peptide and polyglycerol polyricinoleate .

Background to the invention

Cardiovascular disease (CVD) is one of the major causes of death in the western world. Elevated serum, total and/or LDL (low density lipoprotein) cholesterol and triacylglycerol levels, as well as low ratio of HDL (high density lipoprotein) cholesterol to LDL cholesterol are some of the major risks for CVD.

Protein hydrolysates have been shown to have greater serum cholesterol lowering effect than the corresponding unhydrolysed proteins (Sugano et al . J.Nutr. 1990 (120) 977-985). The cholesterol lowering effect is attributed to the formed peptides.

WO01/24789 discloses that β-peptides are very suitable agents to reduce cholesterol up-take and lipid uptake. EP1836906 WO2007/110296 disclose peptides that are able to inhibit uptake of cholesterol.

Hypertension or high blood pressure is considered to be one of the main risk factors for cardio vascular disease (CVD) .One of the mechanisms which regulate blood pressure is the rennin- angiotensin system. This cascades of reactions leading to formation of angiotensin II, which has strong vasoconstrictive and hence blood pressure increasing effect. Inhibition of one of the key enzymes in this cascade, i.e. Angiotensin I

converting enzyme (ACE) , reduces formation of angiotensin II and thus has blood pressure lowering effect.

Protein hydrolysates and fermentates may contain peptides that decrease blood pressure, probably by their inhibitory activity on ACE .

WO2006/084560, WO2006/114195, WO2004/098309, WO2004/098310, WO2004/060073, and WO02/071854 disclose compositions with ACE- inhibiting peptides.

Edible emulsions are used in a variety of ways, e.g. spreadable products, frying products, baking ingredients, drinks, diary type products. They are therefore suitable vehicles for incorporation of peptides.

Unfortunately protein hydrolysates and peptides have a bitter taste and the amount of hydrolates and peptides needed in food products to fortify in order to obtain a beneficial effect from it gives the food product a bitter taste which is not liked by consumers .

One option is to encapsulate the protein hydrolysate or peptides to prevent the contact of the ingredient with the mouth. A drawback of the encapsulation is that a suitable encapsulate should be found that works well in the food product. Furthermore encapsulates are often more expensive than the bare ingredient it self.

Another way of masking the bad taste of protein hydrolysate and peptides is to add another taste that overtakes the bitter taste. However often a lot of the masking taste should be added to mask the undesired taste and not much flexibility in taste

is left, and another strong taste is left, which leaves out neutral tasting food products.

WO2006/084573 discloses food products comprising hydrolysed milk solids. The undesirable off taste of the hydrolysates in the compositions is remedied by the addition of sterol or stanol fatty acid esters.

A drawback of sterol or stanol fatty acid esters is that they are expensive. There is therefore a need for compositions with protein hydrolysates and/or peptides that do not have an undesirable taste of the hydrolysate or peptide that are less expensive than the compositions of WO2006/084573.

Polyglycerol polyricinoleate (PGPR) is a strong water-in-oil emulsifier and has been used in many spread and margarine applications and is commonly used for chocolate compositions. Examples are WO 03/51135, WO 01/91570, EP 0997074, WO03/051136, US 6,159,526, WO 2005/004642, and WO 02/49443 however in none of the applications the use protein hydrolysates and/or peptides is disclosed. Milk proteins may be used, however, intact, unhydrolysed milk proteins do not suffer from the bad taste of hydrolysed proteins and peptides.

WO03/055324 discloses a combination of protein hydrolysates and plant sterols that improves serum lipid profiles. Emulsifiers are optional ingredients and polyglycerol polyricinoleate is mentioned as a possible emulsifier. Emulsifiers are used to complex the protein hydrolysate and/or the plant sterol. No emulsion with protein hydrolysate and polyglycerol polyricinoleate was disclosed.

WO 03/049548 discloses water-continuous emulsified food compositions comprising water-soluble tastants among which peptides are mentioned. The compositions of WO 03/049548 are W1/O/W2 duplex emulsions wherein water phases Wl (dispersed water phase) and W2 (continuous water phase) comprise at least one water-soluble tastant in both of the water phases and are substanstially isotonic for the tastant. These duplex emulsions give a reduced taste impression of the tastant when compared to single water-continuous emulsions. Unfortunately duplex emulsions are inherently more complex than single emulsions. Furthermore at least 2 waterphases need to be prepared each containing the tastant and being isotonic for the tastant. In addition, only water-continuous emulsions are disclosed. The internal emulsion (Wl/O) is stabilised by an emulsifier with hydrophobic lipophilic balance (HLB) of less than or equal to 6. An example of such an emulsifier is polyglycerol polyricinoleate . No specific disclosure of an emulsion with peptides and polyglycerol polyricinoleate is made.

WO 02/089594 discloses all vegetable emulsions wherein the aqueous phase comprises a protein containing cereal base and the fat phase comprises a fractionated vegetable oil. The examples show comparative spreads with polyglycerol polyricinoleate (PGPR) . The spreads were tested by a taste panel. The results show that spreads with fractionated oat oil have better taste feel for salt and sourness in comparison with spread without oat oil but with PGPR. There is however no difference tasted in spreads for rancid, old, metallic and bitterness taste. Furthermore spreads with PGPR and with oat base had a more salty taste than spread without PGPR and without oat base and the same sourness taste for both. No mention of hydrolysed protein or peptides is made.

US2003/0108591 discloses ingestable products for lowering blood total cholesterol including isoflavone, soy protein and phytosterol. Spreads are mentioned as a beneficial form for ingestion. Furthermore, polyglycerol polyricinoleate is described as an advantageous emulsifier. No examples are disclosed containing polyglycerol polyricinoleate. No mention of hydrolysed protein or peptides is made.

In WO 00/64276 discloses water-in-oil spreads including phytoestrogens and calcium salts. The spreads have good taste despite the presence of often-bitter tasting isoflavones. Polyglycerol polyricinoleate is said an optional ingredient. No examples are disclosed containing polyglycerol polyricinoleate. No mention of hydrolysed protein or peptides is made.

WO 00/64268 discloses water in oil emulsions with a variegate composition having a pronounced sweet taste. The variegate composition can be based on vegetable extracts or fruit extracts. Polyglycerol polyricinoleate is mentioned in a long list of emulsifiers. No examples are disclosed containing polyglycerol polyricinoleate. No mention of hydrolysed protein or peptides is made.

It is therefore an object of the current invention to provide a food product which comprises peptides. In addition, it is an object of the present invention to provide a food product which comprises peptides in an amount that gives a health effect. Furthermore an object of the invention is to provide a food product with peptides wherein the bitter taste of the peptide is not noticed by consumers. Another object of the invention is to provide a food product which is stable under storage at ambient and higher temperatures. Further food products with

good organoleptic properties are envisioned with the current invention .

Summary of the invention One or more of the above objects are attained by an edible emulsion comprising a peptide, 0.1 to 1 wt% polyglycerol polyricinoleate, and 15 to 85 wt% fat.

It was surprisingly found that emulsions with peptide do not have the flavour or taste of the peptide if polyglycerol polyricinoleate (PGPR) is used in the emulsion.

Detailed description of the invention

The present invention relates edible emulsion comprising peptide.

Peptide is defined herein as a chain of at least two amino acids that are linked through peptide bonds. Preferably a peptide has less than 30 amino acid residues. A polypeptide is defined herein as a chain containing more than 30 amino acid residues. The peptide formula or sequences are written from left to right in the direction from amino-terminus to carboxy- terminus, in accordance with common practice. The one and three letter code of amino acids used in herein is commonly known in the art and can be found in Sambrook et al . (Molecular cloning: A laboratory Manual, 2 ^nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 1989) .

Protein hydrolysate are also known to have a positive effect on cardiovascular diseases. Protein hydrolysates contain peptides. Protein hydrolysates may be prepared via enzymatic hydrolysis by methods known in the art and by other known hydrolytic techniques such as acid hydrolysis.

Proteolytic enzymes suitable for hydrolysing protein may be selected from the group consisting of proteases, peptidase, exopeptidases, endopeptidases .

The protein substrate may be selected from the group consisting of milk protein, soy, casein, milk protein fragments, casein fragments, whole milk, skimmed milk, acid casein, rennet casein, whey protein, collagen based animal protein such as gelatine, wheat, gluten, milled barley, rice, corn.

Milk ferments may also contain protein hydrolysates and peptides. Milk, fermented with Lactobacillus helveticus contain bioactive peptides (VPP and IPP) responsible for in vitro ACE inhibition, which are produced by proteolysis of casein. Other microorganisms than Lb. helveticus have been reported to produce peptides in milk, which give ACE inhibition. For instance Lb. delbrueckii subsp. bulgaricus, Lb. delbrueckii subsp. lactis, Lactococcus lactic subsp. cremoris .

Flavour is the sensory impression of a food or other substance, and is determined mainly by the chemical senses of taste and smell. Peptides are known to give a bitter, cardboard, medicinal, acid dairy or cheese off-taste or flavour. The flavour of the peptide may be determined by a sensory panel. The present invention is especially suited for compositions comprising a peptide, 0.1 to 1 wt% polyglycerol polyricinoleate, and 15 to 85 wt% fat wherein in the peptide has a bad taste. The present invention is even more suitable for peptides with a taste that is selected from bitter, cardboard, medicinal, acid dairy or cheese off-taste or flavour .

The taste or flavour of an ingredient is dependent on the concentration of the ingredient. Therefore the present invention preferably relates to an emulsion with a peptide in an amount that gives an undesired flavour in the emulsion without polyglycerol polyricinoleate . The emulsion with peptides is tested by a sensory panel that evaluates the flavour of the emulsions with and without polyglycerol polyricinoleate. The amounts of peptides that show a different in taste or flavour between emulsions with and without polyglycerol polyricinoleate are envisioned by the present invention .

Sensory panels consist of trained human assessors that qualify and quantify sensory properties of foods. The responses made by the sensory panels are recorded and may be analysed by statistical methods e.g. ANOVA, multivariate or univariate data analyses. Within food research, sensory panels are commonly used to qualify and quantify sensory properties, such as taste, odour or smell, flavour, mouthfeel, and other organoleptic properties.

Preferably the edible emulsion of the present invention has a good melting behaviour. Melting behaviour influences the organoleptic properties of an emulsion. If the emulsion doesn't melt fast enough a waxy mouthfeel becomes present and this is not appreciated by consumers.

In addition, suitably the edible emulsion of the present invention has overall a good taste impression, suitable for the product, despite the presence of peptides in an amount that gives an undesired flavour. The emulsion preferably has a good melting behaviour in the mouth and a creamy and/or dairy taste is appreciated.

The present invention is especially suited for peptides in an amount needed to obtain a health benefit. High doses are necessary to provide a health benefit, the so-called effective amount. The present invention is specifically suited for peptides in effective amounts.

Preferably the effective amount of peptides is such that it would give a health benefit in a daily serving size of the food product. The daily serving size is the amount of a food product typically eaten in a day. The daily serving size need not to be taken in 1 go, i.e. it may be divided up in several portions a day. Different food products have different daily serving sizes. Margarine for example has a daily serving size of about 10 to 30 g per day, which may be divided up in 4 portions, together giving the daily serving size.

In a suitable embodiment of the present invention, the health effect is selected from the group comprising reduction of risk for cardiovascular disease, reduction of blood pressure, increased resistance to disease, improved immune response, improved blood flow, lowering plasma triglycerides, antiinflammatory effect, inhibition of platelet aggregation, antithrombotic effect and healthy growth for children. More specifically, the composition of the present invention are efficacious for the prevention and treatment of a disease selected from the group consisting of hypertension, heart failure, angina pectoris, myocardial infarction, stroke, peripheral arterial obstructive disease, atherosclerosis, nephropathy, renal insufficiency, erectile dysfunction, endothelial dysfunction, left ventricular hypertrophy, diabetic vasculopathy, fluid retention and hyperaldosteronism. The composition of the present invention may also be useful in the

prevention and/or treatment of gastrointestinal disorders (diarrhoea, irritable bowel syndrome) , inflammation, diabetes mellitus, obesity, dementia, epilepsy, geriatric confusion, and meniere's disease. Furthermore the compositions may enhance cognitive function and memory (including Alzheimer's disease), satiety feeling, limit ischemic damage and prevent reocclusion of an artery after by-pass surgery or angioplasty.

The present invention is especially suitable for non- encapsulated peptides. The use of polyglycerol polyricinoleate in the edible emulsion of the present invention circumvents the need for encapsulation of the peptides with an undesired flavour. A preferred embodiment of the present invention provides for an edible emulsion with a peptides that is not encapsulated.

The present invention is also very suitable for edible emulsions with peptides and another tasty ingredient. Even though a taste or flavour of an ingredient might be acceptable to a food product, the addition of another tasting ingredient might give a combination of flavours that is not desired, e.g. a chocolate flavour with an onion flavour. Because the edible emulsion with polyglycerol polyricinoleate somehow masks the taste of a tasty ingredient, 2 or more tasty ingredients with different taste would still give a food product with an acceptable taste when applying the present invention. A preferred embodiment of the present invention provides for an edible emulsion with peptide and at least one other tasty ingredient. Even more preferred the present invention provides for an edible emulsion with a peptide and at least one other tasty ingredient wherein at least one other ingredient gives a health benefit.

Another suitable embodiment of the present invention is an emulsion with a neutral taste. The present invention avoids the need of the addition of another flavour to mask the taste, thereby providing an ability to have neutral tasting products. The neutral taste of a food product may be assessed by a sensory panel.

The amount of peptide depends on the amount needed for an effect to be obtained and on the quantity of a serving size and on the emulsion type and can be determined by the skilled person .

A preferred amount of peptide is at least O.lg/kg, preferably 1 to 200 g/kg, more preferably 2 to 30 g/kg food product. Preferably the amount of peptide is 0.1 to 20 g of peptide in a daily amount, more preferably 0.5 to 10 g, and most preferably 1 to 5 g.

Suitable daily amount are 10 to 50 g, more preferably about 20 g to 40 g for spreads. Suitably 1 to 5 servings are consumed to provide the daily dosage of the peptide.

Suitably the emulsion of the present invention comprises 0.1 to 10 wt% of peptide, more preferably 1 to 5 wt% and most preferably 2 to 4 wt%.

Preferably the emulsion of the present invention comprises 0.1 to 15 g of protein hydrolysate, more preferably 0.5 to 10 g and more preferably 0.8 to 5 g of protein hydrolysate.

Suitable peptides are selected from the group consisting of IPP, VPP, MAP, ITP, LPP, VY, VF, IW, VAP, AW, LW, HLP, IY, FY, LP, PPL, YP, TPP, MPP, VLP, LPV, LPVP, DKIHPF, HLPLP, KVLPVP,

KVLPVPQ, I IAEK, IPAVF, IPAVFK, RRRR, RRRRRR, RRRRRRRR, KKKK, KKKKKK, KKKKKKKK, VRWRMR, RARWRY, HHHH, HHHHHH, HHHHHHHH, AAAAAA, AAAAAAAA, LLLLLL, LLLLLLLL, peptides having 6-20 amino acids and wherein the peptide is substantially water insoluble, beta peptides and salts thereof. Suitably the emulsion of the present invention comprises combinations of the mentioned peptides and/or peptides with less than 30 amino acids comprising the amino acid sequence of the mentioned peptides.

Suitable protein hydrolysates are hydrolyzed proteins selected from the group consisting milk protein, soy, casein, milk protein fragments, casein fragments, whole milk, skimmed milk, acid casein, rennet casein, whey protein, collagen based animal protein such as gelatine, wheat, gluten, milled barley, rice, corn.

Suitably the proteins are hydrolyzed by proteolytic enzymes selected from the group consisting of proteases, peptidase, exopeptidases, and endopeptidases .

Suitable ferments are selected from the group consisting of milk or milk protein containing compositions fermented by Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii subsp. lactis, Lactococcus lactic subsp. cremoris, and Lactobacillus helveticus .

Products according to the invention comprise polyglycerol polyricinoleate which is commercially available amongst others under the name PGPR 90 ex Danisco, and under the name of Admul WOL ex Kerry. This ingredient is generally known to be excellent water-in-oil emulsifier.

The amount of polyglycerol polyricinoleate in the products of the invention is from 0.1 to 1% wt% on total product weight. Higher amounts lead to products which do not easily de-emulsify in the mouth upon consumption and will hence not show the desired organoleptic properties. Preferably the amount of polyglycerol polyricinoleate in food products according to the invention is from 0.2 to 0.4 wt%.

The emulsion of the invention may comprise thickeners. For stability reasons it may be useful to include thickeners in the emulsion, for example very low spreads, with 20 to 30 wt% of fat, often improve by addition of thickeners. Whether or not a thickener should be added and in what amount depends on factors as stability and application and may be determined by the skilled person.

Thickener may be any known thickener and are preferably selected from the group comprising gums, like xanthan, guar, and locust bean, carrageenan, polysaccharides, alginate, pectin, starch, and gelatine.

In preferred food products according to the invention, the aqueous phase comprises a native or a modified fully gelatinised starch which may be cook-up or pre-gelled, selected from any of the main starch groups: wheat, potato, rice, maize, waxy rice or waxy maize.

Examples of suitable starches include Remyrice™, Resistamyl™, Merigel™, Purity LFS™.

The amount of starch in the food product according to the invention depends somewhat on the type of chosen starch and is

preferably from 0.2 to 5 wt%, more preferred from 0.7 to 3 wt%, most preferred from 1 to 2 wt%.

In a preferred embodiment the emulsion is fat continuous. In another preferred embodiment the emulsion is not a duplex emulsion. Even more preferred the emulsion is a single emulsion. A preferred embodiment of the present invention is a spread. Preferably the emulsion of the present invention is not chocolate or a chocolate containing emulsion.

The emulsion according to the invention comprises from 15 to 85 wt% of a fat, preferably from 20 to 70 wt% more preferably from 25 to 60 wt%, most preferably from 30 to 40 wt% fat. The fat consists predominantly of triglycerides. The fat can be a single fat or a combination of fats.

The fat or fat blend may comprise vegetable or animal fats which may be hydrogenated, interesterified or fractionated and combinations thereof. Suitable animal fats may consist of butterfat or tallow. Suitable vegetable fats can for example be selected from the group comprising bean oil, sunflower oil, palm kernel oil, coconut oil, palm oil, rapeseed oil, cotton seed oil, maize oil, or their fractions, or a combination thereof. Interesterified fat blends of these fats or optionally with other fats are also encompassed in the invention.

Advantageously, long chain poly unsaturated fatty acids (LC- PUFA), e.g. omega-3 and omega-6 fatty acids are incorporated in the edible emulsion of the present invention. Suitably these LC-PUFAs come from sources like fish oil and/or algae oil.

To ensure homogeneous distribution of the aqueous phase in the continuous fat phase, the droplet size distribution D ₃ , ₃ of the

dispersed aqueous phase is preferably less than 8 μm, more preferably from 4 to 8 μm, more preferred even lower than 4 μm. The method to determine D ₃ , ₃ is illustrated in the examples. It will be appreciated that the droplet size can be controlled by adjusting the processing conditions in the unit operations: e.g. higher rotational speed in a scraped surface heat exchanger will produce correspondingly smaller water droplet size distributions.

In addition to the water-in-oil emulsifier polyglycerol polyricinoleate, the food product according to the invention comprises another emulsifier, the co-emulsifier . This co- emulsifier is preferably also a water-in-oil emulsifier. More preferably this co-emulsifier is selected from the group comprising distilled monoglycerides, citric acid esters of monoglycerides, di-acetyl acetic acid esters of monoglycerides, lactic acid esters of monoglyceride, mono-diglycerides, polyglycerol esters of fatty acids or sorbitan esters of fatty acids .

The most preferred co-emulsifier is a distilled monoglyceride. Even more preferred are monoglycerides with unsaturated fatty acids or combinations of a monoglyceride comprising a saturated fatty acid residue and a monoglyceride comprising an unsaturated fatty acid residue.

The amount of co-emulsifier depends on the type and effectiveness of the emulsifier selected and can be determined by the person skilled in the art. Other factors influencing the amount of emulsifier that is required to obtain storage stable products are the amount of fat and the amount of polyglycerol polyricinoleate.

As a general guidance the amount of emulsifier is preferably from 0.05 to 1.5 wt%, more preferred from 0.1 to 0.7 wt%, most preferred from 0.15 to 0.5 wt%.

In another preferred embodiment the emulsion does not contain an emulsifier/protein hydrolysate complex.

The pH of the aqueous phase can be set to the desired value, among others to influence acidic or basic taste impression and to influence microbial stability. Preferably the pH of the aqueous phase in food products according to the invention is from 4.3 to 5.5.

Optionally some protein is added to the product according to the invention. Protein may be added to beneficially influence the taste, flavour and nutritional value of the food product and also may be added to increase browning of food stuff when the current composition is used as a medium for shallow frying. Preferably the protein source is selected from the group comprising milk powders such as skim milk powder, butter milk powder, sodium caseinate, sour whey, denatured whey, or a combination thereof. It should be noted that normal, unhydrolysed dairy protein, is not encompassed in the definition of peptide.

Preferably at least 0.3 wt% of protein is present in the emulsion, more preferably from 0.3 to 1 wt%. In a preferred embodiment the emulsion according to the invention does not comprise a protein containing oat base such that the protein content of the emulsion is 0.01-0.2% by weight. In another preferred embodiment the emulsion is not a water-in-oil spread comprising isoflavones, soy protein, and phytosterols .

The emulsion according to the invention optionally contain other ingredients such as preservatives, vitamins, taste and flavour components, colorants such as beta-carotene, antioxidants .

The emulsion according to the invention can be prepared by any suitable process to prepare such products. A preferred process is a so-called inversion process; a fat phase containing polyglycerol polyricinoleate and a waterphase are provided and mixed to obtain a water-continuous pre-mix containing the peptide. The water-continuous premix is later inverted to a fat-continuous emulsion.

In some cases to obtain a health effect, the effective amount of the peptides is so large that a fat-continuous mix of all the ingredients is too thick and the pressure in the system is too high. It was surprisingly found that first a water- continuous premix could be made that did not have the high pressure issues. Polyglycerol polyricinoleate is a very strong water-in-oil emulsifier (HLB ^~ 1) and it was not expected that a water-continuous premix would not have the pressure problems.

Preferably the emulsion is a food product.

Experimental:

Storage stability test

Food product was stored in a plastic container at 10, 20, 30, 35 and 40 °C for up to 26 weeks. After storage the amount of phase separation was determined by visual examination of the product surface. Storage stable products show a phase separation of less than 5 wt% upon storage at 35 °C for at

least 10 weeks, preferably at least 26 weeks. Preferably the phase separation is less than 5 wt% upon storage at 40 °C.

D3,3 value measurements and E-sigma: Samples were filled to a height of 15 mm in NMR tubes of 10 mm diameter, and thermally equilibrated for 30 min at 20 ⁰ C. A restricted diffusion-based droplet size was obtained by means of pfg-NMR using a Bruker Minispec MQ20. The details of the technique are discussed by Goudappel et al (Journal of Colloid and Interface Science 239, (2001) 535-542) . A measurement yields values for the volume weighted geometric mean diameter d3,3 and the width of the droplet size distribution when plotted as a function of the logarithm of the diameter σ (E-sigma) . Measurements were carried out in triplicate and results are expressed in terms of average d3,3 values. Definitions of droplet sizes are given by Alderliesten (Particle and Particle Systems Characterization 7 (1990) 233-241, and ibid 8 (1991) 237-241) .

Example 1-3 and comparative example A

Ingredients are listed in table 1 Table 1; ingredients in wt%

Processing

In a vessel a mixture was prepared of the fat, monoglyceride, PGPR, antioxidant, and colorant at a temperature of about 60 ⁰ C. In another, separate vessel a mixture was made of starch and water which was heated to a temperature of 92 ⁰ C for 25 minutes. This mixture was cooled to 60 ⁰ C and completed with all the other water soluble ingredients, such as salt, protein, etc. and subsequently mixed with the oil phase in a pre-mix tank at 60 ⁰ C as a water-continuous emulsion, followed by cooling and shearing in a series of A- and C-units ^tm where the dimensions and energy requirements are suitable to deliver a fat-continuous end product and achieve a plastic structure which could be easily packed at around 10 ⁰ C in a suitable packaging material.

Taste scores were determined by a test panel of 10 persons; scores were given at a scale of 1 - 5. 1 indicates a good tasting score 3 is not acceptable for a consumer ready spread

5 is regarded as very bitter and a fully unacceptable product to offer to consumers.