FiELD OF THE lNVENTlON

[0001] The invention relates to a process for improving the taste of a milk product containing at least one sterol and/or stanol ester by means of a biologically active peptide. The invention also relates to the use of a biologically active peptide for preventing the oxidation of a sterol and/or a stanol ester and/or for reducing, eliminating and/or covering an off-taste caused by the oxidation of a sterol and/or a stanol ester in a milk product. The invention also relates to a process for preparing a milk product containing at least one sterol and/or stanol ester and at least one biologically active peptide.

BACKGROUND OF THE INVENTION

[0002] The preparation of therapeutically usefu! and particularly anti-hypertensive peptides by fermentation or enzymatically, and the properties, effect and mechanisms of action of said peptides have been abundantly studied, and literature relating to the field abounds. Peptides obtained from milk have been the object of special interest.

[0003] Anti-hypertensive peptides, particularly tripeptides, such as WPP (valine-proline-proϋne), IPP (isoieucin-proline-proline) and LPP (leucine- proline-proline), may be prepared either by fermenting milk with proteolytic lactic acid bacteria, such as Lactobacillus helveticυs strains or by hydrolyzing milk protein by selective proteases (WO 99/16862, US 5 449 661 , EP 1 231 279 B1 ). The problem in both processes is the off-tastes generated in the product.

[0004] In the fermentative process, not only tripeptides, but also lactic acid and aromatic substances are generated in the product. The milk raw materia! should be fermented to as long a degree as possible in order for a sufficiently high content of active tripeptides to be produced. At the same time, much lactic acid and other metabolic products are generated in the product. Lactic acid renders the product very sour and the taste unpleasant. In addition, aromatic substances are generated in the product whose taste is sensed as unpleasant. The acidity and low pH of a product prepared by conventional fermentation are unpleasant to some consumers. The process described in patent EP 1 226 267 B1 provides a solution to this problem by means of its nano- filtration steps. In the process, precipitated milk protein is separated from tripeptides-containing whey. The whey is then nanofiltered, allowing most of the lactic acid to be washed off into the permeate of the nanofiitration, and the tripeptides, in turn, are concentrated into the retentate of the nanofiftration. The purified whey is then concentrated by evaporation and optionally dried into powder. Alternatively, the purified whey is dried into powder. As the end result in the process, concentrate or powder having a mild and good taste is generated. In addition, said process allows the tripeptides to be concentrated into an as concentrated form as possible. Chromatographic purification of peptides prepared by fermentation is also known, and described in the above- mentioned US patent 5 449 661 , for example.

[0005] Alternative processes have also been presented for improving the yield of therapeutically useful peptides, and they are generally associated with the use of enzymes. In processes wherein milk protein is hydrolyzed by selective proteases, as in the process disclosed in the above EP patent 1 231 279 B1 , peptides having a bitter taste are generated. The generation of bitter peptides is a common problem in enzymatic hydrolysis.

[0006] Publication WO 2006/084573 discloses the use of a sterol fatty acid ester for improving the taste of food products containing tripeptides.

[0007] In studies, food products containing sterol and/or stand esters, such as sour milk preparations, have been found to possess a cholesterol-lowering property. In sterol and/or stanol ester preparations and consumer products containing them, the problem is oxidation reactions and the generation of secondary oxidation products, particularly in heat-treatment and/or during prolonged storage. Oxidation reactions and the generation of secondary oxidation products cause the product to have an unpleasant rancid flavor. Sterol fatty acid esters have been found to oxidize more easily than stanol fatty acid esters (Soupas, L., et al., Eur. J. Lipid Sci. TechnoL, 107 (2) (2005) 107-118).

[0008] Sterol/stanol esters are esterified with one or more plant oil- based fatty acids originating particularly from sunflower oil, rapeseed oil or a mixture thereof. Said fatty acids are often unsaturated, typically linolic acid and alpha linolenic acid. In commercial sterol/stanol ester preparations, the proportion of easily oxidized unsaturated, mainly mono-unsaturated fatty acids is high, typically more than 90%, They are easily oxidized at a low temperature in a sterol/stanol ester preparation and in the end product, such as a milk product, into which they are mixed. Particularly oxidization is a problem if said unsaturated fatty acids are allowed to react with oxygen in the presence of a catalyst (Fe ₁ Cu) or light and/or at an elevated temperature (more than 8 ⁰ C). Industrial use requires that the steroϊ/stanol preparation be heated in order to ensure an even mixing in the end product. This further increases the problems associated with the oxidation of sterol/stanol fatty acid esters.

[0009] Conventional dairy processes do not include oxygen-free conditions or the use of a protective gas especially in the packaging stage, and the product is exposed to oxygen, particularly in the step when the sterol/stanol preparation is mixed into the milk raw material. In addition, as regards mϋk products, the packages most generally used, such as a plastic-coated cardboard package, polystyrene or polyethylene beakers, partly permeate both oxygen and light.

[0010] Retail trade demands the prolongation of the sales period of fresh products and in the future, the sales period wii! continue to lengthen. The reasons include for instance the globalization of trade and also the increase in transports of fresh products between countries, among other things, which requires time. Accordingly, particularly the exposure of fresh products to oxidation has increased and will continue to increase.

[0011] In addition, the packages are not always dose packages, but the consumer of the product may use the same, opened package during several days, it is not either always possible to ensure that the cold chain (0 to 8 ⁰ C) of a milk product containing a plant sterol/plant stano! is continuous. An elevated temperature caused by a breakage of the chain also increases the risk of oxidation of the fatty acids and, thus, also the problems caused by oxidation in the product.

[0012] Processes are known for implementing oxygen-free conditions in commercial sterol/stanol ester preparations. The use of antioxidants is a!so known as an additive in the preparation of commercial sterol/stanol ester preparations for preventing oxidation. In the process disclosed in the above- mentioned publication WO 2006/084573, 200 ppm of vitamin E (tocopherol) was added to an end product, drinkable yoghurt, for example. However, there exists a continuous and evident need to develop natural processes for ensuring the stability of the end product, particularly by preventing the oxidation of fatty acids, without the use of additives.

[0013] On the other hand, it is known that the reactive -SH radicals contained in the β-lactoglobu!in of denatured whey protein prevent the oxidation of fats (Liu, H. C ₁ et al., J. Dairy. Sci. 90 (2) (2007) 547-555). [0014] Anti-hypertensive milk products may aiso contain many minerals, particularly potassium, calcium and magnesium, which intensify the antihypertensive effect. In an end product, said minerals are present in concentrations of up to 300% compared with the concentrations in normal milk, in order for an advantageous effect to be obtained, the amount added minerals has to be high, which causes a tangy (musty, salty) flavor in a milk product, such as miSk, for example.

[0015] Heart and vascular diseases are among the most common diseases in the world and are among the five most common life-threatening diseases in many countries. Unfortunately, a higher standard of living further increases the risk of contracting these diseases, so that their significance is increasing in the future. In addition to conventional medicines, functional products are marketed that offer consumers a lucrative addition and/or alternative to the treatment of heart and vascular diseases.

[0016] Consequently, there is a continuous and obvious need for functional, palatable products that can be used as part of the regular diet. In addition, there is a special need for the manufacture thereof by a natural process that is economic, industrialiy applicable and does not cause additional costs.

[0017] It has now been surprisingly found that biologically active low-molecular peptides, such as (isoleucin-proline-proline) IPP and/or (valine- proline-proline) VPP tripeptides or mixtures thereof, and/or concentrates or other preparations containing mixtures thereof, prevent the oxidation of sterol and/or stanol fatty acid esters. No off-flavor and/or off-taste has been detected in a milk product in which low-molecular peptides had been added in addition to the sterol and/or stanol esters that generally cause a musty off-taste.

BRIEF DESCRIPTION OF THE INVENTION

[0018] The invention relates to a process for improving the taste of a milk product containing at least one sterol and/or stanol ester by means of a biologically active low-molecular peptide. The invention also relates to a process for preventing the oxidation of a sterol and stanol fatty acid ester by means of a biologically active low-molecular peptide, and to a process for reducing, eliminating and/or covering an off-taste caused by the oxidation of a sterol and/or stano! ester by means of a biologically active low-molecular peptide. The invention further relates to the use of at least one biologically active pep- tide for preventing the oxidation of a sterol and/or a stanol ester and/or for reducing, eliminating and/or covering an off-taste caused by the oxidation of a sterol and/or a stanol ester in a milk product.

[0019] The invention also relates to a process for preparing a milk product containing at least one biologically active peptide and at least one sterol and/or stanol ester.

[0020] Accordingly, the invention provides a process for preparing dairy and/or milk products, the process avoiding the off-tastes caused by the oxidation of a sterol and stano! ester(s).

[0021] The inventive biologically active low-molecular peptides include, for instance, tripeptides, such as isoieucin-proline-proline (IPP) or valine-profine-proline (VPP) and mixtures produced thereof.

[0022] The mϋk product, preferably a fermented milk product, prepared by the process of the invention, is free from off-tastes caused by the oxidation of a sterol and stanol ester(s). In addition, the milk product prepared by the process of the invention possesses an effect that promotes heart and vascular health.

DETAILED DESCRIPTION OF THE INVENTION

[0023] The present invention provides a solution for avoiding problematic off-tastes caused by the oxidation of sterol and stanol esters and the generation of secondary oxidation products in dairy and/or milk products containing sterol and/or stanol esters.

[00241 The terms sterol ester and sterol fatty acid ester refer to a fatty acid ester of a plant sterol, i.e. a compound generated by the reaction of a sterol with a fatty acid. Correspondingly, the terms stanoi ester and stanol fatty acid ester refer to the fatty acid ester of a plant stanol, i.e. a compound generated by the reaction of a stanol with a fatty acid.

[0025] It has now been surprisingly found that biologically active low-molecular peptides prevent the oxidation of a sterol and/or stanol ester(s).

[0026] Biologically active low-molecular peptides include dipeptides and tripeptides, such as isoleucin-proline-proline (IPP) or valine-proline-proline (VPP) and mixtures produced thereof, in studies, tripeptides IPP and VPP have been found to possess anti-hypertensive properties. [0027] In organoleptic tests, an educated organoleptic/sensory panel detected a rancid off-taste in a sour milk product containing sterol and/or stano! ester. Surprisingly, in a sour milk product, whereto tripeptides had been added in addition to a sterol and/or stanol fatty acid ester, said off-taste was not detected at all; instead, the taste was experienced as pleasantly refreshing and sour.

[0028] The invention provides a process for preparing mifk products, particularly sour milk products, having a flawless taste without special additional costs, in addition, the process of the invention provides a product that promotes heart and vascular health. The cholesterol-lowering property of the sterol and/or stanol esters of the product and the anti-hypertensive property of biologically active peptides both serve as compounds promoting heart and vascular health. The process of the invention is simple and suitable for iarge- scale production.

[0029] The product to be prepared by the process of the invention is a dairy and/or a milk product, preferably a sour milk product. Typical sour milk products include yoghurt, fermented milk, 'villi' and sour cream, smetana, and quark, cottage cheese and feta (type) cheeses. Sour milk products also include milk-based, so-cailed dairy shot drinks.

[0030] The amounts of sterol and/or stanol ester to be added to foodstuffs vary. The milk products of the invention contain sterol and/or stanol about 0.1 to 12 g/100 g of the milk product, preferably about 0.3 to 5 g/100 g of the milk product. Based on current studies, the European Food Safety Authority recommends a maximum dose of 3 g plant sterol/day for humans.

[0031] The amount of biologically active peptides, such as IPP and VPP, also varies in food products. The milk products of the invention contain biologically active peptides about 0.5 to 25 mg/100 g of the product, preferably about 0.8 to 7 mg/100 g of the product and most preferably about 2 to 6 mg/100 g of the product.

[0032] According to an embodiment of the invention, at least some low-molecular peptides are produced into the product by fermentation. According to another embodiment, the low-molecular peptides are produced into the product by fermentation. According to still another embodiment at least some low-molecular peptides are produced into the product by addition of said peptides thereto during manufacture for instance as a concentrate and/or powder form concentrate containing them. [0033] in the preparation of a sour mϋk product according to an embodiment of the invention, such as yoghurt, fermented mϋk, 'viili' or dairy shot drink, part of the peptides is added as a concentrate or powder containing biologically active tripeptides and the milk in the end product is fermented with the Lactobacillus helveticus strain only to such a degree that the taste of the product is pleasantly sour and refreshing. In the fermentation of the product, other starters having a mild acidity and aroma may afso be used, either as such or together as a mixed starter together with the L, helveticus strain. In the tests of an expert organoleptic panel, the taste of the product prepared by the process of the invention was experienced as pleasantly sour and refreshing. In a concentrate or powder form, the addition of tripeptide did not cause unpleasant off- tastes in the consumer product when evaluated organoleptically.

[0034] In the preparation process of a concentrate or powder containing tripeptides, it is preferable to heat-treat the product in such a manner that under the action of the heat-denaturation reaction, free -SH radicals are generated in the end product, which, when concentrate or powder is subsequently added to the milk product base containing sterol and/or stanol ester, serve to efficiently prevent the oxidation of the sterol and/or stanol esters.

[0035] In addition, it was surprisingly found that the prevention of oxidation was still enhanced by the use of a low-lactose or totally lactose-free milk base.

[0036] The preparation of low-lactose milk products is known. A plurality of processes has been presented for the removal of lactose from milk. There is a generally known conventional enzymatic process for the removal of lactose in the field, the process comprising a step of adding mould- or yeast- based lactase to milk, whereby more than 80% of the lactose is converted into monosaccharides, i.e. glucose and galactose. Herein the problem is the excessively sweet taste of the milk, which is caused by the monosaccharides. Especially a milk product wherein the lactose is hydrolyzed into glucose and galactose contains twice as much reducing sugars as a milk product containing non-hydrolyzed lactose (Evangelisti F. et al., J. Dairy Res. 66 (2) (1999) 237- 243). Consequently, a low-lactose or lactose-free milk base assists in protecting the sterol/stanol ester against oxidation. [0037] in the preparation of sour milk products prepared by the process of the invention, a technique, known per se for these sour milk products, is used as regards starters, rennet, fermentation conditions (temperature, time, mixing) and heat-treatments.

[0038] Fermentation/acidification is performed by means of addition of a biological starter, chemica! starter/acid ifier or organic acids or inorganic acids combined with the addition of rennet or without addition of rennet.

[0039] Heat-treatments are performed as is known in the art: UHT treatment (e.g. milk 138 ⁰ C, 2 to 4 s), ESL treatment (e.g. milk 13O ⁰ C, 1 to 2 s) or pasteurization (e.g. milk 72 ⁰ C, 15 s).

[0040] According to an embodiment of the invention, the milk product is prepared as foliows: a) mixing of mϋk raw material, steroi/stanol ester and tripeptide concentrate, b) homogenizing the mixture obtained, c) heat-treatment, and d) cooling into fermentation temperature, e) addition of starter culture to the cooled mixture, f) fermentation (souring), g) optionally adding fruit preparation, h) cooling, i) packaging and storage.

[0041] The preparation process is economical and does not include unnecessary process steps.

[0042] According to another embodiment of the invention, the milk product containing at least one sterol and/or stanol ester and at least one biologically active peptide, is prepared by the process wherein the above steps a) to i) are conducted in the presented order and/or consecutively. According to a further embodiment of the invention, the milk product containing at feast one sterol and/or stanoi ester and at least one biologically active peptide, is prepared by the process consisting essentially of the steps a) to i) as described above.

[0043] In the context of the present invention, milk raw material refers to milk, whey and combinations of milk and whey as such or as a concentrate. The milk raw material may be supplemented with ingredients generally used in the preparation of milk products, such as fat, protein or sugar fractions and the like. Accordingly, the milk raw material may be e.g. full milk, cream, low-fat milk or fat-free milk, ultrafiltered milk, diafiltered milk, microfiltered milk or mifk prepared from milk powder by reconstitution, organic milk, or a combination thereof. Preferably, the milk raw material is fat-free milk.

[0044] in step (a), other milk-based raw materials may be added to the mixture for instance for increasing dry matter and/or for standardizing the fat, if desired. The tripeptide concentrate may be added as a concentrate or a powder.

[0045] The pasteurized fat-free milk used in the preparation process, as well as the other milk-based raw materials, may be low-lactose or lactose-free depending on whether the end product is to be low-lactose (HYLA) or lactose-free.

[0046] The process of the invention is also suitable for modern component production, wherein milk components having different fat and protein concentrations are known to be combined only before packaging.

[0047] The process of the present invention may be applied to both batch and continuous production. The process of the invention is preferably implemented as a batch process.

[0048] The following examples describe the implementation of the invention but do not restrict the invention only to said product applications.

Example 1 - Stirred yoghurt

[0049] Fat-free low-lactose milk powder, sterol ester in sunflower oil, peptide concentrate (prepared by the process of European Patent 1 226 267) and, when required, low-iactose cream (38% fat) were mixed rigorously into warm (more than 6O ⁰ C) fat-free, hydrolyzed milk. The mixture was homogenized (pressure 200 bars), pasteurized and cooied to fermentation temperature 42 ⁰ C. Yoghurt starters were added and allowed to sour until pH reached a value of about pH 4.5. The composition is shown in Table 1.

[0050] The precipitate was mixed well and cooled to about +20 ⁰ C. The product was packed in beakers, closed with lids and transferred into a 5 ⁰ C storeroom for storage. The product obtained contained tripeptides (IPP and VPP) a total of 5 mg/100 g of the product.

[0051] An educated expert panel evaluated the yoghurts organolep- tically as fresh (at the age of 2 to 3 days) and at 4 weeks. [0052] The product according to the invention was refreshing and pleasantly sour (average 4.5) and had a slight corn-like flavor. The organoleptic properties of the product were good also after a storage of 4 weeks (Table 2).

[0053] The results show that yoghurt containing sterol ester (yoghurt A) was sensed as worst, particularly after a longer storage. The yoghurt containing tripeptides (yoghurt B) was considered best. The organoleptic quality of the yoghurt containing sterol ester improves when peptides are added thereto (yoghurt D).

Table 1. Composition of stirred yoghurt

Table 2. Organoleptic evaluation

Example 2 - Fermented milk

[0054] Fat-free low-lactose milk powder, sterol ester in sunflower oil and peptide concentrate were mixed vigorously into warm fat-free hydrolyzed milk, as is described in Example 1 excluding the addition of milk powder (increasing dry matter) and cream (standardizing fat). The composition is shown in Table 3. Instead of a yoghurt starter, the starter used was a conventional mesophil starter (composed of i.a. Lactococus lactis ssp. cremoris, L. lactis ssp. lactis and L. diacetilactis). The fermentation temperature was 25 ⁰ C. The fermented milk was packaged in a 1 -liter cardboard package and transferred to a 5 ⁰ C storeroom for storage. The obtained fermented milk, prepared by the process of the invention, contained thpeptides (IPP and VPP) a total of 5 mg/100 g of the product.

[0055J An educated expert panel evaluated the fermented milk or- ganoleptically as fresh (at the age of 2 to 3 days) and at 3 weeks (Table 4). Table 3. Composition of fermented milk

Example 3

Anti-hypertensive effect of the inventive product

[0056] The anti-hypertensive effect of the product obtained in accordance with the invention was studied by giving the inventive sour milk product, which contained sterol in an amount corresponding to a daily dose of 2 g/day and tripeptides IPP and VPP in total an amount corresponding to a daily dose of 5 mg/day to 6-week old SHR rats spontaneously developing a high blood pressure. The reference product contained tripeptides in an amount corresponding to a daϋy dose of 5 mg/day in total.

[0057] Two corresponding groups of rats served as control, to which water or a sour milk drink (milk base), respectively, was given.

[0058] The blood pressure of all rats was measured at one-week intervals during 8 weeks. The mean systolic pressure of both rat groups at 0, 2, 4, 6 and 8 weeks is shown in Table 5.

[0059] The results clearly show that the inventive product is capable of significant prevention of hypertension compared with the control product. The sterol ester did not affect the anti-hypertensive effect of the tripeptides.

Table 5. Btood pressure (mmHg)