WO2003070267A1

WO2003070267A1 - Enhancing the availability of minerals by using biologically active peptides

Info

Publication number: WO2003070267A1
Application number: PCT/FI2002/001051
Authority: WO
Inventors: Mirkka Narva; Riitta Korpela; Olli Tossavainen; Annika Mäyrä-Mäkinen
Original assignee: Valio Ltd
Priority date: 2002-02-25
Filing date: 2002-12-20
Publication date: 2003-08-28
Also published as: FI20020360A0; JP2005517424A; JP4242293B2; FI112031B; EP1485119A1; AU2002352303A1; RU2004128448A

Abstract

The invention relates to enhancing the availability of minerals and strengthening the skeletal system by using biologically active peptides and products containing them. The peptides and the products containing them are useful for instance in preventing and treating various disorders and illnesses related to bone mineral absorption. A product having a high content of short-chain peptides and a high calcium content has been found especially effective in the use according to the invention.

Description

ENHANCING THE AVAILABILITY OF MINERALS BY USING BIOLOGICALLY ACTIVE PEPTIDES.

FIELD OF THE INVENTION

[0001] The invention relates to enhancing the availability of minerals and to strengthening the skeletal system by using biologically active peptides and products containing them. Peptides and products containing them are useful for instance in preventing and treating different disorders and illnesses related to bone mineral absorption. A product having a high short-chain peptide content and a high calcium content has been found especially effective for use in accordance with the invention.

BACKGROUND OF THE INVENTION

[0002] Proteins in food provide the source of essential amino acids and nitrogen for the organ system. In addition, the proteins in food contain peptides that may be significant regulators of the functions of the organ system. Biologically active peptides can for instance affect the absorption of nutri- ents (phosphopeptides and casomorphines), the secretion of hormones (casomorphines) and the immunity (immunopeptides, casokinins, casomorphines). Casein in milk is the most significant source of biologically active peptides in food (Schlimme E. & Meisel H. Biologically active peptides derived from milk proteins. Structural, physiological and analytical aspects. Die Nah- rung 1995; 39:1-20; Steijns J. Dietary proteins as the source of new health promoting biologically active peptides with special attention to glutamine peptide. Food Tech Europe 1996; March/April: 80-84).

[0003] Phosphopeptides have been found to affect the absorption of minerals in the body. Phosphopeptides are mainly formed from α and β ca- seins through trypsin-caused proteolysis. Lee with his research group has observed that a diet containing 200 g/kg of casein increased the formation of phosphopeptides (Lee Y et al. Phosphopeptides and soluble calcium in small intestine of rats given a casein diet. Br J Nutr 1980; 43:457-67). When casein was the only source of food, phosphopeptides were, however, not formed. The scientists concluded that the formation of phosphopeptides requires not only casein but also other sources of food, such as starch or fat. This could be due to the fact that other nutrients increase the activity of proteolytic enzymes in the small intestine.

[0004] Animal experiments show that phosphopeptides formed from milk casein increase the amount of soluble calcium in the intestine and may thus improve the absorption of calcium (see for instance Lee Y et al. Phosphopeptides and soluble calcium in small intestine of rats given a casein diet. Br J Nutr 1980; 43:457-67; Lee Y et al. Intestinal absorption of calcium in rats given diets containing casein or amino acid mixture: the role of casein phos- phopeptides. Br J Nutr 1983; 49:67-76; Li Y et al. Indirect effect of casein phosphopeptides on calcium absorption in rat ileum in vitro. Reprod Nutr Dev 1989; 29:227-233; Mykkanen HM, Wasserman RH. Enhanced absorption of calcium by casein phosphopeptides in rachitic and normal chicks. J Nutr 1980; 110:2142-2148; Tsuchita H at al. The effect of casein phosphopeptides on cal- cium utilization in young ovaryectomized rats. Z Emahrungswiss 1993; 32:121- 130; Yuan YV, Kitts DD. Calcium absorption and bone utilization in spontaneously hypertensive rats fed on native and heat-damaged casein and soya- protein. Br J Nutr 1994; 71 :583-603) and the calcium content of the organ system (Tsuchita H. et al. ibid.). All results do not, however, support this argument (see for instance Brommage R et al. Influence on casein phosphopeptides and lactulose on intestinal calcium absorption in adult female rats. Lait 1991 ; 71 :173-180).

[0005] The influence of phosphopeptides on calcium absorption in humans has been examined in a few studies. In Hansen et al. randomised cross-over study, 22 healthy adults were given rice- or whole grain-based infant food with 0, 1 or 2 grams of phosphopeptides and labelled calcium added. The absorption of calcium was verified through radioisotopic retention and was noted to increase due to the phosphopeptides by approximately 30% in the persons who received rice-based food. In the persons who received whole grain-based food, the phosphopeptides did, however, not increase calcium absorption, which according to the scientists may have been caused by the high fytate content of the food (Hansen M et al. Casein phosphopeptides improve zinc and calcium absorption from rice-based but not from whole-grain infant cereal. J Pediatr Gastrolog Nutr 1997; 24:56-62). Heaney et al. gave 35 healthy women 250 g of labelled calcium and 87.5 mg of phosphopeptides or placebo with their food. Calcium absorption was significantly improved in the women who received phosphopeptides and whose calcium absorption was otherwise weak. In the women whose calcium absorption was high and those receiving placebo, phosphopeptides did not markedly increase calcium absorp- tion (Heaney RP et al. Effect of caseinophosphopeptide on absorbability of coingested calcium in normal postmenopausal women. J Bone Miner Metab 1994; 12:77-81 ).

[0006] The use of phosphopeptides could be beneficial for instance to babies, the elderly, and in illnesses, in which the absorption of minerals, es- pecially calcium, is impaired or the need for them is increased. Adding phosphopeptides to foodstuffs could be a safe way to increase calcium absorption (Hansen M. et al. 1997, ibid.).

[0007] Biologically active peptides also include peptides affecting blood pressure. ACE, i.e. the enzyme converting angiotensin I, is a key en- zyme in controlling blood pressure. It converts angiotensin I to angiotensin II, which strongly contracts blood vessels. ACE inhibitors can prevent this action and thus act as agents lowering blood pressure. Peptides serving as ACE inhibitors are present in milk casein and fish and corn proteins (Schlimme et al. 1995, ibid.). Blood-pressure affecting peptides disclosed in the literature of the art include, for instance: Asp-Glu-Leu-Gln-Asp-Lys-lle-His-Pro-Phe-Ala-Gln- Thr-Gln-Ser-Leu-Val-Tyr-Pro-Phe-Gly-Pro-lle-Pro-Asn-Ser and Leu-Leu-Tyr- Gln-Gln-Pro-Val-Leu-Gly-Val-Arg-Gly-Pro-Phe-Pro-lle-lle-Val (Yamamoto N et al. Antihypertensive effect of the peptides derived from casein by an extracellular proteinase from Lactobacillus helveticus CP790. J Dairy Sci 1994; 77:971- 922), Thr-Thr-Met-Pro-Leu-Trp (Maryama S et al. Angiotensin l-converting enzyme inhibitory activity of the C-terminal hexapeptide of α_sι-casein. Agric Biol Chem 1987; 51 :2557-61 ), and Phe-Phe-Val-Ala-Pro (Maryama S et al. Agric Biol Chem 1985; 49:1405).

[0008] Sour milk products are also reported to contain peptides with a blood-pressure lowering effect. It is assumed that the peptides are formed in the milk products as a result of hydrolysis by lactic acid bacteria and especially hydrolysis of milk proteins caused by their extracellular proteinases.

[0009] Yamamoto et al. describe the purification and characterization of the proteinase originating from the micro-organism Lactobacillus helve- ticus CP790 in publication J. Biochem. 114 (1993) 740. Yamamoto et al. have also reported a study in which _sι and β caseins were hydrolysed with said proteinase and the obtained peptides were examined with respect to their ACE-inhibiting effect (J Dairy Sci 1994; ibid.). A total of 25 peptides were examined and they were very different in molecular size and effect. The most effective peptides were three peptides obtained from β casein and containing 8, 18 and 27 amino acids, respectively. The ACE activity of milk fermented with Lactobacillus helveticus strain CP790 and its variant CP791 that is deficient with respect to its proteinase activity were also compared, and the former was found effective in SHR rats developing spontaneous high blood pressure, but not in ordinary rats, whereas the latter lacked any activity. [0010] Nakamura et al. describe the use of a souring agent containing Lactobacillus helveticus and Saccharomyces cerevisiae in the preparation of two ACE inhibitors in publication J Dairy Sci 1995; 78:777 - 783. Skimmed milk was fermented with said souring agent, after which the ACE inhibitors were purified chromatographically and analysed. Both active compounds were tripeptides, Val-Pro-Pro and lle-Pro-Pro.

[0011] US patent 5,449,661 , Nakamura et al., describes the preparation of a peptide containing the tripeptide sequence Val-Pro-Pro and its use in lowering high blood pressure. The peptide is prepared by fermenting skimmed milk powder with Lactobacillus helveticus strain JCM-1004, after which the peptide is purified chromatographically and freeze-dried.

[0012] International patent application WO99/16862, Yamamoto et al., describes Lactobacillus helveticus strain CM4, FERM BP-6060 that is capable of producing a large amount of tripeptide Val-Pro-Pro and/or lle-Pro-Pro and that has a high extracellular protease activity. The publication also de- scribes fermented milk products that contain the above-mentioned tripeptides and bacterium, and a process for their preparation by fermenting products containing the tripeptide sequences with said bacterium.

[0013] Peptides acting as ACE inhibitors have been estimated to possibly have an effect on immunity based on the fact that bradykinins, which are stimulated by ACE inhibitors, are capable of increasing the activity of macrophages and lymphocytes (Migliore-Samour D et al. Biologically active milk peptides implicated in immunomodulation. J Dairy Res 1989; 56:357-362). The effect of these peptides on mineral absorption has not been studied or described in the literature of the art. [0014] Injuries and illnesses related to the weak mineral level of bone are becoming commoner all the time. They are present in all age groups. The problem is the worst in the older age groups, in which a generally weaker metabolism, less physical exercise and scant intake of important foodstuffs, vitamins and minerals and especially weaker calcium absorption may cause osteoporosis. As the population ages, it is feared that the occurrence of illnesses and injuries related to the weakening mineral level of bone will further increase. Calcium absorption is also weakened in postmenopausal women. It is also feared that with the changing nutritional, exercise and other cultural habits, the problem will worsen with time and move to younger population groups. Calcium intake remains below recommendations with lactose- intolerants, vegans and weight-watchers, for instance. A second risk group comprises persons with an increased need of calcium, such as young girls and breast-feeding and pregnant women.

[0015] Other minerals, too, affect in versatile ways the well being of a person. For instance, magnesium transmits muscle and nerve impulses and serves as building material for bones and teeth. Magnesium has also been found to improve immune response. Magnesium deficiency is rare, but dehydration may lead to magnesium depletion. One of the tasks of potassium is to control fluid balance and contract muscles. Potassium deficiency may cause muscle weakness. The intake of magnesium and potassium is important in illnesses that involve disturbances in fluid balance or dehydration, and for athletes to recover after performance.

[0016] Mineral intake in the form of preparations containing inorganic salts is not very efficient; only about 40% of inorganic calcium, for instance, is absorbed in the intestines. In addition, the mineral balance of the body is sensitive and may change abruptly, which is why dosing a specific mineral as such is not always a good alternative. An excessive intake of calcium, for instance, may impair the absorption of other minerals, such as iron. Thus, it is important to maintain or improve the good balance between other minerals while increasing the content of the mineral, the presence or absorp- tion of which is found to be too weak or the need for which is increased. Administering peptides affecting the absorption of minerals as such, as drugs or functional agents is then worth considering.

[0017] As an alternative to medical treatment or in addition to it, it is also possible to utilize other ways of strengthening the skeletal system both as a preventive measure and to provide (re)mineralization. One good way is to increase physical exercise. One of the newer methods is to use functional nutrients as part of a normal diet, which is a welcome alternative to consumers. Adding biologically active agents to foodstuffs has been found a safe method and thus, there are products lowering cholesterol and blood pressure, for in- stance, already on the market. [0018] Novel methods are thus needed to enhance the availability of minerals, prevent, alleviate and cure osteoporosis, improve mineral absorption and support bone metabolism. Functional products that enhance mineral absorption and bone formation would provide a very welcome addition to the selection in stores. It is important that the products do not contain undesirable or even harmful substances, that they are easy to prepare on an industrial scale, and that they appeal to the consumers and are familiar to them and thus easy to use for instance as part of their regular diet.

BRIEF DESCRIPTION OF THE INVENTION [0019] It is thus an object of the present invention to make available a product that enhances the availability of minerals and is thus capable of preventing, alleviating or curing disorders, illnesses and injuries related to mineral availability. The effect of the product on calcium absorption, reduction in the formation of osteoclasts, the development of osteoblasts and thus a healthy skeletal system, and the prevention and alleviation of osteoporosis and its prestages is especially important.

[0020] The product to be used according to the invention has a high content of peptides enhancing the availability of minerals. In a preferred embodiment, the product also has an optimised salt content, which means that the number of monovalent cations is low and the number of beneficial bivalent cations is high in comparison with a known product of corresponding type.

[0021] A most preferred embodiment makes available a product that has a high content of peptides enhancing the availability of minerals and a high calcium content. [0022] It is a further object of the present invention to make the product available either as such, as an agent enhancing the availability of minerals, or for use in the preparation of functional foodstuffs or drugs intended for consumption.

[0023] A yet further object of the present invention is to provide a method for enhancing the availability of minerals and/or for strengthening bones by administering to an individual in need of such treatment peptides enhancing the availability of minerals or a product containing them in a sufficient amount to produce the desired effect.

[0024] A yet further object of the present invention is to provide a method for the prevention, alleviation or cure of osteoporosis by administering to an individual in need of such treatment peptides enhancing the availability of minerals or a product containing them in a sufficient amount to produce the desired effect.

[0025] A yet further object of the present invention is to provide a method for enhancing the availability of calcium by administering to an individual a product that has a high content of casein-derived, small-molecular peptides and a high calcium content and that has been prepared by fermenting a casein-containing starting material with Lactobacillus helveticus strain LBK- 16H, DSM 13137, optionally removing partly or entirely casein and/or other milk proteins and/or lactose, in a sufficient amount to produce the desired effect.

[0026] In a preferred embodiment, the fermented product is also subjected to nanofiltration.

BRIEF DESCRIPTION OF THE FIGURES [0027] Figure 1 shows the effect of a product to be used according to the invention and that of various reference samples on the bone density of a rat.

[0028] Figure 2 shows the effect of a product to be used according to the invention and that of various reference samples on the bone mineral content of a rat.

[0029] Figure 3 shows the effect of different concentrations of VPP (Val-Pro-Pro) used according to the invention on bone formation.

[0030] Figure 4 shows the effect of different concentrations of IPP

(lle-Pro-Pro) used according to the invention on bone formation. In Figures 3 and 4, BL=Baseline, C=Control, -10, -8, -6 and -4 = 10^"10, 10^"8, 10^"6 and 10^"4 M

VPP or IPP peptide, respectively. * (p < 0.05), ** (p < 0.01 ) and *** (p < 0.001 ) represent statistically significant differences from baseline.

[0031] Figure 5 shows the effect of a product to be used according to the invention and that of a reference sample on parathyroid hormone (PTH) concentration.

[0032] Figure 6 shows the effect of a product to be used according to the invention and that of a reference sample on serum calcium concentration. DETAILED DESCRIPTION OF THE INVENTION

[0033] It has been found according to the present invention that the above objects can be achieved by using peptides enhancing the availability of minerals or a product containing them. Di-, tri- and tetrapeptides and their mix- tures are above all considered peptides enhancing the availability of minerals. These peptides can advantageously be prepared from casein or casein- containing starting material by fermentation. Especially preferable is to use a method, in which a product containing biologically active peptides is formed by fermentation, after which it is concentrated and the composition is finished off by nanofiltration. The preferred embodiment provides excellent possibilities to use starting materials of different type and to modify the composition of the end product as desired, as described in detail in the following.

[0034] The invention thus relates to the use of casein-derived, small-molecular peptides in the preparation of a product enhancing the avail- ability of minerals.

[0035] The invention further relates to the use of a product having a high content of casein-derived, small-molecular peptides in the preparation of a product enhancing the availability of minerals.

[0036] Preferably, the casein-derived, small-molecular peptides comprise di-, tri-, and tetrapeptides and their mixtures. Most preferably, the IPP, VPP and calcium contents are high.

[0037] The invention further relates to the use of a product having a high content of casein-derived, small-molecular peptides and a high calcium content that has been prepared by fermenting casein-derived starting material with a lactic acid bacterium, and possibly by nanofiltration of the fermented peptide-containing product obtained, in the preparation of an end product enhancing the availability of minerals.

[0038] As a preferred embodiment, the invention relates to the use of a soured composition that contains casein-derived peptides, minerals and living lactic acid bacterium, in the preparation of a product enhancing the availability of minerals.

[0039] The invention also relates to a method for enhancing the availability of minerals and/or for strengthening bones by administering to an individual peptides enhancing the availability of minerals or a product contain- ing them in a sufficient amount to produce the desired effect. [0040] The individual is primarily human. The positive effects of the products used in accordance with the invention are naturally also beneficial to animals, especially pets and production animals. Examples of these include dogs, cats, rabbits, horses, cows, pigs, goats, sheep and poultry. [0041] Within the scope of this publication, enhancing the availability of minerals refers to a complex effect that comprises especially the absorption and effect of minerals on bone cells.

[0042] Biologically active peptides for use in enhancing the availability of minerals in accordance with the present invention can thus be formed by fermentation. The starting material in fermentation can be any product that contains the sequences of the desired biologically active peptides as part of its own peptide or protein sequence. Milk protein, especially casein, is preferably used as such or in the form of different preparations. Suitable starting materials include preferably also various casein-containing milk products, such as skimmed milk or milk with varying fat contents as such or in the form of a corresponding milk powder and sour milk products, such as sour milk, buttermilk, yoghurt, curdled milk, unripened cheese, etc.

[0043] Fermentation can be carried out with any lactic acid bacterium that is capable of producing small-molecular peptides enhancing the availability of minerals from the starting material. Suitable lactic acid bacteria can be found among species belonging to the Lactobacillus, Lactococcus, Leuconostoc, Streptococcus and Bifidobacterium genera, for instance. The most proteolytic of the lactic acid bacteria is Lactobacillus helveticus and it is thus considered especially suitable for this purpose. A preferable Lactobacillus helveticus strain is L. helveticus LBK-16H, DSM 13137, which is described in detail in patent publication WO 01/32836. The lactic acid bacteria can be used as pure cultures or mixed cultures, separately or together the conventionally used and commercially available souring agents. The lactic acid bacteria can also be used together with other micro-organisms. Microbe combinations are appropriately selected to achieve the best possible taste for the end product and to prevent the risk of contamination.

[0044] The fermentation conditions are selected to meet the requirements of the used strain so as to form a sufficient amount of biologically active peptides to produce the desired effect. The selection of suitable condi- tions, such as temperature, pH and aeration, is part of the know-how of a person skilled in the art. The temperature can be 30 to 45°C, for instance. [0045] The fermentation is allowed to continue until the desired amount of biologically active peptides has formed. Normally, this takes approximately 20 to 30 hours, preferably 22 to 24 hours.

[0046] A mixture of various peptides is formed during fermentation. When the fermentation continues long enough, mainly relatively small di- and tripeptides, such as Val-Pro-Pro (VPP) and lle-Pro-Pro (IPP), are obtained.

[0047] After fermentation, the cell suspension is recovered. It can be used as such or the peptides can be separated and purified using conventional methods. The cell suspension can also be concentrated, for instance by evaporating or drying it partly or completely, such as spreading it on a plate, drying and finally grinding it to produce a well-preserving dry powder.

[0048] Further treatment of the cell suspension by nanofiltration has proven to be a good method that makes it possible to produce the peptides enhancing the availability of minerals and the desired mineral composition in the same end product.

[0049] In some cases, it may be appropriate to subject the fermented product to pre-treatment, for instance to remove casein or all milk proteins from it, before nanofiltration. Methods suited for pre-treatment are known in the art and comprise various precipitation and filtering methods, for instance. One useful method comprises the adjustment of the pH of the fermented product to an area, in which casein precipitates, for instance to approximately 4.6 at 37°C, after which the precipitated casein is separated using a curd separator, casein separation sieve, decanter, by sedimentation or some other suitable method. A second method comprises ultrafiltration of the soured product at pH 3 to 3.5, whereby all proteins are retained on the membrane and nearly protein-free whey is obtained as the permeate. Coprecipitation producing nearly protein-free, peptide-containing whey can be achieved by CaCI₂ addition, thermal treatment or acid addition, for instance. If necessary, possible casein dust is removed by centrifugation. Other agents, such as lactose, can also be removed prior to nanofiltration for instance by enzyme hydrolysis or fermentation.

[0050] The fermentation product that is possibly pretreated in an appropriate manner, such as the whey obtained as described above, is then subjected to nanofiltration, if desired. The nanofiltration membrane can be a conventional NF membrane, such as Nanomax-50 (Millipore) or Desal 5 (Desal

Inc., USA), and the conditions are selected according to the requirements and instructions of the membrane manufacturer. It is possible to significantly influence the composition of the obtained product, especially the salt and sugar composition, by selection of the nanofiltration membrane type and process conditions. [0051] Nanofiltration is performed up to a desired dry content or volume concentration ratio, which is usually as high as possible. The dry content is in the range of 20 to 40% and the volume concentration ratio is approximately 5 to 20. The (whey) concentrate can be diluted with water, whereby a larger amount of salts and lactic acid can be removed from the con- centrate during nanofiltration. This is a simple and efficient way of adjusting the amount of salts in the concentrate to a desired level.

[0052] The small peptides enhancing the availability of minerals and formed during fermentation, such as tripeptides IPP and VPP of approximately 350 D, are completely retained by the nanofiltration membrane. The nanofiltra- tion method also provides the possibility of removing lactose entirely; lactose can be degraded enzymatically before the nanofiltration step, whereby mono- saccharides are removed to a large extent. In nanofiltration, lactic acid, small nitrogen compounds, such as urea, and monovalent salts permeate the membrane. Thus, by means of nanofiltration, the peptide content of the product in- creases. Through concentration, its content of bivalent ions, especially the desired calcium and magnesium ions, also increases, whereas the relative proportion of monovalent ions, such as sodium, potassium and chloride ions, decreases. Sodium ions, in particular, are known to have a significant effect on the fluid balance of the body, and a decrease in the number of these harmful ions can thus be taken as a significant advantage. Certain bivalent ions, especially calcium ions, are known to improve the mineralization of bone, for instance, and thus a high content of them is considered highly desirable.

[0053] The content of certain selected minerals in the product can also be increased by adding the desired mineral in the form of a suitable salt, for instance. Such minerals include calcium and magnesium in particular.

[0054] Table 1 shows the behaviour of the components contained in the prepreparation formed during the fermentation step of the above two-step method based on fermentation and nanofiltration in the nanofiltration step. The used exemplary composition is formed by fermenting skimmed milk with a Lac- tobacillus helveticus strain. As the above description clearly indicates, other products can also be used as starting materials. In addition, the fermentation conditions, the optional nanofiltration treatment and possible other pre- treatments and additional treatments affect the outcome, so the composition of the end products (and the nanofiltration results) naturally change and may differ from what is stated herein.

Table 1

[0055] The behaviour of components of whey containing biologically active peptides during nanofiltration

[0056] Membrane: Nanomax 50, conditions: 40°C, 30 bar, volume concentration ratio K=9, pH 4.6

[0057] The product prepared as described in example 2 of this pub- lication has been found to be extremely suitable for the purpose of the invention. The total composition of the product has been found optimal in the conducted comparative tests. Many factors influence the physiological activities of the product; for instance the extended proteolysis of the product that provides a selection of different active peptides, the excellent mineral content of the product, the living microbe cells in the product and the acidity of the product. All these properties can be influenced and they can be maximized by changing the different parameters of the preparation method in an appropriate manner. The proteolysis conditions, such as enzyme activity and reaction time, affect the quality and amount of obtained peptides. It is also possible to influence these by nanofiltration, and especially to raise the peptide content of the product significantly. In addition, the salt contents and their ratios can easily be adjusted to the desired level. The content of the desired bivalent ions, in particu- lar, can be increased and the content of monovalent ions decreased. The acidity and the number and type of microbe cells can also easily be maximized in manners know to a person skilled in the art.

[0058] The products described above can be used as such to achieve the desired effect. The products can also be dried and used in the form of a powder or lyophilized preparation. The products can also preferably be used in the preparation of functional foodstuffs or other products.

[0059] The concept 'foodstuff' has a wide meaning in this publication, covering all consumable products that can be in a solid, jellied or liquid form, and covering both ready-made products and products to which the bio- logically active peptides or a product containing them are added during consumption as an additive or part of the product. Foodstuffs can be products of dairy industry, meat processing industry, prepared food industry, beverage industry, bakery industry and confectionery industry, for instance. Typical products include milk products, such as yoghurt, curdled milk, curd, sour milk, but- termilk, other sour milk products, unripened cheeses and ripened cheeses, fillings of snack bars, etc. A second important group includes beverages, such as whey beverages, fruit beverages, and beers.

[0060] According to the invention, the biologically active peptides or a product containing them are used in a sufficient amount to achieve the de- sired effect enhancing the absorption of minerals. When using the product obtained by the two-step method described above, the amount to use depends mainly on the concentration degree of the whey and is for instance 0.1 to 30%, preferably approximately 5 to 15% as calculated from the weight of the end product. [0061] Biologically active peptides or a product containing them can be added to a food product during its preparation or to a finished food product. The food products in question thus contain peptides enhancing the availability of minerals, or a product containing them, in addition to other components contained in corresponding food products and fully correspond in taste and behav- iour with these conventional products. [0062] The invention will be described in greater detail by means of the following examples. These examples are provided only to illustrate the invention and should not be considered to restrict its scope of protection in any way. The product marked with X in the reference examples and figures is prepared according to examples 2 and 5.

Reference example 1

The proteolysis degree and mineral content of different products [0063] To determine the proteolysis degree of different sour milk products, their protein content (protein), total nitrogen content (Tot.N), non- protein nitrogen content (NPN; urea, amino acids, small peptides, etc.), amount of titrated free amino acids (Titr.f.aa) and pH were determined. The proteolysis degree can be roughly determined by calculating the ratio of non- protein nitrogen to total nitrogen (NPN / Tot.N); the amount of titrated free amino acids mmol/g protein is obtained by calculating Titr.aa/Prot.

[0064] The results are shown in Table 2. They show clearly the higher proteolysis degree of the Evolus beverage, and thus the higher number of short-chain peptides, in comparison with the Japanese commercial Calpis sour milk product of Calpis Food Industry Co Ltd.

Table 2 Proteolysis degree of different products

I-¹

The product of the invention, Evolus beverage (80% sour milk base) with added peptide concentrate, NPN / Tot.n then does not show the proteolysis degree

** Titrated free amino acids could not be determined

[0065] To determine the mineral content of different sour milk products, their sodium, calcium, magnesium and potassium contents were determined. High sodium content in serum has been found detrimental in many respects, for instance in terms of electrolyte balance, high blood pressure and cardiovascular diseases, so lowering the sodium content in food is advantageous. With the other minerals, the situation is the opposite, and increasing their content is advantageous. The measurements were made in the Environmental Laboratory (Environment Centre, Helsinki). The results are shown in Table 3. The mineral content of the Evolus product is optimal with respect to the object of the invention.

Table 3 Mineral content of different products

Reference example 2 The effect of different products on the skeletal system

[0066] The study determined the effect of water, milk, sour milk and two IPP and VPP peptide-containing beverages, the Japanese commercial Calpis sour milk product, Calpis Food Industry Company Ltd, and product X (Evolus, Valio Oy) of example 2 on the skeletal system. [0067] Six-week old SH rats were used in the study and were given one of the test beverages ad libitum for 14 weeks. After this, the bones of the rats were removed and the bone mineral densities (BMD) and bone mineral content (BMC) were determined with Dexa (dual energy X-ray absorptiometry). Dexa is a technique based on low-energy x-rays that measures bone density. The results were calculated with the Fisher LSD method (Sahai H. and Ageel M. 2000. The analysis of variance: Fixed, random and mixed models, Birk- hauser, Boston). The supply of calcium and energy differs in the groups during the test and therefore, the results are standardized in relation to these variables. The results are shown in Table 4 and in Figures 1 and 2. Figure 1 shows that the bone density of rats that received the Evolus beverage was higher than the bone density in the other groups. Figure 2 shows the distinct enhancing effect of the Evolus beverage on the bone mineral content. There is a statistically significant difference in the bone mineral content between the rats that received the Evolus beverage and water and also between the rats that received the Evolus beverage and the Calpis beverage. The results thus clearly show that the Evolus product has an optimal composition for the object of the invention.

[0068] Fl application 992360 describes the preparation of a corresponding Evolus product and its activity as a blood-pressure lowering agent. As described in the publication, the first studies were made on rats, after which they were repeated on humans. In terms of blood pressure, both studies showed corresponding, clear results. Therefore, there is no reason to believe that the currently on-going further studies concerning the absorption of minerals in humans would not produce similar results as in the animal tests described above. Table 4

The effect of different products on the skeletal system

Reference example 3

The effect of IPP and VPP and a peptide fraction formed with L. helveticus on the formation of bone cells

[0069] The object of the study was to determine the active factor responsible for the result obtained in reference example 2. In the study, the peptide fraction of a milk product soured with L. helveticus, the peptide fraction of Neo sour milk (Valio Oy, Helsinki, Finland), and pure peptides formed with L. helveticus, IPP (isoleucine-proline-proline) and VPP (valine-proline-proline), were added to bone forming cells. Bone formation was measured by the amount of calcium accumulated during the test. The mean amount of calcium released in the baseline group was given the value 100%. Baseline (BL, negative control) and BMP-4 (C, bone morphogenetic protein 4), which is an agent increasing bone formation, were used a controls in the test. The effects of VPP and IPP in bone formation are shown in Figures 3 and 4, showing the amount of formed calcium with different VPP and IPP contents (-10, -8, -6 and -4 = 10^" ¹⁰, 10^-8, 10^"6 and 10^"4 M VPP or IPP peptide, respectively. * (p < 0.05), ** (p < 0.01 ) and *** (p < 0.001 ) represent statistically significant differences from baseline. [0070] The results show clearly that pure peptides markedly increase bone formation. The peptide fraction of a milk product soured with L. helveticus had a small effect on bone formation, * (p < 0.05) with 10^"5 and 10^"3 diluted whey from sour milk acidified with Lactobacillus helveticus, ** (p < 0.01 ) with 10^"4 diluted whey. This effect can be explained by the IPP and VPP con- tent of the peptide fraction. The fraction of Neo sour milk had no effect on bone formation.

Reference example 4

The effect of a milk product soured with L helveticus on calcium absorption [0071] The study determined the effect of a milk product soured with

L. helveticus (Evolus) and Neo sour milk, Valio Oy, Helsinki, Finland, on indirect calcium absorption in postmenopausal women. Twenty persons participated in the study and were given both products on separate days. The products contained 500 mg of calcium. Blood and urine samples were taken during the test day. The absorption of calcium was measured indirectly by using as main variables the parathyroid hormone (PTH) in serum and calcium in urine, the amounts of which change during calcium absorption already within a few hours, and the total calcium (S-Ca) in serum and ionised calcium (iCa). The task of PTH is to ensure a sufficient calcium content in blood circulation. The more calcium is absorbed, the more PTH decreases. The absorption of calcium increases the calcium content in urine and serum.

[0072] In the study, administration of the Evolus product lowered the PTH value significantly more than the administration of Neo sour milk after one hour from the administration. Similarly, the calcium content of serum increased markedly after the administration of the Evolus product. Other markers showed no significant change between the products.

[0073] The results concerning the PTH value and the calcium content in serum are shown in Figures 5 and 6, respectively. [0074] The results thus show clearly that calcium absorption is better from a milk product soured with L. helveticus than from Neo sour milk that is prepared by conventional souring.

Example 1

The preparation of a product to be used according to the inven- tion

[0075] Lactobacillus helveticus strain LBK 16-H was grown in MRS broth at 37°C for 24 hours and inoculated into reconstituted milk (10%) to form an inoculum. After two growth cycles, the inoculum (15%) was inoculated into a fermentor medium made up of 9 to 10% skimmed milk powder milk and sterilized at 110° for 10 minutes. The fermentation was performed at 37°C for 22 to 24 hours under continuous strong agitation. The product can be used as such, in a dry and/or ground form, or the desired peptides can be separated from it using methods known per se.

Example 2 The preparation of a product to be used according to the invention

[0076] A product soured according to example 1 was nanofiltrated as follows.

[0077] The pH of the sour milk was raised to approximately 4.6 with KOH and casein was removed with a diamond mesh. The residual casein dust was removed by centrifugal clarifying. GLL Cone, lactase (Biocon Ltd, Japan) was added to the whey and allowed to hydrolyze at 5°C for 24 hours to degrade lactose into monosaccharides. The thus pretreated whey was nanofiltrated through a Nanomax-50 membrane (Millipore). [0078] The filtration took place at 40°C at a pressure of 30 bar. The whey was filtrated until the volume concentration ratio was 9. The compositions of the obtained concentrate and the dry powder formed from it are shown in Table 5. Table 5 The composition of the finished concentrate and dry powder

Composition Concentrate Dry powder

Dry matter (%) 30.0 94.5

Ash (%) 3.69 13.3

Na (mg/kg) 130 470

K (mg/kg) 1600 5600

Ca (mg/kg) 10000 36000

Mg (mg/kg) 1700 6300

CI (mg/kg) 550 2000

P (mg/kg) 6700 24500

IPP (mg/L) 510 2500

VPP (mg/L) 540 2700

[0079] The removed casein was neutralized to caseinate and dried.

Example 3

The preparation of a product used according to the invention [0080] The fermentation described in example 1 was repeated using (a) rich milk and (b) butter milk instead of skimmed milk. [0081] The pH of the product of example 3(b) was raised to approximately 4.5 and casein was removed with a curd separator. Lactase was added to the whey and the dust removed with a bactofuge. The whey was then nanofiltrated through an NF membrane (Desal-5, Desal Inc.), evaporated and dried. The obtained nanofiltrated concentrate was closely similar in composi- tion to the one described in Table 5.

[0082] Removed casein contains a great deal of whey that contains as much biologically active peptides as fermented sour milk. The casein was used as such in the curd, to which biologically active peptides were added in this manner. Example 4

The preparation of a product to be used according to the invention

[0083] Lactobacillus helveticus strain LBK 16-H was grown in MRS broth at 37°C for 24 hours to form an inoculum, as described in example 1. The inoculum was inoculated into a fermentor medium that was made up of an aqueous solution of acid precipitated casein (2.8%) and glucose (2.5%). Casein was dissolved by raising the pH to 6.7 with 10% KOH. Culturing was performed at 37°C for 24 hours. Corresponding amounts of biologically active peptides VPP and IPP as when fermenting milk were produced in the product, but separating the peptides from the casein solution was easier than from milk. [0084] The pH of the fermentation solution was raised to 4.6 with KOH at 37°C, whereby casein precipitated. Casein was separated in a curd separator and the resulting whey was recovered. Whey contains peptides, lac- tic acid and salts, but no lactose. By nanofiltration of whey, it is possible to remove a great deal of the lactic acid, monovalent salts and glucose residue from it. The nanofiltration retentate can be evaporated to a concentrate or dried to powder, whereby a well-concentrated and long-lasting preparation containing biologically active peptides is obtained.

Example 5

The preparation of a sour milk product

[0085] Sour milk containing peptides that enhance the absorption of minerals was prepared by adding 3.5% of a peptide concentrate obtained according to example 1 to a commercially available sour milk. The composition of the resulting product is shown in Table 6 that also shows for comparison the composition of a second commercially available sour milk product, AB sour milk, Valio Oy. Table 6 The composition of an Evolus product and a commercial sour milk product

Claims

1. Use of casein-derived, small-molecular peptides in the preparation of a product enhancing the availability of minerals.

2. The use according to claim 1, characterized by using a product having a high content of casein-derived, small-molecular peptides in the preparation of a product enhancing the availability of minerals.

3. The use according to claim 2 of a product that has a high content of casein-derived, small-molecular peptides and a high calcium content and is prepared by fermenting a casein-containing starting material with a lactic acid bacterium in the preparation of an end product enhancing the availability of minerals.

4. The use according to claim 3 of a product that has a high content of casein-derived, small-molecular peptides and a high calcium content and has been prepared by fermenting a casein-containing starting material with Lactobacillus helveticus strain LBK-16H, DSM 13137, optionally removing partly or entirely the casein and/or other milk proteins and/or lactose, and nan- ofiltering the resulting peptide-containing fermented product, in the preparation of a product enhancing the availability of minerals.

5. Use of a soured composition containing casein-derived, small- molecular peptides, minerals and a living lactic acid bacterium in the preparation of a product enhancing the availability of minerals.

6. The use of a product according to any one of claims 1 to 5, characterized in that the casein-derived, small-molecular peptides comprise a mixture of short-chain peptides.

7. The use of a product according to claim 6, characterized in that the peptides are di-, tri- and tetrapeptides.

8. The use according to claim 7, characterized by using a product that has a high lle-Pro-Pro and/or Val-Pro-Pro content and a high calcium content.

9. The use according to any one of claims 1 to 8, characterize d in that it enhances the availability of calcium.

10. A method for enhancing the availability of minerals by administering to an individual small-molecular peptides enhancing the availability of minerals or a product containing them in a sufficient amount to achieve the desired effect.

11. A method for preventing, alleviating or curing osteoporosis by administering to an individual in need of such treatment small-molecular peptides enhancing the availability of minerals or a product containing them in a sufficient amount to achieve the desired effect.

12. A method for enhancing the availability of calcium by administering to an individual a product that has a high content of casein-derived, small- molecular peptides and a high calcium content and that has been prepared by fermenting a casein-containing starting material with Lactobacillus helveticus strain LBK-16H, DSM 13137, optionally removing partly or entirely the casein and/or other milk proteins and/or lactose, in a sufficient amount to achieve the desired effect.

13. A method according to claim 12, c h a r a c t e r i z e d in that the fermented product is also subjected to nanofiltration.