CN1997750A - Blood pressure lowering oligopeptides - Google Patents

Abstract

The present invention describes a process to produce IPP from a protein source whereby the ratio of IPP and VPP produced from the protein is at least 5, preferably at least 10 and more preferably at least 20, which comprises the use of a proline specific endoprotease.

Description

oligopeptides that lower blood pressure

field of invention

The present invention relates to the production of IPP.

Background of the invention

Hypertension is a relatively common disease state in humans that represents a prevalent risk factor for cardiovascular disease, renal failure and stroke. The effectiveness of a large number of pharmaceutical products (e.g., calcium blockers (blockers), beta blockers, diuretics, alpha blockers, central alpha antagonists, angiotensin II antagonists, and ACE inhibitors), demonstrating high The underlying physiological mechanisms of blood pressure are multifaceted.

The physiological mechanisms of hypertension, especially the renin-angiotensin mechanism, have attracted much scientific attention. In this mechanism, angiotensin is secreted by the liver and cleaved by the peptidase renin to produce the decapeptide angiotensin I, which is biologically inactive. As angiotensin I passes through the pulmonary capillaries, another peptidase called angiotensin-converting enzyme (hereafter ACE) acts by removing the last two residues of angiotensin I (His-Leu) From angiotensin I, the octapeptide angiotensin II is formed. Angiotensin II octapeptide exhibits potent vasoconstrictor activity, thereby increasing blood pressure. ACE inhibition leads to a decrease in angiotensin II levels, which prevents vasoconstriction and thus hypertension.

In addition to cleaving angiotensin I, ACE also hydrolyzes bradykinin, a nonapeptide that is also involved in blood pressure regulation. In the latter mechanism, ACE inhibition leads to increased levels of bradykinin, which promotes vasodilation and also lowers blood pressure. Inhibition of ACE thus leads to a reduction in blood pressure through at least two separate pathways.

It is also known that the octapeptide angiotensin II stimulates aldosterone release through the adrenal cortex. The target organ of aldosterone is the kidney, where aldosterone promotes increased tubular sodium reabsorption. Through this third mechanism, inhibition of ACE lowers blood pressure, but in this case by eliminating sodium reabsorption.

Because of its multiple physiological roles, inhibition of ACE proteolytic activity is an effective approach to lowering blood pressure. This concept has led to a large number of effective drug-lowering products such as captopril and enalapril (Ondetti, M.A. et al., 1977, Science, Washington DC, 196, 441-444) . Because high blood pressure is a relatively common disease state, it would be advantageous to manage the unwanted consequences of this modern lifestyle with natural ingredients that are mildly active. Especially mild active natural ingredients that can be incorporated into food or drink products as such products are regularly consumed. Alternatively, such mildly active natural ingredients can be included in dietary supplements. In recent decades, it has been found that specific peptides present in fermented milk have ACE-inhibitory capacity and induce a reduction in blood pressure in hypertensive patients. Recently, numerous in vitro experiments and some animal experiments have demonstrated the ACE inhibitory effect of different peptides obtained from various protein sources. Although in vitro ACE inhibition assays have revealed many different peptide sequences, it must be emphasized that ACE inhibitory peptides need to circulate in the blood to exert an effect in vivo. This suggests that potent ACE-inhibiting peptides should be resistant to degradation by the gastrointestinal proteolytic digestive system and should remain intact during subsequent transit across the gut wall.

Structure-function studies of various ACE inhibitory peptides have suggested that they usually have Pro-Pro, Ala-Pro or Ala-Hyp at their C-terminal sequences (Maruyama, S. and Suzuki, H., 1982; Agric Biol Chem ., 46(5):1393-1394). This finding is partly explained by the fact that ACE is a peptidyl dipeptidase (EC 3.4.15.1) that cannot cleave peptide bonds involving proline. Therefore, the dipeptide Pro-Pro cannot be removed from the tripeptide with the Xaa-Pro-Pro structure because the Xaa-Pro bond cannot be cleaved. It is therefore conceivable that if a tripeptide with the Xaa-Pro-Pro structure is present at a relatively high concentration, it will inhibit ACE activity. Since not only ACE but almost all proteolytic enzymes have difficulty cleaving Xaa-Pro or Pro-Pro bonds, the presence of (multiple) proline residues at the carboxy terminus of the peptide would lead to relatively proteolytic molecules in view of It's almost self-evident. Similarly, peptides containing hydroxyproline (Hyp) instead of proline are relatively protein resistant. From this it can be deduced that peptides carrying one or more (hydroxy)proline residues at their carboxyl terminus may be protected from proteolysis in the gastrointestinal tract. These conclusions will help us understand the remarkable in vivo hypotensive effect of specific ACE-inhibiting peptides: they not only fulfill the structural requirements for ACE inhibition, but also resist degradation by the gastrointestinal proteolytic digestive system and remain intact during subsequent transport across the gut wall.

The potent ACE inhibitory activity of the tripeptides Leu-Pro-Pro (JP02036127), Val-Pro-Pro (EP0583074) and Ile-Pro-Pro (J. Dairy Sci., 78:777-7831995) has been reported. All ACE inhibitory peptides were initially analyzed based on their in vitro effects on ACE activity, the tripeptides Ile-Pro-Pro (hereinafter referred to as IPP), Val-Pro-Pro (hereinafter referred to as VPP) and Leu-Pro-Pro (hereinafter referred to as LPP) were selected because of their potent ACE inhibitory effects, resulting in relatively low IC50 values. Later, the putative antihypertensive effects of VPP and IPP tripeptides were verified in spontaneously hypertensive rats (Nakamura et al., J. Dairy Sci., 78:12531257 (1995)). In these experiments, inhibitory tripeptides were obtained from milk fermented by lactic acid bacteria. During milk fermentation, the desired peptides are produced by proteases produced by growing lactic acid bacteria. A disadvantage of this fermentation method is that lactic acid bacteria are living organisms for which it is difficult to control the type and amount of enzymes secreted. The production of ACE-inhibiting peptides is thus difficult to reproduce and it is not possible to generate an optimal set of enzymes to ensure maximum yield of the desired peptide. Furthermore, the relatively long fermentation times required, which together with the low yields imply an unfavorable cost structure for bioactive peptides. Furthermore, fermented products are less suitable for direct incorporation into a.o. solid foods and can create severe sensory limitations. The poor palatability of such fermented milk products, and the many processing difficulties encountered in recovering ACE-inhibiting peptides from such fermentation broths have been described in US 6,428,812. Despite these drawbacks, fermented milk products have been put into practical use as oral vasopressors. Concentration of ACE-inhibiting peptides from fermented milk products following electrodialysis, hollow fiber membrane dialysis or chromatographic methods has made it possible to sell them in the form of concentrated dietary supplements (eg, tablets or lozenges).

The aforementioned disadvantages of the fermentative production route were recognized in a.o. patent applications WO01/68115 and EP1231279. In the latter application, a purely enzymatic process is described to recover Val-Pro-Pro and Ile-Pro-Pro from milk casein. The application claims a process for the production of these tripeptides by digestion of a material containing casein of milk with proteases and peptidases via intermediate peptides. Each of these enzyme incubations may last as long as 12 hours and is performed under conditions that favor the growth of microbial contaminants. The intermediate peptide is preferably purified prior to incubation with the peptidase, and high final concentrations of ACE-inhibiting peptides are obtained only after an additional chromatographic purification step on the intermediate peptide. In view of the above-mentioned multiple disadvantages, there is a need for simpler and more microbiologically reliable enzymatic pathways to produce odorless products with high yields and reproducible antihypertensive peptides.

Contents of the invention

The present invention relates to a method for producing a composition comprising IPP from a protein source, wherein the ratio of IPP to VPP produced from the protein is at least 5:1, preferably at least 10:1, more preferably at least 20:1 ( wt/wt), the method preferably comprises the use of a proline-specific endoprotease or prolyl oligopeptidase. The invention also relates to the use of prolyl oligopeptidases or proline-specific endoproteases which do not have aminopeptidase activity. Proline-specific endoproteases are preferably capable of hydrolyzing large protein molecules, such as polypeptides or proteins themselves. The method according to the invention generally has an incubation time of less than 24 hours, preferably an incubation time of less than 10 hours, more preferably less than 4 hours. The incubation temperature is generally above 30°C, preferably above 40°C, more preferably above 50°C.

Preferably, a protease that cleaves at the end of a proline, such as a proline-specific endoprotease, does not contain any contaminating endoprotease activity. Preferably, a protease that cleaves at the end of a proline, such as a proline-specific endoprotease, is free of contaminating carboxypeptidase activity. Preferably, a protease that cleaves at the end of a proline, such as a proline-specific endoprotease, is free of contaminating aminopeptidase activity. A prolyl oligopeptidase or proline specific endoprotease free of contaminating endoprotease activity is an enzyme preparation preferably having an Endo/Prol specific activity ratio of less than 1, more preferably less than 0.01.

A prolyl oligopeptidase or proline specific endoprotease free of contaminating carboxypeptidase activity is an enzyme preparation preferably having a CPD/Pro specific activity ratio of less than 10, more preferably less than 1.

A prolyl oligopeptidase or proline specific endoprotease free of contaminating aminopeptidase activity is an enzyme preparation preferably having an AP/Pro specific activity ratio of less than 1, more preferably less than 0.1.

During the production of IPP, LPP is also advantageously formed. Another aspect of the invention is a method of purifying or isolating peptides from hydrolyzed proteins, preferably non-aspartic protease hydrolyzed, more preferably serine protease hydrolyzed. The hydrolyzed protein can be precipitated under selected pH conditions. Purification or isolation methods include changing the pH to a pH at which the hydrolyzed protein is precipitated, and separating the precipitated protein from the peptide in solution.

Accordingly, the present invention relates to a method for the preparation of a composition comprising a soluble peptide, preferably IPP, comprising altering the pH of the composition produced by hydrolysis of a suitable protein source such that the pH of the hydrolyzed protein A portion becomes pH insoluble, and the insoluble portion is separated from the soluble peptide to produce a composition comprising the soluble peptide.

The present invention also relates to a peptide composition produced by hydrolysis of a protein having an IPP to VPP ratio of at least 5:1, preferably at least 10:1, more preferably at least 20:1, said composition preferably comprising LPP , or, the invention relates to a composition comprising a soluble peptide according to the invention for use as a nutraceutical, preferably as a medicament. The present invention also relates to the use of these peptide compositions for the production of nutraceuticals (preferably, medicaments) for the promotion of health or for the prevention and/or treatment of diseases, or for the production of medicaments for the treatment or prevention of hypertension (high blood pressure, also referred to as Hypertension), heart failure, pre-diabetes or diabetes, obesity, impaired glucose tolerance or stress.

Preferably, the peptide composition of the present invention is in the form of a dietary supplement, in the form of a personal care application (including topical application in the form of an aqueous solution, gel or emulsion), or as a food, beverage, feed or pet food ingredient. use.

Detailed description of the invention

According to prior art, potent ACE inhibitory peptides may include one or two proline residues at the carboxyl terminus of the peptide. The same structural requirements also apply to peptides with increased resistance to proteolytic degradation, thereby increasing the likelihood that the intact peptide will eventually reach the bloodstream. To obtain peptides having at least one, but preferably multiple, proline residues at their carboxy-terminus, the use of proteases capable of cleaving at the carboxy-terminus of proline residues offers an interesting option. So-called prolyl oligopeptidases (EC 3.4.21.26) have the unique possibility of cleaving peptides with a preference for the carboxyl side of proline residues. In all sufficiently analyzed proline-specific proteases isolated from mammalian as well as microbial sources, a unique peptidase domain has been identified that excludes large peptides from the active site of the enzyme. The fact that these enzymes cannot degrade peptides containing more than about 30 amino acid residues has led to these enzymes being now termed "proline oligopeptidases" (Fulop et al.: Cell, vol. 94, 161-170, July 24, 1998). As a result, these prolyl oligopeptidases require prehydrolysis by other endoproteases before they can exert their hydrolytic action. However, as described in WO02/45523, even the combination of prolyl oligopeptidases with such other endoproteases leads to hydrolysates characterized by a significantly increased proportion of peptides with a carboxy-terminal proline residue . For this reason, such hydrolysates form excellent starting points for the isolation of peptides with ex ACE inhibition and increased resistance to gastrointestinal proteolytic degradation. Despite these potential benefits, we are not aware of applications specifically using proline-specific endoproteases for the recovery of ACE-inhibiting peptides, let alone the selective production of IPP.

The present invention relates to a method for producing a composition comprising IPP from a protein source, wherein the weight ratio of IPP to VPP produced from the protein is at least 5:1, preferably at least 10:1, more preferably at least 20:1 , said method comprising using an enzymatic activity having proline-specific endoprotease activity or prolyl oligopeptidase activity and an enzymatic activity capable of hydrolyzing the bond on the amino-terminal side of the -I-P-P-sequence. Advantageously, the enzymatic activity with proline-specific endoprotease activity or prolyl oligopeptidase activity and the activity capable of hydrolyzing the bond on the amino-terminal side of the -I-P-P-sequence is present in an enzyme, preferably the enzyme is a proline-specific endoprotease or a prolyl oligopeptidase, more preferably, the enzyme is a proline-specific endoprotease. Furthermore, the present invention relates to a method for the preparation of a composition comprising a soluble peptide, preferably IPP, said method comprising changing the pH of the hydrolysis conditions to a pH such that a portion of said hydrolyzed protein becomes insoluble , and separating the insoluble fraction from the soluble peptide, resulting in a composition comprising the soluble peptide. The temperature used for the separation step is preferably between 0 and 20°C, more preferably between 1 and 10°C.

The present invention also relates to a method for preparing a food product, a beverage product or a dietary supplement, the method comprising producing a composition comprising the aforementioned IPP, and adding the IPP-containing composition to the food product, beverage product or dietary health product.

Furthermore, the present invention relates to a method for producing a composition comprising a soluble peptide, said composition being produced by the following steps:

- First, proteolysis to 5-30% DH with a proline-specific endoprotease,

- optionally carrying out a second enzymatic treatment, preferably with a protease, and

- Afterwards, the insoluble fraction of the hydrolyzed protein is separated from the soluble fraction at selected pH conditions, preferably at acidic pH conditions, more preferably at a pH between 3.5 and 6, most preferably between 4 and 5 pH to produce compositions comprising soluble peptides.

The present invention therefore provides a composition obtainable by the latter method and comprising a soluble peptide wherein at least 70 wt%, preferably at least 80 wt%, most preferably at least 90 wt% of the peptide is at 3.5 pH between 4 and 6, preferably between 4 and 5, most preferably pH = 4 is soluble (measured at 4 ° C), and, wherein, none of the soluble peptides is larger than the soluble peptide The soluble peptide is present in an amount of 40 wt%, preferably, none of the soluble peptides is present in an amount greater than 30 wt% of the soluble peptide, most preferably none of the soluble peptides is present in an amount greater than 20 wt% of the soluble peptide Quantity exists.

The latter method and the latter composition are a consequence of the idea that a composition with a high content of soluble peptides can be obtained which, because of the use of a proline-specific endoprotease, also contains a high content of Peptides with a carboxy-terminal proline.

As described above, the protein is first hydrolyzed with a proline-specific endoprotease. In case the hydrolyzed protein is subsequently hydrolyzed by an additional (second) protease, the second enzyme will further hydrolyze the peptide with the carboxy-terminal proline. Preferably, the second enzymatic treatment is performed with pure and selective enzymes. The second enzyme is preferably an aminopeptidase (eg CorolaseLAP, see Examples 12 and 13) or an endoprotease, most preferably an aminopeptidase is used.

It is shown in Examples 12 and 13 that additional IPP and VPP are formed from casein based on the highest IPP selectivity.

The choice of this second enzyme will always be related to the protein used. In this way, it may be possible to manufacture tailor-made compositions comprising soluble peptides from the proteins used. This second enzymatic treatment may preferably be performed after proline specific endoprotease hydrolysis, but alternatively the second enzymatic treatment is performed simultaneously with proline specific endoprotease hydrolysis. According to another embodiment of the invention, this second enzymatic treatment may take place after the acid precipitation step. In this case, the soluble peptide composition is further hydrolyzed by the second enzyme to again obtain a soluble peptide-containing composition.

Preferably, at least 10 mole %, more preferably at least 20 mole %, even more preferably at least 30 mole % of the soluble peptide has a carboxy-terminal proline. In patent application WO 02/45523 it is described how this mole % can be determined.

The present invention relates to compositions containing the peptides of the present invention as nutraceuticals (preferably pharmaceuticals). The present invention also relates to the use of the composition containing the peptide of the present invention as a nutraceutical (preferably a medicament), the use of a composition containing the peptide of the present invention for the production of a nutraceutical (preferably a medicament), and the use of a composition containing the peptide of the present invention Use of a composition of peptides for promoting health and/or treatment of diseases, to the use of compositions containing the peptides of the present invention for the production of nutraceuticals (preferably medicaments), to compositions containing the peptides of the present invention for the treatment of The use of cardiovascular diseases such as hypertension and heart failure, the use of compositions containing the peptides of the invention for the treatment of pre-diabetes or diabetes, the use of compositions containing the peptides of the invention for the treatment or prevention of obesity, the use of The composition containing the peptide of the present invention is used to increase plasma insulin or is used to improve the application of the sensitivity to plasma insulin, relates to the use of the composition containing the peptide of the present invention for increasing plasma insulin in patients with type 2 diabetes or pre-diabetes or for improving Their use against plasma insulin sensitivity relates to the use of compositions containing the peptides of the invention for reducing postprandial glucose concentrations in the blood of patients with type 2 diabetes or pre-diabetes, to the use of compositions containing the peptides of the invention for increasing 2 The use of postprandial insulin secretion in the blood of patients with type 2 diabetes or pre-diabetes, relates to the use of the composition containing the peptide of the present invention, wherein the composition containing the peptide of the present invention is in the form of a dietary supplement, relates to the use of the composition containing the peptide of the present invention Use of a composition of the peptide of the invention for the production of a functional food product for medical treatment of the effects of stress relates to the use of a composition containing the peptide of the invention in topical application (preferably in personal care applications ), and to the use of compositions containing the peptides of the present invention in feed and pet food.

Furthermore, the present invention relates to a method for the treatment of type 1 and type 2 diabetes, and for the prevention of type 2 diabetes in individuals with prediabetes or impaired glucose tolerance (IGT), which method comprises providing Administration of a composition comprising a peptide of the invention to a treated individual, and also a method for treating or preventing hypertension and heart failure in a population suffering from hypertension or heart failure, said method comprising administering to a subject in need thereof Such treated individuals are administered compositions containing the peptides of the invention, thus exhibiting a blood pressure lowering effect. Inhibition of ACE results in reduced vasoconstriction, increased vasodilation, improved salt and water excretion, which in turn leads to reduced peripheral vascular resistance and blood pressure, and improved regional blood flow. Accordingly, the peptide-containing hydrolysates of the present invention are particularly effective in the prevention and treatment of diseases that may be affected by ACE inhibition, including but not limited to: hypertension, heart failure, angina, myocardial infarction, stroke, peripheral arterial occlusive disease, arteriosclerosis , renal disease, renal insufficiency, erectile dysfunction, endothelial insufficiency, left ventricular hypertrophy, diabetic vasculopathy, edema, and hyperaldosteronism. The compositions are also useful for the prevention and treatment of gastrointestinal disorders (diarrhea, irritable bowel syndrome), inflammation, diabetes, obesity, dementia, epilepsy, Alzheimer's and Meriere's disease. In addition, the compositions can improve cognitive function and memory (including Alzheimer's disease), satiety, limit ischemic damage, and prevent arterial reocclusion after bypass surgery or angioplasty.

Diabetes is a widespread chronic disease for which there is as yet no cure. The incidence and prevalence of diabetes has grown exponentially, making it one of the most common diseases in both developed and developing countries. Diabetes mellitus is a complex multifactorial disease characterized by impaired carbohydrate, protein and fat metabolism associated with defects in insulin secretion and/or insulin resistance. This results in elevated fasting and postprandial blood glucose concentrations which, if left untreated, can lead to complications. There are two main classes of the disease, insulin-dependent diabetes mellitus (IDDM, T1DM) and non-insulin-dependent diabetes mellitus (NIDDM, T2DM). T1DM = "Type 1 Diabetes Mellitus" (type 1 diabetes). T2DM = "Type 2 Diabetes Mellitus" (type 2 diabetes).

T1DM and T2DM diabetes are associated with high blood sugar, high blood cholesterol and high blood lipids. In T1DM and T2DM, absolute insulin deficiency and insulin insensitivity lead to decreased glucose utilization by liver, muscle, and adipose tissue, and increased blood glucose levels, respectively. Uncontrolled hyperglycemia is associated with increased and premature mortality due to an increased risk of microvascular and macrovascular disease, including renal disease, neurological disease, retinopathy, hypertension, stroke and heart disease. Emerging evidence shows that tight glycemic control is a major factor in preventing these complications in both T1DM and T2DM. Therefore, optimizing blood sugar control through drugs or treatments is an important means of treating diabetes.

Treatment of T2DM initially involves dietary and lifestyle changes, and when these do not maintain adequate glycemic control, patients are treated with oral hypoglycemic agents and/or exogenous insulin. Oral pharmaceutical agents currently used to treat T2DM include those that promote insulin secretion (sulfonylureas), those that promote the action of insulin in the liver (biguanides), insulin sensitizers (thiazolidinones) and those that act on Agents that inhibit glucose uptake (alpha-glucosidase inhibitors). However, currently available agents are often unable to maintain adequate glycemic control over the long-term due to progressive exacerbations of hyperglycemia. The proportion of patients maintaining target blood glucose levels decreased significantly over time necessitating the administration of additional/alternative pharmaceutical agents.

In addition, drugs can have unwanted side effects and are associated with high rates of primary or secondary failure. Finally, the use of hypoglycemic drugs may be useful in controlling blood sugar levels, but may not prevent all complications of diabetes. Therefore, current treatments for all types of diabetes do not achieve the desired effect of normalizing blood sugar and preventing diabetic complications.

Therefore, although the choice of therapy in the treatment of T1DM and T2DM is basically based on the administration of insulin and oral hypoglycemic drugs, there is a need for safe and effective nutritional supplements with minimized side effects to treat and prevent diabetes. Many patients are interested in alternative therapies that minimize the side effects associated with high-dose medications while yielding additional clinical benefits. Patients with diabetes are of particular interest in treatments that are considered "natural", have mild antidiabetic effects and do not have major side effects, which can be used as adjunct therapy. T2DM is a progressive, chronic disease that is often not recognized until significant damage has occurred to the pancreatic cells responsible for insulin production (β-cells of the islets of Langerhans). Therefore, there is increasing interest in developing dietary supplements that can be used to prevent β-cell damage in at-risk populations, especially elderly populations at high risk of developing T2DM, and thus prevent significant T2DM process. Protection of pancreatic β-cells can be achieved by lowering blood glucose and/or lipid levels, since glucose and lipids can cause damage to β-cells. The reduction in blood glucose levels can be obtained by different mechanisms, for example by increasing insulin sensitivity and/or by reducing hepatic glucose production. Lowering blood lipid levels can also be achieved by different mechanisms, for example, by increasing lipid oxidation and/or lipid storage. Another possible strategy for protecting pancreatic β-cells would be to reduce oxidative stress. Oxidative stress also leads to β-cell damage with subsequent decreased insulin secretion and progression of overt T2DM.

T2DM is thus a complex disease resulting from coexisting defects at multiple organ sites: resistance to insulin action in muscle and adipose tissue, defective pancreatic insulin secretion, unrestricted hepatic glucose production. These defects are often associated with lipid abnormalities and endothelial dysfunction. Combination therapy is an attractive means of managing T2DM in the presence of multiple pathogenic lesions.

The present invention relates to novel nutraceutical compositions comprising the peptide-containing compositions of the present invention. Nutraceutical compositions comprising peptide-containing compositions of the invention may also comprise unhydrolyzed proteins and carbohydrates as active ingredients for the treatment or prevention of diabetes or other conditions associated with glucose tolerance training, such as Syndrome X. In another aspect, the invention relates to the use of such compositions as nutritional supplements for said treatment or prophylaxis, for example, as a supplement to a multivitamin preparation comprising Vitamins and minerals that cannot be synthesized in the body. In yet another aspect, the present invention relates to a method for treating type 1 and type 2 diabetes, and for preventing type 2 diabetes in individuals with prediabetes or impaired glucose tolerance (IGT) or obesity, said method Administration of peptide-containing compositions and protein hydrolysates or unhydrolyzed proteins and/or carbohydrates of the invention to an individual in need of such treatment is included.

The compositions of the invention are particularly useful for the treatment of T1DM and T2DM, and for the prevention of T2DM in individuals with prediabetes or impaired glucose tolerance (IGT).

We have found that compositions containing the peptides of the invention can be used in type 2 diabetes or pre-diabetes, preferably for lowering postprandial glucose concentration or for increasing postprandial insulin secretion in the blood.

Compositions comprising peptides and optionally carbohydrates stimulate insulin secretion, increase glucose dispatch to insulin-sensitive target tissues such as adipose tissue, skeletal muscle and liver, thus providing a synergistic effect in the treatment of diabetes.

It is generally recognized that stress-related diseases, as well as the negative effects of stress on the body, play a significant role in many populations. In recent years, the role of stress and its role in the development of a variety of diseases and conditions has become more widely recognized in the medical and scientific community. Consumers are now increasingly aware of these potential problems and are increasingly interested in reducing or preventing the possible negative effects of stress on their health.

Another object of the present invention is to provide a food product, or an ingredient that can be included in a food product, that is suitable for helping the body deal with the effects of stress.

Yet another object is to provide a food product comprising a composition comprising a peptide of the invention which provides health benefits such as helping the body deal with the negative effects of stress.

The term "nutraceutical" is used herein to refer to both nutritional applications and the field of pharmacy. Therefore, the new nutraceutical composition can be used as food and beverage supplement, and also as pharmaceutical preparation and medicament for enteral or parenteral application, which can be a solid preparation, such as capsule or tablet, or a liquid preparation, For example a solution or a suspension. As can be seen from the foregoing, the term "nutraceutical composition" also encompasses food and beverages comprising compositions comprising the peptides of the invention and optionally carbohydrates, as well as supplement components, e.g. dietary supplements comprising the aforementioned active ingredients.

The term "dietary supplement" refers herein to a product for ingestion by mouth which contains "dietary ingredients" to supplement the diet. "Dietary ingredients" in these products can include: vitamins, minerals, herbal or other botanical substances, amino acids and substances such as enzymes, organ tissues, glands and metabolites. Dietary supplements can also be extracts or concentrates, which can be found in many forms such as tablets, capsules, soft gels, gel capsules, liquids or powders. They can also be in other forms, such as bars, but if they are, the information on the dietary supplement label will generally not indicate that the product is used as a traditional food or as the only item of a meal or meal.

A multivitamin and mineral supplement may be added to the nutraceutical composition of the present invention to obtain sufficient amounts of some essential nutrients that are missing from the diet. Multivitamin and mineral supplements can also be used for disease prevention and protection against deficiencies and nutritional losses due to lifestyle and inadequate dietary patterns sometimes observed in diabetes. Furthermore, oxidative stress has also been implicated in the development of insulin resistance. Reactive oxygen species can impair insulin-stimulated glucose uptake by disrupting the insulin receptor signaling cascade. Control of oxidative stress with antioxidants such as alpha-tocopherol (vitamin E), ascorbic acid (vitamin C) may be of value in the treatment of diabetes. Therefore, the intake of multivitamin supplements can be added to the active substances mentioned above to maintain a well-balanced nutrition.

Furthermore, compositions containing the peptides of the invention in combination with minerals such as magnesium (Mg ²⁺ ), calcium (Ca ²⁺ ) and/or potassium (K ⁺ ) can be used for health promotion as well as prophylaxis and/or treatment Diseases including but not limited to cardiovascular disease and diabetes.

In a preferred aspect of the present invention, the nutraceutical composition of the present invention comprises: a composition containing the peptide of the present invention. IPP is suitably present in the composition according to the invention in a daily dosage providing from about 0.001 g per kg body weight to about 1 g per kg body weight of the individual to which it is administered. The food or drink suitably contains from about 0.05 g to about 50 g of IPP per serving. If the nutraceutical composition is a pharmaceutical formulation, such formulation may contain IPP in an amount of about 0.001 g to about 1 g per dosage unit (e.g., per capsule or tablet), or for liquid formulations, about 1 g per daily dose. 0.035g to approximately 70g per daily dose. Compositions containing a peptide of the invention are suitably present in a composition according to the invention in a daily dosage providing from about 0.01 g per kg body weight to about 3 g per kg body weight to the individual to which it is administered. The food or drink suitably contains from about 0.1 g to about 100 g of protein hydrolyzate per serving. If the nutraceutical composition is a pharmaceutical formulation, such formulation may contain the peptide-containing composition in an amount of from about 0.01 g to about 5 g per dosage unit (eg, per capsule or tablet), or for a liquid formulation, per serving The daily dose is about 0.7 g to about 210 g per daily dose.

In yet another preferred aspect of the invention, the composition comprises the peptides of the invention specified above and optionally carbohydrates. Carbohydrates are suitably present in the composition according to the invention in a daily dosage providing from about 0.01 g per kg body weight to about 7 g per kg body weight of the individual to whom it is administered. The food or drink suitably contains from about 0.5 g to about 200 g of carbohydrates per serving. If the nutraceutical composition is a pharmaceutical formulation, such formulation may contain the peptide-containing composition in an amount of about 0.05 g to about 10 g per dosage unit (e.g., per capsule or tablet), or for a liquid formulation, per serving The daily dose is about 0.7 g to about 490 g per daily dose.

Dose range (for a 70kg person)

IPP: 0.005-70g/day

Protein hydrolyzate: 0.07-210g/day

Unhydrolyzed protein: 0.07-210g/day

Carbohydrates: 0.1-490g/day

It is an object of the present invention to provide edible materials that can be used to provide health benefits to individuals consuming them. Another object is to provide the above-mentioned edible material which can be conveniently ingested in isolated form or included in a food product.

Yet another object of the present invention is to provide a food product or ingredients contained therein which are suitable for use in a weight management regimen.

Yet another object of the present invention is to provide a food product or ingredients contained therein which are suitable for helping to maintain cardiovascular health, for example, through ACE inhibition.

Yet another object of the present invention is to provide a food product or ingredients contained therein which have acceptable stability and/or organoleptic properties, in particular good taste, e.g. no or only acceptable levels of of bitterness.

Yet another object is to provide a food product that has a high concentration of ingredients that provide health benefits, eg aid in obesity prevention/weight management and/or help maintain cardiovascular health.

Surprisingly, according to the invention, one or more of these objects is achieved by the use of a composition comprising a peptide of the invention for the preparation of a food product which, when consumed, provides a health benefit.

According to a first aspect, the present invention provides the use comprising a peptide of the invention for the production of a functional food product for obesity prevention or weight management.

According to a second aspect, the present invention provides the use comprising a peptide of the invention for the production of a functional food product for the maintenance of cardiovascular health.

It is especially preferred according to the invention that maintenance of cardiovascular health comprises inhibition of angiotensin converting (ACE) enzymes and/or control of blood glucose levels.

According to a third aspect, the present invention provides a functional food product capable of providing its consumer with a health benefit selected from the group consisting of obesity prevention, weight control and maintenance of cardiovascular health, and wherein Said food product comprises a composition comprising a peptide of the invention.

Another benefit of the peptide-containing composition according to the present invention is that the peptide-containing composition can be conveniently incorporated into food products to create functional food products without unacceptably affecting its stability and/or organoleptic properties nature.

According to the present invention, "an agent providing health benefits" is a substance that can provide health benefits, which is a substance that has a positive effect on a certain aspect of health when ingested, or an aspect that can help maintain good health, good health These areas are obesity prevention, weight control and maintenance of cardiovascular health. "Health benefit" means an aspect of health that has a positive effect on or helps maintain good health.

A "functional food product" according to the present invention is defined as a food product (for the avoidance of doubt, beverages are also included) suitable for human consumption, wherein the peptide-containing composition of the present invention is used in an effective amount to make the food product consumer Ingredients for significant health benefits.

The term "comprising" when used herein is not limited to denote any subsequently stated elements, and it also includes elements of major or minor functional importance not specifically stated. In other words, the steps, ingredients or options listed are not necessarily exclusive. Where "comprising" or "having" is used, these terms are equivalent to "comprising" as defined above.

A "peptide" or "oligopeptide" is defined herein as a chain of at least two amino acids linked by peptide bonds. The terms "peptide" and "oligopeptide" are considered synonymous (as generally recognized) and each term is used interchangeably where the context so requires. A "polypeptide" is defined herein as a chain containing more than 30 amino acid residues. In accordance with common practice, all (oligo)peptide and polypeptide formulas or sequences herein are written from amino-terminus to carboxy-terminus in a left-to-right direction. The single letter numbering of amino acids used herein is generally known in the art and can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual, ^2nd , ed. Cold Spring Harbor Laboratory, Cold Spring Harbor laboratory Press, Cold Spring Harbor, NY, 1989).

The internationally recognized system from the IUMB for the classification and nomenclature of all enzymes includes proteases. An updated IUMB text for protease EC numbers can be found at the internet site http://www.chem.qmw/ac.uk/iubmb/enzyme/EC3/4/11/ . In this system, enzymes are defined by the fact that they catalyze a single reaction. This has the important implication that several different proteins are all defined as the same enzyme, and proteins that catalyze more than one reaction are treated as more than one enzyme. This system classifies proteases into endo and exoproteases. Endoproteases are those that hydrolyze internal peptide bonds, exoproteases hydrolyze peptide bonds adjacent to the terminal amino group ("aminopeptidases") or between the terminal carboxyl group and the penultimate amino acid ("carboxypeptidases") ). Endoproteases are divided into subgroups based on their catalytic mechanism. The following subgroups exist: serine endoproteases (EC 3.4.21), cysteine endoproteases (EC 3.4.22), aspartate endoproteases (Ec 3.4.23), metallo endoproteases (EC 3.4. 24) and threonine endoprotease (EC 3.4.25).

Aminopeptidases are in group 3.4.11. Subgroup classification was based on the relative efficiency of removal of 20 different amino acids. Aminopeptidases with narrow and broad specificities can be distinguished. Aminopeptidases sequentially remove single amino-terminal amino acids from protein and peptide substrates. Aminopeptidases with narrow specificity display a strong preference for the type of amino acid residue at the P1 position (released from the substrate peptide). Aminopeptidases of broad specificity release a series of different amino acids at the N-terminus or at the P1 position (according to Schechter's nomenclature: Schechter, I. and Berger, A. 1967. Biochem Biophys Res Commun 27:157-162). Carboxypeptidases sequentially remove single carboxy-terminal amino acids from protein and peptide substrates. Similar to the case of endoproteases, carboxypeptidases can be divided into subgroups based on their catalytic mechanism. Serine-type carboxypeptidases are in group EC 3.4.16, metallocarboxypeptidases are in group EC 3.4.17, and cysteine-type carboxypeptidases are in group EC 3.4.18. The values of the EC list for proteases are used to provide standard nomenclature for various types of protease activity, and in particular to assign unique nomenclature numbers and recommended names to each protease.

In EP1231279 a purely enzymatic method is described for the recovery of the tripeptides VPP and IPP from milk casein. The latter application claims a method for the production of tripeptides by digestion of a material containing casein of milk with proteases and peptidases through so-called "intermediate peptides" Selected from the group consisting of: peptides containing the sequence -V-P-P- but apart from these in this sequence no longer containing Pro, and peptides containing the sequence -I-P-P- but apart from these in this sequence no longer containing Pro. As described in the examples of EP1231279, the method involves a two-step process. First, an intermediate peptide comprising VPP or IPP is generated. This is achieved by incubating casein with a suitable protease. According to one of the embodiments, a period of 12 hours is carried out at 37 degrees Celsius. The protease used was then inactivated by heating this first hydrolyzate to 100° C. for 3 minutes and, after cooling again, another enzyme preparation (in fact, a preparation with exoproteolytic activity) was added. After a further 12 hours of incubation at 37°C with this other enzyme preparation, the presence of the tripeptides VPP and IPP could be revealed. To obtain higher yields of these ACE inhibitory peptides, EP1231279 further proposes to purify and concentrate the intermediate peptides before exposure to exogenous proteolytic activities. EP1231279 also suggests that after obtaining the intermediate peptide, and before contacting the intermediate peptide with the peptidase in the process, optionally, various manipulations can be carried out, such as removal by, for example, centrifugation at 5000 to 20000 rpm for 3 to 10 minutes unreacted protein. So, the desired tripeptide was obtained industrially rather than a clumsy two-step enzymatic process. Since each enzymatic incubation may be carried out for up to 12 hours at a pH of 4.5 to 7.0 and a temperature of 25 to 50 degrees Celsius, it is clear that this procedure is not acceptable from a microbiological point of view. Long incubation times combined with low incubation temperatures of 25 to 50°C may easily lead to infection of protein-containing solutions.

WO02/45524 describes a proline-specific endoprotease available from Aspergillus niger. The enzyme of A. niger origin preferentially cleaves at the carboxy-terminus of proline, but also at the carboxy-terminus of hydroxyproline, and with less efficiency at the carboxyl-terminus of alanine. WO02/45524 also teaches that there is no significant homology between this A. niger derived enzyme and known prolyl oligopeptidases from other microbial or mammalian sources. In contrast to known prolyl oligopeptidases, the A. niger enzyme has an acidic pH optimum. Although the known prolyl as well as A. niger derived enzymes are so-called serine proteases, we show here (Example 1) that A. niger enzymes belong to a completely different subfamily. The secreted A. enzyme appears to be a member of the S28 family of serine peptidases rather than the S9 family into which most cytosolic prolyl oligopeptidases are classified (Rawling, N.D. and Barrett, A.J.; Biochim. Biophys. Acta 1298 (1996) 1-3). In Example 2 we demonstrate the pH and temperature optima of an A. niger-derived proline-specific endoprotease. In Example 3 we demonstrate that the A. niger-derived enzyme preparation used in the method of the invention is highly pure, meaning that in addition to the endoproteolytic activity inherent in a pure proline-specific endoprotease There is no significant internal proteolytic activity attached to the outside. We also show that our A. niger derived enzyme preparations used according to the invention do not contain any exoproteolytic, more particularly aminopeptidolytic, side activities. All positively identified protease samples described in EP1231279 were complex mixtures of different enzymes exhibiting different proteolytic activities. Those skilled in the art will appreciate that the methods described in EP1231279 vary with the combination of endoproteolytic activity with one or more exoproteolytic enzyme activities. Such exoproteolytic activity is absent in the A. niger-derived enzyme preparations used in the methods of the invention. Vice versa, the enzymes used in the method according to the invention are also not present in the complex protease samples described in EP1231279. Experimental support for the idea that this enzyme is substantially absent in non-recombinant Aspergillus strains can be found in WO 02/45524. Because the method of the present invention may use only proline-specific endoproteins, optimal incubation conditions (such as temperature, pH, etc.) can be easily selected without having to be fixed as when two or more enzymes are used. suboptimal conditions. Having more freedom in choosing the reaction conditions makes it easier to choose against possible other criteria. For example, it is now much easier to select conditions that are less susceptible to microbial infection and to optimize pH conditions relative to the subsequent protein precipitation step. In Example 4, we show that Aspergillus enzymes are not oligopeptidases, but are true endopeptidases, capable of hydrolyzing intact proteins, large peptides, and smaller peptide molecules without the need for auxiliary endoproteases. This new and surprising discovery opens up the possibility of using the A. niger enzyme to prepare hydrolysates in which peptides with carboxy-terminal proline residues are present in unprecedentedly high levels because no Any auxiliary endoproteases. Such new hydrolysates can be prepared from different protein starting materials from plants or from animal sources. Examples of such starting materials are whey protein, whey beta-lactoglobulin, whey alpha-lactalbumin, casein, gelatin, fish or egg protein, potato protein, wheat and corn bran, soybean and pea protein, Rice protein and lupine protein. Of course, the starting protein should have at least the sequence -I-P-P-. Preferably, the protein also comprises the sequence -L-p-p- in its protein sequence. As explained above, this protein may have a -V-P-P- sequence in its protein. Hydrolysates having an unprecedentedly high content of peptides with carboxy-terminal proline or hydroxyproline residues can be produced from substrates with high levels of proline and hydroxyproline, such as gelatin. Because A. niger enzymes (such as known prolyl oligopeptidases) cannot cleave Pro-Pro or Pro-Hyp, Hyp-Pro or Hyp-Hyp bonds, this approach will also produce peptides containing unprecedentedly high levels of Hydrolysates, the peptides have two, three or even more carboxy-terminal proline or hydroxyproline residues. Clearly, the nature and proline content of the protein starting material dictated the likelihood of producing such peptides. Preferred substrates are those containing more than 6% proline (ie, more than 6 grams of this amino acid per 100 grams of protein), such as casein, gelatin, wheat and corn bran. Given that peptides carrying such carboxy-terminal amino acid sequences are expected to have a fair chance of surviving gastrointestinal hydrolytic activity, incubation with A. niger-derived prolyl endoproteases provided excellent starting material, using For the isolation of known biologically active peptides and for the identification of new biologically active peptides. Because sodium is known to play an important role in hypertension, the preferred substrates for the production of ACE inhibitory peptides are the calcium and potassium salts of these proteins, rather than the sodium salts.

The pH optimum of A. niger-derived prolyl endoprotease is about 4.3 (see Figure 1). Since this pH optimum is low, it is not self-evident to incubate bovine caseinate with A. niger derived prolyl endoprotease. If the pH is lowered below 6.0, bovine caseinate will precipitate and at this pH the A. niger enzyme has only limited activity. However, we show in Example 5 that even under these rather unfavorable conditions, several ACE-inhibiting peptides can still be produced with an A. niger-derived prolyl endoprotease. According to the present invention, the ACE-inhibiting tripeptides IPP and LPP are produced in yields corresponding respectively to 10% and approximately 60% of the amount theoretically present in casein. According to the explanation provided in Example 5, the yield of IPP arises from the fact that IPP can only be released from kappa casein. If this is taken into account, a yield of about 40% is obtained. Preferably, acid-precipitated casein is used as substrate in the method of the invention. Quite surprisingly, although the VPP precursor VVVPP was produced in LPP-like yields (ie, approximately 60% of the theoretical value present), no VPP was produced.

Preferably at least 20%, more preferably at least 30%, even more preferably at least 40%, most preferably at least 60% of the -L-P-P- sequences present in the protein sequence are converted to the peptide LPP.

Preferably at least 20%, more preferably at least 30%, even more preferably at least 40%, most preferably at least 60% of the -I-P-P- sequences present in the protein sequence are converted to the peptide IPP. Proline-specific proteases are preferably capable of hydrolyzing large protein molecules, such as substrate proteins themselves.

These results were obtained by incubating caseinate with an A. niger-derived endoprotease in a one-step enzymatic process. Aqueous solutions containing protein are susceptible to microbial infection, especially if kept at a pH above 5.0 and at a temperature of 50°C or lower for several hours. In particular, microbial toxins may be produced during the above-mentioned prolonged incubation steps, which may survive subsequent heating steps and pose a potential threat to food-grade processes. Unlike the conditions described in EP1231279, the method according to the invention preferably uses an incubation temperature above 50 degrees Celsius. In combination with a one-step enzymatic process, wherein the enzyme incubation is carried out for a period of less than 24 hours, preferably less than 8 hours, more preferably less than 4 hours, the method according to the invention offers the advantage that the stability of the microorganism is improved.

Bovine casein includes a number of different proteins, including beta casein and kappa casein. According to the known amino sequence, beta casein contains ACE inhibitory tripeptides IPP (Ile-Pro-Pro), VPP (ValPro-Pro) and LPP (Leu-Pro-Pro). kappa casein contains only IPP. In Example 5 we show that incubation of an A. niger-derived prolyl endoprotease with potassium caseinate produces the known ACE-inhibiting peptides IPP and LPP in high yield. Using the enzyme-substrate ratios of the invention, combined with high temperature conditions, cleavage of IPP and LPP was accomplished during a 3 hour incubation. Quite surprisingly, no significant amount of concomitant production of the tripeptide VPP was demonstrated. The fact that the A. niger-derived enzyme does not contain any measurable aminopeptidase activity strongly suggests that the IPP formed is released from the -A ₁₀₇ -I108-P ₁₀₉ -P ₁₁₀ -sequence present in kappa casein. We speculate that the carboxy-terminal of the peptide bond of IPP is cleaved by the main activity of prolyl endoprotease derived from A. niger, while the cleavage of the preceding Ala-Ile bond is accomplished by its Ala-specific side activity. Accordingly, the present invention provides a method for producing IPP from a protein source, wherein the ratio of IPP to VPP produced is at least 5, preferably at least 10, more preferably at least 20, said method comprising using proline-specific An enzyme with endoprotease activity and an enzyme capable of hydrolyzing the amino-terminal bond of IPP. Preferably, the enzyme that is able to hydrolyze the amino-terminal bond of -IPP- is at the same time unable to hydrolyze the amino-terminal bond of -VPP-, or has only low activity therefor. Although two enzymatic activities are mentioned for the method of the invention, it is also possible to use in the method of the invention having both activities (acid-specific endoprotease activity and activity to hydrolyze-IPP-amino-terminal bond respectively) An example of an enzyme is a proline-specific endoprotease as described herein, preferably derived from A. niger.

Because of the hydrolysis method chosen according to the present invention, smaller amounts of water-soluble peptides will be formed than in prior art methods. In these water-soluble peptides, IPP and optionally LPP are present in major amounts. This is especially important where high concentrations of IPP and optionally LPP compounds are desired and there are not many other compounds, usually less active compounds.

According to the method of the invention, preferably at least 20%, more preferably at least 30%, most preferably at least 40% of the -A-I-P-P- or -A-L-P-P- present in the protein is converted to IPP or LPP, respectively. Furthermore, according to the method of the invention, preferably at least 20%, more preferably at least 30%, still more preferably at least 40%, most preferably at least 50% of the -P-L-P-P- or -P-I-P-P- present in the protein is converted into LPP or IPP, respectively .

In Example 6 we demonstrate a 5-fold purification of IPP by a novel and surprising purification step. In this method, hydrolysates are formed during a brief enzyme incubation at 55°C, pH 6.0, and then heated to temperatures above 80°C to kill any contaminating microorganisms and to render A. niger-derived preserved Aminoacyl endopeptidase inactivation. Subsequently, the hydrolyzate is acidified to lower the pH to 4.5 or at least below 5.0. This pH cannot be used to inactivate A. niger-derived prolyl endopeptidase (because it represents optimal conditions for this enzyme), at which pH all large The peptides precipitate so that only small peptides remain in solution. Since precipitated caseinate can be easily removed by decantation or filtration steps or centrifugation at low speed (ie below 5000 rpm), the aqueous phase contains a high proportion of bioactive peptides relative to the protein present. According to Kjeldahl data, 80 to 70% of the caseinate protein was removed by the low speed centrifugation step, indicating a four to five fold purification of the ACE inhibitory peptide. We have found that this purification principle can be advantageously applied to obtain bioactive peptides obtained from sources of proteinaceous material other than casein. According to the present invention, not only enzymatically produced hydrolysates but also proteins fermented by suitable microorganisms can be isolated and purified. Incubating the enzyme and substrate at a pH close to the pH at which the substrate will precipitate while the enzyme is still active will allow this purification step to occur. Due to the low pH optimum of A. niger-derived prolyl endoproteases, a pH range of 1.5 to 6.5 could be considered for substrate precipitation. Considering their special sedimentation behavior, wheat bran precipitates above pH 3.5, whey protein precipitates above pH 3.5 but below pH 6.0, and egg white precipitates above pH 3.5 but below pH 5.0 form Examples of conditions under which hydrolyzed proteins can be precipitated can be separated from hydrolyzed proteins or peptides. This soluble portion of the hydrolyzate is also included within the term hydrolyzate. The acidic soluble hydrolyzate is formed by hydrolyzing the protein according to the invention, followed by adjusting the acidic conditions so that the insoluble hydrolyzed fraction can be separated from the soluble peptide. This separation can be achieved, for example, by sedimentation or centrifugation of the insoluble fraction. For the wheat bran hydrolyzate, the acidic separation condition is preferably pH=4, for the whey hydrolyzate, the acidic condition is preferably pH=4.5, for the casein hydrolyzate, the acidic condition is preferably pH=4.5, for For egg whites, acidic conditions are preferably pH=5.0. In general, the preferred acidic conditions for separation are pH = 4.5.

Hydrolyzate means a product formed by hydrolysis of a protein (alternatively, simply referred to as protein hydrolyzate or protein hydrolyzate), acid soluble hydrolyzate is the soluble fraction of a protein hydrolyzate, which is also described herein as containing soluble peptides Compositions or compositions comprising soluble peptides) or mixtures of protein hydrolysates and acidic soluble hydrolysates.

The hydrolysates of the invention are advantageously used in nutraceutical applications and in food and beverage applications. Protein hydrolysates, acid soluble hydrolysates and mixtures thereof can be used in nutraceutical applications, food applications or beverages. Preferably, the acidic soluble hydrolysates are used in nutraceutical applications, food applications or beverages because of the high content of active peptides present. Although a similar principle is used in the cheesemaking process to separate casein curds from whey proteins, only aspartic endoproteases (EC 3.4.23) are used in this cheesemaking process. This group of enzymes (EC 3.4.23) includes well-known cheese-making enzymes such as rennet and various pepsins, such as mammalian pepsin, and various microbial pepsins, such as aspergillopepsin and mucosal pepsin (mucorpepsin). For the purposes of the present invention, curds and whey from the cheesemaking process are not defined as hydrolysates. Furthermore, in the hydrolysis method of the invention, advantageously, no aspartic endoprotease (EC 3.4.23) is used. Furthermore, as discussed previously, the purification steps according to the invention are not known for hydrolysates produced by non-aspartic endoproteases.

Cheese-making processes or curd/whey separation processes are excluded from the purification method of the present invention, so the purification method of the present invention is used to obtain soluble peptides, and with the following preconditions: The method is not a cheese-making process or curdling process Part of the milk/whey separation process.

While this purification step bears superficial similarities to the cheesemaking process, they are quite different. In cheese making, curd formation is initiated by an enzymatic step ("renneting") or an acidification step. However, the rennet clotting process does not depend on acidification, and the cheese curd clumping caused by acidification does not depend on enzymes.

In another method of purification, according to the method of the present invention, water-miscible solvents, such as ethanol, acetone, propanol-1, propanol-2, methanol or mixtures thereof, are conveniently and efficiently obtained from hydrolyzed proteins Peptides are recovered. In this approach, preferably, the protein hydrolyzate is carefully mixed with 30-60% (v/v) of a water-miscible solvent at a pH selected to allow precipitation of larger proteins, Smaller peptides, such as IPP, remain in solution.

After separation (eg, decantation, filtration, or low-speed centrifugation), the supernatant containing the bioactive peptide can be recovered in a purified state. Optionally, the resulting peptide-containing composition (or soluble peptide-containing composition) is treated with additional enzymes, for example, to increase the levels of active ACE-inhibiting peptides (see Examples 12 and 13), or peptide combinations The material can be contacted with a selective binder such as activated carbon, chromatography resins from the Amberlite XAD range (Rohm) or butyl sepharose resin supplied by Pharmacia. Subsequent evaporation and optional spray drying steps will result in an economical route for obtaining food grade highly bioactive pastes or powders. After digestion of the caseinate, a white odorless powder is obtained which has a high concentration of ACE-inhibiting peptides, rich in IPP and LPP. If properly diluted to the correct tripeptide concentration, a versatile starting material with excellent mouthfeel is obtained, suitable for imparting ACE-inhibiting properties to all kinds of foods and beverages. Concentration of the biologically active components can be increased, if desired, by subsequent purification, taking advantage of the hydrophobic character of the peptides IPP and LPP. Preferred purification methods include microfiltration (nanofiltration), extraction (e.g. with hexane or butanol) followed by evaporation/precipitation, or combining the obtained acidified hydrolyzate with an adhesive (e.g. activated charcoal or sorbent from the Amberlite XAD range (Roehm) Chromatography resin) contact. Butyl Sepharose resin supplied by Pharmacia can also be used. Desorption of ACE inhibitory peptides from such materials can be performed with organic solvents such as methanol/ethanol mixtures or with propanol.

The peptides obtained before or after additional (eg chromatographic) purification steps can be used for incorporation into food or drink products, which are routinely consumed. Examples of such products are vegetable butters, spreads, various milk products (eg butter or yoghurt) or beverages containing milk or whey, preferably yoghurt or milk based products such as yoghurt and milk. In other beverages, such as fruit juice drinks or soy drinks, the hydrolyzate of the present invention can also be used. Another option is to use hydrolysates in health products such as fruit bars, protein bars, energy bars, cereal based products such as breakfast cereals.

Another aspect of the present invention relates to a food product, beverage product or dietary supplement obtainable by a method or process as hereinbefore described, said method for preparing the food or beverage product comprising the steps of: producing from a protein source containing A composition of IPP, wherein the weight ratio of IPP to VPP produced from the protein is at least 5:1, preferably at least 10:1, more preferably at least 20:1, said method comprising the use of a proline-specific endoprotease; and (b) adding the IPP-containing composition to a food product, beverage product or dietary supplement. Preferably, said food product, beverage product or dietary supplement according to the present invention comprises 0.05 to 10 wt%, more preferably 0.1 to 5 wt%, most preferably 0.2 to 4 wt% of said IPP-containing composition or protein hydrolyzate. Also preferably, the food or drink product or dietary supplement according to the invention comprises 0.05 to 50 mg of IPP per 100 g of product, more preferably, 0.1 to 40 mg, most preferably, 0.2 to 30 mg. Also preferably, in the food or beverage product or dietary supplement of the invention, the weight ratio of IPP to VPP is from 5:1 to 100:1, more preferably from 5:1 to 48:1. Also preferably, the food product, beverage product or dietary supplement according to the invention comprises IPP and LPP, wherein the weight ratio of IPP to LPP is from 1:10 to 1:1, more preferably from 1.5:7.1 to 4.8:7.1 .

Preferably, the food or drink product or dietary supplement is selected from the group consisting of vegetable butter, spreads, butter, milk products or whey-containing beverages, preferably yoghurt or milk based products, such as yoghurt or milk, wherein the Said food or beverage product or dietary supplement comprises the hydrolyzate in the amount as described above or the IPP in the amount as described above.

Especially preferred are the food or drink products or dietary supplements described above for reducing hypertension in humans. For food or drink or dietary supplements, preferred serving sizes are eg 5-350 grams per serving, eg 5 to 150 grams. Preferably, the number of servings per day is 1-10, such as 2-5.

Although such compositions are typically administered to humans, they can also be administered to animals, preferably mammals, to reduce hypertension. Furthermore, the high content of ACE inhibitors or other bioactive peptides in the obtained products makes these products very useful for incorporation into pills, tablets or dietary supplements in highly concentrated solutions or in paste or powder form. Slow-release dietary supplements that ensure sustained release of ACE-inhibiting peptides or other bioactive peptides are of particular interest. The ACE-inhibiting peptides or other biologically active peptides according to the present invention can be formulated as dry powder, eg in pills, tablets, granules or sachets or capsules. Alternatively, the enzymes according to the invention may be formulated as liquids, eg in syrups or capsules. Compositions containing enzymes according to the invention for various preparations may also comprise at least one compound of the group consisting of physiologically acceptable carriers, adjuvants, excipients, stabilizers, buffers, Agents and diluents, which terms are used in their ordinary sense to denote substances that aid in packaging, delivery, absorption, stabilization or, in the case of adjuvants, enhance the physiological action of an enzyme. Relevant background on various compounds that can be used in combination with the enzymes according to the invention in powder form can be found in "Pharmaceutical Dosage Forms", second edition, Volumes 1, 2 and 3, ISBN 0-8247-8044-2 Marcel Dekker, Inc. Although the ACE-inhibiting peptide formulated as a dry powder according to the present invention can be stored for a considerable period of time, it should be protected from contact with water or moist air by choosing suitable packaging, such as aluminum foil. A relatively new form of oral application is the use of various types of gelatin capsules or gelatin-based tablets.

Given the relevance of natural ACE-inhibiting peptides to combat hypertension, the new cost-effective route of the present invention provides an attractive starting point for mildly hypotensive foods or even veterinary products. Because the approach of the present invention also includes a surprisingly simple purification step, the possibility of a concentrated dietary supplement that lowers blood pressure is also increased.

The method according to the invention can be carried out using any proline-specific oligoprotease or endoprotease. Proline-specific oligopeptidases according to the invention or used according to the invention represent enzymes belonging to EC 3.4.21.26.

Proline-specific endoproteases according to the invention or used according to the invention represent the polypeptides mentioned in claims 1-5, 11 and 13 of WO 02/45525. Accordingly, the proline-specific endoprotease is a polypeptide having proline-specific endoproteolytic activity selected from the group consisting of the following polypeptides:

(a) a polypeptide having an amino acid sequence having at least 40% amino acid sequence identity to amino acids 1 to 526 of SEQ ID NO: 2 or a fragment thereof;

(b) a kind of polypeptide, it is encoded by following polynucleotide, and described polynucleotide can under the condition of low stringency and (i) SEQ ID NO: The nucleotide sequence of 1, or its 60, preferably 100 A fragment that is at least 80% or 90% identical in nucleotides, more preferably a fragment that is at least 90% identical in 200 nucleotides, or (ii) hybridizes to a nucleic acid sequence complementary to the nucleic acid sequence of SEQ ID NO:1. SEQ ID NO: 1 and SEQ ID NO: 2 are shown in WO02/45524. Preferably, the polypeptide is in isolated form.

Preferred polypeptides for use according to the present invention comprise the amino acid sequence of SEQ ID NO: 2 or have an amino acid sequence having at least 50%, preferably at least 60%, of the amino acid sequence at positions 1-526 of SEQ ID NO: 2, Preferably at least 65%, preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95%, even more preferably at least about 97% identical.

Preferably, the polypeptide is encoded by a polynucleotide capable of matching (i) SEQ ID NO under conditions of low stringency, more preferably under conditions of medium stringency, most preferably under conditions of high stringency: 1 or a fragment thereof, or (ii) a nucleic acid sequence complementary to the nucleic acid sequence of SEQ ID NO: 1 hybridised.

The term "hybridization" means that the target polynucleotide of the present invention can be used as a probe nucleic acid (for example, the nucleotide sequence shown in SEQ ID NO: 1, or a fragment thereof, or the complementary sequence of SEQ ID NO: 1 sequence) hybridized at levels significantly higher than background. The present invention also includes the polynucleotide encoding the proline-specific endoprotease of the present invention, and its complementary nucleotide sequence. The nucleotide sequence can be RNA or DNA, including genomic DNA, synthetic DNA or cDNA. Preferably, the nucleotide sequence is DNA, most preferably, a genomic DNA sequence. Typically, a polynucleotide of the present invention comprises a contiguous sequence of nucleotides capable of hybridizing under selective conditions to the coding sequence of SEQ ID NO: 1 or the complementary sequence of the coding sequence. Such nucleotides can be synthesized according to methods well known in the art.

The polynucleotides of the present invention can hybridize to the coding sequence of SEQ ID NO: 1 or the complement of the coding sequence at a level significantly above background. Background hybridization may occur due to the presence of other cDNAs in the cDNA library. The interaction between a polynucleotide of the invention and the coding sequence of SEQ ID NO: 1 or the complement of a coding sequence produces a signal level typically at least as strong as the interaction between other polynucleotides and SEQ ID NO: 1. 10 times, preferably at least 20 times, more preferably at least 50 times, even more preferably at least 100 times. The strength of the interaction can be measured, for example, by radiolabeling the probe (eg with 32P). Typically, selective hybridization can use low stringency conditions (0.3 M sodium chloride and 0.03 M sodium citrate, about 40 ° C), medium stringency conditions (for example, 0.3 M sodium chloride and 0.03 M sodium citrate, about 50°C) or high stringency conditions (eg, 0.3M sodium chloride and 0.03M sodium citrate, approximately 60°C).

The UWGCG Package provides the BESTFIT program, which can be used to calculate identities (eg with its default settings).

The PILEUP and BLASTN algorithms can be used to calculate sequence identity or to line up sequences (e.g. to identify identical or corresponding sequences, e.g. using their default settings).

Software for performing BLAST analyzes is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). The algorithm includes: firstly determine a high scoring sequence pair (high scoring sequence pair, HSP) by determining a short word (shortword) of length W in the sequence being queried, and the short word has the same length as that in the database sequence Words match or can reach a certain threshold score T which is positive. T is called the neighborhood word score threshold. These initial neighborhood word hits act as seeds to initialize the search to find the HSP containing them. The word hits are extended in both directions for each sequence until the cumulative alignment score can be increased. When: the cumulative alignment score is reduced by the amount X from its maximum value; the cumulative score falls to zero or below due to the accumulation of one or more negative-scoring residue alignments; or one of the ends of the sequence is reached , the extension of the word pair in each direction is interrupted. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The default word length (word length, W) used by the BLAST program is 11, BLOSUM62 scoring matrix alignments (BLOSUM62 scoring matrix alignments, B) is 50, the expected value (E) is 10, M=5, N=4, By default two chains are compared.

The BLAST algorithm performs a statistical analysis of the similarity between two sequences. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance . For example, a first sequence is considered to be compatible with another sequence if the smallest sum probability when compared with the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001. resemblance.

Strains of the genus Aspergillus have food-grade status and enzymes obtained from these microorganisms are known to form an unquestionable food-grade source. According to another preferred embodiment, the enzyme is secreted by the producing bacterium rather than a non-secreted so-called cytosolic enzyme. In this way, the enzyme can be recovered in a substantially pure state from the cell culture fluid without costly purification steps. Preferably, the enzyme has a high affinity for its substrate under prevailing pH and temperature conditions.

Description of drawings

Figure 1: Schematic diagram of the pH optimum of prolyl endoprotease derived from A. niger

Figure 2: Specificity of prolyl endoprotease derived from A. niger

Figure 3: Intact ovalbumine and synthetic 27 peptide with chromatographically purified A. SDS-PAGE after incubation with niger-derived proline-specific endoprotease.

Figure 4: Measured relative activities at pH 6.0 against enzyme preparations containing three commercially available aminopeptidases on the synthetic substrates F-pNA, Q-pNA and V-pNA.

Materials and methods

Material

Edible potassium caseinate (88%) was obtained from DMV International, The Netherlands. Synthetic chromogenic peptides were obtained from Pepscan Systems B.V. The Netherlands or from Bachem, Switzerland.

Flavourzyme 1000L Batch HPN00218 was obtained from Novozymes (Denmark), Sumizyme FP was obtained from Shin Nihon (Japan), and Corolase LAP Ch.: 4123 was obtained from AB Enzymes (UK).

Proline-specific endoprotease from A. niger.

The proline specific endoprotease from Aspergillus niger was overproduced as described in WO02/45524. Enzyme activity was detected on the synthetic peptide Z-Gly-Pro-pNA in citric acid/disodium phosphate buffer, pH 4.6, at 37°C. The reaction product was monitored spectrophotometrically at 405 nm. One unit is defined as the amount of enzyme that releases 1 μmol of p-nitroanilide per minute under the above detection conditions.

Chromatographic purification of endoproteases derived from A. niger

Broth obtained from an overproduced A. niger strain was used for chromatographic purification of the protease to remove any contaminating endo- or exo-proteolytic activity. For this purpose, the fermentation broth is first centrifuged to remove fungal biomass, and the supernatant is then passed through a large number of filters with progressively decreasing pore sizes to remove all cell fragments. Finally, the obtained ultrafiltrate was diluted 10-fold in 20 mmol/L sodium acetate (pH 5.1) and loaded onto a Q-Sepharose FF column. Proteins were eluted using a 0-0.4 mol/L NaCl gradient in 20 mmol/L sodium acetate, pH 5.1. Peak fragments exhibiting cleavage activity against Z-Gly-Pro-pNA were collected and pooled according to the method described in World Journal of Microbiology & Biotechnology 11, 209-2125 (1995), but with slightly modified experimental conditions. Considering the acidic pH optimum of the A. niger-derived proline-specific endoprotease, enzyme assays were performed at 37°C, pH 4.6, in citric acid/bisphosphate buffer. Pooling of active fragments followed by centrifugation finally yielded a preparation that showed only a single band on SDS-PAGE and one peak on HP-SEC. Further analysis by hydrophobic interaction chromatography verified the purity of the obtained enzyme preparation.

LC/MS/MS analysis

HPLC using an ion trap mass spectrometer (Thermo Electron, Breda, the Netherlands) coupled to a P4000 pump (Thermo Electron, Breda, the Netherlands) was used to quantify target peptides, including enzymes produced according to the method of the invention Peptides IPP (M=325.2), LPP (M=325.2), VPP (M=311.2), VVVPP (M=509.3) and VVVPPF (M=656.4) in the proteolytic hydrolyzate. 0.1% formic acid in Milli Q water (Millipore, Bedford, MA, USA; solution A) and 0.1% formic acid in acetonitrile (solution B) were combined using an Inertsil 3 ODS 3, 3 μm, 150*2.1 mm column (Varian Belgium, Belgium) A gradient is used for elution to separate the formed peptides. Gradient started at 100% solution A, held for 5 minutes, linearly increased to 5% solution B in 10 minutes, then linearly increased to 45% solution B in 30 minutes, followed by immediate return to starting conditions, held for 15 minutes to stabilize. The injection volume used was 50 microliters, the flow rate was 200 microliters per minute, and the column temperature was maintained at 55°C. The protein concentration of the injected samples was approximately 50 μg/ml.

Detailed information on individual peptides was obtained by using dedicated MS/MS for the target peptide using an optimal collision energy of approximately 30%. Quantification of the individual peptides was performed using an internal calibration using ^N15 , ^C13 labeled IPP (M=332.2) for all isoleucines, which was performed in MS/MS mode for all relevant peptides analyzed The most abundant fragment ion observed was performed. All peptides used were synthesized by Pepscan (Lelystand, The Netherlands).

The tripeptide LPP (M=325.2) was used to fine-tune for optimal sensitivity in MS mode and for optimal fragmentation in MS/MS mode, which exhibited a constant infusion rate of 5 μg/ml, in MS Protonated molecules are generated in MS/MS mode, with approximately 30% of the optimal collision energy in MS/MS mode, producing B- and Y-ion series.

Before LC/MS/MS, the enzymatic protein hydrolyzate was centrifuged at ambient temperature and 13,000 rpm for 10 minutes, filtered through a 0.22 μm filter, and demineralized water (filtered through a Millipore water filtration device) (MilliQ water) to dilute the supernatant 1:100.

degree of hydrolysis

Incubation with various proteolysis mixtures was performed using the rapid OPA test (Nielsen, P.M.; Petersen, D.; Dambmann, C. Improved method for determining food protein degree of hydrolysis. Journal of Food Science 2001, 66, 642-646). The degree of hydrolysis (DH) obtained during this period was monitored.

Kjeldahl nitrogen

Total Kjeldahl nitrogen was measured by Flow Injection analysis. Ammonia released from protein-containing solutions was quantified at 590 nm using a Tecator FIASTAR 5000 Flow Injection System equipped with a TKNMethod Cassette 5000-040, a Pentinum4 computer (with SOFIA software) and a Tecator5027 Autosampler. A sample amount corresponding to the kinetic range of the method (0.5-20 mg N/l) was placed in a digestion tube together with 95-97% sulfuric acid and Kjeltab was digested at 200°C for 30 minutes followed by 90 minutes at 360°C program to process. After injection, the nitrogen peak is measured in the FIASTAR 5000 system, from which the amount of protein measured can be deduced.

Nutraceutical Products

The nutraceutical product according to the invention may be any food type. In addition to food products, they may contain common food ingredients such as spices, sugar, fruit, minerals, vitamins, stabilizers, thickeners, etc. in suitable amounts.

Preferably, the nutraceutical product comprises 50-200 mmol/kg K ⁺ and/or 15-60 mmol/kg Ca ²⁺ and/or 6-25 mmol/kg Mg ²⁺ , more preferably 100-150 mmol/kg K ⁺ and/or 30-50 mmol/kg Ca ²⁺ and/or 10-25 mmol/kg Mg ²⁺ , most preferably, 110-135 mmol/kg K ⁺ and/or 35-45 mmol/kg Ca ²⁺ and/or 13-20 mmol/kg Mg ²⁺ . When included in the nutraceutical product according to the invention, these cations have the beneficial effect of further reducing blood pressure.

Advantageously, the nutraceutical product comprises one or more B vitamins.

The B vitamin folic acid is known to be involved in the metabolism of homocysteine, an amino acid in the human diet. For several years, high levels of homocysteine have been linked to a high incidence of cardiovascular disease. Lowering homocysteine is thought to reduce the risk of developing cardiovascular disease.

Vitamins B6 and B12 are known to interfere with purine and thiamine biosynthesis, participate in methyl synthesis during homocysteine methylation for the production of methionine, and in several growth processes. Vitamin B6 (pyridoxine hydrochloride) is known as a vitamin supplement. Vitamin B12 (cyanobalamin) promotes nervous system health, which is involved in the production of red blood cells. It is also known as a vitamin in food supplements.

For their combined positive effect on reducing the risk of cardiovascular diseases, preferably, the product according to the invention comprises vitamin B6 and vitamin B12 and folic acid.

The amount of B vitamins in nutraceutical products can be calculated by those skilled in the art based on the daily amounts of these B vitamins given herein: folic acid: 200-800 μg/day, preferably, 200-400 μg/day; vitamin B6: 0.2 - 2 mg/day, preferably 0.5-1 mg/day; and vitamin B12: 0.5-4 μg/day, preferably 1-2 μg/day.

Preferably, the nutraceutical product comprises 3 to 25 wt% sterols, more preferably 7 to 15 wt% sterols. The benefit of including sterols is that it will lead to a reduction in LDL-cholesterol levels in the human blood, which will lead to a reduction in cardiovascular risk.

Reference to sterols includes saturated stanols and sterol/sterol esterified derivatives or any mixtures thereof.

In this application, reference to sterol esters also includes their saturated derivatives (hydrosterol esters) and combinations of sterol esters and hydrogenated sterol esters.

Sterols or phytosterols (also plant sterols or vegetable sterols) can be divided into three groups: 4-desmethylsterols, 4-monomethylsterols, and 4,4'-dimethylsterols. In oils, they are mainly present as free sterols or sterol esters of fatty acids, although sterol glucosides and acylated sterol glucosides are also present. There are three major phytosterols, beta-sitosterol, stigmasterol, and campesterol. A schematic diagram of the meaning of these ingredients is given in "Influence of Processing on Sterols of Edible Vegetable Oils", S.P. Kochhar; Prog. Lipid Res. 22: pp. 161-188.

The respective 5alpha-saturated derivatives, such as sitosterol, campesterol and ergosterol and their derivatives, are referred to in this document as hydrosterols. Preferably, the (optionally esterified) sterol or sterol is selected from the group consisting of β-sitosterol, β-sitosterol, campesterol, campesterol, stigmasterol, brassicasterol , the group of brassicasterol or mixtures thereof.

Optionally, the sterol or hydrosterol is at least partially esterified with a fatty acid. Preferably, the sterol or hydrosterol is esterified with one or more _C2-22 fatty acids. For the purposes of the present invention, the term _C2-22 fatty acid means any molecule comprising a _C2-22 backbone and at least one acid group. The _C2-22 backbone may be partially substituted, or side chains may be present, although this is not preferred in this context. Preferably, however, the _C2-22 fatty acid is a linear molecule comprising one or two acid groups as terminal groups. Most preferred are linear _C8-22 fatty acids such as those found in natural oils.

Suitable examples of any such fatty acids are acetic acid, propionic acid, butyric acid, caproic acid, caprylic acid, capric acid. Other suitable acids are eg citric acid, lactic acid, oxalic acid and maleic acid. Most preferred are myristic acid, lauric acid, palmitic acid, stearic acid, arachidic acid, behenic acid, oleic acid, erucic acid, docosenoic acid, trans oleic acid (elaidic acid), linoleic acid acid and linolenic acid.

Mixtures of fatty acids can be used for esterification of sterols or hydrosterols, if desired. For example, esterification can be carried out by transesterification using naturally occurring fats or oils as the source of fatty acids.

The aforementioned nutraceutical ingredients, K ⁺ , Ca ²⁺ and Mg ²⁺ , B vitamins (folic acid, B6, B12) and sterols that act to increase cardiovascular health are collectively referred to as heart health ingredients herein.

Example 1 Enzymes obtained from A. niger represent a new class of proline-specific enzymes

The protein sequence of 526 amino acids can be determined from the complete coding sequence of the A. niger-derived proline-specific endoprotease provided in WO02/45524. Enzyme novelty was verified by BLAST searches against databases such as SwissProt, PIR and trEMBL. To our surprise, no significant homology was detected between this A. niger enzyme and known prolyl oligopeptidases. But closer examination of the amino acid sequence revealed low correlations with Pro-X carboxypeptidase (EC 3.4.16.2), dipeptidyl aminopeptidase I (EC 3.4.14.2) and thymus-specific serine protease but significant homology. All these enzymes have been classified into the S28 family of the SC group of serine peptidases (Handbook of Proteolytic Enzymes; Barrett A.J.; Rawlings N.D.; Woessner J.F., Eds.; Academic Press, London, UK, 1998, 369-415). Furthermore, the GxSYxG conformation around the active site serine is conserved in these enzymes and A. niger-derived endoproteases. In addition, members of the S28 family have an acidic pH optimum, have specificity for cleavage at the carboxy-terminal side of proline residues, and are signal sequences similar to A. niger-derived proline-specific endoproteases. and propeptide synthesis. Furthermore, the A. niger enzyme is similar in size to members of the S28 family. Thus, the A. niger proline-specific endoprotease appears to be a member of the S28 family of serine proteases rather than the S9 family into which most cytosolic prolyl oligopeptidases (including those obtained from Flavobacterium meningosepticum) are assigned a member of. On the basis of these structural and physiological features, we have deduced that the A. niger enzyme belongs to the S28 family, rather than the S9 family, of the SC group of serine proteases. An additional feature that distinguishes A. niger-derived enzymes from prolyl oligopeptidases belonging to the S9 family is the fact that, unlike the cytosolic prolyl endoproteases belonging to the latter family, the newly identified A. The niger enzyme is secreted into the growth medium.

Example 2 The optimal value of DH and mixing degree of the proline-specific endoprotease obtained from A.niger

To establish the pH optimum of the A. niger-derived proline-specific endoprotease, buffers with different pH values were prepared. Use 0.05 mol/l sodium acetate and 0.02 M _CaCl to prepare buffers at pH 4.0-4.5-4.8-5.0-5.5 and 6.0; use _0.05 M Tris/HCl buffer with 0.02 M CaCl to prepare pH 7.0 and 8.0 buffer. The pH was adjusted using acetic acid and HCl, respectively. The chromogenic synthetic peptide Z-Gly-Pro-pNA was used as substrate. The "pNA" (p-nitroanilide) substrate causes a color change if the X-pNA peptide bond is cleaved. The buffer solution, substrate solution and prolyl endoprotease pre-dilution (with an activity of 0.1 U/mL) were heated to exactly 37.0°C in a water bath. After mixing, the reaction was then monitored spectrophotometrically at 37.0° C. at 405 nm for 3.5 minutes, with measurements every 0.5 minutes. From the results shown in Table 1, it is clear that the A. niger-derived proline-specific endoprotease has a pH optimum of about 4.

In addition, the temperature optimum of prolyl endoprotease was established. For this purpose, at different temperatures, Caseine Resorufine (Roche Diagnostics, Alrnere, TheNetherlands) was used as substrate in 0.1 mol/l sodium acetate containing _0.02 mol/l CaCl at pH 5.0 against purified Enzyme preparations were incubated for 2 hours and enzyme activity was quantified by measurement at 574 nm. According to the results obtained, the proline-specific endoprotease from A. niger has a temperature optimum of approximately 50 degrees Celsius.

The specific conductance of the proline-specific endoprotease of embodiment 3 A.niger source

Crude enzyme samples obtained from A. niger strains containing multiple copies of the expression cassette (see WO02/45524) and chromatographically purified enzyme samples were tested against pools of chromogenic peptide substrates to establish the endoprotease specificity. The endoproteolytic activity of the enzyme was tested on the AAXpNA substrate, where "X" represents different natural amino acid residues.

Stock solutions of AAX-pNA substrate (150 mmol/l) were diluted 100X in 0.1 M acetate buffer (pH 4.0) containing ₂₀ CaCl2. Kinetic measurements at 405 nm in a TECAN Genios MTP Reader (Salzburg, Vienna) for 10 minutes at 40°C recorded the increase in optical density. Further processing of the resulting data in Excel resulted in the graph shown in Figure 2. From the results, it is clear that the A. niger-derived endoprotease is highly specific for prolyl peptide bonds, and it has side activity for alanyl bonds. Crude and chromatographically purified preparations showed similar activity profiles.

The proline-specific endoprotease of embodiment 4 A.niger source can hydrolyze large albumen and small peptide, which is thus a true endoprotease

Due to specific structural features, prolyl oligopeptidases of the S9 family belonging to the SC group of serine proteases cannot digest peptides larger than 30 amino acids. This limitation is a clear disadvantage for enzymes which are supposed to hydrolyze different proteins as quickly and efficiently as possible. To check whether A. niger-derived proline-specific endoproteases exhibit the same limitations related to the molecular size of the substrate, we combined the chromatographically purified prolyl endopeptidase from A. niger with the small Synthetic peptides were incubated with large ovalbumin molecules to study the degradation kinetics of these substrate molecules.

The synthetic peptide used was the 27 peptide of the sequence NH2-FRASDNDRVIDPGKVETLTIRRLHIPR-COOH, a gift from the company Pepscan (Lelystad, The Netherlands). As shown by its amino acid sequence, the peptide contains 2 proline residues, one in the middle and one at the very end of the peptide.

The whole ovalbumin molecule used (Pierce Imject, vial containing 20 mg lyophilized material) consisted of 385 amino acids and had a molecular weight of 42750 Da. The molecule contains 14 proline residues, one of which is located at the last position of the C-terminus of the molecule, which cannot be cleaved by proline-specific endoproteases.

The purified A. niger-derived proline-specific endoprotease was incubated with ovalbumin and oligopeptides at 50°C, respectively. Samples were taken at several time intervals and analyzed using SDS-PAGE.

A 100-fold dilution of the chromatographically purified A. niger-derived proline-specific endoprotease having an activity of 4.5 units/ml was performed with 0.1 M acetate buffer (pH 4) containing 20 mM CaCl ₂ . Ovalbumin was dissolved in acetate buffer (pH 4) to a concentration of 1 mg/ml (22 μM). Peptide 27 was dissolved in the same buffer to a concentration of 0.48 mg/ml (152 μM). The molar concentrations of the ovalbumin and 27 peptide solutions were chosen such that both solutions contained the same molar concentration of cleavable proline residues. Ovalbumin contains 13 possible proline cleavage sites, while 27 peptide has only two. 0.5 ml of each of the two substrate solutions was incubated with 10 μl (0.45 mU) of enzyme solution in an Eppendorf thermomixer at 50°C. At several time intervals, 10 μl samples were taken from the incubation mixture and kept at 20° C. until SDS-PAGE. All materials used for SDS-PAGE and staining were purchased from Invitrogen (Breda, The Netherlands). Samples were prepared using LDS buffer according to the manufacturer's instructions and separated on 12% Bis-Tris gels using the MES-SDS buffer system according to the manufacturer's instructions. Staining was performed using Simply Blue Safe Stain (Collodial Coomassie G250).

As can be seen in Figure 3, ovalbumin was cleaved by the Aspergillus-derived enzyme into a discrete band of approximately 35-36 kD during the first 4.75 hours of incubation (lane 3). Prolonged incubation periods lead to further fragmentation into smaller products of various molecular weights (lane 7).

Furthermore, incubation of the 27 peptide with the enzyme resulted in rapid degradation as judged by the slightly dimmer and more indistinct bands in lanes 4, 6 and 8. Because the 27 peptide fragments at the same rate as the much larger ovalbumin molecule, it can be inferred that, unlike the known prolyl oligopeptidases belonging to the S9 family, the much larger protein, A. niger origin The proline-specific endoprotease has no particular preference for small-sized peptides. This observation confirms that A. niger derived enzymes represent true endoproteases, and preferred enzymes for hydrolyzing different types of proteins. This discovery led to the surprising use of this enzyme as illustrated in the Examples below.

Example 5 Warming of potassium caseinate with proline-specific endoprotease from A. niger Rapid production of IPP and LPP but not VPP

In this experiment, overproduced, substantially pure proline-specific endoprotease from A. niger was incubated with potassium caseinate to examine the release of the ACE inhibitory peptides IPP, VPP and LPP. The endoprotease used was essentially pure, indicating that, in addition to the intrinsic endoproteolytic activity (i.e., carboxy-terminal cleavage of proline and alanine residues) of a pure proline-specific endoprotease No other significant proteolytic activity was present (see Example 7). To limit as much as possible sodium uptake as a result of uptake of ACE inhibitory peptides, potassium caseinate was used as substrate in this incubation.

The caseinate was suspended in 65°C water at a concentration of 10% (w/w) protein, after which the pH was adjusted to 6.0 with phosphoric acid. The suspension was then cooled to 55 °C and A. niger-derived proline-specific endoprotease was added at a concentration of 4 units/gram protein (see Materials and methods section for unit definition). The mixture was incubated for 24 hours with constant stirring. No further pH adjustments were made during this period. Samples were taken after 1, 2, 3, 4, 8 and 24 hours of incubation. Enzyme activity was terminated for each sample by rapidly heating the sample to 90°C for 5 minutes. After cooling, the pH of each sample was rapidly lowered to 4.5 using phosphoric acid, after which the suspension was centrifuged at 3000 rpm for 5 minutes in a Hereaus benchtop centrifuge. The fully clarified supernatant was used for LC/MS/MS analysis to quantify the peptides VPP, IPP, LPP, VVVPP and VVVPPF in the supernatant (see Materials and methods section).

Bovine casein includes several different proteins, including beta casein and kappa casein. According to the known amino acid sequence, beta casein contains ACE inhibitory tripeptides IPP, VPP and LPP. In beta casein, IPP is contained in the sequence -P ₇₁ -O ₇₂ -N ₇₃ -I ₇₄ -P ₇₅ -P ₇₆ - and VPP is contained in the sequence -P ₈₁ -V ₈₂ -V ₈₃ -V ₈₄ - In P ₈₅ -P ₈₆ -, LPP is contained in the sequence -P ₁₅₀ -L ₁₅₁ -P ₁₅₂ -P ₁₅₃ -. The kappa casein is present in an acid-precipitated caseinate formulation containing only IPP at a molar concentration of approximately 50% of the beta casein concentration. In kappa casein, IPP is contained in the sequence -A ₁₀₇ -I ₁₀₈ -P ₁₀₉ -P ₁₁₀ -. The other protein components of casein do not contain IPP, VPP or LPP.

Tables 1 and 2 show the concentrations of peptides present in the acidified and centrifuged supernatants, which were quantified by LC/MS/MS. The concentrations of the various peptides presented in these tables are calculated per gram of potassium caseinate added to the incubation mixture. As shown in Table 1, IPP reached its maximum concentration after 1 hour of incubation. The IPP concentration does not increase further above it. The formation of the pentapeptide VVVPP exhibited the same kinetics as IPP production. As expected from theory, the molar yield of VVVPP was similar to that of the LPP peptide. The yields of LPP and VVVPP reach about 60% of theoretically feasible. The fact that the maximum concentration of LPP is reached after only 3 hours of incubation suggests that cleavage of certain parts of the beta casein molecule may be slightly more difficult. In contrast to VVVPP, the hexapeptide VVVPPF is not formed at all. This observation indicates that the proline-specific endoprotease efficiently cleaves the -PF- bond, thereby generating VVVPP. The tripeptide IPP was formed immediately, but at a molar yield no higher than about one third of the maximum molar yield of VVVPP or LPP. This result was unexpected since the IPP tripeptide is present in both beta and kappa casein. A possible explanation for this observation is that the proline-specific endoprotease can generate IPP, but only from the kappa casein fraction of caseinate. Considering the relevant amino acid sequence of kappa casein, this suggests that the -A ₁₀₇ -I ₁₀₈ -peptide bond is cleaved by the alanine-specific activity of the enzyme. If this is true, the amount of IPP released amounts to about 40% of the amount present in kappa casein, but does not exceed about 10% of the amount theoretically present in beta plus kappa casein. This cleavage mechanism for IPP release also explains why VPP cannot be formed from its precursor molecule VVVPP: simply, the required endoproteolytic (or aminopeptidase) activity was not present in the A. niger-derived enzyme preparation used .

Table 1

Molar peptide content in acidified supernatant calculated per gram of protein added

micromole/gram protein IPP LPP VPP VVVPP VVVPPF K-cas 1 hour K-cas 2 hours K-cas 3 hours K-cas 4 hours K-cas 8 hours K-cas 24 hours 2.82.62.62.32.12.0 4.26.18.48.09.49.5  ＜0.2＜0.2＜0.2＜0.2＜0.20.4 8.49.19.18.37.25.5  ＜0.2＜0.2＜0.2＜0.2＜0.2＜0.2

Table 2

Peptide concentration in acidified supernatant calculated in mg/g protein added

mg/g protein IPP LPP VPP VVVPP VVVPPF K-cas 1 hour K-cas 2 hours K-cas 3 hours K-cas 4 hours K-cas 8 hours K-cas 24 hours 0.90.80.80.80.70.7 1.42.02.72.63.03.1  ＜0.05＜0.05＜0.05＜0.05＜0.050.1 4.34.64.64.23.62.8  ＜0.05＜0.05＜0.05＜0.05＜0.05＜0.05

Example 6 Incorporation of an acidic casein precipitation step resulted in a 5-fold concentration of ACE inhibitory peptides

Potassium caseinate at a protein concentration of 10% (w/w) was used for incubation with A. niger derived proline specific endoprotease at pH 6.0 as described in Example 5. After several different incubation cycles, the samples were heated to stop other enzymatic activity, after which the pH was lowered to 4.5 to minimize casein solubility. Insoluble casein molecules were removed by low speed centrifugation. In Tables 1 and 2 we have provided the calculated concentrations of ACE inhibitory peptides based on a starting concentration of 10% protein. However, most of the added protein had been removed as a result of the acidification and subsequent centrifugation steps. To take these reduced protein contents of the acidified supernatant into account, a nitrogen (Kjeldehl) analysis was performed. From the latter data, the different supernatants were found to contain the following protein levels.

table 3

Protein content of acidified supernatant

sample Protein content (g/L) K-cas 1 hour twenty one K-cas 2 hours 27 K-cas 3 hours 30 K-cas 4 hours 34 K-cas 8 hours 40 K-cas 24 hours 48

Taking these data into account, we recalculated the concentration of ACE-inhibiting peptides present in each supernatant, but this was using their true protein content. The results of these recalculations are shown in Table 4.

Table 4

Peptide concentration in acidified supernatant calculated per gram of protein present

mg/g protein IPP LPP VPP VVVPP VVVPPF K-cas 1 hour K-cas 2 hours K-cas 3 hours K-cas 4 hours K-cas 8 hours K-cas 24 hours 0.10.10.10.10.10.3 4.83.43.12.41.91.5 7.18.010.08.58.47.1 22.518.917.013.710.06.4  ＜0.05＜0.05＜0.05＜0.05＜0.05＜0.05

A comparison of Tables 2 and 4 clearly shows that a simple acidification step followed by an industrially feasible step of decantation, filtration or low speed centrifugation leads to a 5-fold increase in the concentration of the specific ACE-inhibiting peptide. Later experiments showed that the efficiency of the acid precipitation step can be increased by overnight storage of the acidified suspension prior to decantation, filtration or low speed centrifugation.

Example 7 Quantification of Contaminating Enzyme Activity in the Production of ACE Inhibiting Peptides

According to the present invention, ACE inhibitory peptides can be obtained in a simple one-step process by incubating a suitable protein substrate with a proline-specific endoprotease. Subsequent enrichment of water-soluble ACE inhibitory peptides was accomplished by precipitation of larger molecular weight protein fragments by acidification. The efficiency of the latter acid enrichment method relies on very selective cleavage of the substrate molecule. The more proteolytic activity present, the more water-soluble peptides are formed thereby, thus reducing the enrichment factor for ACE-inhibiting peptides. For example, during incubation with the substrate, the presence of additional non-proline or non-alanine specific endoproteases results in the solubility of more non-biologically active peptides, thereby diluting IPP and LPP in the final concentrate. relative concentration in . In addition, the presence of contaminating exoproteases (such as carboxypeptidases or aminopeptidases) leads to the release of free amino acids. These additional free amino acids also dilute the relative concentration of ACE-inhibiting peptides and, moreover, give the culture broth a bad taste as a result of the increased Maillard reaction. To minimize any of these unwanted side reactions, it is preferred to use a substantially pure proline-specific endoprotease. Substantially pure means that the activity of contaminating endoproteases and contaminating exoproteases is minimal or preferably absent under the culture conditions employed. The assay procedure described below was used to quantify such contaminating activity.

The basis of this assay protocol is formed by a collection of several selective chromogenic peptides. In all of these chromogenic peptides, "Z" stands for benzyloxycarbonyl and "pNA" stands for the chromophore p-nitroanilide. Since only proline-specific oligo- and endoproteases can release colored pNA from peptides whose N-terminus is blocked by a "Z" group, the peptide Z-AAAP-pNA was used to target the desired proline-specific The proteolytic activity was quantified. Since many endoproteases release pNA from Z-AAAF-pNA and Z-AAAR-pNA, these two peptides were used to quantify contaminating, non-proline-specific endoproteolytic activity. Because the conversion of the peptides QNIPP and VVVPP to IPP and VPP, respectively, requires aminopeptidases capable of efficiently removing Gln(Q) and Val(V) residues, Q-pNA and V-pNA were used to target such contaminating aminopeptides. Enzyme activity was quantified. Because many carboxypeptidases are capable of releasing Phe (F) and Arg (R) residues from peptides, peptides containing these residues are candidates for quantification of contaminating carboxypeptidase activity. However, no suitable chromophores were available for the measurement of carboxypeptidases and an alternative method using synthetic peptides Z-AF and Z-AR had to be developed. This alternative is provided below.

All chromogenic peptides were obtained from Pepscan (Lelystad, The Netherlands). Peptides Z-AF and Z-AR were purchased from Bachem (Switserland). All incubations were performed at 40°C. To illustrate the method, commercially available enzyme preparations Flavourzyme 1000L (BatchHPN00218, obtained from Novozymes (Denmark)), Sumizyme FP from Shin Nihon (Japan) and Corolase LAP from AB Enzymes (UK) (Ch.: 4123) and compared with the proline-specific endoprotease from A. niger. The tested commercial enzyme preparations were diluted to produce optimal incubation conditions, but were recalculated to the commercial product concentrations in the presentation of the final data (see Table 5).

Measuring Contaminating Aminopeptidase Activity

Dilute 150mmol/l V-pNA and Q-pNA stock solutions in 100% DMSO 80-fold in 0.1M BisTris buffer (pH6) to make 3.75mmol containing V-pNA and Q-pNA in a 1:1 ratio /l V-pNA+Q-pNA substrate solution. The aminopeptidase substrate solution was pipetted in 200 [mu]l aliquots into individual wells of a microtiter plate (MTP). MTPs were previously incubated at 40°C in TecanGenios MTP (Salzburg, Vienna), run under Magellan4 software. Reactions were initiated by adding 50 μl of the appropriate enzyme solution such that incubation occurred at a substrate concentration of 3 mM. Typically, a 1:50 dilution of liquid enzyme samples of Flavourzyme, Corolase LAP, and proline-specific endoprotease is used. For dry Sumizyme FP products, use a 1% solution.

The yellow color was measured by Tecan Genios MTP development at 405 nm as a result of cleavage of the amino acid-pNA bond, followed by at least 20 kinetic cycles (approximately 10 minutes). The software generates data acquired as OD ₄₀₅ /min.

Measuring proline-specific enzyme activity

The measurement was carried out essentially as in the aminopeptidase assay, but in this assay Z-AAAP-pNA was used as the sole substrate at a final concentration of 3 mmol/l. The substrate was dissolved by heating the suspension in pH 6 buffer to 50-55°C, resulting in a clear solution at room temperature. Measurements were performed at 40°C.

Typically, a 1:50 dilution of the liquid enzyme sample Flavourzyme, Corolase LAP is used. Sumizyme FP is available as a 1% solution. Typically, the proline specific endoprotease is used at a 1:5000 dilution.

The software generates data expressed as _OD405 /min.

Measurement of contaminating non-proline-specific endoprotease activity

The measurement was also carried out in essentially the same manner as described for the aminopeptidase assay, however, in this assay a 1:1 ratio and a final concentration of 3 mmol/l of Z-AAAF-pNA and Z-AAAR-pNA were used. as a substrate. The substrate Z-AAAF-pNA was poorly soluble under the assay conditions of pH 6.0 used, but assay incubation with subtilisin resulted in rapid dissolution of the substrate with concomitant release of pNA. Measurements were performed at 40°C. However, to compensate for this poor solubility, the MTP reader was programmed to shake between kinetic cycles. The software still generates data expressed as _OD405 /min.

Measuring Contaminating Carboxypeptidase Activity

Since no sensitive chromogenic peptides were available for measuring carboxypeptidase activity, carboxypeptidase C was quantified using a method based on the Boehringer protocol.

A 150 mmol/l Z-A-F and Z-A-R stock solution in ethanol was diluted 80-fold in 0.1 M Bis Tris buffer (pH 6) to make a 3.75 mmol/l Z-A-F+Z-A-R substrate containing Z-A-F and Z-A-R in a 1:1 ratio solution. 200 μl of this substrate solution was taken, transferred into an eppendorf tube, and pre-incubated at 40°C. Reactions were initiated by adding 50 μl of the appropriate enzyme dilution. Typically, a 1:50 dilution is used for Flavourzyme and Corolase LAP and proline specific endoprotease. For Sumizym FP, a 1% solution is used. After 5 minutes, the reaction was stopped by adding 250 [mu]l of ninhydrine reagent. The ninhydrin reagent was prepared from 60 mg reduced ninhydrin (hydrindantin) and 400 mg ninhydrin (Merck) dissolved in 15 ml DMSO, to which 5 ml 4.0 mol/l lithium acetate buffer (pH 5.2) was added. A 4.0 mol/l lithium acetate buffer was prepared by dissolving LiOH (Sigma) and then adjusting the pH to pH 5.2 with glacial acetic acid (Merck).

After terminating the reaction, each sample was heated at 95° C. for 15 minutes to promote color formation, and then diluted 10-fold with pure ethanol. The color formed was measured in a Uvikon spectrophotometer at 578 nm. Blanks were made in the same manner as active samples, but the addition of ninhydrin reagent and enzyme was reversed. To quantify free amino acids produced by carboxypeptidase activity, the amino acid L-phenylalanine was used to generate a calibration curve. Solutions containing 0.1875, 0.375, 0.75, 1.5 and 3.0 mol/l L-phenylalanine (Sigma) in buffer (pH 6) were treated in the same manner as the samples, ie 250 μl in vials. From the obtained OD578 values, a curve was constructed in Excel. This curve was used to calculate the concentration of free amino acids present in samples containing Z-A-F and Z-A-R substrates. From the values obtained, the carboxypeptidase activity can be calculated, expressed in micromoles per minute relative to the enzyme content of each assay.

Calculate activity ratio

To establish the suitability of various enzyme preparations for use in the methods of the invention, quotients for the relevant enzyme activities were calculated. In the MTP reader-based assay, enzyme activity was analyzed by pNA release over time, ie as _ΔOD405 /min. The coefficient of enzyme activity obtained by the MTP reader was calculated by simply dividing by the ΔOD/min value obtained for the same amount of enzyme.

However, in the case of the carboxypeptidase assay, the OD produced cannot be directly compared to the ΔOD/min produced by the MTP-pNA based assay. At this point, the measured OD should first be converted into amino acids released per minute (μmol/min). For this purpose, a calibration curve was generated in an MTP reader in which pure pNA (Sigma) was diluted at 0.25, 0.125, 0.0625, 0.0312 and 0.015 mmol/l and 250 μl was measured per well. A calibration curve was constructed from the obtained data in Excel. From this calibration curve, ΔOD/min was converted to μmol/min so that the pNA-based measurements were comparable to the ninhydrin-based measurements.

On the basis of the data generated in the assays described above, the various enzyme preparations used were analyzed for the desired proline-specific and contaminating endoprotease, aminopeptidase and carboxypeptidase activities. The desired proline-specific activity present in each enzyme preparation is shown in Table 5 in the column "Proline-specific activity". Regarding contaminating aminopeptidase activity (aminopeptidase/proline specific activity) and contaminating carboxypeptidase (carboxypeptidase/proline specific activity) and endopeptidase activity (endopeptidase/proline specific activity) specific activity) data are shown relative to the proline specific activity present. The activity of the contaminating aminopeptidase relative to the contaminating carboxypeptidase present in each enzyme preparation is shown as (aminopeptidase/carboxypeptidase).

Notably, none of the commercial enzyme preparations examined contained any significant proline-specific oligo- or endo-proteolytic activity. Furthermore, all commercial enzyme preparations examined contained contaminating endo- and exoproteolytic activities.

Table 5: Quantification of contaminating proteolytic activity in various enzyme preparations

Proline specific activity ^* Carboxypeptidase/Proline specific activity Aminopeptidase/Proline specific activity Endoproteolytic/proline specific activity Aminopeptidase/Carboxypeptidase Sumizyme FP 0.004 21.7 1.2 1.7 0.06 Flavourzyme 0.0007 253.5 25.6 35.5 0.10 Corolase LAP 0.0 0.74 Proline-specific A. niger 100 0.005 0.00001 0.000004 0.00

^* Sumizyme is measured in a 1% solution, Flavourzyme and Corolase are measured as a 1:50 dilution. Proline-specific activity obtained from A. niger was measured as a 1:5000 dilution. The data are then converted to the activity present in the supplied product.

A. The pharmaceutical composition can be prepared using the following ingredients through traditional formulation procedures:

Example 8

soft gelatin capsules

Soft gelatin capsules are prepared by the conventional procedure using the following ingredients:

Active ingredient: protein hydrolyzate 0.3g

Other Ingredients: Glycerin, Water, Gelatin, Vegetable Oil

Example 9

hard gelatin capsule

Hard gelatin capsules are prepared by the conventional procedure using the following ingredients:

Active ingredient: protein hydrolyzate 0.7g

Other ingredients:

Filler: Lactose or cellulose or cellulose derivatives in sufficient quantity

Lubricant: if desired, magnesium stearate (0.5%)

Example 10

tablet

Tablets are prepared by the conventional procedure using the following ingredients:

Active ingredients: protein hydrolyzate 0.3g, unhydrolyzed protein 0.4g

Other Ingredients: Microcrystalline Cellulose, Silicon Dioxide (SiO ₂ ), Magnesium Stearate, Croscarmellose Sodium.

B. Food products may be prepared according to conventional procedures using the following components:

Example 11

Soft drink with 30% fruit juice

Typical Serving Size: 240ml

Active ingredient:

Protein hydrolysates and maltodextrin as a carbon source are added to this food product:

Protein hydrolyzate: 1.5-15g/serving

Maltodextrin: 3-30g/per serving

I. Prepare the soft drink complex with the following ingredients:

Juice Concentrates and Water Soluble Flavors

[g]

1.1 Orange juice concentrate

60.3°C Brix, 5.15% acidity 657.99

lemon concentrate

43.5°C Brix, 32.7% acidity 95.96

Water Soluble Orange Flavor 13.43

Water-soluble apricot flavor 6.71

Water 26.46

1.2 Pigment

Beta-Carotene 10% CWS 0.89

Water 67.65

1.3 Acids and Antioxidants

Ascorbic acid 4.11

Anhydrous citric acid 0.69

Water 43.18

1.4 Stabilizer

Pectin 0.20

Sodium benzoate 2.74

Water 65.60

1.5 Oil-soluble spices

Oil-soluble orange flavor 0.34

Distilled sweet orange oil 0.34

1.6 Active ingredients

The active ingredients (this means the above mentioned active ingredients: protein hydrolyzate and maltodextrin) are present in the concentrations mentioned above.

The juice concentrate and water-soluble flavor are mixed without air incorporation. Pigments are dissolved in deionized water. Ascorbic acid and citric acid are dissolved in water. Sodium benzoate is dissolved in water. Add the pectin while stirring and bring to a boil to dissolve it. The solution was cooled. Oil soluble flavor and sweet orange oil are pre-mixed. The active ingredients mentioned under 1.6 are mixed dry and then added, preferably with stirring, to the fruit juice concentrate mixture (1.1).

To prepare the soft drink compound, all parts 3.1.1 to 3.1.6 were mixed together and then homogenized with a Turrax and then with a high pressure homogenizer (p ₁ =200 bar, P ₂ =50 bar). quality.

II. Prepare the bottled syrup with the following ingredients:

[g]

Soft Drink Compound 74.50

Water 50.00

Syrup, Brix 150.00 at 60°C

The ingredients of the bottled syrup are mixed together. The bottled syrup is diluted to 1 L with water for a ready-to-use beverage.

Variety:

Instead of using sodium benzoate, the beverage can be pasteurized. The beverage can also be carbonated.

The aminopeptide hydrolysis activity of embodiment 12 different commercial enzyme preparations

In beta casein, IPP is contained in the sequence -P ₇₁ -Q ₇₂ -N ₇₃ -1 ₇₄ -P ₇₅ -P ₇₆ - and VPP is contained in the sequence -P ₈₁ -V ₈₂ -V ₈₃ -V ₈₄ - In P ₈₅ -P ₈₆ -, LPP is contained in the sequence -P ₁₅₀ -L ₁₅₁ -P ₁₅₂ -P ₁₅₃ -. Of the other protein components of casein, only kappa casein includes sequences containing IPP. From the specificity of the proline-specific endoprotease, it can be deduced that incubation of beta casein with enzymes derived from A. niger will form Q ₇₂ -N ₇₃ -I ₇₄ -P ₇₅ -P ₇₆ -, V ₈₂ -V ₈₃ -V ₈₄ -P ₈₅ -P ₈₆ - and L ₁₅₁ -P ₁₅₂ -P ₁₅₃ -. In contrast to LPP, the two pentapeptides formed exhibited only low ACE inhibitory activity. For example, EP0583074 reports that VVVPP has an _IC50 value of 873 micromol/l, whereas the truncated VPP molecule has an _IC50 value of 9 micromol/l. Clearly, therefore, to generate the full ACE inhibitory capacity of casein hydrolysates, VVVPP and QNIPP formed during incubation with proline-specific endoproteases must be converted to the tripeptides VPP and IPP, respectively. Because the aminopeptidase can then remove amino acids from the N-terminus of the peptide, it is necessary to efficiently release the two valine ("V") residues preceding the VPP sequence and the glutamine ("Q") and glutamine ("Q") residues preceding the IPP sequence. Aminopeptide hydrolase activity of paragine ("N") residues. Since the XP and PP peptide bonds present in the XPP tripeptide are known to be resistant to enzymatic cleavage, such aminopeptidolytic activity would likely convert these two pentapeptides into the desired VPP and IPP tripeptides.

Three commercial enzyme preparations were tested for their aminopeptidolytic activity: Flavourzyme 1000L Batch HPN00218 (Novozymes, Denmark), Sumizyme FP (Shin Nihon, Japan) and Corolase LAP Ch.: 4123 (AB Enzymes, UK). Flavourzyme and Sumizyme FP are known to be complex enzyme preparations which contain several aminopeptidohydrolase activities in addition to nonspecific endopeptidolytic and carboxypeptidolytic activities. Corolase LAP exhibits relatively pure, cloned and overexpressed leucine aminopeptidase activity from Aspergillus.

The presence of aminopeptidolytic activity in these three commercial preparations was examined using the chromogenic substrates F-pNA (control), Q-pNA and V-pNA. For this purpose, a stock solution of 150 mMX-pNA in DMSO was diluted 100x in Bis-Tris buffer (pH 6). 200 [mu]l buffered substrate solution was loaded per well in a microtiter plate and pre-incubated at 40[deg.] C. in a Tecan Genios MTP reader (controlled by Magellan4 software). The reaction was initiated by adding 50 μl of the appropriate enzyme solution (Sumizyme FP powder was dissolved in Bis-Tris buffer (pH 6) at a concentration of 100 mg/ml before use). Release of yellow pNA was then monitored at 405 nm for 10 minutes. The software calculates OD/min. The activity of each enzyme preparation against various substrates was normalized to their activity against F-pNA. The data obtained are shown in FIG. 4 . Clearly, all three enzyme preparations displayed the highest activity against F-pNA, but Q-pNA and V-pNA also formed substrates for these enzymes. These results suggest that each of the commercial preparations should be able to form the desired ACE-inhibiting tripeptides IPP, VPP, and LPP if combined with a proline-specific endoprotease, since the activity of the aminopeptidases converts proline Peptides QNIPP and VVVPP formed by acid-specific proteases are converted to IPP and VPP, respectively. This hypothesis was tested in the experiments described in Example 13.

Example 13 Hydrolysis with different aminopeptides using proline-specific endoprotease from A. niger Enzymes to incubate caseinate to produce high yields of IPP, LPP and VPP

In this example, we investigated the effect of combining a proline-specific protease from A. niger with aminopeptidolytic activity in the same incubation step on the formation of various ACE-inhibiting peptides. For this purpose, a caseinate solution was prepared by dissolving 50 g of sodium caseinate in 506 g of 70°C water, resulting in a solution containing 81 g protein/l. The solution was cooled to 50°C before the pH was lowered to 6.0 (measured at 20°C) using phosphoric acid before adding the various enzyme combinations. Proline-specific protease was added to all samples (10 ml each) to a concentration of 4 units per gram of protein (see Materials and methods section for unit definition). Sample A1 contained only the proline-specific protease. Sample B1 contained proline specific protease plus 38 microliters of a solution containing 1140 mg of concentrated Flavourzyme liquid diluted in 5 grams of water. In sample B2, the proline-specific protease was combined with 8 microliters of this Flavourzyme solution. In sample C1, the proline-specific protease was combined with 100 microliters of Corolase LAP liquid. In sample C2, the proline-specific protease was combined with 10 μl of a 10-fold diluted liquid sample of Corolase LAP. In all 5 samples, incubation was allowed to proceed for 6 hours at 50°C. The enzymatic reaction was terminated by heating the mixture to 90 °C for 5 min. The clear supernatant obtained after centrifugation for 10 min in Eppendorf centrifuge tubes was collected and kept frozen until LC/MS/MS analysis. The data obtained after LC/MS/MS analysis are shown in Table 5.

As previously described, the incubation conditions for sample A1 (proline-specific protease only) efficiently produced LPP as well as VVVPP, but not significant amounts of VPP. The absence of peptide VVVPPF indicates that the proline-specific protease efficiently cleaves the carboxyl terminus of proline residues under the conditions used. In sample A1, the yield of IPP was approximately one third of the yield of VVVPP. However, combining the proline-specific protease with Flavourzyme (sample B1 or B2) or with Corolase LAP (sample C1 ) had a clear stimulating effect on the IPP yield. In sample C2 (with a low concentration of Corolase LAP), the yield of IPP was not increased, presumably because the concentration of aminopeptidase was insufficient to convert all of the QNIPP formed by the proline-specific protease. Since 1 gram of casein theoretically yields 6.9 mg (21.1 micromoles) of IPP (produced by beta casein plus kappa casein), the levels of IPP present in samples B1 and C1 each account for 70% of the maximum achievable yield and 55%. Consistent with our expectations, increased aminopeptidolytic activity resulted in a decrease in VVVPP concentration and an increase in VPP concentration. The fact that a small amount of the intermediate peptide VVPP was detectable in samples B2 and C2 suggests that in these samples the aminopeptidolytic activity was insufficient to completely convert VVVPP formed by the proline-specific protease to VPP. Since 1 gram of casein can theoretically produce 4.58 mg (14.7 micromole) of VPP, in samples B1, B2 and C1, the maximum VPP yield was achieved. Altogether, this experiment clearly demonstrates that the combination of a proline-specific protease with appropriate aminopeptidolytic activity can efficiently produce high concentrations of ACE-inhibiting peptides. The combination of proline-specific protease and aminopeptidase is best performed after incubation with the proline-specific enzyme. Incubation with additional enzymes can occur before or after purification using low pH conditions or water-miscible solvents, depending on specific process requirements. Alternatively, incubation may be performed with an aminopeptidase activity capable of removing unwanted amino acid residues preceding the amino acid sequence having an ACE-inhibiting effect, or it may be performed with a suitable endoproteolytic activity. A suitable example of such endoproteolytic activity is represented by papain (available as Collupulin from DSMFood Specialties, Delft, The Netherlands), which efficiently cleaves the carboxyl terminus of Asn (N) and Val (V) residues. If the incubation with additional enzymes is performed prior to the acidification or ethanol precipitation step, it is best performed with a relatively pure aminopeptidase, such as Coralase LAP (see Example 7).

Table 6

Peptide concentration in supernatant calculated as mg/(g protein added)

sample IPP LPP VPP VVPP VVVPP VVVPPF A1B1B2C1C2 1.04.83.03.81.4 3.41.33.03.83.5 0.14.65.04.81.8 ＜0.05＜0.05 0.7 not detected 0.9 4.60.050.6＜0.051.3 <0.05<0.05<0.05<0.05<0.05

Claims (43)

1. method, be used to produce the composition that comprises from proteic IPP, wherein, be at least 5: 1 from the IPP of described albumen generation and the weight ratio of VPP, preferably at least 10: 1, more preferably at least 20: 1 (wt/wt), described method comprises uses the enzymic activity with proline specific endo-protease activity or the active enzymic activity of prolyl oligopeptidase and key that can hydrolysis-I-P-P-sequence N-terminal side.

2. the method for claim 1, wherein, described enzymic activity with key of proline specific endo-protease activity or the active enzymic activity of prolyl oligopeptidase and described energy hydrolysis-I-P-P-sequence N-terminal side is present in a kind of enzyme, preferably, this enzyme is proline specific endo-protease or prolyl oligopeptidase, more preferably, this enzyme is the proline specific endo-protease.

3. as any described method in the claim 1 to 3, wherein, described proline specific endo-protease does not contain aminopeptidase activity and/or carboxypeptidase activity.

4. method as claimed in claim 1 or 2, wherein, the incubation time preferably less than 10 hours, was more preferably less than 4 hours less than 24 hours.

5. any as described above described method of claim, wherein, heated culture temperature is higher than 30 ℃, preferably is higher than 40 ℃, more preferably is higher than 50 ℃.

6. any as described above described method of claim wherein, also produces LPP.

7. any as described above described method of claim wherein, is used milk proem, preferably uses casein.

8. method as claimed in claim 7, wherein, exist in the described protein sequence-A-I-P-P-and/or-P-I-P-P-sequence at least 30% is converted into peptide IPP.

9. method as claimed in claim 6, wherein, exist in the described protein sequence-P-L-P-P-and/or-A-L-P-P-sequence at least 40% is converted into peptide LPP.

10. method, be used for preparation and comprise solvable peptide, the composition that preferably comprises IPP, described method comprises that the pH by the composition that produces with suitable dietary protein origin hydrolysis, preferably produce by any described method in the claim 1 to 9 is changed into a part that makes protolysate becomes insoluble pH, and described insoluble part separated with solvable peptide, obtain comprising the composition of solvable peptide.

11. a method is used to prepare the composition that comprises solvable peptide, described composition is produced by following step:

-at first, with the proline specific endo-protease with the DH of proteolysis to 5 to 30%,

-alternatively, carry out second kind of enzyme and handle, and

-afterwards, the soluble part of protolysate is separated under selected pH condition with soluble part, preferably under condition of acidic pH, more preferably under the pH between 3.5 to 6, most preferably under the pH between 4 to 5, comprise the composition of solvable peptide with generation.

12. composition, described composition comprises solvable peptide, wherein, at least the 70wt% of described peptide, preferred 80wt% at least, the pH of 90wt% between 3.5 to 6 at least most preferably, be soluble under the pH between preferred 4 to 5, and, wherein, exist with amount without any a kind of peptide in the described solvable peptide greater than described solvable peptide 40wt%, preferably, exist with amount without any a kind of peptide in the described solvable peptide, most preferably greater than described solvable peptide 30wt%, exist with amount without any a kind of peptide in the described solvable peptide, and described composition can obtain by the described method of claim 11 greater than described solvable peptide 20wt%.

13. the proline specific endo-protease is used for from the purposes of protein production IPP, wherein, the IPP of generation (weight) is at least five times of the VPP that produces.

14. a composition, described composition produces by proteolysis, and described composition comprises IPP, and wherein, IPP is at least 5: 1 to the weight ratio of VPP in the described composition, preferably at least 10: 1, more preferably at least 20: 1.

15. composition as claimed in claim 14, it also comprises LPP.

16. as claim 14 or 15 described compositions, it exists as nutrient drug, preferably exists as medicament.

17. as any described composition in the claim 12 to 16, it is produced by any described method in the claim 1 to 10.

18. the composition that produces as any described composition among claim 12 and the 14-17 or according to any described method in the claim 1 to 11 is as nutrient drug, purposes preferably as medicament, described purposes is to be used to produce promote health or prevent and/or treat the nutrient drug that disease is used, preferably, medicament, perhaps be used for producing and be used for the treatment of or preventing disease, the nutrient drug of hypertension, heart failure, preceding diabetes or diabetes, obesity, impaired glucose tolerance or pressure for example, preferably, medicament.

19. purposes as claimed in claim 18, wherein said composition exists with the form of dietary supplements, form with personal care application exists, and described application comprises the topical application that the form with aqua, gel or emulsion exists, and perhaps exists as food, feed or pet food composition.

20. the purposes as any described composition in claim 12 and 14 to 17 is used to produce the functional food product, described foodstuff products is used for prevention of obesity or is used for weight management.

21. the purposes as any described composition in claim 12 and 14 to 17 is used to produce the functional food product, described foodstuff products is used for cardiovascular health and keeps.

22. purposes as claimed in claim 21, wherein said cardiovascular health keeps comprising the inhibition angiotensin-converting enzyme.

23. purposes as claimed in claim 21, wherein said cardiovascular health keeps comprising the control blood cholesterol levels.

24. functional food product, it can provide the benefit of healthy aspect to its human consumer, the benefit of described healthy aspect is selected from prevention of obesity, weight management and cardiovascular health and keeps, and described foodstuff products comprises as any described composition in claim 12 and 14 to 17.

25. functional food product as claimed in claim 24, wherein, the benefit of described cardiovascular health maintenance aspect comprises and suppresses angiotensin-converting enzyme and/or control blood cholesterol levels.

26. as claim 24 or 25 any described functional food products, it comprises the K of 50-200mmol/kg ⁺And/or the Ca of 15-60mmol/kg ²⁺And/or the Mg of 6-25mmol/kg ²⁺

27. functional food product as claimed in claim 26, it comprises the K of 110-135mmol/kg ⁺And/or the Ca of 35-45mmol/kg ²⁺And/or the Mg of 13-20mmol/kg ²⁺

28. as any described functional food product of claim 24 to 27, it comprises one or more B family microorganisms.

29. functional food product as claimed in claim 28, it comprises folic acid, vitamin B6 and vitamin B12.

30. as any described functional food product of claim 24 to 29, it comprises 3 to 25wt% sterol, more preferably, and 7 to 15wt% sterol.

31. a method is used to produce foodstuff products, drink product or dietary supplements, described method comprises:

(a) produce the composition that comprises IPP according to any described method in the claim 1 to 9;

(b) composition with the described IPP of containing joins in foodstuff products, drink product or the dietary supplements.

32. method as claimed in claim 31 wherein, in step a, is used milk proem, preferably uses casein.

33. method as claimed in claim 32, wherein in step a, exist in the described protein sequence-A-I-P-P-or-P-I-P-P-sequence at least 10%, more preferably at least 30% be converted into peptide IPP.

34. method as claimed in claim 32, wherein in step a, exist in the described protein sequence-P-L-P-P-or-A-L-P-P-sequence at least 10%, more preferably at least 40% be converted into peptide LPP.

35. method as claimed in claim 31, also comprise step c), with the peptide of purifying from protolysate, described albumen is preferably by non-aspartate protease hydrolysis, more preferably by the serine stretch protein enzymic hydrolysis, described protolysate can precipitate under selected pH condition, and described step comprises pH changed into can make the sedimentary pH of described protolysate, and sedimentary albumen is separated with described protolysate.

36. method as claimed in claim 35, wherein step c takes place after step a) and before step b).

37. method as claimed in claim 31, wherein, described foodstuff products, drink product or dietary supplements are selected from by plant butter, spread, butter, milk preparation or contain the group that whey beverage constitutes, preferably, based on the product of sour milk or milk, for example sour milk or milk.

38. foodstuff products, drink product or the dietary supplements that can obtain by one or multinomial described method in the claim 31 to 37.

39. foodstuff products as claimed in claim 38, drink product or dietary supplements, it comprises 0.05 to 10wt%, and more preferably 0.1 to 5wt%, most preferably 0.2 to 4wt% the described composition that contains IPP.

40. as claim 38 or 39 described foodstuff productss, drink product or dietary supplements, it comprises the IPP of per relatively 100 gram products 0.05 to 50mg, more preferably 0.1 to 40mg, and most preferably 0.2 to 30mg.

41. as one or multinomial described foodstuff products, drink product or dietary supplements in the claim 38 to 40, wherein, IPP is 5: 1 to 100: 1 to the weight ratio of VPP, more preferably 5: 1 to 48: 1.

42. as one or multinomial described foodstuff products, drink product or dietary supplements in the claim 38 to 41, it comprises IPP and LPP, wherein, IPP is 1: 10 to 1: 1 to the weight ratio of LPP, more preferably 1.5: 7.1 to 4.8: 7.1.

43., be used to alleviate human hypertension as one or multinomial described foodstuff products, drink product or dietary supplements in the claim 38 to 42.

Images