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EP1480525A2 - Nouveaux produits a base de proteines de soja - Google Patents

Nouveaux produits a base de proteines de soja

Info

Publication number
EP1480525A2
EP1480525A2 EP03702932A EP03702932A EP1480525A2 EP 1480525 A2 EP1480525 A2 EP 1480525A2 EP 03702932 A EP03702932 A EP 03702932A EP 03702932 A EP03702932 A EP 03702932A EP 1480525 A2 EP1480525 A2 EP 1480525A2
Authority
EP
European Patent Office
Prior art keywords
soy protein
cholesterol
reducing
preventing
eliminating
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP03702932A
Other languages
German (de)
English (en)
Inventor
Lars Henrik Hoie
Karl Johan Nadland
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nutri Pharma ASA
Original Assignee
Nutri Pharma ASA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Nutri Pharma ASA filed Critical Nutri Pharma ASA
Priority to EP03702932A priority Critical patent/EP1480525A2/fr
Publication of EP1480525A2 publication Critical patent/EP1480525A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/168—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/14—Vegetable proteins
    • A23J3/16—Vegetable proteins from soybean
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/185—Vegetable proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae

Definitions

  • the present invention concerns novel soy protein products containing soy protein in a form which result in maximum cholesterol-lowering when ingested and also concerns methods for manufacturing said soy protein preparations. Furthermore, the present invention relates to soy protein products suitable for preventing, treating and/or alleviating cardiovascular diseases such as hypercholesterolemia, hypertriglyceridemia, hyperlipidemia, arteriosclerosis, hypertension and related cardiovascular diseases, for preventing and/or treating type 2 diabetes and/or the metabolic syndrome, and for preventing, treating and/or alleviating pulmonary diseases. The present invention also pertains to the use of such soy protein products in the prevention and/or treatment of a cardiovascular disease in a subject suffering from type 2 diabetes
  • a soy protein product according to the present invention is particularly useful in preventing and/or reducing the influx of triglycerides and/or cholesterol into the arterial wall and/or reducing the accumulation of cholesterol in the arterial wall of subjects at high risk of developing cardiovascular disease or subjects already suffering from a cardiovascular disease such as atherosclerosis or diabetic subjects.
  • a soy protein product according to the present invention is also useful for lowering serum levels of total cholesterol and/or LDL-cholesterol and/or triglycerides and/or homocystein and/or for increasing serum levels of HDL-cholesterol and/or for improving the serum HDL/LDL-ratio in subjects at risk for developing cardiovascular diseases and in subjects already suffering from an arteriosclerotic condition such as e.g.
  • a soy protein product according to the present invention is also useful in lowering serum levels of total cholesterol and/or LDL-cholesterol and/or triglycerides and/or glucose and/or increasing serum levels of HLDL-cholesterol in diabetic subjects.
  • a soy protein product according to the present invention is also useful in treating e.g. chronic obstructive pulmonary disease (COPD), inflammation of the airways, asthma, bronchoconstriction, bronchitis, and small airways disease.
  • COPD chronic obstructive pulmonary disease
  • the present invention relates to the use of these soy protein products as a medicament and/or in the manufacture of a medicament for treating a subject suffering from cardiovascular diseases, more particularly hypercholesterolemia, hypertriglyceridemia, hyperlipidemia, arteriosclerosis, hypertension and/or related cardiovascular diseases.
  • the present invention also relates to the use of these soy protein products as a medicament and/or in the manufacture of a medicament for treating type 2 diabetes and/or the metabolic syndrome and/or a cardiovascular disease in a subject suffering from type 2 diabetes.
  • the present invention also relates to the use of these soy protein products as a medicament and/or in the manufacture of a medicament for treating a subject suffering from a pulmonary disease, more particularly chronic obstructive pulmonary disease (COPD), inflammation of the airways, asthma, bronchoconstriction, bronchitis, and/or small airways disease.
  • COPD chronic obstructive pulmonary disease
  • the present invention also concerns use of a soy protein product according to the present invention in the prevention and/or treatment of said diseases and disorders and for lowering serum levels of total cholesterol and/or LDL-cholesterol and/or triglycerides and/or homocystein in subjects.
  • the present invention also provides methods for preventing, treating, prophylactically treating and/or alleviating by therapy said diseases and disorders.
  • soy protein Intake of soy protein, either alone or in combination with other soybean components, has been shown to have several beneficial effects on humans. These benefits include improvements in plasma lipid and lipoprotein concentrations (i.e. lower LDL cholesterol, lower triglycerides and possibly higher HDL cholesterol).
  • the beneficial effects of soy protein on plasma lipoprotein concentrations have been recognised in 1999, by the U.S. Food and Drug Administration's approval of a health claim that "25 g of soy protein a day, as part of a diet low in saturated fat and cholesterol, may reduce the risk of coronary heart disease".
  • the soy protein could be consumed by eating soybeans, but this has little appeal, as soybeans are conceived by some to have an objectionable flavour and furthermore are not part of a traditional diet in the Western world.
  • An alternative way of consuming soy protein is as part of a food product containing soy protein.
  • Soy protein is used as a food ingredient in the form of products such as soy flour, soy protein concentrate and isolated soy protein.
  • the beans are initially cracked, dehulled and flaked.
  • the material is also conditioned, e.g. by boiling them in water for 20-30 minutes to destroy most of the activity of the trypsin inhibitors.
  • the resulting flakes are then extracted with an inert solvent, such as a hydrocarbon solvent, typically hexane, in one of several types of counter-current extraction systems to remove the soybean oil.
  • the defatted flakes which are the starting material for most commercial protein ingredients, have a protein content of approximately 50%. Moisture content has typically been reduced to 3 to 5% during this process.
  • Any residual solvent may be removed by heat and vacuum, often by employing a deodorising vessel to enable live steam to contact the flakes to remove additional residual solvent.
  • Conditions in the deodorising vessel can be adjusted to produce flakes of varying protein solubility depending on the end-use requirement.
  • the soy protein isolates are typically produced by extracting the protein from defatted soybean flakes with water or a solution of water and ethanol at a pH of 6-11, at temperatures up to 80°C to 90°C.
  • the extracted soybean flakes enter a first centrifuge to separate the soluble fraction from the insoluble fibrous residue.
  • the resulting soluble fraction (containing extracted protein and oligosaccharides) is acidified to a pH of about 4,5 corresponding to the isoelectric point of the protein with a food-grade acid.
  • a second centrifuge is used to separate the whey (which includes the soluble oligosaccharides) from the protein curd that typically involve cooling to below room temperature.
  • the curd is then washed with water in a washing tank using any number of multiple washings.
  • the washed curd is commonly neutralised to a pH of about 7 with water and an alkali such as sodium or calcium hydroxide in a neutralisation tank to make the product pH neutral.
  • the protein curd is normally spray-dried in a dryer at about 150-170°C inlet air temperature and about 80-85°C outlet temperature, to yield the soy protein isolate or soy proteinate having a moisture content of about four (4) to six (6)% and a soluble protein content greater than about 90%.
  • the resulting products from the conventional manufacturing processes have physical properties and flavour characteristics, which render them useful as food additives.
  • a number of alternative manufacturing processes aimed at obtaining products with specific characteristics have been described. These are aimed at obtaining a product with certain specified characteristics such as good dispersion properties, low tendency to crystallise, a high content of phytoestrogens etc.
  • US5994508 relates to a process for providing an isoflavone rich protein isolate, along with the isoflavone rich protein isolate produced thereby.
  • a vegetable material containing protein and at least one isoflavone compound is extracted with an aqueous extractant having a neutral pH.
  • the pH of the extract is adjusted to about the isoelectric point of the protein, in order to precipitate the protein.
  • the protein is separated from the extract. The cool separation temperatures increase the concentration of isoflavones recovered in the protein, while the neutral pH inhibits loss of protein normally observed at cool or cold separation temperatures.
  • US6013771 relates to a process for providing an isoflavone rich protein isolate, along with the isoflavone rich protein isolate produced thereby.
  • a vegetable material containing protein and at least one isoflavone compound is extracted with an aqueous extractant having a pH above the isoelectric point of the protein, and preferably an alkaline pH.
  • the pH of the extract is adjusted to about the isoelectric point of the protein in order to precipitate the protein.
  • the protein is separated from the extract. Washing of the separated protein is avoided, or is conducted with minimum amounts of water. The cool separation temperatures and the low wash conditions significantly increase the concentrations of isoflavones recovered in the protein.
  • US6140469 relates to the production of an isoflavone enriched vegetable protein isolate in which the weight ratio of material to extractant is controlled and washing of the acid precipitated protein curd is avoided or minimised to provide an increased level of isoflavones in the protein isolate.
  • soy proteins have thus far not been concerned with what effect the process might have on the proteins with regard to their cholesterol lowering effect. This is probably due to the fact that soy proteins are believed to be relatively heat-stable. Furthermore, the cholesterol-lowering effect attributed to the proteins themselves or part thereof has been considered as being independent on the conditions by which soy protein products were manufactured.
  • US6005076 which relates to processes for the preparation and purification of proteinaceous materials from oil seeds and protein meals in a substantially non- denatured form.
  • a process is provided in which a protein isolate is obtained from fat- contaminated oil seed material by carrying out an aqueous salt extraction to solubilise protein and fat and subsequently removing the fat by applying a chilling process. Protein is retrieved as micelles upon decreasing the ionic strength of the solution. The resulting protein is described as having improved functional properties.
  • US5674548 which relates to soybean milk having a high transparency and a soybean protein material having excellent oil retention and minimised mouthfeel change and also relates to soybean protein providing transparent solutions and a strong and transparent gel.
  • the process involves an aqueous protein extraction step in which the temperature is kept below 40°C.
  • the proteins obtained are characterised in that 85% of the protein is able to permeate a 0,22 ⁇ membrane and by optical density at 600 nm.
  • US4737014 relates to a process for isolation of proteins from soybeans with high yield and in a substantially undenatured form.
  • the process involves an aqueous extraction step in which the pH is adjusted to from about 4.8 to about 5.4 prior to dilution.
  • the protein obtained is substantially undenatured as opposed to proteins obtained by conventional isoelectric precipitation and the yield is better than described in the state of the art.
  • US5936069 provides a method for producing an improved soy protein concentrate having low raffinose, stachyose; and fiber content, as well as minimal lipoxygenase activity and/or good flavour.
  • the improved soy protein concentrate is high in naturally occurring vitamins, minerals and isoflavones, and may also be high in phytoestrogens.
  • the resulting soy protein concentrate has a protein content of about 60% to about 80% of available protein. Further, the improved soy protein concentrate contains less than 0.3% fibre. Also, since it is not necessary to use any alcohol or acid to produce the improved soy protein concentrate, the final product has not been denatured and is highly functional.
  • the final product is high in water-soluble minerals, proteins and isoflavones, and may generally be higher in phytoestrogens.
  • soy proteins can lose their ability to lower blood cholesterol and to promote other health benefits when ingested, if excessive heat or other denaturing conditions are applied in the manufacturing process of the soy protein product and/or in subsequent processes where the soy protein is for example incorporated into food products.
  • soy protein products containing soy protein in a form which retain the cholesterol-lowering effect and also to provide a process for the manufacture of said protein products.
  • the present invention provides soy protein products which retain the ability to lower blood cholesterol levels upon ingestion, characterised by having more than a further specified minimum content of intact 7S ( ⁇ + ⁇ ' + ⁇ ) and/or 11S subunits (A + B), a minimum 7S enthalpy peak or a minimum solubility.
  • the present invention further provides a method for manufacturing soy protein products which retain the ability to lower blood cholesterol upon ingestion, characterised by having more than a further specified minimum content of intact 7S ( ⁇ + ⁇ ' + ⁇ ) and/or 11S subunits (A + B), a minimum 7S enthalpy peak or a minimum solubility.
  • Said method is further characterised by process conditions which render the 7S and 11S subunits intact.
  • soy proteins with characteristics such as minimum content of intact 7S ( ⁇ + ⁇ ' + ⁇ ) and/or 11S subunits (A + B), ), a minimum 7S enthalpy peak or a minimum solubility facilitate the formation of breakdown products in the digestive tract, in the form of peptides which elicit an effect in the form of cholesterol lowering.
  • the present invention also provides the use of soy protein products which retain the ability to lower blood cholesterol levels upon ingestion, characterised by minimum content of intact 7S ( ⁇ + ⁇ ' + ⁇ ) and/or 11S subunits (A + B), a minimum 7S enthalpy peak or a minimum solubility as a food ingredient, and also provides for the use of the soy protein products which retain the ability to lower blood cholesterol levels upon ingestion to obtain a health benefit.
  • the globulins are the major part of the soy proteins. These can be divided in 7 S and 11 S globulins. The ratio between 7S and 11 S can vary with the cultivars from 0.5-2.0.
  • the 7 S is a glycoprotein.
  • the main component is ⁇ -Conglycinin, which is a trimer, molecular mass of 150-200 kDa.
  • 11S is a glycosylated protein, glycinin. It is a hexamer with ba globular mass of 300- 380 kDa. Each sub-unit (out of six)is composed of a acidic polypeptide (A), 35 kDa and a basic polypeptide (B), 20 kDa. The two polypeptides are linked together by a disulfide bond.
  • hypocholesterolemic effect is directly correlated to the patient's cholesterolemia.
  • Minimal effect or little reductions occur at cholesterol levels of 6 mmol/l or less and with most benefits with cholesterol of greater than 7 mmol/l ( Sirtori, Current Atherosclerotis Reports 2001, 3: 47-53 and J. Anderson, meta-analysis 1995.
  • glycosides corresponds to (specified as mg isofl arms, glycoside forms per g soy protein) 0.2, 1.8, 2.5 and 4.2.
  • the results were as average: 2.5 %, 2.5 %, 4.2 % and 6 % reduction in LDL to placebo.
  • the figures were: 3.8 %, 4.5 %, 8.3 % and 9.8 %.
  • the reduction was not significant.
  • the unfolding of the proteins is accompanied by enthalpic changes and can easily be followed by differential scanning calorimetry (DSC), which is the most used technique.
  • DSC differential scanning calorimetry
  • the protein aggregation can be followed by gel electrophoresis (SDS-PAGE).
  • SDS-PAGE gel electrophoresis
  • the denaturation will adversely affect the solubility of the protiens and this may be measured by standard techniques.
  • the effect of heat treatment on structure, through denaturation, aggregation and effect on the properties of the ISP are studied (Petrocceili and An ⁇ n, J. Agric. Food Chem. 1991 , 39: 1386- 1391; 1991, 39: 1029-1032; 1994, 42: 2161-2169; 1995, 43: 3035-3041).
  • the denaturation temperature for both 7S and 11S is influenced by thermal energy, pH, water concentration and to some degree also ionic strength.
  • the denaturation temperatures increase a lot with reduced water content in the suspension of the soy proteins. High and low pH will also reduce the denaturation temperature.
  • the 7S protein is the most vulnerable and will normally start denaturation at around 70 °C for 10 % dispersion in water, but approx. 130 °C at a moisture content of around 30 %. At 80 °C it will take only a few seconds before all 7S will be completely denatured and the structure unfolded (J. Renkema & al, J. of Biotechnology 2000, 79:223-230; S.Petruccelli and M. Afion, J. Agric. Food Chem., 1996, 44: 3005-3009; A. Morales and J.L Kokini, J. Rheol. 1999, 43: 315-325).
  • the hydrophobic part is towards the inside of the disc, where the isoflators are linked to this hydrophobic part of the amino acids by hydrogen bonds. Even in denatured 7S part of the soy protein, the hydrophobic part is still protected inside the protein molecules, still linked to the isoflators. Used in this form and status, it is possible that the enzymes in the gastric and intestinal juices will not brake up the polypeptides which are linked to the some part of the 7S part of the proteins.
  • the present invention for the first time offers a solution as to how high amounts of soy protein which has not been processed in a way which deanture the soy proteins, can be incorporated in food products without resulting in objectionable flavour or odour.
  • processed soy protein product as used throughout the present specification and the appended claims shall be taken to mean any product containing soy protein obtained by processing of soy beans.
  • intact subunit as used throughout the present specification and the appended claims shall be taken to mean a protein subunit which has the molecular weight of the full-length protein subunit as well as a protein subunit which is associated with other subunits by e.g. disulfide bridges or non-covalent bonding.
  • the content of such "intact subunits” can for example be performed by analysis by SDS-PAGE under reducing conditions.
  • 7S subunits and 11 S subunits as used throughout the present specification and the appended claims shall be taken to mean the subunits which constitute the globulins in legume plants. These are in soy beans also referred to as ⁇ -conglycinin which is a trimer of ⁇ , ⁇ ' and ⁇ and glycinin which is a hexamer comprising A and B subunits.
  • the term "ability to lower blood cholesterol" as used throughout the present specification and the appended claims shall be taken to mean being effective in lowering serum levels of total cholesterol and/or LDL-cholesterol and/or triglycerides and/or in increasing the serum HDL/LDL-cholesterol ratio and/or increasing serum levels of high-density lipoproteins (HDL) and/or in generating a decrease in serum levels of low-density lipoproteins (LDL). It is desirable to achieve an elevated serum HDL LDL-cholesterol ratio since this may result in an increased reverse cholesterol transport and a subsequent excretion.
  • the present invention provides soy protein products which retain the ability to lower blood cholesterol levels upon ingestion, characterised by a minimum content of intact 7S ( ⁇ + ⁇ ' + ⁇ ) and/or 11S subunits (A + B), a minimum 7S enthalpy peak or a minimum solubility.
  • the soy protein products more specifically are believed to facilitate the formation of peptides which has a cholesterol lowering effect.
  • the present invention provides a method for manufacturing a soy protein product which retain the ability to lower blood cholesterol levels upon ingestion and characterised by a minimum content of intact 7S ( ⁇ + ⁇ ' + ⁇ ) and/or 11S subunits
  • the present invention provides a soy protein product prepared by a method according to the present invention.
  • the present invention provides for the use of a soy protein product according to the invention as a food ingredient.
  • the present invention provides for the use of a soy protein product according to the invention to obtain a health benefit, such as cholesterol lowering.
  • the preferred starting material for the process of the invention is soy beans. Normally the soybeans are heated in almost boiling water after de-hulling to reduce the activity of Trypsin inhibitors (TI), Bowman-Birk inhibitor and Kunitz Trypsin inhibitor. This heating is typically carried out for only a very short time, preferably less than 15 minutes.
  • TI Trypsin inhibitors
  • Bowman-Birk inhibitor and Kunitz Trypsin inhibitor. This heating is typically carried out for only a very short time, preferably less than 15 minutes.
  • the level of active Trypsin inhibitors in the final soy protein product is less than 20 % of the original activity in the beans, such as e.g. less than 19%, eg. less than 18%, such as less than 17%, eg. less than 16%, such as less than 15%, eg. less than 14%, such as less than 13%, eg. less than 12%, such as less than 11%, eg. less than 10%, such as less than 9%, eg. less than 8%, such as less than 7%, eg. less than 6%, such as less than 5%.
  • this preferred level of trypsin inhibitors is in a particularly preferrd embodiment, reached by selecting processing conditions which allow for the separation of TI from the globular proteins. It is thus possible to obtain soy protein products with low levels of TI without applying heating, chemical means or separation using e.g. membranes. If such measures are applied it may be in the form of e.g. the NADP/thioredoxin system (from Escherichia coli or wheat germ) (e.g. as described by Jin-an Jiao et al, J. Agric.Food Chem, 40:2333-2336) or by combining the initial heat treatment with small quantities of Sodium Sulphite (Na 2 SO 3 ). This treatment will break the S-S bonds in the globular protein molecules and thus open the protein structure.
  • NADP/thioredoxin system from Escherichia coli or wheat germ
  • Na 2 SO 3 Sodium Sulphite
  • soy beans also soy flakes or flour may be used as a starting material for the process according to the invention.
  • a presently preferred starting material for the process of the invention is soy flakes or flour from which the oil has been removed by solvent or mechanical extraction, and which may be produced from soybeans according to conventional processes.
  • soy flour starting material although other soy protein and vegetable protein containing starting materials e.g. flakes may also be used.
  • the general process combines several precipitation steps under conditions which do not denature the globular 11S and 7S proteins.
  • the process allows for two important objectives to be met. Firstly, it allows for removal of TI from the globular soy proteins without applying traditional methods that might denature the globular proteins and complicate the process. Secondly, the process allows for a partial fractionation of the globular proteins.
  • the first precipitate obtained at modestly acidic pH is thus enriched in 11S globular proteins
  • the precipitate obtained at more acidic pH is enriched in the 7S globular proteins and thus also in 7S ⁇ '. It is thus possible to obtain soy protein products with very high amounts of specific globular proteins.
  • Further processing of a fraction from the acidic precipitation which is enriched in 7S ⁇ ', by for example size fractionation to remove molecular species of small size, would therefore provide a convenient method to obtain soy protein comprising very high amounts of the 7S ⁇ ' subunit. Given the current focus on the effects of the 7S ⁇ ' subunit soy protein products with increased amounts of this subunit may prove to be of particular interest.
  • a cultivar with a natural relation between 7 and 11s of up to 2:1 can be selected and/or further separation steps may be applied.
  • Membrane filtration technology could for example be used to concentrate the 7 S fraction without denaturing the globular structure further.
  • An example of such a technology is "crossflow" membrane filtration as the liquid is flowing tangential to the membrane surface and thus inhibits formation of deposits.
  • globular proteins in the form of ISP obtained by the method according to the invention This could for example be a product produced as described in example 4 or a fraction precipitated out at pH 3.5 and partly or fully neutralized afterwards (pH 3.5-8).
  • the proteins in these two ISPs can be separated according to molecular size in three fractions:
  • the separation can be carried out in two steps:
  • Step 1 Removal of molecules with a molecular weight below 100 kDa and collection of a product fraction with the higher molecular weights (concentrate).
  • Step 2 Removal of the 11S fraction by applying a membrane with a cut-off at approximately 250 kDa.
  • the product fraction, which is the permeate is enriched in 7S proteins.
  • the equipment is typically tubular with a housing of stainless steel.
  • the tubes are equipped with the suitable membrane for the separation.
  • the first step can be an ultrafiltration step with an operating pressure below 10 bars.
  • the second step can be a microfiltration with an operating pressure below 3 bars. (Two different membranes is used in the two membrane filtration steps).
  • the ISP As the separation has to be performed on a molecular level, the ISP must be completely dissolved in the water phase.
  • the separation is normally carried out at at pH 6.5-7 and the ISP is dissolved in water in less than 4 % w/v at from 5-25°C, preferably at from 15 to 20 °C.
  • the permeate, in step 2 above, must be concentrated before spray drying. This is preferably done by reverse osmosis to take out water and concentrate the protein up to a level 20-40 % dry matter.
  • the soy material preferably soy flour
  • an aqueous solution preferably water
  • a concentration of less than 40 % w/v such as less than 30% w/v, e.g. less than 25% w/v, such as less than 20% w/v, e.g. less than 15% w/v.
  • a combination of sodium and calcium chloride is dissolved in enough water to make a maximum 0.1 molar solution in order to increase the denaturation temperatures of both 7S and 11S part of the soy protein.
  • a first extraction is then performed using gentle mixing for at least part of the extraction period.
  • the pH is adjusted up to 6,0- 9.5, preferably 6,5-9.0, more preferably 7-8,5 and most preferably around 8,0 by e.g. addition of KOH or NaOH and the temperature is adjusted to a maximum of 68 °C, preferably lower than 66°C, e.g. lower than 64°C, preferably lower than 62°C, e.g. lower than 60°C preferably lower than 58°C, e.g. lower than 56°C preferably lower than 54°C, e.g. lower than 52°C preferably lower than 50°C, e.g.
  • lower than 48°C preferably lower than 46°C, e.g. lower than 44°C preferably lower than 42°C, e.g. lower than 40°C preferably lower than 38°C, e.g. lower than 36°C, preferably lower than 34°C, e.g. lower than 32°C, preferably lower than 30°C, e.g. lower than 28°C, preferably lower than 26°C, e.g. lower than 24°C, preferably lower than 22°C, e.g. lower than 20°C, preferably lower than 21 °C, e.g. lower than 20°C, preferably lower than 19°C, e.g. lower than 18°C, preferably lower than 17°C, e.g.
  • lower than 16°C preferably lower than 15°C, e.g. lower than 14°C, preferably lower than 13°C, e.g. lower than 12°C, preferably lower than 11°C, e.g. lower than 10°C, preferably lower than 9°C, e.g. lower than 8°C, preferably lower than 7°C, e.g. lower than 6°C preferably lower than 5°C, e.g. lower than 4°C.
  • the extraction time should be restricted to a maximum of 180 minutes, e.g. be less than 150 minutes, such as 120 minutes, e.g. be less than 90 minutes, such as 75 minutes, e.g. be less than 60 minutes, . such as 45 minutes, e.g. be less than 44 minutes, such as 43 minutes, e.g. be less than 42 minutes, such as 41 minutes, e.g. be less than 40 minutes, such as 39 minutes, e.g. be less than 38 minutes, such as 37 minutes, e.g. be less than 36 minutes, such as 35 minutes, e.g. be less than 34 minutes, such as 33 minutes, e.g. be less than 32 minutes, such as 31 minutes, e.g.
  • ⁇ minutes such as 29 minutes, e.g. be less than 28 minutes, such as 27 minutes, e.g. be less than 26 minutes, such as 25 minutes, e.g. be less than 24 minutes, such as 23 minutes, e.g. be less than 22 minutes, such as 21 minutes, e.g. be less than 20 minutes, such as 19 minutes, e.g. be less than 18 minutes, such as 17 minutes, e.g. be less than 16 minutes, such as 15 minutes, e.g. be less than 14 minutes, such as 13 minutes, e.g. be less than 12 minutes, such as 11 minutes, e.g. be less than 10 minutes, such as 9 minutes, e.g. be less than 8 minutes, such as 7 minutes, e.g. be less than 6 minutes, such as 5 minutes.
  • the un-dissolved part of the soy bean material is according to a process of the present invention separated from the extract by decantation, filtering or centrifugation or a combination of these.
  • a second extraction from the un-dissolved material may be carried out in an analogous manner to produce a second extract.
  • These two extracts may be further processed in parallel or processed together.
  • the pH from the extract(s) is reduced to 4.8-5.5 and kept at a temperature below 25°C as short as possible and less than 3.5 hours.
  • the pH of the solution is reduced to 4.9-5.5, e.g. 5.1-5.5, such as 5.3-5.5, for example 5.4.
  • the solution is cooled to a temperature below 20°C, such as below 15 C C, e.g. below 10°C, such as between 3-8°C.
  • the solution is kept at the conditions specified for 0-3 hours, such as 1-2 hours, e.g. 1 ,5 hours.
  • sedimentation of the material may be obtained by e.g. centrifugation and the obtained ISP fractions (ISP1 and 2) may then be dried as described below.
  • the pH of the solution is then further reduced to 2,5-4,5 and kept at a temperature of below 10°C for less than 2 hours.
  • the pH of the solution is reduced to 3-4 for example 3.5.
  • the solution is kept at a temperature below 8°C, such as below 7°C, e.g. below 6°C, such as below 5°C, for example 4°C.
  • sedimentation of the material may be obtained by e.g. centrifugation and the obtained ISP fractions (ISP 3 and 4) may then be dried as described below.
  • the pH of the ISP fractions are optionally increased to 5-8 prior to drying.
  • this drying is done by freeze or spray drying or any other conventional drying method such as for example vacuum drying, drum drying, air drying in a zigzag drier, on continous belt driers, cabinet tray driers or rotational driers.
  • this drying is done by spray drying at an air temperature below 120 °C, such as below 115 °C, e.g. below 110°C, such as below 105 °C, e.g. below 100°C, such as below 95 °C, e.g. below 90°C, such as below 85 °C, e.g. below 80°C, such as below 75 °C, e.g. below 70°C, such as below 65 °C, e.g. below 60°C.
  • the product temperature does not exceed 60 °C.
  • the above parameters are set to ensure that the soy protein structure is only denatured to a minimal extent i.e. meaning that preferably at least 50%, such as at least 52%, preferably at least 54%, such as at least 56%, preferably at least 58%, such as at least 60%, preferably at least 62%, such as at least 64%, preferably at least 66%, such as at least 68%, preferably at least 70%, such as at least 72%, preferably at least 74%, such as at least 76%, preferably at least 78%, such as at least 80%, preferably at least 82%, such as at least 84%, preferably at least 86%, such as at least 88%, preferably at least 90%, such as at least 92%, preferably at least 94%, such as at least 96%, preferably at least 98%, such as at least 99%, of the 7S and 11S subunits are preserved in their undenatured form, thereby referring either to the individual subunits of 7S and 11S alone or all subunits of
  • the globular structure is mainly folded with the hydrophobic part of the molecules inside the globular structure (a disk with room for all fat soluble substances inside the molecule including the isofl arms, which are linked to the hydrophobic side of the proteins by hydrogen bonds).
  • the soy protein products comprise more than 50% protein of dry matter, such as at least 55%, e.g. at least 60%, e.g at least 65%, e.g. at least 70%, such as 75%, e.g. at least 80%, such as at least 85%, e.g. at least 90%, such as at least 95% protein of dry matter.
  • the amount of intact 7S subunits ( ⁇ + ⁇ ' + ⁇ ) and 11S subunits (A + B) in soy protein products according to the invention preferably constitute more than 5 % of the total soy protein content, such as more than 10 %, for example more than 15 %, , such as more than 20 %, for example more than 25 %, such as more than 30 %, for example more than 31 %, such as more than 32 %, for example more than 33 %, such as more than 34 %, for example more than 35 %, such as more than 36 %, for example more than 37 %, such as more than 38 %, for example more than 39 %, such as more than 40 %, for example more than 41 %, such as more than 42 %, for example more than 43 %, such as more than 44 %, for example more than 45 %, such as more than 46 %, for example more than 47 %, such as more than 48 %, for example more than 49 %, such as more than 50
  • the amount of intact 7S subunits ( ⁇ + ⁇ ' + ⁇ ) in soy protein products according to the invention preferably constitute more than 1 % of the total soy protein content, such as more than 2 %, for example more than 3 %, such asmore than 4 %, for example more than 5 %, such as more than 6 %, for example more than 7 %, such as more than 8 %, for example more than 9 %, such as more than 10 %, for example more than 11 %, such as more than 12 %, for example more than 13 %, such as more than 14 %, for example more than 15 %, such as more than 16 %, for example more than 17 %, such as more than 18 %, for example more than 19 %, such as more than 20 %, for example more than 21 %, such as more than 22 %, for example more than 23 %, such as more than 24 %, for example more than 25 %, such as more than 26 %, for example more than 27 %, such as
  • the amount of intact 7S subunit ⁇ in soy protein products according to the invention preferably constitute more than 1 % of the total soy protein content, such as more than 2 %, for example more than 3 %, such as more than 4 %, for example more than 5 %, such as more than 6 %, for example more than 7 %, such as more than 8 %, for example more than 9 %, such as more than 10 %, for example more than 11 %, such as more than 12 %, for example more than 13 %, such as more than 14 %, for example more than 15 %, such as more than 16 %, for example more than 17 %, such as more than 18 %, for example more than 19 %, such as more than 20 %, for example more than 21 %, such as more than 22 %, for example more than 23 %, such as more than 24 %, for example more than 25 %, such as more than 26 %, for example more than 27 %, such as more than 28 %, for example more
  • the amount of intact 7S subunit ⁇ ' in soy protein products according to the invention preferably constitute more than 1 % of the total soy protein content, such as more than 2 %, for example more than 3 %, such asmore than 4 %, for example more than 5 %, such as more than 6 %, for example more than 7 %, such as more than 8 %, for example more than 9 %, such as more than 10 %, for example more than 11 %, such as more than 12 %, for example more than 13 %, such as more than 14 %, for example more than 15 %, such as more than 16 %, for example more than 17 %, such as more than 18 %, for example more than 19 %, such as more than 20 %, for example more than 21 %, such as more than 22 %, for example more than 23 %, such as more than 24 %, for example more than 25 %, such as more than 26 %, for example more than 27 %, such as more than 28 %, for example
  • the 7S enthalpy peak in a soy protein product to be incorporated in a food product according to the invention preferably constitute at least 0.1 J/g protein, such as 0.2, e.g. 0.3, such as 0.4, e.g. 0.5, such as 0.6, e.g. 0.7, such as 0.8, e.g. 0.9, such as 1.0, e.g. 1.1, such as 1.2, e.g. 1.3, such as 1.4, e.g. 1.5, such as 1.6, e.g. 1.7, such as 1.8, e.g. 1.9, such as 2.0 e.g. 2.2, such as 2.4, e.g. 2.6, such as 2.8, e.g.
  • 3.0 such as 3.5, e.g. 4.0, such as 4.5, e.g. 5.0, such as 6.0, e.g. 7.0, such as 8.0, e.g. 9.0, such as 10.0, e.g. 11.0, such as at least 12.0 J/g protein.
  • the soluble proteins preferably constitute more than 55 %, such as more than 60%, e.g. more than 65%, such as more than 70%, e.g. more than 75%, such as more than 80%, e.g. more than 82%, such as more than 84%, e.g. more than 86%, such as more than 88%, e.g. more than 90%, such as more than 92%, e.g. more than 94%, such as more than 95%, e.g. more than 96%, e.g more than 97%, such as more than 98%, e.g. more than 99%.
  • Phytoestrogen compounds according to the present invention are defined as naturally occurring plant substances, said substances being either structurally or functionally similar to 17-estradiol or generating estrogenic effects.
  • Phytoestrogens consist of a number of classes including isoflavones, coumestans, lignans and resorcylic acid lactones.
  • isoflavones according to the present invention are genistein, daidzein, equol, glycitein, biochanin A, formononetin, and O-desmethylangolesin.
  • the phytoestrogen compounds of a soy protein product according to the present invention are preferably isoflavones, more preferably genistein, daidzein, glycitein and/or equol, yet more preferably genistein and/or daidzein, and even more preferably genistein.
  • a preferred soy protein product according to the present invention may accordingly comprise a single isoflavone, such as genistein, daidzein, glycitein or equol, or it may comprise at least one isoflavone selected from the group comprising at least genistein, daidzein, glycitein and equol.
  • the isoflavones When present in the plant the isoflavones are mainly in a glucoside form, i.e. attached to a sugar molecule. This glucoside form can be deconjugated to yield a so-called aglycone form, which is the biologically active species.
  • a soy protein product according to the present invention may comprise isoflavones in glucoside and/or aglycone forms regardless of whether the deconjugation to the aglycone form has taken place biologically, in vitro or by any other means whereby the isoflavones are included in a soy protein product according to the present invention or if the aglycone forms are the native form of the isoflavones.
  • a soy protein product according to the invention may optionally also contain phytoestrogens, such as isoflators.
  • the phytoestrogen compound is preferably present in an amount, referring to the aglycone forms, of at least about 0.10 weight percent of the soy protein content, such as at least about 0.11 weight percent, for example at least about 0.12 weight percent of the soy protein content, such as at least about 0.14 weight percent, for example at least about 0.16 weight percent, such as at least about 0.18 weight percent, for example at least about 0.20 weight percent, such as at least about 0.22 weight percent, for example at least about 0.24 weight percent, such as at least about 0.25 weight percent, for example more than about 0.25 weight percent, such as at least about 0.26 weight percent, for example at least about 0.28 weight percent, such as at least about 0.30 weight percent, for example at least about 0.32 weight percent, such as at least about 0.33 weight percent, for example more than about 0.33 weight percent, such as at least about 0.35 weight percent, for example at least about 0.40 weight
  • a soy protein product according to the invention may optionally have the trypsin inhibitors partly or fully destroyed or removed.
  • the amount of ACTIVE trypsin inhibitor in a soy protein product according to the invention may preferably be less than 50% of the amount in the original soy bean, such as less than 40%, for example less than 30%, such as less than 25%, for example less than 20%, such as less than 15%, for example less than 10%, such as less than 5 %, for example less than 1%.
  • soy protein having a high arginine to lysine ratio effects serum lipid levels and alleviates symptoms of cardiovascular diseases to a greater extent than soy protein having a lower or normal arginine to lysine ratio. Consequently, isolated, potentially processed, soy protein having a high arginine to lysine ratio is a particularly preferred soy protein product according to the present invention.
  • the soy protein product according to the present invention should have an arginine to lysine ratio of at least about 1.0, such as at least about 1.1 , for example at least about 1.2, such as at least about 1.3, for example at least about 1.4, such as at least about 1.5, for example at least about 1.6, such as at least about 1.7, for example at least about 1.8, such as at least about 1.9, for example more than about 2, such as at least about 2.1, for example at least about 2.2, such as at least about 2.5, for example at least about 2.75, such as at least about 3, for example more than about 3.3, such as at least about 3.6, for example at least about 4, such as at least about 4.5, for example at least about 5, such as at least about 6, for example at least about 7, such as at least about 8, for example at least about 9, such as at least about 10, for example at least about 11, such as at least about 12, for example at least about 13, such as at least about 14.
  • arginine to lysine ratio of at least about 1.0,
  • the present invention also provides the use of such a soy protein product as a medicament and/or in the manufacture of a medicament effective in preventing, treating, prophylactically treating, alleviating and/or eliminating a cardiovascular disease in a subject.
  • the present invention also provides the use of such a soy protein product as a medicament and/or in the manufacture of a medicament effective in preventing, treating, prophylactically treating, alleviating and/or eliminating arteriosclerosis or a related cardiovascular disease in a subject.
  • the present invention also provides the use of such a soy protein product as a medicament and/or in the manufacture of a medicament for treating diabetic subjects, said treatment being effective in lowering serum levels of glucose and/or insulin and/or lipids.
  • the present invention also provides the use of such a soy protein product as a medicament and/or in the manufacture of a medicament effective in treating and/or alleviating type 2 diabetes, the metabolic syndrome as defined herein and/or cardiovascular diseases associated therewith in a subject.
  • the present invention also provides the use of such a soy protein product as a medicament and/or in the manufacture of a medicament effective in treating subjects diagnosed as having a pulmonary disease, said treatment being effective at least in increasing FEV1 of a subject, as measured by forced expiratory volume in the first second of expiration.
  • the present invention also provides the use of such a soy protein product as a medicament and/or in the manufacture of a medicament effective in treating and/or alleviating pulmonary diseases in a subject.
  • the present invention also provides the use of such a soy protein product and/or such a soy protein product for use in treating arteriosclerosis or a related cardiovascular disease in a subject.
  • the present invention also provides the use of such a soy protein product and/or such a soy protein product for use in the treatment of diabetic subjects, said treatment being particularly effective in lowering serum levels of glucose and lipids in a subject.
  • the present invention also provides the use of such a soy protein product and/or such a soy protein product for use in the treatment and/or alleviation of a pulmonary disease in a subject, said treatment and/or alleviation resulting in an increased FEV1 of a subject, as measured by forced expiratory volume in the first second of expiration.
  • a soy protein product according to the present invention may optionally comprise a carbohydrate source, flavoring agents, vitamins, minerals, electrolytes, trace elements and other conventional additives.
  • the nutritional soy protein product according to the present invention may in one embodiment also comprise one or more flavoring agents such as cocoa, vanilla, lime, strawberry or soup flavors, such as mushroom, tomato or bouillon and/or sweeteners such as aspartame as well as other additives such as xanthan gum.
  • a carbohydrate source When a carbohydrate source is present in a soy protein product according to the present invention, it is preferably present in an amount of less than 30 weight percent such as less than 25 weight percent of the soy protein product. Preferably, the amount of carbohydrate amounts to at least 5 weight percent, more preferred at least 10 weight percent, and most preferred at least 15 weight percent, of the soy protein product.
  • the preferred carbohydrates for use in a soy protein product according to the present invention are dextrose, fructose and/or maltodextrin, or glucose. Skimmed milk and lecithinated fat reduced cacao are other possible carbohydrate sources.
  • a soy protein product according to the present invention is for use in the prevention and/or treatment of type 2 diabetes
  • the metabolic syndrome and associated cardiovascular diseases lecithinated fat reduced cacao
  • Other preferred carbohydrates for use in a soy protein product according to the present invention for use in the prevention and/or treatment of type 2 diabetes, the metabolic syndrome and associated cardiovascular diseases are polydextrose or saccharose, but these should be limited using other sweeteners like e.g. aspartame.
  • Vitamins and minerals may optionally be added to a soy protein product according to the present invention in accordance with the limits laid down by health authorities.
  • a soy protein product according to the present invention may comprise all recommended vitamins and minerals.
  • the vitamins will typically include A, B1 , B2, B12, folic acid, niacin, panthotenic acid, biotin, C, D, E and K.
  • the minerals will typically include iron, zinc, iodine, copper, manganese, chromium and selenium. Electrolytes, such as sodium, potassium and chlorides, trace elements and other conventional additives may also be added in recommended amounts.
  • a soy protein product according to the present invention may be used for special dietary use, preferably for lowering serum levels of total cholesterol and/or LDL- cholesterol and/or triglycerides in subjects such as hyperlipidemic patients or normocholesterolemic patients suffering from a cardiovascular disease, and/or for lowering serum levels of glucose and/or insulin and/or total cholesterol and/or LDL- cholesterol and/or triglycerides and/or for increasing glucose tolerance and/or insulin sensitivity and/or for preventing, treating and/or alleviating impaired glucose tolerance and/or insulin secretory failure in diabetic subjects and/or for preventing, treating and/or alleviating an arteriosclerotic condition by reducing the influx of lipoproteins and/or cholesterol and/or triglycerides into the endocelium of the arterial wall of a diabetic subject suffering from a cardiovascular disease.
  • soy protein product for example, from one to three daily meals of ordinary food can be supplemented or replaced by a soy protein product according to the present invention.
  • the soy protein product may provide from about 50 to about 250 kcal per serving.
  • the daily dose of a soy protein product according to the present invention may comprise an energy content of from 400 to 800, in particular from 450 to 800 kcal/day, which is considered to be a very low calorie diet (VLCD), or it may comprise an energy content of from 800 to 1200 kcal/day, which is considered to be a low-calorie diet (LCD).
  • the energy content may correspond to the daily energy requirement of a normal person.
  • the present invention also provides a soy protein product according to the invention in the form of a micronutrient.
  • a micronutrient is a nutritional supplement and/or a pharmacological soy protein product and/or a medicament comprising i) a synthetic phytoestrogen-like compound capable of binding to an estrogen receptor or an estrogen-like receptor, and/or ii) a naturally occurring, plant- extractable compound in an amount, on a weight per weight basis, in excess of the amount of said compound, when it is present in a natural host such as a plant cell from which the compound can be extracted or isolated, iii) soy peptides obtainable from a partial hydrolysis of soy protein and iv) soy lecithin.
  • the naturally occurring, plant-extractable compound is preferably but not limited to compounds capable of binding to an estrogen receptor, an estrogen-like receptor, a beta-2-adrenergic receptor or a receptor belonging to the class of beta-2-adrenergic receptors.
  • the naturally occurring compounds are isolated from plants such as soybeans, they may be selected from the group at least containing phytoestrogens such as soybean phytoestrogens such as soybean isoflavones, soy protein or fragments thereof, e.g.
  • peptides or amino acid sequences soybean fibers, lecithin, linolenic acid, an antioxidant, a saponin, a lignan, a protease inhibitor, a trypsin inhibitor, and a tyrosine kinase inhibitor.
  • Additional constituents of the micronutrient may preferably be selected among a DNA topoisomerase inhibitor, a ribosome kinase inhibitor, a growth control factor such as e.g. epidermal growth factor, transforming growth factor alpha, platelet derived growth factor, and preferably any growth control factor controllable by a tyrosine kinase activity.
  • the micronutrient may also comprise ormeloxifene and/or levormeloxifene as described by among others Holm et al. (1997) in Arteriosclerosis, Thrombosis, and Vascular Biology 17 (10), 2264 - 2272, and in Clinical Investigation, 100 (4), 821 - 828.
  • the isoflavone may have been deconjugated to the aglycone form either biologically or in vitro prior to the incorporation in the micronutrient.
  • the present invention provides a soy protein product or a micronutrient according to the present invention in combination with a functional food ingredient comprising a sterol, preferably an ingredient selected from the group comprising a stanol ester, a tocotrienol, a mevinolin, and a phytosterol compound such as e.g. campesterol, sitosterol or stigmasterol, or a combination thereof.
  • a functional food ingredient comprising a sterol, preferably an ingredient selected from the group comprising a stanol ester, a tocotrienol, a mevinolin, and a phytosterol compound such as e.g. campesterol, sitosterol or stigmasterol, or a combination thereof.
  • a soy protein product or a micronutrient according to the present invention is for use as a functional food ingredient.
  • a soy protein product or a micronutrient according to the present invention may also be administered as a probe or by intravenous administration, or in tablet or capsule form.
  • the present invention also provides a pharmaceutical preparation comprising the a soy protein product or a micronutrient according to the present invention, use of the a soy protein product or a micronutrient according to the present invention in therapy and/or a diagnostic method performed on the human or animal body, use of a soy protein product or a micronutrient according to the present invention in the manufacture of a medicament, use of a soy protein product or a micronutrient according to the present invention in the manufacture of a medicament for treating a subject suffering from cardiovascular diseases, use of a soy protein product or a micronutrient according to the present invention in the manufacture of a medicament for treating a subject suffering from type 2 diabetes, the metabolic syndrome and/or cardiovascular diseases associated therewith in a diabetic subject and use of a soy protein product or a micronutrient according to the present invention in the manufacture of a medicament for treating a subject suffering from pulmonary diseases.
  • the micronutrient is particularly useful in preventing, treating, prophylactically treating and/or alleviating hypercholesterolemia, hypertriglyceridemia, other hyperlipidemias, arteriosclerosis, atherosclerosis and/or related cardiovascular diseases and in preventing and/or treating type 2 diabetes, the metabolic syndrome and/or cardiovascular diseases associated therewith in a diabetic subject and in preventing, treating, prophylactically treating and/or alleviating a pulmonary disease such as e.g. a disease selected from the group comprising inflammation of the airways, bronchoconstriction, bronchitis, asthma, and small airways diseases.
  • the present invention provides a soy protein product according to the present invention for use as a medicament or as a dietary preparation.
  • a soy protein product according to the present invention for use as a medicament or as a dietary preparation may preferably be used in preventing, treating, prophylactically treating and/or alleviating cardiovascular diseases such as e.g. a disease selected from the group comprising hypercholesterolemia, hypertriglyceridemia, other hyperlipidemias, arteriosclerosis, atherosclerosis, arteriolosclerosis, coronary heart disease, angina pectoris, thrombosis, myocardial infarction, and hypertension, in a subject, preferably for use in preventing, treating, prophylactically treating and/or alleviating arteriosclerosis and/or atherosclerosis in a subject .
  • cardiovascular diseases such as e.g. a disease selected from the group comprising hypercholesterolemia, hypertriglyceridemia, other hyperlipidemias, arteriosclerosis, atherosclerosis, arteriolosclerosis, coronary heart disease, angina pectoris, thrombosis, myocardial infarction, and hypertension, in
  • a soy protein product according to the present invention for use as a medicament or as a dietary preparation may also preferably be used in preventing, treating, alleviating and/or eliminating type 2 diabetes.
  • a soy protein product according to the present invention for use as a medicament or as a dietary preparation may also preferably be used in preventing, treating, alleviating and/or eliminating a cardiovascular disease, such as e.g. hypercholesterolemia, hypertriglyceridemia, hypertension, hyperglycemia, hyperinsulinemia, arteriosclerosis, atherosclerosis, arteriolosclerosis, coronary heart disease, angina pectoris, thrombosis, and myocardial infarction, in a diabetic subject.
  • a cardiovascular disease such as e.g. hypercholesterolemia, hypertriglyceridemia, hypertension, hyperglycemia, hyperinsulinemia, arteriosclerosis, atherosclerosis, arteriolosclerosis, coronary heart disease, angina pectoris, thrombosis, and myocardial
  • a soy protein product according to the present invention for use as a medicament or as a dietary preparation may also preferably be used for preventing and/or treating pulmonary diseases, such as preferably a disease selected from the group comprising inflammation of the airways, bronchoconstriction, bronchitis, asthma, and small airways diseases, in a subject.
  • pulmonary diseases such as preferably a disease selected from the group comprising inflammation of the airways, bronchoconstriction, bronchitis, asthma, and small airways diseases, in a subject.
  • the present invention also provides the use of a soy protein product according to the present invention as a medicament and/or in the manufacture of a medicament for preventing, treating, prophylactically treating and/or alleviating cardiovascular diseases such as e.g. a disease selected from the group comprising hypercholesterolemia, hypertriglyceridemia, other hyperlipidemias, arteriosclerosis, atherosclerosis, arteriolosclerosis, coronary heart disease, angina pectoris, thrombosis, myocardial infarction, and hypertension, particularly a disease selected from the group comprising arteriosclerosis and atherosclerosis, in a subject.
  • a disease selected from the group comprising hypercholesterolemia, hypertriglyceridemia, other hyperlipidemias, arteriosclerosis, atherosclerosis, arteriolosclerosis, coronary heart disease, angina pectoris, thrombosis, myocardial infarction, and hypertension particularly a disease selected from the group comprising arteriosclerosis and atherosclerosis, in a subject
  • the present invention also provides the use of a soy protein product according to the present invention as a medicament and/or in the manufacture of a medicament for preventing, treating and/or alleviating type 2 diabetes and/or the metabolic syndrome in a subject and/or a cardiovascular disease in a diabetic subject.
  • the present invention also provides the use of a soy protein product according to the present invention as a medicament and/or in the manufacture of a medicament for preventing, treating, prophylactically treating and/or alleviating pulmonary diseases such as e.g. a disease selected from the group comprising inflammation of the airways, bronchoconstriction, bronchitis, asthma, and small airways diseases, in a subject.
  • the soy protein product according to the present invention is effective in lowering levels of cholesterol in normocholesterolemic patients by at least 2%, for example at least 5%, such as at least 8%, for example at least 10%, such as at least 12%, for example at least 14%, such as at least 16%, for example at least 18%, such as at least 20%, for example at least 25%, such as at least 30%.
  • the soy protein product according to the present invention is effective in lowering levels of triglycerides in normocholesterolemic patients by at least 10%, such as at least 12%, for example at least 14%, such as at least 16%, for example at least 18%, such as at least 20%, for example at least 25%, such as at least 30%.
  • the soy protein product according to the present invention is effective in lowering levels of cholesterol in mildly hypercholesterolemic patients by at least 3%, for example at least 5%, such as at least 8%, for example at least 10%, such as at least 12%, for example at least 15%, such as at least 20%, for example at least 25%, such as at least 30%, for example at least 35%, such as at least 40%, for example at least 45%.
  • the soy protein product according to the present invention is effective in lowering levels of triglycerides in mildly hypercholesterolemic patients by at least 15%, such as at least 20%, for example at least 25%, such as at least 30%, for example at least 35%, such as at least 40%, for example at least 45%.
  • the soy protein product according to the present invention is effective in lowering levels of cholesterol in severely hypercholesterolemic patients by at least 3%, for example at least 5%, such as at least 8%, for example at least 10%, such as at least 12%, for example at least 15%, such as at least 20%, for example at least 25%, such as at least 30%, for example at least 35%, such as at least 40%, for example at least 45%, such as at least 50%, for example at least 55%, such as at least 60%.
  • the soy protein product according to the present invention is effective in lowering levels of triglycerides in severely hypercholesterolemic patients by at least 20%, for example at least 25%, such as at least 30%, for example at least 35%, such as at least 40%, for example at least 45%, such as at least 50%, for example at least 55%, such as at least 60%.
  • a soy protein product according to the present invention for use as a medicament and/or the use of a soy protein product according to the present invention as a medicament and/or in the manufacture of a medicament for preventing, treating, prophylactically treating and/or alleviating cardiovascular diseases in a subject may be effective in reducing the influx of cholesterol and/or triglycerides into the arterial wall and/or causing dilation of blood vessels and/or reducing the amount of oxidized LDL- cholesterol present in the arterial wall and/or lowering serum levels of total cholesterol and/or LDL-cholesterol and/or homocystein and/or triglycerides and/or increasing the serum HDL LDL-cholesterol ratio and/or increasing serum levels of HDL-cholesterol in a subject and/or preventing, reducing or eliminating fatty streak formation and/or preventing, reducing or eliminating fibrous plaque formation and/or preventing, reducing or eliminating complicated lesion formation and/or reducing or eliminating the risk of a subject contracting angina pectoris and/
  • a soy protein product according to the present invention for use as a medicament and/or the use of a soy protein product according to the present invention as a medicament and/or in the manufacture of a medicament for preventing and/or treating diabetes and/or the metabolic syndrome and/or a cardiovascular disease associated therewith in a subject may be effective in i) lowering serum glucose levels and/or ii) reducing the influx of cholesterol and/or triglycerides into the arterial wall and/or causing dilation of blood vessels and/or reducing the amount of oxidized LDL- cholesterol present in the arterial wall and/or iii) lowering serum levels of total cholesterol and/or LDL-cholesterol and/or triglycerides and/or homocystein and/or increasing the serum HDL LDL-cholesterol ratio and/or serum HDL-cholesterol levels and/or iv) increasing glucose tolerance and/or insulin sensitivity and/or v) alleviating impaired glucose tolerance and/or insulin secretory failure and/or improving insulin secretion and
  • hypercholesterolemia hypertriglyceridemia, other hyperlipidemias, arteriosclerosis, atherosclerosis, arteriolosclerosis, coronary heart disease, angina pectoris, thrombosis, myocardial infarction, hypertension, hyperglycemia, and hyperinsulinemia, in a diabetic subject and/or preventing, reducing or eliminating fatty streak formation and/or preventing, reducing or eliminating fibrous plaque formation and/or preventing, reducing or eliminating complicated lesion formation and/or reducing or eliminating the risk of a diabetic subject contracting angina pectoris and/or reducing or eliminating the risk of a diabetic subject contracting a myocardial infarction and/or in treating a procoagulant state and/or an increased activity of clotting factors, insulin resistance, glycosidation and/or oxidation and/or chemical modification of lipoproteins, as well as impaired glucose tolerance.
  • a soy protein product according to the present invention for use as a medicament and/or the use of a soy protein product according to the present invention as a medicament and/or in the manufacture of a medicament for preventing, treating, prophylactically treating and/or alleviating pulmonary diseases may be effective in i) preventing, treating, prophylactically treating and/or alleviating asthma and/or ii) reducing and/or eliminating mucus hypersecretion and/or dyspnea in a subject suffering from asthma and/or iii) increasing FEV1 of a subject as measured by forced expiratory volume in the first second of expiration and/or iv) preventing, treating, prophylactically treating, alleviating and/or reducing inflammation of the airways and/or v) preventing, treating, prophylactically treating and/or alleviating bronchoconstriction.
  • the present invention provides the use of a soy protein product according to the present invention in the treatment of cardiovascular diseases in the human or animal body in an amount effective in lowering serum levels of total cholesterol and/or LDL-cholesterol and/or triglycerides and/or homocystein and/or increasing the serum HDL LDL-cholesterol ratio and/or serum HDL-cholesterol levels and/or reducing the influx of cholesterol and/or triglycerides into the arterial wall and/or reducing the amount of oxidized LDL-cholesterol present in the arterial wall and/or preventing, reducing or eliminating fatty streak formation and/or preventing, reducing or eliminating fibrous plaque formation and/or preventing, reducing or eliminating complicated lesion formation and/or reducing or eliminating the risk of a subject contracting angina pectoris and/or reducing or eliminating the risk of a subject contracting a myocardial infarction, and/or alleviating the clinical condition of patients contracting a myocardial infection.
  • the cardiovascular disease is preferably a cardiovascular disease selected from the group comprising hypercholesterolemia, hypertriglyceridemia, other hyperlipidemias, arteriosclerosis, atherosclerosis, arteriolosclerosis, coronary heart disease, angina pectoris, thrombosis, myocardial infarction, and hypertension and more preferred selected from arteriosclerosis and atherosclerosis.
  • the present invention provides the use of a soy protein product according to the present invention in the treatment of type 2 diabetes and/or the metabolic syndrome in an amount effective in lowering serum levels of total cholesterol and/or LDL-cholesterol and/or triglycerides and/or glucose and/or increasing serum levels of HLDL-cholesterol and/or homocystein and/or reducing the influx of cholesterol and/or triglycerides into the arterial wall and/or reducing the amount of oxidized LDL-cholesterol present in the arterial wall and/or improving glucose tolerance and/or increasing insulin sensitivity and/or alleviating impaired glucose tolerance and/or improving insulin secretion and/or reducing or eliminating fatty streak formation and/or preventing, reducing or eliminating fibrous plaque formation and/or preventing, reducing or eliminating complicated lesion formation and/or preventing, reducing or eliminating the risk of a subject contracting angina pectoris and/or preventing, reducing or eliminating the risk of a subject contracting a myocardial infarction and/or
  • the present invention provides the use of a soy protein product according to the present invention in the treatment of a pulmonary disease in a human or animal body, preferably a disease selected from the group comprising inflammation of the airways, bronchoconstriction, bronchitis, asthma, and small airways diseases, in an amount effective in preventing, treating, prophylactically treating and/or alleviating inflammation of the airways and/or bronchoconstriction and/or bronchitis and/or small airways diseases and/or asthma and/or reducing and/or eliminating mucus hypersecretion and/or dyspnea in a subject suffering from asthma and/or increasing FEV1 of a subject as measured by forced expiratory volume in the first second of expiration.
  • a soy protein product according to the present invention in the treatment of a pulmonary disease in a human or animal body, preferably a disease selected from the group comprising inflammation of the airways, bronchoconstriction, bronchitis, asthma, and small airways diseases, in an amount
  • the present invention also provides a method of preventing, treating, prophylactically treating and/or alleviating by therapy a cardiovascular disease in the human or animal body such as an arteriosclerotic condition of a human or animal body, said method comprising administration of a soy protein product according to the present invention in an amount effective in lowering serum levels of total cholesterol and/or LDL- cholesterol and/or triglycerides and/or homocystein and/or increasing the serum HDL LDL-cholesterol ratio and/or serum HDL-cholesterol levels and/or reducing the influx of cholesterol and/or triglycerides into the arterial wall and/or reducing the amount of oxidized LDL-cholesterol present in the arterial wall and/or preventing, reducing or eliminating fatty streak formation and/or preventing, reducing or eliminating fibrous plaque formation and/or preventing, reducing or eliminating complicated lesion formation and/or reducing or eliminating the risk of a subject contracting angina pectoris and/or reducing or eliminating the risk of a subject contracting
  • the cardiovascular disease is preferably a cardiovascular disease selected from the group comprising hypercholesterolemia, hypertriglyceridemia, other hyperlipidemias, arteriosclerosis, atherosclerosis, arteriolosclerosis, coronary heart disease, angina pectoris, thrombosis, myocardial infarction, and hypertension and more preferred selected from arteriosclerosis and atherosclerosis.
  • the present invention also provides a method of preventing and/or treating by therapy type 2 diabetes and/or the metabolic syndrome in a human or animal body, said method comprising administration to said human or animal body of a soy protein product according to the present invention in an amount effective in lowering serum levels of total cholesterol and/or LDL-cholesterol and/or triglycerides and/or glucose and/or increasing serum levels of HLDL-cholesterol and/or homocystein and/or reducing the influx of cholesterol and/or triglycerides into the arterial wall and/or reducing the amount of oxidized LDL-cholesterol present in the arterial wall and/or improving glucose tolerance and/or increasing insulin sensitivity and/or alleviating impaired glucose tolerance and/or improving insulin secretion and/or reducing or eliminating fatty streak formation and/or preventing, reducing or eliminating fibrous plaque formation and/or preventing, reducing or eliminating complicated lesion formation and/or preventing, reducing or eliminating the risk of a subject contracting angina pectoris and/or preventing,
  • the present invention also provides a method of preventing, treating, prophylactically treating and/or alleviating by therapy a pulmonary disease in a human or animal body, preferably a disease selected from the group comprising inflammation of the airways, bronchoconstriction, bronchitis, asthma, and small airways diseases, said method comprising administration to said human or animal body of a soy protein product according to the present invention in an amount effective in preventing, treating, prophylactically treating and/or alleviating, inflammation of the airways and/or bronchoconstriction and/or bronchitis and/or asthma and/or small airways diseases and/or reducing and/or eliminating mucus hypersecretion and/or dyspnea in a subject suffering from asthma and/or increasing FEV1 of a subject as measured by forced expiratory volume in the first second of expiration.
  • a pulmonary disease in a human or animal body preferably a disease selected from the group comprising inflammation of the airways, bronchoconstriction, bronchitis
  • the period of treatment is preferably in the range of from 1 to 12 months or more, such as from 2 weeks to 9 months, for example from 3 weeks to 6 months, such as from 4 weeks to 4 months, such as from 6 weeks to 3 months.
  • the period of treatment shall not be limited to these periods and may e.g. be longer than 12 months, such as e.g. a lifelong treatment in order to prevent cardiovascular diseases or in order to prevent and/or alleviate type 2 diabetes and/or a cardiovascular disease in connection therewith or in order to prevent pulmonary diseases.
  • the present invention provides a pharmaceutical preparation comprising a soy protein product according to the present invention.
  • the pharmaceutical preparation can be prepared in any way known to the skilled person.
  • the present invention provides the use of a soy protein product according to the present invention as a nutritional preparation and/or in the manufacture of a nutritional preparation for lowering serum levels of glucose and/or total cholesterol and/or LDL-cholesterol and/or triglycerides and/or homocystein and/or increasing the serum HDL LDL-cholesterol ratio and/or serum levels of HDL- cholesterol in a subject, including a diabetic subject, and/or for alleviating a pulmonary condition such as e.g. asthma.
  • the nutritional preparation may take any form, which is suitable for human or animal consumption.
  • the soy protein product is a powdery mixture, which is suspendable, dispersible or emulsifiable in a liquid for human or animal consumption.
  • the liquid is preferably a water- containing liquid such as e.g. water, coffee, tea or juice, including fruit juice.
  • the soy protein product may be packed in a package intended for covering part of or the total nutritional requirement for a defined period of time, such as a period of e.g. three days or a week.
  • the present invention also provides the nutritional preparation in the form of a dietary supplement.
  • the nutritional preparation in one embodiment of the present invention is preferably a functional food or drink, i.e. a readily obtainable edible or drinkable substance that is supplemented with a soy protein product according to the present invention to provide a medical or pharmaceutical effect. Accordingly, the present invention provides a soy protein product according to the present invention for use as a functional food ingredient.
  • Functional foods and drinks are preferably selected from the group comprising dairy products, such as yoghurt and yoghurt ice cream, juice, such as orange juice or tomato juice, ready made liquids for drinking, a spreadable product such as e.g. a margarine or a vegetable or plant extracted oil, a cereal product, such as a traditional breakfast cereal product, nutritional bars, biscuits, bread, soups, such as tomato soup, a meat product, such as a hamburger, a meat substitute product, and a vegetable product.
  • dairy products such as yoghurt and yoghurt ice cream
  • juice such as orange juice or tomato juice
  • a spreadable product such as e.g. a margarine or a vegetable or plant extracted oil
  • a cereal product such as a traditional breakfast cereal product, nutritional bars, biscuits, bread
  • soups such as tomato soup
  • a meat product such as a hamburger, a meat substitute product, and a vegetable product.
  • a nutritional preparation according to the present invention may be in the form of a ready made liquid or in a powder form or in the form of a troche, a solid soy protein product such as a nutritional bar, a fruit bar, a cookie, a cake, a bread or a muffin.
  • a soy protein product according to the present invention is a liquid nutritional preparation in a water-containing liquid, in which the solid ingredients are suspended, dispersed or emulgated in an amount of from 10 to 25 weight percent.
  • the liquid nutritional preparation When the liquid nutritional preparation is intended for drinking, it will usually comprise a flavoring agent as discussed above. However, the liquid nutritional preparation may also be used for probe administration.
  • the present invention relates to the use of a soy protein product according to the present invention as a partial or total diet for an overweight subject, an overweight subject suffering from an arteriosclerotic condition or an overweight subject suffering from a diabetic condition.
  • Obesity is believed to be one of the major causes of diabetes including type 2 diabetes.
  • Overweight subjects, including overweight diabetic subjects often have increased serum cholesterol levels and increased triglyceride levels and are therefore more likely to develop cardiovascular diseases.
  • the present invention is not limited to treating subjects with an increased risk of contracting a cardiovascular disease, i.e. subjects likely to have increased serum levels of cholesterol and/or triglycerides, or to treating obese diabetic subjects with an increased risk of contracting a cardiovascular disease, i.e.
  • a soy protein product according to the present invention also has substantial serum cholesterol, serum LDL-cholesterol and serum triglyceride lowering effects in subjects having a more normal lipid profile and in diabetic subjects that do not also suffer from overweight.
  • the medical use of a soy protein product according to the present invention is not limited to overweight or obese subjects, including diabetic subjects, but may be used for normal weight subjects having increased serum levels of cholesterol and/or LDL-cholesterol and/or triglycerides or for subjects with a cardiovascular condition such as e.g. arteriosclerosis or a related condition who have normal serum levels of cholesterol and/or LDL-cholesterol and/or triglycerides.
  • Such increased serum levels of cholesterol and/or LDL-cholesterol and/or triglycerides may be caused by intake of a diet rich in fats or it may be genetically related.
  • subjects having an initial total serum cholesterol level of 5.7 mmol/l or below are considered to have a normal or hypocholesterolemic level, whereas subjects having a total serum cholesterol level above 5.7 mmol/l are considered to be hypercholesterolemic. Accordingly, by treating normocholesterolemic subjects, it is possible to prevent the development of cardiovascular diseases arising from serum cholesterol levels below a concentration of 5.7 mmol/l in subjects, including diabetic subjects, particularly sensitive to developing e.g. arteriosclerosis, or prevent further development of cardiovascular diseases in patients, including diabetic patients, with previous cardiovascular events.
  • cardiovascular diseases arising from serum cholesterol levels above a concentration of 5.7 mmol/l in subjects sensitive to developing e.g. arteriosclerosis under such conditions.
  • subjects having a total serum cholesterol level of from 5.7 mmol/l to 7.9 mmol/l are considered to be mildly hypercholesterolemic. Accordingly, by treating these hypercholesterolemic subjects, it is possible to prevent the development of cardiovascular diseases arising from serum cholesterol levels of from 5.7 to 7.9 mmol/l. Subjects having a total serum cholesterol level of more than 7.9 mmol/l are considered to be severely hypercholesterolemic. Accordingly, by treating these hypercholesterolemic subjects, it is possible to prevent the development of cardiovascular diseases arising from serum cholesterol levels of more than 7.9 mmol/l. It has also been shown that a soy protein product according to this invention has a potentiating effect to the effect of medications such as e.g.
  • statins and/or niacin By combining a soy protein product according to the present invention with e.g. statins, such as HMG-CoA-reductase-inhibitors, niacin, bile acid resins, fibrates, nicotinic acid derivatives, oat products, such as oat meal, rye products, such as rye meal and various fish oil concentrates with a high content of D-3-fatty acids, it is possible to achieve a further 5 to 15% reduction in serum levels of total cholesterol and/or LDL- cholesterol and/or triglycerides.
  • statins such as HMG-CoA-reductase-inhibitors, niacin, bile acid resins, fibrates, nicotinic acid derivatives, oat products, such as oat meal, rye products, such as rye meal and various fish oil concentrates with a high content of D-3-fatty acids, it is possible to achieve a further 5
  • the present invention also provides a soy protein product according to the present invention in combination with a statin, preferably an HMG-CoA-reductase-inhibitor, niacin, bile acid resins, fibrates, oat products, rye products, nicotinic acid derivatives and various fish oil concentrates with a high content of omega-3-fatty acids.
  • a statin preferably an HMG-CoA-reductase-inhibitor, niacin, bile acid resins, fibrates, oat products, rye products, nicotinic acid derivatives and various fish oil concentrates with a high content of omega-3-fatty acids.
  • Beans were dehulled and ground. Coarse powder was extracted three times with petroleum spirit (1:4 ratio). Ground with solid carbon dioxide to pass through a 0.5mm sieve. The flour was suspended in a 100mM Tris-HCI buffer of pH 8.0 in a 1:10 ratio (w/v) and stirred for 1 h at room temperature. Centrifuged (30 min: 9000Xg;10°C) and the supernatant was brought to pH 4.8 with 2 M HCI to induce precipitation. After 2 h at 4°C the dispersion was centrifuged (30 min; 9000Xg: 10°C). The precipitate obtained was washed twice with 10 mM sodium acetate buffer at pH 4.8 in a 1 :8 ratio (W/v) and freeze-dried.
  • the soy beans preferably in the form of flakes will be added to water in an amount less than 15 % w/v.
  • An amount of a combination of sodium and calcium chloride is dissolved in the water in order to make a solution of no more than 0.1 molar. This will increase the denaturation temperatures of both 7S and 11S part of the soy protein.
  • the extraction will be carried out with gentle mixing.
  • the pH is adjusted up to 5.5-7.5 and the temperature should be maximum 68 C C, preferable lower.
  • the time will be restricted to maximum 45 minutes.
  • the un-dissolved material from the beans will be removed by filtration or by centrifugation.
  • the pH is reduced to 4.8-5.5 and the solution is slowly cooled to 5-10 °C during a 1.5 -2 hours period.
  • the solute is removed from the curd. Carbohydrates will be removed by washing once with water.
  • the curd can now be dried carefully by different drying technologies e.g. spray drying/ air temperature below 120 °C.
  • the end temperature of the product should not exceed 60 °C regardless of which drying method is applied.
  • the Flow-sheet for Procedure 3 is shown in figure 1.
  • the washed residue called Fibre, pure isolates, IS1& IS2 and Tl-rich isolate IS3& IS4 and soluble solids SS1& SS2.
  • the samples were examined by chemical analysis for protein, fat and fibre, Trypsin Inhibitor (TI) analysis, Differential Scanning Calorimetry (DSC) and Polyacrylamide electrophoresis (PAGE).
  • TI Trypsin Inhibitor
  • DSC Differential Scanning Calorimetry
  • PAGE Polyacrylamide electrophoresis
  • the precipitated protein fractions IS 1-3 contained 60-65% of the total protein from the flour.
  • TIU trypsin inhibitor units TlU/mg of sample 1.9 TIU equals 1 mg of trypsin inhibited.
  • procedure 3 gave a recovery of 60-62% of the protein in fractions IS 1-3 with only 13% of the original TI. In comparison, gave procedures 1 and 2 higher values of respectively 29% and 25% TI in the IS 1-3 fractions. It is therefore clear that procedure 3 offers the best separation of the globular soy proteins from TI.
  • the DSC measures the heat of denaturation in proteins as they are heated over a regime from 25 to 180°C.
  • the DSC results showed that the isolate formed at pH 5.4 were recovered from soya flour without causing any further denaturation to the proteins. It was furthermore found that the 7 and 11S proteins were partially fractionated by the precipitations, with a relatively higher yield of 11S at pH 5.4 and of 7S at pH 3.5.
  • the 7 fractions from each process were run at an equivalent protein level on a separate gel with standards for molecular size. All the tracks on the gels were stained with a blue dye and also analysed by densitometry. This technique is very sensitive and reveal traces of 7 and 11 S globulins that can not be measured by DSC. The 11S bands were stronger in the IS1. and IS2 fractions and the 7S in the IS3 fraction, in agreement with the DSC findings.
  • soy flour and soy isolate FM are plotted on the same graph with the same scale for the peaks.
  • the difference in baseline due to sample mass has been reduced by shifting the flour graph downwards by 7.0 units.
  • Figure 2 Comparison of DSC graphs of soya flour and isolate FM on same protein basis, showing the 7S (75-85°C) and 11S (95-105°C) enthalpy peaks.
  • the curves are without peaks, indicating that there is not any un-denatured globular protein structure left in the products.
  • OD is the optical density reading of the scanning device used to "read” the gels.
  • Table 8 Selected values from Table 13 for comparison on total isoflavon level
  • the isolate was run at 3 concentrations of material added to the gel. In general terms the O.D. values are linear from 0.01 to 0.8 and it is unwise to use higher values. The most diluted sample gave the best separation of the peaks and the densitogram from this was consequently used for further analysis. This was for the isolate obtained by mixing 0.025ml of the extract with 0.375ml of Laemmli buffer.
  • Table 10 Composition of the basic components in 7S and 11S of the ISP
  • Potassium hydroxide (56.1g/mol.) was purchased from BDH and tap water was used to make the slurry.
  • a foam depressing reagent, antifoam FDP was obtained from Basildon Chemical Company Ltd., Kimber Road Abingdon Oxon., OX14 1RZ.
  • a paddle mixer was used to make the flour/water slurry and for mixing during pH adjustment.
  • a Silverson mixer (without shearing element) model D Silverson Machine Ltd, Waterside, Chesham, Bucks, HP5 1 PQ was used to mix the isolates during pH adjustment.
  • APV 454 litres capacity steam jacketed kettles were used as holding tanks.
  • a NORD, model SK 20R150U90L/433, CIP lobe pump was used to feed slurry to the separator.
  • Freeze drying was chosen as a suitable method in this case. To avoid microbiological growth in all produced ISP in the established pilot process was frozen on trays immediately after production. Instead of having to defrost all material again with increased microbiological risk, we decided to use freeze drying in this particular case. Freeze drying is also a rather gentle drying method.
  • the Verum trial substance (Abacor, reg.trade mark, see table below) or the placebo trial substance (see table below) was stirred into long-life chocolate milk (defatted milk, fat content 0.2%).
  • the resulting chocolate milk preparations contain 25 g of soy protein respectively casein per liter.
  • the treatment at very high temperature was performed as follows: The chocolate milk preparations were preheated to 80°C by a plate heat exchanger for approximately 1-2 minutes. The preheated preparations were then treated at very high temperature by direct steam injection to 142°C and kept at this temperature for 4-8 seconds. Following cooling by vacuum to approximately 80°C the preparations were homogenized at 30 bar and subsequently at 150 bar for a maximum of 30 seconds followed by cooling to approximately 20°C over 60-90 seconds.
  • the profile shows distinct bands for the 7S and 11S globular proteins.
  • the trial subjects each drank one liter (dosage group 1), or half a liter (dosage group 2) of a preparation according to EXAMPLE 9, corresponding to 25 g and 12.5 g of soy protein respectively.
  • a placebo group was included in which each subject ingested a corresponding amount of the placebo substance according to EXAMPLE 9.
  • Blood samples were taken from the trial subjects at the on-set of the experiment (week 0), at 2 weeks and at 4 weeks (end of the experiment), and total serum cholesterol, LDL cholesterol and HDL cholesterol determined.
  • the first and third examination comparisons are interpreted by means of the t-test for paired observation and description on the level of a 5% probability level.
  • the data were recorded according to GCP requirements with double data entry and simultaneous value comparison in dBase IV. Study design, performance and documentation of the study was according to the guidelines "good clinical practice" CPMP/ICH/135/95. The primary objective was the reduction of total cholesterol in serum - secondary objectives were LDL and HDL cholesterol.
  • the 20 trial subjects each drank one liter of a preparation according to EXAMPLE 10 (25 g of soy protein) daily, for a period of 4 weeks, half a liter with the morning meal and half a liter with the evening meal.
  • Drop-outs During the study the following drop outs occurred: Group A: 8 cases, group B: 10 cases and group C: 11 cases, from those only partial results are available. Therefore the analysis was done for the per protocol group, in addition a intention-to- treat analysis was performed.
  • the new ISP formulation of Nutri Pharma has proven to be significantly more effective to reduce total cholesterol than SuproSoy ISP, LDL cholesterol was significantly lowered with Nutri Pharma ' s ISP but not for SuproSoy, in 91 patients with hypercholesterolemia in 8 weeks in a randomized placebo-controlled trial.
  • the percentage improvement of total cholesterol of Nutri Pharmas ISP was 9.8% compared to SuproSoy ISP with 5.4%, an improvement of over 80%.
  • LDL cholesterol was 8.2% for Nutri Pharmas ISP compared to 3.9% for SuproSoy, an improvement of over 100%.
  • Protein solubility 2g of isolate was dissolved in 100ml water. The pH was in all cases adjusted to 7.0. Samples were mixed for 1 hour. For total protein samples were collected and for soluble protein, centrifuged at 32,000 rpm, or 18,000 rpm in another centrifuge in the Chemistry Department. The supernatant was used for soluble proteins and solubility was determined as: Dissolved protein x 100% / total protein. The method is described in detail by Renkema, J.M.S in J. Biochemistry 79,(3) 223- 230, (1976).
  • Samples for DSC were prepared by weighing x mg of solids and adding 4x mg of water to give the equivalent of 5mg of protein in the DSC pans, and allowed to equilibrate for 60 minutes after mixing the two with a spatula.
  • the total amount of wet sample used in each test is given in the table below.
  • the samples were scanned in the temperature range of 25 to 150°C at a scanning rate of 10°C per minute.
  • Portions of the samples (50 mg) were extracted with aqueous ethanol at 60°C for 1h with shaking.
  • Phytoestrogen glycone extracts were defatted with hexane and then hydrolysed to the aglycons with dilute HCL.
  • the hydrolysed aglycons were extracted with ether.
  • Analysis of aglycons was performed by liquid chromatography with isotope dilution mass spectrometric detection (LC-MS). All data are reagent blank and spike recovery corrected.
  • Peak(OD) Optical Density or intensity/height of a peak
  • Band% % area of that peak/band in relation to total area of all the peaks/bands detected within a single lane. I have averaged the duplicate values of the corresponding lanes MW(kd): Molecular weight obtained by using a calibration with standards of known values
  • Rf Retardation Factor, which means the relative mobility of a peak/band within a lane.
  • the top of the gel lane is 0, and the bottom of the lane is
  • each of the Isolate or flour components have Rf values telling the mapped position within a lane, and a calculated MW in the next column. Both these parameters help you to identify the peak band and compare them to the corresponding components within the other lanes.
  • the gels were documented as tif images using a video camera documentation system.
  • the densitometric analysis was performed with the Phoretix 1 D Advanced software.
  • a 14-step Optical Density Calibration strip was included alongside each gel image, to convert pixel readings of band intensities to optical density, and to correct for variations in the image capture process.
  • a background subtraction step is included within the densitometric analysis and molecular weights of protein bands are determined using a calibration with standard protein markers run on each gel. Over-loading distorts the migration of protein bands and lowers the apparent MW value of the protein. Peak height is expressed in Optical Density (OD) units, and Lane % is the area of a particular band relative to the total area of all the bands in that lane, i.e. a percentage of the total protein fraction.
  • OD Optical Density

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Abstract

L'invention concerne des nouveaux produits à base de protéines de soja contenant des protéines de soja sous une forme entraînant une baisse maximale du taux de cholestérol lorsqu'elles sont ingérées. L'invention concerne également des procédés permettant de produire ces préparations à base de protéines de soja. L'invention concerne en outre des produits à base de protéines de soja permettant de prévenir de traiter et/ou d'atténuer les maladies cardio-vasculaires telles que l'hypercholestérolémie, l'hyperlipémie, l'artériosclérose, l'hypertension et les maladies cardio-vasculaires apparentées, de prévenir et/ou de traiter le diabète de type 2 et/ou le syndrome métabolique, et de prévenir, de traiter et/ou d'atténuer les maladies pulmonaires. L'invention concerne par ailleurs l'utilisation de produits à base de protéines de soja pour la prévention et/ou le traitement d'une maladie cardio-vasculaire chez un sujet souffrant du diabète de type 2.
EP03702932A 2002-02-23 2003-02-24 Nouveaux produits a base de proteines de soja Withdrawn EP1480525A2 (fr)

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