PatentDe  


Dokumentenidentifikation EP1469741 23.11.2006
EP-Veröffentlichungsnummer 0001469741
Titel VERFAHREN ZUR EXTRAKTION, REINIGUNG UND ENZYMATISCHEN VERÄNDERUNG VON SOJA 7S GLOBULIN ALPHA' UNTEREINHEIT ZUR VERWENDUNG ALS HYPOCHOLESTEROL-MISCHER-WIRKSTOFF
Anmelder Indena S.p.A., Mailand/Milano, IT
Erfinder DURANTI, Marcello, I-20131 Milano, IT;
MORAZZONI, Paolo, I-20139 Milano, IT
Vertreter derzeit kein Vertreter bestellt
DE-Aktenzeichen 60308994
Vertragsstaaten AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HU, IE, IT, LI, LU, MC, NL, PT, SE, SI, SK, TR
Sprache des Dokument EN
EP-Anmeldetag 27.01.2003
EP-Aktenzeichen 037063872
WO-Anmeldetag 27.01.2003
PCT-Aktenzeichen PCT/EP03/00798
WO-Veröffentlichungsnummer 2003063608
WO-Veröffentlichungsdatum 07.08.2003
EP-Offenlegungsdatum 27.10.2004
EP date of grant 11.10.2006
Veröffentlichungstag im Patentblatt 23.11.2006
IPC-Hauptklasse A23J 3/16(2006.01)A, F, I, 20051017, B, H, EP
IPC-Nebenklasse A23J 1/14(2006.01)A, L, I, 20051017, B, H, EP   A23L 1/305(2006.01)A, L, I, 20051017, B, H, EP   

Beschreibung[en]

The present invention relates to a process for the extraction, purification and enzymatic modification of &bgr;-conglycinin &agr;' subunit.

According to the invention, &bgr;-conglycinin is selectively extracted from ground, defatted soy, then precipitated by treatment with aqueous ethanol; the enriched fraction is then subjected to Metal Affinity Chromatography (MAC) in denaturant conditions to obtain the &agr;' subunit. The latter is treated with chymotrypsin, then subjected to a further affinity chromatography step to recover the amino-terminal region of this polypeptide (MW 28,000 Da).

TECHNICAL BACKGROUND

The known cholesterol lowering properties of soy and derivatives thereof are related with the content in isoflavones (Kirk et al., 1998) and in proteins (Anderson et. al, 1995).

Soy proteins mainly consist of glycinins (11S fraction) and &bgr;-conglycinins (7S fraction), the latter consisting of three subunits, named &agr;, &agr;' and &bgr; (Thanh and Shibasaki, 1976). Studies carried out on soy proteins have established that the 7S fraction (Lovati et. al, 1992, 1996), particularly the &agr;' subunit (Manzoni et. al, 1998) is capable of activating LDL receptor and is therefore the main responsible for the reduction of cholesterol plasma levels. In fact, treatment of an hepatic cell line with 7S globulin induces extensive degradation of the &agr; and &agr;' subunits and stimulation of LDL receptor activity, whereas &bgr; subunits are not degraded and the receptor is not activated. Moreover, soy mutants in which 7S fraction lacks &agr;' subunit are not able to modify the receptor activity, even at high concentrations.

As a consequence of these experimental observations, methods are needed to obtain &bgr;-conglycinin in the pure form, as well as recovering and purifying the &agr;' subunit, from which specific amino acidic sequences could subsequently be obtained by enzymatic treatment, without making use of peptide synthesis.

The process suggested by Than et al. (1975 and 1976) and subsequently modified by O'Keefe et al. (1991) allows to separate glycinins and &bgr;-conglycinins based on their different solubilities at different pH; however, cross-contamination is still high and gel filtration or affinity chromatography are required, which are costly and difficult to carry out on an industrial scale. Also the modification suggested by Nagano et al. (1992), although allowing to increase the fractions purity, is still an expensive method which can be used only on laboratory scale.

Recently, Wu et al. (1999) have described a method for separating glycinins and conglycinins on a pilot-plant scale. Glycinins are precipitated by two subsequent aqueous extractions at pH 8.5, followed by treatment of the supernatant with a 0.98 g/L bisulfite solution, while conglycinins are precipitated by adding 0.25 M NaCl to the mother liquors from the glycinins precipitation, then adjusting pH to 4.8. The process allows to treat high amounts of starting material and also provides high yields in protein, but the fractions purity is still unsatisfactory; &bgr;-conglycinin, in particular, undergoes degradation, apparently during diafiltration with water, which is a treatment necessary to reduce the bisulfite ions excess and to remove salts.

The above cited methods not only do not yield pure &bgr;-conglycinin, but above all do not envisage separation and purification of the &agr;' subunit.

According to the invention, a solid fraction enriched in &bgr;-conglycinin is prepared by extracting a defattedground soy in an aqueous medium according to conventional procedures and subsequently precipitating the supernatant with aqueous ethanol; the resulting fraction is then purified by Metal Affinity Chromatography (MAC) in denaturant conditions to yield the pure &agr;' subunit, which is subjected to enzymatic treatment with chymotrypsin to obtain the amino-terminal region which has apparently the highest LDL receptor-activating activity.

DETAILED DISCLOSURE OF THE INVENTION

The present invention relates to a process for the selective extraction, purification and enzymatic modification of soy &bgr;-conglycinin &agr;' subunit, which process comprises the following steps:

  • a) extraction of a defatted ground with a sodium bisulfite aqueous solution at slightly acidic pH to obtain a &bgr;-conglycinin -enriched soluble protein fraction;
  • b) precipitation of the &bgr;-conglycinin fraction from step a) by treatment with ethanol;
  • c) purification of the precipitated fraction from step b) by Metal Affinity Chromatography (MAC) under denaturant conditions, to isolate the &agr;' subunit;
  • d) precipitation of the &agr;' subunit with organic solvents;
  • e) enzymatic treatment of the &agr;' subunit from step d) with a proteolytic enzyme and further purification by MAC chromatography.

&bgr;-Conglycinin is enriched as shown in Figure 1. The starting material is soy flour, defatted by removing the lipid fraction with solvents. The material is extracted with a sodium bisulfite aqueous solution at slightly acidic pH. A solution volume ranging from 14 to 16 times the weight of the starting material, preferably from 14.5 to 15.5 times, is used. The bisulfite concentration ranges from 0.80 to 1.20 g/L, preferably from 0.90 to 1.10 g/L, most preferably from 0.95 to 1.05 g/L. The extraction is carried out for a time ranging between 14 and 18 hours at a temperature ranging from -2 to 8°C. According to a preferred embodiment of the invention, the extraction is carried out for 16 hours with 15 volumes of a 0.98 g/L bisulfite solution at pH 6.4, at temperatures ranging from 0 to 4°C.

Under these pH and temperature conditions, glycinins solubility is very low, therefore these precipitate together with other insoluble material. The precipitate is separated by centrifugation and the soluble fraction is treated with 35 - 60% (vol/vol) aqueous ethanol, preferably 40% aqueous ethanol, at temperatures ranging from 20 to 30°C, preferably at room temperature, 25°C. The supernatant is centrifuged off and the precipitate, mainly consisting of &bgr;-conglycinin, is freeze-dried. The resulting powder is subjected to the subsequent step (Figure 2).

The choice to separate and purify the &agr;' subunit by means of MAC (Ostrove and Weiss, 1990) depends on its ability to coordinate metal ions such as Zn2+ and Ni2+, as this subunit has higher histidine content than the &agr; and &bgr; subunits (Thanh and Shibasaki, 1978).

A matrix conjugated with zinc or nickel, preferably zinc, is used. According to a preferred embodiment of the invention, the matrix consists of iminodiacetic acid-agarose. The freeze-dried protein material is suspended in a denaturing buffer consisting of 50 mM Tris, 0.5 M NaCl, pH 7.2 and containing 5 to 8 M urea, preferably 5 M. In these conditions, the &agr;' subunit selectively binds to the matrix, and the &agr; and &bgr; subunits can be removed by elution with the above buffer; the &agr;' subunit is subsequently eluted with 0.1 M imidazole in the same buffer or in distilled water.

The protein fraction enriched in &agr;' subunit is collected and treated with organic solvents which precipitate the proteins, preferably with cold acetone. Acetone is used in a volume ranging from 2 to 5 times the fraction volume, preferably 3 to 4 volumes, at a temperature ranging between -10 and -30°C, preferably between -15 and -25°C. According to a preferred embodiment of the invention, 3 volumes of acetone at -20°C are used. The resulting precipitate is separated by centrifugation, resuspended in ethanol, preferably 95% ethanol, further centrifuged and freeze-dried. The lyophilizate contains 94% of protein material and is 10 times more enriched in the &agr;' subunit than the starting material.

Table 1 shows the extraction yields in &bgr;-conglycinin and &agr;' subunit from soy flour. Table 1 Protein fraction Starting material Extraction yield (% by weight) &bgr;-Conglycinin Defatted flour 18.7 &agr;' Subunit &bgr;-Conglycinin 11.0 &agr;' Subunit Defatted flour 2.1

Polypeptide fragments of the &agr;' subunit are prepared by subjecting the lyophilizate from the previous step to enzymatic treatment with a proteolytic enzyme. According to a preferred embodiment of the invention, the proteolytic enzyme is chymotrypsin and the resulting fragment mainly consists of the amino-terminal region having MW 28,000 Da.

The procedure is as follows: the lyophilizate from the previous step is dissolved at a concentration of 5 mg/ml in 0.2 M NH4HCO3 containing 1.6 M urea at pH ranging from 7.5 to 8.5. Chymotrypsin is added in a 1:10 to 1:50 ratio, preferably 1:25 w/w to the substrate, incubating at 37°C with stirring for 24 hours. A step on MAC is subsequently carried out, as described above.

The material eluted with imidazole contains three polypeptide fragments, the main having MW 28,000 Da, and constituting the N-terminal region of the &agr;' subunit.

The administration of the &agr;' subunit and of chymotrypsin fragment to rats (table 2) proved that both are capable of remarkably decreasing cholesterol and total triglycerids plasma levels. In particular, the chymotrypsin fragment proved not only more effective than the other soy components, but also than clofibrate, in reducing cholesterol levels, and it afforded comparable results on triglycerids.

The results of the biological experimentation suggest that the products obtainable according to the process of the present invention, in particular the &agr;' subunit and the fragments thereof, can be used as medicaments, in particular for the treatment of those pathologies which require lowering of cholesterol and/or triglycerids plasma levels. Said compounds will be used, alone or in combination with other active principles and in admixture with suitable carriers, for the preparation of pharmaceutical compositions, in particular for the treatment of hyperlipidemias. Furthermore, they can also be used for the preparation of supplements or food products for dietary regimens to be followed in the above mentioned conditions.

EXAMPLES First step: Purification of 7S globulin from soy

The starting material was ground soy, defatted according to the Soxhlet method, using pentane as solvent.

Proteins were extracted with a 0.98 g/L NaHSO3 solution in amounts 15 times the volume of the defatted ground soy, for 16 hours at temperatures ranging from 0 to 4°C, keeping pH at 6.4. After centrifugation, the supernatant was treated with 40% ethanol (vol/vol) at room temperature. The resulting precipitate, enriched in &bgr;-conglycinin and containing the &agr;' subunit at a double concentration than the starting material, was freeze-dried.

Second step: Purification of the a' subunit

The &bgr;-conglycinin enriched fraction was resuspended in denaturing buffer (50 mM Tris, 0.5 M NaCl, pH 7.2) containing 5 M urea and purified by MAC on an agarose-iminodiacetic acid matrix (Sigma) conjugated with zinc. The unbound protein material was eluted with the same buffer as above, whereas the bound protein material, mainly consisting of the &agr;' subunit, was eluted with 0.1 M imidazole in the same buffer or in distilled water.

The &agr;' subunit- enriched fractions were treated with 3-4 volumes of acetone at -20°C; the resulting precipitate was suspended in 40% ethanol at room temperature, then centrifuged and freeze-dried. The resulting powder contains 94% of proteins and is 10 times more enriched in &agr;' subunit than the starting material.

Third step: Enzymatic treatment of the &agr;' subunit

The lyophilizate from the above step was dissolved at a concentration of 5 mg/ml in 0.2 M NH4HCO3 containing 1.6 M urea, at pH ranging from 7.5 to 8.5. The solution was then treated with chymotrypsin in a 1:25 w/w ratio to the protein substrate and incubated at 37°C with stirring for 24 hours, then purified by MAC as described above. The material retained by the resin and eluted with 0.1 M imidazole contains three polypeptide fragments, the major one having molecular weight 28,000 Da and consisting of the N-terminal region of the &agr;' subunit.

BIOLOGICAL EXPERIMENTATION Animals

Male rats CD SPF/VAF, weighing 75-100 g, were used. The animals were housed in makrolon cages (4-5 animals per cage) in environment with automatic control of light (12 hour light/ 12 hour darkness cycles), temperature (21 ± 1 °C) and humidity (60 ± 5%).

Experimental protocol

After 7 day housing, the animals were randomly divided into seven groups of 20 rats each (Table 2). During 28 days, one group was fed with normal diet (cod. 014RF25C; Mucedola S.r.l., Settimo Milanese, MI, Italy), whereas the others were fed with hypercholesterolemic diet consisting of 1% cholesterol, 0.5% cholic acid and 25% hydrogenated coconut oil (batch 332000, preparation 01.09.2000; Laboratorio Dottori Piccioni, Gessate, MI, Italy), with access to water ad libitum. The diet was given daily (40 g, 09.00 a.m.) and the unconsumed amount was weighed. Treatment was carried out as follows.

Group 1 (control):
animals fed with normal diet and treated orally for 28 days with a 0.5% carboxymethylcellulose solution.
Group 2:
animals fed with hypercholesterolemic diet and treated orally for 28 days with a 0.5% carboxymethylcellulose solution.
Group 3:
animals fed with hypercholesterolemic diet and treated orally for 28 days with clofibrate at a dose of 200 mg/kg.
Group 4:
animals fed with hypercholesterolemic diet and treated orally for 28 days with the soy total protein extract (TPE) at a dose of 200 mg/kg.
Group 5:
animals fed with hypercholesterolemic diet and treated orally for 28 days with &bgr;-conglycinin at a dose of 50 mg/kg.
Group 6:
animals fed with hypercholesterolemic diet and treated orally for 28 days with the &agr;' subunit at a dose of 10 mg/kg.
Group 7:
animals fed with hypercholesterolemic diet and treated orally for 28 days with the &agr;' subunit chymotrypsin fragment at a dose of 1 mg/kg.

Total cholesterol and triglycerids plasma levels were measured at the end of the 28 day treatment and after 16 hour fasting. The animals were anaesthetized with ethyl ether and blood was drawn from the inferior vena cava in tubes containing EDTA (1 mg/ml). After centrifugation for 15 min at 4°C at 3000 rpm, plasma was recovered, frozen and stored at -20°C until measurements.

Total cholesterol and triglycerids plasma concentrations (reported in Table 2) were determined according to conventional enzymatic assays. Table 2 TREATMENT Total cholesterol (mg/dL) Total triglycerids (mg/dL) GROUP 1 55.4±3 105.1±7.2 GROUP 2 284.1 ± 10.3 226.9 ± 12.6 GROUP 3 191.2 ± 8.0 139.1 ± 5.8 GROUP 4 236.1 ± 10.2 176.9 ± 8.1 GROUP 5 196.4 ± 7.6 146.7 ± 5.9 GROUP 6 182.2 ± 12.1 150.1 ± 9.8 GROUP 7 175.8 ± 7.9 140.3 ± 7.4

REFERENCES

  • Anderson J.W., Bryan M.J., Cook-Newall M-E., 1995 N. Engl. J. Med. 333, 276-282.
  • Kirk E.A., Sutherland P., Wang S.A., Chait A., LeBoeuf R.C., 1998 Journal of Nutrition. 128, 954-959.
  • Lovati M R., Manzoni C., Corsini A., Granata A., Frattini R., Fumagalli R., Sirtori C., 1992 J. Nutr. 122, 1971-1978.
  • Lovati M.R., Manzoni C., Corsini A., Granata A., Fumagalli R., Sirtori C., 1996 J. Nutr. 126, 2831-2842.
  • Manzoni C., Lovati M.R., Gianazza E., Morita Y., Sirtori C., 1998 J. Agric. Food. Chem. 46, 2481-2484.
  • Nagano T., Hirotsuka M., Mori H., Kohyama K., Nishinari K., 1992 J. Agric. Food Chem. 40, 941-944.
  • O'Keefe S.F., Wilson L.A., Resurreccion A.P., Murphy P.A., 1991 J. Agric. Food. Chem. 39, 1022-1027.
  • Ostrove S., Weiss S., 1990 Methods in Enzimology 182, 371-379.
  • Thanh V.H., Okubo K., Shibasaki K., 1975 Plant Physiol. 56:19-22.
  • Thanh V.H., Shibasaki K., 1976 J. Agric. Food. Chem. 24, 1117-1121.
  • Thanh V.H., Shibasaki K., 1978 J.Agric. Food Chem. 26, 695-698.
  • Wu S., Murphy P.A., Johnson L.A., Fratzke A.R., Reuber M.A. 1999 JAOCS 76, 285-293.


Anspruch[de]
Verfahren zur selektiven Extraktion, Reinigung und enzymatischen Modifizierung von Soja-&bgr;-conglycinin-&agr;'-Untereinheit, wobei das Verfahren die nachstehenden Schritte umfasst: a) Extraktion von entfetteter zerkleinerter Soja mit einer wässrigen Natriumbisulfitlösung bei leicht saurem pH-Wert, um eine mit &bgr;-Conglycinin angereicherte Fraktion löslichen Proteins zu erhalten; b) Ausfällung der &bgr;-Conglycininfraktion von Schritt a) durch Behandlung mit Ethanol; c) Reinigung der ausgefällten Fraktion von Schritt b) durch Metall-Affinitäts-Chromatographie (MAC) unter Denaturant-Bedingungen, um die &agr;'-Untereinheit zu isolieren; d) Ausfällung der &agr;'-Untereinheit mit organischen Lösungsmitteln; e) enzymatische Behandlung der &agr;'-Untereinheit von Schritt d) mit einem proteolytischen Enzym und weitere Reinigung durch MAC-Chromatographie. Verfahren nach Anspruch 1, wobei die Extraktion mit 15 Volumen von einer 0,98 g/l Natriumbisulfitlösung bei einem pH-Wert von 6,4 ausgeführt wird. Verfahren nach Anspruch 1, wobei die Ausfällung von Schritt b) mit 40 %igem Ethanol ausgeführt wird. Verfahren nach Anspruch 1, wobei MAC in Schritt c) an einer Matrix, die mit Zink oder Nickel konjugiert ist, ausgeführt wird. Verfahren nach Anspruch 4, wobei die Matrix mit Zink konjugiert ist. Verfahren nach Ansprüchen 4 oder 5, wobei die Matrix aus Agarose-Iminodiessigsäure besteht. Verfahren nach Anspruch 1, wobei das bei der MAC von Schritt c) verwendete Denaturant Harnstoff ist. Verfahren nach Anspruch 1, wobei das proteolytische Enzym für die enzymatische Behandlung von Schritt e) Chymotrypsin ist. Verfahren nach Anspruch 1, wobei das Ausfällungslösungsmittel für die &agr;'-Untereinheit von Schritt d) Aceton ist. Verfahren nach Anspruch 1, wobei die mit &bgr;-Conglycinin angereicherte Fraktion und die &agr;'-Untereinheit durch Gefriertrocknen stabilisiert werden. Polypeptidfragmente von Soja-&bgr;-conglycinin- &agr;'-Untereinheit, erhältlich mit dem Verfahren nach Ansprüchen 1 bis 10. Amino-terminales Polypeptidfragment, erhältlich nach dem Verfahren von Anspruch 8. Polypeptidfragmente von Soja-&bgr;-conglycinin- &agr;'-Untereinheit von Anspruch 11 zur Verwendung als Medikament. Amino-terminales Polypeptidfragment nach Anspruch 11 zur Verwendung als Medikament. Verwendung der Polypeptidfragmente der &bgr;-Conglycinin-&agr;'-Untereinheit aus Soja von Anspruch 11 zur Herstellung von Medikamenten für die Behandlung von Hyperlipidämien. Verwendung des Polypeptidfragments nach Anspruch 11 zur Herstellung von Medikamenten für die Behandlung von Hyperlipidämien. Pharmazeutische Zusammensetzungen, die die Polypeptidfragmente von Ansprüchen 11 oder 12, einzeln oder in Kombination mit anderen Wirkstoffen, in Beimengung mit geeigneten Trägern enthalten. Ergänzungsmittel oder Nahrungsprodukte, die die Polypeptidfragmente von Ansprüchen 11 oder 12 enthalten.
Anspruch[en]
A process for the selective extraction, purification and enzymatic modification of soy &bgr;-conglycinin &agr;' subunit, which process comprises the following steps: a) extraction of a defatted ground soy with a sodium bisulfite aqueous solution at slightly acidic pH to obtain a &bgr;-conglycinin -enriched soluble protein fraction; b) precipitation of the &bgr;-conglycinin fraction from step a) by treatment with ethanol; c) purification of the precipitated fraction from step b) by Metal Affinity Chromatography (MAC) under denaturant conditions, to isolate the &agr;' subunit; d) precipitation of the &agr;' subunit with organic solvents; e) enzymatic treatment of the &agr;' subunit from step d) with a proteolytic enzyme and further purification by MAC chromatography. A process as claimed in claim 1 wherein the extraction is carried out with 15 volumes of a 0.98 g/L sodium bisulfite solution at pH 6.4. A process as claimed in claim 1 wherein precipitation of step b) is carried out with 40% ethanol. A process as claimed in claim 1 wherein MAC in step c) is carried out on a matrix conjugated with zinc or nickel. A process as claimed in claim 4 wherein the matrix is conjugated with zinc. A process as claimed in claims 4 or 5 wherein the matrix consists of agarose-iminodiacetic acid. A process as claimed in claim 1 wherein the denaturating agent used in MAC of step c) is urea. A process as claimed in claim 1 wherein the proteolytic enzyme for the enzymatic treatment of step e) is chymotrypsin. A process as claimed in claim 1 wherein precipitating solvent for the &agr;' subunit in step d) is acetone. A process as claimed in claim 1 wherein the &bgr;-conglycinin- enriched fraction and the &agr;' subunit are stabilized by freeze-drying. Polypeptide fragments of soy &bgr;-conglycinin &agr;' subunit obtainable with the process of claims 1-10. Amino-terminal polypeptide fragment obtainable according to process of claim 8. Polypeptide fragments of soy &bgr;-conglycinin &agr;' subunit of claim 11 for use as medicament. Amino-terminal polypeptide fragment of claim 11 for use as medicament. The use of the polypeptide fragments of the &bgr;-conglycinin &agr;' subunit from soy of claim 11 for the preparation of medicaments for the treatment of hyperlipidemias. The use of the polypeptide fragment of claim 11 for the preparation of medicaments for the treatment of hyperlipidemias. Pharmaceutical compositions containing the polypeptide fragments of claims 11 or 12, alone or in combination with other active ingredients, in admixture with suitable carriers. Supplements or alimentary products containing the polypeptide fragments of claims 11 or 12.
Anspruch[fr]
Procédé d'extraction sélective, de purification et de modification enzymatique d'une sous-unité &agr;' de la &bgr;-conglycinine de soja, lequel procédé comprend les étapes suivantes : a) extraction d'un soja moulu dégraissé avec une solution aqueuse de bisulfite de sodium à un pH légèrement acide pour obtenir une fraction de protéine soluble enrichie en &bgr;-conglycinine ; b) précipitation de la fraction de &bgr;-conglycinine provenant de l'étape a) par traitement à l'éthanol ; c) purification de la fraction précipitée provenant de l'étape b) par Chromatographie d'Affinité sur Métal (MAC ; Metal Affinity Chromatography) dans des conditions dénaturantes, pour isoler la sous-unité &agr;' ; d) précipitation de la sous-unité &agr;' avec des solvants organiques ; e) traitement enzymatique de la sous-unité &agr;' de l'étape d) avec une enzyme protéolytique et purification supplémentaire par chromatographie MAC. Procédé selon la revendication 1 dans lequel l'extraction est réalisée avec 15 volumes d'une solution de bisulfite de sodium à 0,98 g/l à pH 6,4. Procédé selon la revendication 1 dans lequel la précipitation de l'étape b) est réalisée avec de l'éthanol à 40 %. Procédé selon la revendication 1 dans lequel la MAC de l'étape c) est réalisée sur une matrice conjuguée avec du zinc ou du nickel. Procédé selon la revendication 4 dans lequel la matrice est conjuguée avec du zinc. Procédé selon les revendications 4 ou 5 dans lequel la matrice consiste en de l'agarose-acide iminodiacétique. Procédé selon la revendication 1 dans lequel l'agent dénaturant utilisé au cours de la MAC de l'étape c) est de l'urée. Procédé selon la revendication 1 dans lequel l'enzyme protéolytique destinée au traitement enzymatique de l'étape e) est de la chymotrypsine. Procédé selon la revendication 1 dans lequel le solvant de précipitation pour la sous-unité &agr;' lors de l'étape d) est de l'acétone. Procédé selon la revendication 1 dans lequel la fraction enrichie en &bgr;-conglycinine et la sous-unité &agr;' sont stabilisées par lyophilisation. Fragments polypeptidiques d'une sous-unité &agr;' de la &bgr;-conglycinine de soja pouvant être obtenus par le procédé selon les revendications 1 à 10. Fragment polypeptidique amino-terminal pouvant être obtenu selon le procédé de la revendication 8. Fragments polypeptidiques d'une sous-unité &agr;' de la &bgr;-conglycinine de soja selon la revendication 11 pour une utilisation en tant que médicament. Fragment polypeptidique amino-terminal selon la revendication 11 pour une utilisation en tant que médicament. Utilisation des fragments polypeptidiques de la sous-unité &agr;' de la &bgr;-conglycinine provenant du soja selon la revendication 11 pour la préparation de médicaments destinés au traitement de l'hyperlipidémie. Utilisation du fragment polypeptidique selon la revendication 11 pour la préparation de médicaments destinés au traitement de l'hyperlipidémie. Compositions pharmaceutiques contenant les fragments polypeptidiques selon les revendications 11 ou 12, seuls ou en combinaison avec d'autres ingrédients actifs, en mélange avec des supports appropriés. Suppléments ou produits alimentaires contenant les fragments polypeptidiques selon les revendications 11 ou 12.






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