WO2006003110A1

WO2006003110A1 - Process for the purification of protein fractions from lupin seeds, active on lipid metabolism

Info

Publication number: WO2006003110A1
Application number: PCT/EP2005/052940
Authority: WO
Inventors: Anna Arnoldi; Cesare Sirtori; Andreas Waesche
Original assignee: Fraunhofer Gesellschaft zur Foerderung der Angewandten Forschung eV
Current assignee: Fraunhofer Gesellschaft zur Foerderung der Angewandten Forschung eV
Priority date: 2004-06-29
Filing date: 2005-06-23
Publication date: 2006-01-12
Anticipated expiration: 2006-12-29
Also published as: ITMI20041308A1

Abstract

In the described process lupin seeds from lupin, for example from Lupinus albus and Lupinus angustifolius, are crushed and transformed into flakes. By taking into account the features of the protein isolates or concentrates desired, and the lipid content of the original material, lipids (oil) are or are not extracted using a suitable solvent (prevalently hexane), by percolation of the solvent through the flakes placed in a tubular reactor. The solvent is then eliminated in controlled temperature conditions. Alternatively, lipids can be removed also by extraction with supercritical CO2 through percolation through a column. After grinding, protein is extracted in a water solution, by alternating treatments in weakly basic and acid environment, in order to obtain the dissolution and subsequent precipitation of the proteins of the different fractions. After suitable centrifugation of insoluble materials, proteins are collected as a water solution. This is then, prevalently, reconcentrated by ultrafiltration. In order to ensure microbiological sterility of the protein ingredients, high temperature short time (HTST) pasteurisation or ultra-high temperature (UI'-TT) sterilisation are carried out. Lastly, protein solutions are transformed into powder by spray-drying (Fig. 1). Significant plasma lipid lowering properties are detected in the total protein isolates (TPE) of lupin, when administered to rats fed a hyperlipidemic diet. Separated fractions, particularly LUPI-F, less so LUPIE, show a significantly higher lipid lowering activity versus TPE.

Description

"Process for the purification of protein fractions from lupin seeds, active on lipid metabolism".

* * * *

The present invention refers to a process for the purification from lupin seed of protein fractions, active on lipid metabolism.

According to this invention, different protein components of lupin seeds, for example of the species Lupinus albus (white lupin) and Lupinus angustifolius (narrow-leafed lupin), are purified in conditions that maintain their biological features and without the use of organic and inorganic reagents or the use of metals that rule out the use for direct human consumption.

Experiments on suitable animal models and extensive clinical investigations have amply demonstrated that vegetable proteins in the diet, in particular proteins of legumes like soy, can markedly reduce cholesterolemia both in animal models and in humans (Anderson et al, 1995,

"Meta-analysis of the effects of soybean protein intake on serum lipids", N. Engl. J. Med. 333: 276-282). On the basis of these findings the Food and Drug Administration of the United States in 1999 authorised the sentence

"may reduce the risk of disease" to be added to the label of soy-based food products for human consumption.

Recently, studies conducted on rats fed with a lipid-rich diet have shown that a daily administration of 50 mg/day for two weeks (the equivalent of approximately 250 mg/kg) of a total protein extract (TPE) of Lupinus albus causes a dramatic decrease in total cholesterolemia (-21%) and of 30% in the cholesterol associated with atherogenic lipoproteins

(VLDL + LDL), with a similarly significant decrease (-17%) of triglyceridemia (Sirtori et al, 2004. "The proteins of white lupin seed- a naturally isoflavone free legume - reduce cholesterolemia in rats and increase LDL receptor activity in HepG2 cells", J. Nutr., 134: 18-23). Lupin seeds contain two classes of protein, which, according to the Osborne classification, belong to the albumins and globulins in a relative ratio of 1 to 9. The globulins in turn consist of two major components, 7S and 1 IS, called beta and alpha conglutin, respectively, and of two minor components, defined gamma and delta conglutin, respectively (Blagrove et al, 1975, "Isolation, purification and characterization of the seed conglutins of Lupinus albus", Aust. J. Plant Physiol. 2: 13-27).

It is also important to underline that lupin proteins, unlike soy proteins, have the significant advantage of being essentially free of isoflavonoid components (''phytoestrogens") (Katagiri et al, 2000. 'ΗPLC analysis of white lupin isoflavonoids", Biosci. Biotechnol. Biochem. 64: 1118-1125;

Sirtori et al, 2004). Potential adverse effects of phytoestrogens have, in fact, been recently described and have led to restrictive regulations in several countries (Sirtori, 2001. "Risks and benefits of soy phytoestrogens in cardiovascular diseases, cancer, climacteric symptoms and osteoporosis. Drug Safety 24: 665-682).

Although the potential applications of lupin proteins in human diet have been widely studied for a long time, the fact that lupin is a legume that is not widely grown; in addition to the limited availability of suitable industrial purification processes, has prevented a widescale use as dietary component. Studies reporting a potential nutraceutical interest of lupin proteins have thus indicated the need to develop industrial processes for the purification of the different protein components, potentially exhibiting significant hypolipidemic properties.

The object of this invention is to develop a process that enables protein fractions active on lipid metabolism to be obtained from lupin seeds.

In view of this objective, the present invention refers to a process for the purification of protein concentrates and isolates from lupin seeds, for example of Lupinus albus and Lupinus angustifolius, comprehensive of the following steps: a) The lupin seeds of one of the two indicated species are coarsely ground and transformed into flakes. During this process the temperature is kept below 60 °C to prevent denaturation of the proteins and non-enzymatic browning of the flakes. At the end of the process, the flakes have a yellowish colour. b) Depending on the lipid content of the original material and the features of the protein isolates or concentrates that one desires to obtain, the lipids are or are not extracted with a suitable solvent (prevalently hexane) by percolation of the solvent through the flakes placed in a tubular reactor using a multistage procedure. The solvent is eliminated in stripping conditions with superheated solvent and steam under vacuum and finally by air blowing. After this stage tihe flakes are almost white and have a protein content exceeding 50 %. c) Alternatively, lipids can also be extracted with supercritical carbondioxide (CO₂) by percolation through the flakes placed in a tubular reactor using a multistage procedure. After this stage the flakes are almost white and have a protein content exceeding 50 %. d) The protein is extracted using acid water solutions (pH 4.3-4.9) to separate a solution enriched with gamma conglutin (extract A) and a partially purified precipitate that contains most of the beta and alpha conglutins (7S and 1 IS globulins) of the lupin seed (precipitate A). e) From extract A, after clarification, ultrafiltration and diafiltration, a water solution is obtained that is enriched with gamma conglutin, which then is HTST pasteurised or UHT treated and spray-dried to give a powder protein concentrate or isolate prevalently containing gamma conglutin (LUPI-F), as confirmed by the SDS-PAGE analysis and two-dimension electrophoresis. f) Precipitate A is extracted in two successive steps with a weakly basic solution (7.0-7.5 pH) and is decanted by centrifugation to obtain two extracts enriched with globulins 7S and 1 IS, which are combined (extract B). g) The globulins are precipitated from extract B by treatment with acid pH, dissolved again at neutral pH, HTSH pasteurised or UHT treated and subsequently transformed into powder by a spray-drying process (LUPI-E). A product is thus obtained that has a composition including:

^■ a source of lupin proteins, selected from native isolated lupin proteins, for example from Lupinus albus (white lupin) or Lupinus angustifolius (narrow-leafed lupin), which can provide a protein content of at least 50 % of the total protein content of the dry matter (calculated on the basis of the nitrogen content, N x 6.25), in such a way that the protein content provides at least 25% of the total energy content of the composition;

^■ at least 50 % of the sweet lupin proteins alpha and beta conglutins;

■ or at least 50 % of gamma conglutin from sweet lupin; ^■ or at least 50 % of a mixture of total lupin proteins: alpha, beta, and gamma conglutins;

■ and less than 4% dietary fibre;

^■ and less than 0.01% lupin phytooestrogens;

^■ and less than 0.02% of lupin alkaloids. This composition may be used for preparing functional and dietary food products for the clinical control of hyperlipidemia, and for the preparation of dietary supplements and nutraceutical products, again for the clinical control of hyperlipidemia.

The process according to the present invention completely eliminates the problems connected with the presence in lupin seeds of bitter quinolizidine alkaloids displaying an antifeedant role in seeds.

All the protein isolates have been characterised by bidimensional electrophoresis (proteomics), in order to assess protein integrity and tested by liquid chromatography-mass spectrometry to confirm the absence of phytoestrogens, in particular genistein glycosides. The identification of the active components of lupin seed was guided by in vitro experiments conducted on HepG2 cells, a human hepatoma cell line, provided with a wide range of enzymes involved in intermediary metabolism. HepG2 cells are widely used to assess the regulatory properties on the lipoprotein system of proteins/peptides of vegetable origin. Studies have been conducted on the modulation of the LDL receptors, responsible for cholesterol homeostasis hi HepG2 cells. LDL receptors bind and internalise circulating LDL cholesterol, thus regulating the levels of LDL and the distribution of cholesterol to tissues and cells. In this model it was possible to define a clear stimulatory activity of LDL receptors at least by one specific lupin protein, gamma conglutin. These in vitro findings indicated the possibility that lupin protein components can regulate plasma lipid levels, even with relatively low daily administrations.

A product obtained with the process described in this invention was subjected to experiments on a universally accepted animal model, i.e. rats on a lipid-rich diet, conducted hi parallel to the administration of a lipid- lowering drug with well known activity hi rats. These experiments have "*' clearly shown the potential of certain lupin-protein isolates to exert a significant lipid-lowering activity. The total lupin protein extract and the different purified fractions were administered as described by Sirtori et al. (2004), i.e. by feeding the different products suspended hi 0.5 % carboxymethylcellulose. A number of experimental groups were thus investigated: 1. standard diet with daily gavage of 0.5% carboxymethylcellulose; 2. hyperlipidemic Nath diet (Nath et al, 1959. "Diet and cholesterolemia, Part 1", J. Nutr. 67:289-293), with daily gavage of 0.5% carboxymethylcellulose; this diet was administered also to all other treatment groups; 3. drug treatment, with 200 mg/kg Clofibrate (Sigma, UK) daily; 4. total protein extract (TPE) of Lupinus albus as described by Sirtori et al (2004) in order to confirm the previous experimental results 5-7. LUPI-E protein isolate, consisting of 7S and 11 S globulins, from L. albus, administered at daily doses of 20, 50 and 100 mg/kg; 8-10. LUPI-F protein isolate, mainly consisting of gamma conglutin, from Lupinus albus, administered at daily doses of 10, 20 and 50 mg/kg;

11-13 LUPI-E protein isolate from L. angustifolius administered at daily doses of 20, 50 and 100 mg/kg;

14-16 LUPI-F protein isolate, mainly consisting of gamma conglutin from Lupinus angustifolius, administered at daily doses of 10, 20 and 50 mg/kg. The major results from the biological experiments can be summarized in the following way: a) the highest lipid lowering activity (significantly greater than that of Clofibrate) was achieved with the protein isolates LUPI-F at the highest doses, the Lupinus albus fraction being slightly more effective than that from L. angustifolius. b) the protein isolates LUPI-E were also very effective at slightly higher doses, again a greater activity being exerted by the Lupinus albus proteins.

The lipid lowering activity was mainly hypocholesterolemic, also considering the modest rise of triglyceridemia in the model. It can be thus reasonably concluded that the lipid lowering activity is almost exclusively exerted on the atherogenic fractions VLDL+LDL, as typically induced by the experimental diet in the rat.

A specific example of the product obtained by the process of purification of the concentrates and protein isolates according to the invention will now be disclosed, together with the relative chemical analysis and the relative biological evaluation.

EXAMPLE

Proteins enriched with alpha and beta conglutin (LUPI-E) and proteins enriched with gamma conglutin (LUPI-F) can be prepared from different species and varieties of sweet and bitter lupins by different embodiments of the process according to the invention.

An example of this type of preparation is described below. All the explanations refer to the process diagram in Figure 1. Coarse material

About 21500 kg of coarse Lupinus albus material of the Kiev variety were delivered by the Agracorp Pty Ltd, Perth, Western Australia 6000 (1999/2000 harvest). Table 1 shows the dry matter contents, the coarse protein, the ashes and the alkaloid content of the delivered material, which had a density of 774 kg/m³. Table 1 . Chemical composition of Lupinus albus (upon delivery)

Shelling lupins and preparing flakes

About 4500 kg of lupins were split using an Underrunner Splitter (Sheller) Type S&S, manufactured by Streckel & Schrader, Hamburg. Subsequently, the shells were separated from the seeds by a jet of air. Thus about 3440 kg of crushed seeds and 1060 kg of husks were collected (phase

I)-

The crushed seeds were transferred to a rotating mill (equipped with wheels for flaking) for flaking (phase 2). During flaking, the temperature of the flaking wheels was kept below 40⁰C to avoid browning and/or protein denaturing. The resulting flakes were yellowish ("yellow flakes") and had an average density of 300-330 kg/m³. Table 2. Composition of the yellow flakes of Lupinus albus

Extraction of the lipids with solvent The extraction of the lipids (essentially oil) (phase 3) from 500-kg batches of yellow flakes was carried out, with said yellow flakes: They formed a 2-metre high column within a vertical tube of 900 mm in diameter was formed. Defatting consisted of percolating hexane through the fixed bed of yellow flakes in 4 stages. Each stage consisted of: a) percolation of hexane until 500 litres of mixture were collected in the tank; b) re-circulation of the mixture for 15 min; c) drainage of the mixture for 15 min after the first three cycles and for 30 min after the final extraction cycle. After defatting, 100-kg batches of flakes (still containing hexane) were completely deprived of solvent by stripping under a vacuum (at 250 mbar) in a stripping apparatus stirred (60 rpm) for 150 min. The internal surface of the filter was heated to make water circulate at 60°C. After the solvent had been eliminated, the hexane content of the flakes had fallen to 250 ppm. This was subsequently reduced to 50 ppm by air blowing.

Table 3a shows the chemical composition of the lupin flakes ("white flakes") that have been defatted by hexane. About 867 kg of white flakes were obtained from 1,027 kg of yellow flakes. Table 3a. Chemical composition of the white flakes of L. albus after extraction with hexane

n.m. = not measurable Alternatively: extraction of lipids with supercritical COi

The lipids were extracted from 250-kg batches of yellow flakes, which formed a 2-metre high column in a vertical tube measuring 900 mm in diameter. Defatting was carried out by percolating supercritical CO₂ (SCF- CO2) through the fixed bed of yellow flakes in 4 stages. The temperature was maintained between 40 and 69°C, preferably 45-5O⁰C. Pressure was controlled between 240 and 300 bar, preferably between 250 and 260 bar. After each stage, the SCF-CO2 was evaporated from the extracted oil and from the defatteS flakes by evaporation at atmospheric pressure. Table 3b shows the chemical composition of the lupin flakes ("white flakes") defatted using SCFCO2.

Table 3b. Chemical composition of the white flakes of Lupinus albus after extraction using SCF-CO2

n.m. = not measurable

Protein extraction

The lupin protein extract was prepared from the white flakes in two extraction phases 4 and 5, the first of which (4) was carried out in an acid water solution and the second (5) was conducted in an alkaline water solution in two stages (5-1 and 5-2).

In phase 4 the white flakes were suspended in acid cold water in order to separate a soluble fraction A enriched with gamma conglutin from an insoluble solid precipitate B containing the proteins with an acid isoelectric point (beta and alpha conglutin).

More precisely, extraction of phase 4 was carried out on a 185-kg batch of white flakes using 1,800 litres tap water acidified at pH 4.5-4.8, in a 2000-litre tank stirred at a controlled temperature between 13.5 and 15.2 ⁰C. A two-arm stirrer was adjusted to 55 rpm. About 23.6 litres of 3 M HCl were used to control the pH during extraction, which lasted for a total of 1 hour.

A centrifugal decanter (CB300. GEA Westfalia GmbH, Oelde/Germany) was used for the separation of the solid residue (approximate speed 4,400 rpm). Starting with 185 kg of white flakes and using 1,800 kg of tap water, approximately 385 kg of residue or solid precipitate B and 1,600 litres of acid extract A were obtained.

In phase 5 the solid precipitate B was suspended in slightly alkaline hot water in order to separate a protein extract C enriched with beta and alpha conglutin and a solid precipitate D, which is insoluble in these conditions.

More precisely, during the first extraction phase 385 kg of solid precipitate B were placed in 900 litres of tap water with 1.2-1 A pH in a 2000-litre tank at a controlled temperature of 28.2 and 31.5°C. A two-arm stirrer was adjusted to 60 rpm. About 19.6 litres of sodium hydroxide solution 3 M were used to control the pH during the total extraction time of 1 hour. About 50 ml of an anti-foaming compound (Struktol SB 2010) were added to the solution. A centrifugal decanter (CB300, GEA Westfalia GmbH, Oelde/Germany) was used for the separation of the solid residue from the extract at a speed of 4,400 rpm.

During the second extraction stage, the solid residue of the first extraction phase was again extracted in a tank equipped in the same way, using 540 litres of tap water with 7.3-7.4 pH and at 29.0-32.0°C. About 0.3 litres of the solution 3 M of sodium hydroxide were used to control pH during the extraction lasting 15 min.

During the course of process 5, from 385 kg of solid residue B and 1,440 kg of tap water about 945 litres of protein solution Cl were obtained in the first stage and about 595 litres of protein solution C2 were obtained in the second stage in addition to 242 kg of solid residue D. Both protein solutions were combined in a single protein solution C, thus obtaining a total of 1,540 litres. Precipitation of the proteins

In a following phase 6, the protein extract C obtained in phase 5 was added with hydrochloric acid to precipitate a protein fraction E that was rich in alpha and beta conglutin.

More precisely, about 16 litres of HCl water solution 3 M were mixed with the protein solution C (1540 litres) in a stirred 2000-litre tank at 31 A- 35.2 °C, the pH being adjusted to 4.6-4.5 in order to precipitate the proteins at their isoelectric point (pH 4.5). About 50 ml of an anti-foaming compound (Struktol SB 2010) were added to the solution. During precipitation, a two- arm stirrer adjusted to 85 rpm was used. The precipitated proteins were enriched in alpha and beta conglutin, whereas the liquid supernatant was enriched in gamma conglutin. Fractioning of the precipitated proteins In a subsequent phase 7, the precipitated proteins F were separated from the protein fraction contained in the supernatant SP.

More precisely, the precipitated protein solution F (about 1,550 litres) was separated using a self-cleaning disc separator with a speed of 6,830 rpm (10,000*g) and a supply speed of 1,800 litres/hour. The solids suspended in the supply fluid varied from 11.0 to 11.5 vol %. The separated protein precipitate F was unloaded at intervals of about 250 seconds. The solids suspended in the clarified extract varied from 0.0 to 0.1 vol %. About 1330 litres of clarified supernatant SP and 213 litres of protein precipitate F were separated. The SP dry matter content varied from 0.4 to 0.5 % and contained about 70 % of total proteins (N*6.25).

Clarification of the acid extract

In the meantime, in phase 8, the acid extract A (1,600 litres) was clarified using a self-cleaning disk separator at a speed of 7,500 rpm (approximately 12,000*g) at a supply speed of 1,800 litres per hour. The solids content of the supply liquid varied between 2 and 2.5 vol %. The separated protein precipitate G was discharged at intervals of about 280 seconds. The solid content of the clarified extract H varied from 0.1 to 0.15 vol %. About 1,500 litres of clarified protein extract H and 100 litres of protein precipitate G were separated. The dry matter content of the extract H varied from 2.2 to 2.5 % and about 25% of total proteins (N*6.25).

Concentration of the extract H and of the supernatant SP

In order to concentrate the extract H and the supernatant SP, a membrane filtration process in two phases, 9 and 10, was applied. In phase 9, the extract H was ultrafiltered (cross-flow membrane filtering). More precisely, 700 litres of extract H were taken from pH 4.5 to a pH value in the range of 6.0 and 7.0 and were placed in the ultrafiltration unit. At this pH the obstruction of the membrane surface by the protein was at a more reduced level than with acid pH. The solution was subsequently recirculated on the membrane unit (Pall, Germany, 2*4.5 m²' cut-off 10,000 Dalton) at a pressure of 3 bar and at a temperature of 40°C, until the volume of clarified acid extract H was reduced to a tenth of the initial volume. The dry matter content of the retentate I was 7 % and had a protein content of approximately 50% (N*6.25).

In the subsequent phase 10 the retentate I was subsequently diluted by adding SP and subjecting the resulting mixture to diafiltration in a second membrane filtration unit.

Each dilution stage was comprehensive of adding 233 litres of (SC) to the retentate I deriving from the ultrafiltration of phase 9 and of recirculating the diluted retentate on the membrane until the retentate volume was returned to the initial value. After the last dilution phase, on the other hand, recirculating was continued until the dry matter content had reached maximum levels.

The dry matter content of the diafiltrated retentate L varied from 14.5 to 15 % and the dry matter contained approximately 84% of total proteins (N*6.25).

The two processes ultrafiltration 9 and diafiltration 10 enabled a fraction L to be obtained that is enriched with gamma conglutin. Readjustment of pH '

In a subsequent phase 11 the pH value of fraction L was changed from 6.5 to an approximate value of 5.2, since between 60⁰C and 65 °C the viscosity is lower at this pH than at neutral pH (fraction M). Pasteurisation and spray drying

For the pasteurisation (phase 12), the fraction with readjusted pH (fraction M) was heated from 40⁰C to 65°C in a small scale heat exchanger with continuous circulation, consisting of a single lined pipe with an internal diameter of 6 mm. The length of this pipe was adjusted in such a way that the residual circulation time was about 1 minute. Alternatively, this material can be subjected to UHT treatment for just a few seconds.

Fraction M, after heating, was then directly introduced into a spray dryer (Niro-Minor, GEA NIRO Ltd, Copenhagen) together with hot air. The inlet air temperature was 195⁰C, the liquid supply speed was 8-10 litres per hour, the resulting air outlet temperature was 77 ⁰C.

The dry powder was separated from the air flow using a 150-mtn cyclonic separator. The dry matter content of the resulting powder varied from 94.0 to 95.2 %. Ignoring the losses of fine particles, starting with 40 kg of fraction L approximately 4.5 kg of dry protein fraction enriched with gamma conglutin (LUPI-F) were obtained.

LUPI-F, prepared according to the described process, contains 84.7% proteins (N*6.25), 0.6 % lipids and 6.4 % ash (of dry matter). In addition, the dry matter contains 8.3 % of nitrogen-free products.

Separation of a fraction enriched with alpha and beta conglutin

(globulins 7S and HS) of precipitates F and G.

A phase 13, consisting of an extract in a slightly basic conditions, enabled a fraction N to be obtained, which was enriched in beta and alpha conglutin, by processing phase 7 solids F and phase 8 solids G.

Approximately 100 litres of protein precipitates F separated with the disc separator in phase 7 and 213 litres of protein precipitates G obtained in phase 8 were soaked in a cooled 800^£litre tank at pH 4.5 and were then kept at 2-10⁰C overnight, preferably at 4-8 ⁰C. Thereafter, the pH was increased to 6-7, preferably 6.5-6.9, using 0.5 M NaOH and afterward the dry matter content was reduced from 30-25% to about 20-25 %, preferably 22-23 %, by diluting with tap water.

Pasteurisation and spray drying of the protein suspension of alpha and beta conglutin After phase 13, the protein solution N was heated from 8 °C to 70-80

⁰C in a small-scale heat exchanger with continuous circulation consisting of a single lined pipe, with an internal diameter of 6 mm. The length of this pipe was adjusted in such a way that the residual circulation time of the protein suspension was about 60 seconds. The hot protein suspension was inserted directly into a spray dryer (Niro-Minor, GEA NIRO Ltd, Copenhagen) together with hot air. The inlet air nozzle temperature was 165-195°C, with a supply speed for the liquid of 25-35 litres an hour, produced an outlet air temperature of 72-78 ⁰C. The dry powder was separated from the air flow using a 150-mm cyclonic separator. The dry matter content of the resulting powder varied from 94 to 96 %. The protein content was greater than 95%. From this process (phase 14) about 50 kg of powdered LUPI-E proteins were obtained. CHEMICAL-ANALYTICAL EXPERIMENTATION Protein constitution All the protein isolates were analysed by a proteomic methodology

(two-dimension electrophoresis, 2DE) to assess purity and integrity of the different protein extracts according to the procedure previously disclosed for soy proteins (Gianazza et al, 2003. "A proteomic investigation of isolated soy proteins with variable effects in experimental and clinical studies", J. Nutr., 133: 9-14). The 2-DE maps were obtained using the IPG-DALT technique. The proteins were separated according to their load at a non¬ linear immobilised gradient of pH 4-10 (TPG), in the presence of 8 M urea, then^* according to the molecular dimension in SDS-PAGE, on a 7.5-17}5 % or 10-23% polyacrylamide gradient. Gels were stained with 0.3% Coomassie Blue.

These analyses confirmed that LUPI-E protein isolates were mainly 7S and 1 IS globulins, whereas the LUPI-F protein isolates are enriched in gamma conglutin. Quantification of isoflavones in HPLC-mass. The extraction of the isoflavones was conducted with the procedure indicated by Wang and Murphy (32) for the isolation from soy seeds. Each sample (1 g) was extracted for 2 h with a mixture of 5 mL acetonitrile, 2.5 mL of distilled water and 1 mL of HCl 0.1 N. After filtration, the solvent was evaporated to dryness, the residue was then dissolved in 1 mL of 50% methanol and filtered on 0.45 micron filters. The analyses were conducted on a 3-micron, 150 x 2.1 mm Alltima Cl 8 microbore column. Conditions: 0.1 % eluent A, acetic acid in acetonitrile, 0.1 % eluent B₅ acetic acid in water, 15 % A to 35 % A gradient in 50 min, then 35 % A for 10 min; flow 0.2 mL/min; temperature 30 °C. The isofiavones were identified by LC-ESI- MS/MS on an Agilent SL 100 Series LC/MSD trap equipped with an

Agilent 1100 binary pump and an Agilent 1100 diode array detector (DAD). Conditions: temperature of source 325 ⁰C, 18 psi spray, drying gas 6 L/min, capillary voltage 4000 V, scan range 60-700 m/z. The analyses were conducted in "negative ion mode", as in this mode greater sensitivity is obtained. This analysis method has variation coefficients between 2 and 10

%.

In these lupin isolates and protein concentrates the concentration of genistein and its glycosides is below the limit of quantification of the method, which in our conditions is about 0.1 nmol/g. Quantification of the alkaloids of quinolizidine in GC-MS.

The alkaloids were extracted and analysed in the manner described by Ruiz and Soleto ("Chemical composition, nutritive value, and toxicology evaluation of Mexican wild lupin", J. Agric. Food Chem.^J2001, 49:5336-9). Each sample (500 mg) was homogenised in a vortex with 5 mL of 5% trichloroacetic acid for 1 minute and was then centrifuged. Extraction was repeated twice. After basification with 1 ml of NaOH 10 M, the samples were extracted with dichloromethane (3 x 5 ml), the solvent was evaporated and the.analyses were conducted by GC-MS by comparison with authentic standards, using a PB-I capillary column (30 m x 0.25 mm id). Equipment used: GC-MS QP-5000 (Shimadzu), with injector temperature 240 °C and detector temperature 290 °C, in EI mode.

In these lupin isolates and protein concentrates the quinolizidine alkaloids were below the limit of quantification of the method. BIOLOGICAL EXPERIMENT In vivo tests on rats

Sprague-Dawley rats (Charles River Italy, Calco, Italy) were used, with mean body weights 200-225 g. They were kept in the laboratory under controlled lighting for 12 hours a day at a constant temperature (18°C) and relative humidity of 55-65%. For a week the animals were fed a standard commercial pellet diet (Piccioni, Gessate, Italia) and were then divided into sixteen experimental groups, all of 12 animals.

Group 1 continued with the commercial pellet diet whereas all the other groups received the Nath diet (1% cholesterol, 0.5% cholic acid, 25% hydrogenated coconut oil) (Piccioni, Gessate, Italia). All the animals had free access to water and fed ad libitum.

After a week, blood was taken from the tail of each animal to evaluate lipid levels. The animals with a cholesterolemia levels below 2 SDs versus the group means were discarded. The final number of the 15 groups being given lipid-rich dietary treatment was thus: 11 for groups 3, 7, 9, 13 10 for groups 2, 6, 8, 10, 11, 12, 15 9 for groups 4, 5, 14, 16. After that, the animals of groups 4 to 16 started to be treated with the protein isolates, which are the subject of this invention, as shown in Table 4. Treatments were administered by individual gavages at 9 am for 27 consecutive days. The animals were sacrificed on the 28th day after 16 hours of fasting. Blood was taken from the jugular vein under light ether anesthesia and the clotted blood samples were spun at 3,000 rpm for 15 min at a controlled temperature (16⁰C). Aliquots were separated for measuring total cholesterolemia and triglyceridemia by enzyme methods (Boehringer SA, Mannheim, Germany) and for lipoprotein fractioning. This was carried out by preparatory ultracentrifugation using pools of three animals. The preparatory ultracentrifugation procedure was performed following the Havel et al. protocol ("The distribution of ultracentrifugally separated lipoproteins in human serum", J. Clin. Invest. 1955, 34: 1345-1353).

Table 4. Type of treatment and concentration of plasma lipids in rats fed a lipid-rich diet and treated with the protein isolates of the present invention

* p <0.05; ** O.01; *** <0.001 FURTHER EXAMPLES

According to a possible variation of the disclosed example, the proteins can be extracted only in an acid environment at pH 4.0-5.0 and at a temperature of 277-340 Kelvin, without beforehand extracting the lipids from the lupin products, i.e. without phase 3 of Figure 1, but at the same time avoiding the formation of emulsions. The crude extract that is thus obtained is then filtered on a membrane to recover the proteins. The operations of washing, pH adjustment, pasteurisation and spray drying then follow as described. Additional recovery of the acid soluble proteins is also possible.

Claims

1) Process for the purification from lupin seeds of protein fractions active on the lipid metabolism, comprehensive of the following steps: a) crushing of lupin seeds, dehusking and separation of the kernels from the husks by air classification, b) preparation of lupin products containing crushed kernels using technologies such as flaking, pre-pressing, expansion, pelleting, milling, c) extraction of the lipids from the products with organic solvent or with supercritical CO2, d) water extraction of the protein products, e) precipitation of the extracted proteins, f) washing of the crude proteins with tap water, g) readjustment of pH to value 6-7, h) pasteurisation of the slurry of native proteins at pH 6-7,

1) drying in bland conditions of the crude protein slurry.

2) Process according to claim 1, wherein the crushing of the lupin seeds is conducted on sweef lupin containing 4-14% of water.

3) Process according to claim 1 or 2, wherein the crushing of the kernels takes place in a flaking mill, a universal mill, a screw press or in an extruder.

4) Process according to claim 1, 2, or 3, wherein the extraction of the lipids is conducted by percolation using an organic solvent (hexane, isohexane or alcohol) at 290-340 Kelvin and at atmospheric pressure. 5) Process according to claim 4, wherein the total elimination of the solvent is achieved through treatment with high-temperature organic vapours under vacuum and subsequently the residual organic solvent is stripped away by steam heated at high temperature under vacuum.

6) Process according to claim 1. 2 or 3, wherein the extraction of the lipids is conducted by percolation with supercritical CO2 at 240-300 bar pressure and at a temperature of 313-342 Kelvin.

7) Process according to any one of the preceding claims, wherein the water extraction of the protein products is conducted at basic pH value between 7.0-9.0 and at a temperature of 277-340 Kelvin. 8) Process according to any one of the preceding claims, wherein precipitation of the proteins is conducted in acid conditions at 4.0-5.0 pH and at a temperature of 277-340 Kelvin.

9) Process according to any one of the preceding claims, wherein extraction of the proteins is conducted in acid conditions at 4.0-5.0 pH and at a temperature of 277-340 Kelvin, without previously extracting the lipids from the lupin products.

10) Process according to claim 9, wherein the proteins are recovered by submitting the coarse extract resulting from said extraction of proteins to membrane filtration. 11) Process according to claim 9. wherein the coarse extract resulting from said extraction of proteins is submitted to a washing procedure conducted using tap water in the ratio 1 :4 to 1 :20, in such a way as to '^"' diminish the presence of substances with a low molecular weight (alpha glucosides, carbohydrates, alkaloids) present in the coarse slurry of native lupin proteins in acid conditions (pH 4-5 and temperature between 277-340

Kelvin).

12) Process according to claim 1, wherein the washing procedure of the extracted proteins is combined with an ultrafiltration procedure at 5-7 pH and at a temperature of 277-340 Kelvin, wherein the washing water is added to the retentate of the ultrafiltration and is recovered through repeated ultrafiltration processes.

13) Process according to claim 12, wherein precipitation of the proteins generates a supernatant that is treated by ultrafiltration to recover the protein fraction that is soluble in acids. 14) Process according to any one of the preceding claims, wherein the adjustment of pH is made at a pH below 7 and at a temperature below 300 Kelvin in such a way as to keep the lipoxygenase activity at a low level, i.e. about 10-20% of the activity in the native coarse proteins.

15) Product active on lipid metabolism obtained by purification of protein fractions from lupin seeds, characterised by a composition including:

■ a source of lupin proteins, selected from isolated native lupin proteins, for example from Lupinus albus (white lupin) oτLupinus angustifolius (narrow-leafed lupin), which can provide a protein content of at least 50 % of the dry matter (calculated on the basis of the nitrogen content, N x 6.25), in such a way that proteins provide at least 25% of the total energy content of the composition;

^■ at least 50 % alpha and beta conglutin proteins from sweet lupin;

■ or at least 50 % gamma conglutin from sweet lupin;

■ or alternatively at least 50 % of a mixture of total alpha, beta, gamma conglutin lupin proteins.

■ dietary fibre in quantity lower than 4%;

■ less than 0.01% lupin phytooestrogens;

■ less than 0.02% lupin alkaloids.

16) Use of the composition as described in claim 15 for the preparation of functional foods and dietary foods for the clinical control of hyperlipidemia.

17) Use of the composition as described in claim 15 for the preparation of dietary supplements and nutraceutical products for the clinical control of hyperlipidaemia.