Method for extraction, purification, and enzyme modification of soy 7s-globulin alpha'-subunit useful as hypocholesteric agent

FIELD: biotechnology, medicals.

SUBSTANCE: invention relates to method for extraction, purification, and enzyme modification of β-conglycinin α'-subunit. Claimed method includes isolation of β-conglycinin by selective extraction from milled defatted soybeans and following deposition by extract treatment with ethanol aqueous solution. Enriched fraction is then subjected to affinity chromatography with metal (MAC) under denaturant condition to produce α'-subunit which further is treated with chymotrypsin and subjected to additional treatment with MAC to isolate amino-terminated region of said polypeptide having molecular weight of 28000 Da.

EFFECT: isolated fraction of increased purity.

10 cl, 2 tbl, 2 dwg, 1 ex

 

The present invention relates to a method of extraction, purification and enzymatic modification α'-subunit β-better.

In accordance with this invention β-conglycinin extracted by the method of selective extraction of ground degreased soybean, followed by precipitation by treatment of the extract with an aqueous solution of ethanol; enriched fraction is then subjected to affinity chromatography using metals (MAC) under denaturing conditions with obtaining α'-subunit. The latter is treated with chymotrypsin, and then subjected to an additional stage affinity chromatography emitting aminobenzoic the area of the specified polypeptide (molecular weight of 28000 Yes).

Prior art

Known cholesterol-lowering ability of soybean and its derivatives is due to the content of isoflavones (Kirk and others, 1998) and proteins (Anderson et. al, 1995).

Soy proteins are composed mainly of glycinin (11S fraction) and konglitsininov (7S fraction), the latter consists of 3 subunits, called α', α and β-subunits (Thanh and Shibasaki, 1976). In studies conducted to study the protein composition of soybean, it was found that the 7S fraction (Lovati et. al, 1992, 1996), in particular α'-subunit (Manzoni et. al, 1998) are able to activate the receptor lipoprotein low is lotnosti (LDL), and thus, it is responsible mainly for the reduction of cholesterol in blood plasma. In fact, when processing cell line liver 7S-globulin is the induction of extensive degradation α' and α-subunits and stimulating the activity of LDL-receptor, while β-subunit does not degrade, and stimulation of the specified receptor does not occur. Moreover, soybean mutants that are missing α-subunit of the 7S fraction, not able to modify the activity of LPS-receptor even at high concentrations.

The result of these experimental observations, it is necessary to obtain β-better in pure form, as well as extraction and purification α'-subunit from which you can then get specific amino acid sequence by enzymatic treatment without the use of peptide synthesis.

The method proposed by Than and others, (1975 and 1976), which was modified later O'keefe and others, (1991) allows you to split glycinin and β-konglitsininy on the basis of their different indicators of solubility at different pH; however, its disadvantage is that the presence of impurities in the samples is still high, and you need to clean them to gel filtration and affinity chromatography, which are costly and difficult to implement them in the industry is certain scale. Also the modification proposed by Nagano and others, (1992), although it allows to increase the purity of the fractions, it remains expensive way, which can only be used in laboratory practice.

In recent publications Wu and others, (1999) reported on the implementation of the method of separation of glycinin and konglitsininov-scale studies in the pilot unit. In accordance with this method glycinin precipitated by carrying out two successive water extraction at pH 8.5 followed by treatment of the supernatant using bisulfite solution at a concentration of 0.98 g/l, while konglitsininy precipitated by adding 0.25 M NaCl to the uterine fluids, obtained by deposition of glycinin, and then bringing the pH to 4.8. This method allows you to process large quantities of source material, and also provides high outputs of products for protein, however, the purity of the obtained fractions are still insufficient; β-conglycinin, in particular, is undergoing degradation, apparently, during diafiltration water, but this processing is necessary to reduce the excess bisulfite ions and removal of salts.

The above methods not only provide a clean β-konglitsininov, but primarily not provide separation and purification α'-subunit.

According to this izopet is of enriched β -conglycinin solid fraction obtained by extraction of fat-free ground soybeans in the aquatic environment in accordance with standard methods with subsequent precipitation of the supernatant using an aqueous solution of ethanol; the resulting fraction is then purified by the method of affinity chromatography with the use of metals (MAC) under denaturing conditions, which gives a clean α'-subunit, which is subjected to enzymatic treatment with chymotrypsin with getting aminobenzoic region, which, as it turns out, has a high activity for the activation of LDL-receptor.

Detailed description of the invention

The present invention relates to a method of selective extraction, purification and enzymatic modification α'-subunit of soybean β-better characterized in that the method comprises the following stages:

a) extraction of fat-free ground soybeans with an aqueous solution of sodium bisulfite at slightly acidic pH and obtaining soluble protein fraction enriched β-conglycinin;

b) deposition of the specified β-conglycinin fraction from stage (a) by treatment with ethanol;

c) purifying the precipitated fraction from stage (b) by the method of affinity chromatography with the use of metals (MAC) under denaturing conditions with selection α'-subunit;

d) enzyme of the processing obtained from stage c) α '-subunit proteolytic enzyme and subsequent purification using MASS chromatography;

e) deposition obtained α'-subunit of organic solvents.

β-conglycinin enrich the method illustrated in figure 1. As the source material used soy flour, fat by removing lipid fraction using solvents. This material is extracted with an aqueous solution of sodium bisulfite at slightly acidic pH. Use a solution that volume is 14-16 times the mass of the initial material, preferably from 14.5 to 15.5 times. The concentration of bisulfite varies in the range from 0.80 to 1.20 g/l, preferably from 0.90 to 1.10 g/l and more preferably from 0.95 to 1.05 g/L. the Extraction is carried out for the period of time from 14 to 18 hours at a temperature in the range from -2 to 8°C. In accordance with the preferred embodiment of the invention, the extraction is carried out for 16 hours with 15 volumes bisulfite solution at a concentration of bisulfite 0,98 g/l, at pH 6.4 and at a temperature in the range from 0 to 4°C.

With these values of pH and temperature the solubility of glycinin is very low, and therefore they are deposited together with other insoluble material. The precipitate is then separated by centrifugation and the soluble fraction is treated 35-0% (vol./about.) water ethanol preferably 40% aqueous ethanol at a temperature in the range from 20 to 30°C, preferably at room temperature, 25°C. the Supernatant centrifuged and separated, and then the formed precipitate, mainly consisting of β-better lyophilizer. The resulting powder was subjected to the following processing stages are shown in figure 2.

The choice for separation and purification α'-subunit with MAC (Ostrove and Weiss, 1990) depends on its ability to form coordination bond with metal ions such as Zn2+and Ni2+because this subunit has a higher content of histidine, than αand β-subunit (Thanh and Shibasaki, 1978).

Use a matrix, conjugate with zinc or Nickel, preferably with zinc. In accordance with a preferred embodiment of the invention, the matrix comprises agarose, modified iminodiacetic acid. Freeze-dried protein material suspended in denaturing buffer consisting of 50 mm Tris, 0.5 M NaCl, pH 7.2 and containing from 5 to 8 M urea, preferably 5 M urea. In these conditions α'-subunit selectively binds with the specified matrix and αand β-subunits can be separated by elution with the above buffer solution; α'-subunit then e is irout using 0.1 M imidazole in the same buffer or distilled water.

The protein fraction enriched in α'-subunit, collect and process organic solvent used to precipitate proteins, preferably with cold acetone. Acetone is used in amounts varying from 2 to 5 times greater than the volume of the protein fraction, preferably from 3 to 4 volumes, at a temperature in the range from -10 to -30°C, preferably in the range from -15 to -25°C. In accordance with the preferred embodiment of the invention using 3 volumes of acetone at -20°C. the resulting precipitate is separated by centrifuging, then resuspended in ethanol, preferably in 95% ethanol, then centrifuged again and dried by lyophilization method. The resulting lyophilisate contains 94% of the protein material, which is 10 times more enriched compared to the original material content α'-subunit.

Table 1 shows the outputs for β-conglycinin and α'-subunit resulting from the extraction of soya flour.

Table 1
Protein fractionsSource materialYield (wt.%) as a result of extraction
β-ConglycininDefatted flour18,7
α'-Subunit&x003B2; -Conglycinin11,0
α'-SubunitDefatted flour2,1

Peptide fragments α'-subunit obtained from the lyophilisate obtained at the previous stage, which is subjected to enzymatic conversion using a proteolytic enzyme. In accordance with a preferred embodiment of the invention, the proteolytic enzyme used chymotrypsin, and the resulting fragment is composed mainly of aminoanisole area, which has a molecular weight of 28000 Yes.

The method consists in the following: the lyophilisate obtained at the previous stage is dissolved at a concentration of 5 mg/ml in 0.2 M solution of NH4HCO3containing 1,6 M urea at pH in the range from 7.5 to 8.5. The substrate is added chymotrypsin at the rate of 1:10 to 1: 50, preferably 1:25 wt./wt., and incubated at 37 °With stirring for 24 hours. After this exercise stage WT according to the method described above.

The product obtained after elution with imidazole contains three polypeptide fragment, and the main fragment has a molecular weight of 28000 Yes, and is N-terminal region α'-subunit.

With the introduction of α'-subunit and chymotrypsinogen fragment rats (table 2) was that the BA is able to significantly reduce the levels of cholesterol and total triglycerides in the blood plasma. In particular, chymotrypsinogen fragment, as it turned out, not only more effective in reducing cholesterol levels in the blood plasma than other components of the soybean, but also in comparison with clofibrate, and it allows to obtain comparable results for triglycerides.

On the basis of the results of biological experiments, we can make the assumption that the products obtained in accordance with the method of the present invention, in particular α'-subunit and its fragments, can be used as medicines, in particular for the treatment of diseases which require reducing the levels of cholesterol and/or triglycerides in the blood plasma. These compounds can be used in pure form or in combination with other active means and in a mixture with suitable media that is used to produce pharmaceutical compositions, in particular for the treatment of hyperlipidemia. Moreover, they can be used to obtain supplements or foods in the dietary supply, appointed under the above conditions.

Examples

The first stage. Cleaning 7S-globulin from soybean

As source material used milled soy, fat in accordance with the Soxhlet method, using pentane as the solvent.

Proteins were extracted using the solution of NaHSO 3at a concentration of 0.98 g/l in quantities of 15 times greater than the amount of fat milled soy, for 16 hours at a temperature in the range from 0 to 4°maintaining at pH of 6.4. After centrifugation the supernatant is treated with 40% ethanol (vol./about.) at room temperature. The resulting precipitate enriched in β-conglycinin and containing α'-subunit at a concentration that is twice higher than that of the source material, lyophilizer.

The second phase. Cleaning α'-subunit

Enriched β-conglycinin fraction resuspended in denaturing buffer (50 mm Tris, 0,5M NaCl, pH of 7.2)containing 5 M urea and purified using a MAC on a matrix of agarose, modified iminodiacetic acid (Sigma)conjugated with zinc ions. Unbound proteins elute with the same buffer described above, while associated protein material, consisting mainly of α'-subunit, elute with 0.1 M imidazole in the same buffer or distilled water.

Enriched α'-subunit fractions treated with 3-4 volumes of acetone at -20°C. the resulting precipitate is suspended in 40% ethanol at room temperature, then centrifuged and dried by lyophilization method. The resulting powder contains 94% protein and is the Wallpaper 10 times more enriched product compared to the original material content α '-subunit.

The third stage. Enzymatic treatment of α'-subunit

The lyophilisate obtained at the previous stage is dissolved at a concentration of 5 mg/ml in 0.2 M solution of NH4HCO3containing 1,6 M urea at pH in the range from 7.5 to 8.5. The resulting solution is then treated with chymotrypsin at a ratio of 1:25 wt./wt. to the protein substrate and incubated under stirring at a temperature of 37°within 24 hours, then do the cleaning using the MAC according to the method described above. The product remaining on the resin and suirvey 0,1M imidazole, contains three polypeptide fragment, and the main fragment of them has a molecular weight of 28000 and is N-terminal region α'-subunit.

Biological experiments

Animals

In the experiments used male rats CD SPF/VAF weighing 75-100, Animals were kept in macroeonomic cells (4-5 animals per cage) under conditions with automatic regulation of light (cycles of 12 hours light and 12 hours dark) at a temperature (21±1°C) and relative humidity (60±5%).

The Protocol experiments

After 7 days the animals were divided randomized method on seven groups of 20 rats in each group (table 2). Within 28 days, one group received normal diet (code 014RF25C; from Mucedola S.r.l., Settimo Milanese, MI,Italy), while others were given a diet with cholesterol-lowering action, containing 1% cholesterol, 0.5% of holeva acid and 25% hydrogenated coconut oil (party 332000 fabricated 01.09. 2000 Laboratorio Dottori Piccioni, Gessate, MI, Italy), while the animals had access to water without restrictions. This diet rats were treated daily (40 g at 09.00 a.m.), while nepotreblenie residue weighed. Treatment was carried out by the following method.

Group 1 (control): animals received a normal diet and oral was administered for 28 days 0.5% solution of carboxymethylcellulose.

Group 2: animals received giperholesterinemiei diet and oral was administered for 28 days 0.5% solution of carboxymethylcellulose.

Group 3: animals received giperholesterinemiei diet and oral was administered within 28 days of clofibrate in the dose of 200 mg/kg

Group 4: animals received giperholesterinemiei diet and oral was administered within 28 days of extract total protein of soybean (TPE) at a dose of 200 mg/kg

Group 5: animals received giperholesterinemiei diet and oral was administered for 28 days β-conglycinin dose of 50 mg/kg

Group 6: animals received giperholesterinemiei diet and oral was administered for 28 days α'-subunit in e is ze 10 mg/kg

Group 7: animals received giperholesterinemiei diet and oral was administered within 28 days of the fragment α'-subunit obtained by treatment with chymotrypsin at a dose of 1 mg/kg

Total cholesterol and triglycerides in plasma were measured at the end of the 28-day treatment period and after 16 hours hungry extracts. The test animals were euthanized with ethyl ether and did the blood from the inferior Vena cava into tubes with EDTA(1mg/ml). After centrifugation for 15 minutes at 4°s speed centrifuge 3000 rpm and the plasma was removed, frozen and stored at -20°to determine the parameters.

The total concentration of cholesterol and triglycerides in plasma (results are shown in Table 2) were determined by standard immunoassay methods.

Table 2
The treatmentTotal cholesterol (mg/DL)Total triglycerides (mg/DL)
Group 155,4±3105,1±7,2
Group 2284,1±10,3226,9±12,6
Group 3191,2 ±8,0139,1±5,8
Group 4236,1±10,2176,9±8,1
Gruppe 196,4±7,6146,7±5,9
Group 6of 182.2±12,1to 150.1±9,8
Group 7175,8±7,9140,3±7,4

Sources of information

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., R.C. LeBoeuf, 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., C. Sirtori, 1998 J. Agric.Food. Chem. 46,2481-2484.

Nagano, T., Hirotsuka, M., Mori H., K. Kohyama, K. Nishinari, 1992 J. Agric. Food Chem. 40,941-944.

O'keefe S.F., L.A. Wilson, 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 VH, Okubo K., Shibasaki K., 1975 Plant Physiol. 56: 19-22.

Thanh VH, Shibasaki K., 1976 J. Agric. Food. Chem. 24, 1117-1121.

Thanh VH, 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.

1. The method of selective extraction, purification and enzymatic modification α'-subunit β-better soybean, comprising the following stages:

a) extraction of fat-free ground soybeans with an aqueous solution of sodium bisulfite in size, 14-16 times the mass of source material containing 0,98 g/l of sodium bisulfite at pH of 6.4, obtaining enriched β-conglycinin soluble protein fraction;

b) is adenia specified β -conglycinin fraction from stage (a) by treatment with 40% ethanol;

c) purifying the precipitated fraction from stage (b) by the method of affinity chromatography with the use of metals (MAC) on the matrix of agarose, modified iminodiacetic acid, conjugated with zinc or Nickel under denaturing conditions with selection α' - subunit, while the denaturing agent is urea;

d) deposition α' - subunit of organic solvents, which is acetone;

e) enzymatic processing α' - subunit from stage C) proteolytic enzyme chymotrypsin followed by purification using (MAC);

enriched β-conglycinin faction and α' - subunit stabilizes lyophilic drying.

2. The method according to claim 1, in which the specified matrix conjugated with zinc.

3. Enriched β-conglycinin faction and α' - subunit of soybean obtained using the method according to claims 1 and 2.

4. Polypeptide fragments α' - subunit β-better soybeans produced using the method according to claims 1 and 2.

5. Polypeptide fragments α' - subunit β-better soybean according to claim 4 for use as a medicine.

6. Aminobenzoic polypeptide fragment obtained using the method according to claim 1.

7. Aminobenzoic polypeptide fra is ment according to claim 6 for use as a medicine.

8. The use of polypeptide fragments α' - subunit β-better from soybeans according to claim 4 to obtain drugs for the treatment of hyperlipidemia.

9. The use of the polypeptide fragments according to claim 6 to obtain drugs for the treatment of hyperlipidemia.

10. Pharmaceutical compositions containing the polypeptide fragments according to claim 4 or 6 in pure form or in combination with other active ingredients, in a mixture with other acceptable media.



 

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EFFECT: semidefatted soybean flour of high organoleptic properties without specific taste and odor of soybean flour; textured soybean protein of high organoleptic properties and storage stability.

FIELD: food industry.

SUBSTANCE: the present innovation deals with food products out of soybeans and technology for obtaining milk substitutes due to reducing and thermal treatment of plant raw material. Soybean food product should be obtained due to hydrodynamic impact upon soybeans due to circulation of the mixture of soybean and water in closed contour. Circulation should be continued till it is possible to obtain the paste as stable gel at weight ratio of soybean : water being restricted to 1:(2-6). Paste content of vitamins and fats against dry product corresponds to their content in the beans of natural soybean. The innovation enables to obtain soybean food product due to hydrodynamic impact upon soybean at compulsory turbulization and cavitation due to circulating the mixture of soybean and water in closed contour for 280-450 cycles to obtain the paste as stable gel. The innovation enables to obtain food product that keeps balanced ratio of structural nutritive components of natural soybean.

EFFECT: higher efficiency.

7 cl, 3 ex

FIELD: food industry, canned food industry.

SUBSTANCE: the present innovation deals with techniques to manufacture vegetable snack-type canned food. One should peel, wash, cut and roast vegetables in vegetable oil at 130-140° C. Roasted raw material should be rubbed through the sieve upon triturators up to product's particle size being about 2-3 mm. Soybean grain should be inspected, washed in water at 20-30° C, placed into a tank filled with water for ¼ to be further put into an autocalve for barothermal treatment. Treated grain should be cooled up and reduced upon a cutter up to paste-like state ( at particles size of reduced raw material being 0.2 mm), mixed with the rest components of the formula at the quantity of not less than 30% against the weight of the main raw material to be boiled up to dry substances content according to refractometer being 12.5%. Ready-to-use product in its hot state should be packed into specially prepared cans to be sealed and sterilized at 120° C for 50 min at counter-pressure of 0.25 MPa. Addition of soybean protein paste as a formula component obtained out of soybean grain due to its barothermal treatment according to the following mode , at intensity of pressure decrease being 0.0067 MPa/min, at the quantity of 30% against the weight of the main raw material enables to increase nutritive value of ready-to-use canned food, improve their organoleptic values and decrease cost price.

EFFECT: higher efficiency of manufacturing.

1 tbl

FIELD: food-processing industry, in particular, soya concentrates with low content of inassimilable oligosaccharides and high content of isoflavones and saponins.

SUBSTANCE: method involves providing ultravibration with the use of membranes having molecular mass passage band of up to 30,000.

EFFECT: improved assimilation of feed.

12 cl, 5 tbl, 5 ex

FIELD: food-processing industry; production of protein products from soy.

SUBSTANCE: soy-beans are cleaned from admixtures, washed in clean water and soaked in water taken in double or triple amount by volume. Swollen beans are ground in 8-10-fold amount of water for obtaining fine suspension. Extract is filtered. Solid phase is separated and soy base is thus obtained. It is boiled for 35-45 min at temperature of 98-100°C. Coagulant is introduced into boiling solution for forming protein coagulum and clear whey. Used as coagulant is calcium chloride solution, calcium or magnesium sulfates and sea salt concentrate. Excessive whey is drained. Protein coagulum is placed in mold, cooled and whey is drained. At moisture content of up to 70%, it is molded in plates or bricks, 1.5-2 cm thick and frozen at temperature not higher than minus 10°C. Then they are thawed out and dried at temperature of 35-45°C to moisture content of 5-6%. Proposed method makes it possible to obtain product with porous structure without "bean taste".

EFFECT: extended storage term; low cost of technological process.

3 cl, 3 ex

FIELD: food-processing industry, in particular, preparing of products with the use of fried chick-pea beans.

SUBSTANCE: method involves providing soaking and thermal processing of chick-pea beans, with soaking process being carried out for 2-2.5 hours at room temperature and thermal processing being performed in vacuum tanks at vacuum extent of 85-95% at temperature of 90-95 C for 40-45 min; removing excessive moisture, followed by drying in drying chambers at temperature of 35-50 C for 1.5-2 hours until moisture content is 12%; mixing chick-pea beans with spices and components. Drying process may be performed with the use of infrared radiation.

EFFECT: provision for obtaining of protein enriched product balanced with regard to amino acid and microelement composition.

1 tbl

FIELD: food processing industry, in particular pulse culture seed reprocessing.

SUBSTANCE: nut seeds are couched for 3-4 days to produce sprouts of 0.5-1.0 cm length, ground in anaerobic conditions in hot water and conditioned for 3 h to produce fermented product. Then product is heated up to 85-92°C for 3-5 min and separated to produce protein emulsion and non-soluble fraction.

EFFECT: emulsion product of increased quality due to improved filtration properties of suspension.

FIELD: food processing industry, in particular foodstuff production from genetically non-modified soybean.

SUBSTANCE: claimed method includes soybean gradation, drying, breakage on four parts, husking, heating up to 55-70°C and moistening; softening, providing flattened flakes of 0.35 mm in thickness, hexane extraction with 70 % ethanol aqueous solution, drying for 4.5 h at 85-95°C to remove rest alcohol and trypcin component and production of food protein. Produces protein is further powered and homogenized in 20 % NaOH solution. Homogenized product is dissolved in water and sprayed into container followed by drying therein.

EFFECT: food protein of high quality obtained without using of additional equipment.

1 tbl

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