RU2337567C2 - Method of manufacturing flax protein isolate - Google Patents

Abstract

FIELD: food products manufacturing technique.

SUBSTANCE: flax protein isolates are manufactured by extraction of flax oilseeds to remove slime before crushing them to produce oil and flour. Then flax seed flour containing protein is treated to produce flax protein isolate.

EFFECT: improved functional properties.

12 cl, 10 tbl, 9 ex

Description

Technical field

The present invention relates to the production of flax protein isolates from flour from oily flax seeds.

Link to related applications

This application claims, in accordance with 35 US Code 119 (e), the priority date indicated in applications for US patents No. 60/491564, filed August 1, 2003, and No. 60/516875, filed November 4, 2003

State of the art

US Patent Application No. 10/266,677, filed October 9, 2002, the assignee of which is the author of this application, and which is included in the list of references to this application, describes the preparation of a flax protein isolate. As noted in this application, it is possible to obtain a protein isolate from oilseed flax seeds having a protein content of at least about 90 wt.%, Preferably at least about 100 wt.%, When determined by Kjeldahl - nitrogen × 6 , 25 (N × 6.25), calculated on the dry matter.

In this method, the yield of flax protein isolate is limited due to the impossibility of concentrating the protein solution to a high protein content due to the presence of water-soluble mucus. Flax seed mucus is a sticky substance consisting mainly of polysaccharides. The presence of mucus in protein products isolated by other methods from flour from oilseeds of flax makes it difficult to produce products with a sufficiently high protein content, which can be classified as isolates.

It is known that flax seeds contain from about 34 to 37 wt.% Proteins; they also identified several different protein components that differ from each other by sedimentation coefficients (S). These proteins include globulin 12S, known as linin, and albumin 2S, known as colinin.

Linric® oilseeds (hereinafter referred to as linole) sold by Agricore United are a mutant oilseed flax seed with a modified fatty acid composition and linolenic acid content (C 18: 3), reduced from about 50% in ordinary oilseed flax seeds to 2 % using traditional inbreeding methods. These modifications were undertaken in order to ensure the production of edible oil enriched with polyunsaturated fatty acids from oilseeds of linole that is substantially identical in fatty acid composition to sunflower oil.

SUMMARY OF THE INVENTION

The authors of this application found that if preliminary extraction of flax seeds is carried out at an elevated temperature using a mildly alkaline solution of sodium bicarbonate in order to remove mucus from them, then a concentrated aqueous protein solution with a much higher concentration can be obtained, which will result in a higher yield Flax protein isolate. In addition, a flax protein isolate can be obtained from protein containing flour from flax seeds by isoelectric precipitation (IEP) or micellar.

It has also been found that the dominant protein component in the flax protein isolates of the present invention is 7S protein, which appears to be half of 12S globulin, known as linin, and has a molecular weight of about 162,000 to 169,000 Da when measured by retention time by HPLC Compared to standard BioRad animal protein solutions. Other protein components of the flax protein isolates of the present invention include lynin having a molecular weight of about 415,000 to 440,000 Da when determined by HPLC by retention time compared to standard animal protein solutions BioRad, and colinin having a molecular weight of about 16,000 to 17,000 Yes when determined HPLC by retention time in comparison with standard solutions of animal protein BioRad.

In addition, it was also found that the relative quantitative content of protein components in the flax protein isolate obtained from protein micellar mass (PMM) and in the flax protein isolate of the present invention obtained from the supernatant is the same.

In particular, it has been found that a flax protein isolate obtained from PMM having a protein content of at least about 90 wt.% (N × 6.25), preferably at least about 100 wt.%, Contains from about 65 up to 95% by weight of 7S protein (1/2 linin), from about 0 to 20% by weight of linin, and from about 0 to 20% by weight of colinine. The flax protein isolate obtained from the supernatant has been found to have a protein content of at least about 90 wt.% (N × 6.25), preferably at least about 100 wt.%, Contains from about 65 to 95 wt. .% protein flax 7S (1/2 lignin), from about 0 to 20 wt.% linin and from about 0 to 20 wt.% colinin.

The identical profile of protein components in flax protein isolates obtained from PMM and from the supernatant determines their identical behavior in different applications of flax protein isolates. The identical protein content profiles allow combining flax protein isolates obtained from PMM and from the supernatant in any desired proportions without changing the composition, which leads to an increase in the productivity of the method.

Flax protein isolates obtained from PMM, from the supernatant and by isoelectric precipitation have a very similar amino acid profile. Established amino acid profiles are discussed below in the examples.

The present invention provides a flax protein isolate having a unique protein profile, and a method for its production, including preliminary extraction of oilseeds. A "protein isolate" is defined herein as a protein containing at least about 90% by weight of a protein as determined by Kjeldahl, i.e. nitrogen multiplied by the conversion factor is N × 6.25. The term "protein content" in the context of the description means the amount of protein in the protein isolate in terms of dry matter.

The flax protein isolate obtained by the method described here can be used in traditional applications of protein isolates, for example, for enriching food products with protein, for emulsifying oils, as structure formers in bakery products and as foaming agents in aerated products. In addition, a protein isolate can be molded into protein fibers used in the production of meat analogues, used as a substitute for egg protein or as a filler added to reduce the cost of foods that traditionally use egg white as a binding agent. Flax protein isolate can also be used as a nutritional supplement. Other uses for flax protein isolate include the production of pet and livestock feed, industrial processing, and the production of cosmetics and personal care products.

Brief Description of the Figures

Figures 1 and 2 show HPLC chromatograms of linole protein isolates, wherein Figure 1 shows a chromatogram of linole protein isolate obtained from PMM, and Figure 2 shows a chromatogram of linole protein isolate obtained from supernatant.

Figures 3, 4 and 5 show thermograms of linole protein isolates obtained by differential scanning calorimetry (DSC), while figure 3 shows a thermogram of linole protein isolate obtained from PMM, figure 4 - linole protein isolate obtained from supernatant , and in Fig.5 - linole protein isolate obtained by IEP.

DETAILED DESCRIPTION OF THE INVENTION

Pre-extraction of flax seeds is carried out using an aqueous solution, usually having a pH of from about 7.5 to 9, preferably at a natural pH of an aqueous solution of alkaline material, at an elevated temperature of from about 30 to 70 ° C, preferably at about 50 ° C. The extraction of oily flax seeds can be carried out at a ratio of seeds to solution from about 1: 1 to 1:20, preferably from about 1: 5 to 1:10, using an aqueous solution containing from about 0.2 to 0.7 M moderately alkaline material. Preferably, an aqueous sodium bicarbonate solution having a concentration of about 0.5 M is used at about 50 ° C. and a flax seed ratio of about 1: 8.

After preliminary extraction of the oilseeds, usually by stirring the oilseeds in an aqueous alkaline solution for about 15 to 60 minutes, preferably about 30 to 60 minutes, the extraction is preferably repeated with fresh aqueous alkaline solution until the oilseeds cease extracted mucus.

After extraction, the oilseeds are subsequently processed to extract the oil and obtain flour from the oilseeds, from which the flax protein isolate can be isolated.

One of the ways in which it is possible to obtain a flax protein isolate from flour from flaxseed oil seeds is isoelectric precipitation (IEP). Without an initial mucus removal as described herein, applicants were unable to obtain flax protein isolate from flour from oilseeds of flax by isoelectric precipitation. Isoelectric deposition is commonly used to produce other protein isolates, such as soy protein isolates.

When isoelectric precipitation of flaxseed oilseed or linole oilseed is extracted with an aqueous alkaline solution, mainly an aqueous solution of sodium hydroxide, having a pH of from about 8 to 12, preferably from about 9 to 11, at a temperature of from about 0 to 40 ° C, preferably about 15 to 25 ° C., and at a flour concentration of about 2.5 to 10% (w / v), preferably about 5% (w / v).

After extraction, the residual flour is separated from the aqueous protein solution by any conventional method, such as vacuum filtration, followed by centrifugation and / or filtration to remove residual flour. The separated residual flour may be dried for later use.

The flax protein solution is then acidified to a pH of about 3 to 5, preferably about 4, using any suitable acid, such as hydrochloric acid, to precipitate a flax or linole protein isolate. The precipitate is separated from the supernatant and dried. The dry isolate of flax protein obtained by isoelectric precipitation has a high protein content of about 90% by weight (N × 6.25), preferably at least about 100% by weight.

Alternatively and preferably, the flax protein isolate is prepared according to the method described in the aforementioned co-pending US Patent Application No. 10/266677. The method can be carried out as a sequence of periodic operations either in continuous or semi-continuous mode.

The initial stage of the production process of flax protein isolate in accordance with the method of the aforementioned application includes the solubilization of protein material from flaxseed oilseed flour. The protein material obtained from flax seed flour may be a native flax seed protein, or the protein material may be a protein modified by genetic manipulation, but possessing hydrophobic and polar properties characteristic of a natural protein. Flaxseed flour can be any flaxseed obtained after removing linseed oil from oilseeds of flax with varying levels of undenatured protein, which is achieved, for example, by hot extraction with hexane or by cold extrusion of oil. The removal of linseed oil from oilseeds of flax is usually carried out as a separate operation from the method for producing protein isolate described here.

Protein solubilization is most effective when using saline, since the presence of salt favors the removal of soluble protein from flour from oilseeds. The salt is usually sodium chloride, although other salts such as potassium chloride may be used. The saline solution has an ionic strength of at least about 0.10 M, preferably at least about 0.15 M, in most cases up to about 0.2 M, i.e. sufficient to solubilize significant amounts of protein. When the ionic strength of the saline solution increases, the degree of solubilization of the protein from the oilseed meal first increases until it reaches its maximum value. Any subsequent increase in ionic strength no longer leads to an increase in the total amount of solubilized protein. The ionic strength of the saline solution, which causes the maximum solubilization of the protein, varies depending on the selected salt and flour from oilseeds.

To achieve the higher degree of dilution required to precipitate the protein with an increase in ionic strength, it is generally preferable to use an ionic strength index of less than about 1.0, and more preferably from about 0.15 to 0.6.

In a batch process, the solubilization of the protein with a salt is carried out at a temperature above about 0 ° C, preferably at a temperature of up to about 35 ° C, and is preferably accompanied by stirring in order to reduce the solubilization time, which is usually from about 10 to 90 minutes. Preferably, the solubilization is carried out in order to extract the maximum possible amount of protein from oilseed flour in order to improve the yield of the product. A temperature of about 35 ° C. is selected as the preferred upper temperature limit, since at elevated temperature levels the batch process is not economical.

In a continuous method, the extraction of protein from flour from oilseeds of flax is carried out by any method suitable for the implementation of continuous extraction of protein from flour from oilseeds of flax. In one embodiment of the invention, flaxseed oil meal is continuously mixed with saline, and the mixture is transported by pipeline, the length of which and the flow rate of the mixture in which provide a residence time sufficient to carry out the required extraction in accordance with the parameters indicated here. In this continuous process, the salt solubilization step is carried out rather quickly - in a time of up to about 10 minutes; preferably, solubilization is carried out in order to extract the maximum possible amount of protein from flour from oily flax seeds. Solubilization in a continuous process is preferably carried out at elevated temperatures, generally up to about 60 ° C. or higher.

Aqueous saline and oilseed flax flour have a natural pH of about 5 to 7, which ensures the formation of the protein isolate in a micellar way, as described in more detail below. The optimal pH for the maximum yield of flax or linole protein isolate varies depending on the selected flour from flaxseed oil seeds.

At limiting and close to limiting pH values in the pH range, the formation of the protein isolate by the micellar route occurs only partially and with a lower yield compared with the yield achieved with the remaining values of the pH range. For these reasons, pH values from about 5.3 to 6.2 are preferred.

In the extraction step, the pH of the saline solution can, if necessary, be adjusted to any desired value in the range of about 4 to 7 with any suitable acid, usually hydrochloric, or alkali, usually sodium hydroxide.

The concentration of flour from oil seeds in saline at the stage of solubilization can vary within wide limits. Typical concentrations are from about 5 to 15% (w / v).

The stage of protein extraction with aqueous saline is characterized by the additional effect of solubilization of fats, which may be present in flour from flax seeds, which leads to the subsequent presence of fats in the aqueous phase.

The protein solution from the extraction step in most cases has a protein concentration of from about 5 to 40 g / l, preferably from about 10 to 30 g / l.

The aqueous phase obtained in the extraction step can then be separated from the residual flour from oilseeds of flax by any convenient method, such as vacuum filtration, followed by centrifugation and / or filtration to remove the residual flour. The separated residual flour may be dried for later use.

If the flaxseed flour contains a significant amount of fat, then the degreasing stages of the separated aqueous protein solution and concentrated aqueous protein solution described in the U.S. Pat. Nos. 5,884,086 and 6,050,076, the successor of which is the author of this application, may be carried out. which are included in the list of links to this application.

As an alternative to extracting flour from oilseeds of flaxseeds with an aqueous saline solution, this extraction can be carried out using only water, although when using only one water, a smaller amount of protein is extracted, as noted, than when using an aqueous saline solution. If this alternative method is used, then after separation of the protein solution from the residual flour from oilseeds of flax, salt can be added in the concentrations discussed above in order to keep the protein in solution during the concentration step described below.

Then, the aqueous protein solution is concentrated to increase the concentration of the protein in it while maintaining its ionic strength, basically constant. Such concentration is usually carried out in order to obtain a concentrated protein solution having a protein concentration of at least about 150 g / l, preferably at least about 250 g / l.

The concentration step may be carried out by any suitable method in batch or continuous mode, for example using suitable selective membrane technology, such as ultrafiltration or diafiltration, using membranes such as hollow fiber or spiral membranes with the required throughput for the compounds with different molecular weights, for example membranes, permeable to compounds with a molecular weight of from about 3,000 to 100,000 Da, preferably from about 5,000 to 10,000 Da, in The dependence on the materials making up the membrane and membrane configuration, and in a continuous process - and, depending on their size, to provide the desired degree of concentration as the aqueous protein solution as it passes through the membrane.

The concentrated protein solution may then undergo a diafiltration step using an aqueous saline solution, usually a sodium chloride solution of the same molarity and at the same pH as the extraction solution. The diafiltration step can be carried out using from about 2 to 20 volumes of diafiltered solution, preferably from about 5 to 10 volumes of diafiltered solution. During diafiltration, additional amounts of contaminants are removed from the aqueous protein solution by passing it through a membrane along with permeate. The diafiltration process can be carried out until a significant amount of impurities remains in the permeate. Such diafiltration can be carried out using a membrane permeable to compounds with a molecular weight of from about 3,000 to 100,000 Da, preferably from about 5,000 to 10,000 Da, depending on the different materials of which the membrane is made and its configurations.

An antioxidant may be present in at least a portion of the diafiltration step in the diafiltration medium. The antioxidant may be any suitable antioxidant, such as sodium sulfite or ascorbic acid. The amount of antioxidant used in the diafiltration medium depends on the materials used and can vary from about 0.01 to 1 wt.%, Preferably about 0.05 wt.%.

The concentration step and the optional diafiltration step can be performed at any suitable temperature, generally about 15 to 60 ° C., and for a period of time sufficient to achieve the desired degree of concentration. The temperature and other conditions used to a certain extent depend on the membrane equipment used to concentrate to the desired protein concentration in the solution.

As is well known, ultrafiltration and similar selective membrane technologies allow the passage of low molecular weight substances through the membranes while retaining high molecular weight substances on the membranes. Low molecular weight substances include not only dietary salt ions, but also low molecular weight materials extracted from raw materials, such as carbohydrates, pigments and anti-nutritional factors, as well as low molecular weight forms of protein. The membrane throughput according to the molecular weight of the substances to be separated is usually selected in order to guarantee the retention of a significant proportion of the protein in the solution while passing contaminants through the membrane, taking into account the various materials from which the membrane can be made, and its configuration.

A concentrated and optionally diafiltered protein solution can be pasteurized in order to kill any bacteria that might have got into the original flour during storage or in some other way, and then extracted from the flour into a solution of flax protein isolate at the extraction stage. Such pasteurization can be carried out at any desired pasteurization conditions. In most cases, the concentrated and optionally diafiltered protein solution is heated to a temperature of about 55 to 70 ° C, preferably about 60 to 65 ° C, for about 10 to 15 minutes, preferably about 10 minutes. Then, the pasteurized concentrated protein solution can be cooled for further processing, as described below, preferably to a temperature of about 25 to 40 ° C.

Depending on the temperature used in the concentration step, the concentrated protein solution can be heated to a temperature of at least about 20 to 60 ° C, preferably about 25 to 40 ° C, in order to reduce the viscosity of the concentrated protein solution so that to facilitate the subsequent stage of dilution and the formation of micelles.

The concentrated protein solution should not be heated above a temperature above which the temperature of the concentrated protein solution will not allow micelles to form when diluted with chilled water. If desired, the concentrated protein solution may undergo a subsequent degreasing step as described in US Pat. Nos. 5,484,086 and 6,050,076.

Then, the concentrated protein solution obtained in the concentration step and optional degreasing step is diluted to initiate micelle formation by mixing the concentrated protein solution with chilled water having the volume necessary to achieve the desired degree of dilution. The concentrated protein solution is diluted about 15 times or less, preferably about 10 times or less.

The chilled water with which the concentrated protein solution is mixed has a temperature below about 15 ° C, mainly from about 3 to 15 ° C, preferably below about 10 ° C, because at these lower temperatures and the above dilution factors an increased yield of protein is achieved isolate in the form of protein micellar mass.

In a batch process, a batch of concentrated protein solution is added to a static column of chilled water having the desired volume, as discussed above. Dilution of the concentrated protein solution and subsequent decrease in ionic strength causes the formation of a flocculent mass of highly associated protein molecules in the form of discrete droplets of protein in micellar form. The batch process provides for the deposition of protein micelles in the thickness of chilled water with the formation of aggregated, coalescent, dense, amorphous, sticky, like gluten, protein micellar mass (PMM). Precipitation can be accelerated by centrifugation. Such induced precipitation reduces the liquid content in the protein micellar mass, thereby decreasing the moisture content in it in most cases from about 70 to 95 wt.% To about 50 to 80 wt.% Of the total micellar mass. Reducing the moisture content in the micellar mass in this way also reduces the content of absorbed salt in the micellar mass and, therefore, the salt content in the dry isolate.

Alternatively, the dilution operation can be carried out continuously by continuously feeding the concentrated protein solution into one inlet of the T-shaped pipe, while the dilution water is supplied to the other inlet of the T-shaped pipe, thereby achieving mixing in the pipe. Water for dilution is fed into the T-shaped pipe at a speed sufficient to achieve the desired degree of dilution of the concentrated protein solution.

Mixing the concentrated protein solution with water for dilution in the pipeline initiates the formation of protein micelles, and the mixture is continuously fed from the outlet of the T-shaped pipe into the sedimentation tank, from which the supernatant is drained when filled. The mixture is preferably fed into the bulk of the liquid in the sedimentation tank in a manner that minimizes turbulence in the bulk of the liquid.

The continuous method involves the deposition of protein micelles in a deposition tank with the formation of an aggregated, coalescent, dense, amorphous, gluten-like, sticky protein micellar mass (PMM), while the process continues until the desired accumulation occurs at the bottom of the deposition tank the amount of PMM, after which the accumulated PMM is removed from the sedimentation tank.

The precipitated isolate is separated from the residual aqueous phase or supernatant, for example, by decantation of the residual aqueous phase from the precipitated mass or by centrifugation. PMM can be used in wet form or can be dried in any convenient way, such as spray drying, freeze-drying or roller vacuum drying, to dry form. The dry flax protein isolate has a high protein content in excess of about 90 wt.%, Preferably at least about 100 wt.% (N × 6.25), mostly undenatured (as determined by differential scanning calorimetry). The dry flax protein isolate obtained from oilseed flour also has a low residual fat content when using the methods described in US patent No. 5844086 and 6005076, which may be less than 1 wt.%.

The supernatant from the stage of PMM formation and precipitation contains significant amounts of flax protein not precipitated at the dilution stage, so the supernatant can be subjected to further processing in order to extract additional quantities of protein from it.

In such a method, the supernatant from the dilution step followed by removal of the PMM can be concentrated to increase the protein concentration in it. Such concentration can be carried out using suitable selective membrane technology, such as ultrafiltration, using membranes with the required throughput for compounds with different molecular weights, for example, membranes capable of passing low molecular weight compounds, including edible salts and other non-protein low molecular weight materials extracted from the starting material raw materials, and at the same time keep flax protein in solution. Ultrafiltration membranes can be used, permeable to compounds with a molecular weight of from about 3,000 to about 100,000 Da, depending on the materials from which the membranes are made and the configuration of the membranes. Concentration of the supernatant in this method also reduces the amount of liquid that needs to be evaporated to extract the protein, and the energy consumption for drying. Before drying, the supernatant is concentrated in most cases to a protein content of about 50 to 300 g / l, preferably about 100 to 200 g / l.

The concentrated supernatant can be dried by any convenient method, such as spray drying, freeze-drying or roller drying, to obtain an additional dry flax protein isolate. This additional flax protein isolate has a high protein content, usually above about 90 wt.% (N × 6.25), preferably at least 100 wt.%, Mostly undenatured (as determined by differential scanning calorimetry). If necessary, wet PMM can be combined with concentrated supernatant before drying the combined protein streams by any suitable method to provide a combined flax protein isolate. The combined flax protein isolate has a high protein content in excess of about 90 wt.% (N × 6.25), preferably at least 100 wt.%, Mostly undenatured (as determined by differential scanning calorimetry).

In another alternative method, only a portion of the concentrated supernatant can be mixed with at least a portion of the PMM, and the resulting mixture is dried. The remainder of the concentrated supernatant can be dried, as well as the remainder of the PMM. In addition, dry PMM and dry supernatant can also be mixed in a dry manner in any desired proportions.

Using this method, it is possible to extract several types of flax protein isolates - in the form of dry PMM, dry supernatant and dry mixtures of PMM and supernatant taken in various ratios by weight, in most cases from about 5:95 to 95: 5 by weight, which may be desirable to provide various functional and nutritional properties.

Alternatively, the canola protein isolate method described in co-pending U.S. Patent Application No. 60 / 544,346, filed February 17, 2004, the assignee of which is the author of this application, and which is included in the list of references to this application. In accordance with the method described in the said application, the concentrated protein solution obtained in the concentration step is immediately dried and not subsequently processed to obtain PMM and a separate processing of the supernatant. Drying the concentrated protein solution, which may also optionally be diafiltered and degreased as described above, can be accomplished by any convenient method, such as spray drying, freeze-drying or roller-vacuum drying.

Protein isolates obtained by drying a directly concentrated protein solution are usually less pure, in particular they have a higher salt content than isolates obtained by the above method, therefore these isolates are preferably used not for food purposes, although they can be subjected to further processing for reducing salt content by any convenient method, such as, for example, dialysis.

The relative amounts of the corresponding proteins in any of the indicated protein isolates can be determined by any suitable analytical method, for example, an analytical separation method. The most commonly used of these methods use selective media in a column that allows size separation. For gel permeation chromatography, spherical materials in the form of a gel are used. In the case of applying pressure, as in high pressure liquid chromatography (HPLC), a harsh environment is used. The latter method is also known as dimensionless chromatography (SEC). The results obtained using such methods on samples of flax protein isolates obtained by the method described here are given in the examples (see below).

The flax protein isolate obtained from PMM and the flax protein isolate obtained from the supernatant mainly consist of 7S protein (1/2 lignin) having a molecular weight of about 162,000 to 169,000 Da, along with minor amounts of lignin having a molecular weight of about 415,000 to 440,000 Da, and colinin having a molecular weight of about 16,000 to 17,000 Da.

In most cases, a flax protein isolate obtained from PMM contains:

from about 65 to 95% by weight of 7S protein (1/2 linin);

from about 0 to 20 wt.% lignin and

from about 0 to 20 wt.% colinine.

In most cases, a flax protein isolate obtained from a supernatant contains:

from about 65 to 95% by weight of 7S protein (1/2 linin);

from about 0 to 20 wt.% lignin and

from about 0 to 20 wt.% colinine.

Information confirming the possibility of carrying out the invention

EXAMPLES

Example 1

This example illustrates the removal of mucus from flour from linoleum oil seeds.

Linole oil seeds were washed with sodium bicarbonate solution at various concentration levels by mixing aqueous sodium bicarbonate with linole oil seeds in a seed: solvent ratio of 1: 8 for one hour at 50 ° C. using a high-speed suspension mixer.

The seeds were washed with an aqueous solution of sodium bicarbonate at each of its tested concentrations in order to compare the amount of mucus removed from the seeds at different solvent concentrations. A total of 500 g of linole was washed in 4 L of sodium bicarbonate at each concentration.

The supernatant from each wash was decanted and 100 ml sampled from each supernatant was diluted 1: 1 with 88% ethanol to precipitate solubilized mucus. Then the mucus was collected and dried so as to calculate the total amount of mucus removed from the seeds.

The amounts extracted at various concentrations of sodium bicarbonate solution are shown in table I.

Table I The mass of mucus removed during the first wash at each concentration Sodium Bicarbonate Concentration Slime mass (g) 0.1 m 16,0 0.2 m 16,4 0.3 m 32,0

As can be seen from table 1, the concentration of sodium bicarbonate of 0.5 M is significantly more effective for removing mucus than its reduced concentration. In addition, at a concentration of 0.5 M, the seeds were still slippery to the touch, due to the presence of mucus. Secondly, an identical flush was carried out, as a result of which 34 grams of mucus were additionally removed. After this second wash, a third wash was carried out and an additional 36.4 grams of mucus were removed. And finally, the fourth wash gave very little mucus, which indicated the complete removal of mucus from 500 grams of linole seeds. The total amount of mucus removed was 102.8 grams.

After these washes, the seeds were no longer slippery to the touch, as was the case with mucus, which was another sign that most of the mucus was removed.

Example 2

This example illustrates the preparation of flax flour in accordance with one embodiment of the invention.

To 25 kg of linole oil seeds, grade 2047, 200 L of 0.5 M sodium bicarbonate was added at 50 ° C. in a 400 L mixing tank. The suspension was thoroughly mixed for 1 hour. After settling, the aqueous phase was decanted, and the precipitate was discarded. A 1 liter portion of the decanted aqueous phase was diluted with an equal volume of ethanol to precipitate mucus in order to make an approximate estimate of the amount of mucus recovered.

After decantation of the aqueous phase, the seeds were washed twice with hot tap water to remove residual wash solution. The operations of extraction with sodium bicarbonate, separation and washing were repeated five times. Then the seeds were additionally washed four times with hot tap water to remove residual wash solution and mucus. It was found that the seeds lost their characteristic mucous appearance, which also served as a sign of mucus removal.

It was found that each subsequent rinse with an aqueous sodium bicarbonate solution removed less mucus than the previous one, and that after the fifth rinse, very little mucus was deposited from the one-liter portion of the washing solution diluted with ethanol, which served as a good sign that most of the mucus was removed from the seeds .

Then the seeds were dried, washed and degreased in order to extract oil from the seeds.

Example 3

This example illustrates the preparation of linole protein isolate from flour with reduced mucus by isoelectric precipitation.

10 kg of fat-free oilseed linole seeds prepared as described in Example 2 were added to 200 L of a 0.15 M NaCl solution at room temperature and the pH of the mixture was adjusted to 11.0 by adding a 50 wt.% Sodium hydroxide solution. The suspension was stirred for one hour, after which the extracted flour was precipitated by settling from the resulting protein solution for one hour.

Then 100 l of a protein solution having a protein content of 13 g / l was discharged from the precipitate and filtered through filters of 20 and 0.2 μm in a filter press in order to clarify the solution. The resulting clarified solution was placed in a refrigerator at 4 ° C for 16 hours so that the oil present in it rose to the surface from where it can be collected. There was very little oil, indicating a very effective degreasing step.

Then, the pH of the protein solution at ambient temperature was adjusted to 4.0 using 3 N HCl, and the protein began to precipitate immediately, changing the color of the solution from golden yellow to milky white. As soon as mixing was stopped, the protein precipitated rapidly from the solution. After a two-hour precipitation period, the supernatant was decanted and assayed for protein content.

After removal of the supernatant, 10 L of lumpy material was centrifuged at 10,000 xg for 5 minutes to reduce the residual supernatant in the precipitated protein. The resulting cake was restored in 4 L of water and dried by spray drying to obtain 293 g of dry linole protein isolate from the IEP stage. The protein content of the spray-dried isolate was 101% by weight (N × 6.25) in dry matter.

Example 4

This example illustrates the functional properties of the linole protein isolate obtained in example 3.

The linole protein isolate from the IEP stage, obtained in accordance with the method of example 3 (linole IEP), was tested for such functional properties as foaming and oil-holding ability, and compared these properties with typical canola protein isolate samples obtained from PMM and from the supernatant by the method described in co-pending application for US patent No. 10/137391, filed May 3, 2002 (WO 02/089597), the assignee of which is the author of this application, and which is included in the list of links to this application.

The testing methods used were similar to those described in co-pending application for US patent No. 10/137306, filed May 3, 2002 (WO 02/089597), the assignee of which is the author of this application, and which is included in the list of links to this application.

To evaluate foaming ability, the technique described by Philips et al. in J. Food Sci., 55: 1441, 1990. 3.75 g protein isolate samples were placed in individual 150 ml beakers. 60 ml of a 0.075 M NaCl solution were added to the protein and a paste was first prepared by dissolving the protein with several ml of liquid. The mixture was stirred on a magnetic stirrer for 10 minutes, the pH of the solution was adjusted to 7.00 using a 0.1 M NaOH solution, and the solution was stirred for another 10 minutes. Then the pH was again adjusted to 7.00, and the liquid volume was adjusted to 75 ml using the required amount of 0.075 M NaCl to obtain a 5% w / v protein solution. 75 ml of the solution was poured into a cup of a Hobart mixer and the total weight of the solution, cups and beater of the mixer was fixed. The protein solution was whipped at a speed of 3 for 5 minutes.

A sufficient amount of foam was carefully selected using a rubber spatula to fill two measuring cups, calibrated to 125 ml. The excess foam was removed using the flat end of a large knife to even the level of foam in the glass along the upper edge of the measuring glass and the foam mass was fixed. The foam was carefully returned to the mixer cup and whipped for another 5 minutes. Then this procedure was repeated. The foam was carefully returned to the mixer cup and whipped for another 5 minutes, for a total of 15 minutes. The procedure was repeated again.

% overrun was calculated by the following equation:

The stability of the foam was also tested. A protein solution was prepared in the same way as for determining% overrun, except that the protein solution was whisked for 15 minutes at speed 3. Using a rubber spatula, the foam was carefully transferred into a funnel with a long end dropped into a 250 ml graduated cylinder . A small amount of glass wool was placed in the opening of the funnel socket to prevent foam from dripping off and at the same time to provide fluid drainage.

The volume of fluid accumulated in the graduated cylinder was measured after 5, 10 and 15 minutes. The volume retained by the cotton was added to the final volume.

The method described by Swift et al. Was used to determine oil retention capacity. at Food Technol. 15, 436-72 (1961).

The emulsion was prepared according to the recipe shown in table II.

Table II Ingredient Added weight (g) Protein isolate 0.5 Vinegar (untitled, 5% acetic acid 55,2 Canola Oil (CSP Foods) N / d Sugar (Rogers, fine granular) 4.1 Salt (Sifto) 1,2 Distilled water 52,4 N / D = not defined.

Sugar, salt and protein isolate were mixed dry in a 600 ml beaker. Next, water and vinegar were mixed, initially preparing a paste by dissolving the protein with several ml of liquid. The mixture was stirred on a magnetic stirrer for 5 minutes. A 2000 ml beaker was filled with canola oil and the mass was fixed. A suction hose was placed in the oil.

The discharge end of the hose was connected to the homogenizer, and the pump was flooded with oil to mark # 1 to ensure a flow rate of 40 to 50 ml / min. At the same time, the homogenizer (Silversen LHRT) was turned on at a speed of 5000 rpm./min, and the pump was turned on to supply oil. The point at which the emulsion was the most viscous was noted visually. At the inversion point, the pump and homogenizer were immediately turned off. The end of the suction hose was intercepted with a clamp to hold the oil in it and the mass of oil poured into a glass of 200 ml was determined.

The results are shown below in tables III and IV.

Table III Comparison of linole protein isolate obtained by IEP and canola protein isolate (CPI) obtained from PMM The consignment % overrun (foam volume) Foam stability (MI expiration in 15 min) Oil Retention Capacity (MI oils / 100 mg protein) Globular Size (μm) CPI-1 1471.8 17.5 147.7 18.9 CPI-2 1030,4 32,7 190.5 29.1 CPI-3 1216.5 24.0 119.8 24.3 CPI-4 1051.2 45.3 115.4 21.6 CPI-5 1091.6 35.3 124.5 28.0 CPI-6 1196.1 34.0 166.9 24.6 IEP Linole Protein Isolate 1770.9 0.67 118.9 59.0

Table IV Comparison of linole protein isolate obtained by IEP and CPI canola obtained from supernatant The consignment % overrun (foam volume) Foam stability (MI expiration in 15 min) Oil Retention Capacity (MI Oils / 100 mg Protein Globular Size (μm) CPI-7 2603.6 22.7 67.2 72.6 CPI-8 1984.8 21.3 53.6 151.7 CPI-9 1924.4 22.0 43.3 151.7 CPI-10 1889.2 17.3 41.3 192.4 IEP Linole Protein Isolate 1170.9 0.67 118.9 59.0

As can be seen from Table III, the linole protein isolate obtained by IEP had a foaming ability superior to the foaming ability of canola protein isolates obtained from PMM, providing a larger foam volume and its lower fluidity (higher stability). The oil-holding ability of the linole protein isolate obtained by IEP was comparable to the oil-holding ability of the canola protein isolate obtained from PMM, but had a large globular size.

As can be seen from Table IV, the volume of the foam formed by the linole protein isolate obtained by IEP was less than the volume of the foam formed by the canola protein isolate obtained from the supernatant, but the foam was significantly more stable. The linole protein isolate had better emulsifying properties compared to the canola protein isolate isolated from the supernatant. The oil holding capacity of the linole protein isolate was approximately two times higher than the canola protein isolate isolated from the supernatant, and had a smaller globular size.

Example 5

This example illustrates the preparation of a linole protein isolate in a micellar manner from flour with a reduced mucus content.

4 kg of fat-free flour from linoleum oil seeds prepared as described in Example 2 was added to 80 L of a 0.5 M NaCl solution at room temperature (5 wt.% / Vol.). The suspension was stirred for one hour, after which it was precipitated by settling for 1/2 hour and the aqueous protein solution was drained from the precipitate. The aqueous protein solution obtained by decantation had a protein content of 7.1 g / l and a volume of 55 l. The solution was filtered through a 20 μm multilayer filter in a filter press to remove suspended solids. The press was washed with 20 L of water in order to provide 75 L of the filtrate having a protein content of 5.28 g / L.

The filtrate was subjected to ultrafiltration using ultrafiltration membranes permeable to compounds with a molecular weight of 300 Da, in order to concentrate the solution to 1.3 l of concentrated aqueous protein solution (retentate) having a protein content of 174 g / l. Then the retentate was diluted in 9 volumes of water with a temperature of 4 ° C, which led to the formation of white turbidity from protein micelles.

After a settling period of 16 hours, the supernatant was decanted and centrifuged to extract as much precipitated material as possible and to provide 11 L of supernatant having a protein content of 1.11 g / L. The precipitate of linole protein isolate obtained in the precipitation step was also centrifuged to reduce its volume to a minimum.

The precipitate of linole protein isolate was dried to obtain 81 g of dry protein, which corresponded to 20 wt.% The yield of protein extracted from flour from linole seeds. The dry linole protein isolate had a protein content of 112% by weight (N × 6.25) in dry matter.

The clarified supernatant was concentrated using membranes permeable for compounds with a molecular weight of 300 Da to 1.25 L of concentrated supernatant containing 63.3 g / L of protein. The concentrated supernatant was dried to obtain 77 g of linole protein isolate (20% yield) having a protein content of 106 wt.% (N × 6.25) in dry matter.

The analysis of two fractions of linole by high pressure liquid chromatography (HPLC) was performed as described in co-pending application for US patent No. 10/413371, filed April 15, 2003 (WO 03/088760), the assignee of which is the author of this application, and which is included to the list of links to this application.

Example 6

This example illustrates the functional properties of linole protein isolates obtained from PMM and from the supernatant according to example 5.

The linole protein isolates from PMM and from the supernatant obtained by the methods of Example 5 were tested for functional properties such as foaming and oil-holding ability, in accordance with the methods described in Example 4, and compared by the indicated properties with typical canola protein isolates (CPI) from PMM and from the supernatant obtained by the method described in the aforementioned application for US patent No. 10/137391 (WO 02/089597).

The results are presented in the following tables V and VI.

Table v Comparison of linole protein isolate and CPI from PMM The consignment % overrun (foam volume) Foam stability (MI expiration after 15 min) Oil Retention Capacity (MI oils / 100 mg protein) Globular Size (μm) CPI-1 1471.8 17.5 147.7 18.9 CPI-2 1030,4 32,7 190.5 29.1 CPI-3 1216.5 24.0 119.8 24.3 CPI-4 1051.2 45.3 115.4 21.6 CPI-5 1091.6 35.3 124.5 28.0 CPI-6 1196.1 34.0 166.9 24.6 PML Linole Protein Isolate 1464.0 2.0 139.5 19.8 Supernatant Linole Protein Isolate 1470.0 0 81.4 11.8

Table VI Comparison of linole protein isolate and CPI from supernatant The consignment % overrun (foam volume) Foam stability (MI expiration after 15 min) Oil Retention Capacity (MI oils / 100 mg protein) Globular Size (μm) CPI-7 2603.6 22.7 67.2 72.6 CPI-8 1984.8 21.3 53.6 151.7 CPI-9 1924.4 22.0 43.3 151.7 CPI-10 1889.2 17.3 41.3 192.4 CPI-11 2776.8 4.0 47.1 118.9 PML Linole Protein Isolate 1464.0 2.0 139.5 19.8 Supernatant Linole Protein Isolate 1470.0 0 81.4 11.8

As can be seen from Tables V and VI, the functional properties of linole protein isolates from PMM and from the supernatant are very similar, which could be expected from the similarity of their HPLC properties, and the main differences in emulsifiability between the two fractions are oil-holding ability and globular size.

In most cases, the functionality of linole protein isolates from PMM and from the supernatant is equally good or superior to the functionality of canola protein isolates from PMM and from the supernatant. The linole protein isolates from PMM and from the supernatant are weaker in terms of the volume of the foam they form, but the linole protein isolates were superior to canola protein isolates in the stability of the foam formed by them.

Example 7

This example illustrates the analysis of linole protein isolates from PMM and from the supernatant obtained in example 5.

An HPLC analysis of two linole protein isolates carried out as described in co-pending application for US patent No. 10/413371, filed April 15, 2003, the successor to which is the author of this application, and which is included in the list of links to this application, showed that each isolate consists mainly of the same components, as can be seen in Figs. 1 and 2. In both linole protein isolates, the main protein component has a molecular weight of about 162,000 to 169,000 Da, and those contained in a smaller amount have mole molecular weight, respectively, from 16,000 to 17,000 Yes and from 415,000 to 440,000 Yes. These results are summarized in tables VII and VIII.

Table VII HPLC linole protein isolate profiles from PMM Protein fractions 12S-linin 78-1 / 2 line 2S-
colinin Other % protein peaks 11.9 78.0 9.2 0.9 M.M. in kDa 415-440 162-169 16-17

Table VIII HPLC supernatant linole protein isolate profiles Protein fractions 12S-linin 7S-1/2 line 2S-
colinin Other % protein peaks 6.4 76.9 12.6 4.1 M.M. in kDa 415-440 162-169 16-17

Example 8

This example illustrates amino acid analysis.

Linole protein isolates prepared as described in Examples 3 and 5 were analyzed for amino acid content.

Amino acid analysis is presented in the following table IX.

Table IX g / 100 g dry matter M.M. (1) amino acids Amino acid Isolate obtained from PMM Isolate obtained from supernatant IEP Isolate 133.1 Aspartic 11.50 11.40 11.90 119.1 Threonine 3.97 3.80 3.77 105.1 Serine 4.74 4.87 4.88 204.2 Tryptophan 1.84 1.81 1.75 146.1 Glutamine 21.10 21.00 19.10 75.1 Glycine 5.61 5.62 5.26 89.1 Alanine 4.67 4.78 4.90 121.1 Cysteine 1.30 1.28 0.96 117.1 Valine 5.30 5.70 5.92 149.2 Methionine 1.47 1.42 1.48 131.2 Isoleucine 4.33 4.49 4.93 131.2 Leucine 5.33 5.36 5.76 181.2 Tyrosine 2.29 2,36 2.25 165.2 Phenylalanine 4.85 4.86 5.86 155.2 Histidine 2.03 2.02 2.09 146.2 Lysine 2.73 2,34 2.69 174.2 Arginine 11.80 12.10 12,20 115.1 Proline 3.73 3.87 4.05 Total content: 98.57 99.08 99.75 Average M.M. (1) amino acids 134.75 135.33 136.43 m.m.b anhydrous state (2) 116.74 117.32 118.41 Note: (1) The molecular weight of the “free” amino acids. (2) Weighted average molecular weight of polymer amino acids. Note: no adjustments have been made for glutamine or asparagine, which are included respectively in glutamic and aspartic acids.

The values presented in table IX reflect the number of amino acids in grams per 100 grams of dry matter. Data were recalculated per 100 grams of amino acid, and the newly obtained values are presented in the following table X.

Table X Total amino acid content: g / 100 g amino acids Amino acid Protein Isolate Obtained from PMM Protein Isolate Derived from Supernatant IEP Protein Isolate Aspartic * 11.7 11.5 11.9 Threonine ^e 4.0 3.8 3.8 Series 4.8 4.9 4.9 Tryptophan ^e 1.9 1.8 1.8 Glutamine * 21,4 21,2 19.1 Glycine 5.7 5.7 5.3 Alanine 4.7 4.8 4.9 Cysteine ^e 1.3 1.3 1,0 Valin ^e 5,4 5.8 5.9 Methionine ^e 1,5 1.4 1,5 Isoleucine ^e 4.4 4,5 4.9 Leucine ^e 5,4 5,4 5.8 Tyrosine 2,3 2,4 2.7 Phenylalanine ^e 4.9 4.9 5.9 Histidine ^e 2.1 2.0 2.1 Lysine ^e 2,8 2,4 2.7 Arginine ^e 12.0 12,2 12,2 Proline 3.8 3.9 4.1 Amount: 100.0 100.0 100.0 Amount of irreplaceable a.k. 45.6 45.6 47.5 e = 11 essential amino acids; A.K. = amino acids;
* Glutamic acid and aspartic acid include glutamine and asparagine, respectively.

As can be seen from table IX and table X, the amino acid profiles of linole protein isolates obtained from PMM, from the supernatant and by IEP are very similar.

Table IX includes the molecular weights of the individual amino acids. The average molecular weights of the “free” amino acids are shown together with their individual amounts in three protein isolates, and in total they are approximately 135 Da.

Weighted average molecular weights (M.M.) are also shown in the anhydrous state, since the proteins are biopolymers of anhydrous amino acids (each minus a water molecule, with the exception of one terminal amino acid in the polypeptide). The average molecular weight of the polymer amino acids in the polypeptide is from about 117 to 118 kDa. Table X also shows the essential amino acids that cannot be synthesized by the human body. The total eleven essential amino acids are very similar in all three protein isolates.

Example 9

This example illustrates the analysis of linole protein isolates by differential scanning calorimetry.

Linole protein isolates prepared as described in Examples 3 and 5 were analyzed by differential scanning calorimetry. Differential scanning calorimetry is an instrumental method that measures the phase transition that occurs in biomolecules. The sample is placed in a hermetically sealed vessel with a certain amount of water or a buffer solution and is heated at a constant speed, for example 10 ° C / min, in a specific temperature range, for example from 20 to 150 ° C. A second vessel containing water or a buffer solution is heated simultaneously and serves as a control. As the temperature rises, a thermogram of energy absorption, called an endothermic heat flux, is recorded. Complex biological compounds, such as proteins, absorb energy, and this energy changes the conformation of the molecule, denaturing or unfolding it. Denaturation occurs specifically in individual proteins or other biomolecules, and analysis allows one to determine the denaturation temperature T _D and the change in enthalpy (ΔН) in joules / g of sample. A thermogram shows an energy “well” representing a phase transition from a native protein to a denatured one. The bottom of the "well" represents the value of T _D. The absence of an energy “well” indicates that the denaturation is complete.

As can be seen from Figs. 3, 4 and 5, linole protein isolates obtained from PMM and from the supernatant are very similar in their thermal stability, which could be expected from their identical properties in HPLC, and the linole protein isolate from PMM has slightly more high stability. The thermogram obtained by differential scanning calorimetry for the linole protein isolate obtained by the IEP method indicates that this protein isolate is more denatured in contrast to linole protein isolates obtained from PMM and from the supernatant, which are essentially undenatured.

Summary of the invention

Briefly, the present invention provides an improved method for the production of flax protein isolates, in which the mucus is preliminarily extracted from oilseeds of flax before the subsequent isolation of oil and the preparation of flour from oilseeds of flax, which provides an increased yield of the obtained protein isolates and greater flexibility in the method of isolation . The present invention also provides novel flax protein isolates having a unique protein profile. Modifications to the scale of the present invention are possible.

Claims (12)

1. A method of obtaining a protein isolate of flax, including removing mucus from oily flax seeds by extracting the seeds with a mildly alkaline aqueous solution of mildly alkaline material at a temperature of 30 to 70 ° C with a seed to solution ratio of 1: 1 to 1:20, crushing extracted oilseeds to extract oil and produce flour, and processing the flour to extract a flax protein isolate from it, comprising isoelectric precipitation of a flax protein solution from an alkaline flax protein solution obtained by extracting the flour with an aqueous alkaline solution, or solubilization of the protein in said flour from oilseeds of flax by extraction using an aqueous solution of sodium chloride having an ionic strength of at least 0.10 M at a pH of 5 to 7 to provide an aqueous solution of a protein having a concentration of from 5 to 40 mg / l concentration of the aqueous protein solution to a concentration of at least 150 g / l using selective membrane technology, diluting the concentrated protein solution with water having a temperature below about 15 ° C to form protein micelles and collection and extraction of these protein micelles in the form of a protein micellar mass of flax protein isolate. 2. The method according to claim 1, in which the moderate alkaline aqueous solution of the moderate alkaline material has a pH of from 7.5 to 9, and preferably the concentration of the moderate alkaline material is from 0.2 to 0.7 M. 3. The method according to claim 1, in which the ratio of seeds to solution is from 1: 5 to 1:10. 4. The method according to claim 1, which is carried out by mixing oilseeds in an aqueous solution for 15 to 60 minutes, preferably 30 to 60 minutes. 5. The method according to claim 1, in which the extraction of oilseeds is carried out repeatedly, until the mucus is no longer extracted from the oilseeds. 6. The method according to claim 1, wherein said moderately alkaline material is sodium bicarbonate, preferably used in the form of an aqueous solution of sodium bicarbonate with its natural pH. 7. The method according to claim 1, in which the protein micellar mass is dried. 8. The method according to claim 7, in which the residual liquid from the stage of extraction of the protein micellar mass is processed to obtain additional amounts of flax protein isolate. 9. Flax protein isolate having a protein content of at least about 90 wt.%, Preferably at least 100 wt.% (N · 6.25) and comprising 7S protein with a molecular weight of from about 162,000 to 169,000 Yes obtained according to the method according to claim 1. 10. Flax protein isolate according to claim 9, which is mainly not denatured, which was established by differential scanning calorimetry. 11. Flax protein isolate according to claim 9 or 10, characterized in that the 7S protein content is from 60 to 95 wt.%, Linin is from 0 to 20 wt.% And colinin is from 0 to 20 wt.%. 12. Flax protein 7S, having a molecular weight of from about 162,000 to 169,000 Da, isolated from the flax protein isolate of claim 9.

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