KR100705282B1 - Making process for protein isolate from rapeseed - Google Patents

Abstract

The present invention relates to a method for producing a separated protein from rapeseed, and more particularly, unlike the skimmed wheat prepared by conventional methods, the content of almost degreasing and phenolic compounds produced using supercritical fluid is reduced. Extracted using the rapeseed skim wheat as a raw material, using a minimum amount of antioxidants and brine, and simply processed and recovered using a cooled alcohol aqueous solution to produce a protein with improved whiteness in high yield.

Rapeseed, skimmed wheat, supercritical fluids, phenolic compounds, protein production

Description

MAKING PROCESS FOR PROTEIN ISOLATE FROM RAPESEED

Figure 1 is a graph showing the extraction rate according to the pressure and time change at a constant temperature (50 ℃), the flow rate of the fluid (4 liter / hour) during the oil extraction using a supercritical fluid.

2 is a graph showing the content of the separated protein according to the concentration rate.

The present invention relates to a method for producing a separated protein from rapeseed, and more particularly, unlike the skimmed wheat prepared by conventional methods, the content of almost degreasing and phenolic compounds produced using supercritical fluid is reduced. Extracted rapeseed skim wheat as a raw material, using a minimum amount of antioxidants and brine, and a simple process using a cooled alcohol solution to recover and recover the protein with improved whiteness.

Protoss is, in Greek, the most important and the most excellent. Animal protein is a recent trend of increasing vegetable protein products because people are reluctant to cow mad cow disease or E-Colli problem.

Until now, soybean and wheat proteins have led the plant protein market. In general, the isolation and production of vegetable proteins such as sunflower, soybean, rapeseed, and peanut have been widely used by extracting them with alkaline solution and then adding acid to the extract. The existing methods of cruciferous and especially rapeseed proteins are as follows.

US Patent 3,758,452 (1973) prepared an isoelectrically precipitated protein by removing an acid from a protein solution extracted using a salt solution instead of an alkaline solution, and US Patent 3,993,636 (1976) prepared an alkaline solution and sodium sulfite (0.5). %) Extract rapeseed protein by using ultrafiltration, add washing solution, and continue ultrafiltration again until dried to 70 ~ 85% of nitrogen and add acid to adjust isoelectric point to precipitate protein. .

In US Patent 4,366,097 (1982), rapeseed protein was extracted by adding water only to a raw material and precipitated by adding water to the extracted protein solution again.

In US Patent 4,889,921 (1989), which is the same as European Patent 0 289 183 (1988), rapeseed protein is extracted with alkaline solution and acid is added to adjust the pH to 3.5 to separate the precipitated isoelectric protein and the solution portion. Impurities were removed through filtration to yield at least 90% soluble isolated protein.

Looking at the above manufacturing method, the protein is extracted by using alkaline solution, salt solution or water, and the protein solution concentrated by direct or ultrafiltration is adjusted with acid to obtain isoelectric point protein and simply by adding water again. Precipitation methods. However, the isoelectrically precipitated protein using alkali or acid is denatured and loses the original protein's functionality although there is a degree of difference. In addition, it is known that lignolanine, which is produced by alkali or heat treatment, is poorly absorbed in the intestine and harmful to the kidneys in animal experiments. In addition, the reduction of lysine, which is an essential amino acid due to lignolananine, will eventually reduce the quality of the protein. The sedimentation method of extracting with water and adding water again is simple, but because it is extracted with water, globulin protein that is well soluble in salt solution is difficult to extract and is obtained by adding water again. There is a problem that the yield is significantly reduced.

On the other hand, US Patent 4,169,090 (1979) proposed a theory that the protein is extracted using a salt solution, concentrated and put in cold water to form a protein mass through a critical protein concentration (critical protein concentration).

Subsequently, a series of patents having an association such as US Patent 4,169,090 (1979), US Patent 4,208,323 (1980) shows the non-denaturation and good functionality of the isolated protein obtained in the micelle form. 4,285,862 (1981) have been used as a binder to replace the functionality of egg whites in food compositions of the present invention, and 5,844,086 (1998) and 6,005,076 (1999) are fat components of Although the manufacturing method of separating fat from the contained wheat to obtain a high content of separated protein is shown, the Murray patents show that the content of rapeseed protein is less than 90%, or the purity and content are not substantially provided. Or rapeseed skimmed wheat contains commercially available skimmed wheat or fat containing about 10% and skimmed wheat by cold press method. In the mill manufacturing method by the cold press method, oil of about 10% is easy to remain in wheat as much as 3 to 8%. This residual oil can be a barrier to the extraction of protein from wheat, and the oil contained in the extract does not facilitate the separation of the protein solution, and the extract must be cooled to low temperature to separate the oil layer floating on the upper layer. There is a problem that there is a cumbersome process.

As a domestic published patent, Korean Patent No. 2004-0026651 covers patents ranging from US Pat. Nos. 4,169,090 (1979) to 6,005,076 (1999), and is obtained by concentrating a protein from a supernatant portion in addition to the precipitated protein in the process of recovering the final separated protein. In other words, the yield is increased by at least 40%, but other parts are shown at the same level. As mentioned earlier, degreasing wheat by cold press requires a cumbersome cold separation process to remove fat components.

In addition, the protein solution is concentrated through ultrafiltration, and then the precipitate is precipitated with cold water to obtain precipitated protein, and the supernatant is reprocessed and concentrated to improve the yield, but it takes a lot of time due to reprocessing. Accordingly, the color may change due to the long concentration time of the protein solution, and the filtration membrane may also become less efficient due to the fine particles of the protein.

Therefore, there is an urgent need for a high yield protein production method while the process is easy and the whiteness is improved.

Therefore, the present inventors have studied to solve the above problems, and as a result, the skim wheat obtained from the rapeseed using supercritical fluid and degreasing the content of the phenolic compound is almost reduced, and the degreasing mill is treated with a minimum amount of antioxidant. The present invention was completed by developing a method of producing rapeseed protein with high yield by easily extracting with brine and recovering with a cooled aqueous alcohol solution.

Accordingly, an object of the present invention is to provide a method for producing a protein with improved whiteness from rapeseed skim wheat in high yield.

The present invention is characterized by a method for producing a protein with improved whiteness from rapeseed skim wheat in high yield.

The present invention will be described in more detail as follows.

The present invention, unlike the skim mill prepared by the conventional method, using a skim skim mill, which is almost degreased and reduced in phenolic compound content using a supercritical fluid, using a minimum amount of antioxidants and brine Extraction, and using a cooled alcohol aqueous solution to simply process the recovery and relates to a method for producing a protein with improved whiteness in high yield.

Briefly describing a method for producing a protein with improved whiteness from rapeseed skim wheat according to the present invention in high yield,

1) obtaining skim wheat using rapeseed seed using supercritical fluid and co-solvent lower alcohol;

2) extracting the skimmed wheat with brine, and adding and extracting a sodium sulfite and sodium hydrogen sulfite mixture having a pH of 5.0 to 6.5 to obtain a protein solution;

3) concentrating the protein solution at 15-20% (w / v);

4) recovering the precipitated protein by mixing the concentrate with an aqueous alcohol solution of 1 ~ 5 ℃

It is made to include, described in detail step by step as follows.

First, the production of degreasing mill produces wheat using supercritical fluids, which means that the oil can be removed almost without applying high heat from pre-ground seeds, and this degreasing supercritical mill Extraction of the protein by means of the present invention facilitates the filtration separation without the cooling separation process of the protein extraction solution, it is easy to obtain a clear and clean protein solution.

Supercritical fluids are easy to change state of low density and high density by using temperature and pressure. That is, the fluid equilibrium (solubility), transfer property (viscosity, diffusion coefficient), and solvation state can be controlled by easy change of fluid properties. Known facts, carbon dioxide, a fluid used in it, is harmless to humans (non-toxic), has little effect on environmental pollution (eco-friendly), prevents oxidation of substances that are susceptible to oxidation, especially organic solvents used in solvent extraction. Unlike those, there is no risk of ignition.

The mill thus produced has less than 3.0% oil remaining, and the biggest factor for this is the supercritical pressure. The pressure on the supercritical fluid is 250 to 650 should be bar. And the extraction temperature is 35 ~ 55 ℃, the flow rate of carbon dioxide is 2 to 6 liters per hour, the extraction time is inversely proportional to the pressure. More than 90% of the oil is extracted in 3 to 6 hours, the remaining oil is less than 3% It should preferably be treated for 5-10 hours [Fig. 1]. In addition, the particle size and moisture content of the ground seeds can also be affected.

In particular, in the present invention The use of lower alcohols as co-solvents in supercritical fluids can reduce oxidizable phenolic compounds in degreasing. This phenolic compound is involved in the taste and color of the protein, resulting in an unpleasant bitter taste, and the color is exposed to air during processing to oxidize into activated quinoid forms, which binds to the protein, reducing digestive absorption. It has a dark brown to black color, which is not good.

In practice, the use of carbon dioxide fluid (solvent) alone in supercritical extraction is difficult to remove phenolic compounds, but by using co-solvent lower alcohols, the content of phenolic compounds in degreasing wheat is reduced by 65 to 50% (first volume). Wherein lower alcohol refers to an alcohol having 1 to 4 carbon atoms, preferably methanol or ethanol. In addition, the content ratio of the co-solvent when mixed with the co-solvent is limited to 10% by volume or less, preferably 5 to 10% by volume with respect to the supercritical fluid, the temperature and pressure is 35 ~ 50 ℃, 350 ~ 650 Bar, and the co-solvent flow rate of 0.1 ~ 0.6 liters per hour, extraction time was carried out for 30 to 150 minutes, preferably 30 to 120 minutes.

Here, glass beads or diatomaceous earth can be used to increase the extraction rate in order to increase the contact of the supercritical fluid with the solute and to avoid the diffusion and penetration of the fluid into the selective and identical routes of the fluid.

Degreasing or reducing phenolic compounds, which are anti-nutrients, is carried out continuously. Since the supercritical fluid is carried out with carbon dioxide, it is blocked from contact with air, thereby preventing the oxidation of phenolic compounds sensitive to air oxidation. Blocking and reducing produces good wheat. This reduces the browning of the protein solution due to the reduced phenolic compounds that reduce protein extraction and ultrafiltration from wheat.

The extracted phenolic compound reduced skim wheat was extracted by stirring with brine, but a vitamin C or a mixture of sodium sulfite and sodium hydrogen sulfite with a minimum amount of antioxidants to maintain a protein solution with improved whiteness during the process, In addition, the protein extract is filtered in a short period of time using a mixture of clay and diatomaceous earth in a suitable ratio to maintain a clear and good color protein solution until recovery to obtain a high quality protein.

Salt water used with the minimum amount of the antioxidant is used in the 0.4 ~ 0.5 molar concentration, it is preferable to extract 10 to 15 times the volume of the skim wheat, less than 0.4 molar concentration or less than 10 times the extraction When the extraction amount of protein is reduced and the extraction amount of protein is more than 0.6 molar concentration or more than 16 times, the extraction amount of protein is similar, so the salt concentration and the brine capacity are increased so there is no advantage.

In this case, a method of extracting protein can generally be agitated or treated with a homomixer, but the latter method can be expected to reduce time and efficiency.

The minimum amount of the antioxidant is the amount that maintains the protein solution of the good color during the process of the protein solution and at the same time there is no abnormality in the protein yield, as mentioned above is prepared by the supercritical method to reduce the skim wheat reduced phenolic compounds Since the protein is extracted, the amount of antioxidants can maintain a good color even with a minimum amount.In the case of vitamin C, 0.01 to 0.1% by weight of the skimmed wheat used for extraction is used.Sodium sulfite and sodium bisulfite It is preferable to adjust the pH within 5.0 to 6.5 by mixing to use 0.005 to 0.05% by weight in the process of protein.

In addition, the crude earth refers to a mixture of clay and diatomaceous earth, the ratio is 1: 1 to 1: 5 weight ratio is appropriate, the amount is preferably used 3 to 5% by weight based on the amount of the treated mill. Filtration under these conditions allows rapid processing in a short time and the protein solution obtained is clear and clean. Here, the short time means that the protein extract is filtered, so that the filter paper is almost clogged with various components mixed with the solution, such as protein, sugar, soluble starch or cellulose debris. Can be. Thus, the use of crude clay mixed in a suitable ratio of white and diatomaceous earth can be filtered very quickly, if necessary re-filtered with 0.45 ~ 0.2 ㎛ room.

Filtration method using activated charcoal can be expected to have similar effect, but because it has the property of adsorbing protein, loss of recovery amount is excluded.

In addition, the present invention provides a method for obtaining a protein by a simple treatment in the process of recovering the protein from the protein concentrate.

First, the concentration of the protein solution is concentrated to 15 ~ 20% (w / v) to slowly pour an aqueous solution of alcohol below 5 ℃ precipitates will occur. At this time, the temperature of the concentrated solution should be adjusted so that the mixed content becomes 1 ~ 5 ℃. The precipitation method of this aqueous alcohol solution dilutes the concentrated solution with cold water to precipitate the protein, which has less molecular weight than the primary precipitated protein. The small amount of the last remaining color, while precipitating even the water-soluble protein, is made to be dissolved in the supernatant. Higher yields can be achieved even at relatively low concentrations, such as% (w / v), which can increase the efficiency of ultrafiltration membranes and reduce the amount of ultrafiltration. The filtration membrane was carried out at room temperature using cutoff 50 (kilo daltons) to 100 (kilo daltons).

The protein concentrate here is that concentrations above 15% (w / v) can be important critical concentrations. The reason for this is that it is not suitable when the protein solution is concentrated and precipitated at a concentration of less than 15% (w / v) because the protein content is less than 90%. Degrades. Therefore, it is desirable to concentrate to 15 ~ 20% (w / v) concentration, and also to increase protein purity and salt content by washing with 0.2 ~ 0.4 mol salt solution (concentration lower than molar concentration during extraction). Results in a lowering. At this time, the ratio of the concentrated solution and the aqueous alcohol solution is also an important factor, and the ratio of the concentrated solution and the aqueous alcohol solution is preferably mixed in a ratio of 1: 1 to 1: 3 (weight ratio). If the use of an aqueous alcohol solution is less than 1: 1, the recovery rate is lowered. If it is greater than 1: 3, the protein content is lowered, and the color material is precipitated together. Moreover, as for aqueous alcohol solution, 80 to 95% of methanol and ethanol are preferable.

The recovery of precipitated protein is 90% or more (concentrate), and the protein content is 90% or more, so that high-quality protein with light yellow color or slight light green color can be easily recovered at once. In addition, the recovered protein was completely dissolved when redissolved in the salt solution.

Therefore, the present invention solves the disadvantages mentioned in the existing method while preparing skimmed wheat using supercritical fluid, maintaining the protein solution extracted from the wheat as a raw material, and light color and high yield by simple protein precipitation treatment. It is a method of separating proteins with a high temperature, and this method is very cost and time efficient because there is no separate process such as multiple cooling separation removal process and reconcentration process.

Hereinafter, the present invention will be described in more detail based on the following examples, but the present invention is not limited thereto.

Example 1 Preparation of Skim Mill Using Supercritical Fluids

The dried 100 g of rapeseed seed was finely ground, put in a 1 liter extractor, tightened with a bolt, and the temperature and pressure were set at 50 ° C. and 500 bar, respectively, at the control panel, and the flow rate of carbon dioxide was maintained at 4 liters per hour. After 40 minutes to reach the normal extraction conditions, the extraction was continued for 3 hours, and then the pressure was increased to 3 hours at 550 bar and extracted. The oil was collected by connecting to a separator, and then extracted while flowing ethanol at a flow rate of 0.4 liters per hour while maintaining a temperature of 45 ° C., a pressure of 600 bar, and a flow rate of carbon dioxide of 5.5 liters per hour. The extraction time was reached under normal conditions and the amount of oil remaining in the skimmed wheat prepared for 1.5 hours and the phenolic compound were measured.

In comparison, the oil content of the rapeseed seed used in the pulverization in Example 1 and the phenolic compound content of the skimmed wheat prepared by the following solvent extraction method were measured.

The oil content was determined by extracting and drying the sample (5 g) for 8 hours using ethyl ether in a soxhlet apparatus with ethyl ether. The amount of phenolic compounds in the degreasing mill and the supercritical mill previously prepared was determined by Method (Folin-Ciocalteu) Sample (1 g) was added to 80% methanol, and the solution extracted by ultrasonic wave for 20 minutes (nitrogen gas treatment) was filtered and dried under reduced pressure, followed by adding a porin-thiocalto phenol reagent and reacting 20% -Sodium carbonate solution was added and absorbance was measured at 730 nm. And the standard curve was measured using gallic acid, and each value was substituted to measure the concentration of the phenolic compound contained in the solution.

Amount of oil remaining in wheat (supercritical): 0.105 g / 5 g (oil amount of rapeseed seed: 41.5%)

Amount of remaining phenolic compound in wheat (supercritical): 0.0112 g / g

                   (The amount of phenolic compounds in wheat by solvent extraction: 1.98%)

Example 2

50 g of skim wheat prepared in Example 1 was added to 500 ml of 0.5 M-salt aqueous solution, and stirred at room temperature over 30 minutes with a stirrer to extract protein. 0.015 g of a mixture of sodium sulfite and sodium bisulfite are added to the saline solution and the pH is 6.0.

The extracted protein solution was filtered by passing through a filter pad of 2.5 g of a mixture of clay and diatomaceous earth (1: 3), and again passed through a filter bed of 0.45 µm to obtain a clear protein solution. This solution was subjected to ultrafiltration, and the ultrafiltration device used was a Millipore's TF-type Pelicon-XL, and the filtration membrane was made of polyethersulfone. It was washed with 0.25M saline solution while treating at room temperature using 50 for 3 hours.

Comparative Example 1

50 g of degreasing mill (using the same seed of Example 2) prepared by the conventional method (hexane extraction method) was extracted, filtered and concentrated in the same manner and time as in Example 2, but without using an antioxidant. Was implemented.

Test Example 1

In order to compare the color of the concentrated protein solution in the process, the concentrates of Example 2 and Comparative Example 1 were diluted 10-fold and the color was measured using a lobby bond.

Measurement was performed at room temperature using a 5.25 inch qubit of a Rovibond PFX995 model.

Example 2 Comparative Example 1 0.5 Red 7.3 Yellow 2.3 Red 19.0 Yellow

As shown in Table 1, it was confirmed that the browning phenomenon was suppressed by Example 2 measured values of the Red series and the Yellow series compared to Comparative Example 1.

In contrast to the brown of Comparative Example 1, Example 2 showed a light yellowish green color, and more time passed.

Example 3

Take 50 ml of the treated concentrated concentrate (16.2% w / v) as in Example 2, cool to 3 ° C and add 75 ml of a pre-cooled 95% ethanol aqueous solution with gentle stirring. Centrifuge the precipitated protein. The precipitated protein was obtained and dried in a freeze dryer to obtain a protein having a light yellow protein content of 99%.

The protein content mentioned above is the value of 6.25 multiplied by the nitrogen level measured by the Buchi automatic Kjeldahl K-370.

Example 4

50 ml of the concentrated concentrate (12.5%, w / v) treated as in Example 2 was treated with a cooled 95% aqueous ethanol solution (120 ml) in the same manner as in Example 3 to give a pale yellowish green 86 Obtained Protein in% Content

Example 5

40 g of wheat containing a protein content of 35.5% made using a supercritical fluid was treated (extracted, filtered and concentrated) in the same manner as in Example 2, and 50 milliliters of the concentrate (17.8% w / v) was taken. 100 ml of an aqueous 90% ethanol solution, which was cooled down in advance, was slowly added with stirring. Then, the precipitated protein was collected and centrifuged (5,000 rpm) and dried in a freeze dryer to obtain 8.15 g of a pale yellow-yellow 103% protein, which showed a recovery rate of 91.6% compared to the concentrate.

Comparative Example 2

After diluting by adding 10 times the volume of water cooled to the protein solution extracted and concentrated in the same manner as in Example 2, the protein becomes cloudy and centrifuged to recover the protein. Seemed.

Test Example 2 Protein Content According to Concentration Rate

As shown in Example 2, the extracted protein solution was concentrated at each concentration, and the content of protein precipitated by putting twice the capacity of the concentrated 95% ethanol aqueous solution compared to the concentrate was shown in FIG. 2.

As described above, the method for producing a protein in a good color and high yield from the rapeseed seed according to the present invention is very cost and time in that it does not go through a separate process, such as multiple cooling separation removal process, reconcentration process, etc. Efficient

Claims (6)

1) extracting the rapeseed seed with a supercritical fluid and an alcohol having 1 to 3 carbon atoms to obtain skim wheat without the phenolic compound; 2) extracting the skimmed wheat with brine adjusted to pH 5.0 ~ 6.5 using sodium sulfite and sodium hydrogen sulfite to obtain a protein solution; 3) filtration of the protein solution with crude earth mixed with clay and diatomaceous earth in a weight ratio of 1: 1 to 1: 5, followed by ultrafiltration and concentrating to 15 to 20% (w / v); 4) recovering the precipitated protein by mixing the concentrate and 80 ~ 95% ethanol aqueous solution of 1 ~ 5 ℃ in a weight ratio of 1: 1 ~ 1: 3 Method for producing a protein with improved whiteness, characterized in that it comprises a high yield. The method of claim 1, wherein the supercritical fluid extraction is carried out under a temperature of 35 to 55 ℃, pressure of 250 to 650 bar, carbon dioxide solvent flow rate of 1 to 5 liters per hour and processing conditions of 5 to 10 hours Way. The method of claim 1, wherein the lower alcohol in step 1) is treated for 30 to 150 minutes at a temperature of 35 to 50 ℃, a pressure of 350 to 650 bar, 0.1 to 0.5 liters per hour. The method of claim 1, wherein the sodium sulfite and sodium hydrogen sulfite are used in an amount of 0.005 to 0.05% by weight based on the skim mill. delete The method of claim 1, wherein the recovery of the protein in step 4) is characterized in that more than 90%.

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