Method for efficiently removing polysaccharide and extracting barley thermostable protein

Abstract

The invention relates to a method for efficiently removing polysaccharide and extracting barley thermostable protein, which comprises the following steps: a) extracting a thermostable protein solution from barley malt through simple processes such as extraction and heating; and b) the extracted protein solution contains a large amount of polysaccharide and polyphenol substances, and is treated by adopting CaCl2 and NaCl with specific doses, so that the malt heat-stable protein solution can be obtained. And c) dialyzing the protein solution obtained in the step b), and freeze-drying to obtain barley malt heat-stable protein powder. The method for separating and extracting the polysaccharide and the protein in the process has the advantages of simplicity, convenience, economy, feasibility, safety, no pollution and the like, and has application potential in food industrial production.

Description

Technical Field

[0001] The invention belongs to the technical field of protein extraction and relates to a method for efficiently removing polysaccharides and extracting heat-stable protein from barley. Background Art

[0002] Heat-stable proteins refer to a class of proteins that exist in organisms and can resist high temperature treatment without denaturation and can maintain their natural biological activity. They are of great significance in the production of new foods and the development and application of functional proteins. The most representative heat-stable protein in malt should be protein Z. According to research reports, this protein has many biological activities, including trypsin and thrombin inhibitory activity, and can be developed as a protein carrier or food supplement for application.

[0003] However, in the separation and purification process of malt heat-stable protein, the traditional alkali-dissolution and acid precipitation extraction method cannot achieve good results, and the environmental pollution caused by the strong acid and alkali used in the alkali-dissolution and acid precipitation extraction method cannot be ignored; the classic ammonium sulfate precipitation method is also limited in its application in food industry production due to the presence of ammonium salts. Without alkali-dissolution and acid precipitation and salting out treatment, the protein solution extracted only by leaching and heating has the defects of high polysaccharide content and low protein content, which limits the effective utilization of the heat-stable protein. Therefore, the heat-stable protein extraction and polysaccharide removal method with simple materials, safety and pollution-free and applicable to food production is of great significance in actual production.

[0004] There is no simple and effective method for removing polysaccharide components from protein solutions in existing patents, monographs and literature reports, so the invention has good application potential. Summary of the invention

[0005] In view of the above technical problems, the purpose of the present invention is to provide a method for efficiently removing polysaccharides and extracting heat-stable protein from barley:

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] A method for efficiently removing polysaccharides and extracting heat-stable protein from barley, the preparation method comprising the following steps:

[0008] a) Extraction of heat-stable protein from barley malt: 100 g of barley malt grains were taken, ground into uniform malt grain powder using a mill, passed through a 100-200 mesh sieve, and uniformly mixed with deionized water at a solid-liquid ratio of 1:4-1:5 (w / v), and extracted by shaking at a speed of 100-200 rpm and a temperature of 37° C. for 2-4 hours. The obtained turbid liquid was filtered to obtain a white emulsion, which was placed in a constant temperature water bath and heated at 80-100° C. for 20-60 minutes. After heating, a large amount of obvious protein precipitation was observed, and the supernatant was taken by centrifugation to obtain a heat-stable protein solution from barley malt;

[0009] b) Removing polysaccharides from the heat-stable protein solution: adding 1-4M CaCl2 and 2-4M NaCl to the heat-stable protein solution of barley malt extracted in a), and letting it stand overnight. After the sample solution has stood, a large amount of gel-like precipitate can be clearly observed. The supernatant is collected by centrifugation to obtain a heat-stable protein solution free of polysaccharides and other substances;

[0010] c) Freeze-drying: The protein solution sample obtained in b) is dialyzed for 48 to 72 hours to remove CaCl2 and NaCl, and then freeze-dried for 48 hours to obtain the barley malt heat-stable protein.

[0011] A polysaccharide removal method for extracting heat-stable protein from barley malt according to the method first achieves separation of heat-stable protein from barley malt through oscillation extraction and heating, and the solution is clear and stable, suitable for further CaCl2 and NaCl removal of polysaccharides.

[0012] In the polysaccharide removal process, 1-4M CaCl2 and 2-4M NaCl are added to the heat-stable protein solution to precipitate the polysaccharide in the heat-stable protein solution to obtain a protein solution after the polysaccharide component is removed. The polysaccharide removal rate of the product obtained in this way is 64.2±4.6%.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] The method for efficiently removing polysaccharides and extracting heat-stable protein from barley introduced in the present invention can be used to simply and efficiently extract heat-stable protein from barley malt, laying a foundation for its subsequent application and having the potential for mass production. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Technical roadmap for polysaccharide removal method to extract heat-stable protein from barley malt;

[0016] Figure 2 The state diagram of heat-stable protein extracted from barley malt after polysaccharide removal and precipitation state diagram;

[0017] Figure 3 Plot of protein and polysaccharide contents in samples before and after polysaccharide removal;

[0018] Figure 4 Electrophoresis (SDS-PAGE) of the heat-stable proteins extracted using this method;

[0019] Figure 5 The state diagram of the heat-stable protein solution extracted by this method (10 mg / ml). DETAILED DESCRIPTION

[0020] The present invention relates to a method designed based on a conventional protein separation and purification method capable of effectively separating heat-stable protein in barley malt and removing polysaccharides. The extracted heat-stable protein maintains a good protein state and can meet production application requirements.

[0021] The following examples are only used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0022] Embodiment 1:

[0023] This embodiment provides a specific method for removing polysaccharides from barley malt by extracting heat-stable protein. The specific steps are as follows:

[0024] a) taking 100 g of barley malt grains, grinding them into fine and uniform malt grain powder with a mill, and uniformly mixing them with deionized water at a solid-liquid ratio of 1:5 (w / v), placing the obtained malt grain suspension in an oscillating incubator at a speed of 200 rpm and a temperature of 37° C. for 2 h, filtering the obtained turbid liquid with four layers of gauze after the oscillation extraction, and obtaining a white emulsion after filtration, placing the emulsion in a constant temperature water bath at 80° C. for 60 min, after which a large amount of obvious protein precipitation can be seen, centrifuging at a speed of 5000 rpm for 15 min and taking the supernatant to obtain a heat-stable barley malt protein solution;

[0025] b) adding 1M CaCl2 or 1M NaCl to the barley malt heat-stable protein solution, allowing it to stand overnight, centrifuging at 10000 rpm for 10 min, and discarding the precipitate after centrifugation;

[0026] c) dialyzing the obtained protein solution sample for 48 hours to remove CaCl2 or NaCl, and freeze-drying the sample for 48 hours to obtain a finished product of heat-stable barley malt protein.

[0027] Embodiment 2:

[0028] This embodiment provides a specific method for removing polysaccharides from barley malt by extracting heat-stable protein. The specific steps are as follows:

[0029] a) taking 100 g of barley malt grains, grinding them into fine and uniform malt grain powder with a mill, and uniformly mixing them with deionized water at a solid-liquid ratio of 1:4 (w / v), placing the obtained malt grain suspension in an oscillating incubator at a speed of 200 rpm and a temperature of 37° C. for 4 h, after the oscillation extraction, filtering the obtained turbid liquid with four layers of gauze to obtain a white emulsion, placing the emulsion in a constant temperature water bath at 90° C. for 30 min, after heating, a large amount of obvious protein precipitation can be seen, centrifuging at a speed of 10000 rpm for 10 min and taking the supernatant to obtain a heat-stable barley malt protein solution;

[0030] b) adding 2M CaCl2 to the barley malt heat-stable protein solution, allowing it to stand overnight, centrifuging at 10,000 rpm for 10 min, and discarding the precipitate after centrifugation;

[0031] c) dialyzing the obtained protein solution sample for 60 hours to remove CaCl2, and freeze-drying the sample for 48 hours to obtain a finished product of heat-stable barley malt protein.

[0032] Embodiment 3:

[0033] This embodiment provides a specific method for removing polysaccharides from barley malt by extracting heat-stable protein. The specific steps are as follows:

[0034] a) taking 100 g of barley malt grains, grinding them into fine and uniform malt grain powder with a mill, and uniformly mixing them with deionized water at a solid-liquid ratio of 1:5 (w / v), placing the obtained malt grain suspension in an oscillating incubator at a speed of 100 rpm and a temperature of 37° C. for 4 h, filtering the obtained turbid liquid with four layers of gauze after the oscillation extraction, and obtaining a white emulsion after filtration, which is placed in a constant temperature water bath and heated at 100° C. for 20 min, after which a large amount of obvious protein precipitation can be seen, and centrifuging at a speed of 10000 rpm for 10 min and taking the supernatant to obtain a heat-stable barley malt protein solution;

[0035] b) adding 4M CaCl2 to the barley malt heat-stable protein solution, allowing it to stand overnight, centrifuging at 5000 rpm for 15 min, and discarding the precipitate after centrifugation;

[0036] c) dialyzing the obtained protein solution sample for 72 hours to remove CaCl2, and freeze-drying the sample for 48 hours to obtain a finished product of heat-stable barley malt protein.

[0037] Test example:

[0038] The samples treated above were characterized for the content of protein and polysaccharide in their components, as well as the composition of protein:

[0039] (1) Protein content determination:

[0040] According to the biuret method, bovine serum albumin (BSA) solution was used as the standard curve to determine the protein content. The proportion of protein components in the system was obtained by dividing the measured solution protein content by the mass of the product lyophilized powder dissolved therein, such as Figure 3 It can be seen that after the polysaccharide removal process, the protein content increased from 9.6% to 60.6%, greatly increasing the protein content in the product.

[0041] (2) Determination of polysaccharide content:

[0042] The polysaccharide content was determined using the phenol-sulfuric acid method with standard glucose solution as the standard curve. The proportion of polysaccharide components in the system was calculated by dividing the polysaccharide content measured in the solution by the mass of the lyophilized powder of the product dissolved therein, such as Figure 4 The polysaccharide removal rate of the obtained product after the polysaccharide removal process was 64.2±4.6%.

[0043] (3) Characterization of protein composition:

[0044] SDS-PAGE was used to characterize the components of the obtained protein subunits, and the extracted protein contained a protein Z characteristic band of about 40 kDa, indicating that we successfully separated the heat-stable target protein we needed.

[0045] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for efficiently removing polysaccharides and extracting heat-stable protein from barley, characterized in that: The preparation method comprises the following steps: a) Extraction of heat-stable protein from barley malt: 100 g of barley malt grains were taken, ground into uniform malt grain powder using a mill, passed through a 100-200 mesh sieve, and uniformly mixed with deionized water at a solid-liquid ratio of 1:4-1:5 (w / v), and extracted by shaking at a speed of 100-200 rpm and a temperature of 37° C. for 2-4 hours. The obtained turbid liquid was filtered to obtain a white emulsion, which was placed in a constant temperature water bath and heated at 80-100° C. for 20-60 minutes. After heating, a large amount of obvious protein precipitation was observed, and the supernatant was taken by centrifugation to obtain a heat-stable protein solution from barley malt; b) Removing polysaccharides from the heat-stable protein solution: adding 1-4M CaCl2 and 2-4M NaCl to the heat-stable protein solution of barley malt extracted in a), and letting it stand overnight. After the sample solution has stood, a large amount of gel-like precipitate can be clearly observed. The supernatant is collected by centrifugation to obtain a heat-stable protein solution free of polysaccharides and other substances; c) Freeze-drying: The protein solution sample obtained in b) is dialyzed for 48 to 72 hours to remove CaCl2 and NaCl, and then freeze-dried for 48 hours to obtain the barley malt heat-stable protein.

2. The method for extracting heat-stable protein from barley malt according to claim 1, characterized in that: The extraction was carried out at a solid-liquid ratio of 1:4 to 1:5 with shaking at 37°C for 2 to 4 hours.

3. The method for extracting heat-stable barley protein according to claim 1, characterized in that: The water extract is heated at 80-100° C. for 20-60 minutes.

4. The method according to claim 1, characterized in that: The removal rate of polysaccharides is above 60%.

Images