CN113552249A - Extraction and identification method of starch shell protein - Google Patents
Extraction and identification method of starch shell protein Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/14—Preparation by elimination of some components
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
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Abstract
本发明提供了一种淀粉壳层蛋白的提取鉴定方法，包括以下步骤：A、配制淀粉乳，并进行不完全糊化得到悬浊液；B、将步骤A所得的悬浊液静置，待其分层，得到上层悬浮液和下层沉淀，随后冻干即得外壳结构和内壳结构；C、在步骤B得到的外壳结构和内壳结构中加入蛋白提取液离心后得到含有外壳蛋白和内壳蛋白的蛋白溶液；D、将步骤C得到的蛋白溶液经过SDS‑PAGE，分离出蛋白条带；E、将步骤D分离出的蛋白条带经LS‑MS鉴定，即可得到淀粉外壳蛋白和内壳蛋白的种类结果。本发明以低耗低成本和简单易行的方法有效分离淀粉外壳和内壳结构，首次提取淀粉内外壳的蛋白并进行鉴定，发现外壳的蛋白含量和种类低于内壳，可以用于淀粉结构的分析。
The invention provides a method for extracting and identifying starch shell protein, comprising the following steps: A. preparing starch milk, and performing incomplete gelatinization to obtain a suspension; B. leaving the suspension obtained in step A to stand, waiting for It is layered to obtain an upper layer suspension and a lower layer precipitation, and then freeze-drying to obtain an outer shell structure and an inner shell structure; C, adding a protein extract to the outer shell structure and inner shell structure obtained in step B and centrifuging to obtain an outer shell protein and an inner shell structure. The protein solution of shell protein; D. The protein solution obtained in step C is subjected to SDS-PAGE to separate protein bands; E, the protein bands separated in step D are identified by LS-MS to obtain starch coat protein and Species results for inner coat proteins. The invention effectively separates the starch shell and the inner shell structure by a low-cost, low-cost and simple method, extracts and identifies the protein of the starch inner shell for the first time, and finds that the protein content and type of the shell are lower than those of the inner shell, and can be used for starch structure analysis.
Description
Technical Field
The invention relates to the technical field of food, in particular to a method for extracting and identifying starch shell protein.
Background
Starch is a natural high polymer with the second content to cellulose and chitin in nature, and different from the former two, the starch can be hydrolyzed by enzyme in human digestive system, so as to provide 70-80% of energy for human body, and play a very important role in human life. The starch has nutritional value, can affect the physical properties of food, is widely applied in food processing industry, and can be used as additives such as adhesive, powder spraying agent, antistaling agent, etc.; meanwhile, starch is an important industrial raw material as a renewable, degradable and cheap natural resource. The application of starch is determined by the property of the starch, the property is determined by the structure of the starch, and the research on the structure of the starch at present lags behind the research on the property and the application, thereby greatly restricting the development of the performance of the starch, hindering the popularization of more application fields of the starch, and comprehensively analyzing the structure of the starch is always a research hotspot and difficulty in the industry.
The structure of the starch can be divided into a molecular structure, a lamellar structure, a Blocklet particle, a growth ring and a particle. Although the hierarchical structure framework of starch is established, the hierarchical structure framework is rough and difficult to provide accurate positioning for the property research of starch. The growth ring of starch granules is also called a shell structure layer, and refers to a concentric ring structure with soft and hard alternation (alternate arrangement of semi-crystalline layers and amorphous layers) around the umbilical core. In recent years, incomplete gelatinization has been used in the industry to separate the outer shell structure from the inner shell structure of starch granules. By properly controlling the gelatinization conditions, such as lowering the processing temperature of the system, incomplete gelatinization of the starch can occur, resulting in limited swelling and localized breakage of starch granules without losing recognizable granule shape. The outer shell of the starch granule is tougher than the inner shell and is more resistant to external conditions such as acid and heat. The findings of these findings strongly suggest that the structure of the inner shell layer of the starch granule is different.
When constructing starch structures based on starch molecules, one typically also contains non-starch components, such as a minor amount of protein, from about 0.1% to about 0.8%, closely associated with the starch granule, referred to as starch granule binding protein. The primary means by which starch granule binding proteins interact with starch granules are hydrophobic interactions, hydrogen bonding, and electrostatic attraction. Starch granule binding proteins are mainly starch synthases, storage proteins and proteins associated with the protection of cereals against biotic and abiotic stresses. Although the content of the starch granule binding protein in the starch is extremely low, the impact on the physicochemical property and the digestibility of the starch is remarkable. However, the distribution of starch granule binding protein on starch structure and the relationship of the structure thereof are only reported, and the analysis of starch structure is limited. If the inner and outer starch shells are used as a model, the distribution and the composition of protein molecules in the shell structure are researched, and the relationship between the protein molecules and the starch structure can be further determined.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a method for extracting and identifying starch shell protein. The method effectively separates the structures of the outer shell and the inner shell of the starch by a low-consumption, low-cost and simple and feasible method, extracts and identifies the protein of the outer shell and the inner shell of the starch for the first time, finds that the protein content and the type of the outer shell are lower than those of the inner shell, and can be used for analyzing the structure of the starch.
The purpose of the invention is realized by the following scheme:
the invention provides a method for extracting and identifying starch shell protein, which comprises the following steps:
A. weighing starch, preparing starch milk, and then carrying out incomplete gelatinization on the starch milk to obtain a suspension; for example, rapid viscometry (RVA-4, Newport Scientific, Australia) can be used for gelatinization;
B. standing the suspension obtained in the step A, naturally layering the suspension to obtain an upper suspension and a lower precipitate, and freeze-drying to obtain an outer shell structure positioned on the lower layer and an inner shell structure positioned on the upper layer;
C. adding a protein extracting solution into the outer shell structure and the inner shell structure obtained in the step B, and centrifuging to obtain a protein solution containing the outer shell protein and the inner shell protein;
D. c, separating a protein band from the protein solution containing the outer shell protein and the inner shell protein obtained in the step C through SDS-PAGE, and cutting the band;
E. and D, identifying the protein band separated in the step D by LS-MS to obtain the type results of the starch coat protein and the starch coat protein.
Preferably, in step a, the starch is low amylose starch, and the amylose content of the low amylose starch is 10.9%.
Preferably, incomplete gelatinization means a degree of gelatinization of 60% to 70%.
Preferably, in step a, the mass concentration of the starch milk is 0.25% to 2%, more preferably 0.5% to 2%, and most preferably 0.5%.
Preferably, in step a, the gelatinization process of the starch milk is as follows: keeping the rotation speed of 960r/min for 10 s, keeping the rotation speed of 160r/min for 1.0min at 50 deg.C, increasing the temperature from 50 deg.C to the maximum treatment temperature for 3.8min, keeping the temperature at the maximum treatment temperature for 2.5min, decreasing the temperature to 50 deg.C within 3.8min, and keeping the temperature at 50 deg.C for 2.0 min.
Preferably, in step A, the maximum treatment temperature is from 75 ℃ to 79 ℃, most preferably 77 ℃.
Preferably, in the step B, after the upper suspension and the lower precipitate are obtained, the mixture is put into an ultra-low temperature refrigerator (-80 ℃) for fast freezing for 2 hours, and then freeze-dried in a freeze-dryer, so as to obtain the outer shell structure (lower layer) and the inner shell structure (upper layer).
Preferably, in the step C, the same amount of the outer shell structure and the inner shell structure are respectively weighed, and the mass of the outer shell structure or the inner shell structure is as follows: protein extract 15 mg: adding 1mL of protein extracting solution, boiling, and centrifuging to obtain supernatant, namely the protein solution containing the coat protein and the inner coat protein.
Preferably, the protein extract comprises 50mM tris (hydroxymethyl) aminomethane (pH 8), 2% sodium dodecyl sulfate and 2% mercaptoethanol.
Preferably, in step E, the protein bands are identified by LS-MS after trypsinization and then compared in the database to obtain the class results of the outer and inner coat proteins.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention adopts an incomplete gelatinization method to effectively separate the shell structure and the inner shell structure of the starch granules; if the degree of gelatinization is 100%, that is, if gelatinization is complete, the outer shell structure is disintegrated and mixed with the inner shell structure, and the inner and outer shell layers cannot be separated. The gelatinization degree measured by the method is between 60% and 70%, and the separation effect is good.
(2) The invention discovers for the first time that the types of the binding proteins of the inner and outer shell layers of the starch are different, and the starch binding proteins are not uniformly distributed in starch granules, thereby promoting the analysis work of the starch structure.
(3) The method has the advantages of simple and efficient operation, low energy consumption and low production cost.
Drawings
Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:
FIG. 1 is a photograph taken by an optical microscope and a scanning electron microscope in example 1 of the present invention;
FIG. 2 is a photograph taken by an optical microscope and a scanning electron microscope in example 3 of the present invention;
FIG. 3 is an optical microscope and scanning electron micrograph of comparative example 1 of the present invention;
FIG. 4 is an optical microscope and scanning electron micrograph of comparative example 2 of the present invention;
FIG. 5 is an SDS-PAGE pattern of the outer and inner coat proteins of starch of example 3 and comparative example 2 of the present invention.
Detailed Description
The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that it would be obvious to those skilled in the art that various changes and modifications can be made without departing from the spirit of the invention. All falling within the scope of the present invention.
The invention provides a method for extracting and identifying starch shell protein, which comprises the following steps: A. preparing a certain proportion of starch milk, and gelatinizing by using a rapid viscosity analyzer (RVA-4 of Newport Scientific company, Australia); B. standing the obtained system suspension, naturally layering to obtain an upper suspension and a lower precipitate, and freeze-drying to obtain an outer shell structure (lower layer) and an inner shell structure (upper layer); C. adding the outer shell and the inner shell structure into the protein extracting solution; D. protein bands are separated from the obtained starch outer shell and inner shell protein samples through SDS-PAGE; E. and (4) identifying the protein band by LS-MS to obtain the identification result of the inner and outer shell protein of the starch. The invention effectively separates the structure of the outer shell and the inner shell of the starch by a low-consumption, low-cost and simple and feasible method, extracts the protein of the outer shell and the inner shell of the starch for the first time and identifies the protein, finds that the protein content and the type of the outer shell are lower than those of the inner shell, and can be used for analyzing the structure of the starch. The present invention will be described in detail with reference to specific examples.
Example 1
This example relates to a method for extracting and identifying starch coat proteins, comprising the steps of:
step 1, weighing 0.56g of low amylose starch (Luhui 1015 rice starch, amylose content 10.9%), preparing 28g of starch milk with concentration of 2% by using distilled water, gelatinizing the starch by using a rapid viscosity analyzer (RVA-4 of Newport Scientific company of Australia) to separate outer and inner shells of the starch to obtain suspension, continuously stirring in the gelatinizing process, keeping the rotation speed of 960r/min for 10 seconds initially and 160r/min all the time, keeping the initial temperature at 50 ℃ for 1.0min, increasing the temperature from 50 ℃ to the maximum treatment temperature after 3.8min, keeping the maximum treatment temperature for 2.5min, then reducing the temperature to 50 ℃ in 3.8min, keeping the temperature at 50 ℃ for 2.0min, and keeping the maximum treatment temperature at 77 ℃;
step 2, standing the system suspension obtained in the step 1 for 1 hour, and naturally layering to obtain supernatant and lower-layer precipitate;
step 3, respectively sucking the supernatant and the lower precipitate obtained in the step 2, dripping the supernatant and the lower precipitate on a glass slide, mixing the supernatant and the lower precipitate with iodine solution, and observing the shape under an upright microscope, wherein as shown in fig. 1(a), the shell structure in the lower precipitate is dark purplish red, is basically wrinkled and has severe shrinkage;
step 4, mixing the lower-layer precipitate obtained in the step 2 with 5 times of volume of absolute ethyl alcohol, centrifuging (3000r/min) for 10min, naturally air-drying at room temperature for 24 hours, and observing the appearance under a scanning electron microscope as shown in fig. 1 (b); the starch outer shell structure is provided with small holes, which indicates that the inner shell structure overflows from the small holes, but whether the inner shell structure completely overflows cannot be judged;
step 5, precipitating the supernatant and the lower layer obtained in the step 2 in a disposable culture dish, quickly freezing the disposable culture dish in an ultralow temperature refrigerator at (-80 ℃) for 2 hours, and freeze-drying the disposable culture dish in a freeze dryer to obtain an outer shell structure (the lower layer) and an inner shell structure (the upper layer);
and 6, standing the system suspension obtained in the step 1 for 1 hour, transferring the system suspension to a 50mL centrifuge tube, washing the residual sample with 10mL of distilled water, pouring the sample into the centrifuge tube, standing until the sample is cooled to room temperature, and centrifuging (3000r/min) for 15 min. The supernatant was pipetted into a measuring cylinder with a disposable pipette to record a volume of 23 ml and the swelling volume of the starch at the set maximum treatment temperature was obtained according to the following equation. The swelling volume of starch milk at 95 ℃ with the same concentration was 35.7mL/g, which is the maximum swelling volume. Thereby obtaining the gelatinization degree of the starch after the starch is processed by a set program.
Pellet volume (mL) — 28mL- (supernatant volume-10 mL);
swelling volume (mL/g) versus precipitation volume (mL)/dry weight of starch (g);
degree of gelatinization (%) — swelling volume at maximum treatment temperature (mL/g)/swelling volume at 95 ℃ (mL/g) × 100%
Step 7, weighing 15mg of the outer shell and the inner shell in the step 5 respectively, adding 1mL of protein extracting solution (50mM tris (hydroxymethyl) aminomethane (pH 8), 2% sodium dodecyl sulfate solution and 2% mercaptoethanol), boiling for 10min, centrifuging (10000r/min) for 10min, and obtaining supernate, namely the protein solution of the outer shell and the inner shell; the protein extracting solution is prepared by dissolving trihydroxymethyl aminomethane, a sodium dodecyl sulfate solution and mercaptoethanol in distilled water, and the final concentration of each substance is as follows: 50mM Tris (pH 8), 12% sodium dodecylsulfate solution and 2% mercaptoethanol.
Step 8, separating protein bands from the starch outer shell and inner shell protein samples obtained in the step 7 through SDS-PAGE;
the implementation effect is as follows: the low amylose starch prepared in this example has poor separation effect between the outer and inner hulls, with 75% gelatinization degree, main bands of outer coat protein of 66,88 and 92kDa, and main bands of inner coat protein of 66,88 and 92 kDa. 30 outer coat proteins and 34 inner coat proteins were identified by LS-MS, as shown in Table 1.
TABLE 1, example 1 classes of outer and inner coat proteins
Example 2
This example relates to a method for extracting and identifying starch coat proteins, which is the same as example 1 except that;
step 1, weighing 0.28g of low amylose starch (Luhui 1015 rice starch, amylose content 10.9%), preparing 28g of starch milk with concentration of 1% by using distilled water, gelatinizing the starch by using a rapid viscosity analyzer (RVA-4 of Newport Scientific company of Australia) to separate an outer starch shell and an inner starch shell, continuously stirring in the gelatinizing process, keeping the rotation speed of 960r/min for 10 seconds initially and 160r/min for all the time, keeping the initial temperature at 50 ℃ for 1.0min, increasing the temperature from 50 ℃ to the maximum processing temperature in 3.8min, keeping the maximum processing temperature for 2.5min, reducing the temperature to 50 ℃ in 3.8min, keeping the temperature at 50 ℃ for 2.0min, and keeping the maximum processing temperature at 77 ℃;
the implementation effect is as follows: the low amylose starch prepared in this example has a good separation effect of outer and inner shells, with a gelatinization degree of 70%, main bands of outer shell protein of 66 and 88kDa, and main bands of inner shell protein of 66,88 and 92 kDa. 20 outer coat proteins and 34 inner coat proteins were identified by LS-MS, as shown in Table 2.
TABLE 2, example 2 classes of outer and inner coat proteins
Example 3
This example relates to a method for extracting and identifying starch coat proteins, which is the same as example 1 except that;
in the step 1, 0.14g of low amylose starch (Luhui 1015 rice starch, amylose content 10.9%) is weighed, 28g of starch milk with concentration of 0.5% is prepared by distilled water, a rapid viscosity analyzer (RVA-4 of Newport Scientific company of Australia) is used for gelatinizing starch to separate an outer starch shell and an inner starch shell, the gelatinizing process is continuously stirred, the rotating speed is 960r/min initially and is kept for 160r/min all the time, the initial temperature is 50 ℃ and is kept for 1.0min, the temperature is increased from 50 ℃ to the maximum processing temperature in 3.8min, the maximum processing temperature is kept for 2.5min, then the temperature is reduced to 50 ℃ in 3.8min and is kept for 2.0min at 50 ℃, and the maximum processing temperature is 77 ℃;
in step 3, observing the shape under an upright microscope, as shown in fig. 2(a), the shell structure is obviously expanded, some is in a balloon shape, the interior is not filled with filler, and some is shrunk;
in step 4, observing the appearance under a scanning electron microscope as shown in fig. 2 (b); the outer shell structure is provided with a hole, and the inner part is obviously observed to be completely overflowed, which indicates that the outer shell structure and the inner shell structure can be completely separated;
the implementation effect is as follows: the low amylose starch prepared in this example has good separation effect between the outer and inner hulls, with a gelatinization degree of 64%, the major coat protein band of 66kDa, and the major inner coat protein bands of 66,88 and 92kDa, as shown in fig. 5. 15 outer coat proteins and 34 inner coat proteins were identified by LS-MS. As shown in table 3.
TABLE 3 example 3 classes of outer and inner coat proteins
Example 4
This example relates to a method for extracting and identifying starch coat proteins, which is the same as example 1 except that;
in the step 1, 0.14g of low amylose starch (Luhui 1015 rice starch, amylose content 10.9%) is weighed, 28g of starch milk with concentration of 0.25% is prepared by distilled water, a rapid viscosity analyzer (RVA-4 of Newport Scientific company of Australia) is used for gelatinizing starch to separate an outer starch shell and an inner starch shell, the gelatinizing process is continuously stirred, the rotating speed is 960r/min initially and is kept for 160r/min all the time, the initial temperature is 50 ℃ and is kept for 1.0min, the temperature is increased from 50 ℃ to the maximum processing temperature in 3.8min, the maximum processing temperature is kept for 2.5min, then the temperature is reduced to 50 ℃ in 3.8min and is kept for 2.0min at 50 ℃, and the maximum processing temperature is 77 ℃;
the implementation effect is as follows: the yield of the inner shell structure of the supernatant obtained in the embodiment after freeze-drying is too low, the supernatant needs to be collected for multiple times, the steps are complicated, the gelatinization degree of the system is only 45%, the outer shell structure is not obviously expanded, the separation effect with the inner shell is poor, the main bands of the outer shell protein are 66,88 and 92kDa, the main bands of the inner shell protein are 66,88 and 92kDa, 33 types of outer shell proteins and 34 types of inner shell proteins are identified by LS-MS. As shown in table 4.
TABLE 4, EXAMPLE 4 classes of outer and inner coat proteins
Example 5
This example relates to a method for extracting and identifying starch coat proteins, which is the same as example 3 except that;
in the step 1, 0.14g of low amylose starch (Luhui 1015 rice starch, amylose content 10.9%) is weighed, 28g of starch milk with concentration of 0.5% is prepared by distilled water, a rapid viscosity analyzer (RVA-4 of Newport Scientific company of Australia) is used for gelatinizing starch to separate an outer starch shell and an inner starch shell, the gelatinizing process is continuously stirred, the rotation speed is 960r/min initially and is kept for 160r/min all the time, the initial temperature is 50 ℃ and is kept for 1.0min, the temperature is increased from 50 ℃ to the maximum processing temperature in 3.8min, the maximum processing temperature is kept for 2.5min, then the temperature is reduced to 50 ℃ in 3.8min and is kept for 2.0min at 50 ℃, and the maximum processing temperature is 79 ℃;
the implementation effect is as follows: the starch prepared in the embodiment has an excessively high gelatinization degree of 76%, an outer shell structure is broken and cannot be separated from an inner shell structure, the separation effect is poor, and after the obtained system suspension is kept still for 1 hour, a good layering effect cannot be obtained, and the next protein extraction cannot be carried out.
Example 6
This example relates to a method for extracting and identifying starch coat proteins, which is the same as example 3 except that;
in the step 1, 0.14g of low amylose starch (Luhui 1015 rice starch, amylose content 10.9%) is weighed, 28g of starch milk with concentration of 0.5% is prepared by distilled water, a rapid viscosity analyzer (RVA-4 of Newport Scientific company of Australia) is used for gelatinizing starch to separate an outer starch shell and an inner starch shell, the gelatinizing process is continuously stirred, the rotation speed is 960r/min initially and is kept for 160r/min all the time, the initial temperature is 50 ℃ and is kept for 1.0min, the temperature is increased from 50 ℃ to the maximum processing temperature in 3.8min, the maximum processing temperature is kept for 2.5min, then the temperature is reduced to 50 ℃ in 3.8min and is kept for 2.0min at 50 ℃, and the maximum processing temperature is 75 ℃;
the implementation effect is as follows: the starch prepared in this example has a low degree of gelatinization of 56%, the major bands of the outer coat protein are 66,88 and 92kDa, and the major band of the inner coat protein is 66 kDa. 39 outer coat proteins and 15 inner coat proteins were identified by LS-MS.
TABLE 6, example 6 classes of outer and inner coat proteins
Example 7
This example relates to a method for extracting and identifying starch coat proteins, which is the same as example 3 except that;
step 7, weighing 15mg of the outer shell and the inner shell in the step 5 respectively, adding 1mL of protein extracting solution (50mM tris (hydroxymethyl) aminomethane (pH 8), 0.2% sodium dodecyl sulfate solution and 2% mercaptoethanol), boiling for 10min, and centrifuging (10000r/min) for 10min to obtain supernate, namely the protein solution of the outer shell and the inner shell;
step 8, separating protein bands from the starch outer shell and inner shell protein samples obtained in the step 7 through SDS-PAGE;
the implementation effect is as follows: the low amylose starch prepared in this example had good separation between the outer and inner shells, with a degree of gelatinization of 64%. However, the types of the outer shell protein and the inner shell protein are less, because the concentration of the sodium dodecyl sulfate in the protein extracting solution is lower, the protein is not completely dissolved in the extracting solution in the extracting process, and the types of the protein are reduced. The major band of the outer coat protein is 66kDa, and the major band of the inner coat protein is 66 kDa. 5 outer coat proteins and 12 inner coat proteins were identified by LS-MS. The outer and inner shell proteins are less diverse.
TABLE 7, example 7 classes of outer and inner coat proteins
Example 8
This example relates to a method for extracting and identifying starch coat proteins, which is the same as example 3 except that;
step 7, weighing 15mg of the outer shell and the inner shell in the step 5 respectively, adding 1mL of protein extracting solution (50mM tris (hydroxymethyl) aminomethane (pH 8), 4% sodium dodecyl sulfate solution and 2% mercaptoethanol), boiling for 10min, centrifuging (10000r/min) for 10min, and obtaining supernate, namely the protein solution of the outer shell and the inner shell;
the implementation effect is as follows: the low amylose starch prepared in this example had good separation between the outer and inner shells, with a degree of gelatinization of 64%. However, the concentration of sodium dodecyl sulfate in the protein extracting solution is high, so that phospholipid and amylose molecules existing in starch granules are dissolved out in the extracting process, the protein concentration in the extracting solution is low, impurities are more, the difficulty is caused to the next step of SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), and protein separation and identification are not carried out
Example 9
This example relates to a method for extracting and identifying starch coat proteins, which is the same as example 3 except that;
in the step 1, 0.14g of low amylose starch (Luhui 276 rice starch, amylose content 14.7%) is weighed, 28g of starch milk with concentration of 0.5% is prepared by distilled water, a rapid viscosity analyzer (RVA-4 of Newport Scientific company of Australia) is used for gelatinizing starch to separate an outer starch shell and an inner starch shell, the gelatinizing process is continuously stirred, the rotating speed is 960r/min initially and is kept at 160r/min, the initial temperature is 50 ℃ and is kept for 1.0min, the temperature is increased from 50 ℃ to the maximum processing temperature in 3.8min, the maximum processing temperature is kept for 2.5min, then is reduced to 50 ℃ in 3.8min and is kept for 2.0min at 50 ℃, and the maximum processing temperature is 77 ℃;
the implementation effect is as follows: the low amylose starch prepared in this example has good separation effect of outer and inner shells, the gelatinization degree is 66%, the main band of the outer shell protein is 66kDa, the main bands of the inner shell protein are 66,88 and 92kDa, 14 kinds of outer shell proteins and 33 kinds of inner shell proteins are identified by LS-MS, as shown in Table 8.
TABLE 8, example 9 classes of outer and inner coat proteins
Example 10
This example relates to a method for extracting and identifying starch coat proteins, which is the same as example 3 except that;
in the step 1, 0.14g of low amylose starch (autumn bright rice starch, amylose content 13.6%) is weighed, 28g of starch milk with concentration of 0.5% is prepared by distilled water, a rapid viscosity analyzer (RVA-4 of Newport Scientific company of Australia) is used for gelatinizing starch to separate an outer shell and an inner shell of the starch, the gelatinizing process is continuously stirred, the rotating speed is 960r/min initially and then is kept at 160r/min, the initial temperature is 50 ℃ and kept for 1.0min, the temperature is increased from 50 ℃ to the maximum processing temperature in 3.8min, the maximum processing temperature is kept for 2.5min, then is reduced to 50 ℃ in 3.8min and kept for 2.0min at 50 ℃, and the maximum processing temperature is 77 ℃;
the implementation effect is as follows: the low amylose starch prepared in this example has good separation effect of the outer shell and the inner shell, the gelatinization degree is 64%, the main band of the outer shell protein is 66kDa, the main bands of the inner shell protein are 66,88 and 92kDa, 14 kinds of outer shell proteins and 31 kinds of inner shell proteins are identified by LS-MS, as shown in Table 9.
TABLE 9, EXAMPLE 10 classes of outer and inner coat proteins
Comparative example 1
The comparative example relates to a method for extracting and identifying starch coat protein, comprising the following steps:
step 1, weighing 0.14g of waxy starch (with amylose content of 0.8%), preparing 28g of starch milk with concentration of 0.5% by using distilled water, gelatinizing starch by using a rapid viscosity analyzer (RVA-4 of Newport Scientific company, Australia) to separate an outer starch shell and an inner starch shell, continuously stirring in the gelatinizing process, keeping the rotation speed of 960r/min for initial 10 seconds and 160r/min all the time, keeping the initial temperature at 50 ℃ for 1.0min, increasing the temperature from 50 ℃ to the maximum processing temperature in 3.8min, keeping the maximum processing temperature for 2.5min, reducing the temperature to 50 ℃ by 3.8min, keeping the temperature at 50 ℃ for 2.0min, and keeping the maximum processing temperature at 77 ℃;
step 2, standing the system suspension obtained in the step 1 for 1 hour, and naturally layering to obtain supernatant and lower-layer precipitate;
step 3, respectively sucking the supernatant and the lower precipitate obtained in the step 2, dripping the supernatant and the lower precipitate onto a glass slide, mixing the supernatant and the lower precipitate with iodine solution, and observing the form under an upright microscope; as shown in fig. 3(a), the particles in the lower layer of sediment did not swell significantly, with overflowing starch molecules distributed around the particles;
step 4, mixing the lower-layer precipitate obtained in the step 2 with 5 times of volume of absolute ethyl alcohol, centrifuging (3000r/min) for 10min, naturally air-drying at room temperature for 24 hours, and observing the appearance under a scanning electron microscope; as shown in FIG. 3(b), the starch granule was split but had no hollow inside, indicating that the inner shell structure did not overflow;
step 5, precipitating the supernatant and the lower layer obtained in the step 2 in a disposable culture dish, quickly freezing the disposable culture dish in an ultralow temperature refrigerator at (-80 ℃) for 2 hours, and freeze-drying the disposable culture dish in a freeze dryer to obtain an outer shell structure (the lower layer) and an inner shell structure (the upper layer);
and 6, standing the system suspension obtained in the step 1 for 1 hour, transferring the system suspension to a 50mL centrifuge tube, washing the residual sample with 10mL of distilled water, pouring the sample into the centrifuge tube, standing until the sample is cooled to room temperature, and centrifuging (3000r/min) for 15 min. The volume of the 26ml supernatant was recorded by sucking it into a measuring cylinder with a disposable pipette, and the swelling volume of the starch at the set maximum treatment temperature was obtained according to the following formula. The swelling volume of starch milk with the same concentration at 95 ℃ is taken as the maximum swelling volume (50 mL/g). Thereby obtaining the gelatinization degree of the starch after the starch is processed by a set program.
Pellet volume (mL) — 28mL- (supernatant volume-10 mL);
swelling volume (mL/g) versus precipitation volume (mL)/dry weight of starch (g);
degree of gelatinization (%) — swelling volume at maximum treatment temperature (mL/g)/swelling volume at 95 ℃ (mL/g) × 100%
Step 7, weighing 15mg of the outer shell and the inner shell in the step 5 respectively, adding 1mL of protein extracting solution (50mM tris (hydroxymethyl) aminomethane (pH 8), 2% sodium dodecyl sulfate solution and 2% mercaptoethanol), boiling for 10min, centrifuging (10000r/min) for 10min, and obtaining supernate, namely the protein solution of the outer shell and the inner shell;
and 8, separating protein bands from the starch outer shell and inner shell protein samples obtained in the step 7 through SDS-PAGE.
The implementation effect is as follows: the starch of this comparative example had a low degree of gelatinization of 56%, with major bands of outer coat protein of 15,25,34,66,88 and 95kDa and major bands of inner coat protein of 15,25,34 and 95 kDa. In the binding proteins identified by LS-MS, there were 600 types of coat proteins and 284 types of coat proteins (the binding proteins of waxy rice starch are many and are the attributes of waxy rice). The starch in this comparative example was waxy rice starch with low amylose content and poor separation of the outer and inner shell structures after 77 c treatment, indicating that this temperature is not suitable as a condition for separating the outer and inner shell structures of waxy starch.
Comparative example 2
This example relates to a method for extracting and identifying starch coat protein, which is the same as in comparative example 1 except that;
in the step 1, 0.14g of waxy starch (amylose content is 0.8 percent) is weighed, 28g of starch milk with concentration of 0.5 percent is prepared by distilled water, a rapid viscosity analyzer (RVA-4 of Newport Scientific company of Australia) is used for gelatinizing starch to separate an outer starch shell and an inner starch shell, the gelatinizing process is continuously stirred, the rotation speed is 960r/min initially and then is kept for 160r/min, the initial temperature is 50 ℃ and is kept for 1.0min, the temperature is increased from 50 ℃ to the maximum processing temperature in 3.8min, the highest processing temperature is kept for 2.5min, then is reduced to 50 ℃ in 3.8min and is kept for 2.0min at 50 ℃, and the maximum processing temperature is 79 ℃;
in step 3, the morphology was observed under an upright microscope, as shown in fig. 4(a), with significant expansion of the shell structure but no significant balloon shape;
in the step 4, the appearance is observed under a scanning electron microscope and is shown in fig. 4(b), the internal structure of the shell structure does not completely overflow, and cracks exist on the surface of the shell structure;
the implementation effect is as follows: in the gelatinization process of the embodiment, the waxy starch outer shell structure is easy to break, the gelatinization degree is 64%, the main bands of the outer shell protein are 15,25,34 and 95kDa, and the main bands of the inner shell protein are 15,25,34,66,88 and 95kDa, as shown in figure 5. 304 outer coat proteins and 601 inner coat proteins were identified by LS-MS. In the comparative example, the gelatinization degree was 64% at a maximum treatment temperature of 79 ℃, which was between 60% and 70%, and the best separation effect was achieved. However, waxy starch is very easy to break during gelatinization, so that the results of the outer shell protein and the inner shell protein are not accurate, and thus the waxy starch is proved to be not used as a raw material for extracting and identifying the shell protein of the starch.
From the above results of examples and comparative examples, it can be seen that (1) for low amylose starch of rice, the highest treatment temperature is 77 ℃, which can achieve the best effect, examples 3, 5 and 6 show that the highest treatment temperature is 75 ℃, which has too low gelatinization degree, which can result in no gelatinization overflow of the inner shell structure, and the separated inner shell structure is extracted, so-called outer shell protein actually contains inner shell protein, example 6, which has the highest treatment temperature of 79 ℃, which has too high gelatinization degree, which can result in breakage of the outer shell structure, and exists together with the inner shell structure, which can not easily separate the two to obtain the protein with the outer and inner shell structures, so the gelatinization degree is between 60% and 70%, which can achieve the ideal effect. (2) Examples 1, 2, 3 and 4 show the effect of different concentrations of starch milk on the results of the experiment, with the best separation being achieved at a concentration of 0.5%. (3) When the amylose is used as a raw material for extracting and identifying the starch shell protein, the gelatinization degree is between 60% and 70%, and the separation effect of the outer shell and the inner shell is good.
The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes or modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention. The embodiments and features of the embodiments of the present application may be combined with each other arbitrarily without conflict.
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