As the main energy source, starch is the main food for humans. Rice starch has become an eye-catching member of the starch family due to its small particle size, white color, and easy digestion, and is increasingly being watched by food researchers and manufacturers. With the increase in the level of agricultural products production and people's demand for high-quality, healthy foods, the demand for rice starch in the food industry is increasing. The main components of rice starch are amylose and amylopectin. Rice starch products with a high amylose content are susceptible to aging after being heat-glued, whereas products with a low content are easy to gelatinize and are not susceptible to aging. Rice with a low amylose content shows a large drillability and a soft and velvety rice. The high-content rice absorbs more moisture and swells when it is cooked. The rice grains are dry, fluffy and dark. It can be seen that amylose in rice starch is one of the important factors affecting the properties of starch gelatinization, gelation, and aging. It not only affects the cooking and processing characteristics of rice starch, but also affects the use of rice starch as an additive and functional component. The nature of the aspect. Therefore, the use of amylose measuring instrument for the effective separation of rice amylose starch, to further understand the molecular structure of amylose in order to establish the relationship between the nature of the ditch, has become the key to distinguish the quality of rice starch products. The research on the relationship between starch molecular structure and quality at home and abroad has a long history. Due to the large molecular weight of amylose, there is no effective solvent to dissolve the sample. At the same time there is no effective means to separate amylose from starch. Therefore, there is almost no report about the molecular weight of amylose in China. The reports on the molecular weight of starch in foreign countries vary greatly depending on the instrumental analysis methods.3 Until the time of tears, the exact molecular weight of amylose and amylopectin has not yet been reported. In this experiment, the rheometer was used to control the gelatinization process of rice starch with different amylose content. The amylose in rice starch was separated by leaching method and HPSEC and multi-angle light were used. The MALLS (MALLS) and Refractive Index Detector (R) systems are used to analyze the molecular weight distribution and the molecular rotation radius, aiming to establish an effective method for the analysis of the molecular weight of amylose, providing the exact molecular structure information of amylose for domestic rice. Industrialization of starch helps. 2.3 Isolation of rice amylose samples Amylose can be freed from starch granules and dissolved in water at a certain temperature, whereas amylopectin can be well dispersed in water only after it is completely gelatinized. The leaching temperature of amylose can be selected from any temperature range between the initial gelatinization temperature in the starch gelatinization curve and the peak temperature. According to this principle, the AR 1000 rheometer is used to control the temperature-temperature gelatinization process of rice starch. Samples are taken and centrifuged at different temperatures between the gelatinization starting temperature and the peak temperature. The resulting supernatant is the leached straight at different temperatures. Chain starch. The gelatinization temperature and peak temperature data of the three types of rice starch were provided by the California Rice Research Fund Association. See Table 1. For Koshi with low amylose content, the gelatinization temperature was lower than that of the other two starches. Therefore, the samples were sampled at 70C, 75'C, 80C, 85°C and 90C, respectively. The L-205 and Cocodrie starches were Samples were taken at 75C, 80C, 85C and 90°C and the resulting samples were immediately centrifuged. The pellet was discarded and the supernatant was taken for analysis. The supernatant reacted with iodine to develop a blue color. After wavelength scanning, the maximum absorption wavelength was 640 nm. These are the characteristics of amylose, which proves that most of the supernatant is amylose. Also, amylose is eluted at each of the above temperatures. Therefore, using a rheometer to control gelatinization and warming to separate amylose is an effective method. 3 Amylose Content Analyzer Analysis Test Conclusion Related Instruments: Experimental Rice Milling Machine Wheat Weight Container Agroforestry Climate Collection System Pesticide Residue Analyzer Henan Zonghai Plastic Industry Co., Ltd. , https://www.hnzonghai.com
1 Materials and Methods
1. 1 test material rice starch samples: Cocodrie (amylose content 21%), L-205 amylose content 17%) and Koshi (amylose content 14%) by the project partner University of California, Davis Department of Food Science provide. 
1.2 Main reagents and instruments Dimethyl sulfoxide (DMSO) (chromatographically pure, Sigma); BP (Analytical Pure, Fishe: Company); Molecular weight of dextran (T40 and T2000, Pharmacia). High Performance Liquid Chromatograph (HPSEC) (United States Hewlett-Packard), ChemStation, Autosampler; Multi-angle Optical Disperser Dawn DSP-F (MALLS) (Wyatt, USA); Refractive Index Detector ERC-7512 (RI) (Japan ERMA Corporation); AR1 000 rheometer (United States TA company); UV1100 spectrophotometer (Beijing Rayleigh company). 
1.3 Test methods
1.3.1 Sample Preparation and Processing
1) Standard sample treatment: Dissolve 20m g glucomium standard sample (T40 or T2000) in 5 mL DMSO/50 mmol/L LiBr solution to prepare a 0.4% solution. After stirring for 2 hours at room temperature, the supernatant was centrifuged at 13500 rmin/min for 10 minutes, filtered through a 0.45 Km microporous membrane and subjected to HPSEC-MALLS-RI analysis.
2) Preparation and Processing of Starch Samples: A 20-m starch sample was dissolved in 5 mL of DMSO/50 mmol/L LiBr solution to prepare a concentration. The starch solution of .400 was mixed with a magnetic stirrer and heated on a magnetic stirrer at 90°C for 1 h followed by stirring at room temperature for 24 h. After centrifugation at 13500 r/min for 10 min, a clear solution was passed through 0.45. The Km microporous membrane was filtered and subjected to HPSEC-MALLS-RI analysis.
3) Preparation and processing of amylose samples: Modified according to the method of Mua et al. [s]. A 3% starch sample was placed on the AR10o0 rheometer platform. Rheometer fixture selection 40,40 mm cone plate, the gap is set to 1 mm, rheometer program selection of steady-state flow (Flowprocedure): 50 C to maintain 1m in, and then from 50 °C in 10C/min program temperature To 75'C, 80C, 85C and 90'C, the shear rate is 200 a-'. After the program is over, the sample on the rheometer platform is completely transferred to the centrifuge tube with a pipette and centrifuged at 13500 r/min for 10 min. The precipitate was discarded and the supernatant was taken as amylose sample. The 0.2 mL supernatant was used for wavelength scan analysis, and the rest of the supernatant passed. A 45-μm microporous membrane was filtered and subjected to HPSEC-MALLS-RI analysis. 
1.3.2 Wavelength scanning of amylose samples According to Cheethan's method, wavelength scanning was performed with appropriate modifications. Take amylose sample (supernatant) 0.2 m L, plus human inclusion. 006mol/L iodine 90% dimethyl sulfoxide solution 8 mL, react at room temperature for 24 h, stir the solution from time to time, and then take 1 mL of solution and 8 mL of deionized water to mix well, and stand for 30 m. After the reagent scan is used as a blank, wavelength scanning is performed in the wavelength range of 250-800 nm Cs7
1.3.3 Color directional and multi-angle light scattering instrument conditions St yra ge l presaturated column, Styragel HMW7, StyragelHMW6 and Styragel HMW2 columns (Waters); mobile phase using dimethyl sulfoxide solution containing 50 mmol/L LiBr ; The flow rate is 0. 6 mL / min; column temperature is maintained at 40'C using the column thermostat; multi-angle light scattering instrument uses helium gas and neon gas light source, the wavelength is selected 623. 8 nmC'7.
1.3.4 Data Processing The data was analyzed and processed using Astra (version 3.4. Wyatt Thchonlogy, Santa Barbara, CA) data analysis software. 
2 Results and Analysis
2.1 Parameter Setting and Instrument Verification
2.1.1 Data Processing and Parameter Setting Currently, gel chromatography and exclusion chromatography are two of the most widely used techniques for determining molecular weight. Standard samples of known molecular weights are usually required, but it is difficult to find a standard with a molecular structure and molecular weight close to that of the starch to be measured in the molecular weight of starch. Exclusion liquid chromatography and multi-angle light scattering and refractive index detectors (HPSEC-MALLS-RI) make it possible to determine the molecular weight of starch. When a beam of light strikes a medium, light intensity can be observed in all directions. The intensity of the scattered light is related to the molecular size, shape, and intermolecular interaction in the solution. The relationship between the weight average molecular weight Mw and the average molecular radius of rotation is expressed by the following formula [CIO]
K' c / R, = [1+ (16, r'/3A 2) 
2.1.2 Accuracy of the Instrument Face Detection Dextran samples T2000 and T40 first entered the HPSEC-MALLS-RI system as a standard to verify the accuracy of the system, and established various parameters and standards for the multi-angle light scattering instrument. Figure 1 shows its HPSEC-MALLS-RI chromatogram. Starch is a polydisperse with a range of molecular weight distributions. The parabolic distribution in Figure 1 shows the concentration of the sample in the different elution volumes of the mobile phase detected by the refractometer. The peak linear distribution shows the corresponding molecular weight distribution at each concentration. It can be seen that the first elution is the highest molecular weight. In part, low molecular weight fractions flow out, and the molecular weights in the figure are linearly distributed from high to low. The weight average molecular weights Mw of T2000 and T40 measured by the HPSEC-MALLS-RI system were 1.95X10' and 3.89X10', respectively, calculated by Astra software analysis, and the actual weight average molecular weight (2.0X10' and 4.0X10') is very close, indicating that the HPSEC-MALLS-RI system combined with the Astra software to determine the molecular weight distribution of the polymer by means of second order Berry extrapolation has a high accuracy. Therefore, the latter determination is based on glucomannan samples. T200. The parameters of the multi-angle light scatterometer are set using the T40 as a standard, that is, the parameters of its side timing as a unified standard.
2.2 HPSEC-MALLS-RI Analysis of Rice Starch Samples The solubility of starch in aqueous solvents is very poor. The most commonly used solvent for the dissolution of starch is dimethyl sulfoxide. Studies have shown that adding a small amount of water to dimethyl sulfoxide or The addition of some electrolytes with smaller molecular weight contributes to the dissolution of starch molecules [3,12], but there have been very large differences in the molecular mass of rice starch that have been reported, approximately 0.39X103-7.510X103 [3.12]. This may be caused by different sources of rice. On the other hand, it also shows that the true dispersion and dissolution of starch still have problems [13]. In the previous study, the experimental group compared the HPSEC-MALLS-RI spectra of different solvents for rice samples of Cocodrie rice. By Astr. The software analysis showed that the weight average molecular weight Mw of Cocodrie rice starch was 9.2×10′, and the average rotation radius of the molecule was 222. 6 nm. It can be seen that there is only one peak in the graph, that is, the two are not well separated in the size exclusion chromatography. Although the molecular weights (Mw) of amylose and amylopectin differ greatly, their radius of rotation (RMS) is very close. Therefore, in the separation by high-performance liquid size exclusion chromatography (HPSEC), the map corresponds to straight The two peaks of the chain and amylopectin did not separate. The molecular weight distribution curve shows that the molecular weight of the sample flowing out at any time is not close to the molecular weight of amylose, and the experimentally obtained molecular weight is the average molecular weight after the aggregation of amylose and amylopectin. Therefore, the real separation of the size exclusion chromatography of linear and amylopectin cannot be achieved. Only the separation of amylose in advance can effectively determine its molecular weight distribution.
2.4 RPSEC-MALLS-RI analysis of rice amylose samples The isolated amylose was analyzed by the HPSECMALLS-RI system. The molecular weight distribution of amylose leached at different temperatures is shown in Figure 3 and can be seen for Cocodrie. Amylose, the molecular weight distribution of amylose leached from 75^90C is very similar. For L-205 and Koshi, the molecular weight distribution of amylose leached at 90°C was significantly different from other temperatures. Because the lower the amylose content, the more conducive to the thermal expansion of the starch granules, and the high content of amylose, will inhibit the thermal expansion of the starch granules. 141 Therefore, at high temperatures, low amylose starch granules are more It is easy to be thermally expanded until it breaks. Not only the amylose will leach and dissolve into the solution, but also the amylopectin will dissolve into the solution. The dissolved amylopectin will be easily aggregated with the longer chain amylose. Therefore, at 90°C, the amylopectin and long-chain amylose aggregates are easily precipitated when sampled and centrifuged. When the supernatant is sampled for molecular weight measurement, a small molecular weight distribution is obtained. The molecular weight distribution deviates downward from the straight line. Therefore, when the temperature is higher than 90°C, it has been unsuitable for the separation of amylose. Below 90°C, the leached amylose has a very similar molecular weight distribution at different temperatures. After calculation and processing by Astra software, the weight average molecular weight (Mw) and molecular average rotation radius (RMS) of the leached amylose can be obtained at different temperatures. See Table 2 and discard the high temperature 90V, as can be seen from Table 2. The weight average molecular weight of the amylose obtained after the separation is in the range of 3.29×10'-2.75×I1F]d, which is much smaller than the weight average molecular weight of the starch measured before the separation of amylose in 2.2. The reported molecular weight of amylose is very close to [-lsl. At the same time, it can be seen that the Mw of the amylose obtained by L-205 and Koshi at 90, C is significantly lower than the Mw of the amylose obtained at its temperature, while Cocodrie is different from the amylose obtained at 900C. The Mw is comparable to the Mw of the amylose obtained at other temperatures, which is consistent with the results of the molecular weight distribution given in FIG. 3 . However, the average molecular radius of RMS does not change significantly with temperature. Because with the increase of temperature, although a small amount of amylopectin dissolves along with amylose and forms aggregates, most of the aggregates have been removed after centrifugation.
1) Although the molecular weights of amylose and amylopectin are very different, their rotation radii are very close. When separated by high-performance liquid size exclusion chromatography (HPSEC), it is impossible to achieve linear and amylopectin. Effective separation. Only the separation of amylose can determine its molecular weight distribution.
2) The use of a rheometer to control the gelatinization of rice starch is an effective method for the separation of amylose by leaching. High temperature (higher than 9D C) is not conducive to the separation of amylose from rice starch with low amylose content by leaching. When the temperature is lower than 90°C and higher than the gelatinization temperature, each temperature can be separated by leaching. Amylose, the resulting amylose has a very similar molecular weight distribution, weight average molecular weight and molecular radius of rotation.
3) The use of a rheometer to separate amylose, combined with HPSEC, MALLS, and RI, can accurately determine rice straight chains. The molecular weight distribution and molecular rotation radius of starch are compared with foreign studies [za], which is more convenient and faster.