A method for quantitatively determining the binding force between rice starch granules and proteins and their components

Abstract

The present invention relates to a method for quantitatively determining the binding force between rice starch granules and proteins and their components in the field of agricultural product analysis technology. It sequentially measures the binding force between albumin, globulin, glutelin, and prolamin and starch granules, and finally calculates the average value to obtain the binding force between starch granules and total protein, and uses this value to evaluate or improve the taste and texture of rice foods. The larger the C value, the greater the binding force between starch granules and proteins, and the better the viscosity and elasticity of starch granules. This method is not limited to measuring the binding force between starch granules and total protein in rice, but can also be used for measuring the binding force between starch granules and total protein in other grains, such as wheat, corn, millet, etc.

Description

Technical Field

[0001] The present invention relates to the technical field of agricultural product analysis, and particularly to the determination technology of the binding force between starch granules and proteins in rice. Background Art

[0002] Rice, as the staple food for half of the world's population, supplies basic energy for more than 60% of the population in China. Starch (90%, existing in the form of starch granules) and protein (8%), as the two most crucial metabolites, account for about 98% of the dry weight of rice. The impacts of these two components on rice are mainly reflected in four aspects: metabolite content, component, structure, and their binding force. Previous studies have established methods for quantitatively describing the content, component, and structure of metabolites, laying a foundation for the formation of rice yield and quality and playing an important role in the development of the rice industry. With the further in-depth research, scholars have further confirmed that the change of the binding force between rice and starch granules and proteins is closely related: proteins affect the gelatinization process of rice flour by competing with starch granules for bound water and through their own disulfide bonds. Excessive proteins will adhere tightly to the surface of amyloplasts. During the cooking process, protein molecules also entangle with long-chain starch, slowing down the disintegration rate of starch structure, inhibiting the release of protons, and reducing the viscosity of cooked rice, resulting in the deterioration of the taste of rice. Although the loose connection between starch and protein will significantly increase the chalkiness, it will also weaken the competition of these two factors for bound water. Many studies on the significant positive correlation between the eating quality of rice and chalkiness can indirectly prove this point. The above shows that the binding force between starch granules and proteins has a significant impact on the taste and quality of rice products. However, there is currently a lack of a method for quantitatively describing the binding force between starch granules and proteins and their components. Summary of the Invention

[0003] The purpose of the present invention is to provide a method for quantitatively determining the binding force between rice starch granules and proteins and their components, and obtaining the binding force between rice starch granules and total proteins through quantitative calculation, so as to further evaluate or improve the taste and texture of rice foods.

[0004] To this end, the present invention provides a method for quantitatively determining the binding force between rice starch granules and proteins and their components, including the following steps:

[0005] Step 1), determining the binding force between starch granules and albumin:

[0006] After defatting rice flour, add ultrapure water and shake evenly. After centrifugal separation by a centrifuge, collect the supernatant, which is the albumin solution A1; continue to add water to the remaining residue for centrifugal separation, separate the supernatant, and repeat this three times. Combine the supernatants of the three repetitions, which is the albumin solution B1; analyze the protein contents of A1 and B1 respectively to obtain A2 and B2, then the binding force C1 between starch granules and albumin is: C1(%) = B2 / (A2 + B2) * 100;

[0007] Step 2), determining the binding force between starch granules and globulin:

[0008] Add the residue at the bottom after step 1) to the NaCl solution, shake well, and after centrifugation and separation by a centrifuge, collect the supernatant, which is globulin solution A3; for the remaining residue, continue to add the NaCl solution for centrifugation and separation, separate the supernatant, repeat three times, and combine the supernatants from the three repetitions to obtain globulin solution B3; analyze the protein contents of A3 and B3 respectively to obtain A4 and B4, and the binding force C2 between starch granules and globulin is: C2(%) = B4 / (A4 + B4) * 100;

[0009] Step 3), determine the binding force between starch granules and glutelin:

[0010] Add the residue at the bottom after step 2) to the NaOH solution and shake well, and after centrifugation and separation by a centrifuge, collect the supernatant, which is glutelin solution A5; for the remaining residue, continue to add the NaOH solution for centrifugation and separation, separate the supernatant, repeat three times, and combine the supernatants from the three repetitions to obtain glutelin solution B5; analyze the protein contents of A5 and B5 respectively to obtain A6 and B6, and the binding force C3 between starch granules and glutelin is: C3(%) = B6 / (A6 + B6) * 100;

[0011] Step 4), determine the binding force between starch granules and prolamin:

[0012] Add the residue at the bottom after step 3) to the ethanol solution and shake well, and after centrifugation and separation by a centrifuge, collect the supernatant, which is prolamin solution A7; for the remaining residue, continue to add the ethanol solution for centrifugation and separation, separate the supernatant, repeat three times, and combine the supernatants from the three repetitions to obtain prolamin solution B7; analyze the protein contents of A7 and B7 respectively to obtain A8 and B8, and the binding force C4 between prolamin and starch granules is: C4(%) = B8 / (A8 + B8) * 100;

[0013] Step 5), determine the binding force between starch granules and total protein:

[0014] Calculate the average value according to the formula C = (C1 + C2 + C3 + C4) / 4. The C value in the formula represents the binding force between starch granules and total protein. The C value is used to evaluate or improve the taste and texture of rice foods. The larger the C value, the greater the binding force between starch granules and protein, and the better the viscosity and elasticity of starch granules.

[0015] Further, when defatting rice flour, place the rice flour in a centrifuge tube, add n-hexane for defatting for a period of time, then centrifuge and separate, and discard the supernatant.

[0016] Furthermore, steps 1)-4) are carried out at a constant temperature of 20°C. The rotational speeds for the centrifugal separation of albumin, globulin, glutelin, and prolamin are 3000 rpm, and the first separation times in the corresponding steps are 6 min, 6 min, 20 min, and 2 min respectively; the remaining residue in each step has a centrifugal separation time of 15 min each time. Based on the differences in the sedimentation coefficients of starch granules and different protein components in different solutions during centrifugation, the present technical invention determines the centrifugation time at a certain rotational speed of the centrifuge. At this time, the magnitude of the binding force between starch granules and total protein can be obtained more accurately and quickly.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: Rice flour is an aggregate composed of starch granules and proteins and their components through adhesion, embedding, and encapsulation. The binding force between starch granules and proteins is manifested as a layer of protein covering the surface of starch granules. This binding can make the starch granules more stable and prevent them from decomposing excessively or losing their shape during cooking or processing. At the same time, the presence of proteins can also increase the viscosity and elasticity of starch granules, improving the taste and texture of food. For example, in bread making, the binding of starch granules to the proteins in the dough can increase the elasticity and extensibility of the dough, making the bread softer and more elastic. The present invention solves the problem that the binding force between starch granules and total protein in rice flour cannot be quantitatively described. It determines the binding forces between albumin, globulin, glutelin, and prolamin and starch granules in sequence through the order of successive steps, avoiding the mutual interference of different reagents on the detection effect during detection, so as to obtain a more accurate binding force between starch granules and total protein. By evaluating or improving the taste and texture of rice-based foods through this quantitative value, the larger the C value, the greater the binding force between starch granules and proteins, and the better the viscosity and elasticity of starch granules. This method is not limited to measuring the binding force between starch granules and total protein in rice, but can also be used for measuring the binding force between starch granules and total protein in other grains, such as wheat, corn, millet, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is an electron micrograph of the combination of rice starch granules and the binding force between starch granules and proteins.

[0019] Among them, A is the state of rice starch granules (when there is no protein); B is the state of the combination of rice starch and proteins (type B rice flour); C is the state of the combination of rice starch and proteins (type C rice flour). DETAILED DESCRIPTION OF THE INVENTION

[0020] The following further illustrates the present invention with specific embodiments.

[0021] A method for quantitatively determining the binding force between rice starch granules and proteins and their components, characterized by comprising the following steps:

[0022] (1)Determine the binding force between starch granules and albumin: After defatting 1 g of rice flour, add 4 ml of ultrapure water and oscillate at a constant temperature of 20 °C for 4 h, then centrifuge at 3000 rpm for 6 min. The supernatant collected is the albumin solution A1; add another 4 ml of ultrapure water to the remaining residue, centrifuge at 3000 rpm for 15 min, repeat three times, and combine the supernatants of the three repetitions to obtain the albumin solution B1. Use the Kjeldahl method or colorimetric method to measure the protein content in solutions A1 and B1, which are A2 and B2 respectively; the binding force C1 between starch granules and albumin is: C1(%) = B2 / (A2 + B2) * 100.

[0023] (2)Determine the binding force between starch granules and globulin: Add 4 ml of 5% NaCl solution to the residue after the treatment in step (1), oscillate at 20 °C for 4 h, centrifuge at 3000 rpm for 6 min, and collect the supernatant as the globulin solution A3; continue to add 4 ml of 5% NaCl solution to the remaining residue, centrifuge at 3000 rpm for 15 min, repeat three times, and combine the supernatants of the three repetitions to obtain the globulin solution B3. Use the Kjeldahl method or colorimetric method to measure the protein content in solutions A3 and B3, which are A4 and B4 respectively; the binding force C2 between starch granules and globulin is: C2(%) = B4 / (A4 + B4) * 100.

[0024] (3)Determine the binding force between starch granules and glutenin: Add 4 ml of 0.02 M NaOH solution (pH = 11.0) to the residue after the treatment in step (2), shake at 20 °C for 30 min, centrifuge at 3000 rpm for 20 min, and collect the supernatant as the glutenin solution A5; continue to add 4 ml of 0.02 M NaOH solution (pH = 11.0) to the remaining residue, centrifuge at 3000 rpm for 15 min, repeat 3 times, and combine the supernatants of the three repetitions to obtain the glutenin solution B5. Use the Kjeldahl method or colorimetric method to measure the protein content in solutions A5 and B5, which are A6 and B6 respectively; the binding force C3 between starch granules and glutenin is: C3(%) = B6 / (A6 + B6) * 100.

[0025] (4)Determine the binding force between starch granules and prolamin: Add 3 ml of 70% ethanol solution to the residue after the treatment in step (3), oscillate at 20 °C for 4 h, centrifuge at 3000 rpm for 2 min, and collect the supernatant as the prolamin solution A7. Repeat the process from adding 3 ml of 70% ethanol solution to centrifuging at 3000 rpm for 15 min three times for the remaining residue, and combine the supernatants of the three repetitions to obtain the prolamin solution B7. Use the Kjeldahl method to measure the protein content in solutions A7 and B7, which are A8 and B8 respectively. The binding force C4 between prolamin and starch granules is: C4(%) = B8 / (A8 + B8) * 100. Step 5), determine the binding force between starch granules and total protein:

[0026] (5) Calculate the average value according to the formula C = (C1 + C2 + C3 + C4) / 4. The C value in the formula characterizes the binding force between starch granules and total protein. The C value is used in the fields of cultivation and breeding to evaluate or improve the taste and texture of rice foods. The greater the C value, the greater the binding force between starch granules and protein, and the better the viscosity and elasticity of starch granules.

[0027] Table 1 is a comparison table of the resolution of the binding force between starch and protein components of different types of rice at different centrifugation times. Rice flour samples with different protein contents and chalkiness degrees were obtained from the same rice variety under different treatments, namely B-type and C-type rice flour (Table 1). B-type rice flour is high-protein and low-chalky rice flour, and C-type rice flour is low-protein and high-chalky rice flour. The rice used for their production was obtained by cultivation in atmospheric environments with CO2 concentrations of 400 and 580 μmol / mol respectively, that is, rice grown in two environments with 400 and 580 μmol of CO2 per mole of atmosphere, and then processed into rice flour. The rice variety is Nanjing 9108; the centrifuge (LXJ-IIB, Shanghai Anting / Anke Scientific Instrument Factory, Shanghai, China) rotates at 3000 rpm. The binding force between starch and protein and their components of the two types of rice flour is shown in Table 1; considering the difference and ratio results of the binding force of the two types of rice flour, the discrimination of albumin, globulin, glutelin and prolamin is the best when centrifuged at 3000 rpm for 6 min, 6 min, 20 min and 2 min respectively. The results in Table 1 show that the binding force between starch granules of C-type rice flour and albumin, globulin, glutelin, prolamin and total protein is 2.21, 3.95, 23.58, 9.29 and 9.76 percentage points higher than that of B-type rice flour respectively, indicating that the binding force between starch granules and protein and protein components of low-protein and high-chalky C-type rice flour is greater. The greater the C value, the greater the binding force between starch granules and protein, and the better the viscosity and elasticity of starch granules. This C value can guide cultivation and breeding and is used to evaluate or improve the taste and texture of rice.

[0028] Table 1 Comparison of the resolution of the binding force between starch and protein components of different types of rice at different centrifugation times

[0029]

[0030] The present invention is not limited to the above embodiments. Based on the technical solutions disclosed in the present invention, those skilled in the art can make some substitutions and deformations of some technical features without creative labor according to the disclosed technical content, such as the recombining of centrifuge speed and centrifugation time, etc. These substitutions and deformations are all within the protection scope of the present invention. The present invention is not limited to rice and can also be used for other grains, such as wheat, corn, millet, etc.

Claims

1. A method for quantitatively determining the binding force between rice starch granules and proteins and their components, characterized in that It includes the following steps: Step 1), determining the binding force between starch granules and albumin: After defatting the rice flour, add ultrapure water and shake evenly. After centrifugal separation with a centrifuge, collect the supernatant as albumin solution A1; for the remaining residue, continue to add water and perform centrifugal separation, then separate the supernatant. Repeat this three times, and combine the supernatants from the three repetitions to obtain albumin solution B1; analyze the protein contents of A1 and B1 respectively to get A2 and B2. Then the binding force C1 between starch granules and albumin is: C1(%) = B2 / (A2 + B2) * 100; Step 2), determining the binding force between starch granules and globulin: Add NaCl solution to the residue at the bottom after Step 1) and shake evenly. After centrifugal separation with a centrifuge, collect the supernatant as globulin solution A3; for the remaining residue, continue to add NaCl solution and perform centrifugal separation, then separate the supernatant. Repeat this three times, and combine the supernatants from the three repetitions to obtain globulin solution B3; analyze the protein contents of A3 and B3 respectively to get A4 and B4. The binding force C2 between starch granules and globulin is: C2(%) = B4 / (A4 + B4) * 100; Step 3), determining the binding force between starch granules and glutelin: Add NaOH solution to the residue at the bottom after Step 2) and shake evenly. After centrifugal separation with a centrifuge, collect the supernatant as glutelin solution A5; for the remaining residue, continue to add NaOH solution and perform centrifugal separation, then separate the supernatant. Repeat this three times, and combine the supernatants from the three repetitions to obtain glutelin solution B5; analyze the protein contents of A5 and B5 respectively to get A6 and B6. The binding force C3 between starch granules and glutelin is: C3(%) = B6 / (A6 + B6) * 100; Step 4), determining the binding force between starch granules and prolamin: Add ethanol solution to the residue at the bottom after Step 3) and shake evenly. After centrifugal separation with a centrifuge, collect the supernatant as prolamin solution A7; for the remaining residue, continue to add ethanol solution and perform centrifugal separation, then separate the supernatant. Repeat this three times, and combine the supernatants from the three repetitions to obtain prolamin solution B7; analyze the protein contents of A7 and B7 respectively to get A8 and B8. The binding force C4 between prolamin and starch granules is: C4(%) = B8 / (A8 + B8) * 100; Step 5), determining the binding force between starch granules and total protein: Calculate the average value according to the formula C = (C1 + C2 + C3 + C4) / 4. The C value in the formula represents the binding force between starch granules and total protein. The C value is used to evaluate or improve the taste and texture of rice in the fields of cultivation and breeding. The larger the C value, the greater the binding force between starch granules and protein, and the better the viscosity and elasticity of starch granules.

2. The method for quantitatively measuring the binding force between rice starch granules and proteins and their components according to claim 1, wherein: When defatting the rice flour, place the rice flour in a centrifuge tube, add n-hexane for defatting for a period of time, then perform centrifugal separation, and discard the supernatant.

3. A method for quantitatively determining the binding force between rice starch granules and proteins and their components according to claim 1, characterized in that: Steps 1) - 4) are carried out at a constant temperature of 20 °C. The rotation speed for the centrifugal separation of albumin, globulin, glutelin, and prolamin is 3000 rpm. The first separation times corresponding to the respective steps are 6 min, 6 min, 20 min, and 2 min; the centrifugal separation time for the remaining residue in each step is 15 min each time.

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