Rice starch ester as well as preparation method and application thereof

Abstract

The invention belongs to the field of food processing, and particularly relates to rice starch ester as well as a preparation method and application thereof. According to the invention, by exploring the action rule of reaction time, reaction temperature, pH value and acid addition amount on modified rice starch ester, the content of resistant starch in rice starch is increased and the GI value is reduced through esterification modification, so that the rice starch with the function of regulating blood sugar level is obtained; the product also can significantly improve inflammation and fat accumulation symptoms caused by hyperglycemia; in a diet intervention experiment, both the CRS and the MRS show an effective reduction effect on the weight growth rate, the blood sugar level, the visceral organ index and the inflammatory factor content of the hyperglycemia mouse. The result shows that the CA / MA rice starch ester not only has the function of regulating the blood sugar level, but also can remarkably improve inflammation and fat accumulation symptoms caused by hyperglycemia, and has a positive effect on maintaining the overall health.

Description

Technical Field

[0001] The present invention belongs to the field of food processing, and particularly relates to a rice starch ester, a preparation method thereof, and an application thereof. Background Art

[0002] With the acceleration of the modern life rhythm and the change of diet structure, the incidence of metabolic diseases such as diabetes has been increasing year by year, posing a serious threat to people's health. As one of the main food crops in the world, rice has a starch content as high as 70%-80%, which is an important source for the human body to obtain energy. However, traditional rice starch has the characteristics of fast digestion speed and high glycemic index (GI). Long-term and large intake may lead to blood glucose fluctuations and increase the risk of diabetes. Therefore, developing new rice starch products with low GI and high resistant starch (RS) content is of great significance for preventing and controlling diabetes.

[0003] As a functional dietary fiber, resistant starch performs excellently in regulating blood glucose, improving insulin sensitivity, promoting intestinal health, etc. Citric acid (CA) and malic acid (MA), as intermediate products of the human tricarboxylic acid cycle, are safe and non-toxic, and have good acidity and chelating ability, and have been widely used in the food industry. Research shows that esterification modification of rice starch with citric acid and malic acid can effectively increase its resistant starch content and reduce the GI value, thereby improving the nutritional value and health function of rice starch.

[0004] At present, the research on citric acid rice starch ester (CRS) and malic acid rice starch ester (MRS) is still in its infancy. Although existing research has confirmed that esterification modification can increase the resistant starch content of rice starch and reduce the GI value, there are still many unsolved mysteries in aspects such as the optimization of the preparation method, the relationship between the structure and physicochemical properties of the modified starch, and the application of the modified starch in the food industry. Summary of the Invention

[0005] The purpose of the present invention is to provide a rice starch ester, a preparation method thereof, and an application thereof, aiming to explore the action rules of reaction time, reaction temperature, pH value, and acid addition amount on the modified rice starch ester, and to increase the resistant starch content of rice starch and reduce the GI value through esterification modification, so as to obtain a product with the function of regulating blood glucose level and significantly improving the symptoms of inflammation and fat accumulation caused by hyperglycemia.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] The present invention provides a preparation method of a rice starch ester, comprising the following steps:

[0008] (1) Use a pulverizer to pulverize the rice sample. After sieving, add sodium hydroxide, mix and stir. Discard the supernatant, wash, dissolve, and then centrifuge again. Repeat the above washing and centrifugation steps to adjust the pH to neutral. After the third centrifugation, discard the supernatant, dry, pulverize, and sieve to obtain rice starch.

[0009] (2) Dissolve citric acid or malic acid, adjust the pH, and then dilute to obtain an acid solution.

[0010] (3) Mix the acid solution prepared in step (2) with the rice starch prepared in step (1) evenly. Take it out, let it stand at room temperature and then dry. After the reaction, wash the reaction mixture, and then dry, grind and sieve to obtain the rice starch ester.

[0011] Further, in step (1), the stirring time is 4 h, the centrifugation parameters are centrifugation at 3000 r / min for 15 min, and the drying is drying in an oven at 50 °C for 8 h.

[0012] Further, in step (2), the dissolution is to dissolve 8 g of citric acid or malic acid in 20 mL of distilled water.

[0013] Further, in step (2), the pH adjustment is to adjust the system pH to 3.5.

[0014] Further, in step (2), the dilution is to dilute to 50 mL with distilled water.

[0015] Further, in step (3), the uniform mixing is to control the acid dosage at 40 g / 100 g based on the dry weight of the starch. The drying after standing at room temperature is to let it stand at room temperature for 16 h, and then put it into a blast drying oven at 50 °C and dry until the water content is 5% - 10%. The reaction temperature is 130 °C and the reaction time is 6 h.

[0016] The present invention also provides a rice starch ester prepared by the above preparation method, and the rice starch ester is a citric acid rice starch ester or a malic acid rice starch ester.

[0017] The present invention also provides an application of the above rice starch ester in the preparation of a product for regulating blood sugar or improving diabetic symptoms, and the product can reduce blood sugar or improve diabetic symptoms through dietary intervention.

[0018] Further, the product can reduce the body weight growth rate, blood sugar level, organ index, and the contents of IL-6, IL-10 and TNF-α in mice.

[0019] Furthermore, the product can gradually restore the damaged liver tissue structure of mice, significantly improve the fat vacuole phenomenon, and thus effectively control the symptoms of liver and kidney hypertrophy in diabetic mice.

[0020] The beneficial effects of the present invention are as follows:

[0021] By optimizing the preparation conditions, including key parameters such as acid addition amount, reaction temperature, reaction time, and reaction pH, the present invention significantly improves the degree of substitution of CA / MA (citric acid / malic acid) rice starch ester. Under the optimal conditions, the degrees of substitution of citric acid starch ester (CRS) and malic acid starch ester (MRS) reach 0.650 and 0.321 respectively. A high degree of substitution means that more carbonyl groups are successfully introduced into the starch molecules. This modification process not only enhances the chemical stability of the starch ester but also significantly improves its physical and chemical properties, providing a solid foundation for subsequent applications.

[0022] The modified CA / MA rice starch ester prepared by the present invention exhibits excellent water-binding ability, thermal stability, anti-aging property, and transparency. Moreover, through in vitro digestion experiments and animal experiments, it is verified that the CA / MA rice starch ester prepared by the present invention can significantly reduce the content of rapidly digestible starch (RDS) in starch while increasing the content of resistant starch (RS). Compared with the original rice starch, the GI values of CRS and MRS decrease by 36.1% and 31.0% respectively, showing excellent anti-digestibility performance. This characteristic is of great significance for the development of low-GI foods, especially healthy foods for patients with diabetes and obesity, helping to control postprandial blood glucose levels and reduce the risk of related metabolic diseases. In the diet intervention experiment, both CRS and MRS effectively reduce the body weight growth rate, blood glucose level, organ index, and inflammatory factor content in hyperglycemic mice. This result indicates that the CA / MA rice starch ester not only has the function of regulating blood glucose levels but also can significantly improve the inflammation and fat accumulation symptoms caused by hyperglycemia, playing a positive role in maintaining overall health. Description of the Drawings

[0023] Figure 1 It is a graph showing the influence of acid addition amount on the degree of substitution of starch ester. Among them, A is the influence of acid dosage on the degree of substitution of citric acid starch ester, and B is the influence of acid dosage on the degree of substitution of malic acid starch ester;

[0024] Figure 2 It is a graph showing the influence of reaction temperature on the degree of substitution of starch ester. Among them, A is the influence of reaction temperature on the degree of substitution of citric acid starch ester, and B is the influence of reaction temperature on the degree of substitution of malic acid starch ester;

[0025] Figure 3It is a graph showing the effect of the pH of the reaction system on the degree of substitution of starch esters. Among them, A is the effect of the pH of the reaction system on the degree of substitution of citric acid starch esters, and B is the effect of the pH of the reaction system on the degree of substitution of malic acid starch esters;

[0026] Figure 4 It is a graph showing the effect of reaction time on the degree of substitution of starch esters. Among them, A is the effect of reaction time on the degree of substitution of citric acid starch esters, and B is the effect of reaction time on the degree of substitution of malic acid starch esters;

[0027] Figure 5 It is a graph showing the effect of CA / MA esterification on the transparency of rice starch. Among them, different letters indicate significant differences (p < 0.05);

[0028] Figure 6 It is a graph showing the effect of CA / MA esterification on the solubility of rice starch. Among them, different letters indicate significant differences (p < 0.05);

[0029] Figure 7 It is a graph showing the effect of CA / MA esterification on the swelling property of rice starch. Among them, different letters indicate significant differences (p < 0.05);

[0030] Figure 8 It is a graph showing the effect of CA / MA esterification on the rheological properties of rice starch esters. Among them, A is the effect of acid concentration on the loss modulus of the two starch esters, B is the effect of acid concentration on the storage modulus of the two starch esters, and C is the effect of acid concentration on the loss factor of the two starch esters;

[0031] Figure 9 It is a graph showing the analysis results of the content of each component of starch. Among them, different letters indicate significant differences (p < 0.05);

[0032] Figure 10 It is a graph showing the change in body weight during the intervention of mice;

[0033] Figure 11 It is a graph showing the change in blood glucose during the intervention of mice. Among them, A is the change in blood glucose during the intervention of mice, and B is the comparison of blood glucose values after four weeks of intervention. Different letters indicate significant differences (p < 0.05);

[0034] Figure 12 It is a graph showing the effect of CRS / MRS on the organ index of mice. Among them, different letters indicate significant differences (p < 0.05);

[0035] Figure 13 It is a graph showing the effect of CRS / MRS on the inflammatory factors of mice. Among them, different letters indicate significant differences (p < 0.05);

[0036] Figure 14Figure showing the effects of CRS / MRS on the histopathology of mouse liver. Among them, A is the normal control group, B is the high-sugar and high-fat control group, C is the natural starch group, D is the citrate starch ester group, and E is the malate starch ester group;

[0037] Figure 15 Figure showing the correlation analysis results between the GI value of esterified starch and the effect of improving hyperglycemia symptoms in mice. Among them, *p<0.05, **p<0.01, ***p<0.001. Detailed implementation methods

[0038] Example 1 Physicochemical properties and structural characterization of CA / MA rice starch ester

[0039] 1. Experimental materials

[0040] The rice used in this example was from Wuchang Jinhe Rice Industry Co., Ltd., Changlixiang; rice starch was extracted and prepared in the laboratory by the alkali method. The specific instruments involved are shown in Table 1, and the specific experimental reagents involved are shown in Table 2.

[0041] Table 1 Specific instruments

[0042] Name Model Manufacturer Electronic balance JA2007 Shanghai Puruisi Scientific Instruments Co., Ltd. pH meter PHS-25 Shanghai Yidian Scientific Instruments Co., Ltd. High-speed multi-functional pulverizer 8000Y Yongkang Boou Hardware Products Co., Ltd. Hot air blast drying oven 101-3B Shangcheng Instrument Manufacturing Co., Ltd. Digital display constant temperature water bath HH-600 Changzhou Tianrui Instruments Co., Ltd. Rapid viscosity analyzer TCW3.17.3.509 Büchi Labortechnik (Beijing) Co., Ltd. UV spectrophotometer A1pHa-1506 Shanghai Puyuan Instruments Co., Ltd. Thermostatic heating magnetic stirrer DF-101Z Xi'an Yibell Instrument Equipment Co., Ltd. High-speed centrifuge M2-16K Changsha Miqi Instrument Equipment Co., Ltd. Standard sieve 80 mesh, 120 mesh Shaoxing Shangyu Zhenghao Spring Sieve Factory Rheometer MCR102 Anton Paar GmbH Differential scanning calorimeter DSC214 NETZSCH X-ray diffraction XRD-6100 Shimadzu Field emission scanning electron microscope SU8010 Hitachi, Ltd. Inverted fluorescence microscope WYS-41XDY Tianjin Weiyi Optical Instruments Co., Ltd. Fourier near-infrared spectrometer Specturm10 Shanghai PerkinElmer Instruments Co., Ltd. Water bath constant temperature oscillator SHA-C Hangzhou Jingfei Instrument Technology Co., Ltd. Circulating water type multi-purpose vacuum pump SHZ-D(III) Shanghai Xiniulaibo Instruments Co., Ltd.

[0043] Table 2 Experimental reagents

[0044]

[0045]

[0046] 2. Experimental methods

[0047] 2.1 Preparation of rice starch

[0048] Use a pulverizer to crush the rice sample, sieve it (120 mesh), weigh 70 g of rice flour, add 0.1 mol / L sodium hydroxide in a ratio of 1:5, mix and stir magnetically for 4 h; centrifuge at 3000 r / min for 15 min, then take out, discard the supernatant and colored precipitate, wash and dissolve with 100 mL of distilled water and centrifuge again; repeat the above washing and centrifugation steps to adjust the pH value to neutral; after the third centrifugation, discard the supernatant, and dry the precipitate in an oven at 50 °C for 8 h; take it out and crush it, sieve it (80 mesh) to obtain rice starch.

[0049] 2.2 Preparation of CA / MA rice starch ester

[0050] Dissolve 8 g of citric acid / malic acid in 20 mL of distilled water, adjust the pH value of the solution to 3.5 with 10 mol / L sodium hydroxide, and dilute it to 50 mL with distilled water; mix the prepared acid solution evenly with 50 g of rice starch, take it out and let it stand at room temperature for 16 h, then put it into a blast drying oven at 50 °C and dry it for 12 h until the water content is 5% - 10%. Place the pulverized mixture in a blast drying oven and react at 130 °C for 6 h. Wash the reacted mixture with a large amount of distilled water to remove unreacted citric acid and malic acid. Dry the washed starch at room temperature and then grind it, and pass it through an 80-mesh sieve to obtain the finished product.

[0051] 2.3 Determination of the degree of substitution of starch ester

[0052] Weigh 1 g of starch sample, add 100 mL of distilled water, mix well in a 250 mL conical flask, add 2 - 3 drops of phenolphthalein reagent, and titrate the mixed solution with NaOH solution (0.1 mol / L, w / v) until the color of the solution turns slightly red and does not disappear. Stop titration. Add 10 mL of NaOH solution (0.5 mol / L, w / v) to the conical flask, saponify it in a shaker at 37 °C for 50 min, and then titrate it with HCl solution (0.2 mol / L, v / v) until the color disappears as the end point. Record the volume of the consumed HCl solution. Weigh 1 g of rice starch and perform the same operation as above, record the volume of the consumed HCl solution, and calculate the degree of substitution of the two starch esters through the formula.

[0053] 2.4 Influence law of citric acid / malic acid esterification modification conditions on the degree of substitution of rice starch ester

[0054] 2.4.1 Influence of acid addition amount on the degree of substitution of starch ester

[0055] Based on the dry weight of starch, prepare starch milk with different acid addition amounts (20 g / 100 g, 30 g / 100 g, 40 g / 100 g, 50 g / 100 g, 60 g / 100 g). Control the pH of the system at 3.5, the reaction time at 6 h, and the reaction temperature at 130 °C to prepare CRS and MRS. Taking DS as the evaluation index, reflect the changing trend of DS with the increase of acid addition ratio, and conduct 3 parallel experiments for each acid addition amount.

[0056] 2.4.2 Influence of reaction temperature on the degree of substitution of starch ester

[0057] Based on the dry weight of starch, prepare starch milk with an acid ratio of 40 g / 100 g. Control the pH of the reaction system at 3.5, the reaction time at 6 h, and prepare CRS and MRS at different reaction temperatures (110 °C, 120 °C, 130 °C, 140 °C, 150 °C). Taking DS as the evaluation index, reflect the changing trend of DS with the increase of reaction temperature, and conduct 3 parallel experiments for each reaction temperature.

[0058] 2.4.3 Effect of pH of Reaction System on Degree of Substitution of Starch Ester

[0059] Based on the dry weight of starch, starch milk with an acid ratio of 40 g / 100 g was prepared. The reaction time was 6 h, the reaction temperature was 130 °C, and CRS and MRS were prepared under different reaction system pH values (2.5, 3.0, 3.5, 4.0, 4.5). Taking DS as the evaluation index, the change trend of DS with the increase of reaction system pH was reflected, and 3 parallel experiments were carried out for each reaction system pH.

[0060] 2.4.4 Effect of Reaction Time on Degree of Substitution of Starch Ester

[0061] Based on the dry weight of starch, starch milk with an acid ratio of 40 g / 100 g was prepared. The reaction system pH was controlled at 3.5, the reaction temperature was 130 °C, and CRS and MRS were prepared at different reaction times (4 h, 5 h, 6 h, 7 h, 8 h). Taking DS as the evaluation index, the change trend of DS with the increase of reaction time was reflected, and 3 parallel experiments were carried out for each reaction time.

[0062] 2.5 Effect of Citric Acid / Malic Acid Esterification on Physicochemical Properties of Rice Starch

[0063] Under the preparation conditions of controlling the reaction temperature at 130 °C, the reaction time at 6 h, and the reaction system pH at 3.5, based on the dry weight of starch, CRS and MRS samples with acid concentrations of 20%, 40%, and 60% were prepared, and natural rice starch was used as a control to explore the effect of different acid concentrations on the physicochemical properties of the two rice starch esters.

[0064] 2.5.1 Determination of Transparency of Rice Starch Ester

[0065] Weigh 1 g of starch sample, add 100 ml of distilled water to make a 1% starch suspension. Heat and stir in a boiling water bath for 30 min, finally take it out and cool to room temperature, and measure the transmittance (T, %) of the starch paste at a wavelength of 650 nm. The size of the transmittance determines the transparency of the modified starch. The larger the transmittance, the higher the transparency of the modified starch.

[0066] 2.5.2 Determination of Solubility and Swelling Capacity of Rice Starch Ester

[0067] Weigh 0.4 g of the starch sample into a 100 mL centrifuge tube, add 20 mL of distilled water to it, mix well, place it in a shaker at 80 °C and shake for 30 min, cool to room temperature, and then centrifuge (3000 r / min) for 20 min. Finally, pour the supernatant into an aluminum box that has been dried to a constant weight, and dry it to a constant weight at a temperature of 105 °C to measure the solubility and swelling of the rice starch ester. The calculation formula is: S = (m1 / 0.4) × 100; SP = m2 / [0.4 × (1 - S)];

[0068] In the formula, S — solubility, %;

[0069] SP — swelling degree, %;

[0070] m1 — mass of dissolved starch, g;

[0071] m2 — mass of the precipitate in the centrifuge tube, g.

[0072] 2.5.3 Determination of the gelatinization properties of rice starch ester

[0073] Dissolve 3 g of the starch sample in 25 g of water to make a 14% starch suspension and conduct the measurement. The RVA measurement process is as follows: The starch suspension is initially heated at 50 °C (1 min), the initial rotation speed is set to 960 r / min (10 s), the test rotation speed is set to 160 r / min, heated to 95 °C (12.16 °C / min), maintained at 95 °C (2.5 min), cooled to 50 °C (12.16 °C / min), and maintained at 50 °C (2 min).

[0074] 2.5.4 Determination of the thermodynamic properties of rice starch ester

[0075] Mix 2 mg of starch and 6 μL of water and seal them in an aluminum crucible. After equilibrating at room temperature for 24 h, use a differential scanning calorimeter (DSC) for measurement.

[0076] 2.5.5 Determination of the rheological properties of rice starch ester

[0077] Accurately weigh 1 g of the starch sample, dilute it with distilled water to a 6% (w / v) suspension, and magnetically stir it at room temperature for 30 min to form a homogeneous emulsion. The suspension is gelated in a boiling water bath for 30 min, placed on a rheometer, and after adding the sample, add the cover plate. Gently scrape off the excess around the paste, and cover the sample edge with silicone oil to prevent water evaporation. At 25 °C, conduct a dynamic oscillation evaluation with a frequency scan from 0.1 - 100 rad / s, the scan strain is 1%, and measure the changes in the storage modulus (G′), loss modulus (G″), and loss coefficient (tanδ) of the sample.

[0078] 2.6 Data statistics and analysis

[0079] The results of this study were calculated in the format of mean ± standard deviation. SPSS 22.0 was used to statistically analyze the experimental data. One-way analysis of variance (ANOVA) was performed at a significance level of p < 0.05, and Origin 2021 was used to plot the obtained data. All experiments were repeated three times.

[0080] 3. Experimental Results

[0081] The results of the effect of acid addition amount on the degree of substitution of starch esters are as Figure 1 shown. It can be seen from Figure 1 that in the range of acid dosage from 20 g / 100 g to 60 g / 100 g, with the increase of acid dosage, the DS of both rice starch esters first increases and then decreases, and reaches the peak when the addition amount is 40 g / 100 g.

[0082] The effect of reaction temperature on the degree of substitution of rice starch esters is as Figure 2 shown. It can be seen from Figure 2 that in the range of esterification reaction temperature from 110 to 130 °C, the DS of both starch esters shows an upward trend with the increase of reaction temperature. However, when the temperature rises in the range of 130 to 150 °C, the reaction temperature is too high, exceeding 130 °C, which will lead to side reactions dominating.

[0083] The effect of the pH of the reaction system on the degree of substitution of rice starch esters is as Figure 3 shown. It can be seen from Figure 3 that the reaction DS increases with the increase of the pH of the reaction system in the range of pH 2.5 to 3.5, and reaches the peak at pH 3.5.

[0084] The results of the effect of reaction time on the degree of substitution of rice starch esters are as Figure 4 shown. It can be seen from Figure 4 that in the range of reaction time from 4 to 8 h, the DS of citric acid starch ester and malic acid starch ester generally shows a trend of first rising and then falling.

[0085] The results of the effect of CA / MA esterification on the transparency of rice starch are as Figure 5 shown. It can be seen from Figure 5 that the control group of natural rice starch gelatinizes under the action of light refraction and reflection, forming a dull, turbid and opaque solution. The transparency of the starch paste is 8.45%. Compared with the control group, the paste transparencies of CRS and MRS increase with the increase of acid concentration in the range of 20% - 40% acid concentration, and are significantly increased by 28.16% and 28.65% respectively. However, when the acid concentration further increases to 60%, the paste transparencies are significantly reduced by 15.15% and 9.27% (p < 0.05).

[0086] The results of the effect of CA / MA esterification on the solubility and swelling property of rice starch are as follows Figure 6-7 As shown, from Figure 6 it can be seen that due to the high binding force between the original rice starch molecules, it is difficult for its amylopectin to dissolve, resulting in a low solubility of 7.96%. Compared with the control group, the solubilities of CRS and MRS increased by 32.08% and 36.82% respectively, significantly increasing the solubility of rice starch (p<0.05). From Figure 7 it can be seen that compared with the rice starch of the control group, at a concentration of 40%, the swelling degrees of ARS and MRS decreased significantly by 32.6% and 26.7% respectively (p<0.05).

[0087] The results of the effect of CA / MA esterification on the gelatinization properties of rice starch esters showed that compared with the original rice starch, the peak viscosity of the modified starch decreased significantly. The peak viscosity of citric acid starch ester decreased from 2678.0 cP to 576.3 cP, a decrease of about 365.7%. The peak viscosity of malic acid starch ester decreased to 1155.3 cP, a decrease of about 131.8%.

[0088] The results of the effect of CA / MA esterification on the thermodynamic properties of rice starch esters showed that the phase transition onset temperature (T0), peak temperature (Tp), termination temperature (Tc), and enthalpy value (ΔH) of natural rice starch were 66.68 °C, 76.75 °C, 84.13 °C, and 28.03 J / g respectively. Compared with the original rice starch, all the thermodynamic parameters of the two modified starches showed a downward trend. The T0, Tp, Tc, and ΔH of the citric acid starch ester sample decreased significantly to 52.53 °C, 63.60 °C, 80.02 °C, and 12.09 J / g respectively (p<0.05), and the T0, Tp, Tc, and ΔH of the malic acid starch ester sample decreased significantly to 61.11 °C, 74.16 °C, 80.06 °C, and 19.07 J / g respectively (p<0.05), indicating that the esterification reaction can reduce the gelatinization parameters of starch.

[0089] The results of the effect of CA / MA esterification on the rheological properties of rice starch esters are as follows Figure 8 As shown, from Figure 8 it can be seen that in the angular frequency range of 0.1 - 100 rad / s, G' and G" increase with the increase of the angular frequency, and the crosslinking effect is further improved, indicating that the crosslinking of a certain amount of acid with starch can enhance the stability of the starch paste, and the Tanδ of the citric acid starch ester is higher than that of other starch samples.

[0090] 4. Summary

[0091] In summary, this example mainly explored the influence rules of the acid addition amount, reaction pH, reaction temperature, and reaction time on the degree of substitution of the product during the preparation of CRS and MRS. Secondly, the effects of different acid concentrations and degrees of substitution on some physicochemical properties such as the transparency, solubility, viscosity, and thermal stability of the two starch esters were studied. Specifically, the degree of substitution of CRS and MRS was significantly affected by the acid addition amount, system pH, reaction temperature, and reaction time (p<0.05). Among them, under the optimal conditions of an acid addition amount of 40 g / 100 g, pH of 3.5, reaction temperature of 130 °C, and reaction time of 6 h, the degrees of substitution of CRS and MRS reached 0.650 and 0.321, respectively. At the same time, the infrared spectroscopy results showed that during the esterification reaction, the acid anhydride of the two successfully underwent a substitution reaction with the hydroxyl group on the starch glucose residue. In addition, by measuring and analyzing the physicochemical properties of CRS and MRS at different acid concentrations, the influence rules of acid concentration and degree of substitution on the hydration, gelatinization, rheological, and thermodynamic properties of CRS and MRS were explored. The results showed that esterification modification could introduce carbonyl groups into the starch molecules, hindering the swelling of starch granules. Compared with the control group, the solubility and transparency of the starch were significantly enhanced (p0.05). Secondly, esterification modification significantly reduced the starch viscosity, but significantly improved the thermal stability and anti-aging properties, etc. (p0.05), and the effect was the best at an acid concentration of 40% and the properties of CRS were better.

[0092] Example 2 Effect of CA / MA rice starch ester digestibility and improvement of mouse blood glucose level

[0093] 1. Experimental materials and instruments

[0094] The citric acid rice starch ester and malic acid rice starch ester used were both prepared by the optimal preparation process in Example 1. The experimental reagents used are shown in Table 3 below.

[0095] Table 3 Experimental reagents

[0096] Name Manufacturer Hydrochloric acid Xilong Chemical Co., Ltd. Amyloglucosidase Beijing Yita Biotech Co., Ltd. Pepsin Beijing Yita Biotech Co., Ltd. Guar gum Beijing Guarlun Technology Co., Ltd. Porcine pancreatic α-amylase Beijing Yita Biotech Co., Ltd. Sodium acetate buffer solution Beijing Yita Biotech Co., Ltd. Glucose test kit Nanjing Jiancheng Bioengineering Institute Streptozotocin Shanghai Yuanye Bio-Technology Co., Ltd. TNF-α ELISA kit Tianjin Alpha Bio-Tech Co., Ltd. IL-6 ELISA kit Tianjin Alpha Bio-Tech Co., Ltd. IL-10 ELISA kit Tianjin Alpha Bio-Tech Co., Ltd. Sterile PBS Beijing Bioleader Technology Co., Ltd. Normal saline Shanghai Yuanye Bio-Technology Co., Ltd. Absolute ethanol Tianjin Fuyu Fine Chemical Co., Ltd.

[0097] The experimental animals used were 40 8-week-old male SPF-grade KM mice, weighing 18 - 22 g, purchased from Changchun Yisi Experimental Animal Technology Co., Ltd. The room temperature was 20±2 °C, the relative humidity was 60%-70%, the light cycle was 12 h each for day and night, and they had free access to water and food. After one week of adaptive feeding, they were randomly grouped. All animal experiments were approved and implemented by the Animal Ethics Committee of Harbin University of Commerce.

[0098] 2. Experimental methods

[0099] 2.1 In vitro digestion determination of starch

[0100] Sample preparation and preliminary treatment: Disperse 200 mg of starch sample and 5 mL of sodium acetate buffer solution (pH 5.2, 0.5 mol / L) in a 50 mL centrifuge tube and mix well. Then place the centrifuge tube in a boiling water bath for 20 min. After that, transfer the tube to a water bath shaker at 37 °C and shake at 170 r / min until it cools down.

[0101] Simulated gastric juice digestion stage: Add simulated gastric juice solution [HCl (10 mL, 0.05 mol / L), pepsin (50 mg), guar gum (50 mg)] to the centrifuge tube. After mixing evenly, shake for 30 min, add 7 glass beads and 5 mL of sodium acetate buffer solution and continue to shake for 30 min.

[0102] Mixed enzyme solution digestion stage: Add 10 mL of mixed enzyme solution [(porcine pancreatic α-amylase (290 U / mL), amyloglucosidase (260 U / mL)] to the tube. Then shake at 170 r / min in a water bath at 37 °C.

[0103] Sampling and enzyme inactivation: Take aliquots (0.5 mL) at intervals of 20 and 120 minutes and mix with 4 mL of 95% ethanol to inactivate the enzyme.

[0104] Centrifugation and measurement: Centrifuge the mixed solution at 10000 r / min for 5 min and measure the amount of glucose released at 20 min and 120 min using a glucose kit (POD-GOD method).

[0105] Calculate the percentages of RDS, SDS, and RS using the following formulas:

[0106] RDS (%) = ((G20 - FG) / TS) × 0.9 × 100;

[0107] SDS (%) = ((G120 - G20) / TS) × 0.9 × 100;

[0108] RS (%) = ((TS - RDS - SDS) / TS) × 100;

[0109] Where:

[0110] G20 - The amount of glucose released within 20 min of hydrolysis, mg;

[0111] G120 - The amount of glucose released within 120 min of hydrolysis, mg;

[0112] FG - The amount of free glucose in starch, mg;

[0113] TS - Total starch weight, mg

[0114] 2.2 Grouping and modeling of mice

[0115] The mouse experiment was divided into 5 groups (n=8), and the specific operation was as follows:

[0116] Grouping: One group of mice was randomly selected to be fed with ordinary feed, and the rest were fed with high-sugar and high-fat feed.

[0117] Modeling: After two weeks, mice fed a high-sugar, high-fat diet were injected intraperitoneally with streptozotocin (STZ) to establish the model. This was repeated three times, once every other day. Three days later, blood was collected from the tail to measure fasting blood glucose. A blood glucose level exceeding 11.6 mmol / L indicated successful modeling. If it was below this value, further STZ supplementation was indicated.

[0118] Grouping treatment: After the model was successfully established, the hyperglycemic mice were divided into four different groups: a high-sugar and high-fat control group, a raw rice starch group, a citrated starch group, and a malate starch group. The dose group was then gavaged with 3g / kg·bw for four weeks, and the normal control group was gavaged with distilled water daily. During the intervention period, the mice were given free access to food and water.

[0119] Effects of CRS / MRS on body weight and blood glucose in mice

[0120] Blood glucose and body weight of mice were measured every other week. The mice were fasted but not deprived of water the night before the measurement, and blood was collected by tail cutting.

[0121] 2.4 Effects of CRS / MRS on Mouse Organ Indexes

[0122] The liver and kidneys of the mice were weighed after autopsy, and the organ index of the mice was estimated based on the body weight on the day.

[0123] Organ index (g / Kg) = organ fresh weight / mouse body weight

[0124] Effects of 2.5CRS / MRS on inflammatory factors in mice

[0125] The eyeball blood of mice was collected and centrifuged (3500r / min, 15min), and serum was extracted for later use. The levels of IL-6, IL-10, and TNF-α in the serum of mice were determined using Elisa kits.

[0126] Effects of CRS / MRS on Mouse Liver Pathology

[0127] Liver histopathology observation involves preparation of pathological sections and HE staining. The procedure can be summarized as follows: sampling, flattening, dehydration and waxing, embedding, wax block trimming, sectioning, baking, hematoxylin and eosin staining, dehydration, sealing, and finally observation of mouse liver sections.

[0128] 2.7 Data statistics and analysis

[0129] Same as the data statistics and analysis in Example 1.

[0130] 3. Experimental results

[0131] The analysis results of the contents of each component of starch are as Figure 9 shown. It can be seen from Figure 9 that compared with the native starch, the esterification treatment with citric acid and malic acid reduced the swelling and gelatinization of starch granules during heating, restricted the binding ability of starch granules to digestive enzymes, and thus reduced the content of RDS. Therefore, compared with starch citrate, the contents of its RDS and SDS decreased from 40.5% to 30.4% and from 39.6% to 7.7% respectively, and the content of RS increased to 62.0%. Compared with starch malate, the contents of its RDS and SDS decreased to 29.0% and 18.4% respectively, and the content of RS increased to 52.7%.

[0132] The results of the effects of CRS / MRS on the body weight and blood glucose of mice are as Figure 10 and Figure 11 shown. It can be seen from Figure 10 that before the dietary intervention on mice, the body weights of each group of mice were similar. After 4 weeks of intervention, it can be seen that the body weight of the mice in the normal group did not change significantly, while the body weight of the mice in the high-sugar and high-fat model group increased relatively fast, which may be due to insulin resistance. The body weight growth rate of the mice in the experimental intervention group was lower than that of the model group. The amount of body weight increase in the intervention group was as follows: starch malate group > starch citrate group, indicating that the dietary intervention with starch citrate can better slow down the increase in body weight of diabetic mice. It can be seen from Figure 11 that before the dietary intervention on mice, the blood glucose levels of the mice in the high-sugar and high-fat control group and the two starch ester intervention groups were between 11.5 - 12.5 mmol / L, with no significant difference (p < 0.05). After 4 weeks of dietary intervention, the blood glucose levels of the mice in the native starch group and the high-sugar and high-fat control group did not change significantly and remained relatively stable. However, the blood glucose levels of the mice in the starch ester intervention group decreased significantly (p < 0.05), and the final blood glucose values after 4 weeks from high to low were: high-sugar and high-fat control group > native starch group > starch malate group > starch citrate group > normal control group. This indicates that both starch citrate and starch malate have significant effects on the blood glucose of diabetic mice, can regulate the blood glucose metabolism of diabetic mice, and reduce the blood glucose level of mice.

[0133] The results of the effects of CRS / MRS on the organ indices of mice are as Figure 12 shown. It can be seen from Figure 12It can be seen that compared with the normal control group, the kidney and liver indices of the high-sugar and high-fat control group mice were significantly increased (p<0.05). The fluctuations in organ indices were closely related to organ congestion, inflammatory responses, and a large accumulation of fat. In terms of the kidneys, the indices of the citrate starch ester and malate starch ester groups both decreased, but there was no obvious difference between these two groups, and they were significantly lower than those of the native starch group (p<0.05). Compared with the native starch group, the liver indices of the citrate starch ester and malate starch ester groups also decreased significantly (p<0.05).

[0134] The results of the effects of CRS / MRS on inflammatory factors in mice are as Figure 13 shown. From Figure 13 it can be seen that compared with the high-sugar and high-fat control group, the contents of IL-6, IL-10, and TNF-α in the native starch group did not change significantly. However, dietary interventions in the citrate starch ester group and the malate starch ester group could significantly reduce the contents of IL-6, IL-10, and TNF-α in the serum of T2DM mice, and the levels of inflammatory factors in the citrate starch ester group were significantly lower than those in the malate starch ester group (p<0.05).

[0135] The effects of CRS / MRS on the histopathology of mouse liver are as Figure 14 shown. From Figure 14 it can be seen that in the normal control group (Figure A), the mouse liver tissue was intact, the hepatocytes were arranged in an orderly manner, the cell nuclei were intact, radiating from the central vein to the surrounding areas and evenly distributed, and there were no fat droplets in the cells. In contrast, in the high-sugar and high-fat control group (Figure B) and the native starch group (Figure C), the liver lobules of the experimental mice showed obvious disorders, accompanied by a large number of fat vacuoles and fatty lesions of hepatocytes. After dietary interventions with the two esterified starches, the damaged liver tissue structure of the mice was repaired to a certain extent, and the fat vacuole phenomenon was also significantly improved. The observation results of the citrate starch ester group (Figure D) and the malate starch ester group (Figure E) were overall closer to the normal group, indicating that these two substances had a significant effect on inhibiting the formation of fatty liver, and the effect of the citrate starch ester group was slightly better than that of the malate starch ester group.

[0136] The correlation analysis results of the GI values of esterified starches and the effects of improving hyperglycemia symptoms in mice are as Figure 15 shown. From Figure 15It can be seen that after four weeks of dietary intervention on mice with native starch and esterified modified starch, there was a basically significant positive correlation (p<0.05) between the blood glucose level of mice and their body weight, organ indices (liver, kidney) and inflammatory factors (IL-6, IL-10, TNF-α), indicating a significant correlation between the blood glucose level of mice under high-fat and high-sugar feeding and liver injury in mice. High blood glucose can cause histological and functional changes in the liver tissue and lead to liver injury. The RS content in the starch samples was significantly negatively correlated with the above indicators and the starch GI value (p<0.05), indicating that resistant starch has a positive effect on improving the inflammation and fat accumulation caused by high blood glucose. At the same time, in the correlation analysis, there was a significant positive correlation (p<0.05) between the starch GI value and each index of mice. Therefore, the starch GI value was negatively correlated with the overall effect of regulating the blood glucose level of mice and improving the symptoms of high blood glucose in mice.

[0137] 4. Summary

[0138] In this example, the digestion performance of two esterified starches and their effects on regulating the blood glucose level of mice and improving diabetic symptoms were analyzed and compared through in vitro simulated digestion experiments and animal experiments. It was found that compared with natural rice starch, the contents of RDS and SDS in citric acid starch ester and malic acid starch ester decreased to varying degrees, but the RS content increased significantly by 42.1% and 32.8%. Both citric acid rice starch ester and malic acid rice starch ester had significant effects on reducing the blood glucose of mice and improving the symptoms of diabetes in mice. Compared with natural rice starch, after 4 weeks of dietary intervention on T2DM mice with CRS and MRS, the growth rate of body weight, blood glucose level, organ indices and the contents of inflammatory factors such as IL-6, IL-10, TNF-α of mice could be significantly reduced, and were significantly lower than those of the high-sugar and high-fat model group (p<0.05). Moreover, it could gradually restore the damaged liver tissue structure of mice, and the fat vacuole phenomenon was significantly improved, indicating that the dietary intervention of the two rice starch esters had a positive effect on improving the inflammation and fat accumulation caused by T2DM, and could effectively control the hypertrophy of the liver and kidney of diabetic mice. In terms of the effect of the two modified starches, citric acid starch ester > malic acid starch ester.

[0139] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered by the scope of the claims of the present invention.

Claims

1. A preparation method of rice starch ester, characterized in that, It includes the following steps: (1) Crush the rice sample using a crusher, add sodium hydroxide after sieving, mix and stir, discard the supernatant, wash, dissolve, and then centrifuge again; Repeat the above washing and centrifugation steps to adjust the pH value to neutral; After the third centrifugation, discard the supernatant, dry, crush, and sieve to obtain rice starch; (2) Dissolve citric acid or malic acid, adjust the pH, and then dilute to obtain an acid solution; (3) Mix the acid solution prepared in step (2) with the rice starch prepared in step (1) evenly, take it out, let it stand at room temperature and then dry. After the reaction, wash the reacted mixture, and then dry, grind, and sieve to obtain the rice starch ester.

2. The preparation method according to claim 1, wherein In step (1), the stirring time is 4 h, the centrifugation parameter is centrifugation at 3000 r / min for 15 min, and the drying is drying in an oven at 50 °C for 8 h.

3. The preparation method according to claim 1, characterized in that, In step (2), the dissolution is to dissolve 8 g of citric acid or malic acid in 20 mL of distilled water.

4. The preparation method according to claim 1, characterized in that In step (2), the pH adjustment is to adjust the system pH to 3.

5.

5. The preparation method according to claim 1, characterized in that, In step (2), the dilution is to dilute to 50 mL using distilled water.

6. The preparation method according to claim 1, characterized in that, In step (3), the uniform mixing is to control the acid dosage at 40 g / 100 g based on the dry weight of the starch. The drying after standing at room temperature is to let it stand at room temperature for 16 h, and then put it into a forced-air drying oven at 50 °C to dry until the water content is 5% - 10%; the reaction temperature is 130 °C, and the reaction time is 6 h.

7. A rice starch ester prepared by the preparation method according to any one of claims 1-6, characterized in that, The rice starch ester is citric acid rice starch ester or malic acid rice starch ester.

8. Use of the rice starch ester as claimed in claim 7 in the preparation of a product for regulating blood glucose or improving symptoms of diabetes, characterized in that, The product can reduce blood sugar or improve diabetes symptoms through dietary intervention.

9. The application according to claim 8, characterized in that, The product can reduce the body weight growth rate, blood sugar level, organ index, and the contents of IL-6, IL-10, and TNF-α in mice.

10. The application according to claim 8, wherein The product can gradually restore the damaged liver tissue structure in mice, significantly improve the fat vacuole phenomenon, and thus effectively control the symptoms of liver and kidney hypertrophy in diabetic mice.

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