Method for detecting resistant starch in rice

Abstract

The invention discloses a rice resistant starch detection method, and belongs to the technical field of resistant starch detection. The rice resistant starch detection method comprises the following steps: grinding rice into rice flour, degreasing the rice flour, adding water, and steaming to obtain cooked rice flour; adding a mixed enzyme buffer solution containing alpha-amylase and glucoamylase into the cured rice flour for treatment, then adding an ethanol solution to terminate the reaction, and centrifuging to obtain a precipitate; adding alkali liquor into the precipitate for treatment, then adding a saccharifying enzyme buffer solution for treatment, and taking supernate after centrifugation; and detecting the content of glucose by using a DNS method, and calculating the content of resistant starch. The method solves the problems that in the prior art, the determination time is long, needed reagents are many and complex, the determination is influenced by starch fermentation due to too long decomposition time of non-resistant starch, the sample pretreatment mode is fuzzy and the like, and the resistant starch content of the rice in the cooking and eating state is accurately reflected.

Description

Technical Field

[0001] The present invention belongs to the technical field of resistant starch detection, and particularly relates to a method for detecting resistant starch in rice. Background Art

[0002] Resistant starch refers to starch substances that cannot be digested in the normal human intestinal tract. Resistant starch has the effects of reducing blood sugar, improving insulin sensitivity, preventing constipation, colon cancer, reducing blood lipids, losing weight by reducing fat, and improving mineral absorption. As a staple food, increasing the content of resistant starch in rice is crucial for improving the nutritional diet structure of residents. Therefore, the genetic physiology of rice resistant starch and the breeding of high-resistant starch rice varieties have become research hotspots. The basis of these studies is the accurate and efficient detection of resistant starch. The determination methods of resistant starch content include the Berry method (1986), Bjorck method (1987), Champ method (1992), Englyst method (1992), Goni method (1996), and AOAC method (2002). Among them, the improved Goni method and AOAC method are the most commonly used methods. In the Goni method, first, pepsin is used to remove proteins, then α-amylase (37 °C) is used to hydrolyze for 16 h, ethanol is used to wash away the sugars formed by the decomposition of non-resistant starch, 2 mol / L KOH is used to hydrolyze resistant starch, and then glucoamylase is used to catalyze the reaction at 60 °C for 30 min to hydrolyze resistant starch into glucose, and then the GOD-POD method is used to detect the glucose content, and the resistant starch content is obtained by conversion. This method can well simulate the physiological environment of the digestive tract, can detect both high-resistant starch and foods with resistant starch content below 1%, and has good repeatability. However, this method has cumbersome operations, long time consumption, a large variety of reagents, and requires the use of highly toxic sodium azide (NaN3). Therefore, some people improved based on the Goni method and proposed the improved Goni method. The biggest change is that a mixed enzyme solution of α-amylase and glucoamylase (pH = 6) is used to decompose non-resistant starch, but the decomposition time is still 16 h. When decomposing resistant starch, an ice bath is used after adding KOH to prevent starch gelatinization, and the catalytic temperature of glucoamylase is adjusted from 60 °C to 50 °C. In 2002, McCleary et al. proposed the AOAC method based on the Goni method, also known as the McCleary method, which is also the method used in the commercial Megazyme resistant starch kit. The biggest difference is that the step of using pepsin to remove proteins is deleted, and a mixed enzyme solution of α-amylase and glucoamylase (pH = 6) is used to decompose non-resistant starch, but the decomposition time is still 16 h. When decomposing resistant starch, an ice bath is used after adding KOH to prevent starch gelatinization, and the catalytic temperature of glucoamylase is adjusted from 60 °C to 50 °C. The improved Goni method and AOAC method are relatively reliable and have good repeatability. Currently, they have been used by the American Society for Analytical Chemistry to detect the resistant starch content in foods and have been adopted by the Chinese industry standard "Determination of Resistant Starch in Rice and Its Products - Spectrophotometry".However, the existing methods have problems such as long determination time, many and complex reagents involved, too long decomposition time of non-resistant starch leading to starch fermentation affecting the determination, and ambiguous sample pretreatment methods.

[0003] To solve the problem that the time for degrading non-resistant starch in the AOAC method is too long (incubating in a 37°C shaker for 16 h) and the starch hydrolysate shows varying degrees of fermentation, the prior art proposes adding a variety of antibiotic compound agents to inhibit the growth of bacteria and avoid the fermentation and decomposition of resistant starch by bacteria to ensure the accuracy of the determination results. However, the proposed antibiotic compound agent contains a wide variety of antibiotics and is relatively complex, which is not conducive to large-scale detection experiments. Summary of the Invention

[0004] In view of the above prior art, the present invention provides a method for detecting resistant starch in rice, which solves the problems in the prior art such as long determination time, many and complex required reagents, too long decomposition time of non-resistant starch leading to starch fermentation affecting the determination, and ambiguous sample pretreatment methods, and accurately reflects the content of resistant starch in rice in the cooked and edible state.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is: to provide a method for detecting resistant starch in rice, including the following steps: (1) Grind rice into rice flour, and then perform degreasing treatment on it to obtain degreased rice flour; (2) Steam the degreased rice flour with water to obtain cooked rice flour; (3) Add a mixed enzyme buffer solution containing α-amylase and glucoamylase to the cooked rice flour, treat it at 37°C for 4 - 6 h, add an ethanol solution to terminate the reaction, and then centrifuge to obtain a precipitate; the concentrations of α-amylase and glucoamylase in the mixed enzyme buffer solution are 40 - 60 U / mL and 80 - 100 U / mL respectively, and the pH of the mixed enzyme buffer solution is 4.5 - 5.5; (4) Add an alkali solution to the precipitate and treat it at 37°C for 30 - 60 min; (5) Add a glucoamylase buffer solution with a pH of 4.5 - 5 and a glucoamylase content of 1000 - 2625 U / mL, and treat it at 60 - 65°C for 1 - 2 h; (6) After centrifugation, take the supernatant, detect the content of glucose in the supernatant by the DNS method, and calculate the content of resistant starch.

[0006] On the basis of the above technical solution, the present invention can also be improved as follows.

[0007] Further, the particle size of the rice flour is 0.38 - 1 mm; in step (1), the degreasing treatment is to reflux and extract with petroleum ether having a boiling range of 30 - 60°C or a methanol solution with a volume fraction of 85% for 4 - 6 h, the reflux extraction speed is 5 - 6 drops / s, and after reflux extraction, it is dried at 40°C for 1 day.

[0008] Further, in step (2), the material-liquid ratio of defatted rice flour to water is 1 g: 10-20 mL, and the steaming time is 20 min.

[0009] Further, in step (3), the treatment time with the mixed enzyme buffer solution is 6 h; the mixed enzyme buffer solution is prepared from α-amylase, glucoamylase, CaCl2 and a 2 M HAC-NaAC buffer solution, and the concentrations of α-amylase, glucoamylase and CaCl2 in the mixed enzyme buffer solution are 40 U / mL, 80 U / mL and 0.032 μmol / mL respectively; the pH of the mixed enzyme buffer solution is 4.75.

[0010] Further, in step (4), the alkali solution is a 2 M KOH solution, and the treatment time is 30 min.

[0011] Further, in step (5), the pH of the glucoamylase buffer solution is 4.75, and the content of glucoamylase in the glucoamylase buffer solution is 2625 U / mL; the treatment temperature is 60 °C, and the treatment time is 1 h.

[0012] Further, the glucoamylase buffer solution is prepared from glucoamylase and a 2 M HAC-NaAC buffer solution, and is dialyzed with a 2 M HAC-NaAC buffer solution at 4 °C for 12 h.

[0013] The beneficial effects of the present invention are as follows: Compared with the prior art, in the detection method of the present invention, the reagents are more conventional and simple, and the detection cost is much lower than that of the Megazyme resistant starch kit. According to the dietary characteristics that both rice and rice flour are cooked and gelatinized before consumption, the present invention clarifies the pretreatment methods such as defatting, solid-liquid ratio, and ripening, which can more accurately reflect the content of resistant starch in rice (the types of resistant starch in cooked rice and cooked rice flour). The standardized pretreatment is beneficial to the screening of a large number of breeding materials or the accurate detection in a large number of comparative tests. The defatting treatment removes the fat components in rice and eliminates the interference of fat on the detection of resistant starch; after defatting, the detected value of resistant starch will be higher. The ripening treatment simulates the cooking process from an experimental perspective, because rice is consumed after cooking, and the definition of resistant starch is also the starch that cannot be digested after consumption. The defatting and ripening treatments make the detected value more in line with the definition of resistant starch. At the same time, the optimal concentration and pH value of the mixed enzyme solution used for the degradation of non-resistant starch are explored, and the decomposition process is reduced from 16 h to 4 - 6 h, effectively avoiding the problem that the long incubation time during the degradation process of non-resistant starch leads to inaccurate detection results due to bacterial fermentation. At the same time, the glucoamylase solution is passed through dialysis technology to remove the sugar carried during the production process of the glucoamylase reagent, avoiding the influence of too high background sugar content on the determination, and adjusting the pH of the glucoamylase buffer to 4.75 to improve the catalytic efficiency. Through the optimization of these key steps, the detection error of resistant starch is significantly reduced. Since the components in the sample are relatively simple after the previous decomposition process and multiple rinses of the precipitate, the detection method of glucose is changed from the GOD-POD method to the DNS method, with simpler reagents and stronger operability. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is the glucose standard curve; Figure 2 is the influence of rice flour particle size on the detection result; Figure 3 is the influence of defatting reagent on the detection result; Figure 4 is the influence of ripening treatment on the detection result; Figure 5 is the iodine color reaction during the degradation process of non-resistant starch; where A is the treatment for 2 h, B is the treatment for 4 h, C is the treatment for 6 h, and D is the treatment for 8 h; Figure 6 is the iodine color reaction during the degradation process of non-resistant starch; where A is the treatment for 10 h, B is the treatment for 12 h, C is the treatment for 14 h, and D is the treatment for 16 h; Figure 7 is the influence of the degradation process of resistant starch on the detection result; Figure 8 is the influence of the reaction temperature of the glucoamylase buffer on the detection result; Figure 9Comparative experiment between the method of the present invention and industry standards. Detailed implementation manners

[0015] The following combines examples to make a detailed description of the specific implementation manners of the present invention.

[0016] Six rice varieties were used as experimental materials, namely Tianjian Junwen Tangmi, Tuntunmai Shitangmi, Yitangmi, Golden Arowana Selected Soft Aromatic Rice, Huazhonghua Yijiaxiangmi, and Huazhonghua Wuchang Rice.

[0017] Example 1 Drawing of a glucose standard curve for determining glucose content by the DNS method, including the following steps: (1) Reagent preparation: 3,5-Dinitrosalicylic acid reagent (referred to as DNS reagent): Weigh 6.5 g of 3,5-dinitrosalicylic acid and dissolve it in a small amount of distilled water. Transfer it to a 1000 mL volumetric flask, add 325 mL of 2 mol / L NaOH solution, add 45 g of glycerol, shake well, and make up the volume to 1 L with distilled water after cooling; Glucose standard solution: Weigh 200 mg of glucose dried to constant weight, dissolve it in distilled water and make up the volume to 100 mL to prepare a glucose standard solution with a concentration of 2.0 mg / mL.

[0018] (2) Preparation of glucose standard curve: Take 6 15 mL plastic test tubes, add glucose standard solution and distilled water according to the amounts in Table 1 and mix well.

[0019] Table 1 Preparation of standards required for drawing glucose standard curve

[0020] (3) Determination of glucose content: Add 2 mL of DNS reagent to each of the test tubes No. 1 - 6, perform color development by boiling water bath treatment for 10 min, quickly cool with running water after taking out, add 9 mL of distilled water to each and shake well; Measure the absorbance value at a wavelength of 540 nm, use test tube No. 6 as a blank for zero adjustment, with the glucose content on the vertical axis and the absorbance value on the horizontal axis, draw the glucose standard curve, as Figure 1 shown.

[0021] Example 2 A method for detecting resistant starch in rice, including the following steps: (1) Weigh an appropriate amount of rice, grind it into powder, and pass it through a 16-mesh sieve; (2) Defatting: Take the rice powder obtained in step (1), defat it with petroleum ether (boiling range 30 - 60 °C), reflux and extract at a speed of 6 drops / s for 4 h, and dry the defatted rice powder in an oven at 40 °C for 1 day; (3) Weigh 100 mg of the defatted rice powder and put it into a 10 mL capped centrifuge tube; (4) Ripening: Add 1.2 mL of distilled water to every 100 mg of defatted rice flour (material-liquid ratio 1 g: 12 mL), cook for 20 min to achieve sufficient gelatinization of starch, and cool to room temperature after taking out; (5) Add 5 mL of mixed enzyme buffer solution (pH = 4.75), and under the condition of 37 °C, treat with a shaker speed of 220 rpm for 6 h; The mixed enzyme buffer solution (pH = 4.75) is prepared with 2 M HAC-NaAC buffer solution (pH = 4.75). Each 5 mL of mixed enzyme buffer solution contains 200 U of medium-high temperature α-amylase, 400 U of glucoamylase, and 40 μL of 4 mM CaCl2 solution; (6) After the treatment in step (5), add 1 mL of 80% (v / v) ethanol and mix evenly, centrifuge at 6000×g for 6 min, and remove the supernatant; Add 4 mL of 80% (v / v) ethanol to wash the precipitate, centrifuge at 6000×g for 6 min, and remove the supernatant; Add 4 mL of 80% (v / v) ethanol to wash the precipitate, centrifuge at 6000×g for 6 min, and remove the supernatant. Note to pour out the supernatant completely, and a small amount of solution can be absorbed with a filter paper strip; (7) Dissolution: Here, a blank tube is added. Add 1 mL of 2 M KOH solution together with the sample, and under the condition of 37 °C, treat with a shaker speed of 220 rpm for 30 min; (8) Add 4 mL of glucoamylase buffer solution (pH = 4.75), and under the condition of 60 °C water bath, treat with a shaker speed of 220 rpm for 1 h; The glucoamylase buffer solution (pH = 4.75) is prepared with 2 M HAC-NaAC buffer solution (pH = 4.75), and the concentration of glucoamylase is 2625 U / mL; It is obtained after dialysis at 4 °C for 12 h, and the dialysis solution is 2 M HAC-NaAC buffer solution (pH = 4.75); (9) After the treatment in step (8), centrifuge at 6000×g for 6 min. After centrifugation, pipette 1 mL of the supernatant into a 15 mL plastic test tube, add 2 mL of DNS solution, and treat in a boiling water bath at 100 °C for 10 min; (10) Quickly cool with cold water after the boiling water bath treatment, add 9 mL of distilled water, and invert and shake well; (11) Zero with the blank tube, measure the absorbance value at a wavelength of 540 nm, and substitute it into the standard curve to obtain the glucose content; The measured glucose content is G, and the content of resistant starch is calculated to be 0.9G according to the glucose standard curve.

[0022] Example 3 A method for detecting rice resistant starch, comprising the following steps: (1) Weigh an appropriate amount of rice, grind it into powder, and pass through a 16-mesh sieve; (2)Degreasing: Take the rice flour obtained in step (1) and degrease it with 85% (v / v) methanol solution. Reflux and extract at a rate of 6 drops / s for 4 h. The degreased rice flour is dried in an oven at 40 °C for 1 day; (3)Weigh 100 mg of the degreased rice flour and put it into a 10 mL centrifuge tube with a lid; (4)Gelatinization: Add 1.2 mL of distilled water to every 100 mg of degreased rice flour (material-liquid ratio 1 g: 12 mL), and cook for 20 min to achieve sufficient gelatinization of starch. After taking it out, cool it to room temperature; (5)Add 5 mL of mixed enzyme buffer solution (pH = 4.75), and under the condition of 37 °C, treat it on a shaker at a rotation speed of 220 rpm for 6 h; The mixed enzyme buffer solution (pH = 4.75) is prepared with 2 M HAC-NaAC buffer solution (pH = 4.75). Every 5 mL of mixed enzyme buffer solution contains 200 U of medium-high temperature α-amylase, 400 U of glucoamylase, and 40 μL of 4 mM CaCl2 solution; (6)After the treatment in step (5), add 1 mL of 80% (v / v) ethanol, mix well, centrifuge at 6000×g for 6 min, and remove the supernatant; Add 4 mL of 80% (v / v) ethanol to wash the precipitate, centrifuge at 6000×g for 6 min, and remove the supernatant; Add 4 mL of 80% (v / v) ethanol to wash the precipitate, centrifuge at 6000×g for 6 min, and remove the supernatant. Note to pour out the supernatant completely, and a small amount of solution can be absorbed with a filter paper strip; (7)Dissolution: Here, a blank tube is added. Add 1 mL of 2 M KOH solution to both the sample and the blank tube, and under the condition of 37 °C, treat it on a shaker at a rotation speed of 220 rpm for 30 min; (8)Add 4 mL of glucoamylase buffer solution (pH = 4.75), and under the condition of a 60 °C water bath, treat it on a shaker at a rotation speed of 220 rpm for 1 h; The glucoamylase buffer solution (pH = 4.75) is prepared with 2 M HAC-NaAC buffer solution (pH = 4.75), and the concentration of glucoamylase is 2625 U / mL; It is obtained after dialysis at 4 °C for 12 h, and the dialysis solution is 2 M HAC-NaAC buffer solution (pH = 4.75); (9)After the treatment in step (8), centrifuge at 6000×g for 6 min. After centrifugation, pipette 1 mL of the supernatant into a 15 mL plastic test tube, add 2 mL of DNS solution, and treat it in a boiling water bath at 100 °C for 10 min; (10)After the boiling water bath treatment, quickly cool it with cold water, add 9 mL of distilled water, and invert and shake well; (11)Use the blank tube to zero, measure the absorbance at a wavelength of 540 nm, and substitute it into the standard curve to obtain the glucose content; The measured glucose content is G, and the resistant starch content is calculated to be 0.9G according to the glucose standard curve.

[0023] Example 4 A method for detecting resistant starch in rice, comprising the following steps: (1) Weigh an appropriate amount of rice, grind it into powder, and pass it through a 40-mesh sieve; (2) Defat: Take the rice powder obtained in step (1), defat it with an 85% (v / v) methanol solution, reflux and extract at a speed of 5 drops / s for 6 h, and dry the defatted rice powder in an oven at 40 °C for 1 day; (3) Weigh 100 mg of the defatted rice powder and put it into a 10-mL capped centrifuge tube; (4) Gelatinize: Add 2 mL of distilled water to every 100 mg of defatted rice powder (material-liquid ratio 1 g:20 mL), cook for 30 min to achieve sufficient gelatinization of the starch, and cool to room temperature after taking out; (5) Add 5 mL of mixed enzyme buffer solution (pH = 5.5), and under the condition of 37 °C, treat it on a shaker at a rotation speed of 220 rpm for 4 h; the mixed enzyme buffer solution (pH = 5.5) is prepared with a 2 M HAC-NaAC buffer solution (pH = 5.5), and each 5 mL of the mixed enzyme buffer solution contains 300 U of medium-high temperature α-amylase, 500 U of glucoamylase, and 40 μL of 4 mM CaCl2 solution; (6) After the treatment in step (5), add 1 mL of 80% (v / v) ethanol and mix evenly, centrifuge at 6000×g for 6 min, and remove the supernatant; add 4 mL of 80% (v / v) ethanol to wash the precipitate, centrifuge at 6000×g for 6 min, and remove the supernatant; add 4 mL of 80% (v / v) ethanol to wash the precipitate, centrifuge at 6000×g for 6 min, and remove the supernatant. Note that the supernatant should be poured out completely, and a small amount of solution can be absorbed with a filter paper strip; (7) Dissolve: Here, a blank tube is set up, add 1 mL of 2 M KOH solution together with the sample, and under the condition of 37 °C, treat it on a shaker at a rotation speed of 220 rpm for 60 min; (8) Add 4 mL of glucoamylase buffer solution (pH = 4.75), and under the condition of a 65 °C water bath, treat it on a shaker at a rotation speed of 220 rpm for 2 h; the glucoamylase buffer solution (pH = 4.75) is prepared with a 2 M HAC-NaAC buffer solution (pH = 4.75), and the concentration of glucoamylase is 1000 U / mL; dialyze at 4 °C for 12 h to obtain it, and the dialysis solution is a 2 M HAC-NaAC buffer solution (pH = 4.75); (9) After the treatment in step (8), centrifuge at 6000×g for 6 min, and after centrifugation, pipette 1 mL of the supernatant into a 15-mL plastic test tube, add 2 mL of DNS solution, and treat it in a 100 °C boiling water bath for 10 min; (10) After the boiling water bath treatment, quickly cool it with cold water, add 9 mL of distilled water, and invert and shake well; (11)Zero the instrument with a zero-adjustment tube, measure the absorbance at a wavelength of 540 nm, and substitute the measured value into the standard curve to obtain the glucose content; the measured glucose content is G, and the resistant starch content is calculated to be 0.9G according to the glucose standard curve.

[0024] Example 5 Effect of pH and degradation time of the non-resistant starch degradation system on degradation (1) Prepare HAC-NaAC buffer solutions with concentrations of 2M and pH values of 4.75, 5.8, and 6.9 respectively; (2) Prepare mixed enzyme solutions with pH values of 4.75, 5.8, and 6.9: Prepare them with HAC-NaAC buffer solutions with concentrations of 2M and pH values of 4.75, 5.8, and 6.9 respectively. Each 5 mL of the mixed enzyme solution contains 200 U of medium- and high-temperature α-amylase, 400 U of glucoamylase, and 40 μL of 4 mM CaCl2 solution; (3) Prepare α-amylase solution with a pH of 6.9: Prepare it with a HAC-NaAC buffer solution with a concentration of 2M and a pH of 6.9. Each 5 mL of the α-amylase solution contains 200 U of medium- and high-temperature α-amylase and 40 μL of 4 mM CaCl2 solution; (4) Treat 6 kinds of rice according to steps (1) - (4) in Example 3; (5) Divide the samples treated in step (4) into 4 groups, with 8 treatments in each group, and each treatment is repeated 3 times; Add the mixed enzyme solutions with pH values of 4.75, 5.8, and 6.9 and the α-amylase solution with a pH of 6.9 to the 4 groups respectively. The 8 treatments are to treat on a shaker at a rotation speed of 220 rpm for 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 14 h, and 16 h respectively; (6) Add iodine solution to the samples treated in step (5) to carry out an iodine color reaction, detect the starch content in the samples, and take pictures for record.

[0025] Comparative Example 1 A method for detecting rice resistant starch. In the flour grinding and sieving process in step (1) of Example 2, sieve through a 100-mesh sieve, and the other steps are the same as in Example 2.

[0026] Comparative Example 2 A method for detecting rice resistant starch. Do not perform the degreasing process in step (2) of Example 2, and the other steps are the same as in Example 2.

[0027] Comparative Example 3 A method for detecting rice resistant starch. Do not perform the ripening process in step (4) of Example 2, and the other steps are the same as in Example 2.

[0028] Comparative Examples 4 - 7 Investigation on the Influence of Resistant Starch Degradation System on the Detection of Resistant Starch Adjust the reagents added in steps (7) and (8) in Example 2. The specific added reagents are shown in Table 1, and the remaining steps are the same as in Example 2 to obtain the detection results of the resistant starch content of different varieties of rice.

[0029] Table 1 Reagents Added in Step (7) and Step (8)

[0030] Comparative Example 8 A method for detecting resistant starch in rice. The saccharifying enzyme reaction temperature in step (8) of Example 2 is 37°C, and the remaining steps are the same as in Example 2.

[0031] Comparative Example 9 Use the method in the industry standard NYT2638-2014 "Determination of Resistant Starch in Rice and Its Products - Spectrophotometry" to detect the resistant starch content of 6 varieties of rice.

[0032] Experimental Results: 1. Influence of Rice Flour Particle Size on the Detection of Resistant Starch The resistant starch contents detected by the methods of Example 2 and Comparative Example 1 are as Figure 2 shown. It can be seen that the rice flour particle sizes with different diameters have a significant influence on the detection of resistant starch. When passing through a 0.15 mm sieve (100 mesh), the resistant starch contents of the 6 varieties all decreased significantly. The reduction ranges of different varieties vary greatly, among which the reduction range of Wengtang rice is the largest. From this, it can be seen that the material structure of the starch granule arrangement may have a greater influence on the formation of resistant starch. To retain more of the original rice starch arrangement structure, a 1 mm sieve aperture is more reasonable; in addition, the difference between the resistant starch content measured by passing through a 1 mm sieve (16 mesh) and the resistant starch obtained in the product information is smaller.

[0033] 2. Influence of Defatting on the Detection of Resistant Starch The resistant starch contents detected by the methods of Example 2, Example 3 and Comparative Example 2 are as Figure 3 shown. Compared with the non-defatted treatment, defatting with different reagents can significantly increase the measured values of the resistant starch content of the 6 varieties. From this, it can be seen that the fat content will interfere with the determination of resistant starch. The influence of different defatting reagents on the determination of resistant starch varies greatly among different types of rice. Among the varieties with high resistant starch content, the measured values of resistant starch after defatting with petroleum ether are significantly higher than those defatted with 85% (v / v) methanol solution. For the rice with low resistant starch content, the measured values of resistant starch defatted with 85% (v / v) methanol solution are higher than those defatted with petroleum ether, and the difference between the treatments of Jinlongyu Zhenxuan Fragrant Soft Rice and Huazhonghua Wuchang Rice reaches a significant level.

[0034] 3. Influence of Rice Flour Gelatinization on Resistant Starch Detection The resistant starch contents detected by the methods of Example 2 and Comparative Example 3 are as Figure 4 shown. The determination method of raw rice flour (Comparative Example 3) is similar to the current in vitro determination method, omitting the cooking process and retaining the original physiological characteristics of rice. The resistant starch was determined using raw and cooked rice flour, and the results showed that the mass fraction of resistant starch in raw rice flour was about 2.5 times higher than that in cooked rice flour.

[0035] 4. Influence of pH and Degradation Time of Non-resistant Starch Degradation System on Degradation The results of the iodine color reaction during the degradation of non-resistant starch in Example 5 are as Figure 5 and 6 shown. During the degradation of non-resistant starch, after the mixed enzyme solution with pH = 4.75 was treated for 4 h, most of the samples showed no color reaction between starch and iodine reagent. After 6 h, all samples showed no color reaction between starch and iodine reagent. After the mixed enzyme solution with pH = 5.8 was treated for 8 h, there was no color reaction. After the mixed enzyme solution with pH = 6.9 was treated for 14 h, there was no color reaction. The α-amylase solution control with pH = 6.9 showed a color reaction of starch throughout the color reaction process. It can also be concluded that a single α-amylase cannot completely decompose non-resistant starch, resulting in a higher measured value of resistant starch. The mixed enzyme solution with pH = 4.75 is the best reagent for non-resistant starch degradation.

[0036] 5. Influence of Resistant Starch Degradation System on Resistant Starch Detection Resistant starch is fully converted into sugar, and the content of resistant starch can be accurately reflected only by detecting the sugar content. Insufficient degradation will lead to a lower measured value. The detection results of the resistant starch content in Example 2 and Comparative Examples 4-7 are as Figure 7 shown. The influence trends of different degradation conditions on the determination of resistant starch in different types of rice are the same. The content of resistant starch measured by treatment with KOH and glucoamylase buffer solution (pH = 4.75) is the highest, significantly higher than other degradation methods. Therefore, the degradation effect of using KOH and glucoamylase buffer solution (pH = 4.75) is the most sufficient.

[0037] 6. Influence of Glucoamylase Reaction Temperature on Resistant Starch Detection After the non-resistant starch in rice is removed, the remaining resistant starch is decomposed into dextrin under strong alkaline conditions, and the dextrin is converted into monosaccharides under the action of glucoamylase. The resistant starch contents detected by the methods of Example 2 and Comparative Example 8 are as Figure 8 shown. When the glucoamylase reaction is carried out at 60 °C, the measured value of resistant starch is significantly higher than that at the reaction temperature of 37 °C; it shows that glucoamylase can better convert dextrin into glucose at 60 °C, resulting in a higher detection value and more accurate detection results.

[0038] 7. Comparative Experiments with Industry Standards As can be seen from Figure 9 , when comparing the detection method in Example 2 with the industry standard method used in Comparative Example 9, the detection result of resistant starch in the industry standard is significantly higher than that of the detection method proposed in the present invention. This is mainly because the industry standard method does not include a gelatinization step (the comparison between Example 2 and Comparative Example 3 shows that the mass fraction of resistant starch in raw rice flour is higher than that in cooked rice flour); the experimental error of the detection method proposed in the present invention is significantly smaller than that of the industry standard.

[0039] Although the specific embodiments of the present invention have been described in detail in conjunction with the examples, it should not be construed as a limitation on the protection scope of this patent. Within the scope described in the claims, various modifications and deformations that can be made by those skilled in the art without creative efforts still fall within the protection scope of this patent.

Claims

1. A method for detecting rice resistant starch, characterized in that, The following steps are involved: (1) grinding rice into rice flour, and then defatting the rice flour to obtain defatted rice flour; (2) adding water to the defatted rice flour and steaming it to obtain cooked rice flour; (3) adding a mixed enzyme buffer containing α-amylase and saccharifying enzyme to the cooked rice flour, treating the mixture at 37° C. for 4 to 6 hours, adding an ethanol solution to terminate the reaction, and centrifuging to obtain a precipitate; the concentrations of α-amylase and saccharifying enzyme in the mixed enzyme buffer are 40 to 60 U / mL and 80 to 100 U / mL, respectively, and the pH of the mixed enzyme buffer is 4.5 to 5.5; (4) adding alkaline solution to the precipitate and treating at 37° C. for 30 to 60 minutes; (5) Add saccharifying enzyme buffer with a pH of 4.5-5 and a saccharifying enzyme content of 1000-2625 U / mL, and treat at 60-65°C for 1-2 h; (6) After centrifugation, the supernatant was collected and the glucose content in the supernatant was detected by DNS method to calculate the content of resistant starch.

2. The rice resistant starch detection method according to claim 1, wherein: The particle size of the rice flour is 0.38-1 mm. The degreasing treatment in the step (1) is to use petroleum ether with a boiling range of 30-60° C. or a methanol solution with a volume fraction of 85% for 4-6 hours under reflux extraction at a speed of 5-6 drops / s, and then dry at 40° C. for 1 day.

3. The rice resistant starch detection method according to claim 1, wherein: In the step (2), the material-liquid ratio of defatted rice flour to water is 1 g:10-20 mL, and the steaming time is 20 min.

4. The rice resistant starch detection method according to claim 1, wherein: The treatment time of adding the mixed enzyme buffer in step (3) is 6 hours; the mixed enzyme buffer is prepared from α-amylase, saccharifying enzyme, CaCl2 and HAC-NaAC buffer with a concentration of 2M, and the concentrations of α-amylase, saccharifying enzyme and CaCl2 in the mixed enzyme buffer are 40U / mL, 80U / mL and 0.032μmol / mL respectively; the pH of the mixed enzyme buffer is 4.

75.

5. The rice resistant starch detection method according to claim 1, wherein: The alkali solution in step (4) is a 2M KOH solution, and the treatment time is 30 minutes.

6. The rice resistant starch detection method according to claim 1, wherein: The pH of the saccharifying enzyme buffer in the step (5) is 4.75, and the content of saccharifying enzyme in the saccharifying enzyme buffer is 2625 U / mL; the treatment temperature is 60° C., and the treatment time is 1 h.

7. The rice resistant starch detection method according to claim 1, characterized in that: The saccharifying enzyme buffer is prepared by saccharifying enzyme and HAC-NaAC buffer with a concentration of 2M, and dialyzed with HAC-NaAC buffer with a concentration of 2M at 4°C for 12h.

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