Preparation and application of a heterologous amyloid fiber-anthocyanin complex

Abstract

The present application relates to a kind of heterogenous amyloid fibril-cyanidin-3-O-glucoside (C3G) complex and its application.The preparation method includes: glutelin and soy protein are dispersed in aqueous phase according to mass ratio 3:7-5:5, and are co-assembled to form heterogenous amyloid fibril under acid heat condition;The heterogenous amyloid fibril solution is mixed with C3G solution, and is dried, to obtain heterogenous amyloid fibril-C3G complex.The complex can reduce the hydrolysis rate of rice starch under in vitro digestion condition after being compounded and gelatinized with rice starch.

Description

Technical Field

[0001] This invention belongs to the field of food processing and functional food ingredients technology, specifically relating to a method for constructing a complex of heterologous amyloid protein fibers co-assembled from rice gluten and soybean protein and cyanidin-3-O-glucoside, and the application of the complex in regulating the in vitro digestion of rice starch. Background Technology

[0002] Cyanide-3-O-glucoside, a natural pigment and functional component among anthocyanins, is easily degraded in food systems by factors such as heat, pH, and oxidation. For rice starch-based food systems that require thermal processing such as gelatinization, the insufficient stability of cyanide-3-O-glucoside leads to a decrease in its effective content and limits its ability to form complexes with the starch matrix.

[0003] Amyloid protein fibers formed from food proteins under acidic and hot conditions have a high specific surface area and adjustable surface chemical characteristics. They can be used to load small molecule active ingredients and form an interfacial shielding layer in the food matrix, thus providing a carrier basis for the stabilization and delivery of small molecules during processing.

[0004] In existing technologies, single plant proteins vary in terms of fiberization efficiency or aggregation control. For example, rice gluten protein has relatively low fiberization efficiency, and soybean protein fiber is prone to aggregation under certain conditions, which may limit the usability of the fiber system (such as dispersibility and effective loading capacity), thereby limiting its application as a cyanidin-3-O-glucoside carrier under food processing conditions.

[0005] Therefore, there is still a need to provide a technical solution that can effectively load cyanidin-3-O-glucoside under food processing conditions and then exert a measurable regulatory effect on the in vitro digestion behavior of rice starch after being compounded and gelatinized with rice starch. Summary of the Invention

[0006] Technical problems to be solved To address the issues of insufficient stability of cyanidin-3-O-glucoside under thermal processing conditions such as rice starch gelatinization, leading to a decrease in effective content, and the limitations of single plant protein fiber systems in terms of conversion rate and aggregation control, thus affecting loading performance, this invention provides a method for preparing a heterologous amyloid protein fiber-cyanidin-3-O-glucoside complex, and uses the complex in combination with rice starch to regulate the in vitro digestion of rice starch.

[0007] Technical solution The technical solution of this invention, as described in the claims, includes the following core components: In one aspect, the present invention provides a method for preparing a heterologous amyloid protein fiber-cyanidin-3-O-glucoside complex, comprising the following steps: (1) Mix rice gluten and soybean protein in a mass ratio of 3:7-5:5 and disperse them in an aqueous phase. Adjust the pH of the system to 2-7 and stir and heat at 50-100℃ for 0.5-24 h to denature the rice gluten and soybean protein and co-assemble them to form heterologous amyloid protein fibers. (2) The heterologous amyloid protein fiber solution obtained in step (1) is mixed with cyanidin-3-O-glucoside solution at a volume ratio of 1-5:1, the pH of the system is adjusted to 2.5-3.5, and the mixture is dried to obtain the heterologous amyloid protein fiber-cyanidin-3-O-glucoside complex.

[0008] In one embodiment, the total protein concentration in the aqueous phase of step (1) is 10-40 mg / mL; in another embodiment, the mass ratio of rice gluten protein to soybean protein in step (1) is 5:5; in another embodiment, the pH of the system in step (1) is 2-3.5, the temperature is 85-95℃, and the stirring and heating time is 8-16 h; in another embodiment, after heating, the resulting system is placed in an ice bath to cool for 10-30 min.

[0009] In one embodiment, the concentration of cyanidin-3-O-glucoside solution in step (2) is 5 mM, and the pH of the system after mixing is 3.0; in another embodiment, step (2) is stirred at room temperature and in the dark for 0.5-4 h; in another embodiment, the drying is freeze drying, wherein when the drying is freeze drying, the pre-freezing temperature is -80°C and the freeze drying time is 48-96 h.

[0010] In another aspect, the present invention also provides a heterologous amyloid fibrous-cyanidin-3-O-glucoside complex, the complex comprising: heterologous amyloid fibrous material co-assembled from rice gluten and soybean protein in a mass ratio of 3:7-5:5, and cyanidin-3-O-glucoside bound to the heterologous amyloid fibrous material.

[0011] In another aspect, the present invention also provides a rice starch composition comprising rice starch and the heterologous amyloid cellulose-cyanidin-3-O-glucoside complex, wherein the mass ratio of the complex to rice starch is (1-4):10.

[0012] In another aspect, the present invention also provides the use of the complex or the rice starch composition in the preparation of food ingredients for delaying the in vitro digestion of rice starch.

[0013] Beneficial effects 1) The examples show that rice gluten and soybean protein co-assemble to form heterologous amyloid fibers under acid-heat conditions. Under the conditions described in the examples, the fiber conversion rate can be improved, with a 5:5 ratio showing a higher fiber conversion rate. This provides a more stable fiber carrier basis for subsequent complex construction (e.g., ...). Figure 1 (As shown).

[0014] 2) Examples show that the heterologous amyloid fibers and cyanidin-3-O-glucoside can form a complex structure and exhibit measurable binding parameters, with the 5:5 heterologous fibers showing a higher binding constant to C3G (as shown in Table 1). Based on this, the C3G loading can be increased and its retention rate under gelatinization conditions can be enhanced (e.g., ...). Figure 2 and Figure 3 (As shown).

[0015] 3) Examples show that combining the complex with rice starch and gelatinizing it can reduce the hydrolysis rate of rice starch under in vitro digestion conditions. The 5:5 heterologous fiber-C3G complex showed a more significant regulatory effect under the same addition conditions (e.g., Figure 4 and Figure 5 As shown in the figure, this provides a feasible solution for developing rice starch-based food ingredients with low digestibility. Attached Figure Description

[0016] Figure 1 This is a comparison chart of the fiber conversion rates of amyloid proteins prepared in Example 1 and Control Examples 1-2; Figure 2 This is a comparison chart of the C3G loading amounts of different protein fibers in Example 2 and Control Examples 3-4; Figure 3 This is a comparison chart of C3G retention rates between Examples 3-5 and Control Example 6 under gelatinization conditions (90°C, 30 min); Figure 4 This is a graph showing the effect of different systems on the in vitro digestion curves of rice starch in Examples 3-5 and Control Examples 5-6; Figure 5 The graph shows the effect of different amounts of the compound added in Example 6, Example 4, and Control Example 5 on the in vitro digestion curves of rice starch. Detailed Implementation

[0017] Terms and Definitions 1) "Rice glutelin" refers to rice glutelin (RG), which can be rice glutelin extracted from rice or commercial rice glutelin products with equivalent amino acid composition and properties.

[0018] 2) "Soy protein" preferably refers to soy protein isolate (SPI), but may also refer to other protein products derived from soybeans with protein content that meets the requirements for fibrosis.

[0019] 3) "Heterologous amyloid fibrils" refer to fibrous aggregates formed by the co-assembly of two proteins from different sources (rice gluten protein and soybean protein) in the same system, which have cross-β-sheet characteristics (characterized by methods such as thioflavin T (ThT) fluorescence, Congo red binding, or Fourier transform infrared spectroscopy (FTIR).

[0020] 4) "Cyanidin-3-O-glucoside (C3G)" refers to a compound in anthocyanins, namely cyanidin-3-O-glucoside.

[0021] 5) "Fiber conversion rate" refers to the proportion of initial protein converted into fibrous aggregates under specified conditions, which can be calculated by separating unconverted protein through ultrafiltration and combining it with protein quantification methods.

[0022] 6) "Rice starch" refers to natural starch derived from rice.

[0023] To facilitate understanding of the present invention, the following description, in conjunction with embodiments, provides further clarification. Those skilled in the art will recognize that equivalent substitutions or optimizations can be made to the raw material sources, process parameters, and testing methods without departing from the spirit and scope of the claims.

[0024] Unless otherwise stated, "mass ratio" in this specification refers to the ratio of the masses of each component, and "volume ratio" refers to the ratio of the volumes of each solution; concentrations are expressed in mg / mL or mM, temperatures are all in degrees Celsius (°C), times are all in hours (h) or minutes (min), and "room temperature" is 20-25°C. "Stirring" in this specification can be magnetic stirring or mechanical stirring; when centrifugation conditions are involved, if expressed in relative centrifugal force (g), the value of g shall be used; if expressed in rotational speed (r / min), equivalent centrifugation conditions corresponding to the radius of the centrifuge rotor used shall be adopted.

[0025] Preparation of heterologous amyloid fibrils In one embodiment, rice gluten and soybean protein are mixed and dispersed in an aqueous phase at a mass ratio of 3:7-5:5 to achieve a total protein concentration of 10-40 mg / mL. The pH of the system is adjusted to 2-7 using an acid solution (e.g., hydrochloric acid solution), and the mixture is stirred and heated at 50-100°C for 0.5-24 h to denature the rice gluten and soybean protein and co-assemble them to form heterologous amyloid fibrous proteins. In some embodiments, after heating, the resulting system is placed in an ice bath and cooled for 10-30 min to obtain a heterologous amyloid fibrous protein solution. For example, the preparation can be carried out under the conditions of pH 2.0, 90°C, stirring speed of 300 r / min, and stirring and heating time of 12 h.

[0026] Preparation of heterologous amyloid fibrils-C3G complex In one embodiment, the obtained heterologous amyloid fibrous solution is mixed with a C3G solution at a volume ratio of 1-5:1, the pH of the system is adjusted to 2.5-3.5, and the mixture is stirred at room temperature and in the dark for 0.5-4 h before drying to obtain a heterologous amyloid fibrous-C3G complex. In some embodiments, the concentration of the C3G solution may be 5 mM. In some embodiments, the drying method may be freeze-drying, wherein when the drying is freeze-drying, the pre-freezing temperature may be -80°C and the freeze-drying time may be 48-96 h.

[0027] Example 1: Co-assembly preparation of heterologous amyloid fibrils Rice gluten and soy protein isolate can be extracted using an alkali-soluble acid-precipitated method, or commercially available protein products can be used. Rice gluten and soy protein were dissolved / dispersed in deionized water to prepare a mixed suspension with a total protein concentration of 20 mg / mL, wherein the mass ratio of rice gluten to soy protein was 7:3, 5:5, or 3:7. The pH of the suspension was adjusted to 2.0 with 2 mol / L hydrochloric acid, and then heated in a 90℃ constant temperature water bath with magnetic stirring at 300 r / min for 12 h. After heating, the suspension was cooled in an ice bath for 20 min to obtain a heterologous amyloid cellulose solution.

[0028] The fiber conversion rate was determined according to the "Fiber Conversion Rate Determination Method" below, and the results are as follows: Figure 1 As shown.

[0029] Comparative Example 1: Rice gluten amyloid fibrils A 20 mg / mL suspension of rice gluten was prepared, and the other conditions were the same as in Example 1, to obtain a rice gluten amyloid cellulose solution.

[0030] Comparative Example 2: Soybean amyloid protein fiber A 20 mg / mL suspension of soybean protein was prepared, and the other conditions were the same as in Example 1, to obtain a soybean protein amyloid cellulose solution.

[0031] Methods for determining fiber conversion rate The cellulose solution was transferred to an ultrafiltration centrifuge tube (molecular weight cutoff 100 kDa) and centrifuged at 5000 r / min for 15 min. The filtrate was collected and washed three times with deionized water at pH 2.0 to remove non-fibrillated proteins. The filtrates were combined and the concentration of non-fibrillated proteins was determined using a BCA protein quantification kit. The cellulose conversion rate was calculated according to formula (1): Formula (1): Fiber conversion rate (%) = (1 - m1 / m) × 100% Where m is the total amount of protein added to the ultrafiltration centrifuge tube, and m1 is the mass of non-fibrinated protein in the filtrate.

[0032] Example 2: Preparation and Binding Parameter Determination of Heterologous Amyloid Fibroid-C3G Complex The heterologous amyloid fibrous solution obtained in Example 1 was used as a carrier. A 5 mM cyanidin-3-O-glucoside (C3G) solution was prepared with deionized water at pH 3.0 and stored at 4°C in the dark. The fibrous solution and C3G solution were mixed at a volume ratio of 1:1, and the pH of the mixture was adjusted to 3.0. After stirring at room temperature in the dark for 1 h, a composite solution was obtained. The composite solution was pre-frozen at -80°C and then freeze-dried under vacuum (e.g., freeze-dried for 48-96 h; in this example, freeze-dried for 3 d (72 h)) to obtain heterologous amyloid fibrous-C3G complex powder.

[0033] C3G load measurement was performed according to the "C3G Load Measurement Method" below, and the results are as follows: Figure 2 As shown in Table 1, the fluorescence quenching / binding parameter was determined according to the "Method for Determination of Fluorescence Quenching and Binding Parameters".

[0034] Control Example 3: Rice gluten fiber-C3G complex The rice gluten amyloid cellulose solution obtained in Comparative Example 1 was mixed with C3G solution in the same manner as in Example 2, the pH of the system was adjusted and reacted, and then freeze-dried to obtain the rice gluten cellulose-C3G complex.

[0035] Comparative Example 4: Soy protein fiber-C3G complex The soybean protein amyloid protein fiber solution obtained in Comparative Example 2 was mixed with C3G solution in the same manner as in Example 2, the pH of the system was adjusted and reacted, and then freeze-dried to obtain soybean protein fiber-C3G complex.

[0036] C3G Load Measurement Method The fiber-C3G composite solution was centrifuged at 10000 g for 10 min, the precipitate was collected and washed with deionized water at pH 3.0; the free C3G content in the supernatant was determined, and the C3G loading was calculated accordingly. The supernatant was filtered through a 0.22 μm filter membrane, and the C3G content was determined by high-performance liquid chromatography (HPLC): the chromatographic column was an SB-Aq C18 column, the detection wavelength was 520 nm; mobile phase A was 2% formic acid aqueous solution, and mobile phase B was acetonitrile; the gradient elution program was: 0-5 min, 5% B; 5-7 min, 5%-10% B; 7-17 min, 10%-15% B; 17-20 min, 15%-25% B; 20-25 min, 25% B decreasing to 5% B; the flow rate was 1 mL / min.

[0037] The C3G load can be calculated using formula (2): Equation (2): Loading capacity (mg / g) = (m0 - m free ) / m fiber Where m0 is the total mass of C3G added to the system, mfree is the mass of free C3G in the supernatant, and mfiber is the mass of amyloid fibrils added to the system.

[0038] Methods for determining fluorescence quenching binding parameters For lyophilized complex samples, the amyloid fibrils were redispersed with deionized water at pH 3.0 before measurement to achieve a final concentration of 0.2 mg / mL. Different volumes of C3G solution (5 mM) were added to the amyloid fibrils (0.2 mg / mL) to achieve final C3G concentrations of 0, 10, 20, 30, 40, and 50 μM. Fluorescence spectra were measured at 25 °C using a fluorescence spectrophotometer with an excitation wavelength of 280 nm and an emission wavelength of 290–450 nm. The excitation and emission slit widths were 5 nm, the scan rate was 1200 nm / min, and the voltage was 600 V. The fluorescence quenching mechanism was analyzed using the Stern-Volmer equation and the double logarithmic equation, and the binding parameters were calculated. Equation (3): F0 / F = 1 + K sv [Q] Formula (4): lg[(F0-F) / F]=lgK a +n·lg[Q] Where F0 and F are the fluorescence intensities in the absence and presence of C3G, respectively; Ksv is the Stern-Volmer quenching constant; [Q] is the C3G concentration; Ka is the binding constant; and n is the number of binding sites. The biomolecule quenching constant Kq can be calculated as Kq = Ksv / τ0, where τ0 is the fluorophore lifetime without quencher, usually taken as 10⁻⁸ s.

[0039] Table 1. Fluorescence quenching / binding parameters of different protein fibrils with C3G As shown in Table 1, under the conditions of the examples, the binding constant Ka of heterologous amyloid fibers and C3G is (10.3628-52.3480)×10^5 M^-1, and the number of binding sites n is 1.1967-1.4535. Among them, the 5:5 heterologous fibers have the highest Ka, which is 52.3480×10^5 M^-1, indicating that they can form a binding complex structure with C3G.

[0040] Example 3: C3G retention rate and in vitro digestion evaluation of rice gluten fiber-C3G complex compounded with rice starch. The composite solution obtained in Comparative Example 3 was freeze-dried to obtain a composite powder. 100 mg of rice starch and 40 mg of rice gluten fiber-C3G composite were weighed and mixed. 10 mL of sodium acetate buffer solution (0.2 mol / L, pH 6.0) was added and the mixture was dispersed evenly. The mixture was gelatinized in a water bath at 90°C for 30 min and then cooled for later use.

[0041] Method for determining C3G retention rate under gelatinization conditions Take the unheated sample and the sample heated at 90℃ for 30 min, respectively, dilute them 10 times with deionized water, centrifuge at 10000 g for 10 min, collect the supernatant, filter the supernatant through a 0.22 μm filter membrane, and determine the C3G content according to the HPLC conditions described in "C3G Loading Determination Method". The C3G retention rate is calculated according to formula (5): Equation (5): Retention rate (%) = (C 30 / C0)×100% Wherein, C0 and C30 represent the C3G concentrations in the samples after no heating and after heating for 30 min, respectively. The results of Examples 3-5 and Control Example 6 are as follows... Figure 3 As shown.

[0042] In vitro digestion evaluation methods After equilibration in a 37°C water bath, 1 mL of digestive enzyme solution was added and timing was started. The digestive enzyme solution was prepared by combining α-amylase (8 U / mL) and amyloglucosidase (40 U / mL). 100 μL samples were taken at 0, 5, 10, 15, 20, 30, 45, 60, 90, 120, and 180 min, and the reaction was terminated by adding 0.9 mL of 0.1 mol / L hydrochloric acid. The supernatant was then collected by centrifugation at 10000 g for 5 min. 6 μL of the supernatant was incubated with 174 μL of glucose oxidase-peroxidase (GOPOD) reagent at 37°C for 15 min, and the absorbance at 510 nm was measured. The glucose concentration in the digestion system at each time point was calculated using a glucose standard curve.

[0043] The glucose concentration at each time point was converted into the glucose mass Gt (mg) released in the digestion system, and the starch digestibility was calculated according to formula (6): Equation (6): Starch digestibility (%) = (0.9 × Gt / M) × 100% Where M is the mass (mg) of rice starch added, and the coefficient 0.9 is the conversion factor between glucose and anhydrous glucose residues.

[0044] Example 4: In vitro digestion evaluation of the heterologous fiber-C3G complex (5:5) combined with rice starch. The rice gluten fiber-C3G complex in Example 3 was replaced with a heterologous fiber-C3G complex prepared from rice gluten and soybean protein in a 5:5 ratio, with the remaining steps identical to Example 3. The in vitro digestion results are as follows: Figure 4 and Figure 5 As shown, under the same addition conditions, the in vitro digestibility of rice starch in the system corresponding to Example 4 is lower.

[0045] Example 5: In vitro digestion evaluation of soybean protein fiber-C3G complex combined with rice starch The rice gluten fiber-C3G complex in Example 3 was replaced with the soybean protein fiber-C3G complex, and the remaining steps were the same as in Example 3. The in vitro digestion results are as follows: Figure 4 As shown.

[0046] Comparative Example 5: Pure Starch 100 mg of rice starch was weighed and gelatinized according to the method in Example 3, but without adding any complex; the remaining steps were the same as in Example 3, and the in vitro digestion curve of pure starch was obtained. Figure 4 and Figure 5 ).

[0047] Compare with Example 6: Free C3G 100 mg of rice starch was weighed and gelatinized according to the method in Example 3. Free C3G (calculated based on the loading amount measured in Example 2) equivalent to the amount of C3G contained in the complex in Example 3 was added, but protein fiber was not added. The remaining steps were the same as in Example 3, and the effect curve of free C3G on the in vitro digestion of rice starch was obtained. Figure 4 ), and used as a control for C3G retention rate under gelatinization conditions ( Figure 3 ).

[0048] Example 6: Effect of different amounts of heterologous fiber-C3G complex on in vitro digestion of rice starch 100 mg of rice starch was weighed and added to 10 mg, 20 mg, or 30 mg of a heterologous fiber-C3G complex prepared from rice gluten and soybean protein in a 5:5 ratio, respectively. The mixture was then dispersed in 10 mL of sodium acetate buffer solution (0.2 mol / L, pH 6.0) and gelatinized in a 90°C water bath for 30 min. The remaining in vitro digestion evaluation steps were the same as in Example 3. The results were as follows: Figure 5 As shown in the examples, under the conditions of the examples, the in vitro digestibility of rice starch decreased as the amount of the complex added increased.

[0049] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the present invention. Equivalent substitutions, modifications, or combinations made by those skilled in the art regarding the source of raw materials, protein ratio, acid-heat treatment conditions, compounding conditions, drying methods, and detection methods without departing from the concept of the present invention shall all fall within the scope of protection of the present invention. The scope of protection of the present invention is determined by the scope defined in the claims, and the specification and drawings can be used to interpret the claims.

[0050] The heterologous amyloid protein fiber-C3G complex and its rice starch composition described in this invention can be used in the preparation of rice starch-based foods or food ingredients, and have industrial applicability.

Claims

1. A method for preparing a heterologous amyloid protein fiber-cyanidin-3-O-glucoside complex, characterized in that, Includes the following steps: Rice gluten and soybean protein were mixed and dispersed in an aqueous phase at a mass ratio of 3:7-5:

5. The pH of the system was adjusted to 2-7. The mixture was stirred and heated at 50-100℃ for 0.5-24 h to denature the rice gluten and soybean protein and co-assemble them to form heterologous amyloid protein fibers. The heterologous amyloid fibrous solution obtained in step (1) was mixed with cyanidin-3-O-glucoside solution at a volume ratio of 1-5:1, the pH of the system was adjusted to 2.5-3.5, and the mixture was dried to obtain the heterologous amyloid fibrous-cyanidin-3-O-glucoside complex.

2. The method according to claim 1, characterized in that, The total protein concentration in the aqueous phase described in step (1) is 10-40 mg / mL.

3. The method according to claim 1 or 2, characterized in that, In step (1), the mass ratio of rice gluten protein to soybean protein is 5:

5.

4. The method according to any one of claims 1-3, characterized in that, In step (1), the pH of the system is 2-3.5, the temperature is 85-95℃, and the stirring and heating time is 8-16 h.

5. The method according to claim 4, characterized in that, In step (1), the system pH is 2.0, the temperature is 90℃, the stirring speed is 300 r / min, and the stirring and heating time is 12 h.

6. The method according to any one of claims 1-5, characterized in that, After heating in step (1), place the resulting system in an ice bath to cool for 10-30 minutes.

7. The method according to any one of claims 1-6, characterized in that, The concentration of the cyanidin-3-O-glucoside solution in step (2) is 5 mM, and the pH of the system after mixing is 3.

0.

8. The method according to claim 7, characterized in that, Step (2) involves mixing at room temperature.

9. The method according to any one of claims 1-8, characterized in that, Step (2) Stir for 0.5-4 hours under dark conditions.

10. The method according to any one of claims 1-9, characterized in that, The drying process is freeze-drying, wherein the pre-freezing temperature is -80℃ and the freeze-drying time is 48-96 h.

11. A heterologous amyloid fibril-cyanidin-3-O-glucoside complex, characterized in that, The complex comprises: heterologous amyloid protein fibers formed by co-assembling rice gluten and soybean protein in a mass ratio of 3:7-5:5; and cyanidin-3-O-glucoside bound to the heterologous amyloid protein fibers.

12. The complex according to claim 11, characterized in that, The mass ratio of rice gluten protein to soybean protein in the heterologous amyloid protein fiber is 5:

5.

13. The complex according to claim 11 or 12, characterized in that, According to the "Method for Determining Fluorescence Quenching Binding Parameters" in the instruction manual, the binding constant Ka between the heterologous amyloid protein fibrils and cyanidin-3-O-glucoside is not less than 5.0 × 10^6 M^-1.

14. A rice starch composition, characterized in that, It includes rice starch and the complex of claim 11, wherein the mass ratio of the complex to rice starch is (1-4):

10.

15. The composition according to claim 14, characterized in that, The mass ratio of the compound to rice starch is 2:10, 3:10, or 4:

10.

16. Use of the complex of claim 11 or the composition of claim 14 in the preparation of a food ingredient for delaying the in vitro digestion of rice starch.

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