Method for preparing slowly digestible starch by double-enzyme method

Abstract

According to the method, corn starch is used as a raw material, maltogenic amylase and transglucosidase are used for dual modification, and the process for preparing the slowly digestible starch comprises the following steps: performing enzymolysis on 300U / g maltogenic amylase at 70 DEG C for 4 hours, performing enzyme deactivation in a boiling water bath, and cooling to room temperature; the preparation method comprises the following steps: adding 350-390U / g glucoside transferase, carrying out enzymolysis at 50-60 DEG C for 2-10 hours, carrying out boiling water bath enzyme deactivation for 10-15 minutes, cooling to room temperature, adding 2-3 times by volume of absolute ethyl alcohol, carrying out alcohol precipitation, centrifuging at 6000-7000r / min for 15-20 minutes, repeating for 2-3 times, and freeze-drying and crushing the obtained precipitate. The slowly digestible starch prepared by the method disclosed by the invention has the mass fraction of (25.96 + / -0.21)%, the hydrolysis index and the eGI of the slowly digestible starch are 25.92 + / -1.17 and 53.95 + / -0.64 respectively, and the slowly digestible starch can be used as a food ingredient with a low glycemic index.

Description

Technical Field

[0001] The invention belongs to the field of food technology, and more specifically, relates to a method for preparing slowly digestible starch by a double enzyme method. Background Art

[0002] Starch is a polyglucose composed of glycogen units connected by α-1, 4 and α-1, 6-glucosidic bonds. After being digested and absorbed by the body, it causes the body's glycemic index (GI) to change rapidly. Studies have found that long-term intake of high-GI foods is closely related to the occurrence of metabolic diseases such as obesity, hyperlipidemia, and diabetes. GI has a certain guiding value in preventing and controlling certain chronic diseases. The change in postprandial glycemic index is positively correlated with the starch digestion rate. According to the digestion rate and degree, it is divided into ready digestible starch (RDS), slowly digestible starch (SDS) and resistant starch (RS). After the human body ingests SDS, it can effectively and for a long time maintain the body's blood sugar balance and improve insulin sensitivity, which can provide beneficial physiological regulation for patients with related chronic metabolic diseases. Therefore, the preparation of slowly digestible starch and the development of its related products have become an important field of current nutrition research and application.

[0003] Compared with traditional physical and chemical preparation methods, the enzymatic preparation of slowly digestible starch has the advantages of simple equipment requirements, no toxic residues, low production costs, and few by-products. The enzymes commonly used in the preparation of slowly digestible starch can be divided into two categories according to the location of their glycosidic bond cleavage, namely enzymes that cut the α-1,4-glycosidic bonds in starch molecules and enzymes that cut the α-1,6-glycosidic bonds in starch molecules, such as α-amylase, β-amylase, branching enzymes, etc. Maltogenic amylase (MA) has the function of cutting α-1,4-glucan into linear chains from the non-reducing end of starch in sequence, promoting the production of soluble dextrins and oligosaccharides; transglucosidase (TG) can catalyze the hydrolysis of oligosaccharides and promote their transfer to obtain more glycosidic bonds with multi-branched structures of side chains, thereby increasing the branch density of starch and helping to delay its digestion. In recent years, there have been many studies on starch modification, but the content of slowly digestible starch obtained is not high. Different enzyme combinations and different addition orders will significantly affect the degradation pattern of corn starch and the properties of the final product. Summary of the invention

[0004] The purpose of the present invention is to overcome the above-mentioned shortcomings and provide a method for preparing slowly digestible starch by a double enzyme method.

[0005] The object of the present invention is achieved in the following ways:

[0006] A method for preparing slowly digestible starch by a dual enzyme method, the method comprising the following steps:

[0007] Corn starch is dissolved in a phosphate buffer solution to prepare a starch slurry solution with a mass fraction of 5%-25%, which is completely gelatinized in a hot water bath, cooled to room temperature, deionized water is added in an equal volume ratio and stirred evenly, 300U / g of maltose amylase is added, enzymolysis is performed at 70°C for 4h, the enzyme is inactivated in a boiling water bath, and the solution is cooled to room temperature; 350-390U / g of glucosyltransferase is added, enzymolysis is performed at 50-60°C for 6-6.5h, the enzyme is inactivated in a boiling water bath for 10-15min, the solution is cooled to room temperature, 2-3 times the volume of anhydrous ethanol is added for alcohol precipitation, centrifugation is performed at 6000-7000r / min for 15-20min, the process is repeated 2-3 times, and the obtained precipitate is freeze-dried and crushed to obtain slowly digestible starch.

[0008] Preferably, the phosphate buffer solution has a concentration of 0.01 mol / L and a pH of 6.0.

[0009] Preferably, the enzymatic hydrolysis time of the glucosyltransferase is 6.5 h.

[0010] Preferably, the enzymatic hydrolysis temperature of the glucosyltransferase is 55°C.

[0011] Preferably, the glucosyltransferase is added in an amount of 350 U / g.

[0012] Preferably, the mass fraction of the starch slurry solution is 12-15%.

[0013] Preferably, the slowly digestible starch has an eGI value of <55.

[0014] Preferably, the mixture is completely gelatinized by bathing in hot water at 90-95°C for 25-35 minutes.

[0015] The amylose content and average chain length of the enzyme-modified corn starch of the present invention are reduced, the segment distribution and crystal structure are changed, and the relative crystallinity and digestibility are reduced. It can be seen that the present invention uses maltogenic amylase and transglucosidase to double-modify starch under certain conditions to effectively improve the digestibility of starch, which is an effective way to prepare slowly digestible starch.

[0016] Compared with the prior art, the present invention has the following beneficial effects: the present invention adopts a combination of MA and TG as enzyme sources to perform double modification treatment on corn starch, the conditions are mild, and there are few by-products; corn starch can be converted into short-chain linear glucan during the MA treatment, and then the linear structure of starch molecules is changed through the transglycosidation of TG, thereby increasing the content of slowly digestible starch in the product; the mass fraction of slowly digestible starch prepared by the method of the present invention is (25.96±0.21)%, and the hydrolysis index and eGI thereof are 25.92±1.17 and 53.95±0.64, respectively, and the corn starch can be used as a low glycemic index food ingredient. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The influence of each single factor on SDS content.

[0018] Figure 2 This is the segment distribution of corn starch before and after enzyme modification.

[0019] in, Figure 2 a is corn starch, Figure 2 b is MA modified starch and Figure 2 c is MA-TG modified starch.

[0020] Figure 3 This is the X-ray diffraction curve of corn starch enzyme before and after modification.

[0021] Figure 4 This is the starch hydrolysis curve.

[0022] The data in the figure are mean±SD (n=3), and different letters in different groups at the same time indicate significant differences (P<0.05). DETAILED DESCRIPTION

[0023] The technical solution of the present invention is further described in detail below in conjunction with several preferred embodiments and drawings. This embodiment is implemented on the premise of the technical solution of the invention, and a detailed implementation method and specific operation process are given, but the protection scope of the present invention is not limited to the following embodiments.

[0024] The experimental methods described in the following examples are conventional methods unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. described in the following examples are all commercially available unless otherwise specified.

[0025] Example 1

[0026] Weigh corn starch and dissolve it in phosphate buffer solution (0.01 mol / L, pH 6.0) to prepare a starch slurry solution with a mass fraction of 15%. Completely gelatinize it in a 95°C water bath for 30 minutes. After cooling to room temperature, add 10 mL of deionized water in an equal volume ratio and stir evenly. Add 300 U / g maltogenic amylase, hydrolyze it at 70°C for 4 hours, inactivate the enzyme in a boiling water bath, and cool to room temperature; add 350 U / g glucosyltransferase and hydrolyze it at 55°C for 6.5 hours, inactivate the enzyme in a boiling water bath for 10 minutes, cool to room temperature, add 2 times the volume of anhydrous ethanol to precipitate, centrifuge at 6000 r / min for 15 minutes, repeat twice, freeze-dry the obtained precipitate, crush it, and pass it through a 100-mesh sieve to obtain slowly digestible starch.

[0027] Example 2

[0028] Weigh corn starch and dissolve it in phosphate buffer solution (0.01 mol / L, pH 6.0) to prepare a starch slurry solution with a mass fraction of 20%, and completely gelatinize it in a 95°C water bath for 30 minutes. After cooling to room temperature, add 10 mL of deionized water in an equal volume ratio and stir evenly, add 300 U / g maltogenic amylase, hydrolyze at 70°C for 4 hours, inactivate the enzyme in a boiling water bath, and cool to room temperature; add 381 U / g glucosyltransferase, hydrolyze at 55°C for 6 hours, inactivate the enzyme in a boiling water bath for 15 minutes, cool to room temperature, add 2 times the volume of anhydrous ethanol to precipitate, centrifuge at 6000 r / min for 15 minutes, repeat twice, freeze-dry the obtained precipitate, and pulverize to obtain slowly digestible starch.

[0029] Example 3

[0030] Weigh corn starch and dissolve it in phosphate buffer solution to prepare a starch slurry solution with a mass fraction of 10%, and completely gelatinize it in a 95°C water bath for 30 minutes. After cooling to room temperature, add deionized water in an equal volume ratio and stir evenly, add 300U / g maltogenic amylase, hydrolyze at 70°C for 4 hours, inactivate the enzyme in a boiling water bath, and cool to room temperature; add 350U / g glucosyltransferase, hydrolyze at 55°C for 6.5 hours, inactivate the enzyme in a boiling water bath for 10 minutes, cool to room temperature, add 2 times the volume of anhydrous ethanol for alcohol precipitation, centrifuge at 7000r / min for 15 minutes, repeat twice, freeze-dry the obtained precipitate, and pulverize it to obtain slowly digestible starch.

[0031] Test Example 1

[0032] 1 Materials and methods

[0033] 1.1 Materials and reagents

[0034] Corn starch: SDS content is 12.67±0.71%.

[0035] Porcine pancreatic α-amylase (enzyme activity 10U / mg); saccharifying enzyme (enzyme activity 1×105U / mL); maltogenic amylase (enzyme activity 1×106U / g); transglucosidase (enzyme activity 5×105U / mL); DNS color developer; disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium hydroxide, anhydrous ethanol, etc. were all of analytical grade.

[0036] 1.2 Instruments and Equipment

[0037] HYG-C combined constant temperature oscillator; Eppendorf 5810r high-speed refrigerated centrifuge; INFINITE 200PRO microplate reader; UV spectrophotometer; LabconcoFreeZone 6L freeze dryer; HH-S 4 digital display constant temperature water bath.

[0038] 1.3 Methods

[0039] 1.3.1 Preparation of SDS

[0040] Weigh corn starch and dissolve it in phosphate buffer solution (0.01mol / L, pH6.0), prepare starch slurry solutions with mass fractions of 5%, 10%, 15%, 20%, and 25%, respectively, and completely gelatinize it in a 95℃ water bath for 30min. After cooling to room temperature, add 10mL deionized water in equal volume and stir evenly. Add 300U / g MA, enzymatically hydrolyze at 70℃ for 4h, inactivate the enzyme in a boiling water bath, and cool to room temperature. Add 100, 200, 300, 400, and 500U / g TG respectively and enzymatically hydrolyze at 45, 50, 55, 60, and 65℃ for 2, 4, 6, 8, and 10h, inactivate the enzyme in a boiling water bath for 10min, and cool to room temperature. Add 2 times the volume of anhydrous ethanol to precipitate, centrifuge at 6000r / min for 15min, repeat twice, freeze-dry the precipitate, crush it, and pass it through a 100-mesh sieve to obtain SDS.

[0041] 1.3.2 Determination of SDS content.

[0042] Enzyme solution (prepared and used immediately): Solution A: 20 mL porcine pancreatic α-amylase solution (6 g porcine pancreatic α-amylase dissolved in 38 mL of deionized water, vortexed at 4°C, centrifuged at 1000 r / min for 8 min, and the supernatant was taken); Solution B: 2 mL deionized water, 2 mL saccharifying enzyme (60 μL diluted to 3 mL).

[0043] Take 0.2g sample and dissolve it in 15mL phosphate buffer solution (0.2mol / L, pH 5.2), keep it warm at 37℃ for 10min, add 5mL AB mixed enzyme solution, react at 37℃ and 200r / min, take 2mL of reaction solution at 20min and 120min respectively, add 2 times volume of anhydrous ethanol to inactivate the enzyme, centrifuge (6000r / min, 10min), aspirate the supernatant to GOPOD kit and determine the hydrolyzed glucose content at 510nm, calculate RDS, SDS and RS contents respectively according to formula (1)(2)(3).

[0044] RDS=(G 20 -FG)×0.9×100%(1)

[0045] SDS=(G 120 -G 20 )×0.9×100%(2)

[0046] RS=1-(G 20 +G 120 )×0.9×100%(3)

[0047] Where: FG, G20 and G120 represent the free glucose content in the solution before hydrolysis and the glucose content released when starch is hydrolyzed for 20min and 120min respectively; 0.9 is the glucose conversion coefficient in starch.

[0048] 1.3.3 Single Factor Experiment

[0049] In order to explore the effects of enzyme addition, enzymatic hydrolysis time, starch slurry mass fraction and enzymatic hydrolysis temperature on the SDS content in the product, each single factor was studied, and the factor levels are shown in Table 1. The specific operations are:

[0050] Effect of enzyme addition on SDS content: Using anhydrous ethanol as the alcohol precipitation solvent, the enzymatic hydrolysis time was fixed at 6h, the starch slurry mass fraction was 15%, the enzymatic hydrolysis temperature was 65℃, the MA addition amount was 300U / g, and the TG addition amount was selected as 100, 200, 300, 400, and 500U / g for the experiment, and each group was repeated 3 times.

[0051] Effect of enzymatic hydrolysis time on SDS content: Using anhydrous ethanol as the alcohol precipitation solvent, fixed MA addition amount 300U / g, TG addition amount 300U / g, starch slurry mass fraction 15%, enzymatic hydrolysis temperature 65℃, and selected enzymatic hydrolysis time 2, 4, 6, 8, and 10h for the experiment, and each group was repeated 3 times.

[0052] Effect of starch slurry mass fraction on SDS content: Using anhydrous ethanol as the alcohol precipitation solvent, fixed MA addition amount 300U / g, TG addition amount 300U / g, enzymatic hydrolysis time 6h, enzymatic hydrolysis temperature 65℃, selected starch slurry mass fraction 5%, 10%, 15%, 20%, 25% for experiment, each group was repeated 3 times.

[0053] Effect of enzymatic hydrolysis temperature on SDS content: Anhydrous ethanol was used as the alcohol precipitation solvent, with fixed MA addition amount of 300U / g, TG addition amount of 300U / g, enzymatic hydrolysis time of 6h, starch slurry mass fraction of 15%, and enzymatic hydrolysis temperatures of 45, 50, 55, 60, and 65℃ for the experiment, and each group was repeated 3 times.

[0054] Table 1 Single factor level table

[0055]

[0056] 1.3.4 Response surface optimization experiment

[0057] According to the optimization results of the single factor experiment, four factors, namely, TG addition amount, enzymatic hydrolysis time, starch slurry mass fraction and enzymatic hydrolysis temperature, were selected for response surface optimization design. The factor levels are shown in Table 2.

[0058] Table 2 Factor levels of response surface experiment

[0059]

[0060] 1.3.5 Determination of physicochemical properties of enzyme-modified starch

[0061] 1.3.5.1 Determination of amylose content and segment distribution

[0062] The content of amylose was determined by iodine colorimetry. 10 mg of sample was weighed and dispersed in 1 ml of distilled water, heated in a water bath at 95 ° C for 15 min, and cooled to room temperature. After adding 2 mL of sodium acetate buffer solution (0.1 mol / L, pH 4), the temperature was kept constant in a water bath at 40 ° C, and 6 μL of isoamylase (180 U / mL) was added to each dispersion, and the mixed solution was oscillated at a constant temperature at 40 ° C for 24 hours. The solution was heated in a boiling water bath for 15 min to inactivate the enzyme. Centrifuged at 2000 r / min for 10 min, the supernatant was taken and passed through a 0.45 μm microporous filter membrane, and then the obtained sample filtrate was injected into the HPAEC-PAD system. The experimental conditions were set as follows: eluent A: 0.25 mol / L NaOH; eluent B: 1 mol / L sodium acetate solution and 0.15 mol / L NaOH solution; elution temperature and flow rate: 30 ° C, flow rate of 0.5 ml / min. The data were collected using Chrominon software (version 6.50). The segment distribution ratio and average chain length of the samples were calculated based on the data results.

[0063] 1.3.5.2 Crystal structure determination

[0064] The crystal structure changes of starch before and after enzyme modification were measured by X-ray diffractometer. Test conditions: the sample to be tested was scanned in the diffraction angle (2θ) range of 5 to 40°, the scanning speed was 2° / min, and the working conditions were set as follows: voltage: 40Kv, current: 40mA.

[0065] 1.3.5.3 Determination of hydrolysis index and estimated glycemic index

[0066] use GARCIA-ALONSO A, SAURA-CALIXTO F. A starch hydrolysis procedure to estimate glycemic index, [J]. Nutrition Research, 1997, 17 (3): 427-437. The disclosed method was used to determine the glycemic index (eGI). An in vitro simulation of the starch digestion process was performed. Samples were taken at 30, 60, 90, 120, 150, and 180 min, and the glucose content was determined by the DNS method. The starch hydrolysis rate was calculated by formula (4). The equation C = C ∞ (1-e -kt ) The first-order kinetic fitting of the starch hydrolysis curve was performed to obtain C ∞ and k value. The hydrolysis index (HI) is calculated by formula (5) (6). Taking white bread as a reference, the glycemic index of white bread is set to 100. There is a high correlation between HI and eGI. The eGI is predicted by HI, and the eGI of the sample to be tested is calculated by formula (7).

[0067] The hydrolysis rate is calculated as follows:

[0068]

[0069] The area under the hydrolysis curve (AUC) is obtained by the formula:

[0070]

[0071] Where: Ct—glucose hydrolysis rate at time t; C ∞ is the glucose hydrolysis rate at the final hydrolysis equilibrium; t f is the final reaction time (180 min); t0 is the initial reaction time (0 min); k is the kinetic constant. The hydrolysis index (HI) is obtained by the formula:

[0072]

[0073] The eGI calculation formula is:

[0074] eGI=39.71+0.549HI(r=0.894)(7)

[0075] 1.3.6 Data Statistics and Analysis

[0076] All experimental measurements were repeated 3 times. The results are expressed as mean ± standard deviation (SD). One-way ANOVE in SPSS statistics 26 was used to perform variance analysis on the experimental data to determine the significance of the difference. The charts were drawn using Origin 2018 and Excel.

[0077] 2 Results and analysis

[0078] 2.1 Effects of various factors on SDS content

[0079] Depend on Figure 1 It can be seen that with the increase of transglucosidase addition, the SDS content gradually increases. When the enzyme addition amount is 300U / g, the SDS content reaches a maximum value of 24.62%, and then gradually decreases with the increase of enzyme addition amount. The main reason may be that the amount of transglucosidase added is low, and the branch of the short starch chain after the action of maltose amylase cannot be fully transferred, resulting in a decrease in starch branch density, and the digestibility of starch cannot be effectively improved; when the amount of transglucosidase added is high, the starch is over-hydrolyzed and a high degree of branching occurs, causing a decrease in the degree of polymerization, and a decrease in the degree of polymerization and a longer outer chain are not conducive to the formation of SDS. Therefore, 300U / g is selected as the optimal enzyme addition amount.

[0080] Effect of enzyme action time on SDS content Figure 1 As shown. When the hydrolysis time of glucosyltransferase increased from 2h to 6h, the SDS content gradually increased; at 6h, it reached a maximum value of 19.16%; but after 6h, the SDS content decreased with the increase of hydrolysis time. The formation of SDS is closely related to the branching density and crystal structure of starch, and transglucosidase can catalyze the hydrolysis and transfer of oligomaltose to make starch produce more cluster-like branching structures, effectively changing the chain structure inside the starch molecule, thereby affecting the starch branching density and crystal structure, which is conducive to the formation of SDS. When the hydrolysis time is insufficient, the long-chain starch cannot be completely hydrolyzed, and the SDS content is low; if the hydrolysis time is too long, hydrolysis products with enzyme inhibition will be produced, and the crystal structure will be hydrolyzed into short chains or small molecules, resulting in a decrease in the content of amylose, leading to a decrease in the SDS content. Therefore, a transglucosidase action time of 6h was selected for further experiments.

[0081] The starch slurry concentration has a significant effect on the SDS content, such as Figure 1As shown in the figure, the SDS content increases first and then decreases with the increase of starch solution concentration. When the starch slurry concentration is 15%, the SDS content reaches a maximum value of 15.47%, and then shows a downward trend. This may be because when the starch slurry solution is too low, the substrate and the enzyme cannot fully react, which is not conducive to the formation of short chains of starch molecules, so the SDS content is low; when the starch slurry concentration is high, the viscosity of the starch solution system increases, the coagulation trend is enhanced, and the contact between starch molecules and enzymes is hindered, resulting in a decrease in the SDS content. Therefore, when the water content is appropriate, it is conducive to the enzymatic reaction, thereby increasing the SDS content. In summary, it is most appropriate to choose a starch slurry concentration of 15%.

[0082] Starch gelatinization is divided into three stages. In the first stage, starch molecules absorb water molecules to undergo a reversible reaction, and the volume of starch molecules expands slightly. When the heating temperature reaches a certain value, the starch molecules absorb water irreversibly and expand, and the volume of starch granules expands rapidly. If the temperature continues to rise, the starch molecules will all disintegrate and diffuse, changing from an ordered state to a disordered state. Figure 1 It can be seen that as the temperature of the enzyme action increases, the reaction system reaches a suitable temperature, and the SDS content gradually increases. At 55°C, the SDS content reaches a maximum value of 17.33%; when the temperature exceeds 55°C, the SDS content gradually decreases. The main reason may be that the temperature is too low, the enzyme activity is low, and it cannot fully react with the starch molecules; if the temperature is too high, the enzyme is easily inactivated, which affects the aggregation of starch molecules and is not conducive to the formation of SDS. When the temperature of the reaction system reaches a suitable state, starch is easy to aggregate into a crystalline structure, the SDS content increases, and at the same time, transglucosidase catalyzes the formation of more side chain branch structures, thereby slowing down the starch digestion rate. Therefore, the enzymatic hydrolysis temperature of 55°C was selected for subsequent experiments.

[0083] 2.2 Response surface analysis

[0084] 2.2.1 Response surface variance analysis

[0085] The quadratic multinomial regression equation for the preparation of slowly digestible starch by enzymatic method was obtained by response surface variance analysis: Y(%) = +27.02 + 1.35 × A + 0.54 × B + 0.50 × C + 0.35 × D + 0.077 ×

[0086] A×B-0.27×A×C-0.31×A×D+0.080×B×C-0.047×B×

[0087] D-0.055×C×D-2.89×A 2 -1.21×B 2 -2.15×C 2 -2.03×D 2

[0088] Table 3 Response surface experimental design and results

[0089]

[0090]

[0091] The results of the response surface experiment are shown in Table 3, using Design-Expert 8.0.6 software. The experimental data in Table 3 were subjected to variance analysis, and the results are shown in Table 4. As can be seen from Table 4, the model P value is less than 0.000 1, indicating that the regression model is highly significant; A, B, C, A in the quadratic multinomial regression equation 2 , B 2 , C 2 , and D 2 The effects on SDS content all reached an extremely significant level; the P value was 0.7028, which was not significant, indicating that the predicted values ​​of the regression model had a good fit with the measured values. 2 It is 0.9609, indicating that the model has a good correlation with the measured value. 2 Adj The value is 0.9218, R 2 Squared The value is 0.834 3, and the difference between the two numbers is 0.087 5 (<0.2), which shows that the model has a good fit and the experimental error is small, and the experimental results can be accurately analyzed and predicted.

[0092] Table 4 Analysis of variance

[0093]

[0094] Note: **. The difference is extremely significant, P<0.01.

[0095] 2.2.2 Analysis of response surface experiment results

[0096] The response surface analysis of the interaction between factors was performed using Design-Expert software. The slope of the response surface can reflect the effect of a single factor on the SDS content. The steeper the slope, the more significant the effect. Changes in enzyme addition, enzymatic hydrolysis time, starch slurry mass fraction and enzymatic hydrolysis temperature will cause corresponding changes in SDS content, and higher SDS content appears near the center of each factor. The amount of transglucosidase added, enzyme action time and starch slurry concentration have a significant effect on the slowly digestible starch content of corn (P<0.05). Among them, the curve of transglucosidase addition is relatively steep, so it has the most significant effect on SDS content. The interaction between enzyme addition and enzymatic hydrolysis time, and the interaction between starch slurry mass fraction and enzyme addition have extremely significant effects on the response value. The enzyme action temperature has no significant effect on the slowly digestible starch content of corn (P>0.05), which is consistent with the results of variance analysis in Table 4.

[0097] 2.2.3 Verification Experiment

[0098] The optimal process prediction parameters obtained by the quadratic multinomial regression equation were: enzyme addition 381.26U / g, enzymolysis time 6.5h, starch slurry mass fraction 15.34%, and enzymolysis temperature 55.1℃. According to the actual experimental operation, the optimal process parameters were adjusted to enzyme addition 350U / g, enzymolysis time 6.5h, starch slurry mass fraction 15%, and enzymolysis temperature 55℃ to prepare enzyme-modified slowly digestible starch. Each group of experiments was repeated 3 times. The SDS mass fractions were 26.05%, 26.11%, and 25.72%, respectively, with an average of (25.96±0.21)%.

[0099] 2.3 Analysis of physicochemical properties of enzyme-modified starch

[0100] 2.3.1 Determination and analysis of amylose content and segment distribution

[0101] As shown in Table 5, the apparent amylose content of common corn starch and modified starch was determined. The amylose content of common corn starch was the highest (24.48±0.11)%. After the starch was modified with MA, the mass fraction of amylose decreased to (23.51±0.21)%. This was attributed to the fact that MA, through hydrolysis, caused the non-reducing end of the α-1,4-glycosidic bond of the starch molecule to break, increasing the intermolecular contact sites and causing the decrease in the amylose content. After the transglucosidase was incorporated into the system reaction, the amylose content decreased to (20.25±0.18)%. TG was more sensitive to amylopectin than amylose. Therefore, TG can preferentially transfer the short sugar chains generated by MA cleavage in the system through transglycosidation, promote the formation of a certain number of short amylopectin branches, and reduce the content of amylose. At the same time, the RDS, SDS, and RS contents in Table 5 showed different changing trends with the decrease of amylose content, among which the SDS and RS contents increased with the decrease of amylose content, indicating that the reduction of amylose content can effectively increase the digestion resistance of starch.

[0102] The molecular segment distribution of starch before and after enzyme modification was characterized by HPSEC-MALLS-RI measurement. Compared with natural corn starch ( Figure 2 a), MA modified starch ( Figure 2 b) and MA-TG modified starch ( Figure 2 c) The proportion of A chain (DP 6-12) increased significantly, increasing by 3.01% and 6.25% respectively. The proportions of B1 (DP 13-24) and B2 chain (DP 25-36) decreased to 51.90%, 47.97% and 16.24%, 15.75% respectively. This is because MA and TG use hydrolysis and transglycosylation reactions to induce starch molecules to produce new α, 1-6 glycosidic bond-connected branch chains, generating branched starch clusters and increasing the amount of short straight chains. In addition, from Figure 2It can be seen that the average chain length of natural corn starch was reduced to 18.39 after MA modification. The main reason is that MA treatment exposes new reaction contact ends of starch, which is conducive to the hydrolysis reaction to produce short linear starch with low molecular weight. Subsequently, TG was added for enzymatic hydrolysis, and the average chain length of starch was reduced to 17.33, indicating that the increase of TG promoted the formation of more broken side chain glycosidic bonds, resulting in more and shorter starch chains.

[0103] Table 5 Amylose content and digestible starch content of corn starch before and after modification by amylase

[0104]

[0105] 2.3.2 Crystal structure determination and analysis

[0106] like Figure 3 The native corn starch shown in the figure presents a typical A-type crystal structure, with strong diffraction peaks at 2θ values ​​of 15.00°, 17.00°, 18.00° and 23.00°, and a relative crystallinity of 35.13%. After being modified by MA and MA-TG enzymes, the relative crystallinity drops to 15.92% and 14.67%, respectively. This is attributed to the fact that the present invention uses anhydrous ethanol as a precipitation solvent during the enzyme modification process, which forms a complex with short linear glucan or lipid to destroy the original crystal structure, promote the formation of a V-type crystal structure, and cause the original diffraction peaks at 15.00°, 17.10° and 18.00° to show a weakening trend, and the original peak at 23.00° no longer appears. On the contrary, a new diffraction peak appears at 15.30°, and then the diffraction peak increases relatively at 19.80°. Therefore, it is shown that the crystal structure of the native starch is completely destroyed. The peaks at 15.30° and 19.80° reflect the formation of amylose-lipid complexes, which are typical peaks of the V-type pattern. Figure 3 It can be seen that compared with natural corn starch, the peak intensity at the 2θ value of 19.80° becomes relatively larger, which shows that after the natural corn starch is modified by enzyme, the A-type crystal form of corn starch can be converted into C-type and V-type complexes.

[0107] 2.3.3 Estimated glycemic index determination and analysis

[0108] Figure 4The starch hydrolysis rate curve of different starches in the simulated internal environment of in vitro digestion within 180 minutes. Taking the ungelatinized original corn starch as the control, the in vitro starch digestibility of maltogenic amylase modified starch, maltogenic amylase and transglucosidase double modified starch (prepared under the optimal preparation process conditions) was measured respectively. Within 0min to 180min, the slope of the starch hydrolysis rate curve decreased significantly (P<0.05); in the first 30min, the hydrolysis rates of original corn starch, single enzyme modified starch and double enzyme composite modified starch increased fastest; the growth rate slowed down from 20 to 120min; when the hydrolysis was carried out for 120min, the hydrolysis rates of single enzyme modified starch and double enzyme modified starch basically tended to be stable. The hydrolysis rate of double enzyme modified starch was lower than that of original corn starch and single enzyme modified starch. After the natural corn starch was double modified by maltogenic amylase and transglucosidase, the glucose hydrolysis rate was lower, which was more conducive to blood sugar control and could be used for food development for special populations such as diabetic patients.

[0109] According to the eGI value, carbohydrate-rich foods can be divided into high eGI foods (above 70), medium eGI foods (55-70), and low eGI foods (GI <55). The characteristic parameters of the in vitro simulated hydrolysis kinetic equation of corn starch are shown in Table 6. As can be seen from Table 6, the eGI values ​​of each sample are from high to low, namely, original corn starch, single enzyme modified starch modified by maltogenic amylase, and double enzyme modified starch modified by maltogenic amylase and transglucosidase (prepared under the optimal preparation process conditions). In addition, compared with single enzyme modified starch and original corn starch, the eGI value of double enzyme modified starch is 53.95±0.64, which is significantly lower (P<0.05) and <55, and belongs to the low eGI category of food. It has low glycemic functionality and is suitable for people with blood sugar control or diabetic patients.

[0110] Table 6 Characteristic parameters of the kinetic equation for in vitro simulated hydrolysis of corn starch

[0111]

[0112] Note: Different lowercase letters indicate significant differences in the same column of data (P<0.05)

[0113] 3 Conclusion

[0114] Through single factor experiments and response surface optimization experiments, the optimal experimental conditions are TG enzyme addition of 350U / g, enzymolysis time of 6.5h, starch slurry mass fraction of 15%, and enzymolysis temperature of 55℃. Under these conditions, the SDS content of the modified starch and ordinary corn starch can reach up to (25.96±0.21)%, and the SDS content is significantly increased compared with ordinary corn starch (SDS content 12.58%). In addition, the amylose content and average chain length of the enzyme-modified corn starch are reduced, the segment distribution and crystal structure are changed, and the relative crystallinity and digestibility are reduced. It can be seen that the use of maltogenic amylase and transglucosidase to double-modify starch can effectively improve the digestibility of starch, which is an effective way to prepare slowly digestible starch. The product prepared under these conditions was subjected to a starch in vitro digestion simulation experiment. The results showed that its eGI value was 53.95±0.64<55, which meets the blood sugar value requirements of low GI products.

Claims

1. A method for preparing slowly digestible starch by a dual enzyme method, characterized in that The method comprises the following steps: Corn starch is dissolved in a phosphate buffer solution to prepare a starch slurry solution with a mass fraction of 5%-25%, which is completely gelatinized in a hot water bath, cooled to room temperature, deionized water is added in an equal volume ratio and stirred evenly, 300U / g of maltose amylase is added, enzymolysis is performed at 70°C for 4h, the enzyme is inactivated in a boiling water bath, and the solution is cooled to room temperature; 350-390U / g of glucosyltransferase is added, enzymolysis is performed at 50-60°C for 6-6.5h, the enzyme is inactivated in a boiling water bath for 10-15min, the solution is cooled to room temperature, 2-3 times the volume of anhydrous ethanol is added for alcohol precipitation, centrifugation is performed at 6000-7000r / min for 15-20min, the process is repeated 2-3 times, and the obtained precipitate is freeze-dried and crushed to obtain slowly digestible starch.

2. The method for preparing slowly digestible starch by dual enzyme method according to claim 1, characterized in that The phosphate buffer solution has a concentration of 0.01 mol / L and a pH of 6.

0.

3. The method for preparing slowly digestible starch by dual enzyme method according to claim 1, characterized in that The enzymatic hydrolysis time of the glucosyltransferase is 6.5h.

4. The method for preparing slowly digestible starch by dual enzyme method according to claim 1, characterized in that The enzymolysis temperature of the glucosyltransferase is 55°C.

5. The method for preparing slowly digestible starch by dual enzyme method according to claim 1, characterized in that The added amount of the glucosyltransferase is 350 U / g.

6. The method for preparing slowly digestible starch by dual enzyme method according to claim 1, characterized in that The mass fraction of the starch slurry solution is 12-15%.

7. The method for preparing slowly digestible starch by dual enzyme method according to claim 1, characterized in that The slowly digestible starch eGI value is <55.

8. The method for preparing slowly digestible starch by dual enzyme method according to claim 1, characterized in that The 90-95° C. hot water bath for 25-35 minutes makes it completely gelatinized.

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