A method for preparing low gi maltodextrin by two enzymes in cooperation

Abstract

The present application relates to a kind of double-enzyme synergistically preparing low GI malt dextrin method, belong to starch biotechnology field.The present application is by starch granule dispersed in water and obtains starch milk;Starch branching enzyme Ro-GBE is added to the obtained starch milk, temperature is raised and gelatinization reaction is carried out, after temperature reduction, constant temperature reaction is continued, and reaction liquid 1 is obtained;After the obtained reaction liquid 1 is cooled, β-amylase and yeast are reacted, and reaction liquid 2 is obtained;The obtained reaction liquid 2 is dried, and the low GI malt dextrin is obtained.The product of the present application is modified by enzyme method, and the degree of branching increases, the relative molecular mass is reduced, and high-branched small molecular structure is formed, so as to delay the digestibility of product, enhance the solubility and solution stability of product.The present application uses starch branching enzyme and β-amylase to carry out synergistic secondary enzymolysis modification to starch, the effect is remarkable, can reduce 42.71% fast-digesting component in product, product cold water is soluble, and the transparency of solution can reach more than 90.00% after being stored at 4 ℃ for 30 d.

Description

Technical Field

[0001] This invention relates to a method for the synergistic preparation of low-GI maltodextrin using two enzymes, belonging to the field of starch biotechnology. Background Technology

[0002] The glycemic index (GI) is an indicator that reflects the postprandial blood glucose response. It is defined as the ratio of the area under the blood glucose response curve (AUC) after consuming a food containing 50 g of available carbohydrates over a period of time (≥2 hours) to the AUC after consuming an equivalent amount of standard food (white bread containing 50 g of starch or 50 g of glucose). Studies have shown that consuming easily digestible high-GI foods leads to a sharp rise in blood glucose, promotes insulin secretion and release, and accelerates the conversion of glucose into fat, resulting in hyperlipidemia and obesity. Obesity is a high-risk factor for diseases such as hypertension and diabetes. Drastic blood glucose fluctuations can also activate the body's oxidative stress pathways, promote local inflammatory responses, and cause damage to vascular endothelial cells, thereby inducing diseases such as coronary heart disease, stroke, retinopathy, and nephropathy. Conversely, consuming low-GI foods prolongs the digestion, absorption, or gluconeogenesis of starch and sugars, releasing energy slowly and continuously, reducing the burden on the pancreas, helping to maintain postprandial blood glucose balance, thereby controlling weight and preventing various chronic diseases.

[0003] Starch, as the most abundant carbohydrate component in the human diet, is a major cause of elevated blood sugar levels when consumed in large quantities. Reducing starch digestibility is beneficial for maintaining postprandial blood glucose homeostasis and has unique effects in preventing chronic diseases. Currently, commonly used methods for reducing starch digestibility mainly include physical modification, chemical modification, enzymatic modification, and combined modification. Among them, bio-enzymatic modification has the characteristics of high reaction efficiency and mild reaction conditions, making it suitable for large-scale industrial production. Current research mainly uses hydrolases such as maltose, α-amylase, β-amylase, and pullulanase, or glycosyltransferases such as starch branching enzymes and 4,6-α-glucosyltransferase to regulate the digestible properties of starch. After being catalyzed by these enzymes alone or in synergistically, the digestion rate of starch in vivo and in vitro is significantly slowed down, the GI value decreases accordingly, and the relative molecular mass also decreases, which is more in line with the definition of "dextrin".

[0004] Low-GI maltodextrin, as a starch derivative with a low glycemic index, has a slow digestion rate, releasing glucose gradually into the bloodstream. This causes moderate postprandial blood glucose changes and insulin response, thereby lowering fasting and postprandial blood glucose levels, regulating insulin homeostasis, and improving glucose metabolism disorders caused by diseases. It is beneficial for lowering postprandial blood glucose and improving blood lipids, and has a certain adjunctive therapeutic effect on some diet-related chronic diseases, such as type 2 diabetes, cardiovascular disease, and metabolic syndrome related to obesity. However, current research on low-GI maltodextrin is still limited. Only a few slow-digesting starch and its derivative products are commercially produced abroad, and the domestic market is virtually nonexistent. Therefore, exploring efficient preparation methods for low-GI maltodextrin and improving its preparation efficiency is of great significance for promoting the industrialization of low-GI maltodextrin.

[0005] In previous research, the inventors' research group disclosed a method for preparing slow-digesting maltodextrin in the patent document with authorization number CN112852906A. The preparation process is as follows: using... Rhodothermus obamensis STB05's amyloid branching enzyme Ro-GBE and derived from Geobacillus thermoglucosidans STB02's starch branching enzyme Gt-GBE acts sequentially on granular starch and gelatinized starch, leveraging the synergistic effect of the two starch branching enzymes to improve the slow digestibility of the product. However, this process requires precise control of the reaction time of the granular starch, and there is still considerable room for improvement in the slow digestibility of the product. Furthermore, it suffers from problems such as process complexity, high reaction viscosity, and poor product solubility and stability, failing to meet practical application requirements.

[0006] Therefore, in order to further improve the slow digestion characteristics of low-GI maltodextrin and enhance its solution stability, it is urgent to explore an efficient method for preparing highly stable low-GI maltodextrin. Summary of the Invention

[0007] To address the aforementioned problems, this invention proposes a method for the synergistic preparation of low-GI maltodextrin using a dual-enzyme approach. This invention establishes a process for the efficient preparation of low-GI maltodextrin from starch through the synergistic modification of starch by starch branching enzyme and β-amylase. The enzymatically modified product exhibits increased branching and decreased relative molecular mass, forming a highly branched small molecular structure, thereby delaying digestibility and enhancing solubility and solution stability. This invention utilizes starch branching enzyme and β-amylase for synergistic modification of starch, achieving significant results. It reduces the rapidly digestible components in the product by 42.71%, ensures the product is soluble in cold water, and maintains a transparency of over 90.00% after storage at 4°C for 30 days.

[0008] This invention is achieved through the following technical solution: The first objective of this invention is to provide a method for the synergistic preparation of low-GI maltodextrin using two enzymes, the method comprising the following steps: (1) Disperse starch granules in water to obtain starch milk; add starch branching enzyme Ro-GBE to the obtained starch milk, heat to carry out gelatinization reaction, cool down and continue to react at a constant temperature to obtain reaction solution 1; (2) After cooling the reaction solution 1 obtained in step (1), add β-amylase and yeast to react and obtain reaction solution 2; the yeast is added for sugar removal treatment. (3) Dry the reaction solution 2 obtained in step (2) to obtain the low-GI maltodextrin.

[0009] In one embodiment of the present invention, in step (1), the starch is selected from one or more of cassava starch, ordinary corn starch, waxy corn starch, potato starch, rice starch and wheat starch.

[0010] In one embodiment of the present invention, in step (1), the concentration of the starch milk is 10% to 30% (w / w), preferably 15% (w / w); the pH value of the starch milk is 6.0 to 8.0.

[0011] In one embodiment of the present invention, in step (1), the starch branching enzyme Ro-GBE is derived from... Rhodothermus obamensis STB05, nucleotide sequence as shown in SEQ ID NO.1.

[0012] In one embodiment of the present invention, in step (1), the amount of starch branching enzyme Ro-GBE added is 100 U / g to 1000 U / g dry starch, preferably 100 U / g to 600 U / g dry starch.

[0013] In one embodiment of the present invention, in step (1), the temperature of the gelatinization reaction is 70°C to 95°C; and the time of the gelatinization reaction is 0 h to 5 h.

[0014] In one embodiment of the present invention, in step (1), the temperature of the isothermal reaction is 60°C to 80°C; the time of the isothermal reaction is 8 h to 48 h, preferably 8 h to 24 h.

[0015] In one embodiment of the present invention, in step (2), the temperature after cooling is 30°C to 60°C.

[0016] In one embodiment of the present invention, in step (2), the amount of β-amylase added is 100 U / g to 1000 U / g dry starch; preferably 100 U / g to 500 U / g dry starch.

[0017] In one embodiment of the present invention, in step (2), the amount of yeast added is 0.5% to 5%.

[0018] In one embodiment of the present invention, in step (2), the reaction temperature is 30℃~60℃, preferably 30℃~37℃; the reaction time is 6 h~72 h, preferably 12 h~60 h.

[0019] In one embodiment of the present invention, step (2) further includes adding β-amylase and yeast again for a secondary reaction after the reaction.

[0020] In one embodiment of the present invention, the amount of β-amylase added is 100 U / g to 1000 U / g dry starch; preferably 100 U / g to 500 U / g dry starch.

[0021] In one embodiment of the present invention, the amount of yeast added is 0.5% to 5%.

[0022] In one embodiment of the present invention, the temperature of the secondary reaction is 30°C to 60°C, preferably 30°C to 37°C; the time of the secondary reaction is 6 h to 72 h, preferably 12 h to 60 h.

[0023] In one embodiment of the present invention, step (2) further includes enzyme inactivation, filtration, decolorization and ion exchange treatment after the reaction.

[0024] In one embodiment of the present invention, in step (2), after the reaction is completed, the temperature is raised to inactivate the enzyme, and the yeast cells and impurities are removed by filtration using a 0.22 μm~0.45 μm filter membrane.

[0025] In one embodiment of the present invention, the decolorization is performed by: adjusting the pH of the clarified solution obtained by filtration, adding activated carbon and stirring to decolorize, thereby obtaining a decolorized solution; And / or, the ion exchange is: using an ion exchange resin to remove metal salts and pigments from the decolorizing solution.

[0026] In one embodiment of the present invention, the ion exchange resin is a strong acid cation-weak base cation-strong acid cation exchange resin; the reaction temperature for ion exchange is 40℃~50℃.

[0027] In one embodiment of the present invention, during decolorization, the pH of the clarified solution is adjusted to 4.0~5.0, the amount of activated carbon added is 1%, and the decolorization is carried out by keeping the solution at 40℃~50℃ for 15 min~30 min.

[0028] In one embodiment of the present invention, in step (3), the drying is selected from one or more of freeze drying, drum drying and spray drying.

[0029] A second objective of this invention is to provide a low-GI maltodextrin prepared by the method described.

[0030] A third objective of this invention is to provide the application of the low-GI maltodextrin in the preparation of probiotic products, health products, or meal replacement products.

[0031] The technical solution of the present invention has the following advantages compared with the prior art: 1. This invention provides a method for the synergistic preparation of low-GI maltodextrin using two enzymes. This method utilizes two amylases with different modes of action to synergistically modify starch to prepare low-GI maltodextrin. The raw materials are readily available, the process is simple and convenient, and the product yield is high. No other chemical groups are introduced, nor are other types of glycosidic bonds generated. Only the hydrolysis of α-1,4-glycosidic bonds within the starch molecule and the reassembly of α-1,6-glycosidic bonds occur, thus resulting in a high product safety.

[0032] 2. This invention fully utilizes the catalytic characteristics of the two amylases to achieve synergistic effects, further enhancing reaction efficiency. It can significantly reduce the rapid digestibility of the product and improve its solubility and solution stability. A low-GI maltodextrin with a slow-digestible component ratio of 20.33% and a resistant component ratio of 36.96% was prepared, reducing the rapid-digestible component ratio to 42.71%. Compared with natural starch, the rapid-digestible component ratio decreased by 56.03%, while the slow-digestible and resistant component ratios increased by 6.85 times and 126.45 times, respectively.

[0033] 3. This invention further improves the branching degree of the product and reduces its relative molecular mass. The resulting product is soluble in cold water with a dissolution rate superior to commercially available maltodextrin. Furthermore, the transparency and viscosity stability of the solution are further improved. After being stored at 4°C for 30 days, the transparency of the solution can still reach over 90.00%. This invention overcomes the shortcomings of natural starch being insoluble in cold water and the poor stability and easy retrogradation of commercially available maltodextrin solutions, achieving a simultaneous improvement in the slow digestibility and stability of the product. Attached Figure Description

[0034] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein... Figure 1 This describes the change in transparency of different dextrin solutions after storage at 4°C for 30 days in the test examples of this invention. Detailed Implementation

[0035] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0036] The detection methods used in the following examples: (1) In vitro simulated digestibility assay: Mix 50 mg of pepsin with 10 mL of HCl (0.05 mol / L) solution, vortex for 5 min to mix thoroughly, and store on ice. Prepare and use immediately. Mix 3 g of porcine pancreatin with 20 mL of deionized water, vortex for 5 min to mix thoroughly, and incubate at 4°C and 3500 °C. g Centrifuge for 10 min, take 15 mL of supernatant, mix thoroughly with 0.8 mL of amylase to prepare mixed enzyme solution, store on ice, and prepare and use immediately.

[0037] The in vitro digestion experiment was performed according to the Englyst method, with slight modifications: 100 mg of the test sample (on a dry basis) was accurately weighed and dispersed in 2.5 mL of sodium acetate buffer (0.25 mol / L, pH 5.2). The sample was heated in a boiling water bath for 30 min to allow for complete gelatinization. Then, 15 glass beads were added, and the mixture was preheated in a water bath shaker at 37°C and 160 r / min for 10 min. Next, 1.67 mL of pepsin solution was added, and the mixture was shaken for 30 min to simulate gastric digestion. After gastric digestion, 2.5 mL of sodium acetate buffer (pH 5.2) was added, and the mixture was shaken for another 30 min. Then, 0.83 mL of mixed enzyme solution was added to simulate small intestinal digestion. At 20 min and 120 min of simulated small intestinal digestion, 200 μL of the digestive solution was dissolved in 5 mL of 66.6% ethanol to terminate the reaction. The mixture was then dissolved in 3500 mL of ethanol. g After centrifugation at room temperature for 5 min, accurately pipette 0.05 mL of the supernatant and determine the glucose content using the glucose oxidase method. Calculate the proportions of rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS). The specific calculation formula is as follows: RDS (%) = G20 × 0.9 × 100 SDS (%) = (G120 – G20) × 0.9 × 100 RS (%) = 100 – RDS – SDS Wherein, G20 represents the proportion of glucose produced after 20 min of sample digestion, and G120 represents the proportion of glucose produced after 120 min of sample digestion.

[0038] (2) Determination of β-amylase hydrolysis rate: Accurately weigh 10 mg of sample and prepare a 5 mg / mL sample solution using phosphate buffer (50 mmol / L, pH 5.5). Heat in a boiling water bath for 30 min to allow the sample to fully gelatinize. After incubating the sample solution in a 50°C water bath for 15 min, add 10 U / mg and 30 U / mg of β-amylase, respectively, and maintain the reaction at 50°C for 24 h. Terminate the reaction by boiling in a water bath for 30 min after completion. Filter the solution through a 0.22 μm aqueous filter membrane and analyze the maltose content using an ICS-5000 high-performance anion exchange chromatography-pulse amperometric detector (HPAEC-PAD) to calculate the β-amylase hydrolysis rate.

[0039] (3) Determination of relative molecular weight: Accurately weigh 10 mg of sample and prepare a 10 mg / mL sample solution with deionized water. Heat in a boiling water bath for 30 min to allow the sample to fully gelatinize. Analyze the molecular weight distribution and weight-average molecular weight of the sample using a Dawn Heleos II high-performance size exclusion chromatography-multi-angle laser light scattering-differential refractive index detector (HPSEC-MALLS-RI). Mw ).

[0040] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available.

[0041] The starch branching enzyme Ro-GBE is derived from Rhodothermus obamensis STB05 (see reference: Wang Z, Xin C, et al. Expression and characterization of an extremely thermophilic 1,4-α-glucan branching enzyme from RhodothermusobamensisSTB05. Protein Expression and Purification [Elsevier]. 2019, 164: 105478-105478).

[0042] The β-amylase (product number M-100H) was purchased from Shandong Yantai Maitel Biotechnology Co., Ltd.; the yeast (high-sugar, high-activity dry yeast) was purchased from Angel Yeast Co., Ltd.

[0043] Example 1: Establishment of a method for preparing low-GI maltodextrin.

[0044] This embodiment provides a method for the synergistic preparation of low-GI maltodextrin using two enzymes, the specific steps of which are as follows: (1) Disperse cassava starch in water to obtain 10% (w / w, on a dry basis) starch milk, stir and preheat at 60°C for 10 min, adjust pH to 7.0, add 100 U / g of starch branching enzyme Ro-GBE to the starch milk, and gelatinize at 95°C for 1 h. (2) Cool the gelatinized product obtained in step (1) to 70°C and react overnight for 12 h; (3) Cool the reaction product obtained in step (2) to 60°C, adjust the pH value to 5.5, add 100 U / g of β-amylase, and react at a constant temperature for 6 h; (4) Cool the reaction product obtained in step (3) directly to 30°C, add 1% of yeast solution activated at 37°C, and co-culture at constant temperature for 48 h before raising the temperature to inactivate the enzyme. (5) Filtration: After the reaction solution is cooled, it is filtered through a filter membrane to remove yeast cells and impurities, and the product solution is obtained. (6) Decolorization: Adjust the pH of the product solution obtained in step (5) to 4.5, add 1% activated carbon at 45℃ according to the mass ratio, and stir for 30 min to decolorize to obtain a decolorized solution; (7) Ion decolorization: At 45°C, the ion exchange resin used has a strongly acidic cation structure. The metal salts and pigments in the decolorized solution obtained in step (6) are removed by a weakly basic cation-strong acid cation exchange resin to obtain a solution containing low-GI maltodextrin.

[0045] (8) The solution containing low-GI maltodextrin obtained in step (7) is spray-dried. The inlet air temperature of the spray dryer is 170°C, the outlet air temperature is 85°C, and the material flow rate is 20 mL / min to obtain the product.

[0046] Comparative Example 1 This comparative example provides a method for the synergistic preparation of low-GI maltodextrin using two enzymes, the specific steps of which are as follows: (1) Disperse cassava starch in water to obtain 10% (w / w, on a dry basis) starch milk, stir and preheat at 60°C for 10 min, adjust pH to 7.0, add 100 U / g of starch branching enzyme Ro-GBE to the starch milk, and gelatinize at 95°C for 1 h. (2) Cool the gelatinized product obtained in step (1) to 70°C and react overnight for 12 h; (3) Cool the reaction product obtained in step (2) to 60°C, adjust the pH value to 5.5, add 100 U / g of β-amylase, react at a constant temperature for 48 h, and then raise the temperature to inactivate the enzyme. (4) Decolorization: Adjust the pH of the product solution obtained in step (3) to 4.5, add 1% activated carbon at 45℃ according to the mass ratio, and stir for 30 min to decolorize to obtain a decolorized solution; (5) Ion decolorization: At 45°C, the ion exchange resin structure used is a strong acid cation-weak base cation-strong acid cation exchange resin to remove metal salts and pigments in the decolorized solution obtained in step (4) to obtain a solution containing low GI maltodextrin. (6) The solution containing low-GI maltodextrin obtained in step (5) is subjected to alcohol precipitation and vacuum drying to obtain the product.

[0047] Comparative Example 2 This comparative example provides a method for preparing low-GI maltodextrin. The specific implementation method is similar to that of Example 1, except that β-amylase is not added in step (3).

[0048] Comparative Example 3 This comparative example provides a method for preparing low-GI maltodextrin, and the specific implementation method is similar to that of Example 1, except that: In step (1), without adding the starch branching enzyme Ro-GBE, the temperature is directly raised to 95℃ for gelatinization for 1 h.

[0049] Comparative Example 4 This comparative example provides a method for the synergistic preparation of low-GI maltodextrin using two enzymes, employing yeast post-treatment. The specific implementation method is similar to that of Example 1, except that: Step (3) is modified as follows: the reaction product obtained in step (2) is cooled to 60°C, the pH value is adjusted to 5.5, 100 U / g of β-amylase is added, the reaction is kept at a constant temperature for 48 h, the temperature is raised to inactivate the enzyme, and then cooled to 30°C. 1% of yeast solution activated at 37°C is added to remove small molecule sugars. Then the solution is filtered through a filter membrane to remove yeast cells and impurities, and a clear solution is obtained. Step (6) is modified as follows: the solution containing low-GI maltodextrin obtained in step (5) is spray-dried. The inlet air temperature of the spray dryer is 170°C, the outlet air temperature is 85°C, and the material flow rate is 20 mL / min to obtain the product.

[0050] The in vitro digestibility of each modified product and untreated cassava starch was analyzed using an in vitro simulated digestibility assay. The results are shown in Tables 1 and 2.

[0051] Table 1. Effects of dual-enzyme synergistic modification

[0052] Table 2. Effects of yeast co-culture

[0053] The above results demonstrate that the modification method established in this invention can fully utilize the catalytic characteristics of the two amylases to achieve synergistic effects. Firstly, by utilizing the high-temperature resistance and hydrolytic and transglycosylation functions of Ro-GBE, the viscosity of the gelatinized starch system can be significantly reduced, and more branching sites can be provided for β-amylase activity. Subsequently, co-culturing with yeast to utilize its sugar-consuming characteristics further enhances the modification efficiency, relieves product inhibition, and significantly reduces the rapid digestibility of the product, showing a clear advantage in the preparation of low-GI maltodextrin. After the above process modification, the proportion of rapidly digestible components in the obtained product can be reduced to 65.09%, the proportion of slowly digestible components increased to 12.40%, and the proportion of resistant components increased to 22.51%. Compared with cassava starch, the proportion of rapidly digestible components decreased by 32.98%, while the proportions of slowly digestible and resistant components increased by 3.79 times and 76.62 times, respectively. The reaction product is soluble in cold water, with a dissolution time of 3 min 21 s.

[0054] Example 2: Effect of gelatinization temperature on synergistic modification effect.

[0055] This embodiment provides a method for the synergistic preparation of low-GI maltodextrin using two enzymes. The specific implementation method is similar to that of Example 1, except that: Step (1) was adjusted as follows: cassava starch was dispersed in water to obtain 10% (w / w, on a dry basis) starch milk, stirred and preheated at 60°C for 10 min, and the pH was adjusted to 7.0. 100 U / g of starch branching enzyme Ro-GBE was added to the starch milk, and the gelatinization reaction was carried out at 70°C, 75°C, 80°C, 85°C, 90°C, and 95°C for 1 h, respectively. Step (3) is modified as follows: the reaction product obtained in step (2) is cooled to 60°C, the pH value is adjusted to 5.5, 100 U / g of β-amylase is added, the reaction is kept at a constant temperature for 6 h, and then cooled to 30°C. 1% of yeast solution activated at 37°C is added for co-culture to remove small molecule sugars. Then the solution is filtered through a filter membrane to remove yeast cells and impurities, and the enzyme is inactivated to obtain a clear solution. Step (6) is modified as follows: the solution containing low-GI maltodextrin obtained in step (5) is spray-dried. The inlet air temperature of the spray dryer is 170°C, the outlet air temperature is 85°C, and the material flow rate is 20 mL / min to obtain the product.

[0056] The in vitro digestibility of each modified product and untreated cassava starch was analyzed using an in vitro simulated digestibility assay. The results are shown in Table 3.

[0057] Table 3 Effect of gelatinization temperature on synergistic modification effect

[0058] The results show that the synergistic modification method established in this invention utilizes the rapid hydrolysis and viscosity reduction advantage of Ro-GBE, which can further lower the gelatinization temperature and thus reduce energy consumption. Specifically, when the gelatinization temperature is 80℃, the in vitro digestibility of the obtained product is further reduced, with the proportion of rapidly digestible components decreasing to 63.37%, the proportion of slowly digestible components increasing to 13.41%, and the proportion of resistant components increasing to 23.22%. Compared to cassava starch, the proportion of rapidly digestible components decreased by 34.76%, while the proportions of slowly digestible and resistant components increased by 4.18 times and 79.07 times, respectively; furthermore, the reaction product is soluble in cold water, with a dissolution time of 3 min 12 s.

[0059] Example 3: Effect of starch milk concentration on synergistic modification effect.

[0060] This embodiment provides a method for the synergistic preparation of low-GI maltodextrin using two enzymes. The specific implementation method is similar to that of Embodiment 2, except that: Step (1) is specifically adjusted as follows: cassava starch is dispersed in water to obtain starch slurries with mass fractions of 10%, 15%, 20%, 25%, and 30% (w / w, on a dry basis), respectively. The slurries are preheated at 60°C with stirring for 10 min, and the pH is adjusted to 7.0. 100 U / g of the starch branching enzyme Ro is then added to the starch slurries. GBE was gelatinized at 80℃ for 1 h, then cooled to 70℃ and reacted overnight for 12 h. Subsequently, the temperature was lowered to 60℃, the pH was adjusted to 5.5, 100 U / g of β-amylase was added, and the reaction was carried out at a constant temperature for 6 h. Then, the temperature was lowered to 30℃, 1% of yeast solution activated at 37℃ was added, and the reaction was carried out at a constant temperature for 48 h. After that, the temperature was raised to inactivate the enzyme and subsequent processing steps were carried out to obtain different low-GI maltodextrin products.

[0061] The in vitro digestibility of each product and untreated cassava starch was analyzed using an in vitro simulated digestibility assay. The results are shown in Table 4.

[0062] Table 4. Effect of starch milk concentration on synergistic modification effect

[0063] The results showed that the synergistic modification method established in this invention can handle high concentrations of starch milk (15%~30%). Specifically, when the starch milk concentration was 15% (w / w, on a dry basis), the in vitro digestibility of the resulting product further decreased, with the proportion of rapidly digestible components decreasing to 62.69%, the proportion of slowly digestible components increasing to 13.93%, and the proportion of resistant components increasing to 23.38%. Compared to cassava starch, the proportion of rapidly digestible components decreased by 35.45%, while the proportions of slowly digestible and resistant components increased by 4.38 times and 79.62 times, respectively; compared to Example 2, the initial starch milk concentration increased by 1.5 times. Furthermore, the reaction product was soluble in cold water, with a dissolution time of 3 min 05 s.

[0064] Example 4: Effect of Ro-GBE addition on synergistic modification effect.

[0065] This embodiment provides a method for the synergistic preparation of low-GI maltodextrin using two enzymes. The specific implementation method is similar to that of Example 3, except that: Step (1) was specifically adjusted as follows: cassava starch was dispersed in water to obtain a 15% (w / w, on a dry basis) starch milk, which was stirred and preheated at 60°C for 10 min, and the pH was adjusted to 7.0. 100 U / g, 200 U / g, 300 U / g, 400 U / g, 500 U / g, and 600 U / g starch branching enzyme Ro-GBE were added to the starch milk, and the mixture was gelatinized at 80°C for 1 h. Subsequent processing steps were then carried out to obtain different low-GI maltodextrin products.

[0066] The in vitro digestibility of each product and untreated cassava starch was analyzed using an in vitro simulated digestibility assay. The results are shown in Table 5.

[0067] Table 5 Effect of Ro-GBE addition amount on synergistic modification effect

[0068] The results showed that the in vitro digestibility of the product first decreased and then increased with increasing Ro-GBE addition. At a Ro-GBE addition of 300 U / g, the in vitro digestibility of the product further decreased, with the proportion of rapidly digestible components dropping to 59.31%, and the proportions of slowly digestible and resistant components reaching 14.17% and 26.52%, respectively. Compared to cassava starch, the proportion of rapidly digestible components decreased by 38.94%, while the proportions of slowly digestible and resistant components increased by 4.47 times and 90.45 times, respectively. Furthermore, the reaction product was soluble in cold water, with a dissolution time of 2 min 39 s.

[0069] Example 5: Effect of Ro-GBE reaction time on synergistic modification effect.

[0070] This embodiment provides a method for the synergistic preparation of low-GI maltodextrin using two enzymes. The specific implementation method is similar to that of Example 4, except that steps (1) and (2) are specifically adjusted as follows: (1) Disperse cassava starch in water to obtain a 15% (w / w, on a dry basis) starch milk, stir and preheat at 60°C for 10 min, adjust the pH to 7.0, add 300 U / g Ro-GBE to the starch milk, and gelatinize at 80°C for 1 h; (2) The reaction product obtained in step (1) was cooled to 70°C and reacted for 8 h, 12 h, 16 h, 20 h and 24 h respectively, and then the subsequent reaction steps were carried out to obtain different low-GI maltodextrin products.

[0071] The in vitro digestibility of each product and untreated cassava starch was analyzed using an in vitro simulated digestibility assay. The results are shown in Table 6.

[0072] Table 6 Effect of Ro-GBE reaction time on synergistic modification effect

[0073] The results showed that, with the increase of starch branching enzyme Ro The extended treatment time of GBE further reduced the in vitro digestibility of the product. When the treatment time with the starch branching enzyme Ro-GBE reached 16 h, the in vitro digestibility of the product decreased further, with the proportion of rapidly digestible components decreasing to 57.82%, the proportion of slowly digestible components increasing to 15.33%, and the proportion of resistant components reaching 26.85%. Compared to cassava starch, the proportion of rapidly digestible components decreased by 40.47%, while the proportions of slowly digestible and resistant components increased by 4.92 times and 91.59 times, respectively. Furthermore, the reaction product was soluble in cold water, with a dissolution time of 2 min 26 s.

[0074] Example 6: Effect of β-amylase addition on synergistic modification effect.

[0075] This embodiment provides a method for the synergistic preparation of low-GI maltodextrin using two enzymes. The specific implementation method is similar to that of Example 5, except that steps (2) and (3) are specifically adjusted as follows: (1) Cool the reaction product obtained in step (2) to 70℃ and react for 16 h; (2) Cool the reaction product obtained in step (3) to 60°C and adjust the pH to 5.5. Add 100 U / g, 200 U / g, 300 U / g, 400 U / g and 500 U / g of β-amylase respectively and react at a constant temperature for 6 h. Then carry out the subsequent reaction steps to obtain different low-GI maltodextrin products.

[0076] The in vitro digestibility of each product and untreated cassava starch was analyzed using an in vitro simulated digestibility assay. The results are shown in Table 7.

[0077] Table 7 Effect of β-amylase dosage on synergistic modification effect

[0078] The results showed that the in vitro digestibility of the product first decreased and then increased with increasing β-amylase addition. At a β-amylase addition of 300 U / g, the in vitro digestibility of the product further decreased, with the proportion of rapidly digestible components dropping to 53.83%, and the proportions of slowly digestible and resistant components reaching 16.98% and 29.19%, respectively. Compared to cassava starch, the proportion of rapidly digestible components decreased by 44.58%, while the proportions of slowly digestible and resistant components increased by 5.56 times and 99.66 times, respectively. Furthermore, the reaction product was soluble in cold water, with a dissolution time of 2 min 13 s.

[0079] Example 7: Effect of β-amylase reaction time on synergistic modification effect.

[0080] This embodiment provides a method for preparing low-GI maltodextrin by synergistic dual-enzyme preparation. The specific implementation method is similar to that of Example 6, except that step (3) is specifically adjusted as follows: the reaction product obtained in step (2) is cooled to 60°C, the pH value is adjusted to 5.5, 300 U / g of β-amylase is added, and the reaction is carried out at a constant temperature for 0 h, 3 h, 6 h, 9 h, and 12 h before proceeding with subsequent reaction steps to obtain different low-GI maltodextrin products.

[0081] The in vitro digestibility of each product and untreated cassava starch was analyzed using an in vitro simulated digestibility assay. The results are shown in Table 8.

[0082] Table 8 Effect of β-amylase reaction time on synergistic modification effect

[0083] The results showed that the longer the reaction time at 60℃, the more significant the recovery in digestibility of the product, which is speculated to be due to the poor thermostability of β-amylase, leading to partial inactivation. When the 60℃ stage was removed and yeast co-culture was performed directly, the in vitro digestibility of the product further decreased, with the proportion of rapidly digestible components decreasing to 51.77%, the proportion of slowly digestible components increasing to 18.53%, and the proportion of resistant components reaching 29.70%. Compared to cassava starch, the proportion of rapidly digestible components decreased by 46.70%, while the proportions of slowly digestible and resistant components increased by 6.15 times and 101.41 times, respectively. Furthermore, the reaction product was soluble in cold water, with a dissolution time of 2 min 09 s.

[0084] Example 8: Effect of yeast co-culture temperature on synergistic modification effect.

[0085] The specific implementation method is the same as in Example 7, except that steps (3) and (4) are specifically adjusted as follows: the reaction product obtained in step (2) is directly cooled to 37°C or 30°C, and the pH is adjusted to 5.5. 300 U / g of β-amylase and 1% of yeast solution activated at 37°C are added. After constant temperature reaction for 48 h, the enzyme is inactivated by raising the temperature and proceeding with the subsequent reaction steps to obtain different low-GI maltodextrin products.

[0086] The in vitro digestibility of each product and untreated cassava starch was analyzed using an in vitro simulated digestibility assay. The results are shown in Table 9.

[0087] Table 9. Effect of yeast co-culture temperature on synergistic modification effect

[0088] The results showed that directly adding yeast solution to the reaction at the initial stage of β-amylase further reduced the in vitro digestibility of the product. When co-cultured at 37℃, the proportion of the rapidly digestible component decreased to 49.40%, while the proportions of the slowly digestible and resistant components were 18.93% and 31.67%, respectively. Compared to cassava starch, the proportion of the rapidly digestible component decreased by 49.14%, while the proportions of the slowly digestible and resistant components increased by 6.31 times and 108.21 times, respectively. Furthermore, the reaction product was soluble in cold water, with a dissolution time of 2 min 02 s.

[0089] Example 9: Effect of yeast co-culture time on synergistic modification effect.

[0090] The specific implementation method is the same as in Example 8, except that steps (3) and (4) are specifically adjusted as follows: the reaction product obtained in step (2) is directly cooled to 37°C and the pH is adjusted to 5.5. 300 U / g of β-amylase and 1% of yeast solution activated at 37°C are added. The reaction is carried out at a constant temperature for 12 h, 24 h, 36 h, 48 h and 60 h respectively, and then the temperature is raised to inactivate the enzyme. The subsequent reaction steps are carried out to obtain different low-GI maltodextrin products.

[0091] The in vitro digestibility of each product and untreated cassava starch was analyzed using an in vitro simulated digestibility assay. The results are shown in Table 10.

[0092] Table 10 Effect of yeast co-culture time on synergistic modification effect

[0093] The results showed that the in vitro digestibility of the product decreased further with increasing co-culture time. When the co-culture time was extended to 60 h, the proportion of rapidly digestible components decreased to 48.63%, while the proportions of slowly digestible and resistant components were 19.07% and 32.30%, respectively. Compared with cassava starch, the proportion of rapidly digestible components decreased by 49.93%, while the proportions of slowly digestible and resistant components increased by 6.36 times and 110.38 times, respectively. Furthermore, the reaction product was soluble in cold water, with a dissolution time of 2 min 02 s.

[0094] Example 10: Effect of secondary enzymatic hydrolysis on synergistic modification The specific implementation method is the same as in Example 9, except that: after reacting for 60 h in step (4), the enzyme is not inactivated, β-amylase and newly activated yeast are added again, and steps (3) and (4) are repeated once. Then, subsequent reaction steps are carried out to obtain different low-GI maltodextrin products.

[0095] The in vitro digestibility of each product and untreated cassava starch was analyzed using an in vitro simulated digestibility assay. The results are shown in Table 11.

[0096] Table 11 Effect of secondary enzymatic hydrolysis on synergistic modification effect

[0097] The results showed that while a single enzymatic hydrolysis could fully hydrolyze the substrate, it was not complete, leaving a small number of long-chain branches unhydrolyzed, which might lead to increased digestibility. Introducing a secondary enzymatic hydrolysis step further ensured complete hydrolysis of the chain segments (β-amylase hydrolysis rate approached 0), achieving a highly branched state that made further hydrolysis by digestive enzymes difficult, thus improving digestibility. Ultimately, the proportion of rapidly digestible components in the product obtained from the secondary enzymatic hydrolysis was reduced to 42.71%, while the proportions of slowly digestible and resistant components were 20.33% and 36.96%, respectively. Compared to cassava starch, the proportion of rapidly digestible components decreased by 56.03%, while the proportions of slowly digestible and resistant components increased by 6.85 times and 126.45 times, respectively. Furthermore, the reaction product was soluble in cold water, with a dissolution time of 1 min 51 s.

[0098] Example 11 Commercially available maltodextrin (control) and the low-GI maltodextrin product solution with significantly reduced rapidly digestible components obtained in Example 10 were stored at 4°C for 30 days. The transmittance was then measured at 620 nm using a spectrophotometer. The results are as follows: Figure 1As shown, commercially available maltodextrin rapidly reverts to white within 3 days, while the sample maintains a transparency of over 90%. This indicates that the stability of the sample solution was greatly improved after dual-enzyme synergistic modification and secondary enzymatic hydrolysis, overcoming the shortcomings of natural starch being insoluble in cold water and the poor stability and easy reversion of commercially available maltodextrin solutions, thus achieving a simultaneous improvement in the slow digestibility and stability of the product.

[0099] Example 12 The relative molecular weights of native cassava starch and products modified by different processes were determined and analyzed. The results are shown in Table 12. It can be seen from the results that since β-amylase is an exonuclease, its degradation efficiency on native starch is low, resulting in small changes in molecular weight. As a result, the product still retains the high viscosity of native starch during the reaction process. Ro-GBE, on the other hand, requires hydrolysis of some chain segments before re-linking them to the starch outer chain in the form of branched chains. It has the advantage of rapid hydrolysis and viscosity reduction. Therefore, adding Ro-GBE first can further improve the hydrolysis efficiency of β-amylase, so that the product is further hydrolyzed into maltodextrin with a small molecular weight, and the stability is further improved.

[0100] Table 12 Relative molecular weights of native starch and modified products

[0101] Example 13 Different types of starch were treated with the above-mentioned modification process (secondary enzymatic hydrolysis), and their in vitro simulated digestibility was measured. The results are shown in Table 13. The results in the table show that this process is applicable to different types of starch and has good application potential.

[0102] Table 13 Applicability of the above modification process to different types of starch

[0103] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for preparing low-GI maltodextrin using a dual-enzyme synergistic approach, characterized in that, The method includes the following steps: (1) Disperse starch granules in water to obtain starch milk; add starch branching enzyme Ro-GBE to the obtained starch milk, heat to carry out gelatinization reaction, cool down and continue to react at a constant temperature to obtain reaction solution 1; (2) After cooling the reaction solution 1 obtained in step (1), add β-amylase and yeast to react and obtain reaction solution 2; (3) Dry the reaction solution 2 obtained in step (2) to obtain the low-GI maltodextrin; In step (1), the starch branching enzyme Ro-GBE is derived from... Rhodothermus obamensis STB05, nucleotide sequence as shown in SEQ ID NO.1; In step (1), the temperature of the gelatinization reaction is 70℃~95℃; the time of the gelatinization reaction is 0 h~5 h.

2. The method according to claim 1, characterized in that, In step (1), the starch is selected from one or more of tapioca starch, ordinary corn starch, waxy corn starch, potato starch, rice starch and wheat starch.

3. The method according to claim 1, characterized in that, In step (1), the concentration of the starch milk is 10% to 30% (w / w); the pH value of the starch milk is 6.0 to 7.

5.

4. The method according to claim 1, characterized in that, In step (1), the amount of starch branching enzyme Ro-GBE added is 100 U / g to 1000 U / g dry starch.

5. The method according to claim 1, characterized in that, In step (1), the temperature of the isothermal reaction is 60℃~80℃; the time of the isothermal reaction is 8 h~48 h.

6. The method according to claim 1, characterized in that, In step (2), the temperature after cooling is 30℃~60℃.

7. The method according to claim 1, characterized in that, In step (2), the amount of β-amylase added is 100 U / g to 1000 U / g dry starch.

8. The method according to claim 1, characterized in that, In step (2), the amount of yeast added is 0.5% to 5%.

9. The method according to claim 1, characterized in that, In step (2), the reaction temperature is 30℃~60℃; the reaction time is 6 h~72 h.

10. The method according to claim 1, characterized in that, In step (2), the reaction is followed by the addition of β-amylase and yeast for a secondary reaction.

11. The method according to claim 10, characterized in that, The amount of β-amylase added is 100 U / g to 1000 U / g dry starch.

12. The method according to claim 10, characterized in that, The amount of yeast added is 0.5% to 5%.

13. The method according to claim 10, characterized in that, The temperature of the secondary reaction is 30℃~60℃; the time of the secondary reaction is 6 h~72 h.

14. The method according to claim 1, characterized in that, In step (2), the reaction is followed by enzyme inactivation, filtration, decolorization and ion exchange treatment.

15. The method according to claim 14, characterized in that, The decolorization process involves adjusting the pH of the clarified solution obtained through filtration, adding activated carbon, and stirring to decolorize, thereby obtaining a decolorized solution. And / or, the ion exchange is: using an ion exchange resin to remove metal salts and pigments from the decolorizing solution.

16. The method according to claim 1, characterized in that, In step (3), the drying process is selected from one or more of freeze drying, drum drying and spray drying.

17. The low-GI maltodextrin prepared by the method according to any one of claims 1-16.

18. The use of the low-GI maltodextrin of claim 17 in the preparation of probiotic products, health products or meal replacement products.

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