A method for preparing resistant dextrin using starch branching enzyme in conjunction with amylase

Abstract

This invention discloses a method for producing resistant dextrin using starch branching enzyme and α-amylase. The specific steps include: preparing pyrodextrin from corn starch via acid-thermal catalysis; then, purifying and resynthesizing the branched chains using starch branching enzyme and α-amylase; further purifying by fermentation with Saccharomyces cerevisiae; and finally, drying to obtain the resistant dextrin product. Through examples, this invention demonstrates that the obtained resistant dextrin possesses excellent glucose binding and retention capabilities, as well as resistance to digestion (resistance component ratio reaches 86.97%), good solubility, and freeze-thaw stability, making it well-suited for applications in the beverage and other industries. This provides a new, efficient, and environmentally friendly approach for the preparation and application of resistant dextrin.

Description

Technical Field

[0001] This invention relates to the field of starch deep processing, specifically to a method for preparing resistant dextrin using starch branching enzyme in synergistic action with α-amylase. Background Technology

[0002] Resistant dextrin is a type of dietary fiber that possesses the physiological functions of dietary fiber and excellent processing properties. It is an FDA-approved and recognized safe material with no intake restrictions. As early as 2012, my country's Ministry of Health listed resistant dextrin as a common food, allowing it to be added to various foods in unlimited quantities. Against the backdrop of a "Healthy China" initiative, rising health awareness is continuously driving the demand for functional foods and dietary supplements. The application of resistant dextrin in the food industry is becoming increasingly widespread.

[0003] The anti-digestive properties of resistant dextrin primarily stem from its complex branched structure, formed by new bonds such as α-1,2-glycosidic bonds and α-1,3-glycosidic bonds, which are difficult for α-amylase and saccharifying enzymes to break down. After ingestion, resistant dextrin's excellent water-absorbing and swelling properties increase stool volume and promote intestinal peristalsis. Its good prebiotic fermentation properties promote the proliferation of beneficial Bifidobacteria in the gut, which, after fermentation, produce short-chain fatty acids (SCFAs). It can regulate intestinal pH, is not digested and absorbed in the small intestine, slows the rate at which sugars enter the bloodstream, prolongs the feeling of fullness, reduces hunger, and delays gastric emptying, thus having a beneficial effect on weight loss. Furthermore, resistant dextrin can promote mineral absorption and improve the bioavailability of minerals such as calcium and magnesium. Its excellent processing performance and health benefits make resistant dextrin a promising candidate for applications in the food industry, with wide applications in functional foods, confectionery, chocolate, infant formula, baby food, flour-based foods, baked goods, nutrition bars, and alcoholic beverages.

[0004] Currently, the preparation of resistant dextrin is mainly based on physical and chemical acid-thermal methods, followed by purification through enzymatic hydrolysis, fermentation, ultrafiltration, and simulated moving bed chromatography. However, traditional acid-thermal methods suffer from low resistant component content, numerous colored byproducts leading to difficult decolorization, complex products, and significant environmental pollution and resource waste. To improve the yield and color quality of resistant dextrin while meeting the requirements of green and sustainable development strategies, exploring efficient methods has become a new research hotspot. In terms of preparation methods, an increasing number of researchers are introducing new enzymes to prepare resistant dextrin, providing strong support for the development of a high-quality, green, and sustainable resistant dextrin industry. Summary of the Invention

[0005] To overcome the problems of insufficient yield and low content of resistant structures in existing resistant dextrin production processes, this invention aims to provide a method for preparing resistant dextrin in synergy with α-amylase and starch branching enzyme. This method has advantages such as mild reaction conditions, less environmental pollution, the ability to replace saccharifying enzymes in the preparation of resistant dextrin, and good product quality. Compared with saccharifying enzymes, it can increase the branching structure of resistant dextrin, thereby increasing its anti-digestion components, thus improving the overall yield and product quality of the resistant dextrin preparation process.

[0006] The objective of this invention is achieved through the following means: A method for preparing resistant dextrin includes the following steps: (1) Starch is mixed with acid, aged, pre-dried, and then baked to obtain caramelized dextrin. (2) Mix the pyrodextrin obtained in step (1) with α-amylase, adjust the pH to 6-7, perform enzymatic hydrolysis modification, and inactivate the enzyme; then add starch branching enzyme. Adjust the pH to 6-7 and perform enzymatic hydrolysis modification.

[0007] (3) Mix the enzymatic hydrolysis product with brewer's yeast, ferment, centrifuge, filter, and dry the filtrate to obtain resistant dextrin.

[0008] Furthermore, the starch described in step (1) is dried.

[0009] Furthermore, the starch mentioned in step (1) includes, but is not limited to, at least one of ordinary corn starch, waxy corn starch, cassava starch, yam starch, mung bean starch, potato starch, wheat starch and taro starch; even further, the starch mentioned in step (1) is ordinary corn starch.

[0010] Further, the mixing with acid mentioned in step (1) is to spray the acid onto the starch and mix it.

[0011] Further, the acid mentioned in step (1) includes at least one of hydrochloric acid, sulfuric acid, citric acid and tartaric acid, and is an acid solution with a mass fraction of 1-2%; even further, it is a dilute hydrochloric acid with a mass fraction of 1-2%.

[0012] Further, the amount of acid added in step (1) is 5-10% of the dry weight of starch.

[0013] Furthermore, the aging conditions described in step (1) are: a temperature of 60-80℃ and a time of 1-2h.

[0014] Furthermore, the pre-drying conditions described in step (1) are: temperature of 100-110℃ and time of 0.5-1h.

[0015] Furthermore, the baking conditions described in step (1) are: temperature of 160-170℃ and time of 3-5h.

[0016] Furthermore, the amino acid sequence of the starch branching enzyme described in step (2) has at least 80% identity with the sequence shown in SEQ ID NO:1; even further, the starch branching enzyme is a starch branching enzyme derived from *Oxytobacter thermoglucosidase*; and still further, the amino acid sequence of the starch branching enzyme is as shown in SEQ ID NO:1.

[0017] Furthermore, the amount of starch branching enzyme used in step (2) is 300-350 U / g dry pyrolysis product.

[0018] Furthermore, the amount of α-amylase used in step (2) is 100-120 U / g dry pyrolysis product.

[0019] Further, the pyrodextrin mentioned in step (2) is first prepared into a 20-30% (w / w) pyrodextrin solution by adding water, and then mixed with α-amylase. In step (2), the conditions for enzymatic hydrolysis modification are: temperature 50-60℃ and time 6-12h.

[0020] Furthermore, the fermentation conditions described in step (3) are: fermentation for 12-36 hours at a temperature of 28-32℃ and a speed of 180-220 rpm.

[0021] Furthermore, the centrifugation conditions described in step (3) are: rotation speed 10000-12000 rpm, time 20-25 min. The main purpose is to remove fermentation cells and macromolecular impurities (such as inactivated enzyme proteins) from the fermentation products.

[0022] Furthermore, the drying described in step (3) is any processing method in the art that can be used for drying resistant dextrin, including at least one of spray drying, vacuum freeze drying, drum drying and fluidized bed drying.

[0023] Further, the drying in step (3) is to make the moisture content of the dried resistant dextrin 5-10% by mass.

[0024] A resistant starch was prepared by the method described above.

[0025] The above-mentioned applications of resistant starch in food preparation.

[0026] Compared with the prior art, the present invention has the following advantages and beneficial effects: The inventors of this invention ingeniously discovered that, based on the traditional acid-heat production process, synergistic modification by starch branching enzyme and α-amylase can effectively obtain high-quality resistant dextrin. This method not only improves the yield and production efficiency of resistant dextrin, but also, through α-amylase and acid-heat treatment, transforms large molecules into small molecule dextrins. This process is more conducive to the breaking and transfer of α-1,4-glycosidic bonds in the pyrodextrin molecules prepared by the acid-heat method, catalyzed by starch branching enzyme, leading to the formation of more branches in the form of α-1,6-glycosidic bonds, etc., thereby improving the overall digestibility of the molecule and resulting in a higher-quality resistant dextrin product.

[0027] Further research revealed that the inventors discovered that pyrodextrin prepared under specific process conditions, after modification with a specific starch branching enzyme, could further improve the yield of resistant dextrin and the content of resistant components in the product, thereby further improving the overall production process and product quality.

[0028] This invention relates to the synergistic preparation of resistant dextrin using an acid-thermal method, an enzymatic method, and a fermentation method. It employs starch branching enzymes instead of saccharifying enzymes for enzymatic purification, cleaving and reforming the glycosidic bonds within the pyrodextrin without introducing other chemical groups. Simultaneously, the starch branching enzymes increase the content of resistant components. This method boasts high preparation efficiency and lower energy consumption. It provides a scientific basis for the development of functional foods and adjuvant preparations for chronic diseases, and strongly promotes the development of the resistant dextrin processing industry. Attached Figure Description

[0029] Figure 1 Electrophoresis diagram of the protein used in the production and purification of starch branching enzyme.

[0030] Figure 2 The image shows the infrared spectrum of the resistant dextrin prepared in the examples. Detailed Implementation

[0031] The invention will now be described in further detail with reference to embodiments and accompanying drawings. Breakpoints and values ​​disclosed herein are not limited to precise ranges or values; these ranges or values ​​should be understood to include values ​​close to them. Reagents or related materials used are not named manufacturers and are commercially available, conventional products and experimentally produced enzymes. The α-amylase used in the examples was purchased from Shanghai Yuanye Biotechnology Co., Ltd., and the amylase was laboratory-produced and purified; its amino acid sequence is shown in SEQ ID NO:1, and its purified protein electrophoresis image is shown below. Figure 1 As shown, the brewing yeast was purchased from Angel Yeast Co., Ltd.

[0032] In this invention, there are no particular restrictions on the specific method of obtaining the starch branching enzyme used. It can be obtained by purchasing or custom-making a starch branching enzyme with the aforementioned characteristics, or by preparing the relevant product according to existing technology. For example, those skilled in the art can obtain the corresponding starch branching enzyme by biological expression using genetic engineering methods based on the amino acid sequence shown in SEQ ID NO:1 and its encoding gene. Furthermore, the sequence can be modified according to the characteristics of the selected genetically engineered bacteria without adversely affecting the function of the starch branching enzyme (e.g., inserting or deleting some amino acid residues without affecting the function of the starch branching enzyme, performing single-point or multi-point mutations to improve the effect of the starch branching enzyme, or adding some common amino acid tags for easier purification, etc.).

[0033] In this invention, there are no particular restrictions on the selection of starch; any starch suitable for the preparation of resistant dextrin can be used. According to some preferred embodiments of the invention, in step (1), the starch is selected from at least one of ordinary corn starch, waxy corn starch, cassava starch, yam starch, mung bean starch, potato starch, wheat starch, and taro starch. Preferably, the starch is ordinary corn starch.

[0034] In some preferred embodiments, the starch is pre-dried before being mixed with the acid. Preferably, the pre-drying conditions are such that the water content in the starch does not exceed 10% by mass.

[0035] Example 1

[0036] A method for preparing resistant dextrin includes the following steps: (1) Mix 1% dilute hydrochloric acid with starch by spraying at a ratio of 10 mL / 100 g dry weight, age at 60-80℃ for 2 h, pre-dry at 105℃ for 1 h, and bake at 160℃ for 4 h to obtain caramelized dextrin.

[0037] (2) Dissolve the pyrodextrin obtained in step (1) in water to form a 30% mass fraction solution, adjust the pH to 6, add α-amylase at 95℃ at an addition amount of 100 U / g pyrodextrin (pyrodextrin is on a dry basis, the same below) for 6 hours, boil in a water bath for 30 minutes, then adjust the pH to 7, add starch branching enzyme at an addition amount of 300 U / g pyrodextrin (pyrodextrin is on a dry basis, the same below) for modification, the reaction temperature is 50℃, the reaction time is 6 hours, and the enzymatically modified product is obtained.

[0038] (3) Boil the enzymatic hydrolysate at 100℃ for 30 minutes to inactivate the enzyme, add 4g / L of food-grade dry yeast powder (brewing yeast) for fermentation, and ferment at 30℃ and 200rpm for 30 hours.

[0039] (4) The fermentation product was centrifuged at 11,000 rpm for 20 min and then freeze-dried under vacuum to finally obtain resistant dextrin A1.

[0040] Its infrared spectrum is as follows Figure 2 As shown.

[0041] Example 2

[0042] A method for preparing resistant dextrin includes the following steps: (1) Mix 1% dilute hydrochloric acid with starch by spraying at a ratio of 10 mL / 100 g dry weight, age at 60-80℃ for 2 h, pre-dry at 105℃ for 1 h, and bake at 160℃ for 4 h to obtain caramelized dextrin.

[0043] (2) Dissolve the pyrodextrin obtained in step (1) in water to form a 30% mass fraction solution, adjust the pH to 7, add starch branching enzyme at 50℃ for 6 hours and boil in a water bath for 30 minutes, then adjust the pH to 6 and add α-amylase at 100U / g pyrodextrin for 6 hours. The reaction temperature is 95℃ and the reaction time is 6 hours to obtain the enzymatically modified product.

[0044] (3) Boil the enzymatic hydrolysate at 100℃ for 30 minutes to inactivate the enzyme, add 4g / L of food-grade dry yeast powder (brewing yeast) for fermentation, and ferment at 30℃ and 200rpm for 30 hours.

[0045] (4) The fermentation product was centrifuged at 11,000 rpm for 20 min, and then the filtrate was freeze-dried under vacuum to finally obtain resistant dextrin A2.

[0046] Example 3

[0047] A method for preparing resistant dextrin includes the following steps: (1) Mix 1% dilute hydrochloric acid with starch by spraying at a ratio of 10 mL / 100 g dry weight, age at 60-80℃ for 2 h, pre-dry at 105℃ for 1 h, and bake at 160℃ for 4 h to obtain caramelized dextrin.

[0048] (2) Dissolve the pyrodextrin obtained in step (1) in water to form a 30% mass fraction solution, adjust the pH to 6.5, add starch branching enzyme at 300 U / g pyrodextrin and α-amylase at 100 U / g pyrodextrin at 52℃ for modification, and the reaction time is 12h to obtain the enzymatically modified product.

[0049] (3) Boil the enzymatic hydrolysate at 100℃ for 30 min to inactivate the enzyme, add 4 g / L of food-grade dry yeast powder (brewing yeast) for fermentation, and ferment at 30℃ and 200 rpm for 30 h.

[0050] (4) The fermentation product was centrifuged at 8000 rpm for 20 min, and the filtrate was freeze-dried under vacuum to finally obtain resistant dextrin A3.

[0051] Comparative Example 1 A method for preparing resistant dextrin includes the following steps: (1) Mix 1% dilute hydrochloric acid with starch by spraying at a ratio of 10 mL / 100 g dry weight, age at 60-80℃ for 2 h, pre-dry at 105℃ for 1 h, and bake at 160℃ for 4 h to obtain caramelized dextrin.

[0052] (2) Dissolve the dextrin obtained in step (1) in water to form a solution with a mass fraction of 30%, adjust the pH to 6.5, add starch branching enzyme at 52℃ for 6 hours, add saccharifying enzyme at 200U / g of dextrin for modification, and react for 6 hours to obtain the enzymatically modified product.

[0053] (3) Boil the enzymatic hydrolysate at 100℃ for 30 min to inactivate the enzyme, add 4 g / L of food-grade dry yeast powder (brewing yeast) for fermentation, and ferment at 30℃ and 200 rpm for 30 h.

[0054] (4) The fermentation product was centrifuged at 8000 rpm for 20 min, and the filtrate was freeze-dried under vacuum to finally obtain resistant dextrin A4.

[0055] Example 4

[0056] In vitro simulated digestion assay The in vitro simulated digestion assay was performed as follows: Weigh 0.2 g of the modified sample and add 10 mL of sodium acetate buffer (0.25 mol / L, pH 5.2). Incubate in a boiling water bath for 30 min to allow the sample to fully gelatinize. After cooling, add 5 mL of sodium acetate buffer, 5 mL of mixed enzyme solution (trypsin 2500 U / mg; glucoamylase 100,000 U / g), and glass beads. Incubate in a 37°C shaking water bath to simulate small intestinal digestion. At 20 min and 120 min, take 200 μL of the digestion solution and dissolve it in 5 mL of 66.6% ethanol to terminate the reaction. Centrifuge the mixture at 3500 g at room temperature for 5 min and determine the glucose content using a glucose assay kit. Calculate the resistant component according to the following formula.

[0057] RDS=G 20 ×0.9×100 SDS=(G 120 -G 20 )×0.9×100 RS = 100 - RDS - SDS Where: RDS—mass fraction of rapidly digestible components, % G 20 —The mass fraction of glucose produced after 20 minutes of sample digestion, % SDS – Mass fraction of slowly digested components, % G 120 —The mass fraction of glucose produced after 120 min of sample digestion, % RS – Mass fraction of the antidigestant component, % Table 1

[0058] The results are shown in Table 1. According to the simulated in vitro digestibility results, the proportions of digestible components in the resistant dextrin prepared in the three different implementation cases were 84.56%, 85.96%, and 86.97%, respectively, which showed good anti-digestion properties. Among them, the proportion of anti-digestion components in Example 3 was the highest, which was higher than that in Comparative Example 1 of the traditional preparation method.

[0059] Glucose adsorption capacity (GAC) determination At 25°C, each sample (0.1 g) was mixed with 20 mL of glucose solution (40 mg / mL) and stirred at 150 rpm for 30 minutes. The mixture was then centrifuged at 3000 × g for 20 minutes. GAC was determined at 540 nm using a UV-9000 spectrophotometer (Shanghai Yuanwei Instrument Co., Ltd., China) via the 3,5-dinitrosalicylic acid (DNS) method. The total acid value was calculated using the following formula: GAC (mmol / g) =

[0060] Where m1 is the weight of glucose in the solution, m2 is the weight of glucose in the remaining solution, M is the relative mass of glucose, and m is the weight of the sample.

[0061] Table 2

[0062] The results are shown in Table 2. Based on the glucose adsorption capacity determination, the resistant dextrins prepared in this case study all exhibited good glucose binding and retention capabilities. Compared to resistant dextrins prepared by traditional enzymatic methods, they showed slightly higher glucose adsorption efficiency.

[0063] Freeze-thaw stability test A certain mass of resistant dextrin sample was weighed and dissolved in distilled water to prepare a 6% solution. The solution was then frozen at -20℃ for 24 hours and thawed at room temperature. The transmittance was measured at a wavelength of 720nm.

[0064] Table 3

[0065] The results are shown in Table 3. All examples exhibited stable transparency after five freeze-thaw cycles, indicating good sample stability. Furthermore, they demonstrated superior transparency compared to traditional enzymatic methods for preparing resistant dextrin. These results further suggest that resistant dextrin can be well-suited for use in the frozen beverage industry.

[0066] Solubility determination Weigh 0.3 g of the sample and dissolve it in 24 mL of deionized water. Stir the solution on a magnetic stirrer for 30 min at room temperature, then centrifuge for 15 min. Take 10 mL of the supernatant and transfer it to an aluminum box dried to constant weight. Dry the box in a drying oven using a two-step drying method (overnight at 60℃ followed by drying at 105℃) to constant weight. Weigh the dry matter in the supernatant, making three copies. Calculate the solubility according to the following formula: Solubility =

[0067] Where: a refers to the sample weight; b refers to the mass of the solids after drying; and 10 refers to the volume of the evaporated supernatant.

[0068] Table 4

[0069] As shown in Table 4, the implemented cases exhibit good solubility, high solubility, low suspended matter production after dissolving in water, and high light transmittance, which is beneficial for forming a homogeneous solution. This result indicates that dextrin can be well used in the beverage industry.

Claims

1. A method for preparing resistant dextrin, characterized in that, Includes the following steps: (1) Starch is mixed with acid, aged, pre-dried, and then baked to obtain caramelized dextrin. (2) Mix the pyrodextrin obtained in step (1) with α-amylase, adjust the pH to 6-7, perform enzymatic hydrolysis modification, and inactivate the enzyme; then add starch branching enzyme, adjust the pH to 6-7, and perform enzymatic hydrolysis modification. (3) Mix the enzymatic hydrolysis product with brewer's yeast, ferment, centrifuge, filter, and dry the filtrate to obtain resistant dextrin.

2. The method according to claim 1, characterized in that, The mixing with acid mentioned in step (1) refers to spraying the acid onto the starch for mixing; The acid mentioned in step (1) includes at least one of hydrochloric acid, sulfuric acid, citric acid and tartaric acid, and is an acid solution with a mass fraction of 1-2%. The amount of acid added in step (1) is 5-10% of the dry weight of starch.

3. The method according to claim 1, characterized in that, The aging conditions described in step (1) are: temperature of 60-80℃ and time of 1-2h; The pre-drying conditions described in step (1) are: temperature 100-110℃, time 0.5-1h; The baking conditions described in step (1) are: temperature of 160-170℃ and time of 3-5h.

4. The method according to claim 1, characterized in that, The amino acid sequence of the starch branching enzyme described in step (2) has at least 80% identity with the sequence shown in SEQ ID NO:

1.

5. The method according to claim 4, characterized in that, The starch branching enzyme is derived from *Oxygenobacterium thermoglucosidase*.

6. The method according to claim 5, characterized in that, The amino acid sequence of the starch branching enzyme is shown in SEQ ID NO:

1.

7. The method according to claim 1, characterized in that, The amount of starch branching enzyme used in step (2) is 300-350 U / g dry weight of pyrodextrin; The amount of α-amylase used in step (2) is 100-120 U / g dry weight of pyrodextrin; The dextrin mentioned in step (2) is first mixed with water to prepare a dextrin solution with a mass fraction of 20-30%, and then mixed with α-amylase.

8. The method according to claim 1, characterized in that, The conditions for enzymatic hydrolysis modification described in step (2) are: temperature 50-60℃, time 6-12h; The fermentation conditions described in step (3) are: fermentation at a temperature of 28-32℃ and a speed of 180-220 rpm for 12-36 hours; The centrifugation conditions described in step (3) are: rotation speed 10000-12000 rpm, time 20-25 min; The drying process described in step (3) aims to achieve a moisture content of 5-10% by mass in the dried resistant dextrin.

9. A method for preparing resistant dextrin by means of any one of claims 1-8.

10. The application of the resistant starch according to claim 9 in the preparation of food.

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