A method for preparing pueraria low oligosaccharide by multi-step sequential enzymatic hydrolysis combined with multi-stage membrane purification

Abstract

This invention discloses a multi-step sequential enzymatic hydrolysis combined with multi-stage membrane purification method for preparing kudzu oligosaccharides, relating to the field of functional food processing technology. The preparation method includes the following steps: adding kudzu powder to water to prepare a starch slurry, then liquefying it using α-amylase, followed by enzymatic hydrolysis using debranching enzymes and glycoside hydrolases to obtain an enzymatic hydrolysate; performing membrane separation on the enzymatic hydrolysate to obtain a fraction with a molecular weight higher than 1 kDa and lower than 5 kDa, followed by drying to obtain the kudzu oligosaccharides; the debranching enzymes are pullulanase and isoamylase; the glycoside hydrolases are β-glucosidase or α-transglucosidase. This preparation method can effectively increase the proportion of the DP3-15 fraction of kudzu oligosaccharides, achieving a product purity of over 91% and a total yield exceeding 22%.

Description

Technical Field

[0001] This invention relates to the field of functional food processing technology, and in particular to a method for preparing kudzu oligosaccharides through multi-step sequential enzymatic hydrolysis combined with multi-stage membrane purification. Background Technology

[0002] Kudzu root is rich in starch, dietary fiber, flavonoids, and various bioactive substances. Its starch structure can be directionally degraded into oligosaccharides (such as isomaltooligosaccharides and xylooligosaccharides). These substances possess multiple physiological functions, including regulating intestinal flora balance, promoting the proliferation of beneficial bacteria, improving intestinal metabolism, enhancing immunity, and lowering blood sugar and lipids. They are currently a research hotspot for prebiotic functional factors. At present, high-purity, high-activity kudzu root oligosaccharides with controllable polymerization degree have become a key development direction.

[0003] Currently, the industrial preparation of kudzu oligosaccharides is mainly based on acid hydrolysis and single enzymatic hydrolysis. Acid hydrolysis is simple to operate and low in cost, but the reaction conditions are harsh; the high-temperature, acidic environment easily leads to the degradation and inactivation of heat-sensitive active ingredients such as flavonoids and puerarin in kudzu. It also suffers from problems such as uncontrollable hydrolysis, a wide distribution of product polymerization degree, high content of monosaccharides and miscellaneous sugars, and a large amount of by-products, seriously affecting product purity and physiological activity. While single enzymatic hydrolysis has milder reaction conditions, its enzyme system is limited and the degradation pathway is narrow, making it difficult to fully dismantle the branched structure of kudzu starch. This results in incomplete enzymatic hydrolysis, low conversion efficiency, low yield of target oligosaccharides, and poor product uniformity. Product purity is generally below 70%, failing to meet the quality requirements of high-end foods and functional products.

[0004] Existing technologies primarily focus on the simple extraction and preliminary separation of kudzu oligosaccharides, lacking precise control over the enzymatic hydrolysis process and the construction of efficient purification systems, thus failing to achieve directional control of the degree of oligosaccharide polymerization. Some processes still rely on traditional decolorizing and desalting materials such as activated carbon and diatomaceous earth, resulting in poor separation selectivity, low purification efficiency, and high risk of solvent residue, making it difficult to obtain high-purity, narrowly distributed target products. Furthermore, traditional processes suffer from long production cycles, high energy consumption, and low resource utilization, with byproducts not being effectively utilized, which does not align with the trends of green, efficient, energy-saving, and emission-reducing modern industrial production. Therefore, the industry urgently needs a highly efficient, precisely purified, controllable, and energy-saving kudzu oligosaccharide preparation technology to overcome existing process bottlenecks and improve product quality and industrial value. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing kudzu oligosaccharides through multi-step sequential enzymatic hydrolysis combined with multi-stage membrane purification, thereby solving the problems existing in the prior art. This preparation method can effectively increase the proportion of kudzu oligosaccharide components with a degree of polymerization (DP3-15), achieving a product purity of over 91% and a total yield exceeding 22%.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides a method for preparing kudzu oligosaccharides through multi-step sequential enzymatic hydrolysis combined with multi-stage membrane purification, comprising the following steps: Kudzu root powder is added to water to make a starch slurry. Then, it is liquefied using α-amylase and then hydrolyzed using debranching enzyme and glycoside hydrolase to obtain the hydrolysate. The enzymatic hydrolysate was subjected to membrane separation to obtain a fraction with a molecular weight higher than 1 kDa and lower than 5 kDa, which was then dried to obtain the kudzu oligosaccharide. The debranching enzyme is pullulanase and isoamylase; the glycoside hydrolase is β-glucosidase or α-transglucosidase.

[0007] Furthermore, the amount of α-amylase added is 0.1-0.3% of the mass of the kudzu root powder.

[0008] Furthermore, the liquefaction process is carried out at a pH of 5.5-6.0, a temperature of 90-95℃, and a time of 20-30 minutes.

[0009] Furthermore, the amount of pullulanase added is 0.1-0.2% of the mass of the kudzu root powder.

[0010] Furthermore, the amount of isoamylase added is 0.05-0.15% of the mass of the kudzu root powder.

[0011] Furthermore, the amount of glycoside hydrolase added is 0.05-0.15% of the mass of the kudzu root powder.

[0012] Furthermore, the enzymatic hydrolysis is performed at a temperature of 58-60℃, a pH of 5.5-6.0, and a time of 23-35 hours.

[0013] Furthermore, the membrane separation process includes microfiltration, ultrafiltration, and nanofiltration.

[0014] Furthermore, the microfiltration treatment uses a microfiltration membrane with a pore size of 0.2 μm; the ultrafiltration treatment uses an ultrafiltration membrane with a molecular weight cutoff of 5 kDa; and the nanofiltration treatment uses a nanofiltration membrane with a molecular weight cutoff of 1 kDa.

[0015] The present invention also provides a kudzu oligosaccharide prepared according to the above preparation method.

[0016] The present invention discloses the following technical effects: This invention achieves efficient preparation and precise separation of kudzu oligosaccharides through a multi-step sequential enzymatic hydrolysis and multi-stage membrane purification process. The complex enzyme system effectively degrades the branched structure of kudzu starch, directionally controlling the degree of polymerization of the product. The target component DP3-15 accounts for over 87%, the product purity exceeds 91%, and the total yield exceeds 22%, significantly superior to traditional acid hydrolysis and single enzymatic hydrolysis processes. Multi-stage membrane separation achieves efficient removal of impurities and precise enrichment of the target product, avoiding the use of organic solvents, making it environmentally friendly and eliminating residue risks. The process cycle is shortened by 40%, energy consumption is reduced by 25%, and it is suitable for continuous industrial production. The prepared oligosaccharide product has concentrated components and stable quality, while retaining the original active ingredients of kudzu. The by-product fiber residue can be further utilized for resource recovery, achieving efficient development of all components. The overall technology comprehensively improves product purity, conversion efficiency, production economy, and environmental friendliness. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 The HPLC chromatogram of the kudzu oligosaccharide product prepared in Example 1 is shown below. Figure 2 The HPLC chromatogram of the kudzu oligosaccharide product prepared in Example 2 is shown below. Figure 3 The image shows the HPLC chromatogram of the kudzu oligosaccharide product prepared in Example 3. Detailed Implementation

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included within the scope of this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0023] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0024] The formula for calculating the total yield of kudzu oligosaccharides used in the following examples is: Total yield (%) = mass of dried finished product (g) / mass of kudzu powder input (g) × 100%, wherein the moisture content of the finished product is determined in accordance with GB 5009.3-2016.

[0025] Purity analysis method: The total sugar in the sample was determined by the phenol-sulfuric acid method.

[0026] Determination of Degree of Polymerization (DUP): The DUP was determined by high-performance liquid chromatography (HPLC). Chromatographic conditions: Sugar-D column, acetonitrile-water (75:25) mobile phase, flow rate 1.0 mL / min, and differential refractive index detector. Purity was expressed as the percentage of oligosaccharide peak area to the total peak area. The DUP was determined by comparing each peak with a reference standard and calculated based on the peak area ratio.

[0027] Example 1 Pulverized kudzu root slices dried to a moisture content ≤8% were pulverized and passed through an 80-mesh sieve to obtain kudzu root powder, which was then sealed and stored for later use. 1 kg of kudzu root powder was taken and mixed with deionized water to prepare a starch slurry with a solid content of 30%, and the pH was adjusted to 5.5. 0.2% (by weight of the kudzu root powder) of α-amylase was added, and the mixture was stirred in a 95℃ water bath for 25 minutes. The resulting liquefied solution was rapidly cooled to 58℃, maintaining a pH of 5.5. The following enzyme preparations (based on the weight of the kudzu root powder) were added sequentially: pullulanase: 0.10%, isoamylase: 0.05%, and β-glucosidase: 0.05%. The enzymatic hydrolysis reaction was carried out at 58℃ with continuous gentle stirring for 23 hours. After the reaction was completed, the reaction solution was heated in a 90℃ water bath for 10 minutes to terminate enzyme activity, and then cooled to room temperature to obtain the enzymatic hydrolysate.

[0028] Multistage membrane separation and purification: The enzymatic hydrolysate was filtered through a 0.2 μm microfiltration membrane to remove suspended particles and large molecular impurities, yielding a clear filtrate. The microfiltration clarified solution was pumped into an ultrafiltration system with a molecular weight cutoff (MWCO) of 5 kDa at an operating pressure of 0.3 MPa, and the permeate was collected. The ultrafiltration permeate was then pumped into a nanofiltration system with a molecular weight cutoff (MWCO) of 1 kDa at an operating pressure of 1.0 MPa, and the nanofiltration retentate (rich in DP3-15 oligosaccharides) was collected.

[0029] The nanofiltration retentate was concentrated under reduced pressure at 60℃ and -0.09 MPa until the solid content was ≥50%. The concentrate was then spray-dried at an inlet air temperature of 180℃ and an outlet air temperature of 85℃ to obtain white kudzu oligosaccharide powder.

[0030] Results: 225 g of kudzu oligosaccharide powder was obtained. Testing showed the product purity was 91.2%, with DP3-15 comprising 87.13% of the total sugar content. Figure 1 The total yield was 22.5% (based on the mass of kudzu root powder input).

[0031] The degree of polymerization distribution of the kudzu oligosaccharide powder obtained in this embodiment was determined. Mass spectrometry (MS) was used to analyze the main chromatographic peaks, confirming that the main signals correspond to the oligosaccharide series ion peaks with a degree of polymerization (DP) of 3 to 15 (Table 1).

[0032] Table 1. Distribution of degree of polymerization in Example 1 (area normalization method) Figure 1 The image shows the HPLC chromatogram of the kudzu oligosaccharide product prepared according to Example 1. Figure 1 As shown, the target oligosaccharide components (degree of polymerization DP3-15) exhibit a concentrated cluster of chromatographic peaks. According to Table 1, DP3-15 accounts for 87.13% of the total, indicating that the product has high purity and a narrow degree of polymerization distribution. The earlier peaks on the left side of the figure represent monosaccharides and some disaccharides (DP1-2), while the trace signals on the right side represent components with a degree of polymerization greater than 15.

[0033] Example 2 Pulverized kudzu root slices dried to a moisture content ≤8% were pulverized and passed through an 80-mesh sieve to obtain kudzu root powder, which was then sealed and stored for later use. 1 kg of kudzu root powder was taken and mixed with deionized water to prepare a starch slurry with a solid content of 30%, and the pH was adjusted to 6.0. 0.25% (by weight of the kudzu root powder) of α-amylase was added, and the mixture was stirred in a 95℃ water bath for 20 minutes. The resulting liquefied solution was rapidly cooled to 60℃, maintaining the pH at 6.0. The following enzyme preparations (based on the weight of the kudzu root powder) were added sequentially: pullulanase: 0.15%, isoamylase: 0.10%, and α-transglucosidase: 0.10%, and the mixture was reacted at 60℃ for 28 hours. After the reaction was completed, the reaction solution was heated in a 90℃ water bath for 10 minutes to terminate enzyme activity, and then cooled to room temperature to obtain the enzymatic hydrolysate.

[0034] Multistage membrane separation and purification: The enzymatic hydrolysate was filtered through a 0.2 μm microfiltration membrane to remove suspended particles and large molecular impurities, yielding a clear filtrate. The microfiltration clarified solution was pumped into an ultrafiltration system with a molecular weight cutoff (MWCO) of 5 kDa at an operating pressure of 0.3 MPa, and the permeate was collected. The ultrafiltration permeate was then pumped into a nanofiltration system with a molecular weight cutoff (MWCO) of 1 kDa at an operating pressure of 1.0 MPa, and the nanofiltration retentate (rich in DP3-15 oligosaccharides) was collected.

[0035] The nanofiltration retentate was concentrated under reduced pressure at 60℃ and -0.09 MPa until the solid content was ≥50%. The concentrate was then spray-dried at an inlet air temperature of 180℃ and an outlet air temperature of 85℃ to obtain white kudzu oligosaccharide powder.

[0036] Results: 248 g of kudzu oligosaccharide powder was obtained. The product purity was 93.5%, the DP3-15 component accounted for 88.06%, and the total yield was 24.8%.

[0037] The degree of polymerization distribution of the kudzu oligosaccharide powder obtained in this embodiment was determined. Mass spectrometry (MS) was used to analyze the main chromatographic peaks, confirming that the main signals correspond to the oligosaccharide series ion peaks with a degree of polymerization (DP) of 3 to 15 (Table 2).

[0038] Table 2. Distribution of degree of polymerization in Example 2 (area normalization method) Figure 2 The image shows the HPLC chromatogram of the kudzu oligosaccharide product prepared according to Example 2 of the present invention. Figure 2 As shown, the target oligosaccharide components (degree of polymerization DP3-15) exhibit a concentrated cluster of chromatographic peaks. According to Table 2, DP3-15 accounts for 88.06% of the total, indicating that the product has high purity and a narrow degree of polymerization distribution. The earlier peaks on the left side of the figure represent monosaccharides and some disaccharides (DP1-2), while the trace signals on the right side represent components with a degree of polymerization greater than 15.

[0039] Example 3 Pulverized kudzu root slices dried to a moisture content ≤8% were pulverized and passed through an 80-mesh sieve to obtain kudzu root powder, which was then sealed and stored for later use. 1 kg of kudzu root powder was taken and mixed with deionized water to prepare a starch slurry with a solid content of 30%, and the pH was adjusted to 6.0. 0.3% (by weight of the kudzu root powder) of α-amylase was added, and the mixture was stirred and reacted in a 90℃ water bath for 30 minutes. The resulting liquefied solution was rapidly cooled to 60℃, maintaining the pH at 6.0. The following enzyme preparations (based on the weight of the kudzu root powder) were added sequentially: pullulanase: 0.20%, isoamylase: 0.15%, and β-glucosidase: 0.15%, and the reaction was carried out at 60℃ for 35 hours. After the reaction was completed, the reaction solution was heated in a 90℃ water bath for 10 minutes to terminate enzyme activity, and then cooled to room temperature to obtain the enzymatic hydrolysate.

[0040] Multistage membrane separation and purification: The enzymatic hydrolysate was filtered through a 0.2 μm microfiltration membrane to remove suspended particles and large molecular impurities, yielding a clear filtrate. The microfiltration clarified solution was pumped into an ultrafiltration system with a molecular weight cutoff (MWCO) of 5 kDa at an operating pressure of 0.3 MPa, and the permeate was collected. The ultrafiltration permeate was then pumped into a nanofiltration system with a molecular weight cutoff (MWCO) of 1 kDa at an operating pressure of 1.0 MPa, and the nanofiltration retentate (rich in DP3-15 oligosaccharides) was collected.

[0041] The nanofiltration retentate was concentrated under reduced pressure at 60℃ and -0.09 MPa until the solid content was ≥50%. The concentrate was then spray-dried at an inlet air temperature of 180℃ and an outlet air temperature of 85℃ to obtain white kudzu oligosaccharide powder.

[0042] Results: 238 g of kudzu oligosaccharide powder was obtained. The product purity was 92.5%, the DP3-15 component accounted for 87.55%, and the total yield was 23.8%.

[0043] The degree of polymerization distribution of the kudzu oligosaccharide powder obtained in this embodiment was determined. Mass spectrometry (MS) was used to analyze the main chromatographic peaks, confirming that the main signals correspond to the oligosaccharide series ion peaks with a degree of polymerization (DP) of 3 to 15 (Table 3).

[0044] Table 3. Distribution of degree of polymerization in Example 3 (area normalization method) Figure 3 The figure shows the HPLC chromatogram of the kudzu oligosaccharide product prepared according to Example 3 of the present invention. As shown in the figure, the target oligosaccharide component (degree of polymerization DP3-15) exhibits a concentrated group of chromatographic peaks. HPLC analysis shows that the peak area of ​​the DP3-15 component in the kudzu oligosaccharide prepared by the present invention accounts for 87.55% of the total peak area (Table 3), indicating that the product has high purity and a narrow degree of polymerization distribution.

[0045] The above examples demonstrate that the "multi-step sequential enzymatic hydrolysis combined with multi-stage membrane purification" method provided by this invention can stably prepare kudzu oligosaccharide products with high purity (≥90%) and high target component content (DP3-15 content ≥85%) from kudzu root powder as raw material. Moreover, the process has good reproducibility and is suitable for large-scale production.

[0046] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing kudzu oligosaccharides through multi-step sequential enzymatic hydrolysis combined with multi-stage membrane purification, characterized in that, Includes the following steps: Kudzu root powder is added to water to make a starch slurry. Then, it is liquefied using α-amylase and then hydrolyzed using debranching enzyme and glycoside hydrolase to obtain the hydrolysate. The enzymatic hydrolysate was subjected to membrane separation to obtain a fraction with a molecular weight higher than 1 kDa and lower than 5 kDa, which was then dried to obtain the kudzu oligosaccharide. The debranching enzyme is pullulanase and isoamylase; the glycoside hydrolase is β-glucosidase or α-transglucosidase.

2. The preparation method according to claim 1, characterized in that, The amount of α-amylase added is 0.1-0.3% of the mass of the kudzu root powder.

3. The preparation method according to claim 1, characterized in that, The liquefaction process is carried out at a pH of 5.5-6.0, a temperature of 90-95℃, and a time of 20-30 minutes.

4. The preparation method according to claim 1, characterized in that, The amount of pullulanase added is 0.1-0.2% of the mass of the kudzu root powder.

5. The preparation method according to claim 1, characterized in that, The amount of isoamylase added is 0.05-0.15% of the mass of the kudzu root powder.

6. The preparation method according to claim 1, characterized in that, The amount of glycoside hydrolase added is 0.05-0.15% of the mass of the kudzu root powder.

7. The preparation method according to claim 1, characterized in that, The enzymatic hydrolysis was performed at a temperature of 58-60℃, a pH of 5.5-6.0, and a time of 23-35 hours.

8. The preparation method according to claim 1, characterized in that, The membrane separation process includes microfiltration, ultrafiltration, and nanofiltration.

9. The preparation method according to claim 8, characterized in that, The microfiltration process uses a microfiltration membrane with a pore size of 0.2 μm; the ultrafiltration process uses an ultrafiltration membrane with a molecular weight cutoff of 5 kDa; and the nanofiltration process uses a nanofiltration membrane with a molecular weight cutoff of 1 kDa.

10. A kudzu oligosaccharide prepared by the preparation method according to any one of claims 1-9.

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