Uniform fructan GEIN and application thereof

Abstract

The present application relates to a kind of uniform fructan GEIN and its application, belong to food processing technical field.The uniform fructan GEIN described in the present application has similar spherical structure of starch granule, can significantly improve the pasting properties of Gastrodia elata starch.And, the uniform fructan GEIN described in the present application also has the effect of improving intestinal flora, can promote the growth of beneficial bacteria in intestinal tract, while not cause postprandial hyperglycemia.The discovery and isolation of the uniform fructan GEIN described in the present application fill the research blank of the starch pasting regulation effect of Gastrodia elata polysaccharide, provide new active ingredient for the depth development of Gastrodia elata in the field of food and medicine, and have important significance for the development of Gastrodia elata food and health care product.

Description

Technical Field

[0001] This invention belongs to the field of food processing technology, specifically relating to a homogeneous fructan GEIN and its applications. Background Technology

[0002] The gelatinization characteristics of starch are a core control indicator in the food processing field. Key indicators such as gelatinization temperature, apparent viscosity, and thermal stability directly affect the adaptability of food processing technology, product texture, taste, and storage characteristics. Therefore, precise improvement of starch gelatinization characteristics is an important research direction for achieving process optimization and product quality improvement in the food processing field.

[0003] Currently, existing methods for improving starch gelatinization properties are mainly divided into two categories: chemical modification and physical modification. Chemical modification utilizes acids, alkalis, oxidants, or cross-linking agents to react with starch molecules, introducing functional groups to alter their functional properties. While this method provides direct improvement, it poses food safety risks due to chemical residues, and the use of some chemical reagents is strictly limited by food additive standards, restricting its applicability to various processing scenarios. Physical modification primarily alters the starch granule structure through mechanical force, heat treatment, irradiation, or ultrasound. While this method avoids chemical residue issues, it suffers from stringent process conditions, high costs associated with specialized equipment and energy consumption, and its modification effect is easily affected by the type of starch raw material, exhibiting poor batch-to-batch stability and hindering widespread application in various food processing scenarios. In summary, existing starch gelatinization improvement methods all have unavoidable application limitations. Therefore, finding a safe, stable solution that can fundamentally improve starch gelatinization properties has become an urgent priority. Summary of the Invention

[0004] The purpose of this invention is to address the current situation where starch gelatinization improvement relies on chemical and physical modification methods, and to provide a natural, safe solution that can significantly improve starch gelatinization properties while also regulating gut microbiota and not causing postprandial hyperglycemia. To this end, this invention provides a homogeneous fructan (GEIN) and its applications.

[0005] This invention provides a homogeneous fructan GEIN, the chemical structural formula of which is shown in Formula I; , Formula I.

[0006] Preferably, the molecular weight of the homogeneous fructan GEIN is 1.66 × 10⁻⁶. 3 Da; The molar ratio of monosaccharides in the homogeneous fructan GEIN is glucose:fructose = 1:7.

[0007] This invention also provides a method for preparing the homogeneous fructan GEIN described in the above technical solution, comprising the following steps: After defatting the tubers of Gastrodia elata with anhydrous ethanol or an ethanol aqueous solution with a volume concentration of ≥85%, they were subjected to water extraction and concentration in sequence to obtain a concentrated water extract. The aqueous extract concentrate was subjected to protein removal to obtain a crude polysaccharide concentrate from Gastrodia elata. The concentrated crude polysaccharide solution of Gastrodia elata was mixed with an aqueous ethanol solution or anhydrous ethanol with a concentration greater than 90%, and the volume concentration of ethanol in the system was adjusted to 30%. After standing, the mixture was centrifuged and the precipitate was discarded to obtain supernatant I. Mix the supernatant I with an ethanol aqueous solution with a volume concentration of 95%, adjust the volume concentration of ethanol in the system to 50%, let it stand, centrifuge, discard the precipitate, and obtain supernatant II; The supernatant II was mixed with an ethanol aqueous solution with a volume concentration of 95%, and the volume concentration of ethanol in the system was adjusted to 70%~90%. After standing, the mixture was centrifuged and the precipitate was collected to obtain a crude polysaccharide component containing homogeneous fructan GEIN. The crude polysaccharide component containing homogeneous fructan GEIN was redissolved in water and centrifuged to obtain the supernatant to be purified. The supernatant to be purified was subjected to ion exchange resin column chromatography, and the eluent was collected. According to the elution order, each eluent was sequentially labeled as Fr-1 to Fr-10. The ion exchange resin column chromatography used was a DEAE-52 ion exchange resin column; The ion exchange resin column chromatography uses water as the eluent. The eluents Fr-4 to Fr-10 were subjected to Sephadex G-75 and Sephacryl S-300 gel column chromatography with water as the eluent. The eluent with a purity of ≥99% was collected and concentrated to obtain homogeneous fructan GEIN.

[0008] The present invention also provides the application of the homogeneous fructan GEIN described in the above-mentioned technical solution and the homogeneous fructan GEIN prepared by the preparation method described in the above-mentioned technical solution in improving starch gelatinization characteristics or preparing foods and / or food additives that improve starch gelatinization.

[0009] Preferably, the starch comprises Gastrodia elata starch; The improvement of starch gelatinization properties includes any one or more of the following: ① to ④: ① Increase the gelatinization temperature of starch; ② Reduce the gelatinization enthalpy of starch; ③ Reduce the apparent viscosity of starch after gelatinization; ④ Improve the thermal stability of starch.

[0010] The present invention also provides the application of the homogeneous fructan GEIN described in the above technical solution and the homogeneous fructan GEIN prepared by the preparation method described in the above technical solution in regulating the intestinal flora of mammals or in preparing products that regulate the intestinal flora of mammals.

[0011] Preferably, the regulation of mammalian gut microbiota includes promoting beneficial bacteria in the gut. Limosilactobacillus , Muribaculum , Lactobacillus reuteri , Muribaculum intestinale and Lactobacillus johnsonii The growth of at least one of them.

[0012] The present invention also provides the application of the homogeneous fructan GEIN described in the above technical solution and the homogeneous fructan GEIN prepared by the preparation method described in the above technical solution in maintaining healthy blood glucose levels or in preparing products that maintain healthy blood glucose levels.

[0013] The present invention also provides a composition that improves starch gelatinization, regulates intestinal flora and maintains healthy blood sugar levels, comprising homogeneous fructan GEIN and gastrodia starch GES as described in the above technical solution; The mass ratio of the homogeneous fructan GEIN to Gastrodia elata starch GES is (1~3):(1~3).

[0014] The present invention also provides the application of the composition described above in the preparation of food and / or health products that are both food and medicine and have any one or more functions as shown in ① to ③; ① Improve starch gelatinization properties; ② Regulate the gut microbiota of mammals; ③ Maintain healthy blood sugar levels.

[0015] Beneficial effects: This invention provides a homogeneous fructan GEIN, the chemical structural formula of which is shown in Formula I. The homogeneous fructan GEIN of this invention has a spherical structure similar to starch granules, which can significantly improve the gelatinization characteristics of Gastrodia elata starch. It can increase the gelatinization temperature, decrease the gelatinization enthalpy, and reduce the apparent viscosity of gelatinized Gastrodia elata starch. Simultaneously, it can inhibit the swelling and decomposition of Gastrodia elata starch, improve its thermal stability, maintain the integrity of some Gastrodia elata starch granules, and effectively improve the defects of easy gelatinization and poor system stability of Gastrodia elata starch components.

[0016] Furthermore, the homogeneous fructan GEIN described in this invention also has the effect of improving intestinal flora and can promote... Limosilactobacillus , Muribaculum , Lactobacillus reuteri , Muribaculum intestinale and Lactobacillus johnsoniiIt promotes the growth of beneficial bacteria without causing a rise in postprandial blood glucose. The discovery and isolation of the homogeneous fructan GEIN described in this invention fills the research gap on the regulatory effect of Gastrodia elata polysaccharides on starch gelatinization, providing a new active ingredient for the in-depth development of Gastrodia elata in the field of food and medicine homology, and is of great significance to the development of Gastrodia elata food and health products. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0018] Figure 1 HPLC chromatograms were constructed from the main components of the crude polysaccharide fractions GEC-30, GEC-50, GEC-70, and GEC-90; among them, Figure 1 In the figure, A represents the HPLC chromatogram of the main components of the crude polysaccharide fraction GEC-30. Figure 1 In the figure, B represents the HPLC chromatogram of the main components of the crude polysaccharide fraction GEC-30. Figure 1 C in the figure represents the main component composition of the crude polysaccharide fraction GEC-30 in the HPLC chromatogram. Figure 1 In the figure, D represents the HPLC chromatogram of the main components of the crude polysaccharide fraction GEC-30. Figure 1 E in the figure represents the standard curve. Figure 2 Chemical composition analysis of crude polysaccharide components GEC-30, GEC-50, GEC-70 and GEC-90; Figure 3 The HPLC chromatograms of the obtained homogeneous GES and GEIN are shown. Figure 4 The NMR spectra of the obtained homogeneous GES and GEIN structures are shown; among them, Figure 4 In the diagram, A represents the NMR spectrum of a homogeneous GES structure characterization. Figure 4 In the diagram, B represents the NMR spectrum of a homogeneous GEIN structure. Figure 5 The diagram shows the effects of gelatinization on the obtained uniform GES and GEIN; where, Figure 5 In the figure, A is a bar chart showing the changes in the gelatinized apparent viscosity of GES and the GES / GEIN mixture at different temperatures. Figure 5 In the figure, B represents the differential scanning calorimetry (DSC) analysis curves of the GES and GES / GEIN mixed systems. Figure 5 C in the figure represents the Fourier transform infrared (FT-IR) spectra of natural GES, gelatinized GES, and GES / GEIN mixed systems. Figure 5 D in the diagram represents the X-ray diffraction pattern of natural GES, gelatinized GES, and a GES / GEIN mixed system. Figure 6 The images show in vitro digestion of artificial gastric juice in homogeneous GES, GEIN, and GES / GEIN mixed systems; among them, Figure 6 In the figure, A represents the HPLC differential chromatograms of GES and GEIN / GES mixed systems at different digestion times in artificial gastric juice (SGF). Figure 6 B in the figure represents the trend of peak area of ​​GES and GEIN / GES mixed system in artificial gastric juice (SGF) with digestion time. Figure 7 The image shows the in vitro digestion analysis of artificial intestinal fluid in homogeneous GES, GEIN, and GEIN / GES mixed systems; among them, Figure 7 In the figure, A represents the HPLC differential chromatograms of GES and GEIN / GES mixed systems at different digestion times in artificial intestinal fluid (SIF). Figure 7 B in the figure represents the trend of peak area of ​​GES and GEIN / GES mixed system in artificial intestinal fluid (SIF) with digestion time. Figure 8 This is a graph showing the analysis of postprandial blood glucose metabolism and gut microbiota regulatory activity in mice using homogeneous GES, GEIN, and GEIN / GES mixed systems; among them... Figure 8 Figure A shows the postprandial blood glucose concentration changes in mice 180 minutes after gavage administration of GES, GEIN, and a GEIN / GES mixture. Figure 8 In the figure, B represents the ACE index box plot of the gut microbiota of mice in the normal control group, pseudo-sterile model group, GES group, and GEIN / GES group. Figure 8 C in the figure represents the stacked bar chart of the relative abundance of species at the phylum level in the gut microbiota of the mice in the above groups; Figure 9 The distribution of gut microbiota in mice after treatment with homogeneous GES and GEIN / GES mixed system; Figure 10 Image of a Gastrodia elata and ham mooncake made with GES, GEIN, and Gastrodia elata residue. Detailed Implementation

[0019] This invention provides a homogeneous fructan GEIN, the chemical structural formula of which is shown in Formula I; , Formula I.

[0020] In one embodiment, the homogeneous fructan GEIN of the present invention has a molecular weight of 1.66 × 10⁻⁶. 3 Da; The molar ratio of monosaccharides in the homogeneous fructan GEIN is glucose:fructose = 1:7. As one embodiment, in Formula I of this invention, m is 1 or 2; n is 7~9.

[0021] In one embodiment, the homogeneous fructan GEIN of this invention has a spherical structure similar to starch granules, which can significantly improve the gelatinization characteristics of Gastrodia elata starch. It can increase the gelatinization temperature, decrease the gelatinization enthalpy, and reduce the apparent viscosity of gelatinized Gastrodia elata starch. Simultaneously, it can inhibit the swelling and decomposition of Gastrodia elata starch, improve its thermal stability, and maintain the integrity of some Gastrodia elata starch granules, effectively improving the defects of easy gelatinization and poor system stability of Gastrodia elata starch components. In another embodiment, the discovery and isolation of the homogeneous fructan GEIN of this invention fills the research gap on the regulatory effect of Gastrodia elata polysaccharides on starch gelatinization, providing a new active ingredient for the in-depth development of Gastrodia elata in the field of food and medicine homology, and is of great significance for the development of Gastrodia elata foods and health products.

[0022] This invention also provides a method for preparing homogeneous fructan GEIN as described above, comprising the following steps: After defatting the tubers of Gastrodia elata with anhydrous ethanol or an ethanol aqueous solution with a volume concentration of ≥85%, they were subjected to water extraction and concentration in sequence to obtain a concentrated water extract. The aqueous extract concentrate was subjected to protein removal to obtain a crude polysaccharide concentrate from Gastrodia elata. The concentrated crude polysaccharide solution of Gastrodia elata was mixed with an aqueous ethanol solution or anhydrous ethanol with a concentration greater than 90%, and the volume concentration of ethanol in the system was adjusted to 30%. After standing, the mixture was centrifuged and the precipitate was discarded to obtain supernatant I. Mix the supernatant I with an ethanol aqueous solution with a volume concentration of 95%, adjust the volume concentration of ethanol in the system to 50%, let it stand, centrifuge, discard the precipitate, and obtain supernatant II; The supernatant II was mixed with an ethanol aqueous solution with a volume concentration of 95%, and the volume concentration of ethanol in the system was adjusted to 70%~90%. After standing, the mixture was centrifuged and the precipitate was collected to obtain a crude polysaccharide component containing homogeneous fructan GEIN. The crude polysaccharide component containing homogeneous fructan GEIN was redissolved in water and centrifuged to obtain the supernatant to be purified. The supernatant to be purified was subjected to ion exchange resin column chromatography, and the eluent was collected. According to the elution order, each eluent was sequentially labeled as Fr-1 to Fr-10. The ion exchange resin column chromatography used was a DEAE-52 ion exchange resin column; The ion exchange resin column chromatography uses water as the eluent. The eluents Fr-4 to Fr-10 were subjected to Sephadex G-75 and Sephacryl S-300 gel column chromatography with water as the eluent. The eluent with a purity of ≥99% was collected and concentrated to obtain homogeneous fructan GEIN.

[0023] In this invention, gastrodia tuber is defatted with anhydrous ethanol or an ethanol aqueous solution with a volume concentration of ≥85%, and then subjected to water extraction and concentration to obtain a concentrated water extract.

[0024] In one embodiment, the *Gastrodia elata* tuber of the present invention can be a fresh *Gastrodia elata* tuber. In one embodiment, the *Gastrodia elata* tuber is cut into small pieces before ethanol defatting. In one embodiment, the aqueous ethanol solution of the present invention comprises an 85% (v / v) aqueous ethanol solution. In another embodiment, the aqueous ethanol solution of the present invention comprises a 95% (v / v) aqueous ethanol solution. In one embodiment, the ethanol defatting is performed three times. In one embodiment, the *Gastrodia elata* tuber, after ethanol defatting, is placed at room temperature and ventilated until no alcohol odor remains. In one embodiment, the water extraction operation of the present invention includes boiling water extraction. In one embodiment, the water extraction time of the water extraction operation of the present invention is 1-4 hours. In another embodiment, the water extraction time of the water extraction operation of the present invention is 2 hours. In one embodiment, the water extraction operation of the present invention is repeated 1-4 times. In another embodiment, the water extraction operation of the present invention is repeated twice. In one embodiment, after the water extraction operation of the present invention is completed, the mixture is filtered, and the filtrates are combined for concentration. In one embodiment, the concentration operation of the present invention includes vacuum concentration. In one embodiment, the concentration operation of the present invention is carried out at 50-70°C. In another embodiment, the concentration operation described in this invention is carried out at 65°C.

[0025] After obtaining the aqueous extract concentrate, the present invention removes proteins from the aqueous extract concentrate to obtain a crude polysaccharide concentrate from Gastrodia elata.

[0026] In one embodiment, the protein removal operation of the present invention includes the Sevage method for protein removal. In one embodiment, the present invention uses a mixture of chloroform and n-butanol for the protein removal operation. In one embodiment, the volume ratio of chloroform to n-butanol in the mixture is 4:1. In one embodiment, the protein removal operation is repeated 2 to 5 times. In another embodiment, the protein removal operation is repeated 3 times. In one embodiment, after the protein removal operation, the present invention performs concentration, the concentration operation including vacuum concentration, the concentration operation being carried out at 65°C.

[0027] After obtaining the concentrated crude polysaccharide solution of Gastrodia elata, the present invention mixes the concentrated crude polysaccharide solution of Gastrodia elata with an ethanol aqueous solution with a volume concentration of 95%, adjusts the volume concentration of ethanol in the system to 30%, lets it stand, centrifuges, discards the precipitate, and obtains supernatant I.

[0028] In one embodiment, after adjusting the volume concentration of ethanol in the system to 30%, the mixture is left to stand overnight, and then centrifuged at 3500 rpm for 20 min. In another embodiment, when the volume concentration of ethanol in the system is adjusted to 30%, the precipitate obtained by centrifugation can be reconstituted with water to obtain a GEC-30 crude polysaccharide solution. In another embodiment, the precipitate of GEC-30 crude polysaccharide obtained by centrifugation can be fully reconstituted with hot water at a temperature above 80°C. In another embodiment, the yield of GEC-30 crude polysaccharide is 19.01%, the total sugar content is 87.38%, and the total protein content is 0.82%. In yet another embodiment, the molecular weight of the main component of the GEC-30 crude polysaccharide is approximately 5.20 × 10⁻⁶. 7 Da represents the high molecular weight polysaccharide component. In one embodiment, the monosaccharide composition of the crude GEC-30 polysaccharide of this invention is predominantly glucose, accounting for 99.39%, with only a small amount of rhamnose, accounting for 0.61%. In another embodiment, the crude GEC-30 polysaccharide of this invention contains a large amount of amylopectin components, but no fructose components, and no enrichment of the target fructan GEIN. In yet another embodiment, the crude GEC-30 polysaccharide of this invention can be purified by subsequent column chromatography to obtain Gastrodia elata homogeneous starch polysaccharide GES.

[0029] As one embodiment, the purification process of the crude polysaccharide GEC-30 of the present invention is as follows: GEC-30 crude polysaccharide is dissolved in water and centrifuged to obtain the supernatant. The supernatant is loaded onto a DEAE-52 ion exchange chromatography column and eluted sequentially with distilled water and 0.5 mol / L NaCl solution. The water elution fractions are combined, dialyzed, concentrated, and freeze-dried to obtain the GEC-30-1 fraction. The GEC-30-1 fraction is subjected to Sephadex G-75 and Sephacryl S-300 gel column chromatography to obtain homogeneous starch polysaccharide GES.

[0030] As one embodiment, the GES derived from the crude polysaccharide GEC-30 of this invention has a molecular weight of 3.53 × 10⁻⁶. 7 Da is a type B starch composed of single glucose molecules, with the chemical structural formula shown in Formula II. As one embodiment, the GES described in this invention can be used in the study of the gelatinization characteristics of Gastrodia elata starch and in the processing of Gastrodia elata foods. As one embodiment, the GES derived from GEC-30 crude polysaccharide described in this invention can be mixed with the target fructan GEIN at a mass ratio of 1:1 to regulate starch gelatinization characteristics, reduce starch digestion rate, and stabilize postprandial blood glucose. As one embodiment, the GES derived from GEC-30 crude polysaccharide described in this invention can be used as a raw material in the preparation of Gastrodia elata foods, including but not limited to the processing and production of Gastrodia elata ham mooncakes.

[0031] After obtaining supernatant I, the present invention mixes supernatant I with an ethanol aqueous solution with an ethanol volume concentration of 95%, adjusts the ethanol volume concentration in the system to 50%, lets it stand, centrifuges, discards the precipitate, and obtains supernatant II.

[0032] In one embodiment, after adjusting the volume concentration of ethanol in the system to 50%, the mixture is left to stand overnight, and then centrifuged at 3500 rpm for 20 min. In another embodiment, when the volume concentration of ethanol in the system is adjusted to 50%, the precipitate obtained by centrifugation can be reconstituted with water to obtain a GEC-50 crude polysaccharide solution. In yet another embodiment, the GEC-50 crude polysaccharide obtained by centrifugation can be fully reconstituted by adding hot water above 80°C.

[0033] In one embodiment, the yield of the crude polysaccharide GEC-50 of the present invention is 11.01%, the total sugar content is 85.37%, and the total protein content is 1.58%. In another embodiment, the molecular weight of the main component of the crude polysaccharide GEC-50 of the present invention is approximately 6.38 × 10⁻⁶. 6 Da represents the high molecular weight polysaccharide component. In one embodiment, the GEC-50 crude polysaccharide of this invention is predominantly composed of glucose, accounting for 92.77%, and also contains small amounts of mannose, ribose, galactose, and arabinose, with relative molar ratios of 4.21%, 1.56%, 0.35%, and 1.11%, respectively. In another embodiment, the GEC-50 crude polysaccharide of this invention contains starch components but no fructose components, and there is no enrichment of the target fructan GEIN.

[0034] As one embodiment, the crude polysaccharide GEC-50 of the present invention can be purified by subsequent column chromatography to obtain homogeneous starch polysaccharide GES from Gastrodia elata. As one embodiment, the purification process of the crude polysaccharide GEC-50 of the present invention is as follows: GEC-50 crude polysaccharide is dissolved in water, centrifuged, and the supernatant is collected. The supernatant is loaded onto a DEAE-52 ion exchange column and eluted sequentially with distilled water and 0.5 mol / L NaCl solution. The water-eluted fractions are combined, dialyzed, concentrated, and freeze-dried to obtain fraction GEC-50-1. Fraction GEC-50-1 is then subjected to Sephadex G-75 and Sephacryl S-300 gel column chromatography to obtain homogeneous starch polysaccharide GES.

[0035] As one embodiment, the GES derived from the crude polysaccharide GEC-50 of this invention has a molecular weight of 3.53 × 10⁻⁶. 7Da is a type B starch composed of single glucose molecules, with the chemical structural formula shown in Formula II. As one embodiment, the GES described in this invention can be used in the study of the gelatinization characteristics of Gastrodia elata starch and in the processing of Gastrodia elata foods. As one embodiment, the GES derived from GEC-50 crude polysaccharide described in this invention can be mixed with the target fructan GEIN at a mass ratio of 1:1 to regulate starch gelatinization characteristics, reduce starch digestion rate, and stabilize postprandial blood glucose. As one embodiment, the GES derived from GEC-50 crude polysaccharide described in this invention can be used as a raw material in the preparation of Gastrodia elata foods, including but not limited to the processing and production of Gastrodia elata ham mooncakes.

[0036] After obtaining supernatant II, the present invention mixes supernatant II with an ethanol aqueous solution with a volume concentration of 95%, adjusts the volume concentration of ethanol in the system to 70%~90%, centrifuges after standing, collects the precipitate, and obtains crude polysaccharide component containing homogeneous fructan GEIN.

[0037] In one embodiment, when mixing the supernatant II with the ethanol-water solution, the ethanol-water solution with a volume concentration of 95% is added using a slow and fast stirring method to avoid uneven polysaccharide separation caused by excessively high local ethanol concentrations. In another embodiment, adjusting the volume concentration of ethanol in the system to 70%~90% can be done in steps, sequentially adjusting the ethanol volume concentration to 70% and then 90%, collecting the precipitates at the corresponding concentrations, and then combining them to obtain a crude polysaccharide component containing uniform fructan GEIN. In yet another embodiment, when adjusting the ethanol volume concentration in the system stepwise, the volume concentration of ethanol is first adjusted to 70%, allowed to stand, and then centrifuged. The precipitate at this concentration is collected to obtain GEC-70 crude polysaccharide, and the supernatant after centrifugation is retained for later use. In one embodiment, when adjusting the ethanol volume concentration in the system stepwise according to the present invention, after collecting the supernatant at a 70% ethanol concentration, an ethanol aqueous solution with a 95% ethanol volume concentration is added to the supernatant to adjust the ethanol volume concentration in the system to 90%. After standing, the mixture is centrifuged, and the precipitate at this concentration is collected to obtain GEC-90 crude polysaccharide. In another embodiment, the crude polysaccharide component containing homogeneous fructan GEIN according to the present invention can be obtained by combining GEC-70 crude polysaccharide and GEC-90 crude polysaccharide. In another embodiment, adjusting the ethanol volume concentration in the system to 70%~90% according to the present invention can be done in a one-step manner, directly adjusting the ethanol volume concentration in the system to 90%, standing, centrifuging, and collecting the precipitate to obtain the crude polysaccharide component containing homogeneous fructan GEIN. In yet another embodiment, after adjusting the ethanol volume concentration in the system to the target value according to the present invention, the mixture is first allowed to stand overnight before centrifugation. In one embodiment, the centrifugation parameters of the present invention are: centrifugation at 3500 rpm for 20 min, followed by separation and collection of the precipitate and supernatant after centrifugation. In another embodiment, the yield of the crude polysaccharide GEC-70 of the present invention is 8.40%, the total sugar content is 76.36%, and the total protein content is 3.61%. In another embodiment, the yield of the crude polysaccharide GEC-90 of the present invention is 9.27%, the total sugar content is 76.63%, and the total protein content is 2.91%. In yet another embodiment, the molecular weight distribution range of the main components of the crude polysaccharide GEC-70 of the present invention is 3.78 × 10⁻⁶. 4 ~ 1.22×10 3 The molecular weight distribution range of the main components of Da,GEC-90 crude polysaccharide is 4.51×10⁻⁶. 4 ~ 8.66×10 2In one embodiment, the monosaccharide composition of the GEC-70 crude polysaccharide of the present invention is mainly glucose, while also containing small amounts of mannose, rhamnose, and fructose; the monosaccharide composition of the GEC-90 crude polysaccharide includes mannose, rhamnose, glucose, galacturonic acid, and fructose. In one embodiment, both the GEC-70 and GEC-90 crude polysaccharides of the present invention turn red after a Seliwanoff colorimetric reaction, and both contain fructose. In one embodiment, the GEC-70 crude polysaccharide of the present invention turns purplish-red after an iodine colorimetric reaction, and contains a small amount of starch; the GEC-90 crude polysaccharide shows no color change after an iodine colorimetric reaction, and does not contain starch, making it the optimal enrichment material for the target homogeneous fructan GEIN.

[0038] After obtaining the crude polysaccharide component containing homogeneous fructan GEIN, the component is reconstituted in water and centrifuged to obtain a supernatant for purification. As one embodiment, when reconstituted in water, hot water above 80°C is used for thorough reconstitution; 3 mL of water is added for every 400 mg of crude polysaccharide component, and the mixture is stirred until completely dissolved. As another embodiment, after reconstitution, insoluble impurities are removed by centrifugation to obtain a supernatant for purification. As another embodiment, the centrifugation parameters are 3500 rpm for 5 min. As another embodiment, the crude polysaccharide component containing homogeneous fructan GEIN includes at least one of GEC-70 and GEC-90 crude polysaccharides. As another embodiment, when the crude polysaccharide component containing homogeneous fructan GEIN is GEC-90 crude polysaccharide, it is the optimal enrichment material for the target homogeneous fructan GEIN, free from starchy impurities.

[0039] After obtaining the supernatant to be purified, the present invention performs ion exchange resin column chromatography on the supernatant to be purified, collects the eluent, and labels each eluent as Fr-1 to Fr-10 in the order of elution; the ion exchange resin column chromatography uses a DEAE-52 ion exchange resin column; the ion exchange resin column chromatography uses water as the eluent; the eluents Fr-4 to Fr-10 are subjected to Sephadex G-75 and Sephacryl S-300 gel column chromatography, using water as the eluent, and the eluent with a purity ≥99% is collected, concentrated, and then homogeneous fructan GEIN is obtained.

[0040] In one embodiment, the Sephadex G-75 and Sephacryl S-300 gel column chromatography of the present invention includes further Sephadex G-75 and Sephacryl S-300 gel column chromatography on the chromatographic product obtained from the Sephadex G-75 and Sephacryl S-300 gel column chromatography. In one embodiment, the further Sephadex G-75 and Sephacryl S-300 gel column chromatography is performed more than twice. In one embodiment, the gel column chromatography operation continues until an eluent with a purity ≥99% is collected. In one embodiment, the purity of the eluent is detected by HPLC-ELSD during the gel column chromatography operation. In one embodiment, ethylene is detected by HPLC-ELSD every other eluent tube. In one embodiment, the DEAE-52 ion exchange resin column of the present invention has a specification of 80 × 4 cm. In one embodiment, the homogeneous fructan GEIN obtained by the purification of the present invention has a molecular weight of 1.66 × 10⁻⁶. 3 Da, the monosaccharide composition has a molar ratio of glucose:fructose = 1:7, and the main chain structure is →3)-α-D-Glcp-(1→ and →1)-β-D-Fruf-(2→, the chemical structural formula is shown in Formula I. As one embodiment, the crude polysaccharide component containing homogeneous fructan GEIN described in this invention can be used in the processing of Gastrodia elata food, or after purification, it can be used in the preparation of functional foods and intestinal flora regulators. As one embodiment, the homogeneous fructan GEIN purified in this invention can be mixed with Gastrodia elata starch GES at a mass ratio of (1~3):(1~3) to regulate starch gelatinization characteristics, reduce starch digestion rate, stabilize postprandial blood glucose, and regulate intestinal flora. As another embodiment, the homogeneous fructan GEIN purified in this invention can be mixed with Gastrodia elata starch GES at a mass ratio of 1:1.

[0041] The present invention also provides the application of the homogeneous fructan GEIN described in the above technical solution and the homogeneous fructan GEIN prepared by the preparation method described in the above technical solution in improving starch gelatinization properties or preparing foods and / or food additives that improve starch gelatinization.

[0042] In one embodiment, the starch of the present invention includes Gastrodia elata starch; The improvement of starch gelatinization properties includes any one or more of the following: ① to ④: ① Increase the gelatinization temperature of starch; ② Reduce the gelatinization enthalpy of starch; ③ Reduce the apparent viscosity of starch after gelatinization; ④ Improve the thermal stability of starch.

[0043] In one embodiment, the homogeneous fructan GEIN of the present invention can interact with GES during gelatinization, ultimately reducing the apparent viscosity of GES and increasing the gelatinization temperature. In another embodiment, the homogeneous fructan GEIN of the present invention can significantly reduce the gelatinization enthalpy ΔHg of GES. In yet another embodiment, the homogeneous fructan GEIN of the present invention contains a large number of hydroxyl groups that can interact with the hydroxyl groups in GES through hydrogen bonding, limiting the stretching vibrations of the hydroxyl groups in GES, ultimately leading to a red shift of the hydroxyl absorption peak in GES. In yet another embodiment, the homogeneous fructan GEIN of the present invention can inhibit the expansion and decomposition of GES, improve the thermal stability of GES, maintain the good integrity of some starch granules in GES, and ultimately enhance the diffraction intensity of the corresponding region of GES.

[0044] The present invention also provides the application of the homogeneous fructan GEIN described in the above technical solution and the homogeneous fructan GEIN prepared by the preparation method described in the above technical solution in regulating the intestinal flora of mammals or in preparing products that regulate the intestinal flora of mammals.

[0045] As one embodiment, the regulation of mammalian gut microbiota according to the present invention includes promoting beneficial bacteria in the gut. Limosilactobacillus , Muribaculum , Lactobacillus reuteri , Muribaculum intestinale and Lactobacillus johnsonii The growth of at least one of the following. As one embodiment, the homogeneous fructan GEIN of the present invention can significantly improve the diversity of the intestinal microbiota in PGF mice, transforming its microbial structure towards that of normal healthy mice. As one embodiment, after using the homogeneous fructan GEIN of the present invention, the intestinal bacterial community structure of PGF mice undergoes significant changes, with a significant increase in the proportion of Firmicutes and Bacteroidetes, similar to the microbial composition of normal mice.

[0046] The present invention also provides the application of the homogeneous fructan GEIN described in the above technical solution and the homogeneous fructan GEIN prepared by the preparation method described in the above technical solution in maintaining healthy blood glucose levels or in preparing products that maintain healthy blood glucose levels.

[0047] In one embodiment, the homogeneous fructan GEIN described in this invention is rapidly digested and degraded in simulated gastric fluid. In another embodiment, after the homogeneous fructan GEIN of this invention is mixed with GES to form a GEIN / GES system, GEIN can still be rapidly digested and degraded, but the digestion process of GES is significantly inhibited. In another embodiment, the homogeneous fructan GEIN of this invention is not further digested and degraded in simulated intestinal fluid. In another embodiment, oral administration of the homogeneous fructan GEIN of this invention does not cause fluctuations in postprandial blood glucose levels in mice; within 3 hours after administration, the blood glucose level in mice remains consistently around 5.2 mmol / L. In another embodiment, after combined administration of the homogeneous fructan GEIN of this invention and GES, the blood glucose level in mice only increases slightly, from 5.0 mmol / L to 7.5 mmol / L, and then quickly recovers; approximately 1 hour later, the postprandial blood glucose level in mice essentially returns to normal. In yet another embodiment, the homogeneous fructan GEIN of this invention can reduce the digestibility and gelatinization rate of GES, thereby inhibiting the sharp increase in postprandial blood glucose caused by GES.

[0048] The present invention also provides a composition that improves starch gelatinization, regulates intestinal flora and maintains healthy blood sugar levels, comprising homogeneous fructan GEIN and gastrodia starch GES as described in the above technical solution; The mass ratio of the homogeneous fructan GEIN to Gastrodia elata starch GES is 1:1.

[0049] The present invention also provides the application of the composition described above in the preparation of food and / or health products that are both food and medicine and have any one or more functions as shown in ① to ③; ① Improve starch gelatinization properties; ② Regulate the gut microbiota of mammals; ③ Maintain healthy blood sugar levels.

[0050] In one embodiment, the starch described in this invention includes Gastrodia elata starch. In another embodiment, the food product of this invention that is both food and medicine includes Gastrodia elata and ham mooncakes.

[0051] To further illustrate the present invention, a homogeneous fructan GEIN and its applications provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0052] Example 1 Extraction and separation of crude polysaccharides from Gastrodia elata 1. Raw material processing Take 4.35 kg of fresh Gastrodia elata tubers, cut them into small pieces, add 95% ethanol aqueous solution for extraction three times, filter, and place the filter residue at room temperature and ventilate until there is no alcohol smell.

[0053] 2. Extraction and Concentration Then, extract with boiling water by adding 20 L of distilled water and extracting for 2 hours, repeating the extraction twice. Filter, combine the filtrates, and concentrate under reduced pressure at 65℃ to 7-10 L to obtain the concentrated aqueous extract of Gastrodia elata.

[0054] 3. Sevage method for protein removal The above-mentioned water extract concentrate was subjected to protein removal treatment using the Sevage method. The reagent used was a mixture of chloroform and n-butanol (volume ratio 4:1), and the treatment was performed three times. After the treatment, the concentrate was concentrated under reduced pressure at 65°C to finally obtain 3L of Gastrodia elata crude polysaccharide concentrate.

[0055] 4. Fractional alcohol precipitation Add a 95% ethanol aqueous solution to the above-mentioned Gastrodia elata crude polysaccharide concentrate, stir slowly and quickly to adjust the ethanol volume fraction in the system to 30%, let it stand overnight, centrifuge (3500 rpm, 20 min), collect the precipitate precipitated at this concentration, add 1 L of hot water (above 80℃) to reconstitute, and concentrate to about 500 mL to obtain GEC-30 crude polysaccharide. Keep the supernatant after centrifugation for later use.

[0056] Add 95% ethanol aqueous solution to the supernatant, stirring slowly and quickly, and adjust the ethanol volume fraction of the system to 50%, 70%, and 90% in turn. After each adjustment to the target ethanol concentration, let stand overnight, centrifuge (3500 rpm, 20 min), and collect the precipitates precipitated at each concentration. The precipitates are reconstituted with 1 L of hot water (above 80℃) and concentrated to about 500 mL to obtain crude polysaccharides GEC-50, GEC-70, and GEC-90 in turn.

[0057] 5. Detection of the yield and molecular weight distribution of crude polysaccharide components According to statistics, a total of 190.124 g of 30% crude polysaccharide (GEC-30) from Gastrodia elata, 110.104 g of 50% crude polysaccharide (GEC-50) from Gastrodia elata, 84.011 g of 70% crude polysaccharide (GEC-70) from Gastrodia elata, and 92.711 g of 90% crude polysaccharide (GEC-90) from Gastrodia elata were obtained, with corresponding yields of 19.01%, 11.01%, 8.40%, and 9.27%, respectively.

[0058] Polysaccharide standards were analyzed using HPLC-ELSD, and standard curves were plotted. The four crude polysaccharides GEC-30, GEC-50, GEC-70, and GEC-90 were analyzed, and the molecular weight distribution range of each component was calculated. Their principal component composition was analyzed, and HPLC chromatograms and standard curves of the principal components of GEC-30, GEC-50, GEC-70, and GEC-90 were obtained as follows: Figure 1As shown in the figure. The test results show that the molecular weight of the main component of GEC-30 is approximately 5.20 × 10⁻⁶. 7 Da, GEC-50 principal component molecular weight ≈ 6.38 × 10 6 Da, GEC-70 principal component molecular weight ≈ 3.78 × 10 4 ~ 1.22 ×10 3 Da, GEC-90 principal component molecular weight ≈ 4.51 × 10 4 ~ 8.66 ×10 2 Da.

[0059] Example 2 Chemical composition analysis of Gastrodia elata crude polysaccharide 1. Determination of total sugar content The total sugar content of the four crude polysaccharides obtained in Example 1 was determined according to the method specified in the 2020 edition of the Pharmacopoeia, using the phenol-sulfuric acid colorimetric method.

[0060] Preparation of standard curve: Glucose was weighed and prepared into aqueous solutions of 0, 0.01, 0.02, 0.03, 0.04, and 0.05 mg / mL, respectively. 2 mL of each solution was taken, and 1 mL of 5% phenol and 5 mL of concentrated sulfuric acid were added sequentially. After mixing, the solutions were heated in boiling water for 20 min, cooled, and 50 μL of each solution was plotted in a 96-well plate. The absorbance was measured at 488 nm. A standard curve was plotted with glucose concentration (μg / mL) on the x-axis and absorbance on the y-axis.

[0061] Sample testing and results: Four crude polysaccharide samples were prepared into 0.05 mg / mL solutions, and the absorbance was measured by repeating the above operation. The total sugar content of GEC-30, GEC-50, GEC-70, and GEC-90 was calculated according to the standard curve. The calculated total sugar contents of GEC-30, GEC-50, GEC-70, and GEC-90 were 87.38%, 85.37%, 76.36%, and 76.63%, respectively.

[0062] 2. Determination of total protein content The total protein content of four crude polysaccharides was determined according to the method specified in the 2020 edition of the Pharmacopoeia, using the Coomassie brilliant blue method.

[0063] Four crude polysaccharide samples were prepared into 1 mg / mL solutions. Following the kit instructions (purchased from Nanjing Jiancheng Company), the samples and working solutions were added to 96-well plates and incubated at 37℃ for 30 min. The absorbance of each well was read at 562 nm using a microplate reader, and the total protein content of GEC-30, GEC-50, GEC-70, and GEC-90 samples was calculated. The final total protein contents of GEC-30, GEC-50, GEC-70, and GEC-90 were 0.82%, 1.58%, 3.61%, and 2.91%, respectively.

[0064] 3. Monosaccharide composition analysis The monosaccharide composition of GEC-30, GEC-50, GEC-70, and GEC-90 was analyzed using the PMP pre-column derivatization method. The results are as follows: Figure 2 As shown in Figure A, the polysaccharides in Gastrodia elata tubers are mainly composed of glucose. Among them, GEC-30 contains a large amount of Glc (99.39%) and a small amount of Rha (0.61%); GEC-50 is composed of five monosaccharides: Man, Rib, Gal, Glc, and Ara, with relative molar ratios of 4.21%, 1.56%, 0.35%, 92.77%, and 1.11%, respectively; GEC-70 is mainly composed of Glc, with small amounts of Man, Rha, Glc, and Fru; and GEC-90 is composed of four monosaccharides: Man, Rha, Glc, GalA, and Fru.

[0065] 4. Qualitative analysis of fructose The fructose content in four crude polysaccharides (GECS) was qualitatively analyzed using the Seliwanoff colorimetric reaction.

[0066] Weigh 1.5 g of resorcinol and dissolve it in 10 mL of concentrated hydrochloric acid. Dilute it with 20 mL of distilled water to prepare Seliwanoff's reagent. Take another 2 mL of the reagent and add it to a 5 mg / mL sample aqueous solution. Heat the solution in a boiling water bath and observe the color change. Analyze the fructose content in the samples (GEC-30, GEC-50, GEC-70 and GEC-90).

[0067] The results are as follows Figure 2 As shown in B, GEC-70 and GEC-90 turn red after reacting with Seliwanoff's reagent, with GEC-90 being a deeper color, indicating that GEC-70 and GEC-90 contain fructose, and that GEC-90 has a higher fructose content. In contrast, GEC-30 and GEC-50 do not change color after the reaction, indicating that GEC-30 and GEC-50 do not contain fructose.

[0068] 5. Qualitative analysis of starch The content of starch polysaccharides in four crude polysaccharides was qualitatively analyzed by iodine colorimetric reaction.

[0069] Preparation of iodine solution: Take 0.22 g of potassium iodide, add water to make up to 50 mL, then add 0.11 g of elemental iodine, dissolve completely and set aside; Sample preparation: Prepare 5 mg / mL aqueous solutions of the four crude polysaccharides separately, and take 4 mL of each solution and place them in 10 mL test tubes; Add 200 μL of iodine solution to the sample solution, shake well and observe the color change (amylose appears blue, amylopectin appears purplish-red), and analyze the content of starch polysaccharides in the sample.

[0070] The results are as follows Figure 2 As shown in C, GEC-30 turns blue-purple after reacting with iodine reagent, indicating that GEC-30 contains a large amount of amylopectin; GEC-50 and GEC-70 turn purple-red after reacting, indicating that they also contain starch; GEC-90 does not change color after reacting, indicating that GEC-90 does not contain starch.

[0071] Example 3 Purification and structural identification of crude polysaccharide from Gastrodia elata The crude polysaccharide components GEC-30 and GEC-90 obtained in Example 1 were systematically purified to prepare homogeneous starch polysaccharide GES and homogeneous fructan GEIN. The structures of the target products were confirmed. The specific operations and experimental results are as follows: 1. Sample preparation and crude purification by DEAE-52 ion exchange column chromatography Sample preparation: Take 400 mg of each GEC-30 and GEC-90 sample, add 3 mL of water to dissolve them completely, centrifuge at 3500 rpm for 5 min, and take the supernatant for later use.

[0072] Column chromatography procedure: The supernatant was loaded onto a DEAE-52 column (80×4 cm) and eluted stepwise with distilled water and 0.5 mol / L NaCl solution. Each eluted fraction was collected in segments using an automatic sample collector.

[0073] Component processing: HPLC-ELSD was used to detect and combine identical components. After dialysis, reduced pressure concentration, and vacuum freeze-drying, three components were obtained from the GEC-30 sample: GEC-30-1 (eluents were labeled Fr-1 to Fr-15 in elution order, and eluents Fr-1 to Fr-10 were collected), GEC-30-2, and GEC-30-3. Similarly, three components were obtained from the GEC-90 sample: GEC-90-1 (eluents were labeled Fr-1 to Fr-10 in elution order, and eluents Fr-4 to Fr-10 were collected), GEC-90-2, and GEC-90-3. Among them, No. 1 is the water eluent obtained by rinsing with distilled water, and Nos. 2 and 3 are the salt eluent obtained by elution with 0.5 mol / L NaCl solution. The content of the salt eluent is small, so it was not further purified. Only the water eluents GEC-30-1 and GEC-90-1 were purified.

[0074] 2. Gel column chromatography purification The water-eluted fractions GEC-30-1 and GEC-90-1 were repeatedly purified by Sephadex G-75 and Sephacryl S-300 gel chromatography (the chromatographic products were then purified again by the same gel column chromatography). Water was used as the eluent, and the eluent was collected in fractions using an automated sampler. Each fraction was analyzed by HPLC-ELSD, and the corresponding target fractions were combined (eluent with a purity ≥99% was collected). Finally, homogeneous starch polysaccharide GES was obtained from GEC-30-1, and homogeneous fructan GEIN was obtained from GEC-90-1. The HPLC chromatograms of homogeneous GES and GEIN are shown below. Figure 3 As shown.

[0075] 3. Structural identification of the target product The purified GES and GEIN were structurally identified by NMR spectroscopy analysis, and the NMR spectra of homogeneous GES and GEIN structures were obtained as follows: Figure 4 As shown.

[0076] according to Figure 4 The results show that GES is a starch polysaccharide composed of a single glucose molecule, with a molecular weight of 3.53 × 10⁻⁶. 7 Da, the chemical structural formula is shown in Formula II; Formula II.

[0077] The main chain structure of GEIN is →3)-α-D-Glcp-(1→ and →1)-β-D-Fruf-(2→), the molar ratio of monosaccharides is glucose:fructose = 1:7, and the molecular weight is 1.66 × 10⁻⁶. 3 The chemical structure of Da is shown in Formula I.

[0078] Example 4 The effect of GEIN on GES gelatinization 1. Gelatinization characteristics determination The GES obtained in Example 3 was mixed with GEIN at a mass ratio of 1:1 to prepare a GES / GEIN mixed sample.

[0079] The gelatinization characteristics of the sample (GES / GEIN) were determined using a rotational viscometer (NDJ-5S).

[0080] The apparent viscosity of the sample suspension before and after gelatinization at different total concentrations (0.2, 0.1, 0.050, 0.0250, 0.0125 g / mL) and at different temperatures (10, 20, 30, 40, 50, 52, 55, 57, 60, 70, 80, 90, 97℃) was measured and the relevant data were recorded.

[0081] The measurement results are as follows Figure 5 As shown in A, the apparent viscosity of gelatinized GES is greater than that before gelatinization, and its gelatinized apparent viscosity first increases and then decreases with increasing temperature. After adding GEIN, the apparent viscosity of GES decreases significantly, and the temperature at which the maximum apparent viscosity occurs increases, indicating that an interaction occurs between GES and GEIN during the gelatinization process, ultimately reducing the apparent viscosity of GES and increasing the gelatinization temperature.

[0082] 2. Gelatinization heat performance determination The effect of GEIN on the gelatinization heat properties of GES was determined using differential scanning calorimetry (DSC). GES (2.5 mg) was added to an aluminum crucible, followed by 10 μL of GEIN solution (0.1 mg / mL). The crucible was sealed and equilibrated at room temperature for 12 h. The sample was programmed to be heated from 20 °C to 120 °C at a rate of 10 °C / min. Using a sealed empty aluminum crucible as a reference, the initial temperature (T0), peak temperature (TP), final temperature (TC), and gelatinization enthalpy ΔH of the sample were recorded.

[0083] The measurement results are as follows Figure 5 As shown in B in the figure, GES undergoes gelatinization during heating, corresponding to the transformation of starch from an ordered structure to an amorphous structure. After the addition of GEIN, the onset temperature (To), peak temperature (Tp), and termination temperature (Tc) of GES all increase, the gelatinization process is delayed, and compared with pure GES, its gelatinization enthalpy ΔHg is significantly reduced after the addition of GEIN.

[0084] 3. Measurement of interaction forces The interaction force between GES and GEIN was analyzed using Fourier transform infrared (FT-IR) spectroscopy. Under an infrared drying lamp, 1.0 mg of sample was mixed with 10 mg of KBr, ground, and pressed into a pellet. The sample was then measured using FT-IR spectroscopy, with a scanning range of 4000–400 cm⁻¹. -1 The resolution is 4 cm. -1 .

[0085] The measurement results are as follows Figure 5 As shown in C. Compared with pure GES, no new infrared absorption peak appeared after the addition of GEIN, indicating that no covalent bonding occurred between GEIN and GES; however, after the addition of GEIN, a new infrared absorption peak appeared at 3400 cm⁻¹. -1 The intensity and wavenumber position of the hydroxyl absorption peak at 3432.05 cm⁻¹ changed; the hydroxyl absorption peak red-shifted, and the wavenumber changed from 3432.05 cm⁻¹. -1 Moved to 3434.45cm -1 The results indicate that GEIN and GES molecules contain a large number of hydroxyl groups, and hydrogen bonding interactions occur between them, which restricts the stretching vibration of the hydroxyl groups, ultimately leading to a red shift of the hydroxyl absorption peak.

[0086] 4. Determination of crystal structure characteristics Take 1.0 g of each prepared sample and test it using a rotating target X-ray polycrystalline diffractometer. The scanning area is 5~35°, the scanning speed is 8° / min, and the sampling step size is 0.02°.

[0087] The measurement results are as follows Figure 5 As shown in Figure D, GES exhibits distinct diffraction peaks at 5.70°, 15.62°, 17.02°, 22.36°, and 24.06°, indicating a typical type B crystal structure. After gelatinization, the diffraction peaks of GES become passivated, and the diffraction intensity decreases, indicating that gelatinization almost completely destroys the crystal structure of GES. After the addition of GEIN, although some diffraction peaks of the gelatinized GES become passivated, the diffraction intensity around 17° increases significantly. Since around 17° is a characteristic diffraction peak of amylose, this result indicates that GEIN can inhibit the expansion and decomposition of GES, improve the thermal stability of GES, maintain the good integrity of some starch granules, and ultimately enhance the diffraction intensity in the corresponding region.

[0088] Example 5 In vitro digestion analysis of homogeneous GES, GEIN and mixed GEIN / GES 1. In vitro simulated gastrointestinal digestion test Take 10 mL of artificial gastric fluid (SGF) or artificial intestinal fluid (SIF) and mix thoroughly with 200 mg of GES, GEIN, or a 1:1 mixture of GEIN and GES (GEIN / GES). Incubate the mixture in a magnetically stirred water bath at 37 °C for 5 h. Take samples at 0, 1, 2, 3, and 4 h during the incubation process for subsequent detection and analysis.

[0089] Sample processing: After sampling, pepsin or trypsin in the system is inactivated, the sample is filtered through a 0.45 μm filter membrane, and then detected and analyzed by liquid chromatography.

[0090] 2. Test Results The results of the digestive behavior analysis of samples in artificial gastric fluid (SGF) and artificial intestinal fluid (SIF) are as follows: Figure 6 and Figure 7 As shown, under the action of SGF, the retention time of GES in liquid chromatography (5.0 min) remained unchanged, but the peak area increased with the incubation time. This phenomenon is due to the gradual degradation of the originally difficult-to-detect highly polymerized starch into detectable water-soluble starch. The retention time of GEIN in liquid chromatography changed from about 8.7 min to about 10.5 min, indicating that GEIN is rapidly digested and degraded in simulated gastric fluid. After the two are mixed to form the GEIN / GES system, GEIN can still be rapidly digested and degraded, but at the same time, the digestion process of GES is significantly inhibited. In contrast, under the treatment of simulated intestinal fluid, the chromatographic retention times of GES, GEIN, and the GEIN / GES mixture remained basically unchanged, indicating that none of the three samples were further digested and degraded in simulated intestinal fluid.

[0091] Example 6 Analysis of postprandial blood glucose and gut microbiota regulatory activity in mice with homogeneous GES, GEIN, and mixed GEIN / GES The two types of Gastrodia elata polysaccharides and their mixture prepared in Example 1 above were evaluated for their postprandial blood glucose and intestinal flora regulation activities according to the following experimental methods.

[0092] 1. Animal Experiment Design Experimental animals: C57BL / 6 mice were randomly divided into 4 groups of 5 mice each: normal control group (saline group), GES group, GEIN group, and GEIN / GES mixed group (GEIN and GES were mixed in a 1:1 ratio).

[0093] Feeding and Adaptation: The experimental mice were acclimatized in an SPF-grade environment for 7 days before the formal experiment. The normal control group was given physiological saline by gavage, while the other groups were given the corresponding polysaccharide samples by gavage. All animals were fasted for 1 day before blood glucose testing.

[0094] Statement: The animal experiments have been approved by the Research Ethics Committee of Kunming Institute of Botany (Approval No. Kib202503016) and have been conducted in accordance with the Chinese Guidelines for the Care and Use of Laboratory Animals.

[0095] 2. Postprandial blood glucose detection in mice Administration method: Mice were administered the drug by gavage according to the experimental animal operation specifications. The dosage was 10 mL / kg, of which the concentration of GES and GEIN was 3.0 g / kg, and the concentration of the GEIN / GES mixture was 6.0 g / kg.

[0096] Detection method: Blood was collected from the tail vein at 0, 0.5, 1, 1.5, 2, 2.5 and 3 h after administration, and the blood glucose concentration of mice was read and recorded using blood glucose test strips provided with a blood glucose meter.

[0097] 3. Detection of gut microbiota regulatory activity Establishment of pseudo-germ-free mouse (PGF) model: To prepare the pseudo-germ-free mouse model, mice were administered vancomycin (10 mg / kg) and neomycin sulfate (10 mg / kg) by gavage daily for 7 consecutive days; all antibiotics used were purchased from Aladdin (Shanghai, China).

[0098] 16S ribosomal RNA (rRNA) sequencing analysis: Two weeks after administration of the drug by gavage, fecal samples were collected from the mice and immediately frozen at -80°C.

[0099] Fecal DNA was extracted from the samples using a DNA extraction kit. The V3-V4 region of bacterial 16S rRNA was amplified using 341F (SEQ ID NO.1: 5'-CCTACGGGGGGCWGCAG-3') and 806R (SEQ ID NO.2: 5'-GGACTACHVGGGTATCTAAT-3') barcode primers. The processing and analysis of sequencing data were performed on the Biomarker Cloud Platform (China).

[0100] To obtain high-quality, clean reads, the original sequence was filtered using FASTP (version 0.18.0) with the following filtering rules: Sequences containing more than 10% unknown nucleotides and those with a quality value >20 accounting for less than 50% of the total were removed. Clean sequences were merged into original tags using FLASH (version 1.2.11), with a minimum overlap length of 10 base pairs (bp) and a mismatch error rate of 2%. Operational taxonomic units (OTUs) were clustered using Uparse version 7.1 with a cutoff of 97% similarity, and chimeric sequences were identified and removed. The minimum classification annotation threshold for OTUs was set to a consensus assignment score ≥70%. The Kruskal-Wallis test was used to assess the significance of α-diversity differences, with a p-value <0.05 considered statistically significant.

[0101] 4. Experimental Results and Analysis The results of postprandial glucose metabolism analysis and gut microbiota regulation analysis in mice treated with uniform GES, GEIN, and mixed GEIN / GES are as follows: Figure 8 As shown; the distribution of gut microbiota in mice after treatment with uniform GES and mixed GEIN / GES is as follows. Figure 9 As shown, oral administration of GES to mice for 30 minutes caused a rapid increase in blood glucose levels, from 5.3 mmol / L to 12.0 mmol / L, followed by a slow decline, with blood glucose returning to normal levels after approximately 3 hours. Oral administration of GEIN did not cause fluctuations in postprandial blood glucose levels in mice; blood glucose levels remained around 5.2 mmol / L for 3 hours after administration. After combined administration of GEIN and GES, blood glucose levels in mice only increased slightly, from 5.0 mmol / L to 7.5 mmol / L, before rapidly recovering, with postprandial blood glucose levels essentially returning to normal levels after approximately 1 hour. These results indicate that the presence of GEIN can reduce the digestibility and gelatinization rate of GES, thereby inhibiting the sharp increase in postprandial blood glucose induced by GES.

[0102] By comparing the gut microbiota α diversity and community composition of mice treated with GEIN / GES for two weeks, blank control (NC) mice, and pseudo-germ-free (PGF) mice, further findings were made: ACE index results showed that GEIN / GES administration significantly improved the diversity of gut microbiota in PGF mice, transforming their microbiota structure into that of normal healthy mice. The results of phylum-level microbial community composition showed that after two weeks of treatment, PGF mice underwent significant changes in bacterial community structure, with a significant increase in the proportion of Firmicutes and Bacteroidetes, which was similar to the microbial community composition of normal mice. The genus-level difference analysis showed that, compared with the PGF group, the genera with the most significant change in abundance were: Limosilactobacillus (Lactobacillus citrica) Muribaculum(Lactobacillus murineis) Lactobacillus reuteri (Lactobacillus reuteri) Muribaculum intestinale (Lactobacillus murineis) and Lactobacillus johnsonii (Lactobacillus johnsonii). The above-mentioned Lactobacillus strains have been widely reported as functional probiotics, possessing various beneficial physiological functions such as pathogen inhibition, intestinal barrier enhancement, and immune system regulation.

[0103] In summary, the carbohydrates in Gastrodia elata are mainly composed of starch-based glucans and non-starch-based fructans. The presence of GEIN can not only improve the gelatinization properties of starch, but also further affect the in vitro digestion process of starch, reduce postprandial blood glucose levels, and at the same time have a probiotic effect on the intestines.

[0104] Example 7 Making Gastrodia elata Mooncakes Using the Gastrodia elata starch (GES) and Gastrodia elata fructan (GEIN) prepared in Example 3, as well as the Gastrodia elata residue after extracting Gastrodia elata polysaccharides, as core raw materials, Gastrodia elata and ham mooncakes were prepared. Figure 10 As shown.

[0105] 1. Mooncake Ingredient Recipe Main ingredients for the oily dough: 100g all-purpose flour, 60g cake flour, 100g Gastrodia elata starch (GES), 60g Gastrodia elata fructan (GEIN), 120g lard, 40g sugar, and 100g water.

[0106] Filling ingredients: 300g minced ham (lean to fat ratio 3:7), Gastrodia elata residue (solid part after polysaccharide extraction), 20g light soy sauce, 10g cooking wine, 10g sugar.

[0107] 2. Production Steps Filling preparation: Add light soy sauce, cooking wine, white sugar and gastrodia elata residue to the minced ham, stir thoroughly until the filling becomes firm, seal and refrigerate to set, then set aside.

[0108] Wrapping and shaping: Divide the oil dough into small dough balls of 30g each, roll them into round wrappers, wrap 40g of shaped meat filling inside, press and flatten with the seam side down to get the mooncake blank.

[0109] Baking and shaping: Place the raw mooncake dough in the oven and bake at 200℃ for 30 minutes. Turn it over halfway through baking to prevent it from burning. After baking, you will get the finished Gastrodia elata and ham mooncake.

[0110] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A homogeneous fructan GEIN, characterized in that, The chemical structural formula of the homogeneous fructan GEIN is shown in Formula I. Equation I.

2. The homogeneous fructan GEIN according to claim 1, characterized in that, The molecular weight of the homogeneous fructan GEIN is 1.66 × 10⁻⁶. 3 Da; The molar ratio of monosaccharides in the homogeneous fructan GEIN is glucose:fructose = 1:

7.

3. The method for preparing homogeneous fructan GEIN as described in claim 1, characterized in that, Includes the following steps: After defatting the tubers of Gastrodia elata with anhydrous ethanol or an ethanol aqueous solution with a volume concentration of ≥85%, they were subjected to water extraction and concentration in sequence to obtain a concentrated water extract. The aqueous extract concentrate was subjected to protein removal to obtain a crude polysaccharide concentrate from Gastrodia elata. The concentrated crude polysaccharide solution of Gastrodia elata was mixed with an aqueous ethanol solution or anhydrous ethanol with a concentration greater than 90%, and the volume concentration of ethanol in the system was adjusted to 30%. After standing, the mixture was centrifuged and the precipitate was discarded to obtain supernatant I. Mix the supernatant I with an ethanol aqueous solution with a volume concentration of 95%, adjust the volume concentration of ethanol in the system to 50%, let it stand, centrifuge, discard the precipitate, and obtain supernatant II; The supernatant II was mixed with an ethanol aqueous solution with a volume concentration of 95%, and the volume concentration of ethanol in the system was adjusted to 70%~90%. After standing, the mixture was centrifuged and the precipitate was collected to obtain a crude polysaccharide component containing homogeneous fructan GEIN. The crude polysaccharide component containing homogeneous fructan GEIN was redissolved in water and centrifuged to obtain the supernatant to be purified. The supernatant to be purified was subjected to ion exchange resin column chromatography, and the eluent was collected. According to the elution order, each eluent was sequentially labeled as Fr-1 to Fr-10. The ion exchange resin column chromatography used was a DEAE-52 ion exchange resin column; The ion exchange resin column chromatography uses water as the eluent. The eluents Fr-4 to Fr-10 were subjected to Sephadex G-75 and Sephacryl S-300 gel column chromatography with water as the eluent. The eluent with a purity of ≥99% was collected and concentrated to obtain homogeneous fructan GEIN.

4. The use of the homogeneous fructan GEIN according to claim 1 or 2, or the homogeneous fructan GEIN prepared by the preparation method according to claim 3, in improving starch gelatinization properties or in preparing foods and / or food additives that improve starch gelatinization.

5. The application according to claim 4, characterized in that, The starch includes Gastrodia elata starch; The improvement of starch gelatinization properties includes any one or more of the following: ① to ④: ① Increase the gelatinization temperature of starch; ② Reduce the gelatinization enthalpy of starch; ③ Reduce the apparent viscosity of starch after gelatinization; ④ Improve the thermal stability of starch.

6. The use of the homogeneous fructan GEIN according to claim 1 or 2, or the homogeneous fructan GEIN prepared by the preparation method according to claim 3, in regulating the intestinal flora of mammals or in preparing products that regulate the intestinal flora of mammals.

7. The application according to claim 6, characterized in that, The regulation of mammalian gut microbiota includes promoting beneficial bacteria in the gut. Limosilactobacillus , Muribaculum , Lactobacillus reuteri , Muribaculum intestinale and Lactobacillus johnsonii The growth of at least one of them.

8. The use of the homogeneous fructan GEIN according to claim 1 or 2, or the homogeneous fructan GEIN prepared by the preparation method according to claim 3, in maintaining healthy blood glucose levels or in preparing products that maintain healthy blood glucose levels.

9. A composition that simultaneously improves starch gelatinization, regulates gut microbiota, and maintains healthy blood glucose levels, characterized in that, Includes the homogeneous fructan GEIN as described in claim 1 and Gastrodia elata starch GES; The mass ratio of the homogeneous fructan GEIN to Gastrodia elata starch GES is (1~3):(1~3).

10. The use of the composition according to claim 9 in the preparation of food products and / or health products having any one or more of the functions shown in ① to ③; ① Improve starch gelatinization properties; ② Regulate the gut microbiota of mammals; ③ Maintain healthy blood sugar levels.

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