Tartary buckwheat starch-ginger exosome compound with high digestion resistance as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of functional food processing, and particularly relates to a tartary buckwheat starch-fresh ginger exosome compound with high digestion resistance and a preparation method and application thereof.According to the compound, fresh ginger exosome is used for increasing the content of resistant starch in tartary buckwheat for the first time, and the compound is prepared by jointly incubating gelatinized tartary buckwheat starch and the fresh ginger exosome; the obtained tartary buckwheat starch-ginger exosome compound not only has a more ordered structure and high digestion resistance, but also can effectively regulate blood glucose indexes and improve lipid metabolism disorder.

Description

Technical Field

[0001] The present application belongs to the field of functional food processing technology, and specifically relates to a tartary buckwheat starch-ginger exosome complex with high digestibility, a preparation method thereof, and an application thereof. Background Art

[0002] Type 2 diabetes mellitus (T2DM), a chronic metabolic disease characterized by insulin resistance and secondary hyperglycemia, accounts for more than 90% of diabetes cases and has become a major global health challenge. Developing a nutritionally and functional diet to regulate blood sugar homeostasis through daily diet and effectively delay the progression of T2DM has become a research hotspot in the interdisciplinary field of food science and medicine in recent years.

[0003] Tartary buckwheat (Fagopyrum tataricum (L) Gaertn.), a medicinal and edible plant in the Polygonaceae family, is rich in nutrients such as starch, amino acids, and flavonoids. With growing interest in natural, pollution-free therapeutic crops, demand for buckwheat in the healthcare and medical markets is increasing. The starch content of buckwheat accounts for 63.5% to 72.5% of the total dry matter, with a higher amylose content than other cereals. This results in a high content of naturally resistant starch, which accounts for a significant proportion of the total starch content. In scientific research and applied practice, to further increase the resistant starch content of buckwheat, traditional physical or chemical modification methods, such as wet heat treatment, autoclave, and extrusion, are generally used to increase the resistant starch (RS) content. However, these methods have limitations that have hindered their industrial production. For example, in the second paragraph of Section 2.1.1 of "Preparation and Properties of Granular Resistant Starch" by Li Suling et al., it is noted that moisture content must be carefully controlled during wet heat treatment. In the second paragraph of Section 3 of their paper "Effects of Debranching and Repeated Heat-Wet Treatment on the Content and Physicochemical Properties of Resistant Starch in Buckwheat," Fu Ziping et al. disclose that autoclaving produces Resistant Starch (RS) with a poor taste and a hard texture, while extrusion may over-shear the buckwheat starch, converting RS into digestible starch. Single-method modification treatments are generally ineffective, and while combining several methods can improve the yield and quality of resistant starch, they are cumbersome, time-consuming, and costly, and the resulting functional health-promoting ingredients are relatively limited.

[0004] Exosomes are small, nanoscale vesicles secreted naturally by both plant and animal cells. Composed of lipids, proteins, and nucleic acids, they play a crucial role as intermediaries in intercellular communication. Plant exosomes (PELNs) offer unique advantages in terms of raw material accessibility, scalability of production processes, and biocompatibility, laying the technical foundation for their industrial application in precision medicine and biomaterials. Ginger-derived exosomes (GELNs), one of the most extensively studied plant exosome samples, have been shown to improve inflammatory bowel disease and effectively deliver drugs, making them a promising natural material. However, existing research has primarily focused on the drug delivery applications of ginger exosomes, while their combination with buckwheat starch to enhance digestibility and synergistically enhance their efficacy in diabetes prevention and treatment has not been reported.

[0005] Therefore, a new method for preparing buckwheat starch complex is needed to increase the resistant starch content of buckwheat starch through ginger exosomes, providing a new technical route for increasing the resistant starch content in buckwheat starch. Summary of the Invention

[0006] The problem that this application aims to solve is to provide a buckwheat starch-ginger exosome complex with high digestibility. This complex uses ginger exosomes for the first time to increase the content of resistant starch in buckwheat. It is made by incubating gelatinized buckwheat starch with ginger exosomes. The obtained buckwheat starch-ginger exosome complex not only has a more ordered structure and high digestibility, but also can effectively regulate blood sugar indicators and improve lipid metabolism disorders.

[0007] In order to solve the above technical problems, this application adopts the following technical solutions: In one aspect, the present application provides a tartary buckwheat starch-ginger exosome complex with high digestion resistance, wherein the tartary buckwheat starch-ginger exosome complex is formed by incubating gelatinized tartary buckwheat starch and ginger exosomes and performing ultrasonic treatment.

[0008] In the above technical solution, the gelatinized buckwheat starch and ginger exosomes self-assemble and reconstruct the gelatinized macromolecular starch structure and functional exosomes through hydrogen bonds, van der Waals forces, and hydrophobic effects to form a complex, thereby increasing the resistant starch content; with the assistance of ultrasonic treatment, the ginger exosomes can further interact with buckwheat starch through hydrogen bonds, increasing the surface porosity and short-range ordered structure of the starch.

[0009] Furthermore, the weight ratio of the tartary buckwheat starch to the ginger exosomes is 50:1 to 50:3.

[0010] In the above technical solution, limiting the weight ratio of buckwheat starch and ginger exosomes can ensure the optimal encapsulation efficiency between buckwheat starch and ginger exosomes, avoiding insufficient ginger exosomes that cannot fully encapsulate buckwheat starch molecules, or excessive ginger exosomes that interfere with the ordered structure of buckwheat starch, affecting the digestibility of the buckwheat starch-ginger exosome complex.

[0011] Furthermore, the incubation temperature is 25° C., and the incubation time is 30 to 75 minutes.

[0012] In the above technical solution, limiting the incubation temperature and time can avoid side reactions, protect the activity of ginger exosomes, effectively promote the interaction between buckwheat starch and ginger exosome molecules, and precisely regulate the structure of the complex. Furthermore, the temperature of the ultrasonic treatment is 25° C., and the time of the ultrasonic treatment is 0 to 15 min.

[0013] In the above technical solution, limiting the temperature and time of ultrasonic treatment can make the starch granules have a smoother and more porous microstructure.

[0014] On the other hand, the present application provides a method for preparing the above-mentioned buckwheat starch-ginger exosome complex with high digestibility, comprising the following steps: Extracting tartary buckwheat starch: hulling tartary buckwheat grains, performing a first drying process until constant weight is achieved, performing a first grinding process, and then passing the tartary buckwheat powder through a first sieve to obtain tartary buckwheat powder; then removing impurities from the tartary buckwheat powder, performing a second drying process, a second grinding process, and a second sieving process to obtain the tartary buckwheat starch; Extracting ginger exosomes: adding the pre-cooled first buffer solution to ginger pieces, crushing them, filtering and centrifuging to remove impurities, obtaining ginger juice, extracting crude ginger exosomes, and purifying the crude ginger exosomes to obtain the ginger exosomes; The complex of tartary buckwheat starch and ginger exosomes: a second buffer solution is added to the tartary buckwheat starch, stirred and gelatinized, and then the ginger exosomes are added and mixed. The mixture is then oscillated, incubated, and ultrasonically treated to obtain a crude complex. The crude complex is centrifuged, and the precipitate after centrifugation is collected and subjected to a third drying, a third grinding, and a third sieve to obtain the tartary buckwheat starch-ginger exosome complex with high digestibility.

[0015] Furthermore, the first sieve, the second sieve and the third sieve are metal sieves with a mesh size of 100-200.

[0016] Furthermore, the first buffer solution and the second buffer solution include PBS buffer solution with a concentration of 0.01 M and a pH of 7.2-7.4.

[0017] Furthermore, the purification method includes filtering using a 0.22 μm microporous membrane.

[0018] Furthermore, the weight ratio of the ginger pieces to the first buffer solution is 1:1 to 1:1.5.

[0019] Furthermore, the solid-liquid ratio of the tartary buckwheat starch, the second buffer solution and the ginger exosomes is 100 mg:5 mL:2 mg to 100 mg:5 mL:6 mg.

[0020] Furthermore, the first drying temperature is 60°C.

[0021] Furthermore, the impurity removal includes flavonoid removal, protein removal and fat removal.

[0022] Furthermore, the second drying temperature is 40°C.

[0023] Furthermore, the crushing is repeated 6 times using an intermittent crushing mode of a juicer.

[0024] Preferably, the model of the juicer is Midea MJ-LZ25Easy119.

[0025] Furthermore, the centrifugal impurity removal method includes differential centrifugation to remove impurities such as fibers, large particles and cell debris.

[0026] Furthermore, the method for extracting crude ginger exosomes includes ultracentrifugation combined with sucrose gradient purification.

[0027] Furthermore, the purification method includes filtering using a 0.22 μm microporous filter membrane.

[0028] Furthermore, the gelatinization temperature is 95° C., and the gelatinization time is 20 min.

[0029] Furthermore, the shaking incubation temperature is 25° C., and the shaking incubation time is 30 to 75 min.

[0030] Furthermore, the temperature of the ultrasonic oscillation is 25° C., and the time of the ultrasonic oscillation is 0 to 15 minutes.

[0031] Furthermore, the centrifugal force of the centrifugation is 8000-10000×g, and the centrifugation time is 10 min.

[0032] Furthermore, the third drying method includes freeze drying at -60°C.

[0033] Furthermore, when the method for extracting crude ginger exosomes is the ultracentrifugation method combined with the sucrose gradient purification method, the steps of extracting crude ginger exosomes include: The ginger juice was centrifuged at 120,000 to 175,000 × g for 60 to 120 minutes at 4°C, and then resuspended in PBS buffer to obtain a ginger exosome suspension. Three sucrose solutions prepared with pre-cooled PBS buffer were then added to the bottom of an ultracentrifuge tube in sequence. The ginger exosome suspension was then added from the top of the ultracentrifuge tube. After centrifugation at 80,000 to 120,000 × g for 30 to 90 minutes at 4°C, the yellow band in the middle of the ultracentrifuge tube was aspirated and resuspended in PBS buffer. After centrifugation at 80,000 to 120,000 × g for 30 to 90 minutes at 4°C, the precipitate was collected and resuspended in PBS buffer to obtain the crude ginger exosome product.

[0034] In the above technical solution, the ultracentrifugation method combined with the sucrose gradient purification method can avoid the introduction of exogenous chemical reagents, thereby effectively maintaining the natural physicochemical properties and biological activity integrity of ginger exosomes.

[0035] Furthermore, the concentrations of the three sucrose solutions are 5% to 60%.

[0036] Preferably, the concentrations of the three sucrose solutions are 8%, 30% and 45%, respectively.

[0037] On the other hand, the present application provides the application of the above-mentioned buckwheat starch-ginger exosome complex with high digestibility or the buckwheat starch-ginger exosome complex with high digestibility prepared by the above method in the metabolic regulation of type 2 diabetes, characterized in that the application includes at least one of reducing food intake, improving blood sugar indicators and improving dyslipidemia.

[0038] This application has the following beneficial effects: (1) This application is the first to use ginger exosomes to significantly improve the physical and chemical properties of buckwheat starch. The resulting highly digestible buckwheat starch-ginger exosome complex has enhanced connectivity, a stable structure, and high digestibility. (2) The preparation method provided in this application is simple in process, does not require complex chemical modification, is natural, green and safe, and can significantly reduce the digestibility of starch, meeting the needs of contemporary health care; (3) The highly digestible buckwheat starch-ginger exosome complex prepared in this application can interfere with the food intake of type 2 diabetes model mice, improve the symptoms of polydipsia and polyphagia, and significantly improve blood glucose indicators and dyslipidemia, providing an innovative strategy for the development of low-glycemic index staple foods and auxiliary hypoglycemic functional foods, and expanding ideas for the development of new strategies for lipid regulation and prevention and treatment of cardiovascular diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Comparison of the effects of different methods for extracting ginger exosomes in this application (A. Ginger exosome standard curve; B. Yield of ginger exosomes extracted by different methods: a. PEG co-precipitation method, b. Ultracentrifugation method combined with sucrose gradient purification).

[0040] Figure 2 Transmission electron microscopy morphological characterization of ginger exosomes extracted by ultracentrifugation combined with sucrose gradient purification in this application (A, B: imaging of ginger exosomes at the junction of 8% and 30% sucrose solutions; C, D: imaging of ginger exosomes at the junction of 30% and 40% sucrose solutions).

[0041] Figure 3 Particle size tracking and potentiometric analysis of ginger exosomes extracted by ultracentrifugation combined with sucrose gradient purification in this application (A. Particle size tracking diagram; B. Potential analysis diagram).

[0042] Figure 4 This is the analytical diagram for the single-factor screening of the buckwheat starch-ginger exosome complex method for this application (A. glucose content standard curve; B. different ginger exosome addition amounts; C. different oscillation incubation times; D. different ultrasonic treatment times).

[0043] Figure 5 In vitro simulated digestion and digestion-hydrolysis curves of the buckwheat starch-ginger exosome complex obtained under the optimal compound conditions of this application (A. In vitro simulated digestion; B. Digestion-hydrolysis curve).

[0044] Figure 6 Scanning electron microscopy morphological characterization of different samples in Example 8 of the present application (A. NTBS; B. TBS; C. TBS-G; D. TBS-GU).

[0045] Figure 7 These are the X-ray diffraction patterns of different samples in Example 9 of the present application.

[0046] Figure 8 2. Fourier transform infrared spectrum analysis diagram of different samples in Example 10 of the present application (A. transmittance of each sample; B. absorbance of each sample).

[0047] Figure 9This is a graph showing changes in physiological indicators of T2DM mice in Example 11 of the present application (A. body weight; B. food intake; C. water intake).

[0048] Figure 10 These are the changes in fasting blood glucose and oral glucose tolerance indices of T2DM mice in Example 12 of the present application (A. FBG index; B. blood glucose value; C. AUCG index).

[0049] Figure 11 This is an analysis of blood lipid levels in T2DM mice in Example 13 of the present application (A. TC index; B. TG index; C. LDL-C index; D. HDL-C index; E. ratio of LDL-C to HDL-C). DETAILED DESCRIPTION

[0050] The following is a clear and complete description of the technical solutions in some embodiments of the present application. Obviously, the embodiments described are only some of the embodiments of the present application, not all of them. Based on the embodiments provided in this application, all other embodiments obtained by ordinary technicians in this field are within the scope of protection of this application.

[0051] Unless the context requires otherwise, throughout the specification and claims, the term "comprising" is to be construed in an open, inclusive sense, that is, meaning "including, but not limited to."

[0052] When describing some embodiments, the expressions "at least one of A, B, and C" and "at least one of A, B, or C" may be used, both of which have the same meaning and include the following combinations of A, B, and C: only A, only B, only C, the combination of A and B, the combination of A and C, the combination of B and C, and the combination of A, B, and C.

[0053] Example 1 Extraction of tartary buckwheat starch Buckwheat starch was extracted from buckwheat grains purchased from the market using the water extraction method disclosed in "The Effect of Different Extraction Methods on the Physicochemical Properties of Buckwheat Starch" by Zhang Weili et al. The specific steps are as follows: (1) After shelling the buckwheat seeds, dry them at 60°C to constant weight, grind them into powder in a mortar, and pass them through a 100-mesh metal sieve to collect the sieved buckwheat powder; (2) 80% ethanol by mass was added to the buckwheat powder at a solid-liquid ratio of 1 mg:20 μL, and the mixture was thoroughly mixed and then treated at 50°C with an ultrasonic instrument at a power of 500 W for 30 min to completely remove the flavonoids in the buckwheat powder. The mixture was then centrifuged at a speed of 5000 r / min for 2 min, and the supernatant was removed to obtain a flavonoid-free buckwheat precipitate. (3) Distilled water was added to the deflavonoidized buckwheat precipitate at a solid-liquid ratio of 1 mg:10 μL, and the mixture was thoroughly mixed. The mixture was then placed in a water bath at 30°C for 18 h. After removing the upper suspension, the mixture was placed in an incubator at 40°C for drying to obtain a deproteinized buckwheat precipitate. (4) Add 30-60°C petroleum ether to the deproteinized buckwheat precipitate at a solid-liquid ratio of 1 mg:10 μL and mix thoroughly. Centrifuge at 5000 r / min for 2 min, collect the precipitate, wash away the residual petroleum ether with 80% by mass ethanol, wash again with distilled water three times, and then centrifuge at 5000 r / min for 2 min. After removing the supernatant, scrape off the gray matter on the surface of the precipitate, dry the remaining white matter in an incubator at 40°C, grind it with a mortar, and then pass it through a 100-mesh sieve. Collect the sieved powder to obtain the buckwheat starch.

[0054] Example 2 Determination of total starch content in tartary buckwheat The buckwheat starch obtained in Example 1 was decomposed into glucose by acid hydrolysis using a plant starch content test kit (Nanjing Jiancheng Bioengineering Institute, A148-1-1). The glucose was then quantified using an anthrone colorimetric method to quantify the total starch content in the buckwheat starch. The specific steps are as follows: (1) Accurately weigh 0.01 g of buckwheat starch sample, add 1 mL of reagent 1 and vortex mix, extract in 80℃ water bath for 20 min, centrifuge at 4000 r / min for 10 min, remove the supernatant and keep the precipitate; (2) Add 0.5 mL of distilled water to the precipitate and gelatinize at 95℃ for 15 min; (3) After gelatinization, cool and let it stand, add 0.35 mL of reagent 2, and incubate in 95℃ water bath for 10 min (take out and mix every 5 min); (4) After the water bath is completed, add 0.85 mL of distilled water, oscillate and mix thoroughly, centrifuge at 4000 r / min for 10 min, and take the supernatant as the sample supernatant; (5) Prepare blank tubes, standard tubes, and assay tubes according to Table 1, mix each tube separately, incubate in 95℃ water bath for 10 min, cool to room temperature, and use at 620℃. The absorbance value of each tube was measured by a spectrophotometer adjusted to zero with distilled water at 0.05 nm. The absorbance value of each tube was substituted into Formula 1 to obtain the total starch content in buckwheat starch.

[0055] Table 1: Operation table Reagents Blank tube Standard tube Assay tube Distilled water (mL) 0.2 0 0 0.05 mg / mL standard application solution (mL) 0 0.2 0 Sample supernatant (mL) 0 0 0.2 Reagent 3 application solution (mL) 1.0 1.0 1.0 Formula 1: Total starch content (mg / g) = (A 测定 -A 空白 ) / (A 标准 -A 空白 )*C 标准 *V 提 *N / W Among them, C 标准 —Concentration of standard solution (0.05 mg / mL); V 提 —Total liquid volume after sample pretreatment (1.7 mL); N—dilution multiple of sample supernatant; W—sample fresh weight (g).

[0056] The results showed that the total starch content of the tartary buckwheat starch extracted in Example 1 was 89.60±0.66%, and the purity was consistent with that reported previously. In subsequent experiments, water extraction was selected as the standard process for extracting tartary buckwheat starch.

[0057] Example 3 Extraction of ginger exosomes using different extraction methods This application uses the PEG co-precipitation method and the ultracentrifugation method combined with the sucrose gradient purification method to extract ginger exosomes from ginger purchased from the market. The specific steps are as follows: In this embodiment, the juicer model used for crushing is Midea MJ-LZ25Easy119, the concentration of PBS buffer is 0.01 M, and the pH is 7.2-7.4, and the pH of the PBS buffer is preferably 7.2.

[0058] Extraction of ginger exosomes by PEG co-precipitation Pre-cooled PBS buffer was added to a juicer, followed by the addition of washed and diced fresh ginger. The juice was repeatedly crushed six times using an intermittent crushing mode, and the residue was filtered through a filter to collect the ginger juice. The ginger juice was mixed with an equal volume of PEG6000 with an initial concentration of 8-30%, and the resulting mixture was incubated at 4°C for 10-14 h to obtain the crude ginger exosomes. The crude ginger exosomes were filtered using a 0.22 μm microporous membrane to obtain the ginger exosomes.

[0059] More preferably, the initial concentration of PEG6000 is 10-25%, most preferably 20%.

[0060] More preferably, the static incubation time is 10 to 14 h, most preferably 12 h.

[0061] (2) Extraction of ginger exosomes by ultracentrifugation combined with sucrose gradient purification Pre-cooled PBS buffer was added to the juicer, and then the washed and chopped fresh ginger was added, the weight ratio of fresh ginger to the PBS buffer was 1:1~1:1.5, preferably 1:1, and the intermittent crushing mode of the juicer was used to repeatedly crush the ginger juice for 6 times, and the residue was filtered with a filter to collect the ginger juice; the collected ginger juice was divided into centrifuge tubes, placed on ice and cooled to below 4°C, and then centrifuged at a centrifugal force of 4000 × g for 20 min, and then the first supernatant was taken and centrifuged at a centrifugal force of 8000 × g for 40 min to remove fiber and large particle impurities, and the supernatant after removing fiber and large particle impurities was collected and centrifuged at a centrifugal force of 12000 × g for 60 min to remove impurities such as cell debris; the supernatant was collected and centrifuged at a centrifugal force of 120000~175000 × g for 60~120 min at 4°C in an ultracentrifuge. min for the first ultracentrifugation, collect the precipitate after the first ultracentrifugation, repackage and balance the mass, and continue to use the ultracentrifuge to perform a second ultracentrifugation at a centrifugal force of 120,000-175,000 × g at 4°C for 60-120 min, collect the precipitate after the second ultracentrifugation and resuspend it with PBS buffer to obtain a ginger exosome suspension; use pre-cooled PBS to prepare three different sucrose solutions with a mass fraction of 5%-60%, and add them in sequence from the bottom of the ultracentrifuge tube. After adding, the concentration of the sucrose solution in the ultracentrifuge tube increases from bottom to top, and then gently add the ginger exosome suspension to the surface of the sucrose solution at the top, and centrifuge at a centrifugal force of 80,000-120,000 × g at 4°C for 30-90 min. After the third ultracentrifugation, the yellow strip between the sucrose solutions of different concentrations in the middle of the centrifuge tube was aspirated, diluted with PBS buffer, and then subjected to a fourth ultracentrifugation at 80,000-120,000 × g for 30-90 min in an ultracentrifuge at 4°C. The precipitate after the fourth ultracentrifugation was collected and resuspended with an appropriate amount of PBS buffer to obtain a crude ginger exosome product. The crude ginger exosome product was filtered using a 0.22 μm microporous filter membrane to obtain the ginger exosomes.

[0062] More preferably, the centrifugal force of the first ultracentrifugation and the second ultracentrifugation is 160,000-175,000 × g, most preferably 170,000 × g.

[0063] Most preferably, the time for the first ultracentrifugation and the second ultracentrifugation is 90 min.

[0064] More preferably, the mass fraction of the sucrose solution is 8-45%, and most preferably, it is 8%, 30%, and 45% in sequence.

[0065] More preferably, the centrifugal force of the third ultracentrifugation and the fourth ultracentrifugation is 100,000-120,000 × g, most preferably 100,000 × g.

[0066] Most preferably, the time for the third ultracentrifugation and the fourth ultracentrifugation is 60 min.

[0067] Example 4 Comparison of the effects of PEG co-precipitation and ultracentrifugation combined with sucrose gradient purification on the extraction of ginger exosomes The protein content of the two was determined according to the instructions of the total protein content test kit (Nanjing Jiancheng Bioengineering Institute, BCA method): the absorbance value of each standard was measured at a wavelength of 562 nm using a spectrophotometer, and the ginger exosome concentration was used as the horizontal axis and the absorbance value as the vertical axis to draw the ginger exosome standard curve (such as Figure 1 A), the equation of the standard curve is Y=0.2807*X+0.008161, R 2 =0.9984; The measured absorbance value was substituted into the equation of the ginger exosome standard curve. The ratio of the mass of ginger exosomes (mg) to the mass of ginger raw material (kg) was taken as the yield of ginger exosomes. The concentrations of ginger exosomes extracted by PEG co-precipitation method and ultracentrifugation method with sucrose gradient purification method were obtained (e.g. Figure 1 B).

[0068] The results showed that ultracentrifugation combined with sucrose gradient purification was significantly more effective (p < 0.01) in extracting ginger exosomes than PEG co-precipitation. Furthermore, ultracentrifugation combined with sucrose gradient purification avoided the introduction of exogenous chemical reagents, thereby effectively maintaining the natural physicochemical properties and bioactivity integrity of ginger exosomes. Based on comprehensive evaluation indicators such as extraction efficiency, product purity, and method reliability, subsequent experiments will use ultracentrifugation combined with sucrose gradient purification as the standard extraction process for ginger exosomes.

[0069] Example 5 Morphological Characterization, Particle Size Distribution, and Surface Potential of Ginger Exosomes Extracted by Ultracentrifugation Combined with Sucrose Gradient Purification The morphology of the yellow bands between sucrose solutions of different concentrations in the middle of the centrifuge tube was characterized by low-voltage transmission electron microscopy ( Figure 2 ), and the particle size distribution and surface potential were identified using nanoparticle tracking analysis technology ( Figure 3 ).

[0070] The results showed that ginger exosome vesicles exhibited typical exosome-like structural characteristics, with bright circles around the edges and darker depressions within, demonstrating a clear membranous structure. The particle size distribution ranged from 100 to 200 nm. Furthermore, no membrane rupture or leakage of contents was observed under an electron microscope, indicating that the structure was well maintained during preparation and storage, making it suitable for subsequent composite experiments.

[0071] Example 6 Screening of optimal compounding conditions for preparing buckwheat starch-ginger exosome complex from buckwheat starch and ginger exosomes (1) The steps for preparing the buckwheat starch-ginger exosome complex are as follows: 200 mg of the tartary buckwheat starch prepared in Example 1 was suspended in 1 mL of PBS buffer with a concentration of 0.01 M and a pH of 7.2-7.4, wherein the pH of the PBS buffer was preferably 7.2. After the tartary buckwheat starch was gelatinized by stirring and heating at 95°C for 20 min, 4-12 mg of the ginger exosomes prepared by ultracentrifugation combined with a sucrose gradient purification method in Example 3 were mixed with the gelatinized tartary buckwheat starch. The mixture was incubated with shaking at 25°C for 30-75 min, and then ultrasonically treated at 25°C for 0-15 min. The composite starch was collected by centrifugation, freeze-dried, ground, and passed through a 200-mesh metal sieve to obtain the tartary buckwheat starch-ginger exosome complex.

[0072] (2) Screening the best composite conditions Referring to the above steps, based on the mass of buckwheat starch of 200 mg, the addition amount of ginger exosomes of 4 mg, the oscillation incubation time of 30 min, and the ultrasonic treatment time of 5 min, a single-factor experiment was conducted to explore the effects of the addition amount of ginger exosomes (4 mg, 6 mg, 8 mg, 10 mg, 12 mg), oscillation incubation time (30 min, 45 min, 60 min, 75 min) and ultrasonic treatment time (0 min, 5 min, 10 min, 15 min) on the in vitro digestibility of buckwheat starch-ginger exosome complex. The same amount of gelatinized freeze-dried buckwheat starch without the addition of ginger exosomes was used as the control group, and the content of hydrolyzed glucose was determined using the GOPOD analysis kit as the in vitro digestibility. The results are shown in Figure 3. Figure 4 B. Figure 4 C. Figure 4 As shown in D.

[0073] According to the results of the single-factor experiment, the more preferred addition amount of ginger exosomes is 8-12 mg, the more preferred oscillation incubation time is 45-75 min, and the more preferred time for ultrasonic treatment is 5-15 min. Among them, the composite effect of buckwheat starch and ginger exosomes is best when the addition amount of ginger exosomes is 10 mg, the oscillation incubation time is 60 min, and the ultrasonic treatment time is 10 min. Further experiments will be carried out on the buckwheat starch-ginger exosome complex prepared under the optimal composite conditions.

[0074] Example 7 Determination of resistant starch content and in vitro digestion curve in buckwheat starch-ginger exosome complex 200 mg of the buckwheat starch-ginger exosome complex prepared under the optimal composite conditions in Example 6 was weighed and dispersed in 10 mL of sodium acetate buffer (0.1 M, pH 5.2). An equal amount of gelatinized freeze-dried buckwheat starch without ginger exosomes was used as a control. After stirring and heating at 95°C for 20 min, the mixture was cooled to room temperature. 4 mL of α-amylase (300 U / mL) and 1 mL of amyloglucosidase (330 U / mL) were added and incubated at 37°C with shaking. The hydrolyzate was collected every 30 min and then mixed with 95% ethanol to terminate the digestion. The mixture was then centrifuged at 12,000 r / min for 15 min. The content of hydrolyzed glucose was determined using a GOPOD analysis kit. The results are shown in Table 2. Figure 5 .

[0075] The results showed that the addition of ginger exosomes slowed the digestion of buckwheat starch, significantly increasing the amount of undigested components at the end of digestion. This was manifested in an increase in the ratio of slowly digestible starch (SDS) and resistant starch (RS), which helped delay postprandial blood sugar rise and has positive implications for blood sugar control. This result suggests that the addition of ginger exosomes may have a beneficial effect on blood sugar metabolism by changing the digestibility of starch and increasing its resistant components.

[0076] Example 8 Morphological Characterization of Buckwheat Starch-Ginger Exosome Complex NTBS, TBS, TBS-G, and TBS-GU were freeze-dried as samples, respectively. NTBS was natural buckwheat starch, TBS was buckwheat starch prepared in Example 1, TBS-G was a buckwheat starch-ginger exosome complex prepared under the optimal composite conditions in Example 6 but without ultrasonic oscillation, and TBS-GU was a buckwheat starch-ginger exosome complex prepared under the optimal composite conditions in Example 6. Each sample was mounted on an aluminum short tube and sputtered with gold powder. Scanning electron microscopy (SEM) imaging was performed at an acceleration voltage of 30 kV and a magnification of 2500X to obtain the following: Figure 6 The microstructure shown.

[0077] The results showed that the addition of ginger exosomes induced the gelatinized starch to form a looser, gelatinized matrix (TBS-G). Especially under ultrasound treatment (TBS-GU), the starch granules became smoother and more porous, and some formed small clumps. This indicates that ginger exosomes significantly changed the microstructure of gelatinized starch.

[0078] Example 9 X-ray diffraction analysis of buckwheat starch-ginger exosome complex XRD patterns were obtained for the freeze-dried samples of NTBS, TBS, TBS-G, and TBS-GU prepared in Example 8 using CuKα radiation. The diffraction angle (2θ) was scanned over a range of 10° to 60° at a scan rate of 10° / min. The relative crystallinity (RC) was calculated using MDI JADE 6 software. The results are shown in Table 1. Figure 7 shown.

[0079] The results showed that after the introduction of ginger exosomes, the diffraction pattern of TBS-G remained isomorphic to that of gelatinized starch (TBS), but two new characteristic diffraction peaks appeared at approximately 27° and 32° (2θ), which were significantly different from the diffraction pattern of TBS. The RC value also increased to 21.81%, indicating that the interaction between ginger exosomes and buckwheat starch may promote the formation of new crystal structures and induce the recrystallization of starch molecular chains. After further ultrasonic treatment (TBS-GU), the crystallographic characteristics of the TBS-GELNs composite system showed further structural optimization. The relative peaks of the new peaks at 27° and 32° (2θ) were enhanced, and the RC value increased to 24.61%, indicating that ultrasonic treatment with appropriate intensity and duration increased the relative crystallinity.

[0080] Example 10 Fourier transform infrared spectroscopy analysis of buckwheat starch-ginger exosome complex The NTBS, TBS, TBS-G, and TBS-GU freeze-dried samples prepared in Example 8 were further prepared as samples to be analyzed using the KBr particle method. The FTIR spectra of each sample to be tested were recorded using an infrared spectrometer, wherein the scanning range was 4000-400 cm −1 A total of 256 scans were recorded with a resolution of 4 cm −1 In addition, the spectrum ranges from 1200 to 800 cm −1 Unequal. Deconvolution, by calculating 1047 and 1022 cm −1 The amplitude ratio at the position determines the short-range ordered structure of starch (R-1,047 / 1,022). Figure 8 shown.

[0081] The results showed that compared to NTBS and TBS, no new absorption peaks appeared in the spectra of all treatment groups with TBS-G addition, indicating that the binding between ginger exosomes and starch molecules occurs through non-covalent interactions, including hydrogen bonds, van der Waals forces, and hydrophobic effects. The TBS-GU spectrum shows that ultrasound-assisted ginger exosomes can interact with buckwheat starch through hydrogen bonds, increasing the surface porosity and short-range ordered structure of the starch, thereby promoting the formation of V-type complexes and reducing the availability of buckwheat starch molecules to water and digestive enzymes, thereby inhibiting starch digestibility.

[0082] Example 11: Improvement of the polydipsia and polyphagia symptoms in type 2 diabetes model mice by buckwheat starch-ginger exosome complex A type 2 diabetes mellitus (T2DM) mouse model was established under the dual effects of streptozotocin (STZ) and a high-fat diet (HFD). From the date of successful model establishment, the T2DM mice were divided into four groups: a model group (MOD), a metformin group (MET), a low-dose buckwheat starch-ginger exosome complex group (L-GTBS), and a high-dose buckwheat starch-ginger exosome complex group (H-GTBS). A CON group (isolated before modeling) was also established. The CON group was fed a normal diet, while the other groups were continuously fed an HFD. The MET group was gavaged daily with 100 mg / kg of metformin, the L-GTBS group with 0.15 g / 100 g of buckwheat starch-ginger exosome complex, and the H-GTBS group with 0.3 g / 100 g of buckwheat starch-ginger exosome complex. The CON and MOD groups were gavaged with an equal volume of distilled water. The intervention period lasted 5 weeks. During the intervention period, food intake, water intake, and body weight were recorded weekly. The results are shown in Table 1. Figure 9 shown.

[0083] The results showed that the intervention of buckwheat starch-ginger exosome complex effectively alleviated the symptoms of weight loss in T2DM mice, while increasing the satiety of T2DM mice and affecting energy consumption, thereby reducing food intake and possibly alleviating diabetes-induced consumption, ultimately improving the symptoms of polydipsia and polyphagia in T2DM mice.

[0084] Example 12 Fasting blood glucose (FBG) and oral glucose tolerance test (OGTT) experiment in T2DM mice During the intervention period of T2DM mice in Example 11, FBG levels were measured once a week. The results were as follows: Figure 10 As shown in A; After 5 weeks of intervention, each group took 2.0 g / kg of glucose orally. Blood glucose levels were measured at 0 min, 30 min, 60 min, 90 min, and 120 min after oral glucose administration (the results are shown in Figure 10 B), respectively as BG0, BG 30 , BG60 , BG 90 and BG 120 Substitute the formula to calculate the area under the glucose curve (AUCG), which is as follows: AUCG (mmol / L) = 1 / 4*BG0 + 1 / 2*BG 30 +3 / 4*BG 60 +1 / 2*BG 120 After sorting, we get Figure 10 C shows the AUCG of each group of mice.

[0085] The results showed that after intervention with the buckwheat starch-ginger exosome complex, blood glucose indicators in T2DM mice were significantly improved. Furthermore, the blood glucose-lowering effect of H-GTBS was closest to that of the MET group, significantly reducing FBG levels and improving the OGTT of T2DM mice. This suggests that the buckwheat starch-ginger exosome complex can improve the hyperglycemic state of T2DM mice by regulating pathways related to glucose metabolism, providing experimental evidence for the development of novel diabetes "dietary therapy" strategies.

[0086] Example 13 Analysis of serum lipid levels in T2DM mice The serum triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) levels of T2DM mice after the intervention in Example 11 were measured using the corresponding detection kits (Nanjing Jiancheng Bioengineering Co., Ltd., Nanjing, China), and the ratio of LDL-C to HDL-C was calculated. The results are shown in Figure 11 .

[0087] As shown in the figure, the buckwheat starch-ginger exosome complex not only improves dyslipidemia in T2DM mice but also reduces the risk of coronary atherosclerosis in these mice, potentially contributing to cardiovascular health. This finding provides strong experimental evidence for further investigation into the mechanisms of the buckwheat starch-ginger exosome complex in regulating lipid metabolism and preventing cardiovascular disease, and also opens new avenues for developing novel strategies for lipid regulation and the prevention and treatment of cardiovascular disease.

[0088] In summary, the composite system constructed by ginger exosomes and buckwheat starch in this application significantly increases the content of slowly digestible starch and resistant starch, and improves the digestibility of buckwheat starch. The resulting buckwheat starch-ginger exosome complex, after ingestion, synergizes the physical barrier and the co-regulation of biological activity, can reduce postprandial blood sugar and serum TC, TG, and LDL-C levels, improve glucose tolerance in T2DM mice, improve lipid metabolism disorders, and alleviate the risk of atherosclerosis. The raw materials used in the buckwheat starch-ginger exosome complex of this application are natural and pollution-free, meet the requirements of clean label food, and have both nutritional carriers and multifunctional delivery properties, providing an innovative strategy for the development of low-GI staple foods and auxiliary hypoglycemic functional foods.

[0089] The foregoing description is merely a preferred embodiment of the present invention. It should be understood that the present invention is not limited to the form disclosed herein and should not be construed as excluding other embodiments. Rather, the present invention can be used in various other combinations, modifications, and environments and can be modified within the scope of the concept described herein through the above teachings or techniques or knowledge in the relevant field. Modifications and variations made by those skilled in the art that do not depart from the spirit and scope of the present invention are intended to be protected by the appended claims.

Claims

1. A buckwheat starch-ginger exosome complex with high digestibility, characterized in that The tartary buckwheat starch-ginger exosome complex is formed by incubating gelatinized tartary buckwheat starch and ginger exosomes and performing ultrasonic treatment.

2. The tartary buckwheat starch-ginger exosome complex according to claim 1, characterized in that The weight ratio of the tartary buckwheat starch to the ginger exosomes is 50:1 to 50:

3.

3. The buckwheat starch-ginger exosome complex according to claim 1, characterized in that The incubation temperature is 25°C, and the incubation time is 30 to 75 minutes; And / or, the temperature of the ultrasonic treatment is 25° C., and the time of the ultrasonic treatment is 0 to 15 min.

4. A method for preparing the tartary buckwheat starch-ginger exosome complex with high digestibility according to any one of claims 1 to 3, characterized in that: The following steps are involved: Extracting tartary buckwheat starch: hulling tartary buckwheat grains, performing a first drying process until constant weight is achieved, performing a first grinding process, and then passing the tartary buckwheat powder through a first sieve to obtain tartary buckwheat powder; then removing impurities from the tartary buckwheat powder, performing a second drying process, a second grinding process, and a second sieving process to obtain the tartary buckwheat starch; Extracting ginger exosomes: adding the pre-cooled first buffer solution to ginger pieces, crushing them, filtering and centrifuging to remove impurities, obtaining ginger juice, extracting crude ginger exosomes, and purifying the crude ginger exosomes to obtain the ginger exosomes; The complex of tartary buckwheat starch and ginger exosomes: a second buffer solution is added to the tartary buckwheat starch, stirred and gelatinized, and then the ginger exosomes are added and mixed. The mixture is then oscillated, incubated, and ultrasonically treated to obtain a crude complex. The crude complex is centrifuged, and the precipitate after centrifugation is collected and subjected to a third drying, a third grinding, and a third sieve to obtain the tartary buckwheat starch-ginger exosome complex with high digestibility.

5. The method according to claim 4, characterized in that The first screen, the second screen and the third screen are metal screens with a mesh size of 100 to 200; And / or, the first buffer and the second buffer comprise PBS buffer with a concentration of 0.01 M and a pH of 7.2-7.4; And / or, the purification method comprises filtering using a 0.22 μm microporous membrane.

6. The method according to claim 4, characterized in that The weight ratio of the ginger piece to the first buffer solution is 1:1 to 1:1.5 And / or, the solid-liquid ratio of the tartary buckwheat starch, the second buffer solution, and the ginger exosomes is 100 mg:5 mL:2 mg to 100 mg:5 mL:6 mg.

7. The method according to claim 4, characterized in that The first drying temperature is 60°C; And / or, the impurity removal includes flavonoid removal, protein removal and fat removal; And / or, the second drying temperature is 40°C; And / or, the crushing is repeated 6 times using an intermittent crushing mode of a juicer; And / or, the centrifugal impurity removal method includes differential centrifugation to remove impurities such as fibers, large particles and cell debris; And / or, the method for extracting crude ginger exosomes comprises ultracentrifugation combined with sucrose gradient purification; And / or, the purification method comprises filtration using a 0.22 μm microporous membrane; And / or, the gelatinization temperature is 95° C., and the gelatinization time is 20 min; and / or, the shaking incubation temperature is 25° C., and the shaking incubation time is 30 to 75 min; And / or, the temperature of the ultrasonic treatment is 25° C., and the time of the ultrasonic treatment is 0 to 15 min; And / or, the centrifugal force of the centrifugation is 8000-10000 × g, and the centrifugation time is 10 min; And / or, the third drying method includes freeze drying at -60°C.

8. The method according to claim 7, characterized in that The step of extracting the crude ginger exosomes by ultracentrifugation combined with sucrose gradient purification method comprises: The ginger juice was centrifuged at 120,000 to 175,000 × g for 60 to 120 minutes at 4°C, and then resuspended in PBS buffer to obtain a ginger exosome suspension. Three sucrose solutions prepared with pre-cooled PBS buffer were then added to the bottom of an ultracentrifuge tube in sequence. The ginger exosome suspension was then added from the top of the ultracentrifuge tube. After centrifugation at 80,000 to 120,000 × g for 30 to 90 minutes at 4°C, the yellow band in the middle of the ultracentrifuge tube was aspirated and resuspended in PBS buffer. After centrifugation at 80,000 to 120,000 × g for 30 to 90 minutes at 4°C, the precipitate was collected and resuspended in PBS buffer to obtain the crude ginger exosome product.

9. The method according to claim 8, characterized in that The concentrations of the three sucrose solutions range from 5% to 60%.

10. Use of the tartary buckwheat starch-ginger exosome complex with high digestibility according to any one of claims 1 to 3 or the tartary buckwheat starch-ginger exosome complex with high digestibility prepared by the method according to any one of claims 4 to 9 in metabolic regulation, characterized in that: The application includes at least one of reducing food intake, improving blood sugar index and improving blood lipid index.

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