Angelica dahurica resistant starch as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of angelica dahurica processing, and particularly discloses preparation and application of angelica dahurica resistant starch, a preparation method of the angelica dahurica resistant starch comprises the following steps: S1, uniformly dispersing angelica dahurica starch in water to obtain an angelica dahurica starch suspension; s2, gelatinizing the radix angelicae starch suspension in a boiling water bath, adding high-temperature alpha-amylase into the gelatinized radix angelicae starch, stirring, adjusting the temperature to 58 DEG C, adjusting the pH to 4.8, and adding pullulanase to induce debranching; and S3, standing the radix angelicae starch paste at 4 DEG C for 12 hours to obtain recrystallized starch, centrifuging the recrystallized starch, collecting, drying, grinding, and filtering with a 200-mesh screen to obtain the radix angelicae resistant starch. The radix angelicae resistant starch is used for reducing postprandial blood sugar, slowing down weight gain and the like; the radix angelicae resistant starch can improve the content of short-chain fatty acids in intestinal tracts, improve the richness, diversity and uniformity of flora, and increase beneficial bacteria in the intestinal tracts. The application provides a theoretical basis and an experimental basis for the development of health-care functional food rich in radix angelicae resistant starch.

Description

Technical Field

[0001] The present application belongs to the field of Angelica dahurica processing technology, and specifically relates to Angelica dahurica resistant starch and its preparation method and application. Background Art

[0002] Angelica dahurica, a plant of the genus Angelica in the Umbelliferae family, is a traditional Chinese medicine used for both food and medicine. Its dried roots are used as medicine, boasting multiple benefits, including dispelling heat, dispelling wind and relieving pain, draining pus and reducing swelling, and clearing the nasal passages. Angelica dahurica has a high nutritional value and is often processed into slices or whole powder, which offer excellent health benefits. Currently, research on the active ingredients of Angelica dahurica has primarily focused on coumarin, polysaccharide, volatile oil, and flavonoid compounds, but little research has been conducted on resistant starch from Angelica dahurica. Some researchers have explored methods for extracting Angelica dahurica starch, studying the effects of factors such as the solid-liquid ratio, soaking agent pH, soaking time, and soaking temperature on starch extraction yield. However, little research has been conducted on the preparation and application of resistant starch from Angelica dahurica.

[0003] Resistant starch is a type of starch that is difficult to digest and absorb in the small intestine. It has functions such as regulating intestinal flora, lowering blood sugar and blood lipids, and is widely used in functional foods and pharmaceuticals. Common extraction methods include using enzymes to hydrolyze non-resistant starch and separate the resistant starch fraction, but enzyme costs are high and enzyme activity is significantly affected by environmental conditions; or treating starch with acids, bases, or chemical reagents (such as ethanol) to separate the resistant starch fraction, but this extraction process may introduce impurities that affect the quality of the resistant starch. Currently, research has been conducted on resistant starches from corn, rice, and kidney beans, but there is no research on the extraction and application of resistant starch from Angelica dahurica.

[0004] Therefore, this application explores the preparation method and application of Angelica dahurica resistant starch, uses the resistant starch in Angelica dahurica starch to conduct a high-fat diet intervention experiment, and explores the effects of Angelica dahurica resistant starch in reducing the risk of obesity, promoting lipid metabolism, and regulating intestinal flora, providing a theoretical basis and relevant experimental basis for the development of health functional foods based on Angelica dahurica starch. Summary of the Invention

[0005] The purpose of the present application is to provide a resistant starch of Angelica dahurica and its preparation method and application. Angelica dahurica has multiple nutritional and functional components and has high nutritional and health value after being processed into food.

[0006] The present application relates to angelica dahurica resistant starch, which is prepared by purification using a double enzyme method.

[0007] In one aspect, the present application provides a method for preparing Angelica dahurica resistant starch, comprising the following steps:

[0008] S1. The angelica starch is evenly dispersed in pure water to obtain an angelica starch suspension;

[0009] S2. The Angelica starch suspension was gelatinized to obtain gelatinized Angelica starch, and high temperature α-amylase was added to the gelatinized Angelica starch, and then pullulanase was added to induce debranching to obtain enzymatic hydrolysis of Angelica starch paste;

[0010] S3. The enzymatically hydrolyzed angelica starch paste is allowed to stand at low temperature for recrystallization, and then the precipitate is collected after centrifugation. The precipitate is dried, ground and sieved to obtain the angelica resistant starch.

[0011] Specifically, the amount of the Angelica dahurica starch in S1 is 15 g, which is dispersed in 100 mL of water;

[0012] Specifically, the gelatinization temperature in S2 is 60° C. to 80° C.;

[0013] Specifically, the stirring speed in S2 is 200 r / min, and the stirring time is 10 min;

[0014] Specifically, after the stirring in S2, the temperature is adjusted to 50°C to 60°C, and the pH is adjusted to 4 to 5;

[0015] Specifically, the debranching reaction time in S3 is 4 to 6 hours;

[0016] Specifically, the low temperature in S3 is 4° C., and the standing time is 8 to 12 hours;

[0017] Specifically, the temperature of the drying process in S3 is 45°C to 55°C, and the filtration uses a No. 4 to 5 sieve;

[0018] The main steps of extracting angelica starch include:

[0019] (1) Wash, peel, and grind Angelica dahurica to obtain an Angelica dahurica sample;

[0020] (2) washing and filtering the Angelica dahurica sample to obtain an Angelica dahurica filtrate;

[0021] (3) allowing the filtrate of Angelica dahurica to stand for 8 to 16 hours, removing the upper layer of yellow pulp water, and obtaining the lower layer of Angelica dahurica starch sediment containing impurities;

[0022] (4) Add appropriate amount of clean water to the angelica starch sediment and stir evenly. Centrifuge at 3000 rpm for 15 minutes. Pour off the supernatant and scrape off the brown impurities on the top to take the white precipitate.

[0023] (5) drying the white precipitate in a 40-50° C. air dryer, grinding and sieving to obtain the angelica starch.

[0024] Furthermore, the resistant starch in Angelica dahurica starch increases the content of short-chain fatty acids in the intestine, which can improve the richness, diversity and uniformity of the bacterial flora and increase the beneficial bacteria in the intestine.

[0025] Furthermore, the angelica starch rich in the resistant starch is used to prepare healthy foods such as angelica noodles and angelica jelly. The noodles and other products prepared have good taste and quality, and have higher digestibility, viscosity and swelling power.

[0026] Among them, Angelica dahurica starch can be modified to eliminate stickiness and produce a loose structure, which provides methods and ideas for the processing of Angelica dahurica starch in health functional foods.

[0027] Furthermore, the angelica resistant starch is also used in the preparation of medicines for inhibiting fat accumulation, lowering blood lipids, improving intestinal flora, and maintaining intestinal health.

[0028] Beneficial effects:

[0029] 1. The resistant starch of Angelica dahurica of the present application can effectively improve the processability of raw materials in the processing of functional foods, while improving the taste and nutritional value of foods.

[0030] 2. The angelica resistant starch of the present application is beneficial to the fermentation and utilization of intestinal flora to produce short-chain fatty acids and a small amount of gas, thereby increasing the consumer's sense of fullness, reducing the risk of obesity, lowering postprandial blood sugar levels, and lowering blood lipids.

[0031] 3. The resistant starch of Angelica dahurica prepared in this application helps to increase the richness of intestinal flora, increase the content of intestinal probiotics, and can effectively improve the balance of intestinal flora and maintain intestinal health. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The physicochemical properties and structural characteristics of Angelica dahurica starch and Angelica dahurica resistant starch (A: thermal characteristic analysis diagram, B: infrared spectrum, C: XRD pattern, D: in vitro digestion, A1-A3: scanning electron micrographs of Angelica dahurica starch morphology, B1-B3 scanning electron micrographs of Angelica dahurica resistant starch, C1-C3: scanning electron micrographs of Angelica dahurica starch after digestion in the mouth, stomach and intestines, D1-D3: scanning electron micrographs of Angelica dahurica resistant starch after digestion in the mouth, stomach and intestines).

[0033] Figure 2 Effects of Angelica dahurica resistant starch on obese mice fed a high-fat diet (A: Body weight change, B: Liver index, C: Serum adiponectin level, D: Serum leptin level, E: Serum triglyceride level, F: Serum total cholesterol level, G: Serum low-density lipoprotein cholesterol level, H: Serum high-density lipoprotein cholesterol level).

[0034] Figure 3Liver and colon sections of mice under different treatments.

[0035] Figure 4 These are the intestinal short chains of mice in different groups (A: acetic acid, B: propionic acid, C: butyric acid, D: isobutyric acid, E: valeric acid, F: isovaleric acid).

[0036] Figure 5 Alpha diversity index analysis of intestinal flora in different groups of mice (G: Chao1, H: Simpson, I: Shannon, J: Plelou_e, K: Observed species, L: Faith_pd, M: Goods coverage, N: abundance rank curve).

[0037] Figure 6 Figure 2 shows intestinal flora analysis and Beta diversity analysis of mice in different groups (O: phylum-level flora composition, P: family-level flora composition, Q: genus-level flora composition, R: PCoA analysis, S: cluster analysis based on Bray-Curtis distance, T: LDA effect value histogram of marker species). DETAILED DESCRIPTION

[0038] The following specific examples are used to further illustrate the content of this application.

[0039] The following is a further detailed description of the present invention in conjunction with specific preferred embodiments, and the specific implementation of this application should not be considered to be limited to these descriptions. For those skilled in the art to which this application belongs, several simple deductions or substitutions can be made without departing from the concept of this application, and all of these should be considered to fall within the scope of protection of this application.

[0040] Example 1. Extraction of Angelica dahurica starch

[0041] The Angelica dahurica samples used in this embodiment were collected in August 2022 at the Sichuan Angelica dahurica Research Demonstration Base (N30°36'59″, 105°18'48″) in Suining City, Sichuan Province. The Angelica dahurica samples used in the other embodiments are the same as those in this embodiment.

[0042] The specific steps are as follows:

[0043] (1) Clean the surface sediment of fresh Angelica dahurica, peel it, cut it into sections, and put it into a pulper for pulping. Filter the pulp with a No. 8 mesh sieve, and wash the residue with appropriate amount of purified water three times.

[0044] (2) Discard the filter residue, combine the filtrates, and let the filtrates settle overnight for 12 hours. After separation, remove the upper yellow slurry; add an appropriate amount of pure water to the precipitate and stir evenly;

[0045] (3) Centrifuge at 3000 rpm for 10 min in a tabletop centrifuge. Discard the supernatant and scrape off the brown impurities on the upper layer. The white impurities on the lower layer are angelica starch.

[0046] (4) The starch precipitate was collected and placed in a 50°C forced air drying oven. After drying, it was ground and passed through a No. 4 sieve to obtain the Angelica dahurica starch sample. The Angelica dahurica starch yield and extraction rate were calculated according to the following formula.

[0047] Starch extraction rate = m1 / m0×100%

[0048] Where m1 is the mass of Angelica dahurica starch, g; m0 is the mass of Angelica dahurica sample, g.

[0049] In this embodiment, the starch of Angelica dahurica was extracted by homogenization method. According to the calculation formula, the starch yield was 11.26%.

[0050] Example 2. Preparation of Angelica dahurica Resistant Starch

[0051] Angelica dahurica starch was purified by a dual enzyme method to prepare Angelica dahurica resistant starch (Ad-RS3). The steps are as follows:

[0052] (1) First, 15 g of Angelica dahurica starch was uniformly dispersed in 100 mL of phosphate buffer (0.1 M, pH 5.5);

[0053] (2) The mixture was fully gelatinized in boiling water for 20 min, high-temperature α-amylase was added, and the mixture was stirred at 200 rpm for 10 min. The temperature was adjusted to 58°C, the pH was adjusted to 4.8°C, and pullulanase was added to induce defatting.

[0054] (3) The starch paste was then placed at 4°C for 12 h to allow degradation. Finally, the recrystallized starch was collected by centrifugation at 3000 rpm, dried at 50°C, ground, and filtered through a 200-mesh sieve.

[0055] (4) The resistant starch content was determined by GOPOD method. The results are shown in Table 1.

[0056] Table 1 Determination of the content of Angelica dahurica starch and Angelica dahurica resistant starch

[0057]

[0058] Example 3. Physicochemical properties and structural characteristics of Angelica dahurica resistant starch

[0059] It mainly includes the determination of the composition, hydration characteristics, particle morphology, particle size distribution, crystal structure, thermodynamic properties and digestibility of Angelica dahurica resistant starch.

[0060] The specific methods for determining the physicochemical properties and structural characteristics of Angelica dahurica resistant starch are as follows:

[0061] (1) Composition of resistant starch in Angelica dahurica: The content of resistant starch was determined according to GB 5009.3-2016; the ash content was determined according to GB / T 22427.8-2008; and the total fat content was determined according to GB 5009.6-2016. The protein content was determined using a BCA protein assay kit (Beyotime, Shanghai, China); the amylose (Am) / amylopectin (Ap) assay kit (Megazyme, Wicklow, Ireland) was used to determine the amylose and amylopectin contents.

[0062] (2) Hydration properties of Angelica dahurica resistant starch: The water absorption index (WAI) and water solubility index (WSI) were calculated by incubating a 10% starch suspension in a 70°C water bath for 20 min, stirring every 5 min, and then centrifuging at 5000 rpm for 15 min. The supernatant was transferred to an aluminum container and dried at 105°C to constant weight. The calculation formulas are as follows (1) to (2):

[0063] WAI(g / g)=W1 / W2 (1)

[0064] WSI(g / g)=W2 / W0 (2)

[0065] Where W0 is the weight of the dried sample, W1 is the weight of the precipitate, and W2 is the weight of the solid dissolved in the supernatant.

[0066] (3) Morphology of Angelica dahurica Resistant Starch Granules: The morphology of Angelica dahurica Resistant Starch samples was characterized using scanning electron microscopy. A small amount of the sample to be tested was adhered to a conductive adhesive, fixed on a stage, and then gold-sprayed. The accelerating voltage was 6.0 kV and the magnifications were 50x, 500x, and 2000x.

[0067] (4) Particle size distribution of Angelica dahurica resistant starch: The particle size and distribution of Angelica dahurica resistant starch were determined using a laser diffraction particle size analyzer (Mastersizer 2000, UK). 50 mg of the sample to be tested was suspended in 15 mL of deionized water, stirred evenly, and then sonicated in an ultrasonicator for 5 min. The sample was then dripped into the sample port until the obscuration reached 8% to 12%.

[0068] (5) Crystal structure determination of Angelica dahurica resistant starch: The crystal structure of Angelica dahurica resistant starch was determined using an X-ray diffractometer (Bruker D8 Advance, Germany) with the following parameters: radiation source: Cu; voltage: 40 kV; current: 100 mA; scanning range: 4° to 45° (2θ); scanning rate: 4° / min. The relative crystallinity was calculated as the ratio of the peak area (crystalline region) under the diffraction curve to the total diffraction pattern area.

[0069] (6) Determination of the short-range ordered structure of Angelica dahurica resistant starch: The infrared spectra of different resistant starches were measured using an infrared spectrometer (Nicolet IS10, USA). 1.0 mg of the sample to be tested and 100 mg of KBr powder were placed in an agate mortar and ground evenly, then pressed into tablets. The tablets were ground and compressed in a continuous dry air atmosphere with a resolution of 4 cm. -1 , the wave number range is 400~4000cm -1 Each sample was measured three times, and the spectral data were background corrected and normalized.

[0070] (7) Determination of the thermodynamic properties of Angelica dahurica resistant starch: The thermodynamic properties of Angelica dahurica resistant starch were determined using a differential scanning calorimeter. 3.0 mg of the sample to be tested was mixed with deionized water at a ratio of 1:2 (w / v). The mixture was sealed and equilibrated for 24 h. The heating temperature was 20°C to 130°C, the heating rate was 10°C / min, and the nitrogen flow rate was 20 mL / min. Using a blank plate as a reference, the thermodynamic properties of the sample were obtained using the platform's built-in software, including the onset temperature (To), peak temperature (Tp), end temperature (Tc), gelatinization temperature range (ΔT), and thermal enthalpy (ΔH).

[0071] (8) Digestion characteristics of Angelica starch: An in vitro digestion model was used to simulate the human digestive system, including the oral cavity, stomach, and small intestine. Digestive fluid electrolytes were prepared according to Table 2, including simulated saliva (SSF), simulated gastric fluid (SGF), and simulated intestinal fluid (SIF).

[0072] Table 2 Digestive fluid configuration table

[0073]

[0074] Oral digestion experiment: Prepare oral digestive fluid (SSF) (40 mL), CaCl2 solution (0.3 M, 0.25 mL), α-amylase solution (1000 U / mL, 7.5 mL), and pure water to a volume of 50 mL. Angelica dahurica resistant starch was mixed with oral digestive fluid at a 50:50 (w / v) ratio, adjusted to pH 6.5, and incubated in a water bath shaker at 37°C and 200 rpm for 5 min. The resulting samples were designated AS-S and RS-S.

[0075] Gastric Digestion Test: Gastric digestive fluid (64 mL) was prepared, along with SGF (64 mL), CaCl₂ solution (0.3 M, 0.04 mL), pepsin solution (40,000 U / mL, 8 mL), and purified water to a volume of 80 mL. To simulate gastric digestion, Angelica dahurica resistant starch was mixed with gastric digestive fluid at a ratio of 80:80 (w / v), the pH was adjusted to 1.0, and the mixture was incubated in a water bath shaker at 37°C and 200 rpm for 2 h. The resulting samples were designated AS-G and RS-G.

[0076] Small Intestinal Digestion Experiment: Prepare small intestinal digestive fluid: SIF (48 mL), CaCl2 solution (0.3 M, 0.24 mL), bile salt solution (200 mM, 12 mL), trypsin solution (800 U / mL, 30 mL), and pure water to a volume of 120 mL and mix thoroughly. To simulate small intestinal digestion, Angelica dahurica resistant starch was mixed with the intestinal digestive fluid at a ratio of 120:120 (w / v). The pH was adjusted to 7.6 and the mixture was incubated in a shaking water bath at 37°C and 200 rpm for 2 hours. The resulting samples were designated AS-I and RS-I.

[0077] Samples were collected after each digestion step and immediately heated at 100°C for 10 minutes to inactivate the enzymes. The resulting samples were freeze-dried and stored at -20°C. The resistant starch content of Angelica dahurica resistant starch after oral, gastric, and small intestinal digestion was determined enzymatically and gravimetrically, and their micromorphology was observed using a scanning electron microscope at 2000x magnification.

[0078] Enzymatic gravimetric method: Disperse 200 mg of sample evenly in 4 mL of distilled water, add 4 mL of sodium acetate solution (0.5 M, pH 5.6), and mix thoroughly. Equilibrate at 37°C for 30 min. Add amyloglucosidase and pancreatin, and incubate at 37°C, 200 rpm, and shake for 60 min. At 0, 20, and 120 min, 0.1 mL of the mixed sample was immediately mixed with 0.9 mL of 90% ethanol to inactivate the enzymes. The mixed solution was then centrifuged at 3000 rpm for 5 min. Glucose content in the supernatant was determined using a GOPOD kit (Beijing Solaibao Technology Co., Ltd., Beijing, China).

[0079] According to 0min(G0), 20min(G 20 )、120min(G 120 The glucose release of rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS) is calculated using the following formulas (3) to (5):

[0080] RDS(%)=(G 20 -G0)×0.9×100 / sample dry weight (3)

[0081] SDS (%) = (G 120 -G 20 )×0.9×100 / sample dry weight (4)

[0082] RS(%)=100%-RDS(%)-SDS(%) (5)

[0083] Results of the determination of the composition, hydration characteristics, particle morphology, particle size distribution, crystal structure, thermodynamic properties, and digestibility of Angelica dahurica resistant starch:

[0084] (1) Composition: The chemical composition of Angelica dahurica resistant starch is shown in Table 3. The yield of Angelica dahurica resistant starch extracted from the dried roots of Angelica dahurica was 10.50%. The moisture content was 8.25%, and the protein content was low at 0.047% to 0.006%. The fat and ash contents in Angelica dahurica resistant starch were both low. The Ap content of Angelica dahurica resistant starch was high, and the Am content increased to 31.66%. During the preparation of Angelica dahurica resistant starch, saccharifying enzymes and pullulanase acted on the α-1,6 glycosidic bond of the Ap molecule, which may be the reason for the increase in the Am content in the Angelica dahurica resistant starch sample. The Am content significantly affects the function and physicochemical properties of starch, such as the gelatinization, gelation, degradation and initial gel hardness of cooked starch. In addition, the Am content of Angelica dahurica resistant starch increased, the structure became denser, and its digestion resistance increased.

[0085] Table 3 Chemical components of resistant starch from Angelica dahurica

[0086]

[0087] (2) Hydration properties: WAI and WSI represent the water absorption capacity (swelling capacity) and solubility (solubility index) of starch granules at high temperatures, respectively. The water absorption capacity (WAI) of Angelica dahurica resistant starch is 2.92 g / g, and the solubility (WSI) of Angelica dahurica resistant starch is 0.72 g / g.

[0088] (3) Morphology of Angelica dahurica resistant starch granules: Figure 2 As shown, Angelica starch ( Figure 1 A1-A3) has a smooth surface, concave, clear outline, and a loose particle structure with large gaps between particles. In contrast, the micromorphology of Angelica dahurica resistant starch ( Figure 2 B1-B3) showed significant differences. Macroscopically, Angelica dahurica resistant starch was connected in blocks, with a smooth film-like surface and irregularly shaped fine particles inside. Compared with ungelatinized starch granules, the shapes and sizes of Angelica dahurica resistant starch granules were more diverse. The Angelica dahurica resistant starch granules had a denser structure and smaller gaps between granules. This may be because the crystal structure of the starch granules was destroyed during the gelatinization process, the intermolecular forces between starch molecules were weakened, and the interactions between granules increased. In addition, due to the intrusion of water molecules during the gelatinization process, the surface of the starch granules appeared to have a moist luster.

[0089] (4) Particle size distribution of Angelica dahurica starch: According to the particle size distribution test results, Angelica dahurica starch showed a unimodal distribution characteristic, with the particle size range concentrated in 3-15 μm, while Angelica dahurica resistant starch showed a bimodal distribution, with the main peak located in 5-17 μm and the secondary peak extending to the range of 50-200 μm. The particle size distribution of Angelica dahurica starch by volume is characterized as follows: 10% of the particles have a particle size <1.93 μm (D 0.1 ), 50% particle size <6.31μm (D 0.5 ), 90% of the particles are <11.51μm; the particle size distribution of Angelica dahurica resistant starch is characterized by: 10% of the particles are <3.26μm (D 0.1 ), 50% particle size <27.75μm (D 0.5 ), with 90% of the particles having a diameter <136.83 μm, indicating a more generalized distribution of Angelica dahurica starch particles. The volume moment average diameter (D[4,3]) for Angelica dahurica starch was 6.66 μm, while that for Angelica dahurica resistant starch increased significantly to 54.38 μm. The surface area average diameter (D[3,2]) for Angelica dahurica starch was 4.09 μm, while that for Ad-RS3 increased to 8.18 μm. These particle size distribution characteristics are highly consistent with the increased surface roughness and secondary aggregation morphology of Angelica dahurica resistant starch particles observed by scanning electron microscopy (SEM).

[0090] (5) Crystal structure characteristics: XRD patterns show that the angelica starch has a typical diffraction peak at 15°, 17°, 22° and 24°, and a typical diffraction peak at 5° (2θ), indicating that the crystal structure of AS is type B ( Figure 1 C). Compared with Angelica dahurica starch, the diffraction peak positions of Ad-RS3 changed significantly, with a decrease in characteristic peaks. This may be due to the expansion of the particles during gelatinization, which destroyed the original crystal structure, and the recrystallization during enzymatic hydrolysis and cooling, which formed a new crystal structure. The change in relative crystallinity reflects the degree of interaction between Ap microfibrils and the extent of microcrystalline filaments. The relative crystallinity of Angelica dahurica starch and Angelica dahurica resistant starch was measured. The relative crystallinity of Angelica dahurica resistant starch (55.94%) was significantly higher than that of Angelica dahurica starch (12.96%), indicating that Angelica dahurica resistant starch has a more ordered Ap double helix structure. This may be due to the release of more low-molecular-weight linear chain fragments, which facilitates the formation of a more organized double helix structure during the regeneration process, ultimately improving the integrity of the crystals. Combined with FT-IR analysis, the results show that dual enzymatic hydrolysis can promote the release and rearrangement of amylopectin chains in AS, forming a more organized double helix structure and improving the perfection of the crystals.

[0091] Table 4 Structural characterization of Angelica dahurica starch and Angelica dahurica resistant starch

[0092]

[0093] (6) Thermal characteristics analysis of Angelica dahurica resistant starch: The gelatinization temperature [To, Tp, Tc], ΔT and ΔH of Angelica dahurica starch and Angelica dahurica resistant starch are different. Angelica dahurica starch has only one exothermic peak ( Figure 1 A), while Angelica dahurica resistant starch has three exothermic peaks ( Figure 1 A), indicating that crystals of varying stabilities exist within the crystalline domains of Angelica dahurica resistant starch granules. The gelatinization parameters To, Tp, Tc, and ΔH of Angelica dahurica resistant starch were 72.07°C, 74.36°C, 81.82°C, and 23.34 J / g, respectively, all higher than those of Angelica dahurica starch, indicating that Angelica dahurica resistant starch has stronger gelatinization resistance. The wide ΔT (9.75°C) of Angelica dahurica resistant starch is associated with a higher relative crystallinity. The thermal properties of Angelica dahurica starch and Angelica dahurica resistant starch are consistent with the XRD results. During the gelatinization process, the gelatinization temperature is affected by the mass fraction of Am starch, Am content, Ap branching distribution, starch structure, additives, and relative crystallinity.

[0094] (7) Digestion characteristics analysis: To determine the digestibility of the samples, we analyzed the contents of RDS, SDS, and RS during in vitro digestion ( Figure 1 D) After double enzymatic hydrolysis and recrystallization, Angelica dahurica starch was converted into Angelica dahurica resistant starch, which had a significantly increased RS content of approximately 54.86%. The RS content of Angelica dahurica starch and Angelica dahurica resistant starch was compared during simulated in vitro digestion. In simulated saliva, Angelica dahurica starch had a higher digestible starch ratio, and its RDS and SDS values ​​were significantly higher than those of Angelica dahurica resistant starch. The RS content of Angelica dahurica resistant starch was approximately 86.76%, indicating strong resistance to α-amylase. In simulated gastric fluid, the digestible starch ratio of Angelica dahurica starch was still higher than that of Angelica dahurica resistant starch, with an RS content of approximately 82.68%, indicating that pepsin and gastric lipase had a weak effect on Angelica dahurica resistant starch. In simulated intestinal fluid, the digestible starch ratio of Angelica dahurica starch was higher, approximately 75%, the highest, indicating that most of the starch was completely digested. However, the digestible starch ratio of Angelica dahurica resistant starch was only approximately 18%, and the RS content was approximately 82.16%, indicating that Angelica dahurica resistant starch remains difficult to digest in intestinal fluid.

[0095] Example 4. Functional verification of angelica starch on high-fat diet obese mice

[0096] Experimental mice: Specific pathogen-free (SPF) C57BL / 6J mice (male, 20 ± 2 g), 7 weeks old, were purchased from Beijing Sibeifu Laboratory Animal Co., Ltd. (No. SCXK 2019-0010, Beijing, China). Before the experiment, all mice were adapted to feeding for 1 week. Laboratory parameter settings: temperature 22 ± 2 ° C; humidity (60 ± 5%); 12-h light / dark cycle, and standard food and water were given. The Institutional Ethics Committee of Chengdu University of Traditional Chinese Medicine approved all procedures (Protocol No. 2020-35), and animal handling followed the Guide for the Care and Use of Laboratory Animals of Chengdu University of Traditional Chinese Medicine. The experimental treatment groups are shown in Table 5:

[0097] Table 5 Experimental treatment groups

[0098]

[0099] Specific steps:

[0100] (1) Sample collection: Fecal samples were collected from the anal area every day starting 5 days before the mice were euthanized and then stored in a -80°C refrigerator for analysis. After 12 weeks of treatment, the mice were fasted for 12 hours, sedated with isoflurane, and blood was collected from the eyeballs. After being placed at room temperature for 60 minutes, the blood samples were centrifuged at 3000xg for 15 minutes in a 4°C centrifuge to obtain serum, which was then aliquoted and placed in a -80°C refrigerator for subsequent analysis. Liver and colon tissue samples were collected, weighed, fixed with 10% (v / v) phosphate-buffered formalin, and fixed with paraffin. The liver index was calculated according to the following formula: Liver index = liver weight / mouse body weight.

[0101] (2) Blood biochemical analysis: The total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-c), and low-density lipoprotein cholesterol (LDL-c) levels in serum were analyzed using a Mindray BS-200 fully automatic biochemical analyzer. The leptin (Elabscience, E-EL-M3008) and adiponectin levels in serum were determined using ELISA kits (R&D Systems, Inc., Mineapolis, MN 55413, USA).

[0102] (3) Tissue Section Analysis: Liver and colon tissues were fixed in 10% (v / v) phosphate-buffered formalin and then paraffin-fixed. Sections (3 μm) were sliced ​​and stained with hematoxylin and eosin. All sections were examined under a light microscope (Olympus D970; Olympus Optical Co., Japan).

[0103] (4) Determination of SCFA content: SCFAs, including acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid, were determined by GC analysis using a Constant 1300 gas chromatograph (Thermo Fisher Scientific, USA).

[0104] (5) 16S rDNA determination: All tissues were tested for 16S rDNA to study the dynamic changes in the composition of the intestinal flora.

[0105] The body weight and serum lipid levels of mice under different treatments were measured to analyze the effects of Angelica dahurica resistant starch on mice. Figure 2 As shown in A, there were significant differences in the weight gain of mice fed a high-fat diet ad libitum among the different intervention groups. The model group had the fastest weight gain, followed by HFD_L, HFD-M, and HFD_H, and all of them were greater than those in the NC group. This experimental result shows that Ad-RS3 can effectively control the weight gain of mice fed a high-fat diet, and the effect is positively correlated with the dose; the application of Angelica starch did not significantly affect the changes in the liver of mice ( Figure 2 B); Serum leptin levels in mice fed a high-fat diet were highest in the group that took a high dose of Angelica dahurica resistant starch, indicating that the addition of Angelica dahurica resistant starch helps increase serum leptin levels and regulates the appetite and metabolism of mice through leptin ( Figure 2 D); Model group mice showed abnormal blood lipids ( Figure 2 E-H) showed significantly increased TC, HDL-C, and LDL-C levels, and significantly decreased TG levels, compared with normal mice. In this study, all three doses of Angelica dahurica resistant starch significantly reduced serum TC, LDL-C, and HDL-C levels in a dose-dependent manner after 12 weeks of treatment, while TG levels did not increase significantly, demonstrating a potential anti-hyperlipidemic pharmacological effect. These results suggest that Angelica dahurica resistant starch is a promising bioactive starch for nutritional intervention strategies to alleviate hyperlipidemia.

[0106] To observe the histopathological changes, H&E staining was performed on liver and colon tissues ( Figure 3 No histological changes were observed in the control group. In contrast, the model group exhibited significant morphological changes in the periphery of the liver tissue, including diffuse vesicular steatosis, abnormal cell arrangement, and enlarged adipocytes, indicating a large accumulation of lipid droplets in the liver. After intervention with Angelica dahurica resistant starch, the fatty lesions in the livers of mice fed a high-fat diet were significantly improved, with hepatocytes rearranging in a regular pattern. In particular, intervention with a high dose of Angelica dahurica resistant starch significantly reduced fat accumulation in the mice.

[0107] The colonic tissue of mice in each group showed a clear layered structure, with tightly packed goblet cells and no significant pathological changes. However, compared with the NC group, the MD group showed more pronounced inflammatory cell infiltration (circular) in the colonic tissue. The intervention effect of angelica dahurica resistant starch was dose-dependent, with medium and high doses of angelica dahurica resistant starch inhibiting the infiltration of inflammatory cells in the colonic tissue. In summary, angelica dahurica resistant starch has a certain effect in inhibiting fat accumulation in mice fed a high-fat diet, preventing liver lesions, alleviating colon inflammation, and maintaining intestinal health.

[0108] like Figure 4 As shown in the results, after long-term high-fat diet consumption, the levels of acetic acid, propionic acid, and butyric acid in the colon of mice decreased significantly, with significant differences. However, these levels increased significantly after intervention with resistant starch, with the HFD_M diet showing a particularly significant effect. Furthermore, the levels of isobutyric acid, valeric acid, and isovaleric acid in the MD group showed slight changes compared to the NC group, and after RS3 intervention, they decreased toward the NC group, but without significant differences. These results suggest that Ad-RS3 can ferment and produce short-chain fatty acids, particularly acetic acid, propionic acid, and butyric acid, in the mouse colon, regulating the levels of these short-chain fatty acids and playing an important role in maintaining intestinal ecological balance.

[0109] like Figure 5 and Figure 6 As shown in the results, Angelica dahurica resistant starch can significantly improve the richness, diversity and uniformity of intestinal flora, maintain a healthy intestinal environment, and prevent diseases related to dysfunction of intestinal microbial communities; it can also help the intestinal flora of obese mice gradually develop towards normal and healthy mice, and increase the abundance of beneficial microorganisms. Compared with the model group, the abundance of Firmicutes in HFD_M and HFD_H was significantly reduced, and the abundance of Bacteroidota was significantly increased, which was consistent with the intestinal flora composition of normal mice in the NC group, indicating that taking Angelica dahurica resistant starch can help gradually restore the intestinal flora of obese mice to a level similar to that of the control group.

[0110] The above description is merely a preferred embodiment of the present application. It should be understood that the present application is not limited to the form disclosed herein and should not be construed as excluding other embodiments. Instead, the present application 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 related fields. Modifications and variations made by those skilled in the art that do not depart from the spirit and scope of the present application should be protected by the claims appended hereto.

Claims

1. Angelica dahurica resistant starch, characterized by: The angelica resistant starch is purified and prepared by adopting a double-enzyme method.

2. The resistant starch of Angelica dahurica according to claim 1, characterized in that: The dual enzyme purification method comprises the following steps: S1. The angelica starch is evenly dispersed in pure water to obtain an angelica starch suspension; S2. The Angelica starch suspension was gelatinized to obtain gelatinized Angelica starch, and high temperature α-amylase was added to the gelatinized Angelica starch, and then pullulanase was added to induce debranching to obtain enzymatic hydrolysis of Angelica starch paste; S3. The enzymatically hydrolyzed angelica starch paste is allowed to stand at low temperature for recrystallization, and then the precipitate is collected after centrifugation. The precipitate is dried, ground and sieved to obtain the angelica resistant starch.

3. The resistant starch of Angelica dahurica according to claim 1, characterized in that: The gelatinization temperature in S2 is 60-80°C.

4. The resistant starch of Angelica dahurica according to claim 1, characterized in that: The stirring speed in S2 is 200-300 r / min, and the stirring time is 10-15 min.

5. The resistant starch of Angelica dahurica according to claim 1, characterized in that: The process further includes adjusting the temperature to 50° C. to 60° C. and the pH to 4 to 5 after the stirring in S2 is completed.

6. The resistant starch of Angelica dahurica according to claim 1, characterized in that: The debranching reaction time is 4 to 6 hours.

7. The resistant starch of Angelica dahurica according to claim 1, characterized in that: The temperature of the low-temperature standing in S3 is 4° C., and the time of the low-temperature standing is 8 to 12 hours.

8. The resistant starch of Angelica dahurica according to claim 1, characterized in that: The drying temperature in S3 is 45° C. to 55° C., and the screening is performed using a No. 4 to No. 5 sieve.

9. The resistant starch of Angelica dahurica according to claim 1, characterized in that: The step of extracting the angelica starch comprises: Wash, peel and grind Angelica dahurica to obtain an Angelica dahurica sample; Washing and filtering the Angelica dahurica sample to obtain an Angelica dahurica filtrate; The filtrate of Angelica dahurica is allowed to stand for 8 to 16 hours, and the upper layer of yellow pulp water is removed to obtain the lower layer of Angelica dahurica starch sediment containing impurities; Add the angelica starch sediment to clean water and stir evenly, centrifuge, pour out the supernatant, scrape off the upper brown impurities, and take the white precipitate; The white precipitate is dried at 40-50° C., ground and sieved to obtain the angelica starch.

10. The use of the resistant starch of Angelica dahurica according to any one of claims 1 to 9, characterized in that: The application includes at least one of lowering blood sugar, avoiding obesity and improving intestinal flora.

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