Preparation method for apios americana medic. resistant starch and use thereof

Abstract

Disclosed in the present invention are a preparation method for Apios americana Medic. resistant starch and use thereof, belonging to the technical field of pharmaceutical preparation. Probiotics (Lactobacillus and Bifidobacteria) are added to an Apios americana Medic. peeled fresh fruit homogenate for fermentation, filtration, aging, centrifugation, alcohol treatment, and drying to obtain the Apios americana Medic. resistant starch. The Apios americana Medic. resistant starch can play a role in preventing and / or treating constipation, significantly promotes the propulsion distance of activated carbon in the small intestine of constipated mice, reduces the excretion time of the first black stool, increases the number of black stool particles excreted within 6 h and the wet weight of the black stools excreted within 6 h, increases the contents of MTL and SP in serum, and increases the contents of lactic acid, acetic acid, propionic acid, and butyric acid in stools. In the present invention, probiotics (Lactobacillus and Bifidobacteria) are used for synergistic aging to prepare the Apios americana Medic. resistant starch, thereby achieving the efficient preparation of the Apios americana Medic. resistant starch. The content of the Apios americana Medic. resistant starch is 76.89%, the process is simple, the product purity is high, the energy consumption is low, and the method is suitable for industrial production.

Description

A method for preparing and applying resistant starch from *Sophora japonica* (Luo Han Shen). Technical Field

[0001] This invention belongs to the technical field of pharmaceutical preparation methods, and in particular relates to a method for preparing and applying resistant starch from *Sargassum fusiforme* (a type of ginseng). Background Technology

[0002] The statements herein provide only background information in relation to this invention and do not necessarily constitute prior art.

[0003] Constipation is a common gastrointestinal disorder characterized by bowel movement disorders, such as straining during defecation, prolonged defecation time, difficulty defecating, and pain and abdominal pain caused by straining. The prevalence in adults worldwide ranges from 2% to 28%. Constipation causes significant pain and inconvenience and may be related to diseases of the respiratory, cardiovascular, and nervous systems. Unhealthy lifestyles lead to intestinal diseases and abnormal bowel movements; however, the pathogenesis of constipation remains unclear. Currently, medications for treating constipation mainly focus on laxatives and prokinetic agents. However, long-term use of these medications may lead to drug dependence and other adverse reactions, such as dehydration, hypotension, tachycardia, dizziness, abdominal distension, and abdominal cramps. Therefore, it is necessary to explore safe and effective alternative treatments. Nutritional therapy, such as dietary adjustments, is a good option for treating constipation. The intake of functional substances from food sources to improve, prevent, and treat constipation is essential. Currently, an increasing number of food-derived functional ingredients are being discovered for their use in suppressing constipation.

[0004] Prebiotics have a wide range of effects in promoting bowel movements and are potential functional factors for improving constipation. Their mechanism involves beneficial gut bacteria, especially Bifidobacteria, fermenting prebiotics to produce large amounts of short-chain fatty acids (SCFAs), which stimulate intestinal peristalsis and increase stool moisture. Polysaccharide prebiotics extracted from traditional Chinese medicine, fruits, vegetables, and fungi have received widespread attention. These sugars have good anti-constipation effects with fewer side effects and have potential clinical application value. Exploring more food-derived active ingredients for inhibiting constipation and conducting in-depth research on their mechanisms of action is of great significance to human health.

[0005] Luo Han Shen (commonly known as taro), mainly cultivated in the southwestern plains of Shandong Province, is a perennial vine-like herb. Its tuberous roots are spherical or irregularly oblong, with a yellowish-brown skin and discontinuous ring-like texture; the flesh is white and tender. Luo Han Shen contains a high amount of resistant starch, making it a promising health resource for development and application. Studies have shown that the resistant starch content in both raw and cooked Luo Han Shen is higher than that of four common tubers: potatoes, taro, sweet potatoes, and yams, making it an ideal raw material for preparing resistant starch. Currently, Luo Han Shen is known to lower blood sugar and improve glucose tolerance in diabetic mice. Currently, Luo Han Shen in the domestic market is mainly used in medicinal cuisine; there is no further processing of Luo Han Shen to prepare active ingredients for use in the preparation of anti-constipation products. Research on the extraction technology of bioactive components from Luo Han Shen is a key focus of its application research. For the preparation of resistant starch, the methods reported in the literature mainly include physical methods, enzymatic hydrolysis, and compound methods. Traditional methods such as pressurization, ultrasound, ultra-high pressure, extrusion, microwave puffing, and single-enzyme hydrolysis are simple and quick, but the resulting products have poor resistant starch content. Physical methods combining multiple pressurization and cooling processes can increase resistant starch content, but the production cycle is long and the production cost is high. Enzymatic hydrolysis using multiple enzymes can significantly improve the content and purity of resistant starch, but the resulting products contain excessive residual hydrolysate and hydrolysis products. These hydrolysate and hydrolysis products mainly contain proteins and inorganic salts. Since proteins are pale yellow, high protein content will affect the whiteness of the starch, resulting in low product whiteness. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the purpose of this invention is to provide a method for preparing and applying resistant starch from *Siraitia grosvenorii* (Luo Han Shen). This invention utilizes a fermentation-assisted aging process to achieve highly efficient preparation of resistant starch from *Siraitia grosvenorii*.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] In a first aspect, the present invention provides a method for preparing resistant starch from *Sophora japonica*, wherein the resistant starch is prepared by a fermentation-assisted aging process, comprising the following steps:

[0009] The product is obtained by adding probiotics to a homogenate of peeled fresh ginseng fruit, followed by fermentation, filtration, aging, centrifugation, alcohol treatment, and drying; the probiotics are Lactobacillus and Bifidobacterium.

[0010] During their research, the inventors unexpectedly discovered that using Lactobacillus and Bifidobacterium together for fermentation could significantly increase the content of resistant starch in the prepared ginseng.

[0011] Preferably, the lactobacillus is selected from one or more of the following: *Lactobacillus reuteri* CICC-6132, *Lactobacillus casei* NRRL B-1922, *Lactobacillus rhamnosus* AS1.2466, *Lactobacillus rhamnosus* HN 001, *Lactobacillus acidophilus* NRRL B-4495, *Lactobacillus brevis* NRRL B-4527, *Lactobacillus reinhardtii* ATCC 7830, *Lactobacillus bulgaricus* ATCC 11842, *Lactobacillus delbrueckii* CICC 6045, *Lactobacillus plantarum* LP 194, *Lactobacillus acidophilus* TYCA 06, and *Lactobacillus helveticus* R0052; the bifidobacterium is selected from one or more of the following: *Bifidobacterium longum* NRRL B-41409, *Bifidobacterium brevis* NRRL B-41408, *Bifidobacterium brevis* M-16V, *Bifidobacterium adolescentis* ATCC 15703, *Bifidobacterium bifidum* ATCC 29521, *Bifidobacterium bifidum* R0071, and *Bifidobacterium lactis* HN. 019, one or more of Bifidobacterium animalis BB-12 and Bifidobacterium infantis R0033.

[0012] In one or more embodiments, the peeled fresh Luo Han Shen fruit is processed according to the following steps: select Luo Han Shen that is free from mold and insect infestation, wash it with clean water to remove surface dirt, peel it, drain the water, and then air dry it naturally.

[0013] Furthermore, the ginseng fruit homogenate is obtained by mixing peeled ginseng fruit with water and then performing a cell wall breaking process.

[0014] Further, peeled fresh Luo Han Shen fruit is mixed with water and homogenized. The solid-liquid ratio of the peeled fresh Luo Han Shen fruit to water is 1:2 to 10, in g / mL; preferably 1:3.

[0015] In one or more embodiments, a pH adjustment treatment is performed before adding probiotics, adjusting the pH value to 3.5–6.

[0016] Furthermore, the total amount of Lactobacillus and Bifidobacterium inoculated is 0.2% to 5% of the weight of the peeled fresh ginseng fruit, preferably 1% to 2%.

[0017] Furthermore, the lactobacillus and bifidobacteria work together, and the mass ratio of lactobacillus to bifidobacteria is 1:10 to 10:1, preferably 2:3 to 3:2.

[0018] In one or more embodiments, the fermentation temperature and time are 30–45°C and 12–80 h, respectively, preferably 35–40°C for 24–48 h.

[0019] In one or more embodiments, the filtration is a 2-4 layer gauze filtration, repeated 2-4 times.

[0020] In one or more embodiments, the aging temperature and time are 0–10°C and 24–120 h, respectively, preferably aging at 0–4°C for 24–48 h.

[0021] In one or more embodiments, after the aging process is completed, the supernatant is removed by centrifugation to collect the precipitate. The centrifugation to remove the precipitate is performed at 8000–15000 rpm for 10–20 min.

[0022] Further, the alcohol is ethanol; the mass ratio of the precipitate to the alcohol is 1:(2-6); preferably 1:(2-3).

[0023] Furthermore, the alcohol treatment is performed at a temperature of 4–25°C for a time of 6–24 h, preferably at 4–10°C for 10–14 h.

[0024] Secondly, the present invention provides resistant starch of Codonopsis pilosula prepared by the above method.

[0025] Thirdly, the present invention provides the application of the resistant starch prepared by the above method in the preparation of products that promote bowel movement and relieve constipation, wherein the products are health foods or functional foods.

[0026] Fourthly, the present invention provides the use of the resistant starch prepared by the above method in at least one of the following aspects:

[0027] 1) Application in the preparation of products for the prevention and / or treatment of constipation;

[0028] 2) Application in the preparation of products that promote intestinal peristalsis;

[0029] 3) Application in the preparation of products that improve bowel parameters;

[0030] 4) Application in the preparation of products that increase serum factor levels;

[0031] 5) Applications in the preparation of products with increased organic acid content;

[0032] The product in question is a pharmaceutical product.

[0033] In one or more embodiments, the constipation is functional constipation or chronic constipation.

[0034] In one or more embodiments, the intestine is the colon, cecum, or small intestine, specifically mouse small intestine tissue.

[0035] In one or more embodiments, the improved defecation parameters refer to improved constipation or constipation-related defecation parameters. Specifically, the defecation parameters may include the time to first black stool passage, the number of black stool particles passed in 6 hours, and the wet weight of black stool passed in 6 hours.

[0036] In one or more embodiments, the serum-improving factor is a constipation-improving serum factor or a constipation-related serum factor. Specifically, the serum factor may be motilin (MTL) and substance P (SP), etc.

[0037] In one or more embodiments, increasing the organic acid content means increasing the organic acid content in feces. The organic acids include lactic acid, acetic acid, propionic acid, and butyric acid, etc.

[0038] When the resistant starch from Luo Han Shen is used as a functional food or health food, it can be used alone or in combination with other ingredients.

[0039] In clinical applications, it can be used as a food component in special medical foods or health foods to help lubricate the intestines and promote bowel movements, thus preventing and / or treating constipation. It can also be prepared using conventional pharmaceutical processes, either alone or in combination with other drugs, into different dosage forms suitable for clinical use. For example, excipients, flavoring agents, and preservatives can be added to create capsules or oral solutions for the prevention and / or treatment of constipation.

[0040] In some embodiments, the dosage form of the drug includes capsules, oral solutions, injections, tablets, powders, or granules.

[0041] Compared with the prior art, the beneficial effects of the present invention are:

[0042] (1) This invention uses probiotics (Lactobacillus and Bifidobacterium) to synergistically age and prepare resistant starch of Scutellaria baicalensis, which realizes the efficient preparation of resistant starch of Scutellaria baicalensis. The content of resistant starch of Scutellaria baicalensis is 76.89%. The process is simple, the product has high purity, low energy consumption and is suitable for industrial production.

[0043] (2) The present invention utilizes probiotics (Lactobacillus and Bifidobacterium) to synergistically age resistant starch prepared from ginseng. In the prevention and / or treatment of constipation, probiotics and aging play a synergistic role, which can significantly prevent and / or treat constipation. It significantly promotes the propulsion distance of activated charcoal in the small intestine of constipated mice, reduces the time of first black stool excretion, increases the number of black stool particles excreted in 6 hours and the wet weight of black stool excreted in 6 hours, increases the content of MTL and SP in serum, and increases the content of lactic acid, acetic acid, propionic acid and butyric acid in feces; it has the effect of lubricating the intestines and relieving constipation.

[0044] (3) The resistant starch prepared by the present invention using probiotics (Lactobacillus and Bifidobacterium) through synergistic aging significantly promoted small intestinal peristalsis in diphenoxylate-induced constipation model mice, improved constipation parameters and serum factor levels, and increased the content of organic acids in feces. The present invention provides a new option for the treatment or prevention of constipation. Attached Figure Description

[0045] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0046] Figure 1 shows the effect of the *Smilax china* resistant starch prepared in Example 1 and Comparative Examples 1 and 2 of this invention on the small intestinal propulsion rate of diphenoxylate hydrochloride-induced constipation mice; where (A) is the small intestinal length, (B) is the activated carbon propulsion distance, and (C) is the small intestinal propulsion rate; data are expressed as mean ± SEM. Compared with the control group, *p<0.05 is significant, and **p<0.01 is highly significant; compared with the model group, #p<0.05 is significant, and ##p<0.01 is highly significant.

[0047] Figure 2 shows the effects of the *Luo Han Shen* resistant starch prepared in Example 1 and Comparative Examples 1 and 2 of this invention on defecation parameters in diphenoxylate-induced constipation mice; where (A) is the time to expel the first black stool, (B) is the number of black stool particles expelled at 6 hours, and (C) is the wet weight of black stool expelled at 6 hours; data are expressed as mean ± SEM. Compared with the control group, *p<0.05 is significant, and **p<0.01 is highly significant; compared with the model group, #p<0.05 is significant, and ##p<0.01 is highly significant.

[0048] Figure 3 shows the effect of the *Luo Han Shen* resistant starch prepared in Example 1 and Comparative Examples 1 and 2 of this invention on the serum factor content of diphenoxylate hydrochloride-induced constipation mice; where (A) is the MTL content and (B) is the SP content; the data are expressed as mean ± SEM. Compared with the control group, *p<0.05 is significant and **p<0.01 is highly significant; compared with the model group, #p<0.05 is significant and ##p<0.01 is highly significant.

[0049] Figure 4 shows the effect of the resistant starch prepared by *Gynostemma pentaphyllum* in Example 1 and Comparative Examples 1 and 2 of this invention on the content of organic acids in the feces of mice with diphenoxylate hydrochloride-induced constipation; where (A) is the lactic acid content, (B) is the acetic acid content, (C) is the propionic acid content, and (D) is the butyric acid content; the data are expressed as mean ± SEM. Compared with the control group, *p<0.05 is significant, and **p<0.01 is highly significant; compared with the model group, #p<0.05 is significant, and ##p<0.01 is highly significant. Detailed Implementation

[0050] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0051] Example 1 - Preparation of Resistant Starch from Ginseng by Probiotic Fermentation and Co-aging

[0052] Resistant starch from *Luo Han Shen* (Siraitia grosvenorii) was prepared using probiotic fermentation and co-aging. The preparation method is briefly described below: After homogenizing peeled fresh *Luo Han Shen* fruit, *Lactobacillus* and *Bifidobacterium* were added. The mixture was then fermented, filtered, aged, centrifuged, treated with alcohol, and dried to obtain the final product. The specific preparation method is as follows:

[0053] Take 10 kg of peeled fresh ginseng fruit and add it to a water reaction container. Add 30 L of distilled water and homogenize it for 5 min using a 500 W blender.

[0054] The pH was adjusted to 3.5–6.0 using citric acid. 1% (based on the weight of peeled fresh ginseng fruit) of *Lactobacillus rhamnosus* AS1.2466 (purchased from Shanghai Xuanke Biotechnology Co., Ltd.) and *Bifidobacterium bifidum* ATCC 29521 (purchased from Shanghai Fuxiang Biotechnology Co., Ltd.) were inoculated at a ratio of 3:2. Fermentation was carried out at 37°C for 48 hours. After fermentation, the mixture was filtered through four layers of gauze to remove the residue, and the filtration was repeated four times. The mixture was then aged at 4°C for 48 hours. After aging, the mixture was centrifuged at 10,000 rpm for 15 minutes, the supernatant was discarded, and the precipitate was collected. The upper protein layer was scraped off, and the protein was removed by repeated centrifugation three times. Three volumes of ethanol were slowly added to the precipitate (while stirring), and the mixture was precipitated at 4°C for 12 hours. Afterward, the mixture was centrifuged at 5,000 rpm for 10 minutes, the supernatant was discarded, and the precipitate was collected.

[0055]

[0056] Starch was prepared by freeze drying (yield 25.6%), with a whiteness of 87.19 and a purity of 99.1%, of which resistant starch content was 76.89%. Its composition is shown in Table 1.

[0057] Table 1

[0058]

[0059] Comparative Example 1 - Preparation of Resistant Starch from Ginseng by Probiotic Fermentation

[0060] The preparation method for resistant starch from Luo Han Shen (Siraitia grosvenorii) using probiotic fermentation is briefly described below: After homogenizing and filtering the peeled fresh Luo Han Shen fruit, probiotics are added for fermentation, followed by filtration, centrifugation, alcohol treatment, and drying. The specific preparation method is as follows:

[0061] Take 10 kg of peeled fresh monk ginseng fruit and add it to a water reaction vessel. Add 30 L of distilled water and homogenize using a 500 W blender for 5 min. Adjust the pH to 3.5–6.0 using citric acid. Inoculate with 1% (based on the weight of peeled fresh monk ginseng fruit) of *Lactobacillus rhamnosus* AS1.2466 and *Bifidobacterium bifidum* ATCC 29521, with a *Lactobacillus* to *Bifidobacterium* mass ratio of 3:2. Ferment at 37℃ for 48 h. After fermentation, filter through two layers of gauze to remove the residue. Repeat the filtration twice. Centrifuge at 10000 rpm for 15 min, discard the supernatant, and collect the precipitate. Scrape off the upper protein layer. Slowly add 3 times the volume of ethanol to the precipitate (while stirring), and precipitate at 4℃ for 12 h. Then centrifuge at 5000 rpm for 10 min, discard the supernatant, and collect the precipitate. Starch was prepared by freeze drying (yield of 18.5%), with a whiteness of 38.54 and a purity of 98.1%, of which resistant starch content was 30.56%. Its composition is shown in Table 2.

[0062] Table 2

[0063]

[0064] Comparative Example 2 - Preparation of Resistant Starch from Ginseng by Aging Method

[0065] Resistant starch from *Luo Han Shen* (Siraitia grosvenorii) was prepared using an aging method. The preparation method is briefly described below: Peeled fresh *Luo Han Shen* fruit is homogenized, aged, centrifuged, treated with alcohol, and dried to obtain the starch. The specific preparation method is as follows:

[0066] Take 10 kg of peeled fresh Luo Han Shen (Ginseng) fruit and add it to a water reaction vessel. Add 30 L of distilled water and homogenize using a 500 W blender for 5 min. Filter through two layers of gauze to remove the residue, repeat the filtration twice, and age at 4℃ for 48 h. After aging, centrifuge at 10000 rpm for 15 min, discard the supernatant, and scrape off the upper protein layer. Slowly add 3 times the volume of ethanol to the precipitate (while stirring), and precipitate at 4℃ for 12 h. Then centrifuge at 5000 rpm for 10 min, discard the supernatant, and collect the precipitate. Freeze-dry to obtain starch (yield 20.6%), with a whiteness of 40.63 and a purity of 98.3%, of which resistant starch content is 28.15%. Its composition is shown in Table 3.

[0067] Table 3

[0068]

[0069] Comparative Example 3: Preparation of Resistant Starch from *Lactobacillus thunbergii* via Fermentation

[0070] The preparation method for resistant starch from *Luo Han Shen* (Siraitia grosvenorii) using lactobacillus fermentation is briefly described below: After homogenizing and filtering the peeled fresh *Luo Han Shen* fruit, lactobacillus is added for fermentation, followed by filtration, centrifugation, alcohol treatment, and drying. The specific preparation method is as follows:

[0071] 10 kg of peeled fresh ginseng fruit was added to a water reaction vessel, along with 30 L of distilled water. The mixture was homogenized for 5 min using a 500 W blender. The pH was adjusted to 3.5–6.0 using citric acid. 1% (based on the weight of the peeled ginseng fruit) of *Lactobacillus rhamnosus* AS1.2466 was inoculated, and fermentation was carried out at 37°C for 48 h. After fermentation, the mixture was filtered through two layers of gauze to remove the residue. This filtration was repeated twice. The mixture was centrifuged at 10,000 rpm for 15 min, the supernatant was discarded, and the precipitate was collected. The upper protein layer was scraped off. Three times the volume of ethanol was slowly added to the precipitate (while stirring), and the mixture was precipitated at 4°C for 12 h. Afterward, the mixture was centrifuged at 5,000 rpm for 10 min, the supernatant was discarded, and the precipitate was collected. The precipitate was freeze-dried to produce starch (yield 16.3%) with a whiteness of 26.54 and a purity of 97.9%, of which resistant starch content was 20.12%. Its composition is shown in Table 4.

[0072] Table 4

[0073]

[0074] Comparative Example 4: Preparation of Resistant Starch from Scutellaria baicalensis by Fermentation of Bifidobacterium

[0075] The preparation method for resistant starch from *Siraitia grosvenorii* using Bifidobacterium fermentation is briefly described below: After homogenizing and filtering the peeled fresh *Siraitia grosvenorii* fruit, Bifidobacterium is added for fermentation, followed by filtration, centrifugation, alcohol treatment, and drying. The specific preparation method is as follows:

[0076] 10 kg of peeled fresh ginseng fruit was added to a water reaction vessel, along with 30 L of distilled water. The mixture was homogenized for 5 min using a 500 W blender. The pH was adjusted to 3.5–6.0 using citric acid. 1% (based on the weight of the peeled ginseng fruit) of *Bifidobacterium bifidum* ATCC 29521 was inoculated, and fermentation was carried out at 37°C for 48 h. After fermentation, the mixture was filtered through two layers of gauze to remove the residue. This filtration was repeated twice. The mixture was centrifuged at 10,000 rpm for 15 min, the supernatant was discarded, and the precipitate was collected. The upper protein layer was scraped off. Three times the volume of ethanol was slowly added to the precipitate (while stirring), and the mixture was precipitated at 4°C for 12 h. Afterward, the mixture was centrifuged at 5,000 rpm for 10 min, the supernatant was discarded, and the precipitate was collected. The precipitate was freeze-dried to produce starch (yield 14.6%) with a whiteness of 24.63 and a purity of 97.6%, of which resistant starch content was 16.43%. The composition is shown in Table 5.

[0077] Table 5

[0078]

[0079] Compared with Example 1, Comparative Examples 3 and 4 showed a significant increase in resistant starch content, and other parameters were also improved. It is speculated that this is because, in this environment, Lactobacillus and Bifidobacterium promote mutual growth and proliferation, producing a large number of extracellular enzymes and metabolites, which promote the formation of resistant starch.

[0080] Application example:

[0081] 1. Effect of resistant starch from *Tradescantia grosvenorii* on small intestinal propulsion rate in diphenoxylate-induced constipation mice.

[0082] Four-week-old female SPF-grade Kunming mice were acclimatized for one week in an SPF-grade animal room (temperature: 22±2℃; humidity: 50±10%; lighting: 12h light-dark alternation, 8:00-20:00 daytime). The mice were divided into 6 groups (n=8 per group): normal group, model group, positive control group, Comparative Example 1 (Luo Han Shen resistant starch group), Comparative Example 2 (Luo Han Shen resistant starch group), and Example 1 (Luo Han Shen resistant starch group). The administration period was 14 days, with mice administered via gavage at regular intervals every morning. The normal group and model group were administered distilled water by gavage; the positive control group was administered bisacodyl aqueous solution at a dose of 0.1 g / kg; the Comparative Example 1 (Luo Han Shen resistant starch group), Comparative Example 2 (Luo Han Shen resistant starch group), and Example 1 (Luo Han Shen resistant starch group) were administered the corresponding Luo Han Shen resistant starch homogenate by gavage at a dose of 1000 mg / kg. Mice were fed and administered samples via gavage for 14 days. On days 15 and 16, they were administered the corresponding samples again via gavage. After the sample gavage, all groups except the normal group were administered 30 mg / kg of diphenoxylate hydrochloride via gavage, while the normal group was administered distilled water via gavage. On day 17, mice were dissected to measure intestinal propulsion rate, time to first melena, wet weight of melena excreted at 6 hours, serum SP, MTL, and other indicators.

[0083] As shown in Figure 1A, the small intestinal length of diphenoxylate-induced constipation mice was significantly reduced compared to the normal group. Compared to the model group, the small intestinal length of mice in the positive control group, Comparative Example 1, Comparative Example 2, and the *Luo Han Shen* resistant starch intervention group of Example 1 increased by 8.0%, 2.0%, 1.0%, and 10.8%, respectively (p<0.05). As shown in Figure 1B, the activated charcoal propulsion distance of mice in diphenoxylate-induced constipation was significantly reduced compared to the normal group. Compared to the model group, the activated charcoal propulsion distance of mice in the positive control group and the *Luo Han Shen* resistant starch intervention group of Example 1 was significantly increased, with increases of 122.9% and 115.6%, respectively (both p<0.01), while the increase in Comparative Example 1 was only 11.2% (p>0.05), and the increase in Comparative Example 2 was only 5.7% (p>0.05). As shown in Figure 1C, the small intestinal propulsion rate of mice in diphenoxylate-induced constipation was significantly reduced. Compared with the model group, mice in the positive control group and the Luo Han Shen resistant starch intervention group of Example 1 showed significant increases, with increases of 129.7% and 128.9% respectively (both p<0.01), while the increase in Comparative Example 1 was only 7.4% (p>0.05) and the increase in Comparative Example 2 was only 1.9% (p>0.05).

[0084] 2. Effects of ginseng-resistant starch on defecation parameters in diphenoxylate-induced constipation mice

[0085] Animal grouping and intervention were the same as above. The results are shown in Figure 2A. Compared with the normal group, the time to expel the first black feces in diphenoxylate-induced constipation mice was significantly increased. Compared with the model group, the time to expel the first black feces in the positive control group and the *Luo Han Shen* resistant starch intervention group (Example 1) was significantly reduced, by 40.6% and 36.5% respectively (both p < 0.01), while the reduction in Comparative Example 1 was only 7.0% (p > 0.05), and the reduction in Comparative Example 2 was only 2.2% (p > 0.05). As shown in Figures 2B-2C, compared with the normal group, the number of black feces expelled at 6 hours and the wet weight of black feces expelled at 6 hours were significantly reduced in diphenoxylate-induced constipation mice. Compared with the model group, the number of black feces excreted at 6 hours and the wet weight of black feces excreted at 6 hours were significantly increased in the positive control group and the Luo Han Shen resistant starch intervention group of Example 1. The increases in Example 1 were 115.4% and 84.6% respectively (both p<0.01), while the increases in Comparative Example 1 were only 19.2% and 7.7% respectively (p>0.05), and the increases in Comparative Example 2 were only 7.8% and 2.3% respectively (p>0.05).

[0086] 3. Effects of ginseng-resistant starch on serum factor levels in diphenoxylate-induced constipation mice.

[0087] Animal grouping and intervention were the same as above. The results are shown in Figures 3A-3B. Compared with the normal group, the serum levels of factors MTL and SP in mice with diphenoxylate hydrochloride-induced constipation were significantly reduced. Compared with the model group, the serum levels of factors MTL and SP in the positive control group and the *Luo Han Shen* resistant starch intervention group (Example 1) were significantly increased. The increases in Example 1 were 30.9% and 69.1%, respectively (both p < 0.01), while the increases in Comparative Example 1 were only 5.6% and 6.9%, respectively (p > 0.05), and the increases in Comparative Example 2 were only 1.4% and 1.2%, respectively (p > 0.05).

[0088] 4. Effect of resistant starch from *Smilax china* on the content of organic acids in feces of mice with diphenoxylate-induced constipation.

[0089] Animal grouping and intervention were the same as above. As shown in Figures 4A-4D, compared with the normal group, the levels of lactic acid, acetic acid, propionic acid, and butyric acid in the feces of mice with diphenoxylate hydrochloride-induced constipation were significantly reduced. Compared with the model group, the levels of lactic acid, acetic acid, propionic acid, and butyric acid in mice in the positive control group and the *Luo Han Shen* resistant starch intervention group of Example 1 were significantly increased. Specifically, the levels in the *Luo Han Shen* resistant starch intervention group of Example 1 increased by 42.4%, 59.2%, 105.0%, and 292.6%, respectively (all p < 0.01), while the increases in Comparative Example 1 were only 4.9%, 9.3%, 13.0%, and 19.2%, respectively (p > 0.05), and the increases in Comparative Example 2 were only 2.0%, 2.1%, 3.2%, and 0.7%, respectively (p > 0.05).

[0090] In summary, the resistant starch prepared by probiotic fermentation and synergistic aging in Example 1 showed a more significant effect in application, far exceeding the effects of Comparative Example 1 and Comparative Example 2, indicating that probiotics and aging had a synergistic effect.

[0091] The principle behind this invention is as follows: During probiotic fermentation, the extracellular enzymes hydrolyze the amorphous and partially crystalline regions of starch granules, reducing the degree of starch polymerization and shortening the chain length. Simultaneously, organic acids produced during probiotic metabolism penetrate into the micelles of the starch granules, breaking the α-1,4-glycosidic bonds of the starch molecules, resulting in the breakdown of the chain-like molecules into shorter starch chains. The reduced molecular weight and lower degree of polymerization enhance the recrystallization ability of the starch molecules, making it easier to generate resistant starch under aging conditions. Therefore, the probiotic fermentation-assisted aging method can significantly increase the content of resistant starch.

[0092] Based on this principle, other lactobacilli and bifidobacteria that secrete extracellular enzymes and produce organic acids can also be used to prepare resistant starch from ginseng.

[0093] In some embodiments, the lactobacillus may be one or more of the following: *Lactobacillus reuteri* CICC-6132, *Lactobacillus casei* NRRLB-1922, *Lactobacillus rhamnosus* AS1.2466, *Lactobacillus rhamnosus* HN 001, *Lactobacillus acidophilus* NRRLB-4495, *Lactobacillus brevis* NRRLB-4527, *Lactobacillus reinhardtii* ATCC 7830, *Lactobacillus bulgaricus* ATCC 11842, *Lactobacillus delbrueckii* CICC6045, *Lactobacillus plantarum* LP 194, *Lactobacillus acidophilus* TYCA 06, and *Lactobacillus helveticus* R0052; the bifidobacterium may be *Bifidobacterium longum* NRRLB-41409, *Bifidobacterium brevis* NRRLB-41408, *Bifidobacterium brevis* M-16V, *Bifidobacterium adolescentis* ATCC 15703, and *Bifidobacterium bifidum* ATCC. One or more of the following: 29521, Bifidobacterium bifidum R0071, Bifidobacterium lactis HN019, Bifidobacterium animalis BB-12, and Bifidobacterium infantis R0033.

[0094] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included in the present invention.

Claims

1. A method for preparing resistant starch from *Sophora japonica*, characterized in that, Includes the following steps: The product is obtained by adding probiotics to a homogenate of peeled fresh ginseng fruit, followed by fermentation, filtration, aging, centrifugation, alcohol treatment, and drying; the probiotics are a combination of lactobacillus and bifidobacteria.

2. The method for preparing resistant starch from *Sophora japonica* according to claim 1, characterized in that, The lactobacilli are selected from one or more of the following: *Lactobacillus reuteri* CICC-6132, *Lactobacillus casei* NRRL B-1922, *Lactobacillus rhamnosus* AS 1.2466, *Lactobacillus rhamnosus* HN 001, *Lactobacillus acidophilus* NRRL B-4495, *Lactobacillus brevis* NRRL B-4527, *Lactobacillus reesei* ATCC 7830, *Lactobacillus bulgaricus* ATCC 11842, *Lactobacillus delbrueckii* CICC 6045, *Lactobacillus plantarum* LP 194, *Lactobacillus acidophilus* TYCA06, and *Lactobacillus helveticus* R0052; the bifidobacteria are selected from one or more of the following: *Bifidobacterium longum* NRRL B-41409, *Bifidobacterium brevis* NRRL B-41408, *Bifidobacterium brevis* M-16V, *Bifidobacterium adolescentis* ATCC 15703, *Bifidobacterium bifidum* ATCC 29521, *Bifidobacterium bifidum* R0071, and *Bifidobacterium lactis* HN. 019, one or more of Bifidobacterium animalis BB-12 and Bifidobacterium infantis R0033.

3. The method for preparing resistant starch from *Smilax china* according to claim 1, characterized in that, Take peeled fresh Luo Han Shen fruit and mix it with water, then homogenize it. The solid-liquid ratio of the peeled fresh Luo Han Shen fruit to water is 1:2 to 10, in g / mL; preferably 1:

3.

4. The method for preparing resistant starch from *Sophora japonica* according to claim 1, characterized in that, Before adding probiotics, perform pH adjustment treatment to adjust the pH value to 3.5-6.

5. The method for preparing resistant starch from *Sophora japonica* according to claim 1, characterized in that, The total amount of Lactobacillus and Bifidobacterium inoculated is 0.2% to 5% of the weight of the peeled fresh fruit of the ginseng, preferably 1% to 2%.

6. The method for preparing resistant starch from *Sophora japonica* according to claim 1, characterized in that, The mass ratio of Lactobacillus to Bifidobacterium is 1:10 to 10:1, preferably 2:3 to 3:

2.

7. The method for preparing resistant starch from *Sophora japonica* according to claim 1, characterized in that, The fermentation temperature and time are 30–45℃ and 12–80 h, respectively, with the preferred temperature being 35–40℃ for 24–48 h.

8. The resistant starch of *Gynostemma pentaphyllum* prepared by the method according to any one of claims 1 to 7.

9. The application of the resistant starch prepared by the method of preparing resistant starch of ginseng according to any one of claims 1 to 7 in the preparation of a product for relieving constipation, wherein the product is a health food or a functional food.

10. The application of the resistant starch prepared by the method of preparing resistant starch of *Tradescantia grosvenorii* according to any one of claims 1 to 7 in at least one of the following aspects: 1) Application in the preparation of products for the prevention and / or treatment of constipation; 2) Application in the preparation of products that promote intestinal peristalsis; 3) Application in the preparation of products that improve bowel parameters; 4) Application in the preparation of products that increase serum factor levels; 5) Application in the preparation of products that increase the content of organic acids in feces, wherein the organic acids are lactic acid, acetic acid, propionic acid and butyric acid; The product in question is a pharmaceutical product.

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