Application of pea type iii resistance starch, preparation method, medicine

Abstract

This application discloses the application, preparation method, and drug of pea type III resistant starch, belonging to the field of biotechnology. It discloses the application of pea type III resistant starch in the preparation of drugs for the prevention and / or improvement of fatty liver disease related to metabolic dysfunction. In addition, this application found through experiments that, although both are resistant starches, RS3 has a more significant inhibitory effect on Akkermansia bacteria than RS2. More importantly, the conclusion of this application, that RS3 inhibits Akkermansia bacteria, breaks the traditional understanding of the prior art and provides a new approach for the inhibition of fatty liver disease related to metabolic dysfunction.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to the application, preparation method, and pharmaceutical of pea type III resistant starch. Background Technology

[0002] Metabolic dysfunction-associated fatty liver disease (MASLD) is the most common chronic liver disease worldwide, characterized by excessive lipid accumulation in the liver and closely associated with obesity, type 2 diabetes, and cardiovascular disease. Dietary intervention, particularly dietary fiber supplementation, has been proven to be an effective strategy for improving MASLD. Resistant starch (RS), as a novel type of dietary fiber, is not digested in the small intestine but can enter the colon and be fermented by gut microbiota to produce beneficial metabolites such as short-chain fatty acids, thereby playing a role in improving glucose and lipid metabolism and regulating gut microbiota.

[0003] Based on their source and anti-digestion mechanism, resistant starch is mainly classified into RS2 (natural resistant starch granules) and RS3 (retrograde starch, usually formed by recrystallization after gelatinization). Studies have shown that different types of resistant starch differ significantly in structural characteristics and physiological functions. RS3 exhibits higher thermal stability and better maintains its anti-digestion properties during food processing.

[0004] In recent years, research on the effects of RS3 on metabolic diseases has been increasing. One study reported that RS3 intervention significantly reduced serum total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C) levels and liver fat content in mice fed a high-fat diet, and increased the relative abundance of Bacteroidetes and the concentration of short-chain fatty acids in the gut. Another study showed that potato RS3 can improve glucose tolerance and insulin resistance in type 2 diabetic mice, alleviate pathological damage to the liver and pancreas, and regulate gut microbiota structure.

[0005] Although some studies suggest that Akkermansia muciniphila (AKK) has antibacterial properties, its abundance increases under low-fiber dietary conditions, and it participates in intestinal barrier disruption. Literature reports indicate that under conditions of dietary fiber deficiency (such as Western diets), the abundance of Akkermansia muciniphila (AKK), a mucin-specific degrading bacterium, is significantly increased in the gut. This bacterium uses intestinal mucus layer proteins as a substitute nutrient source, continuously upregulates the expression of mucin-degrading enzymes, and directly degrades the colonic mucus layer, leading to mucus layer thinning and impaired intestinal barrier function.

[0006] Excessive proliferation of *Amycosis ulmoides* (AKK) can increase intestinal permeability, promote bacterial product translocation, and exacerbate liver inflammation and the progression of MASLD. In a MASLD-related dietary model (co-exposure to fructose and food preservatives), *Amycosis ulmoides* can exhibit more than a four-fold overproliferation. Its mucin degradation properties disrupt the intestinal mucus layer, increase intestinal permeability, and promote the translocation of bacterial products (such as lipopolysaccharide) to the liver, thereby exacerbating liver inflammation and driving the occurrence and development of MASLD. These studies suggest that increased abundance of *Amycosis ulmoides* is closely related to the pathological process of MASLD, and reducing *Amycosis ulmoides* abundance may be an important intervention strategy for improving MASLD.

[0007] Furthermore, the interaction between starch and AKK bacteria has been studied to some extent in existing technologies, such as Luk-In S, Leepiyasakulchai C, Saelee C, et al. Impact of resistant starch type 3 on fecal microbiota and stool frequency in Thai adults with chronic constipation randomized clinical trial[J]. Scientific reports, 2024, 14(1): 27944. It records: "Changes in bacterial abundance were evaluated using linear discriminant analysis effect size (LEfSe) and linear discriminant analysis (LDA) coupled with effect size analysis. Results from the LEfSe analyses are shown as bar plots for the phylum and genus levels, displaying a significant taxon for each particular group (Fig. 6). A higher LDA score indicates that there is a significantly higher relative taxon abundance after 12 weeks of intervention compared to that at baseline. The bacterial phylum of Euryarchaeota had a significantly higher relative phylum abundance in particular group of the cohort on RS-3 with an LDA score of 3.61 (Fig.6b).At the genus level, Bifidobacterium showed a significantly higher relativeabundance after 12 weeks on RS-3, with the highest LDA score (5.53) followed by Akkermansia (4.7), Fusobacterium (4.6), Catenibacterium (4.49) and Megamonas (4.43), as shown in Fig. 6d. They also had significantly lower relative taxon abundances of Bacteroides (-5.2), Alistipes (-4.79), Parabacteroides (4.62) and Eubacterium hallii group (-4.47). However, in thecohort on placebo, five significantly higher relative genera abundances were found (Fig. 6c): Collinsella (4.82), Catenibacterium (4.71), Holdemanella (4.48), Streptococcus (4.46) and Escherichia / Shigella (4.44) states: “Changes in bacterial abundance were assessed using a combination of linear discriminant analysis (LEfSe) and linear discriminant analysis (LDA). LEfSe results were presented as bar graphs at the phylum and genus levels, showing the taxonomic units of significance in each specific group. Higher LDA scores indicated a significant increase in the relative abundance of the taxonomic unit after 12 weeks of intervention compared to baseline. In the RS-3 group, the relative abundance of the specific taxonomic unit Archaea increased significantly, with an LDA score of 3.61. At the genus level, Bifidobacterium showed a significant increase in relative abundance after 12 weeks of RS-3 exposure, with the highest LDA score (5.53), followed by Akkermansia (4.7), Freund's (4.6), Streptococcus (4.49), and Megacoccus (4.43). In addition, the relative abundance of these genera was significantly reduced, such as Bacteroides (-5.2), Alisteria (-4.79), Parabacteroides (4.62), and Eubacterium halys (-4).47). However, in the placebo group, the relative abundance of five genera was significantly increased: *Colincera* (4.82), *Streptobacter* (4.71), *Holdmanna* (4.48), *Streptococcus* (4.46), and *Escherichia / Shigella* (4.44). This indicates that the literature suggests that treatment with RS3 starch leads to an increase in Akkermania abundance.

[0008] The technical problem to be solved by this application is: how to provide a method to protect the intestinal barrier from the perspective of inhibiting the abundance of Akkermansia to prevent and / or improve fatty liver disease associated with metabolic dysfunction. Summary of the Invention

[0009] The purpose of this application is to provide a way to enhance the intestinal barrier by suppressing the abundance of Akkermansia, thereby preventing and / or improving fatty liver disease associated with metabolic dysfunction.

[0010] To address the aforementioned issues, this application provides the use of pea type III resistant starch in the preparation of medicaments for the prevention and / or improvement of fatty liver disease associated with metabolic dysfunction.

[0011] It should be noted that this application also attempted to test with type II resistant starch in the experiment. The test results showed that the type III resistant starch prepared in this application had a more significant inhibitory effect on the abundance of Akkermansia compared with type II resistant starch. Moreover, regardless of whether it was type II resistant starch or type III resistant starch, the test in this application showed that it had an inhibitory effect on Akkermansia, which is contrary to the phenomenon described in the prior art.

[0012] Preferably, the pea type III resistant starch prevents and / or improves fatty liver disease associated with metabolic dysfunction by inhibiting the abundance of Akkermansia in the gut.

[0013] In addition, this application also discloses the use of pea type III resistant starch in the preparation of drugs that inhibit the abundance of Akkermansia in the intestine.

[0014] In addition, this application also discloses a method for preparing pea type III resistant starch, comprising the following steps: Step 1: Add water to pea starch to form starch milk, then gelatinize it once. After gelatinization, cool it and then let it stand at 2-6℃ for 10-14 hours to age it, thus obtaining aged starch. Step 2: Dry, pulverize and sieve the primary aged starch, then add water again to form a starch milk and perform secondary gelatinization. After secondary gelatinization, cool and place at a temperature of 2-6℃ for static aging for 10-14 hours to obtain secondary aged starch. Step 3: Dry, pulverize and sieve the secondary aged starch to obtain pea type III resistant starch.

[0015] Preferably, step 1 specifically includes the following sub-steps: Step A1: Mix pea starch with water to obtain a starch milk with a starch concentration of 0.18-0.22 kg / L; Step A2: Heat the starch milk obtained in step A1 at 118-123℃ to gelatinize for at least 30 minutes to obtain primary gelatinized starch;

[0016] Step A3: Cool the primary gelatinized starch obtained in step A2 and then place it at a temperature of 2-6°C for static aging for 10-14 hours to obtain primary aged starch.

[0017] Preferably, step 2 specifically includes the following sub-steps: Step B1: Dry, pulverize and pass the once aged starch through a 40-mesh sieve, then mix it with water to obtain a starch milk with a starch concentration of 0.18-0.22 kg / L; Step B2: Heat the starch milk obtained in step B1 at 118-123℃ to gelatinize for at least 30 minutes to obtain secondary gelatinized starch; Step B3: Cool the secondary gelatinized starch obtained in step B2 and then place it at a temperature of 2-6°C for static aging for 10-14 hours to obtain secondary aged starch.

[0018] In addition, this application also discloses an Akkermansia inhibitor containing pea type III resistant starch prepared by the above method.

[0019] The beneficial effects of this application are: This application provides a pea type III resistant starch that enhances the intestinal barrier by inhibiting the abundance of Akkermansia, thereby preventing and / or improving fatty liver disease associated with metabolic dysfunction. Furthermore, this application found through experiments that, although both are resistant starches, RS3 has a more significant inhibitory effect on Akkermansia than RS2. More importantly, the conclusion of this application, that RS3 inhibits Akkermansia, breaks with the traditional understanding of the prior art and provides a new approach to the inhibition of fatty liver disease associated with metabolic dysfunction. Attached Figure Description

[0020] Figure 1 Scanning electron microscope images of GRS3 and MRS2; Figure 2 Infrared spectra of GRS3 and MRS2 (without deconvolution); Figure 3 Infrared spectra of GRS3 and MRS2 (deconvolutioned); Figure 4 X-ray diffraction patterns of GRS3 and MRS2; Figure 5Oil Red O staining image of mouse liver sections; Figure 6 Figure showing the results of Oil Red O staining of mouse liver sections; Figure 7 This is a graph showing the relative content analysis of alanylhistidine; Figure 8 This is a graph showing the relative content analysis of isopentenyl adenine; Figure 9 This is a graph showing the relative content of tyrosine. Figure 10 The graph shows the relative content analysis of N-acetyl-L-phenylalanine. Figure 11 The graph shows the relative content analysis of dopamine 4-O-β-D-glucuronide. Figure 12 Principal coordinate analysis (PCoA) diagram of cecal microbiome metagenomics; Figure 13 Venn diagram of metagenomics of the cecal microbiota; Figure 14 This is a graph showing the species abundance analysis between sections at the Akkermansia genus level. Figure 15 A plot showing the intergroup species abundance analysis at the species level for Akkermansia_sp. Figure 16 A graph illustrating the contribution of cecal microbial species and functions; Figure 17 A graph showing the contribution of Akkermansia to metabolic pathways; Figure 18 A graph showing the contribution of Akkermansia to the amino acid biosynthesis pathway; Figure 19 This is a graph showing the contribution of Akkermansia to the cofactor biosynthesis pathway. Detailed Implementation

[0021] In the description of this invention, it should be noted that unless specific conditions are specified in the examples, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0022] Example 1: Preparation of Pea Type III Resistant Starch (1) Raw material processing: Pea starch is selected as raw material and 20% starch milk is prepared according to the ratio of starch (w): distilled water (v) = 1:5. For example, 1 kg of starch corresponds to 5 L of water. (2) Gelatinization: The starch milk is heated at 121°C for 30 minutes to gelatinize it, so that the starch granules are completely broken down; (3) Retrogradation: After the gelatinized starch paste is cooled to room temperature, it is placed in an environment of 4℃ for 12 hours for aging treatment, so that the starch molecules recrystallize to form resistant starch. (4) Drying: Dry, pulverize, and pass the aged sample through a 40-mesh sieve; (5) Secondary gelatinization: The sieved sample is mixed with distilled water to make a 20% starch milk, and then the starch milk is heated at 121℃ for 30 minutes to gelatinize it so that the starch particles are completely broken. (6) Secondary aging and retrogradation: After cooling the starch paste after secondary gelatinization to room temperature, place it in an environment of 4℃ for 12 hours for secondary aging treatment; (7) Secondary drying: The sample after secondary aging is dried, crushed and passed through a 40-mesh sieve to obtain pea type III resistant starch.

[0023] Example 2: Structural characterization of pea type III resistant starch Pea RS3 was prepared using the same method as in Example 1, and its structure was characterized as follows: Resistant starch content determination: The resistant starch content was determined using the Megazyme resistant starch assay kit. The results showed that the resistant starch content of the pea RS3 of this invention was 18.26%.

[0024] Scanning electron microscopy (SEM): The sample is sputter-coated with gold and then observed under a scanning electron microscope. For example... Figure 1 The results show that GRS3 is in the form of irregular granules with a diameter of about 8 μm, and the surface morphology is irregular fragments or flakes with folded structures, which are typical characteristics of starch structural reorganization during gelatinization and aging. MRS2 is in the form of ellipsoidal granules with a diameter of about 7 μm and a relatively smooth surface.

[0025] Fourier Transform Infrared Spectroscopy (FT-IR) Analysis: A Bruker Vertex 70 FT-IR spectrometer was used. The KBr pellet method was employed, with approximately 100 mg of KBr mixed thoroughly at a 1% ratio, ground, and then pelleted for analysis. Test parameters: Scan wavenumber range 4000–800 cm⁻¹. -1 The resolution is 4cm. -1 The scan was performed 64 times, with a KBr blank film used as the background. The infrared spectrum of the sample was deconvolved using Fourier transform software (OMNIC), with a full width at half maximum (FWHM) of 20 cm⁻¹. -1 With an enhancement factor of 1.9, calculate the corresponding peak area. (Reference) Figure 2-3 1022cm -1 The nearby absorption peaks indicate the structural characteristics of the amorphous region of starch, corresponding to the random coil structure of the starch macromolecule; 995 cm⁻¹ -1 The nearby absorption peak is mainly due to the bending vibration of C-OH, corresponding to the hydrogen bond structure formed between the hydroxyl groups of the starch macromolecule; 930 cm⁻¹-1 The nearby absorption peaks represent vibrational absorption peaks of the glucose ring. The results show that there is no significant difference between the two in the relative proportions of the crystalline and amorphous phases.

[0026] X-ray diffraction (XRD) analysis: Powder diffraction was used to analyze resistant starch using X-ray diffraction scanning. The sieved sample powder was filled into the grooves of the sample plate, leveled with a glass slide to ensure uniformity and complete coverage, guaranteeing the sample surface was flush with the sample plate without any protrusions, depressions, or cracks. The plate was then placed on the sample stage for analysis. Test parameters: characteristic X-ray CuKa, measurement voltage 40 kV, current 40 mA, diffraction angle (2θ) 5°–60°, scanning speed 5° / min, step size 0.02°. Relative crystallinity was calculated using Jade 6.0 analysis software. (Reference) Figure 4 GRS3 formed a more stable B-type crystal structure. The relative crystallinity levels of the two were similar, with MRS2 slightly higher than GRS3, consistent with the trend of the 1047 / 1022 ratio results in infrared spectroscopy analysis. B-type starch generally exhibits higher thermal stability and digestibility. The B-type crystal structure of GRS3 may constitute the structural basis for its superior performance compared to MRS2 in subsequent biological effects.

[0027] Example 3: Animal experiments on the improvement of MASLD by pea RS3 1. Animal grouping and treatment Seven-week-old male C57BL / 6J mice were selected and, after one week of acclimatization feeding, were randomly divided into the following four groups (n=8 per group): Normal control group (N group): fed with ordinary maintenance diet.

[0028] Model control group (Group H): fed a high-fat, high-fructose, and high-cholesterol diet (the diet contained 20% fat, 20% fructose, and 2% cholesterol).

[0029] MRS2 group (M group): fed a high-fat, high-sugar, and high-cholesterol diet supplemented with 4% MRS2 (type II resistant starch).

[0030] GRS3 group (G group): fed a high-fat, high-sugar, and high-cholesterol diet supplemented with 4% GRS3 (type III resistant starch).

[0031] All mice were allowed free access to food and water and were fed continuously for 10 weeks. After the experiment, the mice were sacrificed, and a portion of the liver tissue was collected for Oil Red O staining. The remaining liver tissue and cecal contents were flash-frozen in liquid nitrogen and stored at -80°C for later use.

[0032] 2. Liver oil red O staining Liver tissue was fixed with 4% paraformaldehyde, then dehydrated using a gradient of 30% sucrose solution until the tissue settled. It was then embedded in OCT and frozen at -20°C to section thickness, with sections of 8-10 μm. After air-drying at room temperature, the sections were fixed with 10% neutral formalin for 5-10 min, briefly washed with 60% isopropanol, and stained in freshly prepared 0.5% Oil Red O working solution (isopropanol:distilled water = 3:2 dilution) for 15-20 min in the dark. Background staining was then removed by differentiation with 60% isopropanol, followed by a brief wash with distilled water. The nuclei were counterstained with hematoxylin for approximately 1-2 min, and after the stain returned to blue under running water, the sections were mounted with glycerol gelatin or aqueous mounting medium. Under a light microscope, lipid droplets appeared orange-red to bright red, and the nuclei appeared blue. (Reference) Figure 5-6 Compared with group N, group H showed a significant increase in average lipid droplet size and lipid droplet number density, while group G showed a significant decrease. Group M showed a decreasing trend, but this was not statistically significant.

[0033] 3. Liver metabolomics analysis Liver samples were collected for non-targeted metabolomics analysis. After pretreatment, liver tissue was analyzed using a Vanquish (Thermo Fisher Scientific) ultra-high performance liquid chromatograph (UPLC) with a Waters ACQUITY UPLC BEH Amide column (2.1 mm × 50 mm, 1.7 μm) to separate the target compounds. Phase A of the HPLC was aqueous, containing 25 mmol / L ammonium acetate and 25 mmol / L ammonia, while Phase B was acetonitrile. Sample tray temperature: 4℃, injection volume: 2 μL. An Orbitrap Exploris 120 mass spectrometer was used for primary and secondary mass spectrometry data acquisition under the control software (Xcalibur, version 4.4, Thermo). Detailed parameters are as follows: sheath gas flow rate 50 Arb, auxiliary gas flow rate 15 Arb, capillary temperature 320℃, full-scan mass spectrometry resolution 60,000, tandem mass spectrometry resolution 15,000, collision energy SNCE 20 / 30 / 40, spray voltage 3.8 kV (positive ion mode) or -3.4 kV (negative ion mode). Reference Figure 7-11 The liver metabolic profile of the RS3 group mice in this invention was significantly different from that of the model group and the commercially available RS2 group. The livers of the G group showed significant upregulation of five key endogenous metabolites: tyrosine, N-acetyl-L-phenylalanine, alanylhistidine, dopamine 4-O-β-D-glucuronide, and isopentenyl adenine, with some even exceeding those in the N group, suggesting that it may be more effective than MRS2 in improving liver metabolic reprogramming.

[0034] 4. Cecal metagenomic analysis Total DNA was extracted from cecal contents samples for metagenomic sequencing analysis. DNA extraction was performed using the FastPure Stool DNAIsolation Kit (Magnetic bead) (MJYH, Shanghai, China). After extraction, DNA concentration and purity were assessed, and DNA integrity was checked using 1% agarose gel electrophoresis. DNA fragmentation was performed using a Covaris M220 (Genetron Health, China), selecting fragments of approximately 350 bp for PE library construction. Quality control, OTU clustering, species taxonomic annotation, and microbial community diversity analysis were performed on the sequencing data using the QIIME2 software package and R (version 4.3.0). References Figure 12-19 β-diversity analysis (PCoA) results showed significant differences in cecal microbial species among groups N, H, G, and M. Further Venn diagram analysis revealed differences in species composition among the groups. Inter-group microbial abundance results showed that at the genus level, compared to group N, the abundance of *Akkermansia* was significantly increased in group H and significantly decreased in group G, but no significant change was observed in group M. At the species level, compared to group N, the abundance of *Akkermansia_sp* was significantly increased in group H and significantly decreased in group G, but no significant change was observed in group M. Genus-level species and functional contribution analysis showed that *Akkermansia*, as the most abundant genus, contributed more to multiple metabolic pathways in group H than in group N, and its contribution significantly decreased in group G, but this phenomenon was not observed in group M.

[0035] The above results indicate that the GRS3 prepared in this invention promotes the generation of beneficial metabolites by more effectively regulating the composition and function of gut microbiota, thereby demonstrating a superior effect to MRS2 in improving MASLD.

[0036] Based on the metabolomics and metagenomics results of Example 3, the following comparative conclusions can be drawn: In summary, the GRS3 of this invention is superior to MRS2 in improving liver metabolism and gut microbiota associated with MASLD.

Claims

1. Application of pea type III resistant starch in the preparation of drugs for the prevention and / or improvement of fatty liver disease associated with metabolic dysfunction.

2. The application according to claim 1, characterized in that, The pea type III resistant starch prevents and / or improves fatty liver disease associated with metabolic dysfunction by inhibiting the abundance of Akkermansia in the gut.

3. Application of pea type III resistant starch in the preparation of drugs that inhibit the abundance of Akkermansia in the intestine.

4. A method for preparing pea type III resistant starch, characterized in that, Includes the following steps: Step 1: Add water to pea starch to form a starch milk, then gelatinize it once. After gelatinization, cool it and then place it at a temperature of 2-6℃ for aging for 10-14 hours to obtain primary aged starch. Step 2: Dry, pulverize and sieve the primary aged starch, then add water again to form a starch milk and perform secondary gelatinization. After secondary gelatinization, cool and place at a temperature of 2-6℃ for static aging for 10-14 hours to obtain secondary aged starch. Step 3: Dry, pulverize and sieve the secondary aged starch to obtain pea type III resistant starch.

5. The preparation method according to claim 4, characterized in that, Step 1 specifically includes the following sub-steps: Step A1: Mix pea starch with water to obtain a starch milk with a starch concentration of 0.18-0.22 kg / L; Step A2: Heat the starch milk obtained in step A1 at 118-123℃ to gelatinize for at least 30 minutes to obtain primary gelatinized starch; Step A3: Cool the primary gelatinized starch obtained in step A2 and then place it at a temperature of 2-6°C for static aging for 10-14 hours to obtain primary aged starch.

6. The preparation method according to claim 4, characterized in that, Step 2 specifically includes the following sub-steps: Step B1: Dry, pulverize and pass the once aged starch through a 40-mesh sieve, then mix it with water to obtain a starch milk with a starch concentration of 0.18-0.22 kg / L; Step B2: Heat the starch milk obtained in step B1 at 118-123℃ to gelatinize for at least 30 minutes to obtain secondary gelatinized starch; Step B3: Cool the secondary gelatinized starch obtained in step B2 and then place it at a temperature of 2-6°C for static aging for 10-14 hours to obtain secondary aged starch.

7. An Akkermansia inhibitor, characterized in that, The pea type III resistant starch is prepared by any of the preparation methods described in claims 4-6.

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