An oral preparation of short-chain amylopectin having a function of relieving colitis

Abstract

The application discloses a short branched amylose oral preparation with a function of relieving colitis, and belongs to the technical field of functional food. The relieving effect of the short branched amylose on colitis mice is further investigated in combination with clinical pathological features and inflammatory factor level changes. The experimental results show that the short branched amylose can improve the pathological state of DSS-induced colitis. In terms of immune regulation, the short branched amylose inhibits the release of proinflammatory factors (IL-6, IL-1beta and TNF-alpha), promotes the expression of anti-inflammatory cytokine IL-10, and reestablishes the balance of intestinal immune microenvironment, so that the short branched amylose can be used as a functional food for relieving colitis.

Description

Technical Field

[0001] This invention relates to an oral preparation of short-branched starch with the function of relieving colitis, belonging to the field of functional food technology. Background Technology

[0002] Colitis is a chronic, nonspecific inflammatory disease affecting the colon and terminal ileum. Its exact pathogenesis is not fully understood, but it is generally believed to be closely related to multiple mechanisms, including genetic susceptibility, abnormal immune regulation, gut microbiota dysbiosis, and environmental factors. Clinical manifestations are mainly characterized by recurrent abdominal pain, diarrhea, and bloody, mucous stools. Some patients may also experience tenesmus, weight loss, and fatigue. Epidemiological surveys show that the incidence and disease burden of colitis remain high in developed countries in Europe and America. In recent years, with changes in lifestyle and improved diagnostic capabilities in my country, the prevalence of colitis in my country has also shown a continuous upward trend, becoming one of the common digestive system diseases. Due to its prolonged course, high recurrence rate, and the potential increase in the risk of colon cancer due to long-term active lesions, patients' long-term quality of life is significantly affected.

[0003] Current clinical treatments primarily rely on aminosalicylic acid preparations, glucocorticoids, and immunosuppressants to control inflammatory activity. While these can relieve symptoms in the short term, long-term use often leads to adverse reactions such as gastric mucosal damage, endocrine disorders, and immunosuppression. Furthermore, the relapse rate after discontinuation of these medications is high, making long-term remission difficult to achieve. Therefore, identifying natural active ingredients or functional food ingredients with significant anti-inflammatory activity, good safety profile, and low toxicity has become an important research direction for improving colitis treatment strategies. Summary of the Invention

[0004] The technical problem that this invention aims to solve is to analyze the main anti-inflammatory components in short-chain starch and provide an anti-inflammatory food that can effectively relieve or help improve colitis with few side effects.

[0005] To address the aforementioned problems, this invention provides a method for preparing short-chain amylopectin using a dual-enzyme method, and clinical evaluation analysis reveals the main components that exert anti-inflammatory activity. This invention utilizes a dual-enzyme method to modify ordinary corn starch in the treatment of DSS-induced colitis mice. Clinical evaluation shows that short-chain amylopectin can improve colonic tissue pathology, reduce serum pro-inflammatory factors, and increase serum anti-inflammatory factors, thereby exerting an anti-inflammatory effect. The first objective of this invention is to provide a method for efficiently preparing short-chain starch using 6-α-glucosyltransferases from two different sources, comprising the following steps: (1) Add ordinary corn starch to deionized water, mix well, heat to gelatinize, and obtain ordinary corn starch pretreatment solution; (2) Adding ingredients derived from ordinary corn starch pretreatment solution to the solution Paenibacillus sp. 598K 6-α-glucosyltransferase (Pa-6 GT) was initially modified; (3) Boil the solution after enzymatic hydrolysis in step (2) to inactivate the enzyme, and add the enzyme-inactivated solution to the solution derived from Sporosacina globispora C11 6-α-glucosyltransferase (Sp-6GT) was modified twice; (4) Boil the modified starch solution obtained in step (3) to inactivate the enzyme, and freeze-dry the enzyme-inactivated solution.

[0006] In one embodiment of the present invention, the concentration of ordinary corn starch substrate in the ordinary corn starch pretreatment solution in step (1) is 5%-8% (w / w).

[0007] In one embodiment of the present invention, the ordinary corn starch is pretreated at 100°C and stirred at 350 rpm for 15 min.

[0008] In one embodiment of the present invention, the source of the encoding step (2) is... Paenibacillus sp. The nucleotide sequence of 598K 6-α-glucosyltransferase is shown in SEQ ID No. 1, and the amino acid sequence of the 6-α-glucosyltransferase is shown in SEQ ID No. 2. The final product of this enzyme acting on ordinary corn starch is short-chain amylopectin.

[0009] In one embodiment of the present invention, the source of the encoding step (2) is... Sporosacina globispora The nucleotide sequence of C116-α-glucosyltransferase is shown in SEQ ID No. 3, and the amino acid sequence of 6-α-glucosyltransferase is shown in SEQ ID No. 4.

[0010] In one embodiment of the present invention, the product includes functional foods, feed, or pharmaceuticals.

[0011] In one embodiment of the present invention, the enzyme inactivation method in step (3) is to boil the enzyme for 15 min.

[0012] A second objective of this invention is to provide the use of short-chain starch in the preparation of products that alleviate or help improve colitis.

[0013] In one embodiment of the present invention, the product includes feed or medicine.

[0014] In one embodiment of the present invention, the dosage of short-chain starch is 100-200 mg / kg.

[0015] In one embodiment of the present invention, the dosage form of the product is any medically recognized form, including tablets, capsules, granules, injections, liposome nanoparticles, sustained-release agents, dispersible tablets, or enteric-coated granules.

[0016] In one embodiment of the present invention, the pharmaceutical product further comprises a drug carrier and / or pharmaceutical excipients.

[0017] In one embodiment of the present invention, the pharmaceutical carrier includes one or more of the following commonly used in medicine: fillers, adhesives, wetting agents, disintegrants, lubricants, and flavoring agents.

[0018] In one embodiment of the present invention, the product is an oral preparation containing 75-80% by weight of short-chain starch, 14-18% of microcrystalline cellulose, and 1-2% of magnesium stearate.

[0019] Beneficial effects: This invention prepares a modified starch with extremely short branches. The chain length distribution results of the modified starch show that, compared with ordinary corn starch, the modified starch increases the content of DP1-12 branches and reduces the content of DP>36 branches. The dual-enzyme modification further increases the content of DP1-12 branches, with the content of DP1-12 branches reaching 94.82%.

[0020] Experimental results show that, compared with the model group, the high-dose short-chain starch group can significantly improve colitis symptoms, reduce inflammatory response, and promote the repair of colonic mucosa. Therefore, short-chain starch can be used as a functional food, medicine or health product to relieve colitis. Attached Figure Description Figure 1 The HPAEC-PAD results of the prepared short-chain starch are shown in the figure.

[0021] Figure 2 The HPAEC-PAD results of modified starch prepared by first using Sp-6GT and then using Pa-6GT are shown in the figure.

[0022] Figure 3 This is a flowchart of the mouse modeling process.

[0023] Figure 4 This is a representative image of a mouse colon.

[0024] Figure 5 A graph showing the length of the mouse colon.

[0025] Figure 6 Image of mouse colon tissue stained with H&E. Detailed Implementation

[0026] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0027] The culture media involved in the following examples are as follows: LB liquid medium: yeast extract 5 g / L, tryptone 10 g / L, NaCl 10 g / L, pH 7.0.

[0028] LB solid medium: yeast extract 5 g / L, tryptone 10 g / L, NaCl 10 g / L, pH 7.0, 1.5% (w / v) agar.

[0029] Fermentation liquid culture medium: yeast extract 24 g / L, tryptone 12 g / L, KH2PO4 2.32 g / L, K2HPO4 16.44 g / L, glycerol 5 g / L, pH 7.0.

[0030] Detection of short-chain starch: The sample of ordinary corn starch modified with Pa-6GT and Sp-6GT dual enzymes was boiled for 15 min to inactivate the enzymes, centrifuged, diluted, and filtered through a 0.22 μm aqueous membrane. The products were then analyzed using a high-performance anion exchange chromatography system (HPAEC-PAD). The HPAEC-PAD conditions were as follows: Dionex CarboPac PA-200 anion exchange column, column temperature 35°C, mobile phase 100 mM NaOH, 100 mM NaOH & 500 mM NaAc, flow rate 0.5 mL / min.

[0031] Example 1: Expression of recombinant 6-α-glycosyltransferase The specific steps are as follows: (1) Strain construction: Primers were designed based on the 6-α-glucosyltransferase gene. For Pa-6GT, the upstream primer was 5'-CCATGGCCGCGGGC-3', and the downstream primer was 5'-GCGCGCCGCTCGAG-3'. Using the artificially synthesized gene described in SEQ ID No. 1 as a template, PCR was performed using these primers to obtain the Pa-6GT gene containing the signal peptide. For Sp-6GT, the upstream primer was 5'-CCATGGCCTATGTGAGCAG-3', and the downstream primer was 5'-GCGTGACCAAACAGCTCGA-3'. Using the artificially synthesized gene described in SEQ ID No. 3 as a template, PCR was performed using these primers to obtain the Sp-6GT gene containing the signal peptide. The nucleotide sequences encoding the 6-α-glucosyltransferase signal peptide are shown in SEQ ID No. 5. Based on the pET-20b(+) vector sequence, upstream primer 5'-TGCCCAGCCGGCGATGGCCATGGCCTATGTGA-3' and downstream primer 5'-CTCGAGCACCACCACCACCACCACTGAGATCCGGCTGC-3' were designed to clone the pET20b(+) vector containing the linker site.

[0032] The PCR system consisted of 25 µL of 2×phanta Max Master Mix (Dye plus), 2 µL of forward primer (20 µM), 2 µL of reverse primer (20 µM), 1 µL of template DNA, and double-distilled water to a final volume of 50 µL. The PCR amplification conditions were: 95℃ pre-denaturation for 3 min; followed by 30 cycles (95℃ for 15 s, 60℃ for 15 s, 72℃ for 3.5 min); and finally, incubation at 72℃ for 5 min. The PCR system for the pET20b(+) vector consisted of 25 µL of 2×phanta Max Master Mix (Dye plus), 2 µL of forward primer (20 µM), 2 µL of reverse primer (20 µM), 1 µL of template DNA, and double-distilled water to a final volume of 50 µL. The PCR amplification conditions were as follows: pre-denaturation at 95℃ for 3 min; followed by 30 cycles (95℃ for 15 s, 60℃ for 15 s, 72℃ for 4 min); and finally incubation at 72℃ for 5 min.

[0033] The PCR products of the 6-α-glucosyltransferase gene and the pET20b(+) vector were subjected to nucleic acid electrophoresis and then recovered by gel extraction. The 6-α-glucosyltransferase gene and the pET20b(+) vector were ligated using homologous recombination. The ligation was then performed on *E. coli* JM109, plated on LB agar plates containing 20 μg / mL ampicillin, and transformants were picked for sequencing and nucleic acid electrophoresis verification to obtain the expression vectors pET-20b(+) / sp and pET-20b(+) / Pa containing the 6-α-glucosyltransferase gene. The expression vectors were then transformed... Escherichia coli BL21(DE3) yielded genetically engineered bacteria. E. coli BL21(DE3)(pET-20b(+)) / sp and E. coli BL21(DE3)(pET-20b(+)) / Pa.

[0034] (2) Activation: 100 μL containing genetically engineered bacteria E. coli BL21(DE3)(pET-20b(+)) / sp and E. coli The glycerol stock solution of BL21(DE3)(pET-20b(+)) / Pa was inoculated into 50 mL of LB medium (1% tryptone, 0.5% yeast extract, 1% sodium chloride, pH 7.0) containing 20 μg / mL ampicillin and cultured overnight in a shake flask at 37°C and 200 rpm to obtain the seed culture.

[0035] (3) Fermentation: The seed culture obtained in step (2) was transferred at an inoculum of 4% to 50 mL of TB medium (1.2% tryptone, 2.4% yeast extract, 0.4% glycerol, 17 mM KH2PO4, 72 mM K2HPO4, pH 6.0) containing 20 μg / mL ampicillin. The culture was incubated in shake flasks at 25°C and 200 rpm for 6 h. Then, IPTG was added to a final concentration of 0.05 mM, and the culture was further induced at 25°C (200 r / min) for 36 h. After fermentation, the fermentation broth was centrifuged to collect the cells. The cells were resuspended in 50 mM PBS (pH 7.0) buffer, sonicated, and the supernatant was collected by high-speed centrifugation. This was the crude enzyme solution of recombinant 6-α-glucosyltransferase. The amino acid sequence of Pa-6GT enzyme is shown in SEQ ID NO.2. SP- The amino acid sequence of the 6-GT enzyme is shown in SEQ ID NO.4.

[0036] Example 2: Preparation of short-chain starch by a two-enzyme method The specific steps are as follows: (1) Weigh 8 g of ordinary corn starch, add it to 90 mL of deionized water, stir for 15 min at 100 ℃ and 350 rpm, mix evenly, and obtain gelatinized starch with a concentration of 8% (w / w); (2) After the water bath temperature is reduced to 35℃, add 130 U / g Pa-6 GT crude enzyme solution, enzymatically hydrolyze for 12 h, and then boil for 15 min to inactivate the enzyme. (3) Add the obtained solution to 130 U / g Sp-6 GT crude enzyme solution, enzymatically hydrolyze at 35℃ for 12 h, and then boil for 15 min to inactivate the enzyme. (4) After freeze-drying the enzyme-inactivating solution, grind it into powder to obtain short-chain starch.

[0037] Example 3: Preparation of short-chain starch by single-enzyme (Pa-6 GT) method For the specific implementation method, refer to Example 2, except that step (3) is omitted and only Pa-6 GT crude enzyme solution is used for enzymatic hydrolysis.

[0038] Example 4: Preparation of short-chain starch by single-enzyme (Sp-6GT) method For the specific implementation method, refer to Example 2, except that step (2) is omitted and only Sp-6GT crude enzyme solution is used for enzymatic hydrolysis.

[0039] Example 5: Preparation of short-chain starch by a two-enzyme method For a specific implementation method, refer to Example 2, except that the order of steps (2) and (3) is reversed.

[0040] Example 6: Characterization of short-chain starch Samples of ordinary corn starch, the single-enzyme (Pa-6GT) modified sample of Example 3, the single-enzyme (Sp-6GT) modified sample of Example 4, the dual-enzyme modified sample of Example 2, and the enzyme-inactivated hydrolysate obtained by adding Sp-6GT first and then Pa-6GT in Example 5 were taken at 1 g each and diluted to 10 mL with sodium acetate buffer at pH 4. 1 mL of 1000 U / g isoamylase was added, and the mixture was reacted at 40 °C and 160 r / min for 24 h to completely debranch the starch. The reaction was terminated by boiling in a water bath for 30 min, then centrifuged at 10000 r / min for 10 min, diluted 10-fold, and filtered through a 0.22 μm aqueous membrane for HPAEC-PAD detection. The results are as follows: Figure 1 As shown, its main product is short-chain starch. In the modified starch obtained in Example 2, the content of DP1-12 reached 94.82%. At the same time, the modified starch contained only trace amounts of monosaccharides, exhibiting extremely high directional modification efficiency.

[0041] Table 1. Chain length distribution of ordinary corn starch before and after modification

[0042] Example 7: Application of short-chain starch in alleviating DSS-induced colitis in mice (1) Animal experiments were conducted using short-chain starch prepared in different doses as raw materials, as described in Example 2: The experimental procedure is as follows Figure 3 Thirty-two mice were randomly divided into four groups of eight mice each: a control group, a model group, a low-dose amylopectin group (SCS-5%), and a high-dose amylopectin group (SCS-10%). All mice underwent a 7-day acclimatization period. The intervention period began on day 1. The control group was administered PBS phosphate buffer daily by gavage. The model group was administered PBS phosphate buffer daily for the first 28 days, followed by 3.0% sodium dextran sulfate (DSS) daily for the next 7 days to establish the model. The low-dose amylopectin group was administered low-dose amylopectin (100 mg / kg) daily for the first 28 days, followed by 3.0% sodium dextran sulfate (DSS) daily for the next 7 days, and then low-dose amylopectin (100 mg / kg) daily for 1 hour. The high-dose amylopectin group was administered high-dose amylopectin (200 mg / kg) daily for the first 28 days, followed by 3.0% sodium dextran sulfate (DSS) daily for the next 7 days. H h later, mice were administered a high dose of short-chain starch (200 mg / kg) by gavage. All mice had free access to food and water. The gavage dose of DSS in each group was 0.012 g. The weight of the mice was measured every 2 days, and relevant indicators were measured. On the 7th day after modeling (the 35th day of the intervention period), the mice were sacrificed, and the distal colon of the mice was taken, fixed in 4% neutral formaldehyde, embedded in paraffin, and then sectioned. A portion of the tissue was stained with hematoxylin and eosin (HE) and analyzed under a light microscope.

[0043] (2) Experimental method: During the experiment, mouse body weight was measured every other day and change curves were plotted to calculate the daily body weight change rate. Fecal samples were collected for phenotypic evaluation and occult blood detection. At the end of the experiment, mice were sacrificed and colon length was measured. Colon tissue was routinely paraffin-embedded, prepared into sections, and stained with hematoxylin and eosin (H&E). H&E staining was used to observe colon tissue morphology, inflammatory cell infiltration, and ulceration. Mucosal thickness was measured, and enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of IL-10, IL-6, TNF-α, and IL-1β in mouse intestinal tissue.

[0044] (3) Experimental results: This experiment investigated the protective effect of short-chain starch (SCS) against DSS-induced colitis in mice. Two dosage groups were set up: 100 mg / kg and 200 mg / kg, with the low-dose group containing 5% SCS and the high-dose group containing 10% SCS. The results comprehensively confirmed the protective effect of SCS: Regarding body weight changes, the control group showed a stable growth trend, while the DSS model group experienced a significant decrease in body weight (p<0.001), and all doses of SCS intervention effectively alleviated this symptom (p<0.05); Disease Activity Index (DAI) assessment results showed that compared with the normal control group, the DSS model group had a significantly higher DAI score (p<0.001), manifested as abnormal fecal characteristics, increased defecation frequency, and severe occult blood. After intervention with different doses of SCS, the DAI score was significantly lower than that of the model group (p<0.05), showing a clear dose-response relationship; colon length measurement revealed… Figures 4-5 DSS resulted in colonic shortening to 5.12 ± 0.15 cm, while the short-chain starch intervention group, especially the high-dose group (6.90 ± 0.13 cm), significantly reversed this pathological change, while the low-dose group showed a weaker effect (5.94 ± 0.23 cm). HE staining results showed ( Figure 6 In the control group, the colonic tissue structure was intact, and the glands were arranged regularly. The DSS model group showed mucosal erosion and ulceration, extensive infiltration of inflammatory cells, and destruction of crypt structures. The degree of colonic tissue damage was significantly reduced in all short-chain starch intervention groups, with the high-dose group showing better improvement than the low-dose group. Inflammatory factor analysis (Tables 1-2) showed that all doses of short-chain starch significantly inhibited the release of pro-inflammatory factors IL-6, IL-1β, and TNF-α, while upregulating the expression of the anti-inflammatory factor IL-10, with the high-dose group showing the most significant effect. These results collectively indicate that short-chain starch has a multi-target protective effect against DSS-induced colitis by improving clinical symptoms, repairing intestinal barrier function, and regulating the balance of inflammatory factors, and this effect is clearly dose-dependent.

[0045] Table 2. Comparison of anti-inflammatory factors in colon tissue of mice in each group (Unit: pg / mL protein; data are expressed as mean ± standard error (SEM) (n = 8))

[0046] Experimental results: DSS-induced mice treated with short-chain starch had higher serum levels of the anti-inflammatory factor IL-10 than DSS mice, with statistically significant differences among the groups, especially in the high-dose group, where the effect was more pronounced. Experiments show that short-chain starch can effectively promote the secretion of anti-inflammatory factors, thereby exerting an anti-inflammatory response.

[0047] Table 3. Comparison of pro-inflammatory factors among mice in each group (Unit: pg / mL protein; Data are expressed as mean ± standard error (SEM) (n = 8))

[0048] Example 8: An oral formulation An enteric coating comprises 200 mg of short-chain amylopectin, 37.5 mg of microcrystalline cellulose, 7.5 mg of hydroxypropyl methylcellulose, and 5 mg of magnesium stearate. The freeze-dried short-chain amylopectin is pulverized through an 80-100 mesh sieve, with moisture content controlled to ≤5% to prevent tablet sticking during compression. AOS and microcrystalline cellulose are mixed in equal increments and stirred in a V-type mixer for 15-20 minutes. A binder solution (HPMC dissolved in pure water) is added, and wet granulation is performed, passing the granules through a 16 mesh sieve. The granules are then fluidized bed dried at 50-60℃ until moisture content is ≤3%, granulated, and then mixed with magnesium stearate for 5 minutes. The mixture is then compressed into tablets and finally coated to obtain an oral formulation of short-chain amylopectin. The prepared oral formulation has the effect of relieving colitis in mice.

[0049] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for preparing modified corn starch, characterized in that, Includes the following steps: (1) Prepare corn starch into a solution with a mass concentration of 5-8%, and stir it at 100℃ and 350-400rpm for 15-20min to obtain gelatinized starch; (2) Add 100-300 U / g 6-α-glucosyltransferase 1 to the gelatinized starch in step (1), and inactivate the enzyme after enzymatic hydrolysis for at least 12 hours to obtain enzymatic hydrolysate 1; (3) Add 100-300 U / g 6-α-glucosyltransferase 2 to the enzymatic hydrolysate 1 described in step (2), and inactivate the enzyme after enzymatic hydrolysis for at least 12 hours to obtain enzymatic hydrolysate 2; (4) The enzymatic hydrolysate 2 described in step (3) is freeze-dried to obtain modified starch.

2. The method according to claim 1, characterized in that, The enzymatic hydrolysis temperature in steps (2) and (3) is 35-40℃.

3. The method according to claim 1, characterized in that, The amount of 6-α-glucosyltransferase added in steps (2) and (3) is 100-150 U / g.

4. The method according to claim 1, characterized in that, The amino acid sequence of 6-α-glucosyltransferase 1 in step (2) is shown in SEQ ID No. 2; the amino acid sequence of 6-α-glucosyltransferase 2 in step (3) is shown in SEQ ID No.

4.

5. Modified starch prepared by any one of the methods described in claims 1 to 4.

6. A product containing the modified starch of claim 5.

7. The product according to claim 6, characterized in that, The products include health supplements, animal feed, or pharmaceuticals.

8. The product according to claim 7, characterized in that, The feed formulation is any one of the following: pelleted feed, extruded feed, powdered feed, crushed feed, liquid feed, or premixed feed.

9. The product according to claim 8, characterized in that, The drug also contains a drug carrier and / or pharmaceutical excipients; the pharmaceutical carrier includes one or more of the following commonly used in medicine: fillers, binders, wetting agents, disintegrants, lubricants, and flavoring agents.

10. The use of the modified starch according to claim 5 in the preparation of health products for regulating intestinal flora.

Images