Starch-based feed for increasing the abundance of akkermansia muciniphila

Abstract

The application discloses a kind of improvement Akkermansia muciniphila The application discloses a starch-based feed for improving bacterial abundance, and belongs to the technical field of functional food. Experimental results show that the starch-based feed can significantly reverse the bacterial flora disorder of mice: PCoA analysis shows that it can restore the overall structure of the disordered bacterial flora to a healthy state; specifically, it improves the ratio of disordered Firmicutes / Bacteroidetes, promotes the proliferation of beneficial bacteria Akkermansia muciniphila, and inhibits the growth of conditional pathogenic bacteria, and the regulatory effect shows a dose-dependent manner. Therefore, the modified starch-based feed is a potential functional food raw material for regulating intestinal flora.

Description

Technical Field

[0001] This invention relates to an improvement Akkermansia muciniphila Starch-based feeds with high microbial abundance belong to the field of functional food technology. Background Technology

[0002] The gut microbiota is a key factor in maintaining host health, and its balance is closely related to digestive, immune, and metabolic functions. In livestock farming, gut microbiota dysbiosis can lead to decreased animal growth performance, increased disease susceptibility, and higher production costs. Recent studies have shown that enrichment of specific beneficial bacteria can effectively improve intestinal barrier function, suppress inflammation, and promote nutrient absorption. Among them, *Akkermansia muciniphila*, a Gram-negative anaerobic bacterium, has been proven to be a key beneficial bacterium in the gut microbiome. It colonizes the intestinal mucus layer, maintains mucosal integrity by degrading mucin, and produces metabolites such as short-chain fatty acids, exhibiting anti-inflammatory, antioxidant, and immunomodulatory effects. However, no feed has yet been able to specifically target and increase the abundance of *Akkermansia muciniphila* in the gut. Summary of the Invention

[0003] The present invention aims to solve the problem that ordinary feeds in the prior art have insufficient ability to regulate the intestinal flora, and provides a modified starch source combination feed that can optimize the composition of intestinal microorganisms, promote the proliferation of beneficial bacteria and improve intestinal health.

[0004] This invention achieves precise nutritional intervention on the gut microbiota, especially the hindgut microbiota, by accurately designing the proportion of modified starch. Changes in key gut microbiota were analyzed through clinical evaluation. This invention uses enzymatically modified ordinary corn starch to create a starch feed for mice. Clinical evaluation showed that the modified starch feed can increase the abundance of *Akkermansia muciniphila* and the content of beneficial bacteria, thus exerting its therapeutic effect.

[0005] The first objective of this invention is to provide a method for preparing modified starch, 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) Modify the ordinary corn starch pretreatment solution by adding 6-α-glucosyltransferase; (3) Boil the solution after enzymatic hydrolysis in step (2) to inactivate the enzyme; (4) Freeze-dry the enzyme-inactivated solution obtained in step (3).

[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 5-10% (w / w).

[0007] In one embodiment of the present invention, the pretreatment temperature of the ordinary corn starch is 100 ℃, and the mixture is stirred at 350-400 rpm for 15-20 min.

[0008] In one embodiment of the present invention, the 6-α-glucosyltransferase in step (2) is derived from... Sporosacina globispora C11 6-α-GT. The final product of this enzyme acting on ordinary corn starch is modified starch.

[0009] In one embodiment of the present invention, the nucleotide sequence encoding the 6-α-glucosyltransferase is shown in SEQ ID No. 1.

[0010] In one embodiment of the present invention, the enzyme inactivation in step (3) is performed by boiling for 20 min.

[0011] A second objective of this invention is to provide modified starch prepared by the above method.

[0012] A third object of the present invention is to provide a product containing the modified starch.

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

[0014] The fourth objective of this invention is to provide a starch feed that can increase the abundance of Akkermansia muciniphila bacteria.

[0015] In one embodiment of the present invention, the dosage of modified starch is 100-400 mg / kg.

[0016] In one embodiment of the present invention, the feed formulation is any one of pelleted feed, extruded feed, powdered feed, crushed feed, liquid feed or premixed feed.

[0017] In one embodiment of the present invention, the feed also contains auxiliary materials.

[0018] In one embodiment of the present invention, the excipients include protein feed, minerals and vitamins, and amino acids.

[0019] In one embodiment of the present invention, preferably, the auxiliary materials include soybean meal, dicalcium phosphate, salt, and amino acids.

[0020] A fifth objective of this invention is to provide the application of the modified starch in the preparation of health products that regulate gut microbiota, wherein the regulation of gut microbiota is targeted at increasing the abundance of Akkermansia muciniphila.

[0021] Beneficial effects: This invention prepares a modified starch with improved branching and good physical properties. The modified starch can significantly improve the intestinal flora and increase the abundance of Akkermansia muciniphila. Therefore, the modified starch-based feed can be used as a functional food, medicine or health product for regulating intestinal flora. Attached Figure Description Figure 1 A process flow diagram for the preparation of modified starch.

[0022] Figure 2 The image shows the RVA results of the product obtained in Example 2.

[0023] Figure 3 The image shows the result of HPAEC-PAD, the product obtained in Example 2.

[0024] Figure 4 The image shows the HPAEC-PAD results of the product obtained in Example 3.

[0025] Figure 5 This is a diagram showing the changes in the gut microbiota in Example 4.

[0026] Figure 6 The images show H&E staining of colon tissue from mice in each group in Example 4. Detailed Implementation

[0027] 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.

[0028] 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.

[0029] LB solid medium: yeast extract 5 g / L, tryptone 10 g / L, NaCl 10 g / L, pH 7.0, 1.5% (w / v) agar.

[0030] Detection of short-chain starch The sample of ordinary corn starch modified with Sp-6GT enzyme was boiled for 20 min to inactivate the enzyme, centrifuged, debranched, diluted, and filtered through a 0.22 μm aqueous membrane. The product was then analyzed using a high-performance anion exchange chromatography system (HPAEC-PAD) and a rapid viscosity analyzer. 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 artificially synthesized 6-α-glucosyltransferase Sp-6GT gene: upstream primer: 5'-CCATGGCCTATGTGAGCAG-3'; downstream primer: 5'-GCGTGACCAAACAGCTCGA-3'. The target gene containing the signal peptide sequence SEQ ID No. 1 (where positions 1-66 encode the signal peptide) was cloned by PCR. Primers were also designed based on the pET-20b(+) vector sequence: upstream primer: 5'-TGCCCAGCCGGCGATGGCCATGGCCTATGTGA-3'; downstream primer: 5'-CTCGAGCACCACCACCACCACCACTGAGATCCGGCTGC-3'. The pET20b(+) vector containing the linker site was cloned.

[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 resulting vector was then transformed into *E. coli* JM109, plated on LB agar plates containing 20 μg / mL ampicillin, and transformed individuals were picked for sequencing and nucleic acid electrophoresis verification to obtain the expression vector pET-20b(+) / sp containing the 6-α-glucosyltransferase gene. The expression vector was then transformed... Escherichia coli BL21(DE3) yielded genetically engineered bacteria. E. coli BL21(DE3)(pET-20b(+) / sp).

[0034] (2) Activation: 100 μL containing genetically engineered bacteria E. coli The glycerol stock solution of BL21(DE3)(pET-20b(+) / sp) 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 medium was then cultured in shake flasks at 25°C and 200 rpm for 6 h. IPTG was then 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 supernatant was the crude enzyme solution of recombinant 6-α-glucosyltransferase (Sp-6GT).

[0036] Example 2: Method for preparing modified starch The specific steps are as follows: (1) Weigh 10 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 10% (w / w); (2) After the water bath temperature is reduced to 35℃, add Sp-6 GT crude enzyme solution with concentrations of 0 U / g and 150 U / g respectively, and after enzymatic hydrolysis for 10 h, boil for 20 min to inactivate the enzyme. (3) The enzyme hydrolysate after enzyme inactivation is freeze-dried, concentrated, and weighed. The process flow diagram is as follows: Figure 1 As shown.

[0037] The obtained modified starch lyophilized powder was subjected to RVA analysis, and the results are as follows: Figure 2 As shown. The total sample volume was 25g. Based on 8% dry starch, the required weight of water was calculated and added according to the moisture content of the freeze-dried sample, and then loaded into the RVA unit for measurement.

[0038] Table 1. RVA results before and after modification of ordinary corn starch

[0039] From the perspective of RVA characteristics, compared with ordinary corn starch, modified starch has a significantly lower gelatinization temperature, meaning that the starch begins to absorb water, swell, and gelatinize at very low temperatures. This facilitates gelatinization at lower conditioning temperatures, saving energy. It is also more easily broken down by enzymes in the animal digestive tract, potentially improving digestibility. Its extremely high peak viscosity indicates that the starch granules have a very strong swelling capacity and can produce extremely high viscosity after absorbing water. This can significantly improve the hardness and durability of granular feed.

[0040] Take 1 g of the enzymatic hydrolysate obtained in step (2) and bring the volume to 10 mL. Add 1 mL of 1000 U / g isoamylase and react at 40 ℃ and 160 r / min for 24 h to completely debranch the starch. Terminate the reaction by boiling in a water bath for 30 min, then centrifuge at 10000 r / min for 10 min. After diluting 20 times, filter through a 0.22 μm aqueous membrane for HPAEC-PAD detection. The results are as follows: Figure 3 As shown, the chain length distribution of the modified starch changed significantly compared with starch treated without the addition of Sp-6 GT crude enzyme solution.

[0041] Example 3: Optimization of the preparation method of modified starch The specific implementation method is the same as in Example 2, except that the amount of Sp-6 GT crude enzyme solution added in step (2) is adjusted to 50 U / g, 100 U / g, 200 U / g, and 300 U / g. The enzymatic hydrolysate obtained in step (2) was subjected to HPAEC-PAD detection, and the results are shown in Table 2. Figure 4 As shown.

[0042] Table 2. Chain length distribution of ordinary corn starch modified with different enzyme dosages

[0043] The data from the charts show that when the enzyme dosage was 50 U / g and 100 U / g, the degree of starch modification was low, and the product still retained a large number of branched segments with a degree of polymerization (DP) of 13-24. When the enzyme dosage increased to 200 U / g, the proportion of segments with DP 1-12 significantly increased to 94.18%, but the excessive enzyme dosage also caused significant hydrolytic side reactions, generating 14.39% monosaccharides. Further increasing the enzyme dosage to 300 U / g, the monosaccharide content further increased to 27.51%, indicating that the hydrolytic effect of 6GT enzyme had become dominant. In summary, choosing an enzyme dosage of 150 U / g can effectively control the formation of short chains while significantly inhibiting the excessive formation of monosaccharides, thereby achieving a higher efficiency in targeted modification.

[0044] Example 4: Application of modified starch feed in regulating intestinal flora (1) Animal experiments were conducted using the modified starch prepared in different doses of Example 2 as raw materials: Forty mice were randomly divided into four groups of ten mice each: a control group, a model group, a low-dose modified starch diet group (L-SCS), and a high-dose modified starch diet group (H-SCS). All mice underwent a 7-day acclimatization period before the experiment. 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 by gavage for the first seven days, followed by 3.0% sodium dextran sulfate (DSS) by gavage for the next seven days to establish the model. The low-dose modified starch diet group was administered low-dose modified starch diet (100 mg / kg) by gavage for the first seven days, followed by 3.0% sodium dextran sulfate (DSS) by gavage for the next seven days, and then low-dose modified starch diet (100 mg / kg) 1 hour later. The high-dose modified starch diet group was administered high-dose modified starch diet (400 mg / kg) by gavage for the first seven days, followed by 3.0% sodium dextran sulfate (DSS) by gavage for the next seven days. H hours later, mice were administered a high dose of modified starch feed (400 mg / kg) by gavage. All mice had free access to food and water. The amount of DSS administered by gavage to 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 14th day of the intervention period), the mice were sacrificed, and intestinal contents samples were collected for analysis. (2) Experimental method: During the experiment, the body weight of mice was measured every other day and the change curve was plotted to calculate the daily body weight change rate. At the same time, fecal samples were collected for phenotypic evaluation and occult blood detection. At the end of the experiment, the mice were sacrificed and the colon tissue was routinely embedded in paraffin. After sectioning, Alcian blue staining was performed to observe the number and distribution of goblet cells. At the same time, 16S rRNA analysis was performed on the collected intestinal contents.

[0045] (3) Experimental results: This experiment investigated the regulatory effect of modified starch diet on gut microbiota in DSS-induced mice, with two dosage groups: 100 mg / kg and 400 mg / kg. The results comprehensively confirmed the regulatory effect of modified starch diet: 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 dosages of modified starch diet intervention effectively alleviated this symptom (p<0.05); the 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 dosages of modified starch diet, the DAI score was significantly lower than that of the model group (p<0.05), showing a clear dose-response relationship; alcinocyanine blue staining analysis showed that ( Figure 6 In the DSS group, the density of goblet cells in the colonic mucosa was significantly reduced and unevenly distributed (p<0.001); while the modified starch diet intervention groups effectively increased the number of goblet cells and improved their distribution, showing a dose-dependent effect, indicating that modified starch can effectively repair the mucus barrier function; 16S rRNA analysis results showed that the modified starch diet can significantly reverse the dysbiosis caused by colitis. Figure 5 The results show a highly significant statistical difference in the microbial community structure between the groups. In the PCoA plot, the sample points in the DSS model group deviated significantly from the control group, while the sample points treated with high-dose modified starch feed moved closer to the control group and were clustered elliptically. This demonstrates that high-dose modified starch can effectively restore the overall structure of the disordered gut microbiota to a healthy state. Observations show that the ratio of Firmicutes to Bacteroidetes was disordered in the DSS group, but improved in the modified starch feed treatment group. The abundance of beneficial bacteria Akkermansia muciniphila increased significantly. The abundance of opportunistic pathogens or pro-inflammatory bacteria (such as Escherichia and Helicobacter) decreased. The figure shows that the degree of improvement in the microbial community structure is closely related to the dosage of modified starch. The improvement effect of the high-dose group is generally significantly better than that of the low-dose group and closer to the healthy control group, indicating that its regulatory effect is dose-dependent.

[0046] Example 5: A pelleted feed A pelleted feed comprises 400 mg of modified starch, 115 mg of soybean meal, 35 mg of dicalcium phosphate, 16.5 mg of salt, and 5.5 mg of amino acids. The freeze-dried modified starch is pulverized through an 80-100 mesh sieve, with a moisture content controlled to ≤10%. The modified starch is mixed with the remaining ingredients 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. The pellets are then dried in a fluidized bed at 50-60℃ until the moisture content is ≤5%, yielding the starch pelleted feed. The prepared starch pelleted feed has the effect of regulating the intestinal flora of mice.

[0047] 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 5-10%, and stir it at 100℃ and 350-400rpm for 15-20min to obtain gelatinized starch; (2) Add 100-300 U / g 6-α-glucosyltransferase to the gelatinized starch in step (1), and inactivate the enzyme after enzymatic hydrolysis for at least 10 hours; (3) The enzyme hydrolysate after enzyme inactivation in step (2) is freeze-dried to obtain modified starch.

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

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

4. Modified starch prepared by any one of the methods described in claims 1 to 3.

5. A product containing the modified starch of claim 4.

6. The product according to claim 5, characterized in that, The products include food, medicine, feed, or health products.

7. The application of the modified starch according to claim 4 in the preparation of feed for regulating or assisting in improving intestinal flora, characterized in that, The regulation or assistance in improving the gut microbiota is to target and increase the abundance of Akkermansia muciniphila.

8. The application 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 application according to claim 7, characterized in that, The feed also contains auxiliary materials; the auxiliary materials include protein feed, minerals and vitamins, and amino acids; preferably, the auxiliary materials include soybean meal, dicalcium phosphate, salt, and amino acids.

10. The application of the modified starch according to claim 4 in the preparation of health products for regulating intestinal flora, characterized in that, The regulation of gut microbiota involves targeting and increasing the abundance of Akkermansia muciniphila.

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