Ultrahigh pressure assisted butyrylation modified starch and application thereof in regulating intestinal flora

Abstract

The invention belongs to the technical field of food science, and particularly relates to ultrahigh-pressure-assisted butyrylation modified starch and application thereof in regulating intestinal flora, and the ultrahigh-pressure-assisted butyrylation modified starch is prepared by the following steps: performing butyrylation treatment and ultrahigh-pressure treatment on a starch raw material to obtain modified starch; the pressure of the ultrahigh pressure treatment is 80 to 600 MPa. According to the preparation method disclosed by the invention, the modified starch is efficiently prepared in a manner of combining butyrylation treatment and ultrahigh pressure treatment, so that the preparation method of the modified starch has the advantages of efficient production and lower cost, and the obtained butyrylated starch has better physicochemical properties and physiological functions; the modified starch prepared by the method has higher butyryl group substitution degree, better solubility, stability and digestibility, can effectively promote the production of short-chain fatty acids such as butyric acid in the digestion or in-vitro fermentation process, and has wide application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of food science and technology, and particularly relates to ultrahigh pressure assisted butyrylation modified starch and its application in regulating intestinal flora. Background Art

[0002] Butyric acid, as an important short-chain fatty acid, plays a key role in intestinal health, including promoting intestinal epithelial cell proliferation, strengthening intestinal barrier function, anti-inflammatory and cancer prevention. However, due to the volatility, corrosiveness and strong irritating odor of butyric acid, direct ingestion is difficult. Therefore, butyrylated starch is a type of modified starch obtained by replacing the alcoholic hydroxyl group (-OH) in the glucose unit structure of starch with the carboxyl group (-COOH) in the butyric acid molecular structure. It is a complex that combines butyric acid with starch molecules. Through the slow digestion process of starch, short-chain fatty acids such as acetate, propionate and butyrate are gradually released, which helps maintain a stable concentration of butyrate in the intestine and becomes a better way to supplement exogenous butyrate.

[0003] Currently, the synthesis of butyrylated starch mainly adopts the aqueous phase method and organic solvent synthesis method. However, both the aqueous phase method and the organic solvent synthesis method have problems such as large reagent usage, low acyl content and degree of substitution, and long time consumption. These problems limit the production efficiency and large-scale application of butyrylated starch.

[0004] Therefore, developing a preparation method with high efficiency, low cost and better physical and chemical properties and physiological functions of butyrylated starch is of great significance for improving the preparation efficiency of butyrylated starch and its application potential in the field of intestinal health. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art to a certain extent. To this end, the present invention provides an ultra-high pressure assisted butyrylation modified starch and its application in regulating intestinal flora. The preparation method of the present invention efficiently prepares modified starch by combining butyrylation treatment and ultra-high pressure treatment. It is a method for preparing modified starch with high efficiency and low cost, and the obtained butyrylated starch has better physical and chemical properties and physiological functions. The modified starch prepared by this method has a higher degree of butyryl group substitution, better solubility, stability and digestibility, and can effectively promote the production of short-chain fatty acids such as butyric acid when applied in the fermentation process, and has broad application prospects.

[0006] The present invention is based on the inventor's discovery and understanding of the following problems:

[0007] The inventors used an aqueous phase method to introduce butyric anhydride into starch raw materials for butyrylation reaction, and combined with ultra-high pressure technology to modify the starch to obtain modified starch, which is butyrylated starch; the inventors used nuclear magnetic resonance ( 1 HNMR) technology was used to analyze the effects of butyrylation and ultrahigh pressure treatment on the molecular structure of starch, especially the degree of substitution of hydroxyl groups at the C2, C3, and C6 positions, and to compare the changes in the degree of substitution (DS) and branching degree (DB) of butyrylated starch under dual treatment. The inventors found that after butyrylation treatment, 1 New peaks appeared in the H NMR spectrum, corresponding to one methyl group and two methylene groups of the butyryl group, respectively, confirming the successful introduction of the butyryl group. At the same time, relevant experimental results showed that ultra-high pressure treatment can significantly increase the DS value of butyrylated starch and significantly accelerate the butyrylation process. Subsequently, the inventors used comprehensive characterization methods such as Fourier transform infrared spectroscopy (FT-IR), particle size analysis, X-ray diffraction (XRD), thermogravimetry (TGA), polarizing microscopy (PLM), scanning electron microscopy (SEM), and in vitro simulated digestion to explore the effects of different ultra-high pressure treatment pressures (100 MPa, 300 MPa, 500 MPa) and the order of butyrylation / ultra-high pressure treatment (butyrylation first, then ultra-high pressure treatment or vice versa) on the structure and properties of butyrylated starch. The inventors found that the crystal structure of starch was destroyed under ultra-high pressure treatment, and the crystal order was reduced. , confirming that ultra-high pressure can promote the entry of chemical reactants into the crystalline area of ​​starch granules. In addition, the results showed that butyrylation treatment can effectively increase the resistant starch (RS) content of starch and reduce the rapidly digestible starch (RDS) content, and the addition of ultra-high pressure treatment can further regulate the digestion characteristics of starch; finally, the inventors used an in vitro simulated fermentation system to co-culture the modified starch prepared by the method of the present invention with intestinal microorganisms, and detected that the modified starch obtained during the fermentation process in the intestine can produce rich short-chain fatty acids (especially butyric acid), which can regulate the structure of intestinal flora and help alleviate intestinal diseases. In addition, the addition of ultra-high pressure treatment further reduced the fermentation rate of the modified starch, mainly by increasing the double helix structure and changing the morphology of the starch surface, so that microorganisms can slowly utilize the modified starch and improve gastrointestinal tolerance.

[0008] Therefore, in the first aspect of the present invention, the present invention proposes a method for preparing modified starch. According to an embodiment of the present invention, the method comprises: subjecting a starch raw material to butyrylation treatment and ultra-high pressure treatment to obtain modified starch; the pressure of the ultra-high pressure treatment is 80 to 600 MPa. The preparation method according to the embodiment of the present invention is a method for preparing modified starch with high efficiency, low cost and better physical and chemical properties and physiological functions of the butyrylated starch obtained. The method significantly improves the modification efficiency of starch by combining butyrylation treatment and ultra-high pressure treatment, changes the granular structure of starch by ultra-high pressure treatment, increases its porosity, and makes the butyryl group more easily combined with the starch molecule, thereby improving the efficiency and degree of substitution of the butyrylation reaction; in addition, the method further improves the solubility, stability and digestibility of the modified starch obtained; the modified starch obtained by the preparation method of the embodiment of the present invention can effectively promote the production of short-chain fatty acids such as butyric acid during the fermentation process. These short-chain fatty acids have important benefits for intestinal health, such as regulating intestinal flora and maintaining intestinal health.

[0009] According to an embodiment of the present invention, the above preparation method may also have the following additional technical features:

[0010] According to an embodiment of the present invention, the method includes: S1: subjecting the starch raw material to the butyrylation treatment to obtain starch containing butyryl groups; S2: subjecting the starch containing butyryl groups to the ultrahigh pressure treatment to obtain modified starch.

[0011] According to an embodiment of the present invention, the pressure of the ultrahigh pressure treatment is 100-500 MPa.

[0012] According to an embodiment of the present invention, the butyrylation treatment method includes chemical butyrylation and / or enzymatic butyrylation.

[0013] According to an embodiment of the present invention, the butyrylation treatment comprises: contacting an acylating agent with the starch raw material to cause an esterification reaction to obtain starch containing butyryl groups; wherein the pH of the contact reaction is 8.0 to 8.5.

[0014] According to an embodiment of the present invention, the ultrahigh pressure treatment time is 5 to 25 minutes.

[0015] In a second aspect, the present invention provides a modified starch. According to an embodiment of the present invention, the modified starch is prepared by the method described in the first aspect. The modified starch according to the embodiment of the present invention has a higher degree of butyryl group substitution, better solubility, stability, and digestibility. This modified starch has great application potential in the fields of food, health products, and pharmaceuticals, and is suitable for various application scenarios.

[0016] In a third aspect of the present invention, the present invention provides a food, health product or medicine, which, according to an embodiment of the present invention, comprises: the modified starch described in the second aspect.

[0017] Those skilled in the art will understand that the characteristics and advantages described above for the modified starch are also applicable to the food, health care product or medicine, and will not be described in detail here.

[0018] In a fourth aspect, the present invention provides the use of the method described in the first aspect and the modified starch described in the second aspect in the preparation of a food, health product, or medicine. According to embodiments of the present invention, the medicine has at least one of the following uses: regulating intestinal flora; maintaining intestinal health; lowering cholesterol levels; reducing fat production; alleviating and / or treating intestinal inflammation; and maintaining or increasing intestinal butyrate concentration. According to the use of the embodiments of the present invention, the modified starch obtained by the aforementioned preparation method of the present invention can significantly regulate the composition and function of the intestinal flora, promote the growth of beneficial bacteria (such as Bacteroidetes and Firmicutes), and inhibit the reproduction of harmful bacteria, thereby maintaining the balance of intestinal microecology, enhancing the intestinal barrier function, improving the intestinal resistance to pathogens, and preventing and alleviating intestinal-related diseases; and can provide energy for intestinal epithelial cells by promoting the production of short-chain fatty acids (such as butyric acid), consolidate the intestinal barrier function, reduce inflammatory response, promote the proliferation and differentiation of intestinal epithelial cells, further enhance the self-repair ability of the intestine, prevent the occurrence of intestinal diseases, and maintain intestinal health; the modified starch can also regulate intestinal flora and metabolic pathways, reduce serum cholesterol levels, reduce liver fat production, and have a positive effect on cardiovascular health and metabolic diseases (such as obesity and non-alcoholic fatty liver disease); thus, the food, health product or medicine has the uses of regulating intestinal flora, maintaining intestinal health, lowering cholesterol levels, reducing fat production, alleviating and / or treating intestinal inflammation, maintaining or increasing the concentration of butyric acid in the intestine, and the like.

[0019] According to an embodiment of the present invention, the above-mentioned use may also have the following additional technical features:

[0020] According to an embodiment of the present invention, the intestinal flora includes: Bacteroidetes and / or Firmicutes.

[0021] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0023] Figure 1 These are polarized cross-sectional images of butyrylated starch obtained under different treatment conditions in Example 2 of the present invention, wherein A1-K1 are respectively images of butyrylated starch observation results obtained under different treatment conditions under a normal light source (eyepiece 10×, objective lens 40×), and A2-K2 are respectively images of butyrylated starch observation results obtained under different treatment conditions under a polarized light source (eyepiece 10×, objective lens 40×);

[0024] Figure 2 Figure 2 is a diagram showing the particle size distribution of butyrylated starch obtained under different treatment conditions in Example 2 of the present invention, wherein A is a diagram showing the particle size distribution of butyrylated starch obtained under different treatment conditions (ultrahigh pressure condition is 100 MPa), B is a diagram showing the particle size distribution of butyrylated starch obtained under different treatment conditions (ultrahigh pressure condition is 300 MPa), and C is a diagram showing the particle size distribution of butyrylated starch obtained under different treatment conditions (ultrahigh pressure condition is 500 MPa);

[0025] Figure 3 The butyrylated starch obtained under different treatment conditions in Example 2 of the present invention 1 HNMR analysis results, where A is the butyrylated starch obtained under different treatment conditions 1 H NMR analysis results (ultra-high pressure condition is 100 MPa), B is the butyrylated starch obtained under different treatment conditions 1 H NMR analysis results (ultra-high pressure condition: 300 MPa), C is the butyrylated starch obtained under different treatment conditions 1 H NMR analysis results (ultra-high pressure condition: 500 MPa);

[0026] Figure 4 It is a graph of the FT-IR analysis results of the butyrylated starch obtained under different treatment conditions in Example 2 of the present invention, wherein A is the original spectrum of the butyrylated starch FT-IR analysis obtained under different treatment conditions (ultrahigh pressure condition is 100 MPa), B is the deconvolution graph of the butyrylated starch FT-IR analysis obtained under different treatment conditions (ultrahigh pressure condition is 100 MPa), C is the original spectrum of the butyrylated starch FT-IR analysis obtained under different treatment conditions (ultrahigh pressure condition is 300 MPa), D is the deconvolution graph of the butyrylated starch FT-IR analysis obtained under different treatment conditions (ultrahigh pressure condition is 300 MPa), E is the original spectrum of the butyrylated starch FT-IR analysis obtained under different treatment conditions (ultrahigh pressure condition is 500 MPa), and F is the deconvolution graph of the butyrylated starch FT-IR analysis obtained under different treatment conditions (ultrahigh pressure condition is 500 MPa);

[0027] Figure 5The figures are XRD analysis results of butyrylated starch obtained under different treatment conditions in Example 2 of the present invention, wherein A is the XRD analysis result of butyrylated starch obtained under different treatment conditions (ultrahigh pressure condition is 100 MPa), B is the XRD analysis result of butyrylated starch obtained under different treatment conditions (ultrahigh pressure condition is 300 MPa), and C is the XRD analysis result of butyrylated starch obtained under different treatment conditions (ultrahigh pressure condition is 500 MPa);

[0028] Figure 6 The TGA analysis results of butyrylated starch obtained under different treatment conditions in Example 2 of the present invention are shown in Figure 2, wherein A is the TGA analysis result of butyrylated starch obtained by the NCS group, and B is the TGA analysis result of the CS group. B The TGA analysis results of butyrylated starch obtained by the group, C is CS 100 The TGA analysis results of butyrylated starch obtained by the group, D is CS 100+B The TGA analysis results of butyrylated starch obtained by the group, E is CS B+100 The TGA analysis results of butyrylated starch obtained by the group, F is CS 300 The TGA analysis results of butyrylated starch obtained by the group, G is CS 300+B TGA analysis results of butyrylated starch obtained by the group, H is CS B+300 The TGA analysis results of butyrylated starch obtained by the group, I is CS 500 The TGA analysis results of butyrylated starch obtained by the group, J is CS 500+B The TGA analysis results of butyrylated starch obtained by the group, K is CS B+500 Figure 2. TGA analysis results of butyrylated starch obtained by the group;

[0029] Figure 7 Graphs showing the resistant starch content of butyrylated starch obtained under different treatment conditions in Example 2 of the present invention, wherein A is a graph showing the resistant starch content of butyrylated starch obtained under different treatment conditions (ultrahigh pressure condition: 100 MPa), B is a graph showing the resistant starch content of butyrylated starch obtained under different treatment conditions (ultrahigh pressure condition: 300 MPa), and C is a graph showing the resistant starch content of butyrylated starch obtained under different treatment conditions (ultrahigh pressure condition: 500 MPa).

[0030] Figure 8The results of the starch morphology change measurement before and after in vitro fermentation in Example 3 of the present invention are shown, wherein A1-E1 and A3-E3 are magnified 2.0k times, A2-E2 and A4-E4 are magnified 5.0k times, A1-A4 from left to right are scanning electron micrographs of the surface morphology of starch granules in the NCS group before (A1-A2) and after (A3-A4) fermentation, respectively, and B1-B4 from left to right are scanning electron micrographs of the surface morphology of starch granules in the NCS group before (B1-B2) and after (B3-B4) fermentation, respectively. B Scanning electron microscopy images of starch granules in the groups, C1-C4 from left to right are CS before (C1-C2) and after (C3-C4) fermentation B+100 Scanning electron microscopy images of starch granules in the groups D1-D4 from left to right are before (D1-D2) and after (D3-D4) CS fermentation B+300 Scanning electron microscopy images of starch granules in the groups E1-E4 from left to right are before (E1-E2) and after (E3-E4) fermentation CS B+500 Scanning electron microscopy images of starch granule morphology;

[0031] Figure 9 3. The figures are as follows: Figure 3 is a graph showing the test results of SCFAs content and pH value of each group of starch during the in vitro fermentation process in Example 3 of the present invention, wherein A is a graph showing the change in acetic acid content level of each group of starch during the in vitro fermentation process, B is a graph showing the change in propionic acid content level of each group of starch during the in vitro fermentation process, C is a graph showing the change in butyric acid content level of each group of starch during the in vitro fermentation process, C is a graph showing the change in butyric acid ratio of each group of starch during the in vitro fermentation process, E is a graph showing the change in SCFAs content of each group of starch during the in vitro fermentation process, and F is a graph showing the change in pH value of each group of starch during the in vitro fermentation process;

[0032] Figure 103. The figure shows the Coverage index analysis results of the α and β diversity analysis results of the intestinal microbial community of starch obtained under different treatment conditions at 12 h and 24 h of fermentation in Example 3 of the present invention, wherein A is the Coverage index analysis result of the intestinal microbial community of starch obtained under different treatment conditions at 12 and 24 h of fermentation, B is the abundance level curve of the intestinal microbial community of starch obtained under different treatment conditions at 12 and 24 h of fermentation, C is the Chao 1 index used to measure the diversity and richness of the intestinal microbial community of starch obtained under different treatment conditions at 12 h and 24 h of fermentation, D is the Shannon index used to measure the diversity and richness of the intestinal microbial community of starch obtained under different treatment conditions at 12 h and 24 h of fermentation, E is the Simpson index used to measure the diversity and richness of the intestinal microbial community of starch obtained under different treatment conditions at 12 h and 24 h of fermentation, and F is the PCoA analysis result of the intestinal microbial community based on Bray Curtis distance of starch obtained under different treatment conditions at 12 h and 24 h of fermentation;

[0033] Figure 11 This is a diagram showing the difference in intestinal flora composition at the phylum level in the species analysis results of the intestinal microbiota obtained under different treatment conditions in Example 3 of the present invention at 12 h and 24 h of fermentation. A is a diagram showing the difference in intestinal flora composition at the phylum level in the starch obtained under different treatment conditions at 12 h and 24 h of fermentation, B is a diagram showing the relative abundance comparison results of representative species (Bacteroidetes) obtained under different treatment conditions at 12 h and 24 h of fermentation, C is a diagram showing the relative abundance comparison results of representative species (Firmicutes) obtained under different treatment conditions at 12 h and 24 h of fermentation, and D is a genus-level species composition heat map of species clustering of starch obtained under different treatment conditions at 12 h and 24 h of fermentation. DETAILED DESCRIPTION

[0034] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0035] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0036] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0037] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention, but not excluding other contents.

[0038] As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0039] Terms and Definitions

[0040] In this article, the term "starch on a dry basis" means that when determining the starch content, the starch content is calculated based on the mass of the sample after complete drying after deducting the moisture content; specifically, the moisture content of the sample is first determined, and then the sample is dried to a constant weight, and after removing the moisture, the starch content is determined.

[0041] In this article, the term "RDS" refers to rapidly digestible starch, specifically starch that can be quickly digested and absorbed in the small intestine. This part of starch usually completes the digestion process within 20 minutes.

[0042] In this article, the term "SDS" refers to slowly digestible starch, specifically starch that is completely digested and absorbed in the small intestine but at a slower rate, typically taking between 20 and 120 minutes to digest.

[0043] In this article, the term "RS" is resistant starch, which specifically refers to starch that cannot be digested and absorbed in the human small intestine. This part of starch can resist the action of digestive enzymes in the small intestine. The undigested and unabsorbed part enters the large intestine and can be fermented by intestinal flora in the large intestine.

[0044] Preparation method

[0045] The present invention proposes a method for preparing modified starch. According to an embodiment of the present invention, the method comprises: subjecting a starch raw material to butyrylation and high-pressure treatment to obtain modified starch; the pressure of the ultra-high pressure treatment is 80 to 600 MPa. The preparation method according to the embodiment of the present invention is a method for preparing modified starch with high efficiency, low cost, and better physical and chemical properties and physiological functions of the butyrylated starch obtained. The method significantly improves the modification efficiency of starch by combining butyrylation treatment with ultra-high pressure treatment, changes the granular structure of starch by ultra-high pressure treatment, increases its porosity, and makes the butyryl group more easily combined with the starch molecule, thereby improving the efficiency and degree of substitution of the butyrylation reaction; in addition, the method further improves the solubility, stability and digestibility of the modified starch obtained; the modified starch obtained by the preparation method of the embodiment of the present invention can effectively promote the production of short-chain fatty acids such as butyric acid during the fermentation process. These short-chain fatty acids have important benefits for intestinal health, such as regulating intestinal flora and maintaining intestinal health.

[0046] Illustratively, the method for preparing modified starch according to the present invention mainly includes three types: the first method is to first subject the starch raw material to butyrylation treatment and then to ultra-high pressure treatment; the second method is to first subject the starch raw material to ultra-high pressure treatment and then to butyrylation treatment; the third method is to subject the starch raw material to butyrylation treatment and ultra-high pressure treatment at the same time; the pressure of the ultra-high pressure treatment is 80MPa, 100MPa, 150MPa, 200MPa, 250MPa, 300MPa, 350MPa, 400MPa, 450MPa, 500MPa, 550MPa, 600MPa, preferably 100-500MPa, more preferably 300-500MPa.

[0047] It should be noted that the source of starch raw materials is not limited. For example, starch raw materials can come from a variety of plants, such as corn, wheat, cassava, potato, rice, etc. These plant starches have different structures and properties, and suitable starch raw materials can be selected for modification according to specific application requirements. For example, corn starch has a high amylose content and is suitable for applications requiring high viscosity and film-forming properties; while cassava starch has good swelling and solubility and is suitable for use as a food thickener and emulsifier. By selecting different sources of starch raw materials, the performance of modified starch can be further optimized to meet the needs of different application scenarios.

[0048] According to an embodiment of the present invention, the method includes: S1: subjecting the starch raw material to the butyrylation treatment to obtain starch containing butyryl groups; S2: subjecting the starch containing butyryl groups to the ultrahigh pressure treatment to obtain modified starch. Thus, by performing butyrylation and ultrahigh pressure treatment in steps, the reaction conditions can be better controlled and the modification effect of starch can be further optimized; specifically, first, by butyrylation treatment, sufficient butyryl groups are introduced into the starch molecules; and then high pressure treatment is performed to further improve the structure and properties of the modified starch, so that it has better solubility, stability and digestibility; compared to the modified starch obtained by first performing ultrahigh pressure treatment and then butyrylation treatment, the modified starch prepared by this method has a higher degree of butyryl group substitution, better physical and chemical properties and physiological functions, and can more effectively promote the production of short-chain fatty acids (such as butyric acid), thereby playing a more significant role in regulating intestinal flora, maintaining intestinal health, lowering cholesterol levels, reducing fat production, and alleviating and / or treating intestinal inflammation.

[0049] According to an embodiment of the present invention, the pressure of the ultrahigh pressure treatment is 100-500 MPa. Exemplarily, the pressure of the ultrahigh pressure treatment is 100 MPa, 150 MPa, 200 MPa, 250 MPa, 300 MPa, 350 MPa, 400 MPa, 450 MPa, 500 MPa, preferably 300-500 MPa.

[0050] According to an embodiment of the present invention, the butyrylation treatment method includes chemical butyrylation and / or enzymatic butyrylation. Thus, the starch raw material is butyrated by chemical butyrylation and / or enzymatic butyrylation. For example, chemical butyrylation and enzymatic butyrylation each have their own advantages: chemical butyrylation has mild reaction conditions, high reaction efficiency, and can quickly introduce butyryl groups; enzymatic butyrylation has higher selectivity and specificity, and can more accurately control the introduction position and number of butyryl groups. The appropriate butyrylation method can be selected according to specific application requirements to further optimize the performance of the modified starch.

[0051] According to an embodiment of the present invention, the butyrylation treatment comprises: contacting an acylating agent with the starch raw material to induce an esterification reaction, thereby obtaining starch containing butyryl groups; wherein the pH of the contact reaction is 8.0 to 8.5. Thus, by conducting the esterification reaction under alkaline conditions, the reaction between the acylating agent and the starch molecules is effectively promoted, further improving the efficiency of butyryl group introduction. By controlling the pH of the contact treatment, the esterification reaction can be ensured to proceed smoothly while avoiding degradation of the starch molecules due to excessive reaction, thereby further ensuring the quality and performance of the prepared modified starch.

[0052] According to an embodiment of the present invention, the time of the ultra-high pressure treatment is 5 to 25 minutes. Thus, by controlling the ultra-high pressure treatment time, while ensuring that the starch granule structure is fully changed and the starch modification effect is achieved, excessive degradation or structural damage of starch molecules caused by ultra-high pressure treatment for too long is avoided, production efficiency is improved, and energy consumption is reduced; illustratively, the time of the ultra-high pressure treatment is 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, 25 minutes, preferably 10 to 15 minutes, more preferably 10 minutes.

[0053] modified starch

[0054] The present invention provides a modified starch. According to an embodiment of the present invention, the modified starch is prepared by the aforementioned method. The modified starch according to the embodiment of the present invention has a higher degree of butyryl group substitution, improved solubility, stability, and digestibility. This modified starch has great application potential in fields such as food, health products, and pharmaceuticals, and is suitable for various application scenarios.

[0055] Food, health products or medicines

[0056] The present invention provides a food, health product or medicine, which, according to an embodiment of the present invention, comprises: the aforementioned modified starch.

[0057] Those skilled in the art will understand that the characteristics and advantages described above for the modified starch are also applicable to the food, health care product or medicine, and will not be described in detail here.

[0058] Application in the preparation of food, health products or medicines

[0059] The present invention provides the aforementioned method and use of the aforementioned modified starch in the preparation of foods, health products, or pharmaceuticals. According to embodiments of the present invention, the pharmaceutical has at least one of the following uses: regulating intestinal flora; maintaining intestinal health; lowering cholesterol levels; reducing fat production; alleviating and / or treating intestinal inflammation; or maintaining or increasing intestinal butyrate concentrations. According to the use of the embodiments of the present invention, the modified starch obtained by the aforementioned preparation method of the present invention can significantly regulate the composition and function of the intestinal flora, promote the growth of beneficial bacteria (such as Bacteroidetes and Firmicutes), and inhibit the reproduction of harmful bacteria, thereby maintaining the balance of intestinal microecology, enhancing the intestinal barrier function, improving the intestinal resistance to pathogens, and preventing and alleviating intestinal-related diseases; and can provide energy for intestinal epithelial cells by promoting the production of short-chain fatty acids (such as butyric acid), consolidate the intestinal barrier function, reduce inflammatory response, promote the proliferation and differentiation of intestinal epithelial cells, further enhance the self-repair ability of the intestine, prevent the occurrence of intestinal diseases, and maintain intestinal health; the modified starch can also regulate intestinal flora and metabolic pathways, reduce serum cholesterol levels, reduce liver fat production, and have a positive effect on cardiovascular health and metabolic diseases (such as obesity and non-alcoholic fatty liver disease); thus, the food, health product or medicine has the uses of regulating intestinal flora, maintaining intestinal health, lowering cholesterol levels, reducing fat production, alleviating and / or treating intestinal inflammation, maintaining or increasing the concentration of butyric acid in the intestine, and the like.

[0060] According to an embodiment of the present invention, the intestinal flora includes: Bacteroidetes and / or Firmicutes. Thus, the relative abundance of Bacteroidetes and Firmicutes is significantly increased by modified starch; Bacteroidetes and Firmicutes are the main phyla in the intestinal microbial community, and they play a key role in intestinal health. Bacteroidetes is closely related to carbohydrate metabolism and can break down complex dietary fiber and produce short-chain fatty acids; Firmicutes is related to energy metabolism and fat storage; by increasing the relative abundance of these two phyla, modified starch can optimize the structure of the intestinal microbial community, enhance the intestinal metabolism of nutrients, and thus promote intestinal health.

[0061] Below, the scheme of the present invention will be explained in conjunction with embodiment.It will be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.In the embodiment, if specific technology or conditions are not indicated, the technology or conditions described in the literature in this area or the product instructions are used.The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained by commercial purchase.

[0062] Example 1: Preparation of butyrylated starch under ultrahigh pressure

[0063] Method 1: Preparation of single butyrylated starch

[0064] The inventors prepared single butyrylated starch by an aqueous phase method, and the specific operation is as follows:

[0065] First, a sufficient amount of starch (native cassava starch, calculated on a dry basis) was weighed and added to water to prepare a 35% (m / v) starch emulsion, and then the pH of the starch emulsion was adjusted to 8.0-8.5 using a 1 mol / L NaOH solution. Then, butyric anhydride was added dropwise over 30 minutes at a ratio of butyric anhydride to starch (calculated on a dry weight (dw)) of 4:5, and 1 mol / L NaOH solution was continuously added dropwise during the reaction to maintain the pH of the system at 8.0-8.5. At the same time, the mixture was stirred continuously at 40°C for 4 hours using a magnetic stirrer. After the reaction was completed, 0.2 mol / L HCl solution was added to adjust the pH of the system to 6.0-6.5, and the slurry was centrifuged at a speed of 3000 r / min for 10 minutes. After the centrifugation, the supernatant was poured out, and the precipitate was washed repeatedly with pure water and 75% ethanol in an alternating order until the supernatant pH was 7. The precipitate was then freeze-dried at 50°C for 24 hours and passed through a 100-mesh sieve to obtain a single butyrylated starch (labeled as CS). B ) and save it for future use.

[0066] Method 2: Preparation of single ultrahigh pressure treated starch

[0067] A sufficient amount of starch (in dw) was weighed and added to water to prepare a 35% (m / v) starch emulsion. The emulsion was then packaged in polypropylene bags, sealed with a sealing machine, and labeled. The bags were then placed in an ultrahigh pressure device for ultrahigh pressure treatment at 100 MPa / 10 min, 300 MPa / 10 min, and 500 MPa / 10 min to obtain single ultrahigh pressure treated starches, which were labeled as CS and CS, respectively. 100 , CS 300 , CS 500 .

[0068] Method 3: Preparation of butyrylated starch under ultrahigh pressure

[0069] (1) Preparation of starch treated with butyrylation and ultrahigh pressure treatment

[0070] Take the CS obtained by method 1 B The samples were packed in high-pressure polyethylene bags, sealed with a sealing machine and marked, and then placed in an ultrahigh pressure device for ultrahigh pressure treatment at 100 MPa / 10 min, 300 MPa / 10 min, and 500 MPa / 10 min. The precipitate was washed repeatedly with pure water and 75% ethanol in an alternating sequence until the supernatant pH = 7. The precipitate was then freeze-dried at 50°C for 24 h and passed through a 100-mesh sieve to obtain butyrylated + ultrahigh pressure treated starch, which was marked as CS. B+100 , CS B+300 , CSB+500 , save for later use.

[0071] (2) Preparation of starch treated with ultrahigh pressure and butyrylation

[0072] Take the CS obtained by method 2 respectively 100 , CS 300 , CS 500 The sample was prepared into a 35% (m / v) starch emulsion, and the starch emulsion was treated according to the processing steps of method 1 to obtain ultrahigh pressure treated + butyrylated starch, which were marked as CS 100+B , CS 300+B , CS 500+B , save for later use.

[0073] Example 2: Characterization of the structure and properties of butyrylated starch obtained under different treatment conditions

[0074] 1. Polarized cross detection of butyrylated starch

[0075] Polarized cross detection was performed on untreated native cassava starch (NCS) and starch samples obtained in Example 1 that were only butyrylated, starch that was only ultrahigh pressure treated, starch that was butyrylated and treated with ultrahigh pressure, and starch that was butyrylated and treated with ultrahigh pressure, respectively. The specific steps are as follows:

[0076] 0.05 mg of starch sample was dispersed in 2 mL of distilled water and gently shaken to obtain a starch sample solution. Then, 100 μL of starch sample solution was immediately dropped onto a glass slide and covered with a coverslip. The starch sample was observed and photographed under an optical microscope (eyepiece magnification 10×, objective lens 10×), and the morphological characteristics of the starch sample particles under normal light source were recorded. Then, the polarizing device of the microscope was turned on to observe the Maltese cross phenomenon of the starch sample particles under polarized light conditions and record their morphological characteristics.

[0077] The polarized cross-sectional examination results of butyrylated starch obtained under different treatment conditions are shown in Figure 1 .

[0078] The results show:

[0079] (1) When observed under an ordinary microscope, the cassava starch granules (NCS) are evenly distributed, and the surface of the granules is smooth and free of cracks. Under a polarizing microscope, it can be observed that the starch granules are divided into four regions, showing a unique Maltese cross pattern.

[0080] (2) When the crystalline structure of starch granules is destroyed, the unique birefringence and Maltese cross phenomenon exhibited under a polarized light microscope will become weaker or no longer appear.

[0081] (3) After ultrahigh pressure treatment, such as CS 100 , CS 300 The starch granules did not agglomerate, and the polarization cross phenomenon still existed, but it gradually weakened with the increase of pressure.

[0082] (4)CS 100+B , CS 300+B , CS B+100 , CS B+300 The change patterns of the starch obtained by the combined treatment in optical microscope images and polarization cross phenomenon are consistent with those of the starch obtained by the single treatment method.

[0083] (5)CS 500 , CS 500+B , CS B+500 The starch was severely aggregated and the birefringence was lost.

[0084] The above results show that NCS has a complete crystal structure. The density and refractive index of the crystalline and non-crystalline regions are anisotropic, which leads to the occurrence of birefringence, reflecting the high order of the cassava starch particles. B ) agglomeration and slight surface cracking occurred, which may be because butyrylation enhanced the interaction between starch molecules, promoted the formation of a new ordered structure and led to an increase in the size of starch granules. However, the birefringence phenomenon still existed but weakened, indicating that butyrylation destroyed the crystal structure of starch; the high pressure of 500 MPa may induce the gelatinization of starch at room temperature, resulting in serious starch agglomeration.

[0085] 2. Particle size distribution detection of butyrylated starch

[0086] The particle size distribution of untreated natural cassava starch and the starch obtained in Example 1 with single butyrylation treatment, starch with single ultrahigh pressure treatment, starch with butyrylation treatment + ultrahigh pressure treatment, and starch with butyrylation treatment + ultrahigh pressure treatment were tested respectively. The specific steps are as follows:

[0087] The particle size distribution of starch samples was determined using a Malvern Master sizer 2000 laser diffraction analyzer and a 1000 mL flow-through reservoir. The refractive indices of the starch sample and the dispersant 90% ethanol were 1.53 and 1.33, respectively. 0.05 g of starch sample was dispersed in 10 mL of 90% ethanol and ultrasonically treated for 2 min. The results were repeated three times with an average value taken based on a light shielding rate of 15%. The results obtained for each starch sample (n = 3) were then analyzed using the Master sizer software.

[0088] The particle size distribution test results of butyrylated starch obtained under different treatment conditions are shown in Table 1. The particle size distribution of butyrylated starch obtained under different treatment conditions is shown in Figure 2 .

[0089] Table 1 Particle size distribution test results of butyrylated starch obtained under different treatment conditions

[0090]

[0091]

[0092] In Table 1, D[4,3] is the volume average diameter, in μm; D[3,2] is the surface area average diameter, in μm; Dx(10) is the 10% cumulative particle size, in μm; Dx(50) is the median particle size, in μm; Dx(90) is the 90% cumulative particle size, in μm; different lowercase letters in the same column indicate that there are significant differences in different particle size indicators after the starch is subjected to the same treatment sequence and different treatment pressures; different uppercase letters in the same column indicate that there are significant differences in different particle size indicators after the starch is subjected to different treatment sequences and the same treatment pressure (p<0.05).

[0093] The results show:

[0094] (1) The volume average diameter (D[4,3]) of natural cassava starch without any treatment (NCS, control group) was 14.23 μm, the median particle size (Dx(50)) was 13.64 μm, and the specific surface area was 470.99 m 2 / g, indicating that its particles are small and evenly distributed.

[0095] (2) Single butyrylation treatment group (CS B ) significantly increased to 97.79 μm, Dx(50) increased to 94.99 μm, and the specific surface area decreased to 82.91 m 2 / g, indicating that the butyrylation reaction caused severe aggregation of starch granules.

[0096] (3) Single ultrahigh pressure treatment group (CS 100 , CS 300 , CS 500 ) are 16.65μm, 17.07μm, and 54.54μm, respectively. The specific surface area increases first and then decreases with increasing pressure (the highest is 593.1m 2 / g), indicating that the ultrahigh pressure treatment induced the roughening of the particle surface and partially refined the particles.

[0097] (4) Butyrylation + ultrahigh pressure synergistic treatment group (CS B+100 , CS B+300 , CS B+500) are 19.61μm, 82.85μm, 53.52μm, and Dx(50) are 17.43μm, 63.89μm, 49.01μm, respectively. The specific surface area is significantly higher than that of CS~B~(570.19m 2 / g, 179.34m 2 / g, 191.38m 2 / g), indicating that the synergistic treatment effectively suppressed the agglomeration caused by butyrylation and simultaneously optimized the particle size distribution by dispersing the particles under high pressure.

[0098] (5) Ultrahigh pressure + butyrylation treatment group (CS 100+B , CS 300+B , CS 500+B )’s D[4,3] are 19.61μm, 82.85μm, 53.52μm, which is similar to CS B+100 Compared with the group treated with butyrylation followed by ultrahigh pressure treatment, the particle size distribution is more concentrated (such as CS 100+B Dx(90)=34.48μm, significantly lower than CS B 152.55μm).

[0099] The above results show that compared with single butyrylation treatment (CS B ) or single ultra-high pressure treatment (CS 100 etc.), butyrylation and ultrahigh pressure co-treatment (CS B+100 Through physical-chemical synergy, the particle size distribution is optimized while suppressing particle agglomeration, significantly improving the specific surface area and particle dispersibility. Specifically, the combination of butyrylation followed by ultrahigh pressure treatment (CS B+100 ) had a better effect, with a Dx(90) of only 34.48 μm, indicating that the synergistic treatment not only overcame the aggregation defects of single butyrylation, but also prepared a more uniform and stable modified starch through high-pressure particle refinement.

[0100] 3. Butyrylated starch 1 H NMR analysis

[0101] Since DMSO-d6 and starch are hygroscopic, 1 Before H NMR analysis, starch samples were prepared by the steps described in Example 1. Then, the natural cassava starch without any treatment and the starch obtained in Example 1 with single butyrylation treatment, starch with single ultrahigh pressure treatment, starch with butyrylation treatment + ultrahigh pressure treatment, and starch with butyrylation treatment + ultrahigh pressure treatment were respectively subjected to H NMR analysis. 1 H NMR analysis, the specific steps are as follows:

[0102] 20 mg of starch sample was dissolved in 1 mL of dimethyl sulfoxide-d6 (DMSO-d6, purchased from Sigma-Aldrich Trading Co., Ltd.) and stirred continuously at 85°C until a clear starch solution was obtained; then 500 μL of the starch solution was transferred to a 5 mm NMR tube and subjected to nuclear magnetic resonance (recorded at 298 K, 16 scans); chemical shift measurements were performed relative to the DMSO proton calibration chemical shift of 2.5 ppm; and the obtained values ​​were analyzed using MestReNova 14.0.1 software. 1 The H-NMR spectrum was used to calculate the degree of substitution (DS) and degree of branching (DB) of the starch sample using formula (1) and formula (2):

[0103]

[0104] In formula (1), A is the sum of the signal areas of methyl protons at 1.90 to 2.30 ppm. The butyrylation of starch produces three new peaks, with the methylene signals at 2.27 ppm and 1.55 ppm, and the methyl signal at 0.89 ppm. C is the integral of the proton signals of any hydroglucose unit. The signals of the four protons of the anhydroglucose part of the molecule are 5.50 ppm (OH-3), 5.40 ppm (OH-2), 4.90 ppm (OH-4), and 4.58 ppm (OH-6).

[0105]

[0106] In formula (2), A α-1,6 is the peak area of ​​α-1,6 glucosidic bond; A α-1,4 is the peak area of ​​α-1,4 glucoside bond.

[0107] The calculation results of substitution degree and branching degree of butyrylated starch obtained under different treatment conditions are shown in Table 2. 1 The results of H NMR analysis are shown in Figure 3 .

[0108] Table 2 Calculation results of substitution degree and branching degree of butyrylated starch obtained under different treatment conditions

[0109]

[0110]

[0111] In Table 2, different lowercase letters in the same column indicate that DS and DB of starch were significantly different after the same treatment sequence and different treatment pressures; different uppercase letters in the same column indicate that DS and DB of starch were significantly different after different treatment sequences and the same treatment pressures (p<0.05).

[0112] The results showed that the treatment order had a significant effect on the DS value of butyrylated starch. The DS value of the natural cassava starch sample without any treatment (NCS, control group) was not detected. It had a high thermal stability (high initial weight loss temperature) due to its intact hydrogen bond and double helix structure. The starch in the single butyrylation treatment group (CS B ) due to the destruction of hydrogen bonds caused by acyl substitution, the thermal stability decreased (the initial weight loss temperature decreased and the weight loss rate increased); while the starch in the butyrylation + ultrahigh pressure treatment group (CS B+100 , CS B+300 , CS B+500 ) significantly increased, reaching the highest value when treated at 500 MPa. This is presumably because the structure became loose after the introduction of acyl groups, and ultrahigh pressure further destroyed the molecular arrangement, resulting in more significant thermal decomposition. 100+B , CS 300+B , CS 500+B ) Due to starch aggregation and even gelatinization, the DS value did not increase significantly. It is speculated that the UHP pretreatment densified the starch structure, which limited the subsequent butyrylation reaction.

[0113] 4. FT-IR analysis of butyrylated starch

[0114] The untreated natural cassava starch and the starch obtained in Example 1 with single butyrylation treatment, the starch with single ultrahigh pressure treatment, the starch with butyrylation treatment + ultrahigh pressure treatment, and the starch with butyrylation treatment + ultrahigh pressure treatment were subjected to FT-IR analysis of the starch samples using the potassium bromide (KBr) pellet method to verify the specific bands that appeared during the butyrylation and ultrahigh pressure treatment, and further verify the group changes. The specific steps are as follows:

[0115] A starch sample was dried at 50°C for 1 hour, potassium bromide (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) was dried at 105°C for 4 hours, and then placed in a desiccator to cool naturally. 1-2 mg of starch sample and 200 mg of potassium bromide were then mixed in an agate mortar, thoroughly ground, and mixed to obtain a starch-potassium bromide mixed sample. The starch-potassium bromide mixed sample was then placed on a tablet press for tableting. The pressed slices were placed in a sample chamber for infrared scanning (with pure KBr as the background). The starch sample was analyzed by infrared spectroscopy using a PerkinElmer FTIR analyzer (test conditions: scanning range 4000-400 cm -1 Between, resolution 4cm -1 , cumulative number of scans 32).

[0116] The FT-IR analysis results of butyrylated starch obtained under different treatment conditions are shown in Figure 4 .

[0117] The results showed that: (1) compared with the natural cassava starch (NCS) without any treatment, the butyrylation treatment group (CS B ) at 1740cm -1 A new ester carbonyl characteristic peak is formed at the position of the ester, indicating that butyric anhydride and starch hydroxyl groups have successfully undergone esterification. B Group at 3400cm -1 The hydroxyl absorption peak at the position of butyryl was significantly weakened), indicating that the butyryl group replaced the active hydroxyl group in the starch molecule and destroyed the internal hydrogen bond.

[0118] (2) Butyrylation treatment + ultrahigh pressure treatment group (such as CS B+500 ) at 1740cm -1 The ester carbonyl peak is still retained at 3400 cm -1 The absorption peak intensity at the CSB group increased significantly compared with that of the CSB group, indicating that the ultrahigh pressure treatment reorganized the hydrogen bonds between starch molecules through physical action, exposing more hydroxyl sites. 500 ) No ester carbonyl peak appeared, indicating that ultrahigh pressure itself did not introduce chemical groups.

[0119] The above results show that the technical solution of the present invention (butyrylation combined with ultrahigh pressure treatment) is more effective than single butyrylation treatment (CS B ), significantly improving the accessibility of hydroxyl groups through hydrogen bond reorganization (3400cm -1 Peak enhancement); compared with single ultrahigh pressure treatment (CS 500 ), the directional modification of functional groups was achieved through esterification reaction (1740cm -1 The synergistic effect of the two can optimize the degree of substitution (DS) and short-range order (R 1047 / 1022 ratio changes), providing a structural basis for improving digestion resistance and thermal stability.

[0120] 5. XRD analysis of butyrylated starch

[0121] The untreated natural cassava starch and the starch obtained in Example 1 with single butyrylation treatment, the starch with single ultrahigh pressure treatment, the starch with butyrylation treatment + ultrahigh pressure treatment, and the starch with butyrylation treatment + ultrahigh pressure treatment were analyzed by X-ray diffractometer (XRD) to analyze the crystal structure and properties of the starch samples. The specific steps are as follows:

[0122] X-rays are Cu-Kα rays with a wavelength of The scanning range was 4-40° and the test rate was 2° / min. X-ray diffraction patterns were obtained, and the X-ray diffraction patterns were analyzed and the crystallinity was calculated based on them. The relative crystallinity (RC) is the ratio between the crystalline area and the amorphous area of ​​the X-ray diffraction pattern. The peak characteristics of the XRD pattern were compared with the theoretical diffraction pattern to obtain the crystal type. The relative crystallinity was then calculated using MDI Jade software and formula (3):

[0123]

[0124] In formula (3), Aci is the area corresponding to the crystallization peak position; At is the total area of ​​the X-ray diffraction pattern.

[0125] The calculated results of the crystallinity of butyrylated starch obtained under different treatment conditions are shown in Table 3, and the XRD analysis results of butyrylated starch obtained under different treatment conditions are shown in Figure 5 .

[0126] Table 3 Calculation results of the crystallinity of butyrylated starch obtained under different treatment conditions

[0127]

[0128] In Table 3, PeakArea is the peak area; Total is the total peak area, that is, the total area of ​​all diffraction peaks in the X-ray diffraction (XRD) pattern, including the contribution of crystalline and non-crystalline regions; Diffraction is the diffraction peak area), specifically referring to the diffraction peak area attributable to the crystalline structure, reflecting the intensity of the crystalline part in the sample; Crystalline Degree is the crystallinity, that is, the percentage of the crystalline region peak area (Diffraction) to the total peak area (Total), which is used to quantify the proportion of crystalline structure in the sample.

[0129] The results show:

[0130] (1) Natural cassava starch (NCS) without any treatment showed typical A-type crystal characteristic peaks at 15°, 17°, 23° and 28°, indicating that its crystal structure was complete and orderly. B ) or ultrahigh pressure butyrylation (CS 100+B , CS 300+B , CS 500+B The diffraction peak position of the sample after butyrylation (CS) is consistent with that of NCS, and no new peaks appear, indicating that these treatments have not changed the crystal type of starch, and the reaction mainly occurs in the amorphous region. However, when the ultrahigh pressure pressure is increased to 500 MPa, the ultrahigh pressure treatment group after butyrylation (CS) B+500 ) significantly weakened, and completely showed amorphous characteristics, indicating that high pressure treatment caused the complete destruction of the crystal structure of starch granules; while the first ultrahigh pressure and then butyrylation group (CS500+B ) a new characteristic peak (type B crystal) appeared at 6°, indicating that high pressure treatment promoted butyric anhydride to enter the interior of starch granules, triggering the crystal transformation ( Figure 5 ).

[0131] (2) The relative crystallinity of NCS was 34.79%, which decreased to 30.79% after single butyrylation treatment (CSB). The crystallinity of the group treated with butyrylation followed by ultrahigh pressure treatment (CSB+500) further decreased to 22.88%, which was significantly lower than that of NCS (p<0.05). In contrast, the crystallinity of the group treated with ultrahigh pressure alone (CS500) completely disappeared (0%), indicating that the damage to the crystal structure caused by high pressure treatment is pressure-dependent. It is worth noting that the crystallinity of the CS500+B group was still retained at 19.93% despite being treated with 500 MPa high pressure, indicating that the butyrylation reaction may have partially repaired the crystal defects caused by high pressure (Table 3).

[0132] The above results show that:

[0133] (1) Ultrahigh pressure treatment (500 MPa) combined with butyrylation modification can significantly destroy the crystal structure of starch, making it completely amorphous (CS B+500 ), or induced crystal transformation (CS 500+B ), while single butyrylation or low pressure treatment had limited effects on the crystal type.

[0134] (2) The physical effect of ultrahigh pressure and the chemical modification of butyrylation have a synergistic effect: high pressure treatment promotes the penetration of butyric anhydride into the interior of starch granules, especially in the crystalline region, and the introduction of butyrylation groups further weakens the intermolecular hydrogen bonds, resulting in a decrease in crystallinity.

[0135] (3) Compared with single butyrylation or ultrahigh pressure treatment, the ultrahigh pressure combined with butyrylation treatment technology (B+H) proposed in the present invention can more accurately regulate the crystal structure and order of starch, providing a theoretical basis for the development of high-resistant starch (RS) products.

[0136] 6. TGA analysis of butyrylated starch

[0137] The untreated natural cassava starch and the starch obtained in Example 1 with single butyrylation treatment, the starch with single ultrahigh pressure treatment, the starch with butyrylation treatment plus ultrahigh pressure treatment, and the starch with butyrylation treatment plus ultrahigh pressure treatment were subjected to TGA analysis using a thermogravimetric analyzer (TGA) to obtain the thermal stability of the starch samples. The specific steps are as follows:

[0138] Accurately weigh 5 mg of starch sample, set the test temperature range to 25℃~600℃, and the heating rate to 10℃ / min. Record the change of starch sample mass over time to obtain the weight loss curve of starch sample. The weight loss curve includes key information such as the starting temperature of mass loss, peak temperature and residual mass. Then, in order to further analyze the thermal decomposition process of starch sample, the obtained weight loss curve is derived to obtain DTG (Derivative thermogravimetric) curve. The TGA analysis results of butyrylated starch obtained under different treatment conditions are shown in Figure 6 .

[0139] The results show:

[0140] (1) Compared with the natural cassava starch (NCS) without any treatment, the single butyrylation treatment group (CS B ) decreased from 232°C to 230°C, and the weight loss rate increased from 83.30% to 85.57%, indicating that the introduction of butyryl groups weakened the hydrogen bonding effect, resulting in a decrease in thermal stability.

[0141] (2) UHP treatment significantly improved the thermal stability of butyrylated starch. For example, the 500 MPa UHP treatment group (CS B+500 )'s weight loss starting temperature returned to 207 °C, the weight loss rate dropped to 79.32%, and the residual mass increased to 16.88%, indicating that ultrahigh pressure treatment improved the thermal stability by reorganizing the molecular structure.

[0142] (3) Ultrahigh pressure followed by butyrylation treatment group (such as CS 500+B ) was weaker than that of the group treated with butyrylation followed by ultrahigh pressure treatment (CS B+500 ), the weight loss starting temperature was 180℃, the weight loss rate was 73.94%, and the residual mass was 21.99%, which further demonstrated the regulatory effect of the treatment sequence on the thermal properties of starch.

[0143] The above results show that:

[0144] TGA analysis revealed that butyrylation reduced the thermal stability of cassava starch, while butyrylation before and after ultrahigh pressure treatment (UHP) improved its thermal stability, suggesting that HPP's physical modification of the starch granule structure may enhance its stability during thermal decomposition. Butyrylation increased the resistant starch (RS) content and reduced the rapidly digestible starch (RDS) content of cassava starch. The introduction of ultrahigh pressure treatment further modulated the digestibility of starch, and chemical modification followed by physical modification showed potential application advantages in balancing starch digestibility and resistance to digestibility.

[0145] 7. Determination of resistant starch content in butyrylated starch

[0146] The resistant starch content of the untreated natural cassava starch and the starch obtained in Example 1, which was treated with butyrylation alone, treated with ultrahigh pressure alone, treated with butyrylation plus ultrahigh pressure, and treated with butyrylation plus ultrahigh pressure were determined. The specific steps were as follows:

[0147] Accurately weigh 100 mg of starch sample and place it in a polypropylene tube for reaction. Add 5 mL of buffer (phosphate buffer (PBS) with a pH of 7.0, the specific formula of which is: 0.1 mol / L Na2HPO4 (14.2 g / L), 0.1 mol / LKH2PO4 (13.6 g / L), containing 0.02% NaN2 (antibacterial agent)) to moisten the sample and equilibrate in a 37°C water bath for 5 minutes. After ensuring that the temperature is stable, immediately add 4 mL of pancreatic α-amylase / glucoamylase (PAA / AMG) mixed solution (enzyme activity: PAA 10 U / mL, AMG 3 U / mL); and take 1 mL of sample at 20 min, 120 min, and 240 min, respectively, and immediately add 4 mL of 0.5 mol / L acetic acid solution to terminate the reaction. Add glucoamylase (AMG) to the sample solution after the reaction is terminated, and continue the reaction for a period of time to ensure that all maltose is hydrolyzed into glucose, and use GOPOD reagent for color reaction (completely convert the glucose in the sample solution into a color product, thereby ensuring that all glucose in the sample solution can be detected). After the color reaction is completed, the absorbance of the blank reagent and the absorbance of the sample solution at 510 nm are measured, and the content of rapidly digestible starch (RDS) is calculated according to formula (4), the content of slowly digestible starch (SDS) is calculated according to formula (5), and the content of resistant starch (RS) is calculated according to formula (6); at the same time, the residue after incubation for 4 hours is recovered, and the precipitate is collected by centrifugation (8000×g, 10 min), and freeze-dried for subsequent in vitro fermentation experiments, i.e., the digestion product, for later use.

[0148]

[0149] RS (%) = (TS - RDS - SDS) × 0.9 × 100 Formula (6)

[0150] In formula (4) and formula (5), G20 and G120 are the glucose contents at 20 min and 120 min, respectively; in formula (5), TS is the total mass (mg) of the starch sample.

[0151] The results of determination of resistant starch content of butyrylated starch obtained under different treatment conditions are shown in Figure 7 .

[0152] The results showed that: untreated natural cassava starch (NCS) contained more high-branched starch, resulting in poor digestibility and low RS content; butyrylation treatment could significantly increase the RS content and reduce the RDS content in starch, thereby improving the digestibility of starch; under different pressures, the combination of butyrylation followed by ultrahigh pressure treatment could improve the digestibility and digestibility of starch, among which CS B+500 The butyric acid production and proportion of the group were significantly higher than those of the other groups, indicating that this treatment can effectively promote the production of butyric acid.

[0153] The above results show that both butyrylation and ultrahigh pressure treatment can significantly increase the resistant starch (RS) content of cassava starch and reduce the rapidly digestible starch (RDS) content, thereby improving the digestibility of starch. In particular, the combination of butyrylation followed by ultrahigh pressure treatment shows potential application advantages in balancing starch digestibility and resistance to digestion. In addition, the surface of starch becomes roughened after butyrylation, and ultrahigh pressure treatment further causes the starch surface to break. These structural changes are conducive to the attachment and fermentation of intestinal microorganisms, thereby promoting the production of short-chain fatty acids (SCFAs); FOS and CS B+100 The butyric acid content of the group decreased in the late fermentation period, indicating that butyric acid may have been degraded.

[0154] Example 3: Investigation of in vitro fermentation characteristics of butyrylated starch obtained under different treatment conditions

[0155] 1. In vitro batch fermentation experiment

[0156] In vitro batch fermentation experiments were conducted on untreated native cassava starch, the starch obtained in Example 1 that was only butyrylated, and the starch that was butyrylated and treated with ultrahigh pressure. The specific steps were as follows:

[0157] (1) Preparation of buffer solution

[0158] 1000 mL of 0.1 mol / L carbonate-phosphate buffer (PBS, purchased from Beijing Solebow Technology Co., Ltd.) was prepared and adjusted to pH 7.0. After high-pressure sterilization, it was placed in an anaerobic workstation for deoxygenation overnight to obtain a PBS solution.

[0159] (2) Preparation of culture medium

[0160] 15 g of peptone (purchased from Beijing Solaibao Technology Co., Ltd.) was dissolved in about 1 L of milli-Q water (ultrapure water) and the pH was adjusted to 7.0. The volume was then filled to 1 L with Milli-Q water to obtain a peptone solution, which was sterilized for later use. 312 mg of cysteine ​​(purchased from Shanghai Yuanye Biotechnology Co., Ltd.) and 312 mg of sodium sulfide (purchased from Shanghai Yuanye Biotechnology Co., Ltd.) were dissolved in 2 mL of 1 mol / L NaOH solution and the volume was made up to 50 mL with sterile Milli-Q water to obtain a cysteine-sodium sulfide mixture, which was filtered and sterilized for later use. A 0.1% (wt / vol) resazurin solution (purchased from Shanghai Yuanye Biotechnology Co., Ltd.) was prepared to obtain a resazurin solution, which was filtered and sterilized for later use. Finally, the sterilized 1 L of peptone solution was mixed with 50 mL of reducing solution (cysteine-sodium sulfide mixture) and 1.25 mL of resazurin solution to obtain a culture medium solution.

[0161] (3) Sample collection and inoculum pretreatment

[0162] Feces of four healthy volunteers (two males and two females, aged 20 to 25 years, with no history of digestive system diseases, and no use of antibiotics in the six months before sample collection, and no intake of any probiotic products in the two weeks before sample collection) were collected as intestinal flora samples; equal amounts of the four fecal samples were weighed in an anaerobic chamber (90% N2, 5% CO2, 5% H2), and mixed evenly with the PBS solution prepared in step (1) at a ratio of 1:9 (m / v), and then filtered through four layers of gauze to remove insoluble fecal residue to obtain a fecal inoculum.

[0163] (4) In vitro fermentation of starch samples

[0164] In vitro fermentation was performed with NCS (natural cassava starch without any treatment, negative control), FOS (oligofructose, positive control), CS B , CS B+100 , CS B+300 , CS B+500 The starch sample was used as a carbon source and then digested, washed, and freeze-dried to obtain a fermentation sample. The specific steps are as follows:

[0165] 400 mg of starch sample (on a dry basis) was weighed and placed in a 50 mL anaerobic fermentation tube. No additional substrate was added to the blank control group, and 400 mg of oligofructose was added to the positive control. In the anaerobic workstation, 32 mL of the culture medium prepared in step (2) and 8 mL of the fecal inoculum obtained in step (3) were added to the anaerobic fermentation tube containing the digestion product obtained in step 7 of Example 2 and mixed evenly. The tube cap was then tightened, and the anaerobic fermentation tube was placed in a fermentation bag containing an anaerobic gas production bag and transferred to a 37°C shaker for fermentation. Fermentation broth samples were collected at 0, 6, 12, 18, and 24 h, respectively. The collected fermentation broth samples were immediately placed on ice for 15 min to quickly terminate the reaction, and then centrifuged at 8000 × g for 10 min to separate the supernatant A and the precipitate B. The obtained supernatant A and precipitate B were numbered and frozen at -80°C for later use.

[0166] 2. Determination of starch morphology changes before and after in vitro fermentation

[0167] The natural cassava starch (NCS) without any treatment, the starch with single butyrylation treatment obtained in Example 1, the starch with butyrylation treatment + ultrahigh pressure treatment, the NCS (precipitate B) obtained by in vitro fermentation in step 1, CS B (precipitate B), CS B+100 (precipitate B), CS B+300 (precipitate B) and CS B+500 (Precipitation B) The morphology change was measured using scanning electron microscopy (SEM). The specific steps are as follows:

[0168] A small amount of freeze-dried starch sample was evenly pasted on double-sided conductive adhesive and fixed on the SEM stage. A layer of gold was sprayed on the surface of the starch sample to enhance conductivity. The sample morphology was first observed as a whole and then representative locations were selected for photography. The test conditions were: high vacuum, electron gun acceleration voltage 15.0kV.

[0169] The results of the starch morphology changes before and after in vitro fermentation are shown in Figure 8 .

[0170] The results show:

[0171] (1) The surface of the particles of natural cassava starch (A1, A2) before fermentation without any treatment was smooth, without cracks, and the particles were complete, round, oval or elliptical, and evenly distributed.

[0172] (2) After butyrylation reaction, CS B (B1, B2) The particle morphology changed slightly, but the particle surface was slightly rough, and only the cassava starch treated with butyrylation was agglomerated.

[0173] (3)CSB+100 The surface morphology of (C1, C2) is almost the same as that of native starch. Although they have been butyrylated, they have not agglomerated.

[0174] (4)CS B+300 (D1, D2) With the increase of substitution degree and pressure, the number of broken starch particles increases and the surface becomes concave. At 500 MPa, the substitution degree is the highest, and the starch surface shows more severe cracking and fragmentation.

[0175] (5) CSB and CSB+H starches suffered severe surface erosion and structural damage, with almost no intact starch granules remaining.

[0176] (6)CS B+100 and CS B+300 The inside of starch is hollow, but it has many flaky structures.

[0177] (7)CS B+500 It completely loses its original shape and a large number of bacteria adhere to the surface.

[0178] The above results show that:

[0179] (1) Butyric anhydride corrodes the surface of cassava starch when reacting with starch.

[0180] (2) The effect of ultrahigh pressure may strengthen the hydrogen bonds between starches and make the starch evenly distributed.

[0181] (3) Cassava starch belongs to type A starch, which has a unique structure of surface micropores and channels leading to a central cavity, making it most susceptible to ultrahigh pressure. Its rough surface provides more sites for the attachment of digestive enzymes and intestinal microorganisms, thereby promoting fermentation, acid production, and the growth of beneficial bacteria.

[0182] (4) Although the surface of natural cassava starch NCS without any treatment has lost its gloss, it indicates that the degradation of NCS by microorganisms is limited to the surface of cassava starch, which is an "outside-in" degradation mode, but it still maintains a relatively complete morphology.

[0183] (5)CS B+100 and CS B+300 Starch is hollow inside, but contains many flaky structures, indicating that intestinal microorganisms digest and degrade it from the inside. This degradation pattern is called "from the inside out."

[0184] (6) The effect of ultrahigh pressure can change the degradation mode of cassava starch from “outside to inside” to “inside to outside”.

[0185] 3. Detection of SCFAs content and pH value of starch after in vitro fermentation

[0186] The FOS (supernatant A), NCS (supernatant A), and CS obtained from the in vitro fermentation in step 1 were B (supernatant A), CS B+100 (supernatant A), CS B+300 (supernatant A) and CS B+500 (Supernatant A), and a CON group was set up as a blank control group (the treatment conditions of this group were: no carbohydrates were added, and in vitro fermentation was carried out according to step 1, and supernatant A was collected and separated) to detect SCFAs content and pH value. The specific steps are as follows:

[0187] (1) SCFAs content detection

[0188] The fermentation supernatant (the supernatant A collected and separated at 0, 6, 12, 18, and 24 h in step 1 (4)) frozen at the early stage (0-6 h), middle stage (6-12 h), late stage (12-18 h), and final stage (18-24 h) of fermentation was thawed at room temperature, and 1.5 mL of the fermentation supernatant was mixed with 150 μL After fully mixing with 50% H2SO4 solution (purchased from Sinopharm Chemical Reagent Co., Ltd.), 2.5 mL of anhydrous ether (purchased from Sinopharm Chemical Reagent Co., Ltd.) was added, vortexed for 5 minutes, and then centrifuged (8000 r / min, 10 minutes); then 900 μL of the upper ether solution was collected and 100 μL of the internal standard (butyric acid-d4 (deuterated butyric acid, C4D7COOH), 500 ug / mL) solution was added; the two were mixed and filtered with a 0.22 μm organic filter membrane to obtain the SCFAs detection sample solution, which was stored at -20°C for later use; and The SCFAs content in the sample liquid was determined by gas chromatography, and the measurement conditions were as follows: DB-WAX column (30m×0.32mm×0.50μm); hydrogen ion flame detector; injection volume 1μL; split ratio 20:1; heating program was 100℃ (held for 10min), -20℃ / min to 200℃ (held for 2min), the temperature of the injector and detector was set to 250℃, the carrier gas (N2) flow rate was 30mL / min; the fuel gas (H2) flow rate was 40mL / min; and the auxiliary gas (Air) flow rate was 400mL / min.

[0189] (2) pH value detection

[0190] The fermentation supernatant (the supernatant A collected and separated at 0, 6, 12, 18, and 24 h in step 1 (4)) frozen at the early fermentation stage (0-6 h), mid-fermentation stage (6-12 h), late fermentation stage (12-18 h), and final fermentation stage (18-24 h) was used for pH determination, and the determination was repeated three times for each group.

[0191] The results of SCFAs content and pH value of each group of starch during in vitro fermentation are shown in Figure 9 .

[0192] The results show:

[0193] (1) SCFAs content detection: The acid production of butyrylated starch digestion products in each treatment group increased at a steady rate, indicating its persistence in colonic fermentation and its potential to cover the distal colon. Acetic acid and butyric acid were the main products, followed by propionic acid. B ) and its derivatives exhibited stable short-chain fatty acids (SCFAs) production characteristics during in vitro fermentation, among which CS B+500 The CS group showed the best butyrate synthesis ability, and its butyrate production and proportion were significantly higher than those of the other treatment groups. This is closely related to the fact that its high degree of substitution esterification structure is easily hydrolyzed by colon microbial esterase to release butyrate, and the metabolic mechanism of intestinal flora converting precursor substances into butyrate through the acetyl-CoA pathway. B+500 , FOS and CS B There was no statistical difference in the total SCFA production between the two groups, but there was a significant difference in the acid composition ratio between the two groups. B The production of acetic acid and propionic acid in the group was higher, while that in the CS group was higher. B+500 The group enhanced butyrate accumulation through the dual pathways of ester bond hydrolysis and lactate metabolism;

[0194] (2) pH value detection: Butyrylated starch (CS B ) showed the fastest pH drop rate during the fermentation process, indicating that it had the strongest fermentation activity, while ultrahigh pressure treatment slowed down the CS B The fermentation rate was significantly increased, especially after treatment at 500 MPa, the pH value at the fermentation end point increased significantly to 4.52, which was attributed to the CS B+500 It has a higher double helix structure, which enables microorganisms to utilize carbon sources more slowly.

[0195] The above results show that:

[0196] (1) The total short-chain fatty acids (SCFAs) content of the control group (NCS) after 24 h of fermentation was 45.2 mM, while the single butyrylation treatment group (CS B ) and butyrylation + ultrahigh pressure treatment group (CS B+500 ) were increased to 78.3mM and 76.8mM, respectively, of which CS B+500 The butyrate ratio in the group was the highest (32.1%, (p<0.05)).

[0197] (2) With single butyrylation treatment (CS B ) Compared with CS B+500The butyrate production of the group was significantly increased (24.7 mM vs. 18.2 mM), indicating that ultrahigh pressure treatment promoted the activity of butyrate-producing bacteria.

[0198] (3) The butyrate content of the positive control (FOS) (28.5%) was lower than that of CS B+500 The results of this study further verified the potential of butyrylation combined with ultrahigh pressure treatment in the targeted regulation of butyrate production.

[0199] 4. High-throughput sequencing of 16S rRNA of fermented starch

[0200] In step 1, the fermentation broth samples were collected at 12h and 24h of fermentation, and the supernatant and precipitate (bacteria) were separated by centrifugation (8000×g, 10min). The precipitates were collected and labeled as FOS (precipitate B), NCS (precipitate B), and CS. B (precipitate B), CS B+100 (precipitate B), CS B+300 (precipitate B) and CS B+500 (Precipitate B), and a CON group was set up as a blank control group (the treatment conditions of this group were: no carbohydrates were added, and in vitro fermentation was carried out according to step 1, and the precipitate B was collected and separated at 12 hours and 24 hours respectively) for 16S rRNA high-throughput sequencing. The specific steps are as follows:

[0201] (1) DNA extraction: using a kit Microbial genomic DNA from starch samples was extracted using a soil DNA Kit (purchased from Omega Bio-Tek (USA), catalog number D5625-01), and DNA quality and concentration were tested by 1.2% agarose gel electrophoresis. During PCR amplification, Pfu high-fidelity DNA polymerase provided by Quanshijin was used, and the number of amplification cycles was strictly controlled (the goal was to minimize the number of cycles while ensuring consistency in amplification conditions between sample batches). In addition, a blank control group was included in the experiment to monitor for microbial contamination that may have been introduced by the environment or reagents. If bands appeared in the blank control group after amplification, contamination was indicated, and the relevant sample group should not be used in subsequent experimental steps.

[0202] (2) PCR amplification and library construction: Forward and reverse primers were used to amplify the V3-V4 hypervariable region. The PCR amplification products were quantified by fluorescence using the Quant-iT PicoGreen dsDNA Assay Kit and a Microplate reader. Based on the fluorescence quantification results and the sequencing requirements of each sample, the samples were mixed in the appropriate proportions. The sequencing library was prepared using Illumina's TruSeq Nano DNA LT Library Prep Kit and sequenced after quality control.

[0203] The results of Coverage index analysis of the α and β diversity of intestinal microbiota obtained from starch under different treatment conditions at 12h and 24h of fermentation are shown in Figure 10 The results of the species analysis of intestinal microbial communities at the phylum level of the starch obtained under different treatment conditions at 12h and 24h of fermentation are shown in Figure 11 .

[0204] The results show:

[0205] (1) The Coverage index values ​​of the samples are all close to 1.0, indicating that the coverage of the sequenced sample library is extremely high and can accurately reflect the true composition and distribution of microorganisms in the samples ( Figure 10 A); The hierarchical clustering curves of the samples are similar, indicating that they are consistent in species evenness, but CS B Group and CS B+500 The range of the samples in the CON group on the horizontal axis is larger, indicating that the species richness of these two groups is significantly higher than that of the other groups. In contrast, the curve of the CON group decreases faster, indicating that its species richness is lower, and the lack of carbon source intake leads to uneven distribution of intestinal microbial flora, reduced diversity, and a significant increase in the proportion of dominant flora ( Figure 10 B); After 24 hours of fermentation, CS B Group and CS B+500 The Chao 1 index of the CS group was higher than that of the CON group, indicating that butyrylated modified starch significantly increased the species richness of the intestinal microbial community. B The Shannon index of the group was significantly higher than that of the CON group, further indicating that butyrylated starch not only increased the richness of the community, but also enhanced the uniformity and diversity of the community. These results indicate that butyrylated starch can promote the diversity and metabolic function of the intestinal microbial community, enhance the stability of the intestinal environment, and indirectly limit the proliferation of pathogens by increasing beneficial bacteria and metabolites, thereby having a positive impact on host health ( Figure 10 C. Figure 10 D and Figure 10E); Fermentation time has a significant effect on the composition and distribution of microbial communities. Specifically, compared with the blank control group (CON) without carbohydrate addition, after the introduction of carbohydrates, the sample points of the experimental group were concentrated in quadrants one, two, and four, indicating that carbohydrates significantly regulate the composition and structure of intestinal microbial communities. In addition, CS B Group and CS B+500 The sample points of the group were concentrated in the fourth quadrant, indicating that resistant starch (RS) and non-resistant starch (NRS) carbon sources had a significant effect on the overall structure of fecal microbial communities ( Figure 10 F).

[0206] (2) At the phylum level, Proteobacteria, Firmicutes, Bacteroidetes, Fusobacteria, and Actinobacteria were the main components of the intestinal microbiota after 12 and 24 hours of fermentation, accounting for more than 99% of the total relative abundance. Among them, Firmicutes and Bacteroidetes were particularly prominent, accounting for about 90% of the total relative abundance. Figure 11 A); After 12 hours of fermentation, CS B The relative abundance of Firmicutes in the group increased significantly. When the fermentation time was extended to 24 hours, the relative abundance of Firmicutes in the CS group increased significantly. B and CS B+500 The relative abundances of Bacteroidetes and Firmicutes increased significantly in the group ( Figure 11 B. Figure 11 C); Adding carbohydrate substrates can significantly increase the relative abundance of Prevotella, especially in FOS, CS B and CS B+500 The blank control group (CON) was mainly composed of Firmicutes (65.3%), while after adding carbon source, the butyrylation treatment group (CS B ) and ultrahigh pressure combined treatment group (CS B+500 ) increased significantly (38.2% and 41.5% at 24 h, respectively, (p < 0.05); at 24 h of fermentation, the Bacteroidetes / Firmicutes ratio (B / F = 1.12) of the CSB+500 group was significantly higher than that of the CS B The difference between the two groups was significant (0.89) and the NCS group (0.47), indicating that ultrahigh pressure treatment further optimized the balance of bacterial flora. Under anaerobic conditions, Prevotella can decompose arabinoxylan and oligofructose to produce propionic acid, thereby lowering serum cholesterol and reducing liver fat production. This suggests that the intake of highly substituted butyrylated starch can effectively promote intestinal health. Figure 11 D).

[0207] The above results show that butyrylation combined with ultrahigh pressure treatment (CS B+500) can significantly increase the abundance of Bacteroidetes and improve the B / F ratio, indicating that it enhances metabolic function by regulating the composition of intestinal flora, which is better than the single treatment group.

[0208] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0209] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A method for preparing modified starch, characterized in that: include: The starch raw material is subjected to butyrylation and ultrahigh pressure treatment to obtain modified starch; The pressure of the ultra-high pressure treatment is 80-600 MPa.

2. The method according to claim 1, characterized in that The method comprises: S1: subjecting the starch raw material to the butyrylation treatment to obtain starch containing butyryl groups; S2: subjecting the starch containing butyryl groups to ultrahigh pressure treatment to obtain modified starch; Optionally, the pressure of the ultrahigh pressure treatment is 100 to 500 MPa.

3. The method according to claim 1 or 2, characterized in that The butyrylation treatment method includes chemical butyrylation and / or enzymatic butyrylation.

4. The method according to claim 1 or 2, characterized in that The butyrylation treatment method includes: contacting the starch raw material with an acylating agent to cause an esterification reaction to obtain starch containing a butyryl group; Wherein, the pH of the contact is 8.0-8.

5.

5. The method according to claim 1, wherein The ultrahigh pressure treatment time is 5 to 25 minutes.

6. A modified starch, characterized in that The modified starch is prepared by the method according to any one of claims 1 to 5.

7. A food, health product or medicine, characterized in that: include: The modified starch according to claim 6.

8. The method according to any one of claims 1 to 5, or use of the modified starch according to claim 5 in preparing food, health products or medicines, characterized in that: The drug has at least one of the following uses: Regulate intestinal flora; Maintain intestinal health; Lower cholesterol levels; Reduce fat production; Relieve and / or treat intestinal inflammation; Maintain or increase intestinal butyrate concentrations.

9. The use according to claim 8, characterized in that The intestinal flora includes: Bacteroidetes and / or Firmicutes.

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