Rhizoma gastrodiae resistant starch with prebiotics as well as preparation method and application of rhizoma gastrodiae resistant starch

Abstract

The invention discloses gastrodia elata resistant starch with prebiotics as well as a preparation method and application of the gastrodia elata resistant starch, and belongs to the technical field of pharmaceutical preparations. The preparation method of the gastrodia elata resistant starch comprises the following steps: (1) preparing gastrodia elata crude starch by taking fresh gastrodia elata as a raw material; and (2) carrying out pre-gelatinization treatment, autoclaving treatment, enzymolysis treatment and aging retrogradation treatment on the solution of the gastrodia elata crude starch to obtain the gastrodia elata resistant starch. According to the method, the gastrodia elata resistant starch is prepared by using a high-pressure enzymolysis method, and by optimizing process parameters, the conversion extraction rate of the gastrodia elata resistant starch is effectively improved, the production cost is reduced, and the safety and the environmental protection property of the product are ensured. In addition, the prebiotic effect of the gastrodia elata resistant starch is deeply researched, and the prebiotics of the gastrodia elata resistant starch are systematically verified. Compared with the traditional resistant starch, the gastrodia elata resistant starch has more outstanding advantages in the aspects of bioavailability and probiotic effect.

Description

Technical Field

[0001] The invention relates to the technical field of pharmaceutical preparations, and in particular to a probiotic gastrodia elata resistant starch and a preparation method and application thereof. Background Art

[0002] Some studies have shown that Gastrodia elata contains a certain amount of resistant starch. As a type of starch that is not easily decomposed by digestive enzymes, resistant starch not only has significant prebiotic effects and can promote the growth of beneficial bacteria in the intestines, but also can provide the human body with more sustained energy release by improving bioavailability, thus showing broad application prospects in the field of functional foods. However, the specific prebiotic effects and mechanisms of Gastrodia elata resistant starch are still unclear, and there is a lack of sufficient experimental data to prove its true value in intestinal health.

[0003] In addition, the existing technology has significant limitations in developing functional foods using Gastrodia starch, which limits the application of Gastrodia starch in the field of functional foods. The preparation of Gastrodia resistant starch with traditional technology has the problems of low yield and high cost. In the process of developing Gastrodia resistant starch, the main challenge is how to optimize its resistant starch process to improve conversion efficiency and product quality. Traditional preparation methods include physical method, chemical method and enzymatic method. Physical method (such as extrusion method, wet heat treatment method, microwave radiation treatment method, ultrasonic treatment method, etc.) has a low loss of active ingredients in Gastrodia, but the preparation process is more complicated and the energy utilization efficiency is low. Chemical method (such as acid hydrolysis, etherification, esterification and cross-linking, etc.) may damage the active ingredients in Gastrodia, affect the functionality and safety of the product, and have potential pollution to the environment and risks to human health. The material cost required for enzymatic hydrolysis is low, there is no excessive energy consumption, but the conversion efficiency is limited. Therefore, improving the extraction and conversion efficiency of Gastrodia elata resistant starch will greatly promote the in-depth research on the prebiotic effects of Gastrodia elata resistant starch and the development of related functional foods. Summary of the invention

[0004] The purpose of the present invention is to provide a probiotic Gastrodia elata resistant starch and a preparation method and application thereof, so as to solve the problems existing in the above-mentioned prior art. The present invention uses a high-pressure enzymatic hydrolysis method to prepare Gastrodia elata resistant starch, and through the optimization of process parameters, effectively improves the conversion and extraction rate of Gastrodia elata resistant starch, reduces production costs, and ensures the safety and environmental protection of the product. In addition, the present invention deeply studies the prebiotic effect of Gastrodia elata resistant starch and systematically verifies the probiotic nature of Gastrodia elata resistant starch. Compared with traditional resistant starch, Gastrodia elata resistant starch shows more outstanding advantages in bioavailability and probiotic effects.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a method for preparing gastrodia elata resistant starch, comprising the following steps:

[0007] (1) Using fresh Gastrodia elata as raw material to prepare Gastrodia elata crude starch;

[0008] (2) subjecting the solution of the crude starch of Gastrodia elata to pregelatinization, autoclave treatment, enzymolysis treatment and aging and retrogradation treatment to obtain Gastrodia elata resistant starch;

[0009] The mass concentration of the solution of Gastrodia elata crude starch is 5%-25%;

[0010] The pre-gelatinization time is 20-40min;

[0011] The enzyme used in the enzymatic hydrolysis treatment is pullulanase, the enzyme dosage is 10-50 ASPU / g, the enzymatic hydrolysis time is 8-14h, and the enzymatic hydrolysis temperature is 50-60°C;

[0012] The time of the aging and rejuvenation treatment is 12-32 hours.

[0013] Preferably, the temperature of the pre-gelatinization treatment is 85-95°C; the temperature of the autoclave treatment is 120-130°C, and the time is 25-35 min; the pH of the enzymatic hydrolysis treatment is 4.5±0.05; and the temperature of the aging and regeneration treatment is 0-4°C.

[0014] Preferably, the mass concentration of the solution of Gastrodia elata crude starch is 15%; and / or,

[0015] The pre-gelatinization time is 25 minutes; and / or,

[0016] The enzyme dosage in the enzymatic hydrolysis treatment is 24.5 ASPU / g; and / or,

[0017] The enzymatic hydrolysis time in the enzymatic hydrolysis treatment is 10 hours; and / or,

[0018] The enzymatic hydrolysis temperature in the enzymatic hydrolysis treatment is 56° C.; and / or,

[0019] The aging and regeneration time is 24h.

[0020] Preferably, the pre-gelatinization temperature is 90°C; and / or,

[0021] The temperature of the autoclave treatment is 121°C; and / or,

[0022] The autoclave treatment time is 30 minutes; and / or,

[0023] The temperature of the aging and regeneration treatment is 4°C.

[0024] The preparation process of the crude Gastrodia elata starch of the present invention adopts a conventional method for preparing plant starch; in some embodiments of the present invention, the preparation process of the crude Gastrodia elata starch is as follows:

[0025] The washed fresh Gastrodia elata sample is crushed into slurry, filtered to obtain a first filtrate and a filter residue; the filter residue is repeatedly washed and filtered with water until the filtrate is clear, and the filtrate in the washing process is collected as a second filtrate; the first filtrate and the second filtrate are mixed, centrifuged at 5000r / min for 5min, and the upper brown impurities are scraped off to obtain a first precipitate; the first precipitate is repeatedly washed and centrifuged with water until the supernatant is clear and the precipitate is free of brown impurities to obtain a second precipitate; the second precipitate is washed and precipitated with anhydrous ethanol, 85% ethanol solution and 75% ethanol solution in sequence until the precipitate is white and free of impurities to obtain the Gastrodia elata crude starch.

[0026] The present invention also provides gastrodia elata resistant starch prepared by the above preparation method.

[0027] The present invention also provides an application of the above-mentioned Gastrodia elata resistant starch in preparing a medicine for protecting intestinal health.

[0028] Preferably, the drugs for protecting intestinal health include drugs that promote the proliferation of intestinal probiotics, drugs that maintain the intestinal barrier, drugs that inhibit the growth of harmful intestinal bacteria, drugs that improve the structure of intestinal flora, or drugs that promote the production of short-chain fatty acids by intestinal probiotics.

[0029] Preferably, the drug for maintaining the intestinal barrier includes a drug for promoting the production of short-chain fatty acids by intestinal probiotics.

[0030] The present invention also provides a medicine for protecting intestinal health, wherein the active ingredient of the medicine is the above-mentioned Gastrodia elata resistant starch.

[0031] Preferably, the drug further comprises pharmaceutically acceptable excipients.

[0032] The present invention discloses the following technical effects:

[0033] The present invention uses high-pressure enzymolysis to prepare Gastrodia elata resistant starch, and through the optimization of process parameters, the conversion and extraction rate of Gastrodia elata resistant starch is effectively improved, the production cost is reduced, and the safety and environmental protection of the product are ensured. In addition, the present invention further studies the prebiotic effect of Gastrodia elata resistant starch, and systematically verifies the probiotics of Gastrodia elata resistant starch. It is found through research that Gastrodia elata resistant starch, as a functional ingredient with significant probiotic effects, has shown excellent potential in promoting intestinal health. Its probiotics are mainly manifested in being able to provide a high-quality nutrient source for the beneficial flora in the intestine, especially key probiotics such as bifidobacteria and lactic acid bacteria, and by fermenting in the intestine to produce short-chain fatty acids (such as butyric acid, propionic acid, etc.), Gastrodia elata resistant starch can not only regulate the intestinal pH value, inhibit the growth of harmful bacteria, but also enhance the intestinal barrier function and improve the intestinal microecological environment. At the same time, the indigestible characteristics of Gastrodia elata resistant starch make it basically remain intact when passing through the small intestine, and after entering the large intestine, it is decomposed and utilized by beneficial bacteria, thereby effectively promoting the proliferation of beneficial bacteria, providing continuous support for intestinal health. Compared with traditional resistant starch, Gastrodia elata resistant starch can not only significantly increase the number of beneficial bacteria in the intestine in the short term, but also improve the overall balance of intestinal flora through long-term intake, showing its great potential as a new prebiotic. The preparation of Gastrodia elata resistant starch and its functional research in the present invention are of great significance to promoting the innovative application of Gastrodia elata as a traditional Chinese medicinal material in modern health food. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0035] Figure 1 The effects of pregelatinization time (a), starch paste concentration (b), aging time (c), enzymatic hydrolysis time (d), enzyme dosage (e) and enzymatic hydrolysis temperature (f) on the content of resistant starch;

[0036] Figure 2 The effects of pregelatinization time (a), starch paste concentration (b), aging time (c), enzymatic hydrolysis time (d), enzyme dosage (e) and enzymatic hydrolysis temperature (f) on the yield of resistant starch;

[0037] Figure 3 ANOVA for regression models;

[0038] Figure 4 is the fitting curve;

[0039] Figure 5 It is the interaction response surface and contour line of enzymatic hydrolysis time and enzymatic hydrolysis temperature;

[0040] Figure 6 It is the response surface and contour line of the interaction between enzyme addition amount and enzymolysis temperature;

[0041] Figure 7 It is the response surface and contour line of the interaction between enzyme addition amount and enzymolysis time;

[0042] Figure 8 It is the gas production of Gastrodia elata resistant starch fermentation liquid within 48 hours of fermentation;

[0043] Fig. 9 The pH of the fermentation liquid of Gastrodia elata resistant starch within 48 hours of fermentation;

[0044] Fig.10 It is the propionic acid yield of Gastrodia elata resistant starch fermentation liquid within 48 hours of fermentation;

[0045] Fig.11 It is the butyric acid yield of Gastrodia elata resistant starch fermentation liquid within 48 hours of fermentation;

[0046] Fig.12 It is the valeric acid yield of Gastrodia elata resistant starch fermentation liquid within 48 hours of fermentation;

[0047] Fig.13 The acetic acid yield of the Gastrodia elata resistant starch fermentation liquid within 48 hours of fermentation;

[0048] Fig.14 It is the isobutyric acid yield of Gastrodia elata resistant starch fermentation liquid within 48 hours of fermentation;

[0049] Fig.15 It is the isovaleric acid yield of the Gastrodia elata resistant starch fermentation liquid within 48 hours of fermentation;

[0050] Fig.16 It is the total acid production of Gastrodia elata resistant starch fermentation liquid within 48 hours of fermentation;

[0051] Fig.17 This is the cluster analysis result of all metabolites of Gastrodia elata resistant starch fermentation broth;

[0052] Fig.18 This is the PCA score result of all metabolites in the fermentation liquid of Gastrodia elata resistant starch;

[0053] Fig.19 This is the principal coordinate analysis (PCoA) diagram of intestinal flora after fermentation of Gastrodia elata resistant starch;

[0054] Fig. 20 This is the LEFSe discriminant analysis diagram between the Gastrodia elata resistant starch group and the control group (log10LDA>2.0);

[0055] Fig.21The changes in bacterial flora composition at the phylum level in the Gastrodia elata resistant starch group (a.) and the control group (b.);

[0056] Fig. 22 Changes in bacterial flora composition at the genus level in the Gastrodia elata resistant starch group (a.) and the control group (b.). DETAILED DESCRIPTION

[0057] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0058] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0059] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0060] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0061] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0062] Example 1 Preparation of Gastrodia elata resistant starch

[0063] 1. Materials and reagents:

[0064] Gastrodia elata (fresh sample, purchased from Zhaotong, Yunnan); citric acid was purchased from Shanghai Haohong Biopharmaceutical Technology Co., Ltd., and pullulanase was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; ether (60-29-7 ≥ 99.5% Greagent) and phosphoric acid (7664-38-2 ≥ 85%) were purchased from Sinopharm Group, and sodium hydroxide, isobutyric acid, propionic acid, acetic acid, butyric acid, isovaleric acid, valeric acid, and caproic acid were purchased from Xilong Scientific Co., Ltd.

[0065] 2. Experimental methods:

[0066] 2.1. Preparation of Gastrodia elata crude starch:

[0067] Wash the fresh Gastrodia elata sample, remove the black and rotten parts, crush it into pulp with a high-speed tissue grinder, filter it with four layers of gauze, and collect the filtrate. Add water to the filter residue repeatedly, mix it evenly, and filter it until the filtrate is clear. Centrifuge the filtrate at 5000r / min for 5 minutes, scrape off the brown impurities on the upper layer, add water and centrifuge repeatedly until the supernatant is clear and transparent, and there is no obvious brown impurities in the precipitate. Repeatedly add anhydrous, 85%, and 75% ethanol solutions to the precipitate to wash the precipitate several times until the precipitate is white and free of impurities, and the crude Gastrodia elata starch is obtained.

[0068] 2.2 Preparation of Gastrodia elata resistant starch:

[0069] Weigh 5g (accurate to 0.01g) of crude starch from Gastrodia elata, place it in a conical flask, use a measuring cylinder to measure 50mL of 0.1MPBS, pour it into the above conical flask, and stir it evenly with the crude starch from Gastrodia elata to prepare Gastrodia elata starch paste; heat and stir the Gastrodia elata starch paste at 90°C for pre-gelatinization for 30min; then use an autoclave to heat at 121°C for 30min; adjust the pH to 4.5 (±0.05) with citric acid solution and sodium hydroxide solution, measure 25μL of pullulanase solution and add it to a conical flask, place it in a 60°C water bath for enzymolysis for 20 hours, and shake it every 3 hours. After the enzymolysis is completed, place it in a 100°C water bath for inactivation for 5min. Place it in a 4°C refrigerator for 24 hours to allow the resistant starch to age and regenerate. Take it out and cool it naturally to room temperature, freeze-dry the sample, crush it through a 200-mesh sieve, and obtain Gastrodia elata resistant starch.

[0070] 2.3. Single factor optimization experiment of the preparation process of Gastrodia elata resistant starch:

[0071] The effects of pregelatinization time (10, 15, 20, 25, 30 min), aging time (12, 16, 20, 24, 30 h), starch paste concentration (5%, 10%, 15%, 20%, 25%), pullulanase dosage (10, 20, 25, 30, 40, 50 ASPU / g), enzymolysis temperature (40, 45, 50, 55, 60 ° C) and enzymolysis time (8, 10, 12, 14, 16, 18, 20 h) on the content and yield of resistant starch in Gastrodia elata were investigated to determine the appropriate level of each factor. The fixed conditions were: starch paste concentration of 10%, pregelatinization of 20 min, autoclave temperature of 121 ° C, autoclave time of 30 min, pullulanase dosage of 25 ASPU / g, enzymolysis time of 16 hours, enzymolysis temperature of 50 ° C, and aging time of 24 h.

[0072] 2.4. Response surface experiment for optimization of preparation process of Gastrodia elata resistant starch:

[0073] Based on the Box-Behnken experimental design principle and the experimental results obtained from the single factor experiment, the factors of enzymatic hydrolysis time, enzyme addition and enzymatic hydrolysis temperature were finally selected as independent variables, and the yield of resistant starch was used as the response value. A three-factor three-level response surface experiment was designed to optimize the conditions for the preparation of Gastrodia elata resistant starch by hot pressing enzymatic hydrolysis. The factors and level designs are shown in Table 1.

[0074] Table 1 Response surface factors and level design

[0075]

[0076] 2.5 Evaluation Method

[0077] Mass of crude resistant starch of Gastrodia elata: weigh the mass of each sample (g, accurate to 0.01 g) and express it as mean ± standard deviation.

[0078] Resistant starch content: The resistant starch content (%) of the prepared crude resistant starch of Gastrodia elata was determined by a kit, and each sample was determined three times, and the results were expressed as mean ± standard deviation.

[0079]

[0080] Resistant starch content = m2 × a(2)

[0081] Wherein: m1 is the mass of Gastrodia elata starch (g); m2 is the mass of Gastrodia elata crude resistant starch (g); a is the resistant starch content of Gastrodia elata crude resistant starch (%).

[0082] 2.6. Data statistics and analysis:

[0083] IBM SPSS Statistics25 and Excel were used for data processing and statistical analysis, and one-way ANOVA and Turkey's multi-factor t-test were used for significance analysis. Origin2018 and Prism were used for graphics drawing.

[0084] 3. Experimental results:

[0085] 3.1. Single factor optimization experimental results of the preparation process of Gastrodia elata resistant starch:

[0086] The pregelatinization time of resistant starch preparation was investigated. Figure 1 A and Figure 2 From a, we can see that the resistant starch content first increases and then tends to be stable. After 25 minutes of pregelatinization, the resistant starch content tends to be stable. It may be because the starch can be fully gelatinized in 25 minutes. As time goes by, the starch is always in a completely gelatinized state, so the resistant starch content no longer increases. Therefore, when the pregelatinization time is 25 minutes, the resistant starch content is the highest.

[0087] Select starch paste with five different concentrations: 5%, 10%, 15%, 20%, and 25%. Figure 1 b and Figure 2 From b, we can see that as the starch paste concentration increases, the resistant starch content continues to increase, and when the starch paste concentration reaches 15%, the content reaches the highest. Afterwards, as the starch concentration increases or decreases, the resistant starch content does not increase. This shows that too high or too low starch paste concentration is not conducive to the formation of resistant starch. When the starch paste concentration is too low, the starch molecules are in a more dispersed state in the solution. When the starch paste concentration is too high, it is difficult for the starch to fully gelatinize. Therefore, when the starch paste concentration is 15%, the resistant starch content is the highest.

[0088] The aging time of resistant starch was investigated. Figure 1 C and Figure 2 It can be seen from the c that the content of resistant starch shows a trend of first increasing and then decreasing with the increase of aging time. When the starch paste is aged at 4°C, the starch granules can be transformed from disordered arrangement to ordered arrangement. As time goes by, the resistant starch ages completely, and the resistant starch content no longer increases. A small decrease may be within the acceptable range. Therefore, when the aging time is 24h, the resistant starch content is the highest.

[0089] The enzymatic hydrolysis time of resistant starch preparation was investigated. Figure 1 D and Figure 2It can be seen from the data that the content of resistant starch shows a trend of first increasing and then decreasing with the increase of enzymatic hydrolysis time. When the enzymatic hydrolysis time is 10h, the content of resistant starch in Gastrodia elata starch reaches the highest, and with the extension of enzymatic hydrolysis time, the content of resistant starch shows a significant downward trend. Because pullulanase specifically acts on a-1,6 glycosidic bonds, a large number of linear starch chains are produced after the debranching action of pullulanase. The linear starch chains have good mobility and are easy to form an ordered double helix structure, which promotes the production of resistant starch. However, with the increase of enzymatic hydrolysis time, Gastrodia elata starch is over-decomposed to produce many shorter straight-chain starches. These short straight-chain starches move more violently, which is not conducive to the formation of double helix structure, so the content of resistant starch decreases. Therefore, when the enzymatic hydrolysis time is 10h, the content of resistant starch is the highest.

[0090] The amount of pullulanase used in the preparation of resistant starch was investigated. Figure 1 E and Figure 2 It can be seen from the data that the content of resistant starch shows a trend of first increasing and then decreasing with the increase of enzyme dosage. When the enzyme dosage is 30ASPU / g, the content of resistant starch reaches the highest. As the enzyme dosage increases, the content of resistant starch no longer increases, and a small decrease may be within an acceptable range. When the enzyme dosage is too low, the starch cannot be completely debranched and it is difficult to form resistant starch; but when the enzyme dosage is too high, it will cause excessive debranching of starch, and the formed straight-chain starch molecular chain will be too short, affecting the recrystallization of resistant starch. Therefore, when the enzyme dosage is 30ASPU / g, the resistant starch content is the highest.

[0091] The enzymatic hydrolysis temperature of resistant starch preparation was investigated. Figure 1 f and Figure 2 It can be seen from the results that the content of resistant starch shows a trend of first increasing and then decreasing with the increase of enzymatic hydrolysis temperature. When the enzymatic hydrolysis temperature is 50℃, the enzymatic hydrolysis effect is the best. As the enzymatic hydrolysis temperature increases, the content of resistant starch no longer increases. Therefore, when the enzymatic hydrolysis temperature is 50℃, the content of resistant starch is the highest.

[0092] 3.2. Response surface experiment results of optimization of preparation process of Gastrodia elata resistant starch:

[0093] 3.2.1. Response surface results:

[0094] The response surface experimental design and results are shown in Table 2. The multivariate quadratic regression equation between factors (A, B, C) and response values ​​(Y) is Y = 13.31-0.2707A+0.7157B+0.3112C+0.8644AB+0.3350AC-0.3160BC-0.4068A2-1.64B2-0.6762C2.

[0095] Table 2 Response surface experimental design and results

[0096]

[0097] 3.2.2. Response surface model significance analysis:

[0098] The experimental data were analyzed using the software Desgin-Expert. As shown in Table 3, the p value of the experimental model is less than 0.01, indicating that the experiment is extremely significant; the lack of fit term P = 0.4177> 0.05, which is not significant, indicating that there is no obvious lack of fit between the regression equation and the actual result, and this model can be used to analyze the yield of Gastrodia elata resistant starch; from the F value of the regression model, it can be seen that the order of factors affecting the content of Gastrodia elata resistant starch is enzymolysis time (B)>enzymolysis temperature (C)>enzyme addition amount (A). Among them, AB, BC, A 2 , B 2 , C 2 It had a significant effect on the resistant starch of Gastrodia elata, while AC had no significant effect on the resistant starch of Gastrodia elata, indicating that the interaction between the enzyme addition amount and the enzymatic hydrolysis temperature had little effect on the resistant starch of Gastrodia elata.

[0099] Table 3 Analysis of variance of regression model

[0100]

[0101] Note: “*” “P<0.05” means the result has a significant effect; “**” “P<0.01” means the result has an extremely significant effect.

[0102] 3.2.3. Response surface model precision analysis:

[0103] Depend on Figure 3 It can be seen that R 2 =0.9675, RAdj=0.9256, CV=2.84%, indicating that the coefficient of variation of the data is low and the model fits well; the experimental precision is 14.8755>4, indicating that the model has good precision.

[0104] 3.2.4. Response surface model variance analysis:

[0105] The experimental data fitting using Desgin-Expert software helps to intuitively show the effects of the pairwise interactions among enzyme addition, enzymolysis time, and enzymolysis temperature on Gastrodia elata resistant starch. The results are shown in Figure 5-Figure 7 ,from Figure 5-Figure 7 It can be seen that the response surface of enzyme addition amount and enzymolysis temperature on Gastrodia elata resistant starch is the steepest, indicating that enzyme addition amount and enzymolysis temperature have the most significant effects on Gastrodia elata resistant starch; followed by enzymolysis time and enzymolysis temperature, and enzyme addition amount and enzymolysis time have the least effect. The order of the three factors affecting Gastrodia elata resistant starch is enzyme addition amount > enzymolysis temperature > enzymolysis time, which is consistent with the results of variance analysis.

[0106] The optimal preparation process conditions obtained by the software Desgin-Expert analysis were enzyme addition 24.66ASPU / g, enzymolysis temperature 55.8℃, enzymolysis time 10.36h, and RS yield prediction result was 13.4152%. In order to be suitable for experimental operation, the process conditions were adjusted to enzyme addition 24.5ASPU / g, enzymolysis temperature 56℃, and enzymolysis time 10h. Under this optimized condition, multiple parallel experiments were carried out, and the actual value of resistant starch yield was (13.2±0.24)%, which was close to the predicted value.

[0107] Example 2 Functional verification of Gastrodia elata resistant starch

[0108] This example studies the effect of the Gastrodia elata resistant starch group on the intestinal flora. The second type of resistant starch, high-amylose corn starch (amylose content of about 72.7%, purchased from Hangzhou Prostar Starch Co., Ltd.), was used as the control group, and an in vitro simulated intestinal digestion and fermentation experiment was used to study the role of resistant starch in the fermentation process of intestinal flora. The fermentation process was compared by measuring gas production, pH, and short-chain fatty acids (SCFAs), and the composition of intestinal flora was determined by 16S rRNA high-throughput sequencing technology to study the effect of resistant starch on intestinal flora.

[0109] The specific test process is as follows:

[0110] 1. Preparation of Gastrodia elata resistant starch:

[0111] 1.1. Preparation of Gastrodia elata starch: same as 2.1 of Example 1.

[0112] 1.2. Preparation of Gastrodia elata resistant starch: Weigh 7.5g of Gastrodia elata starch (accurate to 0.01g), place it in a conical flask, use a measuring cylinder to measure 50mL of 0.1M PBS, pour it into the above conical flask, and stir it evenly with Gastrodia elata starch to prepare a Gastrodia elata starch paste with a mass concentration of 15%; heat and stir the Gastrodia elata starch paste at 90°C, pre-gelatinize for 25min; then use an autoclave at 121°C for 30min; adjust the pH to 4.5 with citric acid solution and sodium hydroxide solution, add pullulanase at an amount of 24.5ASPU / g, place it in a 56°C water bath for enzymolysis for 10h, and shake it every 3 hours. After the enzymolysis is completed, place it in a 100°C water bath for inactivation for 5min. Place it in a 4°C refrigerator for 24h to allow the resistant starch to age and regenerate. Take it out and cool it naturally to room temperature, freeze-dry the sample, crush it through a 200-mesh sieve, and obtain Gastrodia elata resistant starch.

[0113] 2. Preparation of simulated digestive fluid:

[0114] 2.1. Preparation of simulated digestion electrolyte solution:

[0115] The simulated digestion method usually includes the oral cavity, stomach and small intestine stages. Simulated saliva (SSF), simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) are composed of corresponding electrolyte stock solutions, enzymes, CaCl2 and water. The preparation method of the electrolyte stock solution is shown in Table 4. The concentration and volume of various electrolytes are added and mixed according to Table 4, and the pH is adjusted with sodium hydroxide and dilute hydrochloric acid. Finally, the volume of the electrolyte stock solution is fixed to 500mL.

[0116] Table 4 Preparation of electrolyte stock solution for simulated digestion fluid

[0117]

[0118] 2.2. Oral fluid simulated digestion products:

[0119] Prepare two oral fluid simulated digestion products: prepare 5mL starch (Gastrodia elata resistant starch or high amylose corn starch or Gastrodia elata starch) sample suspension (10%, m / v), add 3.5mL simulated salivary electrolyte stock solution to the above solution, vortex oscillate to mix, continue to add 0.5mL simulated salivary electrolyte stock solution containing 1500U / mL α-amylase, then add 25μL of 0.3mol / L CaCl2 and 975μL ultrapure water, and mix thoroughly. All solutions need to be preheated to 37°C before adding, and then the mixed solution is reacted in a 37°C water bath for 2min, with a stirring speed of 100r / min, and finally 0.15mL of 1.0mol / L hydrochloric acid solution is added to inactivate α-amylase.

[0120] One of the oral fluid simulated digestion products was stored for the next step; the other oral fluid simulated digestion product was centrifuged (5000×g, 5 min), washed with water 3-5 times, and finally dried at 40°C to obtain the oral digestion product SRS.

[0121] 2.3. Gastric juice simulated digestion products

[0122] Prepare two portions of gastric fluid simulated digestion products: add 7.5 mL of simulated gastric fluid original electrolyte solution to 10 mL of oral fluid simulated digestion product obtained in 2.2, then add 1.6 mL of simulated gastric fluid original electrolyte solution without porcine pepsin, continue to add 5 μL of 0.3 mol / L CaCl2, 695 μL of ultrapure water and 0.2 mL of 1.0 mol / L hydrochloric acid solution, adjust the solution pH to 3.0, then react at 37 ° C and 100 r / min for 2 h, add 0.14 mL of 1.0 mol / L sodium hydroxide solution to terminate the reaction.

[0123] One portion of the gastric juice simulated digestion product was saved for the next step; the other portion of the gastric juice simulated digestion product was centrifuged at 5000×g for 5 min, washed with water 3-5 times, and dried in an oven at 40°C to obtain the oral-gastric digestion product GRS.

[0124] 2.4. Intestinal fluid simulated digestion products

[0125] Add 11 mL of simulated intestinal fluid electrolyte solution to the 20 mL of oral-gastric digestion product obtained in 2.3, then add 5.0 mL of pancreatic enzyme (4000 U according to the activity of trypsin, weigh 500 mg in 5 mL of simulated intestinal fluid electrolyte solution), 0.1 mL of saccharifying enzyme (add 0.2 mL of saccharifying enzyme per gram of dry starch), and continue to add 40 μL of 0.3 mol / L CaCl2, 0.15 mL of 1.0 mol / L sodium hydroxide solution and 1.31 mL of ultrapure water. After mixing, the mixed solution reaches pH 7.0, and then reacts at 37°C and a rotation speed of 100 r / min for 2 h, centrifuges at 5000×g for 5 min, washes with water 3-5 times, and finally dries at 40°C to obtain oral-gastric-small intestinal digestion product IRS.

[0126] 2.5. Feces collection and preparation

[0127] Feces of healthy (hormone-free) SD rats were collected, and the collected fresh feces samples were immediately transferred to an anaerobic incubator. PBS culture medium solution (containing 8.0 g / L sodium chloride, 0.2 g / L potassium chloride, 1.15 g / L disodium hydrogen phosphate, 0.2 g / L sodium dihydrogen phosphate, 0.5 g / L L-cysteine, and 1 mg / L resazurin) with a weight of 3 times the feces was added, and the feces suspension was filtered through four layers of gauze to obtain a 25% (w / w) feces suspension.

[0128] 3. In vitro fermentation culture

[0129] The preparation method of fermentation blank medium is as follows: 10.0g tryptone, 2.5g yeast extract, 1.0g L-cysteine, 0.01g hemin chloride, 0.001g resazurin, 0.9g NaCl, 0.45g KH2PO4, 0.45g K2HPO4, 0.09gCaCl2·6H2O, 0.09g MgSO4·7H2O, 0.2mL multivitamin, make up to 1000mL with deionized water, and then sterilize at 121℃ for 30min for use.

[0130] The resistant starch of Gastrodia elata, high-amylose corn starch and Gastrodia elata starch treated with oral-gastric-small intestine simulated fluid were fermented in feces in vitro. 500 μL of fecal suspension was drawn from the fecal sample box and injected into each vial of culture medium in turn, and then 5 mL of blank culture medium and 20 mg of starch were added. After inoculation, the corresponding sample number, inoculation date and time were marked on the vial shelf. The culture medium vials were placed in a 37°C incubator for fermentation.

[0131] At 0, 6, 12, 24 and 48 hours of culture, 5 mL of fermentation liquid was extracted with a 10 mL syringe and placed in an ice bath for 20 minutes to stop the fermentation reaction and wait for measurement. Three replicates were set for each culture time, and the results were averaged.

[0132] 4. Determination of gas production and pH value during fermentation

[0133] The gas production was determined as follows: serum bottles of the corresponding time were taken out from the incubator at 0, 6, 12, 24, and 48 h, and a needle connected to a graduated syringe was inserted into the rubber stopper to measure gas production, i.e., the volume represented by the displacement of the syringe plunger was the amount of gas produced (mL). In addition, a pH meter was used to measure the pH value of the fermentation broth.

[0134] 5. Determination of SCFAs content

[0135] Take 500 μL of liquid culture medium sample in a 1.5 mL centrifuge tube, add 100 μL of crotonic acid metaphosphate solution, and then centrifuge at 10,000 × g and 4 ° C for 3 min to remove impurities such as protein. Take the supernatant, filter it with a 0.22 μm filter membrane, and use it for gas chromatography determination.

[0136] Use a 100μL microinjection needle to draw 80μL acetic acid, 60μL propionic acid, 40μL each of butyric acid, isobutyric acid, valeric acid, and isovaleric acid, dissolve and dilute to 10mL ultrapure water, this is the standard stock solution. When using, use ultrapure water to gradient dilute to 1 / 2, 1 / 4, 1 / 20, 1 / 100, 1 / 500, 1 / 1000 of the stock solution. After dilution, add 100μL of metaphosphoric acid-crotonic acid solution to each 500μL of each concentration solution to prepare a series of standard working solutions containing 12.514mmol / L crotonic acid internal standard. Use standard samples of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid for determination and make a standard curve.

[0137] The chromatographic column used was Agilent FFAP 30m×0.25mm×0.25μm; the heating steps were as follows: the column temperature was 100°C, increased to 200°C at a rate of 20°C / min, and maintained for 1 min; the injection port temperature was 250°C, the injection volume was 1.0μL, and the split ratio was 5:1; the carrier gas was high-purity nitrogen at a flow rate of 2.5mL / min, maintained for 6min, increased from 2.8mL / min to 3mL / min, and maintained for 2min; the detector was FID; the temperature was 250°C; the tail blow was 20mL / min; the hydrogen flow rate was 30mL / min; and the air flow rate was 300mL / min.

[0138] The peak area internal standard method is used for quantitative determination. The determination results can be automatically calculated by the workstation according to the standard curve equation internal standard method, and the sample determination results are automatically converted to the target SCFA amount in the sample. The relative standard deviation of this method is ≤10%. The detection limit of this method is: acetic acid 0.05mmol / L; propionic acid 0.02mmol / L; isobutyric acid, butyric acid, isovaleric acid, valeric acid 0.01mmol / L.

[0139] 6. High-throughput sequencing of intestinal flora

[0140] TransGen AP221-02: TransStart Fast pfu DNA Polymerase was used for PCR. The PCR product was detected by 1% agarose gel electrophoresis to detect the size of the amplified target band and purified by Agencourt AMPure XP nucleic acid purification kit. DNA was quantitatively detected by UV spectrophotometer.

[0141] A series of PCR primers designed by Sanshu Bio were used to amplify two highly variable regions including V3 and V4 on the 16SrDNA of prokaryotes. Then, a connector with an index was added to the end of the PCR product of 16SrDNA by PCR for NGS sequencing. The self-joined fragments of the connector were removed by magnetic bead screening, and the concentration was detected by an ELISA instrument and the fragment size was detected by agarose gel electrophoresis. The library concentration was detected by an ELISA instrument, and PE250 / FE300 double-end sequencing was performed according to the instruction manual of the Illumina MiSeq / Novaseq instrument, and the sequence information was read by the MiSeq Control Software (MCS) / Novaseq Control Software (NCS) that comes with MiSeq / Novaseq.

[0142] The chimeras and problematic sequences in the original sequence were removed by QIIME2 software, and the UCLUST algorithm was called to merge similar sequences. The characteristic sequences were merged and divided at a similarity of 97%. After further streamlining and removing rare sequences, the optimized sequences were obtained, and the representative sequences of the operational classification units (OUT) and their abundance information were obtained. The OTU sequences were homologously compared with the RDP database to obtain the OTU classification status. According to the results of OTU division and classification status identification, the specific composition of each sample at the phylum, genus and other classification levels can be obtained. According to the characteristics of the sample data, the Bray-curtis method was used to calculate the distance between samples, and the principal coordinate diagram (PCoA) was drawn to study the overall effect of different types of resistant starch on the microbial community. Based on the annotation results, the classification information at each level can be obtained, so as to conduct correlation analysis of sample composition and community structure differences between samples. The Sanshu Biological Platform (https: / / omic.sanshugroup.com / ) was used to visualize the taxonomic composition of the community and interactively display it.

[0143] 7. Data statistics and analysis:

[0144] IBM SPSS Statistics25 and Excel were used for data processing and statistical analysis, and one-way ANOVA and Turkey's multi-factor t-test were used for significance analysis. Origin2018 and Prism were used for graphics drawing.

[0145] 8. Experimental results:

[0146] 8.1. Gas production analysis:

[0147] Depend on Figure 8 It can be seen that with the increase of fermentation time, the amount of gas produced by the fermentation of Gastrodia elata resistant starch gradually increased, the gas production increased sharply within 0-6 hours, and the gas production tended to be stable after 12 hours. At each sampling time point, the gas production of Gastrodia elata resistant starch was significantly higher than that of the control group, indicating that Gastrodia elata resistant starch was utilized by intestinal flora. In the initial stage of Gastrodia elata starch, less gas was produced (mainly in the starch decomposition stage). As aerobic respiration proceeds, the amount of carbon dioxide generated gradually increases. Finally, the gas production tends to stabilize and reaches a state of equilibrium.

[0148] 8.2 pH value analysis:

[0149] Depend on Fig. 9 It can be seen that the pH value of the control group maintained a slow downward trend from 0 to 48 hours; while the pH value of the fermentation broth of Gastrodia elata resistant starch decreased rapidly from 0 to 6 hours and remained stable after 12 hours. During the fermentation process, the pH value of the fermentation broth of Gastrodia elata resistant starch was significantly lower than that of the Gastrodia elata starch group, indicating that Gastrodia elata resistant starch can be fermented and utilized by intestinal flora to produce SCFAs such as acetic acid, propionic acid, and butyric acid, resulting in a decrease in intestinal pH, which is consistent with the results that traditional resistant starch can promote the production of SCFAs and reduce intestinal pH. In the initial stage of Gastrodia elata starch, the pH remained slightly stable; as glycolysis proceeded, acidic metabolites (such as lactic acid) accumulated, causing the pH to decrease; eventually, the pH gradually stabilized and slightly rebounded.

[0150] 8.3. Changes in the production of various SCFAs during fermentation:

[0151] 8.3.1. Impact on propionic acid production:

[0152] Fig.10The effect of Gastrodia elata resistant starch and the control group on the propionic acid production during fecal fermentation. It can be seen that the propionic acid production of each group showed a significant upward trend during the fermentation process. At 6 hours of fermentation, there was no significant difference in the propionic acid production between the Gastrodia elata resistant starch group and the control group. After 12 hours of fermentation, the gap between the two further widened. The propionic acid production of the Gastrodia elata resistant starch group was significantly higher than that of the control group. At 24 hours of fermentation, the propionic acid production of the Gastrodia elata resistant starch group was much higher than that of the control group, indicating that Gastrodia elata resistant starch has a strong promoting effect on the production of propionic acid in the later stage of fermentation.

[0153] 8.3.2 Impact on butyric acid production:

[0154] Fig.11 The effect of the Gastrodia elata resistant starch group and the control group on the butyric acid production during fecal fermentation. It can be seen that the butyric acid production of each group of resistant starch basically showed a significant increasing trend during the fermentation process. Starting from 6 hours, the butyric acid production of the Gastrodia elata resistant starch group gradually exceeded that of the control group. As the fermentation time increased, at 24 hours and 48 hours, the butyric acid production of the Gastrodia elata resistant starch group was significantly higher than that of the control group, indicating that it has a significant promoting effect on the production of butyric acid. Gastrodia elata resistant starch has an advantage in promoting butyric acid production.

[0155] 8.3.3 Impact on Valeric Acid Production:

[0156] Fig.12 The effect of the Gastrodia elata resistant starch group and the control group on the production of valeric acid during fecal fermentation. It can be seen that the valeric acid production of the resistant starch in each group basically showed a significant increasing trend during the fermentation process. At 6 hours of fermentation, the valeric acid production of the resistant starch in each group was relatively low, with little difference, and the Gastrodia elata resistant starch group was slightly higher than the control group. As the fermentation time increased, after 12 hours of fermentation, the growth rate of valeric acid production in the Gastrodia elata resistant starch group was lower than that in the control group, and slightly lower than that in the control group. At 48 hours of fermentation, the valeric acid production in the Gastrodia elata resistant starch group increased rapidly, significantly higher than that in the control group. Finally, it was shown that the Gastrodia elata resistant starch group and the control group can significantly promote the production of valeric acid, and the production of valeric acid gradually increased in the later stage of fermentation, and the promotion effect was best at 48 hours of fermentation.

[0157] 8.3.4 Impact on acetic acid production

[0158] Fig.13 The effect of Gastrodia elata resistant starch and the control group on the production of acetic acid during fecal fermentation. It can be seen that the production of acetic acid in each group showed a significant upward trend during the fermentation process. The production of acetic acid was not much different between the Gastrodia elata resistant starch group and the control group at 6 hours, but as the fermentation time increased, the acetic acid content in the Gastrodia elata resistant starch group was significantly higher than that in the control group, especially at 24 hours and 48 hours. The results show that Gastrodia elata resistant starch can promote the production of acetic acid, and the promotion effect is better.

[0159] 8.3.5. Impact on Isobutyric Acid Production:

[0160] Fig.14 The effect of the Gastrodia elata resistant starch group and the control group on the isobutyric acid production during fecal fermentation. It can be seen that the isobutyric acid production of the resistant starch in each group basically showed a significant increasing trend during the fermentation process. At 6 hours of fermentation, the isobutyric acid production of the resistant starch in each group was basically the same. At 12 hours of fermentation, the production of the Gastrodia elata resistant starch group increased less and was lower than that of the control group. When the fermentation time increased to 24 hours, the isobutyric acid production of the Gastrodia elata resistant starch group increased significantly, but in the end, the isobutyric acid production of the Gastrodia elata resistant starch group was lower than that of the control group. This shows that the Gastrodia elata resistant starch group can promote the production of isobutyric acid, but their promotion effect is not as good as that of the control group. This shows that the promoting effect of Gastrodia elata resistant starch on the production of isobutyric acid is relatively weak.

[0161] 8.3.6. Impact on isovaleric acid production:

[0162] Fig.15 The effect of the Gastrodia elata resistant starch group and the control group on the isovaleric acid production during fecal fermentation. It can be seen that the isovaleric acid production of the resistant starch in each group basically showed an increasing trend during the fermentation process. At 6 hours of fermentation, there was no significant increase in the isovaleric acid production of the resistant starch in each group. At 12 hours of fermentation, the isovaleric acid production of the Gastrodia elata resistant starch group increased significantly, and the Gastrodia elata resistant starch group gradually increased above the control group. As the fermentation time increased, at 24 hours of fermentation, the isovaleric acid production of the Gastrodia elata resistant starch group was the highest, and the difference reached the largest at 48 hours. Finally, it was shown that the Gastrodia elata resistant starch group can promote the production of isovaleric acid.

[0163] 8.3.7 Impact on total acid production

[0164] Depend on Fig.16 The effect of the Gastrodia elata resistant starch group and the control group on the total acid production during fecal fermentation. It can be seen that at 6 hours of fermentation, the total acid production of the resistant starch in each group was slightly different. At 12 hours of fermentation, the total acid production of the Gastrodia elata resistant starch group was significantly higher than that of the control group. As the fermentation time increased, at 24 hours of fermentation, the total acid production of the Gastrodia elata resistant starch group was much higher than that of the control group, indicating that the Gastrodia elata resistant starch group can significantly promote the production of total SCFA, especially in the late fermentation period, which is likely to be closely related to the dense and enzymatically resistant structure of the recrystallized resistant starch.

[0165] 9. Cluster analysis of all metabolites:

[0166] Depend on Fig.17It can be seen that the SCFA content of the Gastrodia elata resistant starch fermentation liquid was low at 0h and appeared blue, indicating that at the beginning of fermentation, the content of various short-chain fatty acids was relatively small. At 6h, as the fermentation of the Gastrodia elata resistant starch fermentation liquid progressed, the SCFA content gradually increased, and the color gradually changed from blue to light gray, indicating that some SCFA had begun to be generated at this time, but had not yet reached a significant level. At 12h, the color of the Gastrodia elata resistant starch fermentation liquid sample became lighter and closer to the value of 0, indicating that the generation of SCFA was in progress and gradually accumulated. At 24h and 48h, the SCFA content of the Gastrodia elata resistant starch fermentation liquid reached the highest level and the color turned red, especially at 48h, the production of propionic acid, butyric acid and isobutyric acid increased significantly.

[0167] The contents of propionic acid, butyric acid and isobutyric acid increased significantly at 24h and 48h, reflecting that the production of these SCFAs was the largest in the later stage of fermentation; acetic acid and valeric acid also increased to a certain extent after 12h, but in comparison, the amount produced was lower than that of propionic acid and butyric acid; the content of caproic acid changed little during the entire fermentation process, and the content was always at a low level.

[0168] The sample clustering at the top of the heat map shows that the 0h and 6h samples are clustered into one category, the 12h samples are independent of the two, and the 24h and 48h samples are clustered into one category. This indicates that with the increase of fermentation time, the production of SCFA changes significantly, and the samples tend to be stable after 24h.

[0169] In the early stage of fermentation (0h and 6h), the SCFA content was low, mainly blue and light gray, indicating that the production of short-chain fatty acids had not increased significantly at this time.

[0170] After 12 hours of fermentation, the SCFA content gradually increased, and the production of various SCFA (especially propionic acid, butyric acid, and isobutyric acid) in the 24h and 48h samples reached the highest peak, and the color was obviously red, indicating that Gastrodia elata resistant starch has a significant promoting effect on the production of short-chain fatty acids in the later stage of fermentation.

[0171] 10. PCA score of all metabolites:

[0172] Depend on Fig.18It can be seen that the first principal component (PC1) explains 97.9% of the variation, and the second principal component (PC2) explains 1.06% of the variation, indicating that most of the differences are reflected in PC1, and the variation contributed by PC2 is relatively small. The three sample points of 0h (RS_0h) are clustered in the lower left corner, indicating that the performance of these three samples in the principal component space is very similar. The sample point of 6h (RS_6h) has moved a little to the right on PC1, indicating that the sample has changed over time. The sample of 12h has moved further to the right on PC1, indicating that the metabolite changes during the fermentation process are more significant compared with 6h. At 24h and 48h, these sample points move further on PC1, and the sample point of 48h is very close to that of 24h, indicating that the fermentation tends to stabilize in the later stage and the changes are no longer as drastic as in the early stage.

[0173] As time went on, the samples gradually moved to the right on PC1, reflecting the significant changes in metabolites during the fermentation process. From 6h to 48h, the metabolic changes in the RS group were more obvious in the early stage, and the samples tended to be stable at 24h and 48h.

[0174] As one of the metabolites of intestinal flora, SCFAs have the functions of maintaining the intestinal barrier and inhibiting the growth of harmful bacteria, especially propionic acid and butyric acid have excellent physiological activity. Therefore, it is necessary and beneficial to introduce appropriate dietary fiber into the intestine to ensure that intestinal bacteria produce higher butyric acid or propionic acid. The SCFAs produced by anaerobic fermentation depend on the substrate. By monitoring the production of acetic acid, propionic acid, butyric acid and total SCFAs during the in vitro fermentation of Gastrodia elata resistant starch, the content of acetic acid, propionic acid, butyric acid and total SCFAs in Gastrodia elata resistant starch increased significantly within 0-6h, and the content of acetic acid, propionic acid, butyric acid and total SCFAs in each group of samples reached the maximum at 48h. During the fermentation process, the content of acetic acid, propionic acid, butyric acid and total SCFAs in Gastrodia elata resistant starch was significantly higher than that in the control group. It is particularly noteworthy that at 48h of fermentation, the content of acetic acid, propionic acid, butyric acid and total SCFAs produced by Gastrodia elata resistant starch was significantly higher than the acid production of other groups during the same period, indicating that Gastrodia elata resistant starch has the ability to improve the ability to promote the intestinal flora to use resistant starch to produce SCFAs.

[0175] 11. 16S rRNA high-throughput sequencing results:

[0176] 11.1. Analysis of differences in intestinal flora

[0177] The principal component analysis (PCoA) diagram of the intestinal flora can intuitively reflect the similarity between the flora of each group. The closer the projection distance of the cluster points of each group on the coordinate axis of the PCoA diagram, the more similar the community composition between the groups in the corresponding dimension. The effect of Gastrodia elata resistant starch on the structure of the intestinal microbial community was studied by β diversity analysis, and the differences in the composition of the sample microorganisms were compared by PCA analysis. The points of different colors in the PCoA diagram represent different time points. Fig.19 It can be seen that PC1 and PC2 accounted for 16.31% and 32.4% of the data variance, respectively. The intestinal flora samples at 6, 12, 24, and 48 hours after Gastrodia elata resistant starch fermentation were close, indicating that the intestinal flora structure was similar after Gastrodia elata resistant starch intervention. There was an obvious separation phenomenon after Gastrodia elata resistant starch fermentation and before intervention, indicating that Gastrodia elata resistant starch can affect the composition of intestinal flora.

[0178] LEFSe analysis is an algorithm for high-dimensional biomarker (gene, pathway or cluster) discovery and interpretation that can identify and characterize genomic features that differ between two or more classes. Fig. 20 ) It can be seen that Bifidobacterium, Lactobacillus, g_Bacteroides, Bacillus, Lachnoclostridium, and Muribaculaceae are the bacterial species with the greatest significant difference in effect on Gastrodia elata resistant starch, indicating that they are biomarkers after intervention.

[0179] 11.2 Analysis of intestinal flora composition

[0180] To further investigate the changes in gut microbiota induced by Gastrodia elata resistant starch intervention, the relative abundance of microbial species was evaluated at the phylum and genus levels.

[0181] The distribution of bacterial flora in the Gastrodia elata resistant starch group and the control group at the phylum classification level is as follows Fig.21As shown in the results, the intestinal flora is mainly composed of microorganisms such as Firmicutes, Bacteroidetes, Actinobacteriota, and Proteobacteria, and there are certain differences in the abundance at the phylum level among the groups. At the phylum level, Firmicutes and Bacteroidetes are the main dominant flora, accounting for more than 90% of the total flora, which is basically consistent with the proportion reported in the past literature. Compared with the control group, the relative abundance of Firmicutes decreased after resistant starch intervention, while the relative abundance of Bacteroidetes increased. Gastrodia elata resistant starch can promote the growth of Bacteroidetes, indicating that it has super prebiotic properties. Comparing the ratio of Firmicutes to Bacteroidetes (F / B) between the fermentation groups, it can be found that the F / B value gradually increases. This is consistent with the research results that prebiotics such as dietary fiber can promote the growth of Bacteroidetes while inhibiting the growth of Firmicutes. Human and animal experiments have verified that individual obesity is closely related to the ratio of intestinal flora. The F / B value is one of the biological indicators of obesity. When the body's obesity is relieved, the F / B value decreases. Therefore, the above results indicate that Gastrodia elata resistant starch may inhibit obesity and prevent obesity by regulating the flora structure of intestinal microorganisms.

[0182] like Fig. 22 As shown in the results, at the genus level, compared with the control group, the Gastrodia elata resistant starch group significantly promoted the growth of Bacteroides, Bifidobacterium, Phascolarctobacterium, Muribaculaceae, Lactobacillus, Blautia, and Ruminococcaceae_unclassified, and reduced the relative abundance of Lachnospiraceae_unclassifed and Enterococcus. This is consistent with the results of SCFAs content. The study found that Bacteroides is the main bacterial group that degrades starch, and its metabolites are mainly acetic acid and propionic acid; Bacteroidaceae is a bacterium that can decompose sugar and produce propionic acid; the metabolites of Bifidobacterium are mainly acetic acid and lactic acid; Ruminococcaceae can promote the production of butyrate. Phascolarctobacterium can produce acetic acid and propionic acid; compared with the control group, Gastrodia elata resistant starch has stronger prebiotic properties.

[0183] Firmicutes and Bacteroidota play an important role in regulating metabolism, improving intestinal immunity and promoting the body's resistance to inflammatory responses. Ruminococcaceae plays an important role in stabilizing the intestinal barrier, improving diarrhea and reducing the risk of colon cancer. Phascolarctobacterium occupies a specific intestinal niche, can stably colonize the host intestine, and participate in cholesterol metabolism and maintain bile acid homeostasis. Blautia has a probiotic effect on obesity, diabetes, cardiovascular disease and other diseases. Muribaculaceae is beneficial to the health of the intestinal epithelium, and this species is more likely to be enriched and grown under a high dietary fiber diet. The control group and the Gastrodia elata resistant starch group have different species, which indicates that the intervention of Gastrodia elata resistant starch has certain differences in regulating the diversity of intestinal flora and improving the abundance of flora, thereby adjusting the structure of the intestinal flora to a beneficial direction, thereby affecting its hypoglycemic and lipid-lowering effects.

[0184] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing gastrodia elata resistant starch, characterized in that: The steps include: (1) Using fresh Gastrodia elata as raw material to prepare Gastrodia elata crude starch; (2) subjecting the solution of the crude starch of Gastrodia elata to pregelatinization, autoclave treatment, enzymolysis treatment and aging and retrogradation treatment to obtain Gastrodia elata resistant starch; The mass concentration of the solution of Gastrodia elata crude starch is 5%-25%; The pre-gelatinization time is 20-40min; The enzyme used in the enzymatic hydrolysis treatment is pullulanase, the enzyme dosage is 10-50 ASPU / g, the enzymatic hydrolysis time is 8-14h, and the enzymatic hydrolysis temperature is 50-60°C; The time of the aging and rejuvenation treatment is 12-32 hours.

2. The preparation method according to claim 1, characterized in that: The temperature of the pre-gelatinization treatment is 85-95°C; the temperature of the autoclave treatment is 120-130°C, and the time is 25-35 min; the pH of the enzymatic hydrolysis treatment is 4.5±0.05; and the temperature of the aging and regeneration treatment is 0-4°C.

3. The preparation method according to claim 1, characterized in that: The mass concentration of the solution of Gastrodia elata crude starch is 15%; and / or, The pre-gelatinization time is 25 minutes; and / or, The enzyme dosage in the enzymatic hydrolysis treatment is 24.5 ASPU / g; and / or, The enzymatic hydrolysis time in the enzymatic hydrolysis treatment is 10 hours; and / or, The enzymatic hydrolysis temperature in the enzymatic hydrolysis treatment is 56° C.; and / or, The aging and regeneration time is 24h.

4. The preparation method according to claim 2, characterized in that: The pre-gelatinization temperature is 90°C; and / or, The temperature of the autoclave treatment is 121°C; and / or, The autoclave treatment time is 30 minutes; and / or, The temperature of the aging and regeneration treatment is 4°C.

5. Gastrodia elata resistant starch prepared by the preparation method according to any one of claims 1 to 4.

6. Use of the Gastrodia elata resistant starch according to claim 5 in the preparation of a drug for protecting intestinal health.

7. The use according to claim 6, characterized in that: The drugs for protecting intestinal health include drugs for promoting the proliferation of intestinal probiotics, drugs for maintaining the intestinal barrier, drugs for inhibiting the growth of harmful intestinal bacteria, or drugs for improving the structure of intestinal flora.

8. The use according to claim 7, characterized in that: The drug for maintaining the intestinal barrier includes a drug for promoting intestinal probiotics to produce short-chain fatty acids.

9. A drug for protecting intestinal health, characterized in that: The active ingredient of the medicine is the Gastrodia elata resistant starch as described in claim 5.

10. The drug according to claim 9, characterized in that The medicine also includes pharmaceutically acceptable auxiliary materials.

Images