Preparation method and application of an anoa composita containing chrysopogon zizyahu

Abstract

This invention relates to the field of intestinal health regulation technology, and discloses a method for preparing and applying a composition containing *Anoectochilus roxburghii* (a type of orchid) to improve intestinal health. The composition, by weight, includes the following components: 8-25 parts of *Anoectochilus roxburghii* complex extract, 5-20 parts of fermented *Anoectochilus roxburghii* prebiotics, 20-60 parts of complex prebiotics, and 1-10 parts of pre-adapted probiotic powder. The *Anoectochilus roxburghii* complex extract is composed of *Anoectochilus roxburghii* polysaccharide fraction and *Anoectochilus roxburghii* alcohol extract composite fraction. The alcohol extract composite fraction is a complex formed by the alcohol extract of *Anoectochilus roxburghii* and phospholipids. The mass ratio of the alcohol extract composite fraction to the polysaccharide fraction is 1:(1-4). This invention overcomes the technical problems of low utilization rate of *Anoectochilus roxburghii* active ingredients, poor probiotic environmental tolerance, and limited dispersibility of active substances in existing intestinal health improvement products. This invention constructs a composite system that combines improved utilization of active ingredients, enhanced stability of live bacteria, and improved overall intestinal health improvement capabilities.

Description

Technical Field

[0001] This invention relates to the field of intestinal health regulation technology, and in particular to a method for preparing and applying a composition containing *Anoectochilus roxburghii* to improve intestinal health. Background Technology

[0002] The balance of the gut microbiota and the integrity of the intestinal mucosal barrier are closely related to human health. Gut microbiota imbalance, impaired intestinal mucosal barrier, and abnormal local inflammation are often associated with constipation, diarrhea, and various chronic diseases. Currently, probiotics, prebiotics, and plant extracts are widely used in gut health regulation products.

[0003] In existing technologies, plant extracts such as Anoectochilus roxburghii are typically added directly to compositions as functional ingredients, utilizing their natural activity to exert regulatory effects. However, most existing compound formulations are produced using a direct dry-mixing method at the point of production, resulting in the plant extracts, prebiotics, and probiotics existing only in a simple physical coexistence state.

[0004] The shortcomings of existing technologies are specifically reflected in the following aspects: First, the different active parts of plant extracts, especially those from *Anoectochilus roxburghii* (such as small molecule parts and polysaccharide parts), cannot be utilized in a stratified manner, resulting in insufficient overall raw material utilization and activity. Second, probiotics are easily inactivated during preparation, storage, and gastrointestinal passage due to the influence of the plant's active environment, gastric acid, bile salts, and processing conditions, resulting in insufficient effective live bacteria reaching the intestines. Finally, the dispersibility, release efficiency, and intestinal utilization of plant active ingredients in the complex system are limited, making it difficult to fully exert their comprehensive regulatory effects on the intestinal inflammatory microenvironment and mucosal barrier. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a method for preparing and applying a composition containing Anoectochilus roxburghii to improve gut health. This method can overcome the technical problems of low utilization rate of active ingredients of Anoectochilus roxburghii, poor environmental tolerance of probiotics, and limited dispersibility of active substances in existing gut health improvement products. This invention constructs a composite system that combines improved utilization of active ingredients, enhanced stability of live bacteria, and improved overall gut health improvement by using multi-level technical means such as dual-part extraction of Anoectochilus roxburghii, synergistic delivery of phospholipids, pre-compounding of Anoectochilus roxburghii and prebiotics, constructing prebiotics after fermentation of Anoectochilus roxburghii, and short-term pre-adaptation culture of probiotics.

[0006] The present invention solves the above-mentioned technical problems through the following technical means:

[0007] In a first aspect, the present invention provides a composition containing Anoectochilus roxburghii to improve intestinal health, comprising the following components by weight: 8-25 parts of Anoectochilus roxburghii compound extract, 5-20 parts of Anoectochilus roxburghii fermented prebiotic, 20-60 parts of compound prebiotic, and 1-10 parts of pre-adapted probiotic powder.

[0008] The Anoectochilus roxburghii complex extract is composed of Anoectochilus roxburghii polysaccharide fraction and Anoectochilus roxburghii alcohol extract complex fraction. The Anoectochilus roxburghii alcohol extract complex fraction is a complex formed by Anoectochilus roxburghii alcohol extract fraction and phospholipids. The mass ratio of Anoectochilus roxburghii alcohol extract complex fraction to Anoectochilus roxburghii polysaccharide fraction is 1:(1-4).

[0009] This invention uses *Anoectochilus roxburghii* as the core functional plant raw material. Through layered extraction and targeted utilization of its different active components, and combined with compound prebiotics, *Anoectochilus roxburghii*-derived postbiotics, pre-adapted probiotics, and phospholipid synergistic components, a multi-level composite system is constructed that simultaneously acts on the release and utilization of active ingredients, regulation of intestinal microecology, protection of intestinal mucosa, and enhancement of probiotic stability. This improves the overall composition's comprehensive ability to improve intestinal health.

[0010] Preferably, the phospholipid contains not less than 20% phosphatidylcholine and is selected from one or more of soybean lecithin, sunflower lecithin, and natural phospholipids rich in phosphatidylcholine.

[0011] Preferably, the compound prebiotics include, by weight, 8-20 parts inulin, 5-15 parts fructooligosaccharides, 3-10 parts galactooligosaccharides, 3-12 parts resistant dextrin or resistant starch, and optionally 0.5-5 parts xylooligosaccharides.

[0012] Preferably, the pre-adapted probiotic powder is obtained by pre-adapting probiotics in a culture system containing 0.01%-0.5% *Anoectochilus roxburghii* polysaccharide fraction and 1%-3% prebiotics for 4-12 hours.

[0013] Preferably, the probiotics include Bifidobacteria and Lactobacillus, wherein the Bifidobacteria are selected from one or more of Bifidobacterium longum and Bifidobacterium infantis, and the Lactobacillus are selected from one or more of Lactobacillus plantarum, Lactobacillus rhamnosus, and Lactobacillus acidophilus.

[0014] Preferably, the protective excipients include one or more of low-ester pectin, sodium alginate, soluble dietary fiber, microcrystalline cellulose, and maltodextrin.

[0015] Secondly, the present invention provides a method for preparing a *Anoectochilus roxburghii*-containing composition for improving intestinal health, the method comprising the following steps:

[0016] S1. Prepare the alcohol extract and polysaccharide fraction of Anoectochilus roxburghii, respectively;

[0017] S2. The alcohol extract of *Anoectochilus roxburghii* and phospholipids were mixed in an ethanol solution at a mass ratio of (2-5):1. After stirring, homogenization, and removal of the solvent, a *Anoectochilus roxburghii*-phospholipid complex was obtained. This complex was then mixed with the polysaccharide extract of *Anoectochilus roxburghii* at a ratio of 1:(1.5-3) to obtain a *Anoectochilus roxburghii* complex extract.

[0018] S3. Disperse the Anoectochilus roxburghii compound extract in water and add compound prebiotics. Stir at 25-45℃ for 0.5-3 hours and then dry to obtain Anoectochilus roxburghii-prebiotic compound matrix powder.

[0019] S4. Add the polysaccharide fraction and / or alcoholic extract of Anoectochilus roxburghii obtained in step S1 to purified water to prepare an extract of Anoectochilus roxburghii with a mass concentration of 2%-8%. Adjust the pH to 5.5-6.8, sterilize, inoculate with probiotics, and ferment at 30-37℃ for 12-36 hours. After fermentation, obtain fermented Anoectochilus roxburghii probiotics.

[0020] S5. Inoculate probiotics into a culture system containing polysaccharide fractions of Anoectochilus roxburghii and prebiotics, culture at 30-37℃ for 4-12 hours, collect the bacterial cells and dry them to prepare pre-adapted probiotic powder;

[0021] S6. Premix the Bifidobacteria in the pre-adapted probiotic powder with prebiotics to obtain the first premixed component; premix the Lactobacillus in the pre-adapted probiotic powder with the complex matrix powder to obtain the second premixed component;

[0022] S7. Mix the components from the above steps with the remaining prebiotics and protective excipients, and mix thoroughly under conditions where the temperature and relative humidity are not higher than 40%.

[0023] Preferably, in step S1, the preparation process of the alcohol extract of *Anoectochilus roxburghii* is as follows: *Anoectochilus roxburghii* powder is added to a 30%-60% ethanol solution at a material-to-liquid ratio of 1:8-1:20, and ultrasonically extracted at 40-60℃ for 10-40 min, and / or refluxed at 50-70℃ for 1-2 times, each time for 1-2 h; the preparation process of the polysaccharide portion of *Anoectochilus roxburghii* is as follows: the residue after alcohol extraction is added to water, and extracted at 85-100℃ for 2-3 times, each time for 1-3 h; after the extract is concentrated, ethanol is added to achieve a final concentration of 70%-85% for alcohol precipitation; after collecting and redissolving the precipitate, it is dialyzed for 24-48 h using a dialysis bag with a molecular weight cutoff of 3000-8000 Da, and finally freeze-dried;

[0024] The specific parameters for preparing the composite in step S2 are as follows: stirring temperature is 30-50℃, stirring time is 20-60min; homogenization speed is 8000-12000rpm, and homogenization time is 5-15min.

[0025] Preferably, in step S4, the microbial strain used for fermentation is selected from one or more of Lactobacillus plantarum, Lactobacillus acidophilus, and Lactobacillus rhamnosus; the fermented biotics of Anoectochilus roxburghii are prepared using any of the following methods.

[0026] (1) Centrifuge the fermentation broth, collect the supernatant, and concentrate and / or freeze-dry the supernatant under reduced pressure;

[0027] (2) The fermentation liquid is subjected to high-temperature instantaneous sterilization, and then the sterilized fermentation liquid is concentrated under reduced pressure and / or freeze-dried;

[0028] In step S5, the pre-adaptation culture system contains 0.01%-0.5% polysaccharide fraction of *Anoectochilus roxburghii* and 1%-3% prebiotics.

[0029] After mixing in step S7, the process further includes preparing the final composition into powder, granules, capsules, tablets, solid beverages, or strip formulations.

[0030] Thirdly, the present invention provides the application of the aforementioned golden thread lotus compound composition in the preparation of food or health food that improves intestinal flora imbalance, helps relieve constipation or diarrhea, helps improve intestinal mucosal barrier function, and helps regulate intestinal inflammatory state.

[0031] The beneficial effects of this invention are:

[0032] (1) This invention overcomes the problem of insufficient utilization of active ingredients caused by traditional single extraction methods by performing layered and targeted extraction of alcohol extract and polysaccharide extract of Anoectochilus roxburghii. This dual-part extraction mode realizes the division of labor and cooperation of components at different active levels. The small molecules extracted from the alcohol extract are responsible for regulating the local inflammatory microenvironment, while the polysaccharide extract provides mucosal protection and prebiotic-like support, thereby improving the overall utilization efficiency and functional integrity of Anoectochilus roxburghii raw materials.

[0033] (2) Phospholipids are introduced as synergistic enhancers into the composite composition containing Anoectochilus roxburghii. By pre-forming a complex with the alcohol extract of Anoectochilus roxburghii, the amphiphilic structure of phospholipids improves the dispersion of small molecule active ingredients. This technique significantly improves the wettability and release efficiency of active ingredients in the gastrointestinal fluid environment and enhances their contact with the intestinal mucosa interface, thereby allowing Anoectochilus roxburghii to exert its regulatory effect on intestinal inflammation more fully.

[0034] (3) This invention uses a liquid-phase pre-composite process to distribute the functional components of *Anoectochilus roxburghii* more evenly in the prebiotic matrix, enhancing the carrier compatibility and functional coupling between the components. Combined with the construction of post-fermentation probiotics in *Anoectochilus roxburghii*, the system forms a dual-pathway mode of "pre-probiotic regulation + post-probiotic regulation", which allows the composition to exert its improving effect without completely relying on the survival of live bacteria, thus enhancing the functional hierarchy and sustainability of the product.

[0035] (4) Regarding the improvement of probiotic stability, the present invention uses a culture system containing Anoectochilus roxburghii polysaccharide and prebiotics to conduct short-term pre-adaptation culture of the strains, and combines multi-strain stepwise feeding technology to provide suitable local microenvironments for different strains, thereby enhancing the probiotics' tolerance to the preparation and processing environment, long-term storage conditions and gastric acid and bile salt environment, and effectively improving the effective viable bacteria retention rate in the final preparation and the amount of viable bacteria reaching the intestine. Detailed Implementation

[0036] The technical solutions of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0037] Unless otherwise specified, all raw materials used in the examples are conventionally available in the art, and all methods used are conventional methods in the art. Unless otherwise specified, all percentages in the examples are by mass.

[0038] Unless otherwise stated, the weight parts of each component in the final product described herein are based on the actual amount of each component added to the final composition. For components introduced in the form of intermediates, such as *Anoectochilus roxburghii*-prebiotic complex matrix powder, premixed components, etc., the *Anoectochilus roxburghii* complex extract, prebiotics, pre-adapted probiotic powder, and other excipients contained therein can be included in the weight parts of the corresponding components in the final composition according to their actual content in the intermediate. In the embodiments of this invention, for the convenience of describing the intermediate preparation process, some steps are described using intermediate names, but their corresponding effective components are all part of the final composition.

[0039] This invention provides a *Anoectochilus roxburghii*-containing composition for improving gut health, which mainly consists of a *Anoectochilus roxburghii* complex extract, fermented *Anoectochilus roxburghii* prebiotics, a complex prebiotic, and pre-adapted probiotics. The *Anoectochilus roxburghii* complex extract is a combination of the alcoholic extract and the polysaccharide extract of *Anoectochilus roxburghii*.

[0040] The "post-fermentation probiotics" mentioned in this article refer to fermentation product components obtained by fermenting Anoectochilus roxburghii extract with probiotics, which exert intestinal regulatory effects without relying on the survival of live bacteria. It can be a product obtained by concentrating and / or drying the fermentation supernatant, or a product obtained by sterilizing the whole fermentation liquid and then concentrating and / or drying it.

[0041] The preferred extract of *Anoectochilus roxburghii* for fermentation is mainly composed of the polysaccharide fraction, or a combination of the polysaccharide fraction and the alcoholic extract fraction. When using a combination of both, the amount of alcoholic extract fraction added should be controlled to avoid adverse effects on the growth of the fermentation strain.

[0042] The preparation method of the composition of the present invention specifically includes the following steps:

[0043] Step S1. Extraction of two parts of Anoectochilus roxburghii.

[0044] S1-1. Preparation of the alcoholic extract of *Anoectochilus roxburghii*: The dried whole *Anoectochilus roxburghii* herb was pulverized and sieved, and a 30%-60% ethanol solution was added at a material-to-liquid ratio of 1:8-1:20. Ultrasonic extraction was performed at 40-60℃ for 10-40 min, and / or reflux extraction was performed at 50-70℃ 1-2 times, 1-2 h each time. The extract was filtered, concentrated under reduced pressure, and dried to obtain the alcoholic extract of *Anoectochilus roxburghii* rich in small molecule active ingredients.

[0045] S1-2. Preparation of Polysaccharide Fraction of *Anoectochilus roxburghii*: The residue after ethanol extraction was mixed with water and extracted 2-3 times at 85-100℃, 1-3 hours each time. The extract was concentrated, and ethanol was added to achieve a final concentration of 70%-85% for ethanol precipitation. The precipitate was redissolved and dialyzed using a dialysis bag with a molecular weight cutoff of 3000-8000 Da for 24-48 hours, followed by freeze-drying to obtain the polysaccharide fraction of *Anoectochilus roxburghii*.

[0046] Step S2. Preparation of Anoectochilus roxburghii-phospholipid complex and complex extract

[0047] The alcoholic extract of *Anoectochilus roxburghii* was mixed with phospholipids (mass ratio 2-5:1) in ethanol, stirred at 30-50℃ for 20-60 min, and homogenized at 8000-12000 rpm for 5-15 min. After removing the solvent, the mixture was dried to obtain the *Anoectochilus roxburghii*-phospholipid complex. This complex was then mixed with the polysaccharide fraction obtained in steps S1-2 at a predetermined ratio to prepare the *Anoectochilus roxburghii* complex extract.

[0048] Step S3. Preparation of the Golden Thread Lotus-Prebiotic Precomposite Matrix

[0049] The compound extract of *Anoectochilus roxburghii* was dispersed in water, and compound prebiotics (such as inulin and fructooligosaccharides) were added. The mixture was stirred at 25-45℃ for 0.5-3 hours to allow the active ingredients and prebiotics to form a pre-compound system. After drying and pulverizing, the *Anoectochilus roxburghii*-prebiotic compound matrix powder was obtained.

[0050] Step S4. Preparation of biotics after fermentation of Anoectochilus roxburghii.

[0051] Extracts of *Anoectochilus roxburghii* (2%-8% solids content) were inoculated with *Lactobacillus plantarum*, *Lactobacillus acidophilus*, or *Lactobacillus rhamnosus*. Fermentation was carried out at 30-37℃ for 12-36 hours. The fermentation broth was then concentrated, sterilized, and freeze-dried to obtain the fermented serotonin from *Anoectochilus roxburghii*.

[0052] Step S5. Preparation of pre-adapted probiotics

[0053] Probiotic pre-adaptation culture involves inoculating probiotics into a culture system containing 0.01%-0.5% *Anoectochilus roxburghii* polysaccharide fraction and 1%-3% prebiotics, and culturing for a short time at 30-37℃ for 4-12 hours. The bacterial cells are then collected and freeze-dried at low temperature to obtain pre-adapted probiotic powder.

[0054] Step S6. Multi-strain stepwise feeding and premixing

[0055] Premixing was performed stepwise according to the characteristics of the bacterial strains: Bifidobacteria were premixed with galactooligosaccharides or inulin; Lactobacilli were premixed with resistant dextrin or a complex matrix powder. The different premixed components were then combined at low temperature.

[0056] Step S7. Final composition mixing

[0057] Mix the components from the above steps, along with the remaining prebiotics and protective excipients, and mix thoroughly at a temperature of 40°C and a relative humidity of 40% to prepare powders, granules, or capsules.

[0058] Example 1

[0059] This embodiment describes the preparation of a composition containing *Anoectochilus roxburghii* to improve intestinal health; the product is a powder.

[0060] S1. Extraction of two parts of *Anoectochilus roxburghii*

[0061] S1-1. Preparation of the alcoholic extract of Anoectochilus roxburghii

[0062] Weigh 1000g of dried whole Anoectochilus roxburghii herb, pulverize it and pass it through a 60-mesh sieve.

[0063] Add 12,000 mL of 40% ethanol solution at a material-to-liquid ratio of 1:12, and extract by ultrasonication at 45°C for 25 min with an ultrasonic power of 350 W; then heat to 60°C and reflux for extraction twice, each time for 1.5 h.

[0064] After extraction, the mixture was filtered, and the filtrates were combined. The ethanol was recovered under reduced pressure at 55°C and concentrated to a relative density of 1.10-1.15 to obtain the concentrated ethanol extract of Anoectochilus roxburghii.

[0065] The concentrated alcohol extract was vacuum dried at 50°C and pulverized to obtain a powder of the alcohol extract of Anoectochilus roxburghii rich in small molecule active ingredients, denoted as component A.

[0066] S1-2. Preparation of polysaccharide fractions from *Anoectochilus roxburghii*

[0067] Take the residue after the above alcohol extraction, add 15 times the mass of purified water, and extract it 3 times at 95℃, 2 hours each time.

[0068] The aqueous extracts were combined, filtered, and then concentrated under reduced pressure at 60°C to 1 / 5 of their original volume.

[0069] Slowly add anhydrous ethanol to the concentrate to achieve a final concentration of 80%, and let it stand at 4°C for 12 hours for alcohol precipitation.

[0070] The precipitate was collected by centrifugation, reconstituted with purified water, and then placed in a dialysis bag with a molecular weight cutoff of 5500 Da. Dialysis was performed in purified water for 36 hours, with the water changed every 8 hours.

[0071] After dialysis, the dialysate was freeze-dried to obtain the polysaccharide powder of Anoectochilus roxburghii, which was designated as component B.

[0072] S2. Preparation of *Anoectochilus roxburghii*-phospholipid complex and compound extract

[0073] Weigh 50g of the powdered alcohol extract of Anoectochilus roxburghii and add an appropriate amount of 95% ethanol to disperse it fully.

[0074] Weigh 20g of soybean lecithin and add it to the above dispersion. Stir at 40℃ for 30min.

[0075] The homogenizer was then used for homogenization for 10 minutes at a speed of 8000-12000 rpm.

[0076] After homogenization, ethanol was recovered under reduced pressure at 45-50℃. The resulting material was then vacuum dried to constant weight, pulverized through an 80-mesh sieve, and the resulting Anoectochilus roxburghii-phospholipid complex was obtained, denoted as component A1.

[0077] Mix component A1 and component B at a mass ratio of 1:2 to obtain a compound extract of Anoectochilus roxburghii.

[0078] S3. Preparation of *Anoectochilus roxburghii*-prebiotic composite matrix powder

[0079] Weigh 80g of the Anoectochilus roxburghii compound extract, wherein the proportion of Anoectochilus roxburghii polysaccharide is not less than 60%.

[0080] Disperse it in 800 mL of purified water and stir for 20 min.

[0081] Then add 150g of inulin, 100g of fructooligosaccharides and 80g of resistant dextrin, and stir at 35℃ for 1.5h to allow the active components of Anoectochilus roxburghii to come into full contact with the prebiotics.

[0082] The resulting mixture was concentrated under reduced pressure at 55°C to a solid content of approximately 30%, then freeze-dried and pulverized through an 80-mesh sieve to obtain the *Anoectochilus roxburghii*-prebiotic composite matrix powder.

[0083] S4. Preparation of biotics after fermentation of Anoectochilus roxburghii

[0084] Weigh 30g of the polysaccharide portion and 10g of the alcoholic extract of Anoectochilus roxburghii, add purified water to prepare an extract of Anoectochilus roxburghii with a total solids content of 4%, and adjust the pH to 6.3.

[0085] After pasteurization and cooling to 37°C, Lactobacillus plantarum was inoculated at a rate of 2%.

[0086] Anaerobic fermentation at 37℃ for 18 hours.

[0087] After fermentation, the supernatant was collected by centrifugation, concentrated under reduced pressure at 50°C, and then freeze-dried to obtain the fermented genistein powder.

[0088] S5. Preparation of pre-adapted probiotic powder

[0089] S5-1. Bifidobacterium preconditioning culture

[0090] Prepare a basic culture medium and add 2.0% galactooligosaccharide and 0.08% Anoectochilus roxburghii polysaccharide fraction.

[0091] Inoculate with Bifidobacterium longum and incubate at 37°C for 6 hours.

[0092] After the culture was completed, the bacterial cells were collected by centrifugation, washed twice with sterile physiological saline, and then freeze-dried after adding an appropriate amount of protectant to obtain pre-adapted Bifidobacterium powder.

[0093] S5-2. Lactobacillus preconditioning culture

[0094] Prepare a basic culture medium and add 1.5% inulin, 1.0% fructooligosaccharide and 0.05% Anoectochilus roxburghii polysaccharide fraction.

[0095] The bacteria were inoculated with Lactobacillus plantarum and Lactobacillus rhamnosus respectively and cultured at 37°C for 8 hours.

[0096] After the culture is completed, the bacterial cells are collected and freeze-dried in the manner described above to obtain pre-adapted Lactobacillus powder.

[0097] S6. Multi-strain stepwise feeding premix

[0098] Pre-adapted Bifidobacterium longum powder was mixed with galactooligosaccharides and inulin at a mass ratio of 1:2:2 to obtain premix component A.

[0099] Pre-adapted Lactobacillus plantarum powder, pre-adapted Lactobacillus rhamnosus powder, resistant dextrin, and Anoectochilus roxburghii-prebiotic complex matrix powder were mixed at a mass ratio of 1:1:2:4 to obtain premixed component B.

[0100] S7. Preparation of the final composition

[0101] Weigh each component according to the following mass proportions: 12 parts of Anoectochilus roxburghii compound extract, 10 parts of Anoectochilus roxburghii fermented prebiotic, 30 parts of Anoectochilus roxburghii-prebiotic compound matrix powder, 10 parts of inulin, 8 parts of fructooligosaccharides, 6 parts of galactooligosaccharides, 8 parts of resistant dextrin, 8 parts of premixed component A, and 8 parts of premixed component B.

[0102] Mix for 20 minutes at a temperature not exceeding 35°C and a relative humidity not exceeding 35%, pass through a 40-mesh sieve, and then package to obtain a powder composition for improving intestinal health.

[0103] Example 2

[0104] This embodiment describes the preparation of a composition containing *Anoectochilus roxburghii* to improve intestinal health, and the product is in granule form.

[0105] S1. Extraction of two parts of *Anoectochilus roxburghii*

[0106] S1-1. Preparation of the alcohol extract of Anoectochilus roxburghii:

[0107] Take 800g of whole Anoectochilus roxburghii herb, crush it through an 80-mesh sieve, add 60% ethanol at a material-to-liquid ratio of 1:20, sonicate for 20 minutes, and then reflux at 70℃ twice for 1 hour each time; filter, concentrate, and dry to obtain the ethanol extract.

[0108] S1-2. Preparation of polysaccharide fractions from *Anoectochilus roxburghii*:

[0109] After ethanol extraction, the residue was added to purified water at a ratio of 1:20 and extracted twice at 100℃ for 2.5 hours each time. After concentration, it was precipitated with 75% ethanol, dialyzed for 48 hours using a dialysis bag with a molecular weight cutoff of 8000 Da, and then freeze-dried to obtain the polysaccharide fraction.

[0110] S2. Preparation of *Anoectochilus roxburghii*-phospholipid complex and compound extract

[0111] Weigh 40g of the alcoholic extract of Anoectochilus roxburghii and 15g of sunflower lecithin, add them to 80% ethanol, stir for 20 minutes and then homogenize at 35℃ for 8 minutes.

[0112] The solvent was removed under reduced pressure, and the product was dried under vacuum to obtain the Anoectochilus roxburghii-phospholipid complex.

[0113] Then, it is compounded with the polysaccharide part of Anoectochilus roxburghii at a mass ratio of 1:3 to obtain the Anoectochilus roxburghii compound extract.

[0114] S3. Preparation of *Anoectochilus roxburghii*-prebiotic composite matrix powder

[0115] Take 100g of the compound extract of Anoectochilus roxburghii and disperse it evenly in 1000mL of purified water;

[0116] Add 120g inulin, 120g fructooligosaccharides, and 60g galactooligosaccharides, and stir at 40℃ for 2 hours;

[0117] The composite matrix powder was obtained by spray drying.

[0118] S4. Preparation of biotics after fermentation of Anoectochilus roxburghii

[0119] The polysaccharide portion of Anoectochilus roxburghii was prepared into a 5% solution, and a mixed bacterial culture of Lactobacillus acidophilus and Lactobacillus plantarum was inoculated. The total inoculation amount was 3%, and fermentation was carried out at 35°C for 24 hours.

[0120] After fermentation, the entire process is sterilized at high temperature and then freeze-dried to obtain the post-fermentation serotonin from *Anoectochilus roxburghii*.

[0121] S5. Preparation of pre-adapted probiotics

[0122] In a culture system containing 0.10% Anoectochilus roxburghii polysaccharide, 2.0% inulin and 1.0% fructooligosaccharide, Lactobacillus plantarum and Bifidobacterium longum were pre-adapted for 6-10 hours.

[0123] After centrifugation and washing, freeze-dry.

[0124] S6. Multi-strain stepwise feeding premix

[0125] Premixed component A and premixed component B were prepared according to the method in Example 1.

[0126] S7. Preparation of the final composition

[0127] By weight, the product comprises: 18 parts of *Anoectochilus roxburghii* compound extract, 15 parts of *Anoectochilus roxburghii* fermented prebiotic, 25 parts of *Anoectochilus roxburghii*-prebiotic compound matrix powder, 8 parts of inulin, 10 parts of fructooligosaccharides, 8 parts of galactooligosaccharides, 6 parts of resistant starch, 3 parts of premix component A, 7 parts of premix component B, and 8.5 parts of protective excipient (microcrystalline cellulose). After mixing, it is formulated into granules.

[0128] Example 3

[0129] This embodiment describes the preparation of a *Anoectochilus roxburghii*-containing composition for improving gut health, and the product is in capsule form.

[0130] S1. Extraction of two parts of *Anoectochilus roxburghii*

[0131] S1-1. Preparation of the alcohol extract of Anoectochilus roxburghii:

[0132] Take 800g of whole Anoectochilus roxburghii herb, crush it through an 80-mesh sieve, add 30% ethanol at a material-to-liquid ratio of 1:8, sonicate for 10 minutes, and then reflux at 50℃ twice for 1 hour each time; filter, concentrate, and dry to obtain the ethanol extract.

[0133] S1-2. Preparation of polysaccharide fractions from *Anoectochilus roxburghii*:

[0134] After ethanol extraction, the residue was added to purified water at a ratio of 1:20 and extracted twice at 85℃ for 2.5 hours each time. After concentration, it was precipitated with 70% ethanol, dialyzed for 24 hours using a dialysis bag with a molecular weight cutoff of 3000 Da, and then freeze-dried to obtain the polysaccharide fraction.

[0135] S2. Preparation of *Anoectochilus roxburghii*-phospholipid complex and compound extract

[0136] The alcohol extract of Anoectochilus roxburghii was mixed with soybean lecithin at a mass ratio of 3:1, dispersed in 50% ethanol, and then subjected to a combined ultrasonic-homogenization treatment, wherein the ultrasonic power was 300W for 15 minutes and the homogenization time was 8 minutes.

[0137] Subsequently, the mixture was dried under reduced pressure to obtain the *Anoectochilus roxburghii*-phospholipid complex.

[0138] Then, it is combined with the polysaccharide part of Anoectochilus roxburghii, and the ratio of the two is adjusted to 1:1.5 to obtain the Anoectochilus roxburghii compound extract.

[0139] S3. Preparation of *Anoectochilus roxburghii*-prebiotic precomposite matrix

[0140] A pre-composite matrix of *Anoectochilus roxburghii* and prebiotics was prepared according to the method in Example 1.

[0141] S4. Preparation of biotics after fermentation of Anoectochilus roxburghii

[0142] The alcoholic extract of Anoectochilus roxburghii was diluted to prepare a fermentation substrate solution with a solid content of 3%, inoculated with Lactobacillus rhamnosus, and fermented at 34℃ for 16 hours. After fermentation, the supernatant was collected, concentrated under reduced pressure, and freeze-dried to obtain the fermented biotic of Anoectochilus roxburghii.

[0143] S5. Preparation of pre-adapted probiotics

[0144] A culture system containing 0.05% *Anoectochilus roxburghii* polysaccharide, 1.5% galactooligosaccharide, and 1.0% inulin was used to conduct a short-term pre-adaptation culture of *Bifidobacterium infantis* for 7 hours.

[0145] Lactobacillus plantarum was pre-adapted for 6 hours using a culture system containing 0.03% Anoectochilus roxburghii polysaccharide and 1.5% resistant dextrin.

[0146] Freeze-dry separately for later use.

[0147] S6. Multi-strain stepwise feeding premix

[0148] Premixed component A and premixed component B were prepared according to the method in Example 1.

[0149] S7. Preparation of the final composition

[0150] By weight, it includes:

[0151] Ten parts of *Anoectochilus roxburghii* compound extract, eight parts of *Anoectochilus roxburghii* fermented prebiotic, 12 parts of inulin, 10 parts of fructooligosaccharides, 10 parts of galactooligosaccharides, 10 parts of resistant dextrin, 20 parts of *Anoectochilus roxburghii*-prebiotic compound matrix powder, five parts of premixed component A, and five parts of premixed component B were mixed evenly, and an appropriate amount of pharmaceutically acceptable protective excipients were added. The mixture was then filled into empty capsules to obtain capsules.

[0152] Comparative Example 1

[0153] The difference from Example 1 is that: instead of preparing the alcoholic and polysaccharide fractions of *Anoectochilus roxburghii*, the *Anoectochilus roxburghii* was directly pulverized and subjected to a single hot water extraction. The resulting extract was then dried and added to the composition as the *Anoectochilus roxburghii* extract. All other conditions were the same as in Example 1.

[0154] Comparative Example 2

[0155] The difference from Example 1 is that, instead of performing a liquid-phase pre-compounding of *Anoectochilus roxburghii* and prebiotics, and preparing a composite matrix powder, the *Anoectochilus roxburghii* composite extract, prebiotics, and probiotics are directly dry-mixed in the final step. All other conditions are the same as in Example 1.

[0156] Comparative Example 3

[0157] The difference from Example 1 is that:

[0158] No prebiotics were prepared from fermented Anoectochilus roxburghii, no probiotic pre-adaptation culture was performed, and no multi-strain stepwise feeding was used; ordinary freeze-dried probiotic powder was directly added to the final formula. All other conditions were the same as in Example 1.

[0159] Comparative Example 4

[0160] The difference from Example 1 is that:

[0161] No phospholipids were added, nor was a *Anoectochilus roxburghii*-phospholipid complex prepared; the alcoholic extract of *Anoectochilus roxburghii* was directly mixed with the polysaccharide extract for subsequent preparation. All other conditions were the same as in Example 1.

[0162] Comparative Example 5

[0163] The difference from Example 1 is that an equal amount of soybean lecithin was added, but the soybean lecithin was not pre-compounded with the alcoholic extract of *Anoectochilus roxburghii*; instead, it was added all at once in the final mixing step. All other conditions were the same as in Example 1.

[0164] To verify the technical effects of the composition of the present invention, the following testing items were set:

[0165] 1. Viable cell count detection and formulation stability testing;

[0166] 2. Testing of tolerance to artificial gastric juice and bile salts;

[0167] 3. In vitro probiotic proliferation promotion test;

[0168] 4. Detection of intestinal inflammation-related cytokines;

[0169] 5. Analysis of gut microbiota structure;

[0170] 6. Release rate of flavonoids / glycosides, the hallmark components of Anoectochilus roxburghii.

[0171] Experimental Example 1. Detection of viable bacterial count and formulation stability

[0172] Samples obtained from Examples 1-3 and Comparative Examples 1-3 were taken immediately after preparation, and the initial viable count was determined. The samples were then stored under the following conditions: Condition A: 4°C, protected from light; Condition B: 25°C, 60% relative humidity; Condition C: 37°C accelerated storage. Samples were taken after 30 days of storage, and the total viable count was determined using the plate count method, with separate tests for Lactobacillus and Bifidobacterium viable counts. To ensure the impartiality of the comparison, the initial total viable count was adjusted to approximately 8.5 × 10⁻⁶ by adjusting the dosage of either pre-adapted probiotic powder or regular probiotic powder in each group of samples. 10 Within the CFU / g range.

[0173] Table 1 Initial viable count and viable count retention after 30 days of storage at 25℃

[0174]

[0175] Table 2 Total viable cell retention rate under different storage conditions for 30 days

[0176]

[0177] As shown in Tables 1 and 2, the viable bacteria retention rates of Examples 1-3 were higher than those of Comparative Examples 1-3 under all storage conditions, with Example 1 showing the highest retention rate. This indicates that the use of dual-part extraction of *Anoectochilus roxburghii*, pre-compounding of *Anoectochilus roxburghii* and prebiotics, fermentation of *Anoectochilus roxburghii* followed by pre-adaptation culture, and stepwise addition of multiple strains can significantly improve the storage stability of probiotics in the preparation. The most significant decrease was observed in Comparative Example 3, indicating that when ordinary probiotics are directly mixed into the final mixture, the viable bacteria are more easily inactivated during storage.

[0178] Experimental Example 2. Tests on tolerance to artificial gastric juice and bile salts

[0179] Probiotic components from samples of Examples 1-3 and Comparative Examples 1-3 were respectively prepared into bacterial suspensions with the same bacterial concentration.

[0180] (1) Artificial gastric juice tolerance test

[0181] The bacterial suspension was added to artificial gastric fluid at pH 2.5 and cultured at 37°C with shaking for 2 hours. The number of viable bacteria before and after treatment was then measured.

[0182] (2) Bile salt tolerance test

[0183] The bacterial suspension was added to a culture medium containing 0.3% bile salts and incubated at 37°C for 4 hours. The number of viable bacteria before and after treatment was then determined.

[0184] Table 3. Data on tolerance to artificial gastric juice and bile salts.

[0185]

[0186] As shown in Table 3, the survival rates of gastric juice and bile salts in Examples 1-3 were significantly higher than those in Comparative Examples 1-3, indicating that the short-term pre-adaptation culture involving Anoectochilus roxburghii polysaccharide, as well as the combined effects of Anoectochilus roxburghii-prebiotic composite matrix and post-biotic, help improve the tolerance of probiotics to gastrointestinal stress environment.

[0187] Experimental Example 3. In vitro probiotic proliferation promotion experiment

[0188] Test methods

[0189] Configure the following culture systems respectively:

[0190] Group A. Basal culture medium;

[0191] Group B: Basal culture medium + compound prebiotics;

[0192] Group C: Basal culture medium + Anoectochilus roxburghii compound extract;

[0193] Group D: Basal culture medium + Anoectochilus roxburghii compound extract + compound prebiotics;

[0194] Group E: Basal culture medium + Anoectochilus roxburghii compound extract + compound prebiotics + Anoectochilus roxburghii fermented post-fermentation prebiotics.

[0195] Lactobacillus plantarum and Bifidobacterium longum were inoculated separately and cultured at 37℃. The OD values ​​and viable cell counts were measured at 0h, 12h, and 24h.

[0196] Table 4. Proliferation data of Lactobacillus plantarum

[0197]

[0198] Table 5. Proliferation data of Bifidobacterium longum

[0199]

[0200] As shown in Tables 4 and 5, Group D was significantly better than Groups B and C, indicating that there is a synergistic promoting effect between the functional components of Anoectochilus roxburghii and the compound prebiotics; Group E was even better than Group D, indicating that the prebiotics after Anoectochilus roxburghii fermentation can further enhance the system's support for beneficial bacteria.

[0201] Experimental Example 4. Detection of Intestinal Inflammation-Related Cytokines

[0202] A DSS-induced colitis mouse model was used for evaluation. SPF-grade male mice were randomly divided into four groups: normal group, model group, Examples 1-3 groups, and Comparative Examples 1-3 groups, with eight mice in each group. Except for the normal group, the other groups were allowed free access to the modeling solution containing DSS for modeling. After successful modeling, each treatment group was administered an equal volume of sample by gavage for 14 consecutive days. After the last administration, serum and colon tissue were collected, and the levels of TNF-α, IL-1β, IL-6, and IL-10 were detected using kits. Colon length and spleen index were also recorded.

[0203] Table 6. Inflammatory factor data in the DSS mouse colitis model

[0204]

[0205] As shown in Table 6, the Example 1 group was superior to the comparative groups in reducing TNF-α, IL-1β, IL-6, increasing IL-10, and improving colon length, indicating that the composition of the present invention can more effectively improve the intestinal inflammatory state.

[0206] Experimental Example 5. Analysis of Gut Microbiota Structure

[0207] 16S rRNA sequencing analysis was performed on animal fecal samples obtained from Experiment 4 to compare the α diversity index, relative abundance of Bifidobacterium, relative abundance of Lactobacillus, relative abundance of opportunistic pathogens, and Firmicutes / Bacteroidetes ratio among the samples in each group.

[0208] Table 7. Relative abundance of major bacterial groups

[0209]

[0210] As shown in Table 7, Examples 1-3 all increased the abundance of Bifidobacteria and Lactobacillus, reduced the proportion of opportunistic pathogens, and made the flora structure closer to the normal group. Among them, Example 1 showed the best effect, indicating that the composition of the present invention has significant advantages in regulating the intestinal flora.

[0211] Experimental Example 6: Release rate of Anoectochilus roxburghii glycosides

[0212] To evaluate the effect of the phospholipid complex technology in this invention on the in vitro release behavior of the active ingredients of *Anoectochilus roxburghii*, the marker glycoside *Anoectochilus roxburghii* was selected as the detection index. Samples obtained from Example 1, Comparative Example 4, and Comparative Example 5 were taken and, based on an equal volume of *Anoectochilus roxburghii* complex extract, were placed in artificial intestinal fluid for in vitro release tests.

[0213] The sample was shaken and released at 37℃. After 2 hours, the sample was collected, centrifuged, and the supernatant was collected. After appropriate pretreatment, the content of roximate in the supernatant was determined by high performance liquid chromatography, and the cumulative release rate over 2 hours was calculated.

[0214] High performance liquid chromatography (HPLC) conditions: A C18 column was used, with acetonitrile-water (15:85) as the mobile phase, and the detection wavelength was 220 nm. Quantification was performed using the external standard method with adenophora stricta glycoside reference standard.

[0215] Table 8

[0216]

[0217] In Example 1, the release rate of roximate was significantly higher than that of Comparative Examples 4 and 5, indicating that phospholipids can promote the dispersion and release of small molecule active sites of Roxithonia spp.

[0218] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the present invention. Equivalent substitutions or modifications made by those skilled in the art without departing from the concept of the present invention should all fall within the scope of protection of the present invention.

Claims

1. A composition containing *Anoectochilus roxburghii* to improve gut health, characterized in that, By weight, it includes the following components: 8-25 parts of Anoectochilus roxburghii compound extract, 5-20 parts of Anoectochilus roxburghii fermented prebiotic, 20-60 parts of compound prebiotic, and 1-10 parts of pre-adapted probiotic powder. The Anoectochilus roxburghii complex extract is composed of Anoectochilus roxburghii polysaccharide fraction and Anoectochilus roxburghii alcohol extract complex fraction. The Anoectochilus roxburghii alcohol extract complex fraction is a complex formed by Anoectochilus roxburghii alcohol extract fraction and phospholipids. The mass ratio of Anoectochilus roxburghii alcohol extract complex fraction to Anoectochilus roxburghii polysaccharide fraction is 1:(1-4).

2. The *Anoectochilus roxburghii*-containing composition for improving intestinal health according to claim 1, characterized in that, The phospholipid contains no less than 20% phosphatidylcholine and is selected from one or more of soybean lecithin, sunflower lecithin, and natural phospholipids rich in phosphatidylcholine.

3. The *Anoectochilus roxburghii*-containing composition for improving intestinal health according to claim 1, characterized in that, The compound prebiotics, by weight, include: 8-20 parts inulin, 5-15 parts fructooligosaccharides, 3-10 parts galactooligosaccharides, 3-12 parts resistant dextrin or resistant starch, and optionally 0.5-5 parts xylooligosaccharides.

4. The *Anoectochilus roxburghii*-containing composition for improving intestinal health according to claim 1, characterized in that, The pre-adapted probiotic powder is obtained by pre-adapting probiotics in a culture system containing 0.01%-0.5% Anoectochilus roxburghii polysaccharide fraction and 1%-3% prebiotics for 4-12 hours.

5. The *Anoectochilus roxburghii*-containing composition for improving intestinal health according to claim 4, characterized in that, The probiotics include Bifidobacteria and Lactobacillus, wherein the Bifidobacteria are selected from one or more of Bifidobacterium longum and Bifidobacterium infantis, and the Lactobacillus are selected from one or more of Lactobacillus plantarum, Lactobacillus rhamnosus, and Lactobacillus acidophilus.

6. The *Anoectochilus roxburghii*-containing composition for improving intestinal health according to claim 1, characterized in that, The composition further includes protective excipients, which include one or more of low-ester pectin, sodium alginate, soluble dietary fiber, microcrystalline cellulose, and maltodextrin.

7. A method for preparing a *Anoectochilus roxburghii*-containing composition for improving intestinal health, used to prepare the *Anoectochilus roxburghii*-containing composition for improving intestinal health as described in any one of claims 1-6, characterized in that, The preparation method includes the following steps: S1. Prepare the alcohol extract and polysaccharide fraction of Anoectochilus roxburghii, respectively; S2. Mix the alcohol extract of Anoectochilus roxburghii with phospholipids in an ethanol solution at a mass ratio of (2-5):

1. After stirring, homogenizing and removing the solvent, Anoectochilus roxburghii-phospholipid complex is obtained. Then, it is mixed with the polysaccharide part of Anoectochilus roxburghii at a ratio of 1:(1.5-3) to obtain the Anoectochilus roxburghii complex extract. S3. Disperse the Anoectochilus roxburghii compound extract in water and add compound prebiotics. Stir at 25-45℃ for 0.5-3 hours and then dry to obtain Anoectochilus roxburghii-prebiotic compound matrix powder. S4. Add the polysaccharide fraction and / or alcoholic extract of Anoectochilus roxburghii obtained in step S1 to purified water to prepare an extract of Anoectochilus roxburghii with a mass concentration of 2%-8%. Adjust the pH to 5.5-6.8, sterilize, inoculate with probiotics, and ferment at 30-37℃ for 12-36 hours. After fermentation, obtain fermented Anoectochilus roxburghii probiotics. S5. Inoculate probiotics into a culture system containing polysaccharide fractions of Anoectochilus roxburghii and prebiotics, culture at 30-37℃ for 4-12 hours, collect the bacterial cells and dry them to prepare pre-adapted probiotic powder; S6. The pre-adapted probiotic powder is premixed with a portion of prebiotics and / or the *Anoectochilus roxburghii*-prebiotic composite matrix powder to obtain a probiotic premixed component; S7. Mix the compound extract of Anoectochilus roxburghii, fermented Anoectochilus roxburghii prebiotic, compound prebiotic, probiotic premixed components and optional protective excipients under low temperature and low humidity conditions to obtain the final composition.

8. The preparation method according to claim 7, characterized in that, In step S1, the preparation process of the alcohol extract of *Anoectochilus roxburghii* is as follows: *Anoectochilus roxburghii* powder is added to a 30%-60% ethanol solution at a material-to-liquid ratio of 1:8-1:20, and ultrasonically extracted at 40-60℃ for 10-40 min, and / or refluxed at 50-70℃ for 1-2 times, each time for 1-2 h; the preparation process of the polysaccharide portion of *Anoectochilus roxburghii* is as follows: the residue after alcohol extraction is added to water, and extracted at 85-100℃ for 2-3 times, each time for 1-3 h; after the extract is concentrated, ethanol is added to achieve a final concentration of 70%-85% for alcohol precipitation; after collecting and redissolving the precipitate, dialyzing is performed for 24-48 h using a dialysis bag with a molecular weight cutoff of 3000-8000 Da, and finally freeze-dried; The specific parameters for preparing the composite in step S2 are as follows: stirring temperature is 30-50℃, stirring time is 20-60min; homogenization speed is 8000-12000rpm, and homogenization time is 5-15min.

9. The preparation method according to claim 7, characterized in that, In step S4, the fermentation strains are selected from one or more of Lactobacillus plantarum, Lactobacillus acidophilus, and Lactobacillus rhamnosus; the fermented phytoestrogen of Anoectochilus roxburghii is prepared using any of the following methods. (1) Centrifuge the fermentation broth, collect the supernatant, and concentrate and / or freeze-dry the supernatant under reduced pressure; (2) The fermentation liquid is subjected to high-temperature instantaneous sterilization, and then the sterilized fermentation liquid is concentrated under reduced pressure and / or freeze-dried; In step S5, the pre-adaptation culture system contains 0.01%-0.5% polysaccharide fraction of *Anoectochilus roxburghii* and 1%-3% prebiotics. After mixing in step S7, the process further includes preparing the final composition into powder, granules, capsules, tablets, solid beverages, or strip formulations.

10. The use of the composite composition containing *Anoectochilus roxburghii* according to any one of claims 1-6 in the preparation of food or health food that improves intestinal flora imbalance, helps relieve constipation or diarrhea, helps improve intestinal mucosal barrier function, and helps regulate intestinal inflammation.

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