Preparation method of intestinal targeting type high-absorption ganoderma lucidum composite fermented drink

Abstract

The invention discloses a preparation method of an intestinal targeting type high-absorption ganoderma lucidum composite fermented beverage, and belongs to the technical field of functional food processing. The method comprises the following steps: separating supernate and mycelium from ganoderma lucidum fermentation liquor; ganoderma lucidum polysaccharides are extracted from mycelia and are subjected to enzymatic compounding with prebiotic oligosaccharides under the catalysis of transglycosidase to form a polysaccharide-prebiotic compound, and triterpenoids are loaded on the polysaccharide-prebiotic compound; taking the supernate and tea polyphenol to form a composite core, coating chitosan and citrus pectin through electrostatic layer-by-layer self-assembly, and constructing pH responsive nanoparticles; and finally, mixing the composite system, the nanoparticles and auxiliary materials, and sterilizing and filling to obtain the product. Through the synergistic effect of enzymatic compounding and nano assembly, targeted release and efficient absorption of active ingredients in intestinal tracts are achieved, bioavailability and product stability are remarkably improved, and the preparation method is suitable for preparing functional food for regulating intestinal flora and enhancing immunity.

Description

Technical Field

[0001] This invention relates to the field of functional food processing technology, specifically a method for preparing a gut-targeted, highly absorbable Ganoderma lucidum compound fermented beverage. Background Technology

[0002] Ganoderma lucidum fermentation products (including mycelium and fermentation broth) are rich in bioactive substances such as polysaccharides and triterpenoids, which have a wide range of health benefits. However, these active ingredients, especially hydrophobic triterpenoids, are easily destroyed by gastric acid after oral administration, and have low absorption efficiency and unstable bioavailability in the intestine. To improve this situation, existing technologies mainly use physical encapsulation (such as microcapsules) or simply mix the active ingredients with prebiotics. The former is mostly suitable for solid dosage forms, but is prone to leakage and sedimentation in liquid beverages. The latter, due to the weak binding force between components, is easy to separate during digestion and cannot achieve simultaneous delivery and synergistic effect.

[0003] Therefore, developing a Ganoderma lucidum product preparation technology that can be used in liquid systems to achieve precise intestinal targeting and efficient absorption of active ingredients has significant industrial value. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide an innovative and effective method for preparing a highly absorbable Ganoderma lucidum compound fermented beverage that targets the intestines, as well as the product obtained by this method.

[0005] To achieve the above objectives, the present invention provides the following technical solution: A method for preparing an intestinal-targeted, highly absorbable Ganoderma lucidum compound fermented beverage includes the following steps: S1. Raw material processing: Take the liquid deep fermentation broth of Ganoderma lucidum mycelium, and after solid-liquid separation, obtain supernatant A rich in triterpenoids and mycelium; extract and purify Ganoderma lucidum polysaccharides from the mycelium.

[0006] S2. Enzymatic complexation: The Ganoderma lucidum polysaccharide and prebiotic oligosaccharide are subjected to a glycosylation reaction catalyzed by transglycosidase. The amount of transglycosidase added is 6-20 enzyme activity units (U) per gram of Ganoderma lucidum polysaccharide. After the reaction, the mixture is purified (e.g., after inactivating the enzyme in the reaction solution, dialysis or ultrafiltration is performed to remove small molecules, and then the mixture is dried) to obtain a polysaccharide-prebiotic complex. The complex is then mixed with the supernatant A to load triterpenoid compounds, resulting in a loaded complex system B.

[0007] S3. In-situ nanoassembly: Take a portion of the supernatant A and mix it with tea polyphenols. Stir at 20-40°C for 0.5-2 hours to form a structure with a triterpenoid-tea polyphenol complex as the core through intermolecular interactions. Under stirring or shearing (3000-9000 rpm), chitosan solution and citrus pectin solution are added sequentially. Through electrostatic layer-by-layer self-assembly, a pH-responsive coating is formed on the outside of the composite core to obtain nanoparticles A.

[0008] S4. Product preparation: Mix the loaded composite system B, the nanoparticles A, the prebiotics and excipients in an aqueous medium (using conventional stirring, such as 100-500 rpm, or low-speed homogenization), then pasteurize (72-85°C, 15-30 seconds) or ultra-high temperature instantaneous sterilize (135-140°C, 4-8 seconds), and finally aseptically fill to obtain the Ganoderma lucidum compound fermented beverage.

[0009] The enzymatic complexation step is not a simple physical mixing of Ganoderma lucidum polysaccharides and prebiotics, but rather a complex with a more compact and stable structure formed by transglycosidase catalysis. Its loading capacity for triterpenoid components and its targeting of probiotics in the intestine are significantly better than those of physical mixtures.

[0010] The excipients mentioned herein are food science acceptable excipients, including but not limited to: sweeteners (such as sucralose, mogroside), acidulants (such as citric acid), stabilizers (such as xanthan gum), preservatives (such as potassium sorbate), etc., and their usage shall comply with the provisions of the National Food Safety Standard for the Use of Food Additives (GB 2760-2014).

[0011] Furthermore, the liquid deep fermentation broth of Ganoderma lucidum mycelium can be prepared using techniques known in the art; specifically, the following process can be referenced: Ganoderma lucidum inoculum is inoculated into a liquid fermentation medium (such as a medium containing glucose, soybean meal powder, yeast extract, and inorganic salts), and deep fermentation is carried out under suitable conditions (such as temperature 26-28°C, stirring speed 150-250 rpm, and aeration rate 0.8-1.2 vvm) for 5-7 days. The fermentation broth contains mycelium and fermentation supernatant.

[0012] In one alternative: in step S2, the prebiotic oligosaccharide is selected from one or two of galactooligosaccharides and fructooligosaccharides; the transglycosidase is β-galactosidase or fructosyltransferase; the mass ratio of Ganoderma lucidum polysaccharide to prebiotic oligosaccharide is 1:(0.5-2), the reaction temperature is 35-55°C, the reaction pH is 5.5-7.5, and the reaction time is 4-10 hours.

[0013] In one alternative: in step S3, the tea polyphenols are selected from epigallocatechin gallate; the degree of deacetylation of the chitosan is ≥80%, and the degree of esterification of the citrus pectin is ≥60%; the mass ratio of the triterpenoids, tea polyphenols, chitosan, and citrus pectin is 1:(0.2-2.5):(0.5-4):(1-7).

[0014] Preferably, the nanoparticles A obtained in step S3 have an average particle size of 50-300 nm, and their structure is stable in an acidic environment with pH ≤ 4.0, while the coating dissociates in a weakly alkaline environment with pH ≥ 6.5.

[0015] It needs to be explained that the stomach environment is highly acidic (typically pH 1.5-3.5). The composite coating formed by chitosan (positively charged) and citrus pectin (negatively charged) through electrostatic interaction, under acidic conditions, causes the amino groups of chitosan to be protonated (-NH3+), which then reacts with the carboxyl anions (-COO-) of the pectin. - The binding is tighter, and the esterification groups of pectin are stable under acidic conditions, which together maintain the dense structure of the coating, thereby effectively protecting the core active ingredients; When the nanoparticles reach the weakly alkaline environment (pH rises to approximately 6.5-7.5) of the terminal small intestine to the colon, the amino groups of chitosan are deprotonated (-NH2), which weakens or even eliminates their positive charge. This significantly reduces the electrostatic attraction between the chitosan and citrus pectin. At the same time, the hydrophilicity of the pectin molecular chains increases under near-neutral conditions, and they may undergo slight hydrolysis. The synergistic effect of these two actions leads to the relaxation and eventual dissociation of the coating structure, enabling the targeted release of the active ingredients.

[0016] The present invention also provides an intestinal-targeted, highly absorbable Ganoderma lucidum compound fermented beverage prepared by the above method.

[0017] Preferably, the fermented beverage is a homogeneous and stable liquid system containing nanoparticles with an average particle size of 50-300 nm and a pH-responsive coating, as well as a complex functional component containing Ganoderma lucidum polysaccharides, prebiotic oligosaccharides and Ganoderma lucidum triterpenes.

[0018] The present invention also provides the application of the intestinal-targeted, highly absorbable Ganoderma lucidum compound fermented beverage in the preparation of functional foods or health foods for regulating intestinal flora and / or enhancing the body's immunity.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. An active target-intestinal probiotic functional unit was constructed through enzymatic compounding, and an intelligent protective unit that responds to the colonic environment was constructed through in-situ nano-assembly. The two work together to achieve efficient delivery of active ingredients from precise release to targeted capture and absorption, resulting in a significant improvement in absorption. 2. This invention combines enzymatic modification with food-grade polyelectrolyte self-assembly technology to directly construct a stable nanodelivery system in a liquid system, perfectly solving the problems of stability and targeting of active ingredients in liquid beverages; 3. The product has excellent colon targeting and high bioavailability. At the same time, the product has stable physicochemical properties during its shelf life and has good commercialization potential. Attached Figure Description

[0020] Figure 1 The flowchart illustrates a method for preparing an intestinal-targeted, highly absorbable Ganoderma lucidum compound fermented beverage provided by this invention. Detailed Implementation

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs; the terminology used herein in the specification of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application; the terms "comprising" and "having," and any variations thereof, in the specification, claims, and foregoing drawings of this application are intended to cover non-exclusive inclusion. The terms "first," "second," etc., in the specification, claims, or foregoing drawings of this application are used to distinguish different objects, not to describe a particular order.

[0022] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0023] Raw materials and preparation instructions: Example 1 Please see Figure 1 In this embodiment of the invention, a method for preparing an intestinal-targeted, highly absorbable Ganoderma lucidum compound fermented beverage includes the following steps: S1. Raw material processing: Prepare Ganoderma lucidum mycelium liquid deep fermentation broth according to existing technology. Take 10L of the fermentation broth and centrifuge at 8000×g for 15 minutes at 4°C to separate the supernatant A and wet mycelium. Extract the wet mycelium twice with hot water at 90°C. Combine the extracts, concentrate them, and add 4 times the volume of 95% ethanol for alcohol precipitation. Redissolve the precipitate in water and remove the protein using the Sevage method (chloroform: n-butanol = 4:1) to obtain a crude Ganoderma lucidum polysaccharide solution. Further purify and concentrate the solution using an ultrafiltration membrane with a molecular weight cutoff of 50kDa to obtain a Ganoderma lucidum polysaccharide solution (solid content of about 5%).

[0024] S2. Enzymatic complex treatment: Take 100g of the above-mentioned Ganoderma lucidum polysaccharide (dry weight) and 150g of oligogalactose, and dissolve them together in 1L of 0.1M phosphate buffer at pH 6.5; add 1000U of β-galactosidase (i.e., 10U / g Ganoderma lucidum polysaccharide) to the solution and react in a 45°C constant temperature water bath shaker for 6 hours; after the reaction is completed, heat the reaction solution in an 85°C water bath for 10 minutes to terminate the enzyme reaction; transfer the reaction solution to a dialysis bag with a molecular weight cutoff of 3500Da and dialyze it in flowing deionized water for 48 hours to remove unreacted sugars and small molecules; freeze-dry the dialyzed solution to obtain a white flocculent solid, i.e., the polysaccharide-prebiotic complex; weigh 10g of this complex and add it to 1L of supernatant A, and stir at 25°C and 200rpm for 2 hours to load it with triterpenoids, obtaining the loaded complex system B.

[0025] S3. In-situ Nanoassembly: 1 L of supernatant A (containing approximately 2.0 g of triterpenoids) was measured and its pH was adjusted to 5.5 with 1 M citric acid solution; 2.0 g of epigallocatechin gallate was added and stirred at room temperature (25°C) in the dark for 1 hour; under high-speed homogenization at 8000 rpm, 200 mL of 1% (v / v) acetic acid solution containing 4.0 g of chitosan (degree of deacetylation ≥90%) was slowly added dropwise using a constant flow pump, and homogenization was continued for 15 minutes after the addition was completed; subsequently, under the same homogenization conditions, 400 mL of aqueous solution containing 8.0 g of highly esterified citrus pectin (degree of esterification 70%) was slowly added dropwise, and homogenization was continued for 10 minutes; finally, the pH of the entire system was slowly adjusted to 7.0 with 1 M sodium bicarbonate solution to obtain nanoparticle dispersion A; the Z-average particle size was (128±5) nm and the polydispersity index (PDI) was 0.19 as determined by dynamic light scattering particle size analyzer.

[0026] S4. Product Preparation: Combine the entire loaded composite system B with the nanoparticle A dispersion, add 200g of fructooligosaccharides, 0.5g of sucralose, and 50g of mogroside as flavor modifiers, and add purified water to a final volume of 10L. Stir with a paddle mixer at 300rpm for 40 minutes to ensure thorough mixing. Use an ultra-high temperature instantaneous sterilization system to treat at 135°C for 5 seconds, and then aseptically fill into sterilized PET bottles to obtain the final product.

[0027] Example 2 The difference from Example 1 lies in the raw materials and parameters in steps S2 and S3: S2. Replace the prebiotic oligosaccharides with 150g of fructooligosaccharides, replace the enzyme with 2000U of fructosyltransferase (i.e., 20U / g Ganoderma lucidum polysaccharide), adjust the pH of the reaction buffer to 7.0, set the reaction temperature to 50°C, and extend the reaction time to 8 hours; the purification method is the same as in Example 1.

[0028] S3. Adjust the feeding mass ratio (based on triterpenoids) to: triterpenoids: epigallocatechin gallate: chitosan: citrus pectin = 1:0.5:3:4.5. Take 1L of supernatant A (containing about 2.0g of triterpenoids), add 1.0g of epigallocatechin gallate, 6.0g of chitosan, and 9.0g of citrus pectin. The remaining operations are the same as in Example 1. The average particle size of the obtained nanoparticles A is (175±8)nm, and the PDI is 0.23.

[0029] Example 3 The difference from Example 1 lies in the characteristics of the raw materials in step S3: S3. Replace the citrus pectin with a variety with an esterification degree of approximately 65%; take 1L of supernatant A (containing approximately 2.0g of triterpenoids), and add 2.0g of epigallocatechin gallate, 4.0g of chitosan, and 4.0g of the above-mentioned citrus pectin according to the feeding mass ratio (based on triterpenoids) of 1:1:2:2; after self-assembly, do not perform the final pH adjustment step, so that the final pH of the system is maintained at approximately 5.0, and the remaining steps and parameters are the same as in Example 1; the obtained nanoparticle A dispersion has an average particle size of (118±6) nm under pH 5.0 conditions; after incubating it in simulated gastric juice (pH 3.0) for 2 hours, the particle size only increases to (127±7) nm, indicating that its structure is stable in an acidic environment.

[0030] Example 4 The difference between this embodiment and Embodiment 1 is that the key feeding ratios in steps S2 and S3 are adjusted to near the lower limit of the range: S2. Adjust the mass ratio of Ganoderma lucidum polysaccharide to galactooligosaccharide to 1:0.5 (i.e., 100g of polysaccharide corresponds to 50g of galactooligosaccharide), reduce the amount of β-galactosidase to 600U, and shorten the reaction time to 4 hours. The remaining conditions are the same as in Example 1.

[0031] S3. Adjust the feeding mass ratio (based on triterpenoids) to: triterpenoids: epigallocatechin gallate: chitosan: citrus pectin = 1:0.3:1:2. Take 1L of supernatant A (containing about 2.0g of triterpenoids), add 0.6g of epigallocatechin gallate, 2.0g of chitosan, and 4.0g of citrus pectin. The remaining operations are the same as in Example 1. The average particle size of the obtained nanoparticles A is (102±12)nm, and the PDI is 0.26.

[0032] Example 5 The difference between this embodiment and Embodiment 1 is that the reaction raw materials in step S2 and the tea polyphenols in step S3 have been adjusted: S2. Replace the prebiotic with 100g of isomaltooligosaccharide and react with β-glucosidase (1000U) at 55°C and pH 5.5 for 8 hours. The mass ratio of Ganoderma lucidum polysaccharide to oligosaccharide is 1:1.

[0033] S3. Replace tea polyphenols with theaflavins. Take 1L of supernatant A (containing about 2.0g of triterpenoids) and add 1.5g of theaflavins, 5.0g of chitosan, and 6.0g of citrus pectin according to the feeding ratio (based on triterpenoids) 1:0.75:2.5:3. The remaining operations are the same as in Example 1. The average particle size of the obtained nanoparticles A is (155±10)nm and the PDI is 0.24.

[0034] Example 6 The difference between this embodiment and Embodiment 1 is that the key feeding ratios in steps S2 and S3 are adjusted to near the upper limit of the range: S2. Adjust the mass ratio of Ganoderma lucidum polysaccharide to galactooligosaccharide to 1:2 (i.e., 100g of polysaccharide corresponds to 200g of galactooligosaccharide), increase the amount of β-galactosidase to 1500U, extend the reaction time to 10 hours, and keep the other conditions the same as in Example 1.

[0035] S3. Adjust the feeding mass ratio (based on triterpenoids) to: triterpenoids: epigallocatechin gallate: chitosan: citrus pectin = 1:2.5:4:7. Take 1L of supernatant A (containing about 2.0g of triterpenoids), add 5.0g of epigallocatechin gallate, 8.0g of chitosan, and 14.0g of citrus pectin. The remaining operations are the same as in Example 1. The average particle size of the obtained nanoparticles A is (235±18)nm, and the PDI is 0.29.

[0036] Comparative Example 1 (Blank Control Group: Basic Physics Mixture) Take an equal amount of Ganoderma lucidum fermentation supernatant A (1L), untreated Ganoderma lucidum polysaccharide dry powder (10g), and galactooligosaccharide (15g) as in Example 1, and mix them directly in a mixing tank; add an equal amount of excipients (200g of fructooligosaccharide, etc.) as in Example 1, add water to make up to 10L, and homogenize, sterilize, and fill under the same conditions as in Example 1.

[0037] Comparative Example 2 (Enzyme-catalyzed complex treatment only) Perform only step S2 of Example 1 to obtain the loaded composite system B; do not perform in-situ nano-assembly of step S3; directly mix the obtained loaded composite system B with excipients (200g of fructooligosaccharide, etc.), add water to make up to 10L, and sterilize and fill.

[0038] Comparative Example 3 (In-situ Nanoassembly Only) Without performing step S2 of Example 1 (i.e., without enzymatic treatment), only step S3 is performed: Take 1L of supernatant A (containing about 2.0g of triterpenoids), add 2.0g of epigallocatechin gallate, 4.0g of chitosan, and 8.0g of pectin according to the mass ratio of the feed (based on the triterpenoids) 1:1:2:4, and perform the remaining operations as in step S3 of Example 1 to prepare nanoparticle A dispersion; mix nanoparticle A dispersion with an equal amount of untreated Ganoderma lucidum polysaccharide dry powder (10g) and galactooligosaccharide (15g) physical mixture, as well as excipients, add water to make up to 10L, sterilize and fill.

[0039] Comparative Example 4 (using only physically mixed nanoparticles, without enzymatic compounding) Preparation method: Raw material processing: Same as step S1 in Example 1, to obtain supernatant A and Ganoderma lucidum polysaccharide solution.

[0040] Physical mixing preparation of the loading system: Weigh 10g of Ganoderma lucidum polysaccharide dry powder (equal to that in Example 1) and 150g of galactooligosaccharide, dissolve them directly in 1L of supernatant A, and stir for 2 hours at 25°C and 200rpm to obtain the physical mixing loading system.

[0041] Nanoparticle assembly: Nanoparticle A dispersion was prepared exactly according to step S3 of Example 1.

[0042] Product preparation: Combine the physical mixing and loading system of step 2 with the nanoparticle A dispersion of step 3, add the same amount of excipients as in Example 1, add purified water to the final volume of 10L, stir, sterilize, and fill.

[0043] Comparative Example 5 (using a non-pH-responsive coating) Preparation method: Raw material processing: Same as step S1 in Example 1.

[0044] Enzymatic complexation: Same as step S2 in Example 1, to obtain the loaded complex system B.

[0045] Preparation of non-pH-responsive nanoparticles: Take 1L of supernatant A (containing about 2.0g of triterpenoids), add 2.0g of epigallocatechin gallate, and stir at room temperature in the dark for 1 hour.

[0046] Dissolve 4.0g of gelatin (Bloom value 250-300) in 200mL of 50°C hot water, and dissolve 8.0g of gum arabic in 400mL of 50°C hot water.

[0047] Under high-speed homogenization conditions of 8000 rpm, the gelatin solution was slowly added dropwise to the supernatant A-tea polyphenol mixture, and homogenization was continued for 10 minutes.

[0048] Subsequently, gum arabic solution was slowly added dropwise under the same conditions, and homogenization was continued for 15 minutes.

[0049] The pH of the system was adjusted to 7.0 with 1M sodium bicarbonate solution to obtain a gelatin-gum arabic composite agglomerated nanoparticle dispersion (nanoparticle C).

[0050] Product preparation: Combine the loaded composite system B with the nanoparticle C dispersion, add an equal amount of excipients, add purified water to 10L, stir, sterilize, and fill.

[0051] Experimental Example 1: Evaluation of In Vitro Simulated Digestion and Targeted Release Performance The internationally recognized INFOOGS 2.0 in vitro static simulated digestion model was used to test all examples and comparative products. 50 mL samples were accurately measured and subjected to three stages: simulated gastric digestion (pH 3.0, pepsin, 2 h), simulated small intestinal digestion (pH 7.0, pancreatic enzymes and bile salts, 2 h), and simulated colonic fermentation (pH 7.5, colonic microbial enzyme solution, 4 h). Samples were taken at the end of each stage, centrifuged, and the supernatant was collected. The total triterpenoid content of Ganoderma lucidum released was determined using the vanillin-perchloric acid colorimetric method, and the cumulative release rate was calculated. The results are shown in Table 1. Table 1. Cumulative release rate of total triterpenes from Ganoderma lucidum at different stages of simulated in vitro digestion (%, mean ± standard deviation, n=3) It can be seen from the above table: In Examples 1-6, the cumulative release rate of total triterpenes from Ganoderma lucidum in the gastric and small intestinal phases was less than 36%, while the release rate in the colonic phase was as high as 85%, demonstrating excellent pH-responsive colonic targeted release characteristics.

[0052] Comparative Example 1 (physical mixing) and Comparative Example 5 (non-pH-responsive coating) showed a large release (>50%) in the gastric phase, and the final release rate of Comparative Example 5 in the colonic phase (78.5%) was significantly lower than that of the embodiments of the present invention, indicating that its coating not only lacks gastric acid protection ability, but also cannot achieve effective enrichment and targeted release of active ingredients in the colon.

[0053] Comparative Example 2 (enzyme-catalyzed complex only) still had a gastric release rate of over 50% due to the lack of a physical barrier.

[0054] The colon phase release rates of Comparative Example 3 (nano-assembled only) and Comparative Example 4 (physically mixed nanoparticles) (72.2% and 82.6%, respectively) were significantly lower than those of the embodiments of the present invention, with Comparative Example 3 showing the worst performance. This demonstrates that nanosystems lacking enzyme-catalyzed composite stable loading have insufficient release efficiency of active ingredients at the target site.

[0055] The release curve of Comparative Example 4 (physical mixture + nano) is between that of Comparative Example 2 and Comparative Example 3, further demonstrating that the enzymatic complexation and nano assembly work synergistically and are indispensable.

[0056] Experimental Example 2: Pharmacokinetic Study in Rats Healthy SD rats were selected and randomly divided into groups. They were administered by gavage the products of Examples 1-6, Comparative Examples 1-5, and the original Ganoderma lucidum fermentation supernatant (raw material control group) at the same triterpenoid dose. The AUC of ganoderic acid A in plasma was measured. 0-24 h, relative bioavailability was calculated with the raw material control group as the baseline, and the results are shown in Table 2: Table 2 Comparison of pharmacokinetic parameters of ganoderic acid A in rats (mean ± SD, n=6) It can be seen from the above table: The relative bioavailability of Examples 1-6 of the present invention all exceed 250%, with the highest reaching 320%, indicating that they have extremely high intestinal absorption efficiency.

[0057] The bioavailability of Comparative Example 1 (physical mixing) and Comparative Example 5 (non-pH-responsive coating) was only 123% and 126%, respectively, which was not substantially improved compared with the raw material control group.

[0058] The bioavailability of Comparative Example 2 (enzyme-catalyzed complex only) and Comparative Example 4 (physically mixed nanoparticles) was improved (152%, 220%), but significantly lower than that of the Example.

[0059] The bioavailability of Comparative Example 3 (nano-assembly only) (201%) was higher than that of Comparative Example 2, confirming the targeted delivery value of the nano-coating, but it was still far lower than that of the complete technical solution, indicating that single physical encapsulation cannot achieve efficient absorption of active ingredients.

[0060] In summary, this invention achieves a significant improvement in absorption efficiency through the synergy of "enzyme-catalyzed compounding" and "nano-assembly".

[0061] Experiment Example 3: Product Stability Study The products of Examples 1-6 and Comparative Examples 1-5 were placed in a 37°C constant temperature chamber for accelerated stability testing. Samples were taken on days 0 and 90 to observe the appearance and determine the retention rate of total triterpenoids in Ganoderma lucidum. The results are shown in Table 3.

[0062] Table 3. Product stability data after 90 days of accelerated storage at 37°C. It can be seen from the above table: After being stored at 37°C for 90 days, the products of Examples 1-6 of this invention exhibited uniform appearance and triterpene retention rates of over 92%, demonstrating excellent physicochemical stability.

[0063] Comparative Example 1 (physical mixing) showed severe stratification and the lowest retention rate (78.5%).

[0064] Comparative Example 2 (enzyme-catalyzed complex only) and Comparative Example 5 (non-pH-responsive coating) showed slight opalescence or flocculent matter, with a retention rate of less than 88%.

[0065] Although the stability (95.1%, 89.8%) of Comparative Example 3 (nano-assembly only) and Comparative Example 4 (physically mixed nanoparticles) was better than that of Comparative Examples 2 and 5, neither reached the level of the best embodiment, indicating that the complete synergistic system contributes more comprehensively to the long-term stability of the product.

[0066] Experiment Example 4: Evaluation of the role of gut microbiota regulation Thirty healthy 6-week-old C57BL / 6 mice were randomly divided into a control group (administered by gavage with physiological saline), Example 1 group (administered by gavage with the final product, dose 10 mL / kg), and Comparative Example 1 group (administered by gavage with the physically mixed product, dose 10 mL / kg), with 10 mice in each group. The mice were administered the product by gavage for 28 consecutive days. Fresh feces were collected from the mice, and the gut microbiota composition was analyzed by 16S rRNA high-throughput sequencing.

[0067] The results showed that, compared with the control group, the abundance of Bifidobacterium in the feces of mice in Example 1 group increased by 2.8 times, the abundance of Lactobacillus increased by 3.1 times, and the ratio of Bacteroides / Firmwallis decreased from 1.2 to 0.7; in Comparative Example 1 group, only the abundance of Bifidobacterium increased by 1.1 times, with no statistical difference; indicating that the product of the present invention can significantly regulate the intestinal flora structure through the synergistic effect of "enzyme-catalyzed compounding + nano-assembly", and the effect is better than that of physical mixing products.

[0068] It should be noted that, for the sake of simplicity, the foregoing embodiments are all described as a series of actions. However, those skilled in the art should understand that the present invention is not limited to the described order of actions, as some steps may be performed in other orders or simultaneously according to the present invention. Furthermore, those skilled in the art should also understand that the embodiments described in the specification are preferred embodiments, and the actions and modules involved are not necessarily essential to the present invention.

[0069] 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 invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on these embodiments, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

Claims

1. A method for preparing an intestinal-targeted, highly absorbable Ganoderma lucidum compound fermented beverage, characterized in that, Includes the following steps: S1. Raw material processing: Take the liquid deep fermentation broth of Ganoderma lucidum mycelium, and separate the solid and liquid to obtain the supernatant A rich in triterpenoids, as well as the mycelium, for later use; extract and purify Ganoderma lucidum polysaccharides from the mycelium; S2. Enzymatic complexation: The Ganoderma lucidum polysaccharide and prebiotic oligosaccharide were reacted under the catalysis of transglycosidase. The reaction solution was purified to obtain a polysaccharide-prebiotic complex. The complex was mixed with the supernatant A to load triterpenoids to obtain a loaded complex system B. S3. In-situ nano-assembly: Take a portion of the supernatant A and mix it with tea polyphenols at 20-40°C for 0.5-2 hours to form a composite core; under stirring or shearing conditions, add chitosan solution and citrus pectin solution to it in sequence, and form a pH-responsive coating on the outside of the composite core through electrostatic layer-by-layer self-assembly to obtain nanoparticles A; S4. Product preparation: The loaded composite system B, the nanoparticles A, the prebiotics and excipients are mixed in an aqueous medium, sterilized and filled to obtain the Ganoderma lucidum compound fermented beverage.

2. The preparation method of the intestinal-targeted, highly absorbable Ganoderma lucidum compound fermented beverage according to claim 1, characterized in that, In step S2, the prebiotic oligosaccharide is selected from one or more of galactooligosaccharides, fructooligosaccharides, and isomaltooligosaccharides; the transglycosidase is selected from one of β-galactosidase, fructosyltransferase, and β-glucosidase; the amount of transglycosidase added is 6-20 enzyme activity units (U) per gram of Ganoderma lucidum polysaccharide; the mass ratio of Ganoderma lucidum polysaccharide to prebiotic oligosaccharide is 1:(0.5-2), the reaction temperature is 35-55°C, the reaction pH is 5.5-7.5, and the reaction time is 4-10 hours.

3. The preparation method of the intestinal-targeted, highly absorbable Ganoderma lucidum compound fermented beverage according to claim 1, characterized in that, In step S2, the purification process includes inactivating the enzyme in the reaction solution, performing dialysis or ultrafiltration to remove small molecule substances, and then drying.

4. The preparation method of the intestinal-targeted, highly absorbable Ganoderma lucidum compound fermented beverage according to claim 1, characterized in that, In step S3, the tea polyphenols are selected from one of epigallocatechin gallate and theaflavins; the degree of deacetylation of the chitosan is ≥80%, and the degree of esterification of the citrus pectin is ≥60%.

5. The preparation method of the intestinal-targeted, highly absorbable Ganoderma lucidum compound fermented beverage according to claim 1, characterized in that, In step S3, the mass ratio of the triterpenoids, tea polyphenols, chitosan and citrus pectin is 1:(0.2-2.5):(0.5-4):(1-7).

6. The preparation method of the intestinal-targeted, highly absorbable Ganoderma lucidum compound fermented beverage according to claim 1, characterized in that, In step S3, the average particle size of nanoparticle A is 50-300 nm, and its structure is stable in an environment with pH ≤ 4.0, while the coating dissociates in an environment with pH ≥ 6.

5.

7. The preparation method of the intestinal-targeted, highly absorbable Ganoderma lucidum compound fermented beverage according to claim 1, characterized in that, In step S3, the process of adding chitosan solution and citrus pectin solution is carried out under shear conditions with a rotation speed of 3000-15000 rpm.

8. A gut-targeted, highly absorbable Ganoderma lucidum compound fermented beverage, characterized in that, Prepared by the method described in any one of claims 1-7.

9. The intestinal-targeted, highly absorbable Ganoderma lucidum compound fermented beverage according to claim 8, characterized in that, The fermented beverage is a homogeneous and stable liquid system containing nanoparticles with an average particle size of 50-300 nm and a pH-responsive coating, as well as a complex functional component containing Ganoderma lucidum polysaccharides, prebiotic oligosaccharides and Ganoderma lucidum triterpenes.

10. The use of the intestinal-targeted, highly absorbable Ganoderma lucidum compound fermented beverage as described in any one of claims 8-9 in the preparation of food or health food for regulating intestinal flora and / or enhancing the body's immunity.

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