Ganoderma lucidum and rhizoma polygonati functional component composition as well as preparation method and application thereof

Abstract

The invention discloses a lucid ganoderma and rhizoma polygonati functional component composition as well as a preparation method and application thereof. The composition is prepared from a lucid ganoderma extract and a rhizoma polygonati aqueous extract, the mass ratio of the lucid ganoderma extract to the rhizoma polygonati aqueous extract is (1-3): (1-3). By optimizing the extraction process, the extraction yield of the lucid ganoderma extract is effectively improved, and a stable and efficient raw material basis is provided for subsequent function development. Compared with the single use of the lucid ganoderma extract or the rhizoma polygonati aqueous extract, the composition can effectively regulate the fermentation rate and promote the synthesis of short-chain fatty acids such as butyric acid. The compounding groups with different mass ratios show differentiated advantages in flora regulation. The invention provides a new direction for the development of ganoderma lucidum and rhizoma polygonati products, promotes the development of high value-added processing industry, and can improve the economic value and market competitiveness of ganoderma lucidum and rhizoma polygonati in medicine processing.

Description

Technical Field

[0001] This invention belongs to the field of natural active ingredient extraction technology, specifically relating to a functional component composition of Ganoderma lucidum and Polygonatum sibiricum and its preparation method, as well as its application in improving intestinal microbiota, promoting short-chain fatty acid production and enhancing prebiotic function. Background Technology

[0002] Reishi mushroom, a precious edible and medicinal fungus belonging to the Ganoderma family and the Ganoderma genus, is listed as a substance that is traditionally both a food and a Chinese medicine. Its fruiting body is rich in polysaccharides, triterpenoids, minerals, and proteins, and possesses various health benefits, including lowering blood pressure and blood sugar, boosting immunity, protecting the liver and relieving pain, and delaying aging. Polygonatum, a perennial herb belonging to the Asparagaceae family and the Polygonatum genus, has rhizomes rich in polysaccharides, flavonoids, alkaloids, and steroidal saponins, and is believed to nourish the spleen and lungs, replenish qi and yin. Both are widely used in the development of health foods and functional foods. Reishi mushroom extract and Polygonatum water extract both exhibit strong biological activities, such as antioxidant, immunomodulatory, and gut health-promoting effects.

[0003] Currently, most research on Ganoderma lucidum extract and Polygonatum sibiricum water extract focuses on their individual extraction and bioactivity evaluation. However, single polysaccharides or oligosaccharides may have problems such as low bioavailability and limited ability to regulate gut microbiota. The traditional Chinese medicine theory of monarch-minister-adjuvant-guide formulas emphasizes the synergistic effect of multiple components, providing a theoretical basis for the combination of these two ingredients. However, research on the regulatory effects of their combination on the gut microbiota is still insufficient. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a composition of Ganoderma lucidum and Polygonatum sibiricum functional components, which, under a certain compounding ratio, has a better probiotic function in improving the structure of the intestinal microbiota.

[0005] This invention provides a composition of functional components of Ganoderma lucidum and Polygonatum sibiricum, which is composed of Ganoderma lucidum extract and Polygonatum sibiricum water extract; The mass ratio of the Ganoderma lucidum extract to the Polygonatum sibiricum water extract is 1:3 to 1:3.

[0006] Preferably, the Ganoderma lucidum extract comprises the following components: mannan, glucan, galactan, rhamnan, and fucoidan.

[0007] Preferably, the Polygonatum extract is composed of aqueous extracts with different degrees of polymerization, ranging from DP2 to DP30.

[0008] This invention provides a method for preparing the functional component composition of Ganoderma lucidum and Polygonatum sibiricum, comprising the following steps: Fresh Ganoderma lucidum fruiting bodies are dried, pulverized, and then ultrasonically treated with ethanol. The ethanol is removed from the residue, and the residue is then gelatinized.

[0009] The gelatinized residue was added to an aqueous solution, and amylase and protease were added for enzymatic hydrolysis. After enzyme inactivation, deionized water was added for extraction to obtain secondary residue and extract A. The secondary residue was then extracted with an alkaline solution to obtain alkaline extract B. Extract A and alkaline extract B were mixed, and polysaccharides were collected by alcohol precipitation to obtain Ganoderma lucidum extract. Fresh Polygonatum rhizomes were dried to form Polygonatum powder, which was then extracted with an aqueous solution. The polysaccharides in the extract were separated by alcohol precipitation, and the supernatant was rotary evaporated to obtain the water extract of Polygonatum. Ganoderma lucidum extract and Polygonatum sibiricum water extract were combined to obtain a composition of functional components of Ganoderma lucidum and Polygonatum sibiricum.

[0010] Preferably, during ethanol ultrasonic extraction, the material-to-liquid ratio of Ganoderma lucidum powder to ethanol solution is 1g:20~40mL, the ethanol ultrasonic extraction time is 60~120min, the ethanol ultrasonic extraction temperature is 20~40℃, and the ultrasonic frequency is 30~50 kHz.

[0011] Preferably, the ratio of the gelatinized residue to the aqueous solution is 1g:10-20mL; the mass percentages of amylase and protease in the aqueous solution are 0.5%-5% respectively; the ratio of the enzyme-inactivated water-leached residue to deionized water is 1g:10-20mL; the ratio of the secondary residue to the alkaline solution is 1g:20-40mL; the concentration of the alkaline solution is 0.5-2M; and during the extraction of Polygonatum sibiricum, the ratio of Polygonatum sibiricum powder to the aqueous solution is 1g:10-30mL. The enzymatic hydrolysis temperature of the amylase is 80-100℃, and the enzymatic hydrolysis time is 40-80 min; the enzymatic hydrolysis temperature of the protease is 40-60℃, and the enzymatic hydrolysis time is 100-150 min; the deionized water extraction temperature is 80-100℃, and the deionized water extraction time is 100-150 min; the alkaline solution extraction temperature is 20-30℃, and the alkaline solution extraction time is 40-80 min; the Polygonatum sibiricum aqueous solution extraction temperature is 80-90℃, and the extraction time is 40-80 min.

[0012] Preferably, during alcohol precipitation, the volume percentage of the alcohol solution is 70% to 85%.

[0013] This invention provides the application of the aforementioned Ganoderma lucidum and Polygonatum functional component composition in the preparation of health foods with the function of regulating intestinal health.

[0014] This invention provides a complex of functional components from Ganoderma lucidum and Polygonatum sibiricum, composed of Ganoderma lucidum extract and Polygonatum sibiricum aqueous extract; wherein the mass ratio of the Ganoderma lucidum extract to the Polygonatum sibiricum aqueous extract is 1-3:1-3. Experiments have shown that the complex of Ganoderma lucidum extract and Polygonatum sibiricum aqueous extract provided by this invention, compared with using Ganoderma lucidum extract or Polygonatum sibiricum aqueous extract alone, can effectively regulate the fermentation rate and promote the synthesis of short-chain fatty acids such as butyric acid. Different mass ratios of the complex exhibit differentiated advantages in gut microbiota regulation, and can respectively promote the growth of beneficial bacteria in the intestine, especially *Faecalibacterium*. (Faecalibacterium) With fecal cocci (Coprococcus) It significantly inhibited the proliferation of Escherichia coli and Shigella spp. (Escherichia-Shigella) With Klebsiella spp. (Klebsiella) The abundance of harmful bacteria is also reduced. Furthermore, the composition obtained in this invention can reduce the original bitterness of Ganoderma lucidum while preserving its functional components as much as possible, and can also obtain multiple nutritional values. This research provides a new direction for the development of Ganoderma lucidum and Polygonatum products, promotes the development of high value-added processing industries, and enhances the economic value and market competitiveness of Ganoderma lucidum and Polygonatum in pharmaceutical processing. At the same time, the comprehensive extraction process of this invention can obtain more Ganoderma lucidum extract compared to existing extraction methods, greatly improving process efficiency. Attached Figure Description

[0015] Figure 1 Line graph showing pH changes in the compound of functional components of Ganoderma lucidum and Polygonatum sibiricum during in vitro simulated fermentation; Figure 2 Bar chart showing the changes in short-chain fatty acid content in the compound of functional components of Ganoderma lucidum and Polygonatum sibiricum during in vitro simulated fermentation; Figure 3 Chromatogram of molecular weight changes of compound functional components of Ganoderma lucidum and Polygonatum sibiricum during in vitro simulated fermentation; Figure 4 Bar chart showing the abundance (genus level) of some microbial communities in the in vitro simulated fermentation of the compound of functional components of Ganoderma lucidum and Polygonatum sibiricum. Detailed Implementation

[0016] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0017] This invention provides a composition of functional components of Ganoderma lucidum and Polygonatum sibiricum, which is composed of Ganoderma lucidum extract and Polygonatum sibiricum water extract; The mass ratio of the Ganoderma lucidum extract to the Polygonatum sibiricum water extract is 1-3:1-3. More preferably, it is 3:1.

[0018] Preferably, the Ganoderma lucidum extract comprises the following components in molar percentage: mannan, glucan, galactan, rhamnan, and fucoidan.

[0019] Preferably, the Polygonatum extract is composed of aqueous extracts with different degrees of polymerization, ranging from DP2 to DP30.

[0020] This invention provides a method for preparing the Ganoderma lucidum extract, comprising the following steps: Fresh Ganoderma lucidum fruiting bodies are dried, ground into powder, and then sonicated with an ethanol-water solution. The residue is gelatinized after removing the ethanol, and then enzymatically hydrolyzed with a solution containing amylase and protease. It is then extracted with deionized water to obtain a secondary residue and extract A. The secondary residue is then extracted with an alkaline solution to obtain an alkaline extract B. A and B are mixed, and polysaccharides are collected by alcohol precipitation to obtain Ganoderma lucidum extract.

[0021] In this invention, the ratio of Ganoderma lucidum powder to ethanol solution during ultrasonication is 1g:20-40 mL, more preferably 1g:30 mL. The ultrasonication time is preferably 60-120 min. The method for removing ethanol is preferably to air-dry the residue in a fume hood.

[0022] In this invention, during extraction, the material-to-liquid ratio of the residue after ethanol removal to the solution containing amylase and protease is preferably 1:10 to 1:20 (g / mL), more preferably 1:15 (g / mL). The final enzyme mass percentage in the solution containing amylase and protease is preferably 0.5% to 5%, more preferably 1% to 4%, and most preferably 2%. The enzymatic hydrolysis temperature of the amylase is preferably 80 to 100°C, more preferably 90°C. The enzymatic hydrolysis time of the amylase is preferably 40 to 80 min, more preferably 50 to 70 min, and most preferably 60 min. The enzymatic hydrolysis temperature of the protease is preferably 40 to 60°C, more preferably 50°C. The enzymatic hydrolysis time of the protease is preferably 100 to 150 min, more preferably 110 to 130 min, and most preferably 120 min.

[0023] In this invention, after enzymatic hydrolysis, the preferred material-to-liquid ratio of deionized water is 1:10 to 1:20 (g / mL), more preferably 1:15 (g / mL). The preferred extraction temperature is 80 to 100°C, more preferably 90°C. The preferred extraction time is 100 to 150 min, more preferably 110 to 130 min, and most preferably 120 min. The preferred material-to-liquid ratio for alkaline extraction is 1:20 to 1:40 (g / mL), more preferably 1:30 (g / mL). The concentration of the alkaline solution is 0.5 to 2 M, more preferably 1 M; the preferred extraction temperature is 20 to 30°C, more preferably 25°C. The preferred extraction time is 40 to 80 min, more preferably 50 to 70 min, and most preferably 60 min.

[0024] In this invention, after mixing extracts A and B, the mixture is preferably concentrated before alcohol precipitation. The concentration method is preferably rotary evaporation. The rotary evaporation temperature is preferably 45–60°C, more preferably 50–55°C, and most preferably 55°C. The rotary evaporation is preferably stopped when the volume is reduced to 1 / 4–1 / 3 of the original volume. During alcohol precipitation, the volume percentage of the alcohol-water solution is preferably 70%–80%. The alcohol is preferably ethanol. The alcohol-water solution is three times the volume of the concentrated extract. The alcohol precipitation time is preferably 10–15 hours, more preferably 12 hours. After alcohol precipitation, it is also preferable to remove small molecule polysaccharides and remove water. The method for removing small molecules is preferably to reconstitute the precipitate obtained from alcohol precipitation with water and then dialyze it. The dialysis bag used for dialysis is preferably a 5–10 kDa dialysis bag, and the method for removing water is preferably vacuum freeze-drying. The vacuum drying temperature is preferably -40 to -75°C.

[0025] In this invention, the molecular weight of the Ganoderma lucidum extract was determined to be 1000-2000 kDa, and it was composed of mannan, glucan, galactan, rhamnan, and fucoidan.

[0026] In this invention, the method for preparing the aqueous extract of Polygonatum odoratum preferably includes the following steps: The dried rhizome powder of Polygonatum odoratum was extracted with hot water, the extract was separated, concentrated and the oligosaccharides were collected by alcohol precipitation, and the supernatant was the water extract of Polygonatum odoratum.

[0027] In this invention, the liquid-to-solid ratio of the Polygonatum powder and hot water is preferably 1:10-30, more preferably 1:20. The extraction time is preferably 40-80 min, more preferably 50-70 min, and most preferably 60 min. The extraction temperature is preferably 80-90℃, more preferably 85℃. The alcohol is preferably ethanol. The alcohol-water solution is three times the volume of the concentrated extract. The alcohol precipitation time is preferably 10-15 h, more preferably 12 h. The alcohol precipitation process preferably includes water removal. The water removal method is preferably rotary evaporation and freeze drying.

[0028] Preferably, the Polygonatum extract is composed of aqueous extracts with different degrees of polymerization, ranging from DP2 to DP30.

[0029] This invention provides the application of the Ganoderma lucidum and Polygonatum functional component complex or the Ganoderma lucidum extract and Polygonatum aqueous extract complex obtained by the preparation method in the preparation of health foods with intestinal health regulation functions such as improving the structure of gut microbiota.

[0030] In this invention, an in vitro simulated fermentation model was used to compare the differences in the effects of a combination of Ganoderma lucidum and Polygonatum functional components on regulating intestinal health compared to individual components. The results showed that the functional component combination can effectively regulate the fermentation rate. The functional component combination can improve the intestinal environment by promoting the production of short-chain fatty acids. The functional component combination can effectively regulate the gut microbiota structure and intestinal health. The Ganoderma lucidum and Polygonatum functional component combination can effectively regulate short-chain fatty acid production and gut microbiota structure, with better effects than individual components. In particular, this functional component combination can significantly promote the growth of beneficial bacteria, specifically *Faecalibacterium*. (Faecalibacterium) and fecal cocci (Coprococcus) It can enrich and effectively inhibit Escherichia coli-Shigella spp. (Escherichia-Shigella) It can inhibit the growth of harmful bacteria, and the compound has a proportion-dependent microbial community regulation characteristic, which can target and promote different functional types of beneficial bacteria while inhibiting harmful bacteria, providing theoretical support for the development of diversified and targeted microecological intervention products.

[0031] The following detailed description, in conjunction with embodiments, illustrates a functional composition of Ganoderma lucidum and Polygonatum sibiricum provided by the present invention, its preparation method, and its application. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0032] Comparative Example 1 A traditional water decoction method for extracting Ganoderma lucidum extract involves the following steps: (1) After washing the fruiting bodies of Ganoderma lucidum (using the same batch of raw materials as in the following examples), place them in a 40°C drying oven and dry for 36 h. Then, crush them with a pulverizer and pass them through a 40-mesh sieve. (2) The obtained Ganoderma lucidum powder was added to deionized water at a ratio of 1:30 (g / mL) and heated and decocted in a water bath at 100℃ for 2 hours. After the decoction was completed, the mixture was cooled and filtered, and the supernatant was collected. (3) Slowly add 3 times the volume of ethanol to the obtained filtrate, mix well, let stand at 4℃ for 12 h for alcohol precipitation, centrifuge at 8000 r / min for 10 min, collect the precipitate, and redissolve it in distilled water. (4) The heavy solution was placed in a 7 kDa dialysis bag for dialysis. After dialysis, the solution was vacuum dried at -75℃ to obtain Ganoderma lucidum extract with a yield of 1.86%.

[0033] Example 1 A combination of functional components from Ganoderma lucidum and Polygonatum sibiricum and its comprehensive extraction process comprises the following steps: (1) After washing the fruiting body of Ganoderma lucidum, place it in a 40℃ drying oven and dry for 36 h. Then, crush it with a pulverizer and pass it through a 40-mesh sieve. (2) The obtained powder was added to ethanol solution at a ratio of 1:30 (g / mL) and ultrasonically treated for 90 min (25℃, 40kHz). Then the ethanol was removed and the powder was air-dried in a fume hood until there was no ethanol smell. PBS was added at a ratio of 1:15 (g / mL) and gelatinized at 100℃ for 10 min.

[0034] (3) After gelatinization, the residue was added to deionized water at a liquid-to-solid ratio of 1:15 (g / mL). α-Thermoresistant amylase was added and hydrolyzed in a 90℃ constant-temperature shaker for 60 min (amylase dosage: 1%). Papain was added and hydrolyzed in a 50℃ constant-temperature shaker for 120 min (papain dosage: 2%). The enzyme was inactivated by a 100℃ water bath for 10 min. The pH of the solution was adjusted to 3, and the solution was placed in a 4℃ refrigerator for 30 min. Subsequently, deionized water was added at a liquid-to-solid ratio of 1:15 (g / mL), and the solution was extracted at 90℃ for 2 h. The solution was then centrifuged (8000 r / min, 10 min, 4℃) to obtain the residue and extract A. The residue was further extracted with 1M alkaline solution (containing 20 mmol NaBH4) at a liquid-to-solid ratio of 1:30 (g / mL) at 25℃ for 1 h, and then centrifuged (8000 r / min, 10 min, 4℃) to obtain the residue and extract B. Mix ingredients A and B, evaporate to one-quarter of their original volume at 55°C, then slowly add three times the volume of ethanol while stirring. Place in a refrigerator at 4°C for 12 hours, then centrifuge at 8000 rpm for 10 minutes. Collect the precipitate and redissolve it in distilled water. Dialyze using a 7 kDa dialysis bag. After dialysis, vacuum dry the solution at -75°C to obtain the Ganoderma lucidum extract, with a yield of 9.50%.

[0035] To verify the effectiveness of the extraction process of this invention, the same batch of dried Ganoderma lucidum powder was used, and parallel extraction was performed using both the process of this invention and the traditional hot water decoction method. The polysaccharide yield of the two methods was compared and analyzed. The results are shown in Table 1: Table 1 As shown in Table 1, the polysaccharide yield per unit of raw material in this invention is increased by more than 410%, which is significantly better than existing conventional extraction methods, demonstrating high raw material utilization efficiency and industrial adaptability.

[0036] (4) The molecular weight of the polysaccharide was determined using the SEC-MALLS-RI system. A 0.15 mol / L NaCl solution (containing 0.02% NaN3) was prepared using ultrapure water as the mobile phase, filtered through a 0.22 μm water film, and degassed by sonication for 20 min. The polysaccharide sample was dissolved in distilled water to prepare a solution with a concentration of 5 mg / mL, filtered through a 0.22 μm water film, and prepared for detection. Detection conditions: flow rate 0.5 mL / min, column temperature 40℃, injection volume 50 μL, data collection for 55 min, and refractive index increment dn / dc of 0.138 mL / g. The molecular weight of the polysaccharide was determined to be 1100 kDa.

[0037] The monosaccharide composition of the Ganoderma lucidum extract was determined using a Waters ACQUITY UPLC H-Class system and a Waters Xevo TQD triple quadrupole mass spectrometer. The sample was weighed, dissolved in deionized water, and after adding trifluoroacetic acid and sealing, hydrolyzed at a specific temperature. After cooling, residual reagents were removed by methanol-nitrogen blowing. PMP derivatization was then performed. After the reaction, appropriate amounts of acetonitrile and dichloromethane were added to extract impurities, and the supernatant was collected by centrifugation for analysis. Separation was performed using an Agilent ZORBAX Eclipse Plus C18 column (2.1 mm × 100 mm, 1.8 μm). Mobile phase A was a weakly basic acetonitrile-buffered saline system, and mobile phase B was a high-proportion acetonitrile system, using a gradient elution program. Column temperature and flow rate were optimized. Mass spectrometry analysis was performed in ESI positive ion mode, and key parameters such as voltage, gas flow rate, and collision energy were configured according to the system's recommended standards.

[0038] The extract of Ganoderma lucidum was found to be composed of mannan, glucomannan, galactan, rhamnan, and fucoidan.

[0039] (5) After washing the Polygonatum, place it in a 60℃ drying oven for 24 hours, then crush it with a pulverizer and pass it through a 60-mesh sieve. (6) The obtained powder was added to deionized water at a material-to-liquid ratio of 1:20 (g / mL), and extracted in a constant temperature shaker at 85℃ for 60 min. The mixture was then centrifuged (8000 r / min, 10 min, 4℃) to obtain residue and extract. Four times the volume of ethanol was slowly added to the extract with stirring. After being placed in a refrigerator at 4℃ for 12 h, the mixture was centrifuged at 8000 r / min for 10 min. The supernatant was evaporated at 55℃ until no ethanol odor remained. The solution was then vacuum dried at -75℃ to obtain the water extract of Polygonatum sibiricum, with a yield of 39%.

[0040] (7) HILIC-MS / MS analysis of the structure of the aqueous extract of Polygonatum odoratum: A suitable amount of low molecular weight carbohydrate solution from the rhizome of Polygonatum odoratum was taken, and an equal volume of acetonitrile was added. The solution was filtered through a 0.22 μm organic filter membrane. Chicory oligofructose (sigma-FOS) was used as a standard for reference. Online separation was performed using a Waters HILICAmide column (1.7 μm, 1.2 × 150 mm), and analysis was performed using a SCiex Triple-TOF 5600+ time-of-flight mass spectrometer. The liquid chromatography conditions were as follows: mobile phase (A) was 20% acetonitrile containing 10 mmol / L ammonium formate and 0.2% formic acid; mobile phase (B) was 80% acetonitrile containing 10 mmol / L ammonium formate and 0.2% formic acid; flow rate was 0.25 mL / min; column temperature was 35℃; time gradient: 0–35 min, 100%–70% B; 35–50 min, 70%–20% B; 50–55 min, 20% B; 55–56 min, 20%–100% B; 56–60 min, 100% B. The injection volume was 2 μL. The mass spectrometry conditions were as follows: scanning mode was negative ion mode; nebulizer gas was 55 psi; curtain gas was 35 psi; ion source temperature was 550°C; ion source voltage was -4500 V; declustering voltage in the first scan was 100 V; focusing voltage was 10 V; acquisition mode in the second scan was IDA mode; CID energy was 40±20 eV.

[0041] The extract of Polygonatum odoratum was found to be composed of water extracts with different degrees of polymerization, ranging from DP2 to DP30.

[0042] (8) The obtained Ganoderma lucidum extract and Polygonatum sibiricum water extract are compounded in a mass ratio of 3:1 to obtain a combination of Ganoderma lucidum and Polygonatum sibiricum functional components.

[0043] Example 2 A combination of functional components from Ganoderma lucidum and Polygonatum sibiricum and its comprehensive extraction process comprises the following steps: (1) After washing the fruiting body of Ganoderma lucidum, place it in a 55℃ drying oven for 24 hours, then crush it with a pulverizer and pass it through a 60-mesh sieve. (2) The obtained powder was added to ethanol solution at a ratio of 1:40 (g / mL) and ultrasonically treated for 120 min (25℃, 30kHz). Then the ethanol was removed and the powder was air-dried in a fume hood until there was no ethanol smell. PBS was added at a ratio of 1:20 (g / mL) and gelatinized at 100℃ for 10 min.

[0044] (3) After gelatinization, the residue was added to deionized water at a liquid-to-solid ratio of 1:20 (g / mL), and α-thermoresistant amylase was added and hydrolyzed in an 80℃ constant temperature shaker for 40 min (amylase dosage was 2%). Papain was added and hydrolyzed in an 80℃ constant temperature shaker for 130 min (protein dosage was 4%). The enzyme was inactivated by a 100℃ water bath for 5 min, the pH of the solution was adjusted to 3.5, and the solution was placed in a 4℃ refrigerator for 60 min. Then, deionized water was added at a liquid-to-solid ratio of 1:20 (g / mL), and the solution was extracted at 80℃ for 150 min. The solution was then centrifuged (8000 r / min, 10 min, 4℃) to obtain the residue and extract A. The residue was then further extracted with 0.5 M alkaline solution (containing 20 mmol NaBH4) at a liquid-to-solid ratio of 1:40 (g / mL), and the solution was extracted at 30℃ for 80 min. The solution was then centrifuged (8000 r / min, 10 min, 4℃) to obtain the residue and extract B. Mix ingredients A and B, evaporate to one-third of their original volume at 55°C, then slowly add four times the volume of ethanol while stirring. Place in a refrigerator at 4°C for 15 h, then centrifuge at 8000 r / min for 10 min, collect the precipitate, and redissolve it in distilled water. Dialyze using a 10 kDa dialysis bag. After dialysis, vacuum dry the solution at -40°C to obtain the Ganoderma lucidum extract with a yield of 9.27%.

[0045] (4) The molecular weight of the polysaccharide was determined using the SEC-MALLS-RI system. A 0.15 mol / L NaCl solution (containing 0.02% NaN3) was prepared using ultrapure water as the mobile phase, filtered through a 0.22 μm water film, and degassed by sonication for 20 min. The polysaccharide sample was dissolved in distilled water to prepare a solution with a concentration of 5 mg / mL, filtered through a 0.22 μm water film, and prepared for detection. Detection conditions: flow rate 0.5 mL / min, column temperature 40℃, injection volume 50 μL, data collection for 55 min, and refractive index increment dn / dc of 0.138 mL / g. The molecular weight of the polysaccharide was determined to be 1100 kDa.

[0046] The monosaccharide composition of the Ganoderma lucidum extract was determined using a Waters ACQUITY UPLC H-Class system and a Waters Xevo TQD triple quadrupole mass spectrometer. The sample was weighed, dissolved in deionized water, and after adding trifluoroacetic acid and sealing, hydrolyzed at a specific temperature. After cooling, residual reagents were removed by methanol-nitrogen blowing. PMP derivatization was then performed. After the reaction, appropriate amounts of acetonitrile and dichloromethane were added to extract impurities, and the supernatant was collected by centrifugation for analysis. Separation was performed using an Agilent ZORBAX Eclipse Plus C18 column (2.1 mm × 100 mm, 1.8 μm). Mobile phase A was a weakly basic acetonitrile-buffered saline system, and mobile phase B was a high-proportion acetonitrile system, using a gradient elution program. Column temperature and flow rate were optimized. Mass spectrometry analysis was performed in ESI positive ion mode, and key parameters such as voltage, gas flow rate, and collision energy were configured according to the system's recommended standards.

[0047] The extract of Ganoderma lucidum was found to be composed of mannan, glucomannan, galactan, rhamnan, and fucoidan.

[0048] (5) After washing the Polygonatum, place it in a 55℃ drying oven for 36 hours, then crush it with a pulverizer and pass it through a 60-mesh sieve. (6) The obtained powder was added to deionized water at a material-to-liquid ratio of 1:30 (g / mL), and extracted in an 80℃ constant temperature shaker for 80 min. The mixture was then centrifuged (8000 r / min, 10 min, 4℃) to obtain residue and extract. Three times the volume of ethanol was slowly added to the extract with stirring. After being placed in a 4℃ refrigerator for 15 h, the mixture was centrifuged at 8000 r / min for 10 min. The supernatant was evaporated at 55℃ until no ethanol odor remained. The solution was then vacuum dried at -40℃ to obtain the Polygonatum odoratum water extract, with a yield of 35%.

[0049] (7) HILIC-MS / MS analysis of the structure of the aqueous extract of Polygonatum odoratum: A suitable amount of low molecular weight carbohydrate solution from the rhizome of Polygonatum odoratum was taken, and an equal volume of acetonitrile was added. The solution was filtered through a 0.22 μm organic filter membrane. Chicory oligofructose (sigma-FOS) was used as a standard for reference. Online separation was performed using a Waters HILICAmide column (1.7 μm, 1.2 × 150 mm), and analysis was performed using a SCiex Triple-TOF 5600+ time-of-flight mass spectrometer. The liquid chromatography conditions were as follows: mobile phase (A) was 20% acetonitrile containing 10 mmol / L ammonium formate and 0.2% formic acid; mobile phase (B) was 80% acetonitrile containing 10 mmol / L ammonium formate and 0.2% formic acid; flow rate was 0.25 mL / min; column temperature was 35℃; time gradient: 0–35 min, 100%–70% B; 35–50 min, 70%–20% B; 50–55 min, 20% B; 55–56 min, 20%–100% B; 56–60 min, 100% B. The injection volume was 2 μL. The mass spectrometry conditions were as follows: scanning mode was negative ion mode; nebulizer gas was 55 psi; curtain gas was 35 psi; ion source temperature was 550°C; ion source voltage was -4500 V; declustering voltage in the first scan was 100 V; focusing voltage was 10 V; acquisition mode in the second scan was IDA mode; CID energy was 40±20 eV.

[0050] The extract of Polygonatum sibiricum was found to be composed of water extracts with different degrees of polymerization, ranging from DP2 to DP30.

[0051] (8) The obtained Ganoderma lucidum extract and Polygonatum sibiricum water extract are combined in a mass ratio of 1:1 to obtain a combination of Ganoderma lucidum and Polygonatum sibiricum functional components.

[0052] Example 3 In vitro fermentation experiment of Ganoderma lucidum and Polygonatum sibiricum functional component composition 1. Experimental Design The experimental groups were set up as Ganoderma lucidum extract group, compound groups 1, 2, and 3 (the mass ratio of Ganoderma lucidum extract to Polygonatum sibiricum water extract was 3:1, 1:1, and 1:3, respectively), and Polygonatum sibiricum water extract group. Inulin was used as a positive control, and fermentation medium without carbohydrate source was used as a blank control. Each experimental group and control group were set up in triplicate.

[0053] 2. In vitro simulated fermentation Preparation of fecal microbiota suspension: Fresh feces were collected from 8 healthy volunteers (4 males and 4 females, no gastrointestinal diseases and no antibiotics taken in the past 3 months, BMI between 18.5 and 24). Equal amounts of feces were weighed and mixed, and sterile PBS (0.1 mol / L, pH 7.2) was added to prepare a 10% fecal microbiota suspension. After homogenization, the suspension was filtered through four layers of sterile gauze and the filtrate was collected for later use.

[0054] Preparation of fermentation medium: Taking the preparation of 1L of anaerobic fermentation medium as an example: Weigh 5.4 g NaCl, 2.7 g KH2PO4, 0.16 g CaCl2·2H2O, 0.12 g MgCl2·6H2O, 0.06 g MnCl2·4H2O, 0.06 g CoCl2·6H2O, and 5.4 g (NH4)2SO4, and dilute to 1000 mL with deionized water to prepare solution A; Weigh 2.7 g K2HPO4 and dilute to 1000 mL with deionized water to prepare solution B; Weigh 500 mg Na2EDTA, 200 mg FeSO4·7H2O, 10 mg ZnSO4·7H2O, 3 mg MnCl2·4H2O, 30 mg H3PO4, 20 mg CoCl2·6H2O, and 1 mg CuCl2·2H2O, and add 2 mg NiCl2·6H2O and 3 mg... Prepare a trace mineral solution by diluting Na2MoO4·2H2O with deionized water to 1000 mL; prepare a water-soluble vitamin solution by diluting 100 mg Thiamin-HCl, 100 mg calcium pantothenate, 100 mg vitamin B6, 5 mg P-aminobenzoic acid, and 0.25 mg vitamin B12 with deionized water to 1000 mL; prepare a folic acid:biotin solution by diluting 10 mg folic acid, 2 mg D-biotin, and 100 mg NH4HCO3 with deionized water to 1000 mL; prepare a riboflavin solution by dissolving 10 mg riboflavin in 5 mM (1.19 g / L) HEPES solution to 1000 mL; prepare a short-chain fatty acid mixture by mixing 2.5 mL valerate, 2.5 mL isovalerate, 2.5 mL isobutyrate, and 2.5 mL α-methylbutyrate; prepare a short-chain fatty acid mixture by diluting 1 g of... Prepare a razor cyan solution by diluting the volume of razor cyan solution to 1000 mL with deionized water.

[0055] Mix 330 mL of solution A, 330 mL of solution B, 10 mL of mineral solution, 5 mL of folic acid:biotin solution, 5 mL of riboflavin solution, 0.4 mL of short-chain fatty acid mixture, 1 mL of resazurin, 0.5 g of yeast extract, 4 g of sodium carbonate (Na₂CO₃), 0.5 g of cysteine ​​HCl-H₂O, and 5 g of casein peptone. Add 285 mL of distilled water and sterilize at 121 °C for 20 min. After cooling to room temperature, place the sterilized mixture, vitamin solution, and heme chloride powder (≈10 mg) in a clean bench for UV sterilization. Filter the vitamin solution using a 0.22 μm membrane filter and add 20 mL of vitamins and heme chloride. Finally, adjust the pH to 6.9-7.1 with 6 M HCl to obtain the anaerobic fermentation medium, which is then sterilized and ready for use.

[0056] In vitro simulated fermentation: The sample was dissolved in the fermentation medium at a final concentration of 5 mg / mL and fermented in an anaerobic environment at 37℃. Samples were taken at 0, 6, 12, 18 and 24 h, and centrifuged immediately (12,000 r / min, 10 min) to separate the supernatant and precipitate. The supernatant and precipitate were stored at -80℃ for the detection of various indicators.

[0057] 3. Detection indicators and measurement methods (1) pH measurement The supernatant of fermentation samples at 0, 6, 12, 18, and 24 h were collected in test tubes, and the pH of the samples was measured using a pH meter.

[0058] (2) Determination of short-chain fatty acid (SCFA) content Supernatants from fermentation samples at 0, 6, 12, 18, and 24 h were filtered through a 0.22 µm aqueous filter membrane, and the composition and content of short-chain fatty acids (SCFAs) were determined by gas chromatography (GC). The chromatographic column was an HP-INNOWAX (No. 19091N-133; 0.32 mm × 0.25 μm × 30 m, Agilent Technologies, Inc., USA). GC conditions were as follows: FID detector, N2 carrier gas at a flow rate of 16.2 mL / min, split ratio of 10:1, detector air flow rate of 260 mL / min, hydrogen flow rate of 40 mL / min, and make-up gas flow rate of 20 mL / min; injection and detector temperatures were both 250 °C; the temperature program was 80 °C (hold for 1 min) to 235 °C (heating rate of 5 °C / min, hold for 4 min); and the sample injection volume was 1 μL. The SCFA content in the samples was calculated based on the calibration curve of SCFA standards. The standard curve equations for each standard are shown in Table 2.

[0059] Table 2 (3) Molecular weight determination Supernatants from fermentation samples at 0, 6, 12, 18, and 24 h were filtered through a 0.22 µm aqueous membrane, and the molecular weight changes of polysaccharides during fermentation were detected using gel permeation chromatography. The chromatographic column was a Shodex 806 HQ column in series with a Shodex 804 HQ column. The mobile phase was 0.2 mol / L NaCl solution, filtered through a 0.45 µm aqueous membrane, and degassed by sonication for 20 min. The flow rate was 0.45 mL / min, and the injection volume was 50 µL.

[0060] (4) Gut microbiota analysis Sample precipitates collected after 24 h of fermentation were used to identify the quality of the microbial community and analyze the composition of the gut microbiota. Total DNA was extracted using the TianGen DNA Extraction Kit (DP336). After passing the tests, 30 ng of each sample was used for PCR amplification. Primers 341F (5'-CCTAYGGGRBGCASCAG-3') and 806R (5'-GGACTACNNGGGTATCTAAT-3') were used, amplifying the V3–V4 region of the 16S rRNA gene. The PCR instrument was a Bio-Rad T100 gradient PCR instrument. After purification, the PCR products were used for library construction using the NEBNext Ultra II DNA Library Prep Kit (E7645B). The libraries were quantified using a Qubit 2.0 fluorometer before sequencing. The sequencing platform was an Illumina NovaSeq 6000. After obtaining the raw Reads, quality control, splicing, and OTU clustering were performed on the QIIME2 platform. Species composition was annotated based on the SILVA database, and the structure and diversity of gut microbiota in each group were analyzed.

[0061] 4. Experimental Results (1) pH change Changes in pH value reflect, to some extent, alterations in the intestinal acidity or alkalinity during fermentation, influencing the production of short-chain fatty acids and the microbial community structure. The pH changes during the fermentation of the sample groups are as follows: Figure 1 As shown, except for the blank group, the other groups showed the fastest decline between 0 and 12 hours, and tended to stabilize after 12 hours. Compared with the Ganoderma lucidum extract group and the Polygonatum sibiricum water extract group, the different compound ratios of the sample groups showed different decline rates, indicating that the compound may have a synergistic effect and may accelerate fermentation at a specific ratio.

[0062] (2) Changes in the content of short-chain fatty acids Short-chain fatty acids are mainly products of fermentation of undigested carbohydrates by intestinal microorganisms in the colon, including acetic acid, propionic acid, butyric acid, isovaleric acid, and valeric acid. They play an important role in regulating the balance of intestinal flora and improving intestinal function. Figure 2 As shown, after 24 h of in vitro fermentation, all groups promoted the production of SCFAs. The compound group showed a higher level of short-chain fatty acid accumulation in the early stage of fermentation (6 h), which was significantly better than the single-addition group, demonstrating a faster metabolic response. Although the total SCFA content of the compound group was not significantly different from that of the Ganoderma lucidum extract group and the Polygonatum sibiricum water extract group at 24 h, its overall level was higher, suggesting a synergistic promoting effect. In particular, the butyric acid content of compound group 2 at 24 h was significantly higher than that of other groups, indicating that this compound ratio has an advantage in promoting the production of functional SCFAs, which may achieve synergistic effects by optimizing substrate structure and enhancing the metabolic activity of gut microbiota. Overall, the compound functional components help to enhance the production of short-chain fatty acids, thereby improving the metabolic function of gut microbiota, providing theoretical support for its application.

[0063] (3) Changes in molecular weight Molecular weight is an important structural feature and a key factor affecting the biological activity of polysaccharides. Figure 3 The results showed that, overall, with the extension of fermentation time, the retention time of each group was delayed, and the corresponding peak area also decreased significantly, indicating that the molecular weight and content of the samples were decreasing, suggesting that they could all be degraded and utilized by intestinal microorganisms. Figure 3 As shown, in samples with high Ganoderma lucidum extract content, the molecular weight retention time and peak area did not change significantly during the 0-6 h fermentation stage, indicating that intestinal microorganisms failed to effectively degrade the large polysaccharides within 6 h. In contrast, after 6 h of fermentation, both retention time and peak area changed significantly, indicating that they were broken down into smaller polysaccharide degradation products. In the sample group with high Polygonatum sibiricum water extract content, the molecular weight decreased rapidly mainly within 0-12 h, indicating that smaller molecular weights are more easily degraded. Compared with whole Ganoderma lucidum extract and Polygonatum sibiricum water extract, different proportions of compound groups showed significant differences in degradation efficiency.

[0064] (4) Changes in microbial community composition The gut microbiota provides nutrients to the host, defends against invading pathogens, and maintains gut homeostasis. The effects of Ganoderma lucidum extract, Polygonatum sibiricum water extract, and compound extracts on the microbial structure at the genus level were analyzed, and the results are as follows: Figure 4 As shown. Based on species differences at the genus level, different groups exhibited different regulatory effects. All compound groups effectively regulated the intestinal microecological environment, demonstrating a combined effect of upregulating various beneficial bacteria and downregulating typical harmful bacteria. Among them, compound group 2 significantly increased the levels of *Faecalibacterium*. (Faecalibacterium) With fecal cocci (Coprococcus) The relative abundance of these two genera is widely believed to be closely related to functions such as short-chain fatty acid production, anti-inflammation, and intestinal barrier repair, showing good potential for microecological regulation. Compound group 1 also significantly increased the abundance of bacteria such as *Rhodotorula*. (Roseburia) genus Streptomyces (Fusicatenibacter) The abundance of functional probiotics suggests a positive role in improving energy metabolism and maintaining gut microbiota homeostasis. Furthermore, all compound groups effectively reduced the prevalence of Escherichia coli-Shigella spp. (Escherichia-Shigella) and Klebsiella spp. (Klebsiella) The abundance of potentially harmful bacteria indicates that the compound combination has synergistic advantages in maintaining the intestinal barrier and inhibiting the inflammatory microenvironment.

[0065] In summary, by adjusting the ratio of Ganoderma lucidum to Polygonatum sibiricum, the structure of the microbial community can be regulated in a targeted manner. Different ratios have different regulatory focuses and potentials, providing a scientific basis for the subsequent development of differentiated microecological intervention products with specific functional positioning.

[0066] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A composition of functional components from Ganoderma lucidum and Polygonatum sibiricum, characterized in that, Composed of Ganoderma lucidum extract and Polygonatum sibiricum water extract; The mass ratio of the Ganoderma lucidum extract to the Polygonatum sibiricum water extract is 1-3:1-3.

2. The composition of Ganoderma lucidum and Polygonatum sibiricum functional components according to claim 1, characterized in that, The Ganoderma lucidum extract includes the following components: mannan, glucan, galactan, rhamnan, and fucoidan.

3. The functional component composition of Ganoderma lucidum and Polygonatum sibiricum according to claim 1, characterized in that, The Polygonatum extract is composed of water extracts with different degrees of polymerization, ranging from DP2 to DP30.

4. The method for preparing the functional component composition of Ganoderma lucidum and Polygonatum sibiricum according to any one of claims 1 to 3, characterized in that, Includes the following steps: Fresh Ganoderma lucidum fruiting bodies are dried, pulverized, and then ultrasonically treated with ethanol. The residue is then treated to remove the ethanol and gelatinize. The gelatinized residue was added to an aqueous solution, and amylase and protease were added separately for enzymatic hydrolysis. After enzyme inactivation, deionized water was added for extraction to obtain secondary residue and extract A. The secondary residue was then extracted with an alkaline solution to obtain alkaline extract B. Extract A and alkaline extract B were mixed, and polysaccharides were collected by alcohol precipitation to obtain Ganoderma lucidum extract. Fresh Polygonatum rhizomes were dried to form Polygonatum powder, which was then extracted with an aqueous solution. The polysaccharides in the extract were separated by alcohol precipitation, and the supernatant was rotary evaporated to obtain the water extract of Polygonatum. Ganoderma lucidum extract and Polygonatum sibiricum water extract were compounded in the specified ratio to obtain the functional component composition of Ganoderma lucidum and Polygonatum sibiricum.

5. The preparation method of the functional component composition of Ganoderma lucidum and Polygonatum sibiricum according to claim 4, characterized in that, During ultrasonic extraction with ethanol, the ratio of Ganoderma lucidum powder to ethanol solution is 1g:20~40mL, the ultrasonic extraction time is 60~120min, the temperature is 20~40℃, and the ultrasonic frequency is 30~50 kHz.

6. The method for preparing the Ganoderma lucidum and Polygonatum sibiricum functional component composition according to claim 4, characterized in that, The ratio of the gelatinized residue to the aqueous solution is 1g:10-20mL; the mass percentages of amylase and protease in the aqueous solution are 0.5%-5% respectively; the ratio of the enzyme-inactivated water-leached residue to deionized water is 1g:10-20mL; the ratio of the secondary residue to the alkaline solution is 1g:20-40mL; the concentration of the alkaline solution is 0.5-2M; during the extraction of Polygonatum sibiricum, the ratio of Polygonatum sibiricum powder to the aqueous solution is 1g:10-30mL. The enzymatic hydrolysis temperature of the amylase is 80-100℃, and the enzymatic hydrolysis time is 40-80 min; the enzymatic hydrolysis temperature of the protease is 40-60℃, and the enzymatic hydrolysis time is 100-150 min; the deionized water extraction temperature is 80-100℃, and the deionized water extraction time is 100-150 min; the alkaline solution extraction temperature is 20-30℃, and the alkaline solution extraction time is 50-70 min; the Polygonatum sibiricum aqueous solution extraction temperature is 80-90℃, and the time is 40-80 min.

7. The method for preparing the Ganoderma lucidum and Polygonatum sibiricum functional component composition according to claim 4, characterized in that, During alcohol precipitation, the volume percentage of the alcohol solution is 70%–85%.

8. The Ganoderma lucidum and Polygonatum functional component composition according to any one of claims 1 to 3, or the Ganoderma lucidum and Polygonatum functional component composition obtained by the preparation method according to any one of claims 4 to 7, in the preparation of products that promote the growth of beneficial bacteria *Faecalibacterium* in the intestines. (Faecalibacterium) spp. of fecal cocci (Coprococcus) Applications in products with increased yield.

9. The functional component composition of Ganoderma lucidum and Polygonatum sibiricum according to any one of claims 1 to 3, or the functional component composition of Ganoderma lucidum and Polygonatum sibiricum obtained by the preparation method according to any one of claims 4 to 7, in the preparation of a mixture that inhibits harmful intestinal bacteria Escherichia coli-Shigella spp. ( Escherichia-Shigella ) and Klebsiella spp. Klebsiella Application in products with increased yield.

Images