Preparation method, product and application of a fermented polygonatum cyrtopodum liquid rich in galactomannan acidic oligosaccharides

Abstract

The present application relates to the food processing and microbial fermentation technology field of medicine homologous food, particularly relates to a kind of preparation method, product and application of fermentation rhizoma polygonati liquid rich in galactomannan acidic oligosaccharide.The fresh rhizoma polygonati is washed, dried and crushed into powder, and is prepared into uniform rhizoma polygonati powder mixture with distilled water, and is subjected to enzymatic hydrolysis and wall breaking by adding cellulase and pectinase;Sterilization and enzyme inactivation are carried out by high-temperature and high-pressure treatment, and after cooling, plant lactobacillus and rhamnose lactobacillus are inoculated to carry out fermentation;The supernatant is collected by centrifugation of fermentation liquor, and the fermentation rhizoma polygonati liquid rich in galactomannan acidic oligosaccharide is obtained.The fermentation rhizoma polygonati liquid prepared by the present application can significantly reduce fasting blood glucose, reduce serum lipid level and reduce liver fatty degeneration, and the effect is better than that of nine steaming and nine drying rhizoma polygonati and unfermented rhizoma polygonati, and has wide application prospect in the functional food, health food or drug field for improving fatty liver and reducing blood sugar.

Description

Technical Field

[0001] This invention relates to the fields of food processing and microbial fermentation technology, and in particular to a method for preparing, a product of, and the application of fermented Polygonatum extract rich in galactomannic acid oligosaccharides. Background Technology

[0002] Polygonatum ( Polygonatum Polygonatum sibiricum (spe.) is a plant with both edible and medicinal properties, often referred to as "the leftover food of immortals." Its dried rhizome is used medicinally to replenish qi and nourish yin, strengthen the spleen and moisten the lungs. Studies have shown that Polygonatum sibiricum possesses various biological activities, including antioxidant, immunomodulatory, hypoglycemic, and anti-aging effects. Traditional processing methods primarily involve steaming, with the "nine steaming and nine drying" method being the most classic. After this process, the properties of Polygonatum sibiricum change from cooling to warming, resulting in a milder taste that reduces irritation to the throat and gastrointestinal tract and facilitates digestion and absorption. However, the traditional nine-steaming and nine-drying process suffers from drawbacks such as long processing time (requiring repeated steaming and drying nine times), high energy consumption, and potential loss of active substances during repeated heating, making it difficult to meet the demands of modern industrial production.

[0003] Modern research has found that the numerous health benefits of Polygonatum sibiricum lie in its rich content of various bioactive components, among which polysaccharides are the most abundant active substances, possessing multiple biological activities such as antioxidation, anti-inflammation, hypoglycemia, and immune regulation. However, Polygonatum sibiricum polysaccharides have a high molecular weight, resulting in high solution viscosity and low permeability, which is not conducive to the full realization of their efficacy. Studies have shown that low molecular weight oligosaccharides can be absorbed by the human body more quickly and exhibit stronger activity than high molecular weight polysaccharides. Therefore, it is necessary to degrade Polygonatum sibiricum polysaccharides into oligosaccharides of suitable molecular weight and develop a functional fermented Polygonatum sibiricum liquid rich in oligosaccharides. This would not only solve the problem of low efficiency in traditional processes but also provide a new dietary intervention option for people with fatty liver and hyperglycemia. Summary of the Invention

[0004] Based on the above, this invention provides a method for preparing, a product of, and the application of fermented Polygonatum sibiricum extract rich in galactomannic acid oligosaccharides. This invention utilizes cellulase and pectinase to enzymatically hydrolyze Polygonatum sibiricum powder, releasing intracellular polysaccharide components. After autoclaving to inactivate the enzymes, fermentation is carried out by inoculating with a compound probiotic composed of *Lactobacillus plantarum* and *Lactobacillus rhamnosus*. Combined with centrifugation and component analysis, a fermented Polygonatum sibiricum extract rich in galactomannic acid oligosaccharides is obtained. In this fermented Polygonatum sibiricum extract, galactomannic acid oligosaccharides with a molecular weight of 1000–10000 Da account for 62.36%–79.26% of the total sugars, and the molar ratio of mannose, galactose, and galacturonic acid is 0.43–1.54 : 0.88–2.83 : 1. Compared to unfermented Polygonatum sibiricum, the fermented Polygonatum sibiricum extract shows increased content of active ingredients such as saponins, total phenols, total flavonoids, and γ-aminobutyric acid. In vivo experiments showed that the fermented Polygonatum extract could significantly reduce fasting blood glucose and serum lipid levels in mice with high-fat diet-induced non-alcoholic fatty liver disease and alleviate hepatic steatosis, indicating that the fermented Polygonatum extract prepared in this invention has broad application prospects in improving fatty liver and metabolic diseases.

[0005] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of this invention is a fermented Polygonatum sibiricum extract rich in galactomannic acid oligosaccharides. The total sugar content of the fermented Polygonatum sibiricum extract is 30.56–39.59 mg / mL, of which galactomannic acid oligosaccharides with a molecular weight of 1000–10000 Da account for 62.36%–79.26% of the total sugar mass. The monosaccharide composition of the galactomannic acid oligosaccharides includes mannose, galactose, and galacturonic acid, with a molar ratio of 0.43–1.54 : 0.88–2.83 : 1.

[0006] The pH of the fermented Polygonatum extract was 3.04–5.23, and the saponin content was 152.84–180.37 μg / mL. Compared with the Polygonatum extract without enzymatic hydrolysis and fermentation, the saponin content was increased by 43.15%–68.93%.

[0007] The total phenol content in the fermented Polygonatum extract was 30.25–45.62 μg / mL, which was 13.42%–71.05% higher than that in Polygonatum extract without enzymatic hydrolysis and fermentation.

[0008] The total flavonoid content in the fermented Polygonatum extract was 70.26–102.86 μg / mL, which was 6.25%–55.54% higher than that in Polygonatum extract without enzymatic hydrolysis and fermentation.

[0009] The fermented Polygonatum extract contains 24-25 kinds of free amino acids, including the 9 essential amino acids required by the human body. The γ-aminobutyric acid is increased by 14%-25% compared with the Polygonatum extract without enzymatic hydrolysis and fermentation, and the free arginine is increased by 7%-18%.

[0010] The second technical solution of this invention is a kind of Lactobacillus plantarum ( Lactiplantibacillus plantarum The Lactobacillus plantarum YZ11 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 39764 and deposit date of July 20, 2026.

[0011] The third technical solution of this invention is a type of Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus The Lactobacillus rhamnosus ZL73 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 39765 and deposit date of July 20, 2026.

[0012] The fourth technical solution of the present invention is the application of the above-mentioned *Lactobacillus plantarum* and / or *Lactobacillus rhamnosus* as described in claim 2 in the fermentation preparation of oligosaccharides.

[0013] The fifth technical solution of this invention is a method for preparing fermented polygonatum extract rich in galactomannic acid oligosaccharides, comprising the following steps: Mix Polygonatum powder with water, add cellulase and pectinase for enzymatic hydrolysis to obtain Polygonatum hydrolysate; The enzymatic hydrolysate of Polygonatum was sterilized, cooled, and then inoculated with compound probiotics for fermentation to obtain the fermentation product. The fermentation product is subjected to solid-liquid separation, and the supernatant is collected to obtain fermented Polygonatum sibiricum liquid rich in galactomannic acid oligosaccharides. The compound probiotic is a mixture of the above-mentioned Lactobacillus plantarum and Lactobacillus rhamnosus.

[0014] In a preferred embodiment of the present invention, the particle size of the Polygonatum powder is 60-200 mesh; the mass ratio of the Polygonatum powder to water is 1:5-1:20; the enzymatic hydrolysis temperature is 40-60℃, and the enzymatic hydrolysis time is 1-4 h; the amount of cellulase added is 0.1%-1.0% of the mass of the Polygonatum powder, and the amount of pectinase added is 0.05%-0.5% of the mass of the Polygonatum powder.

[0015] In a preferred embodiment of the present invention, the volume percentage of Lactobacillus plantarum in the compound probiotic is 30% to 70%, with the remainder being Lactobacillus rhamnosus; the inoculation amount of the compound probiotic is 1% to 10% (V / V).

[0016] In a preferred embodiment of the present invention, the fermentation temperature is 30-37°C and the fermentation time is 12-72 hours.

[0017] In some embodiments of the present invention, the fermentation liquid of Polygonatum sibiricum can also be concentrated, freeze-dried or spray-dried to obtain concentrated fermented Polygonatum sibiricum liquid or freeze-dried fermented Polygonatum sibiricum powder.

[0018] In some embodiments of the present invention, the fermented liquid of Polygonatum sibiricum can be mixed with pharmaceutical or food excipients to prepare oral liquid, tablets, capsules, granules or beverages.

[0019] The sixth technical solution of the present invention is the application of the above-mentioned fermented Polygonatum extract in the preparation of products for improving fatty liver and / or metabolic diseases.

[0020] In a preferred embodiment of the present invention, the fatty liver and / or metabolic disease includes reducing fasting blood glucose, reducing liver triglyceride content, reducing serum total cholesterol and / or low-density lipoprotein cholesterol levels, increasing serum high-density lipoprotein cholesterol levels, alleviating liver steatosis, and improving non-alcoholic fatty liver disease; the product includes functional food, health food, medicine, or special medical food.

[0021] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a method for preparing fermented Polygonatum sibiricum broth rich in galactomannic acid oligosaccharides. The method employs a three-stage process: enzymatic hydrolysis to break cell walls, high-pressure sterilization to inactivate enzymes, and probiotic fermentation. Intracellular polysaccharides are released through the synergistic enzymatic hydrolysis of cellulase and pectinase. After high-pressure sterilization to inactivate enzymes, the polysaccharides are then directionally converted into the target oligosaccharides through a combined fermentation process using *Lactobacillus plantarum* and *Lactobacillus rhamnosus*. The method is simple to operate, operates under mild conditions, and is easy to scale up. The resulting fermentation broth has a high content of galactomannic acid oligosaccharides and contains no glucose or fructose, making it safe and suitable for people with high blood sugar and high cholesterol. It can also significantly improve non-alcoholic fatty liver disease induced by a high-fat diet, providing a new technical solution for the industrial development of functional fermented Polygonatum sibiricum products. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 The molecular weight distribution of Polygonatum sibiricum fermentation broth under different treatment conditions in Example 3; Figure 2 Example 5 shows the changes in body weight and blood glucose levels in mice under different treatment groups; Figure 3 The liver index of mice in different treatment groups in Example 5; Figure 4 The levels of triglycerides (TG), total cholesterol (TC), high-density lipoprotein (HDL-C), and low-density lipoprotein (LDL-C) in the serum of mice in different treatment groups in Example 5; Figure 5 The liver tissue morphology of mice in different treatment groups in Example 5; Figure 6 HE-stained sections of mouse livers from different treatment groups in Example 5. Detailed Implementation

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0026] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of this invention.

[0027] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0028] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0029] Unless otherwise specified, the "%" in this invention refers to a percentage by mass.

[0030] In the extraction and transformation of active ingredients in Polygonatum sibiricum, enzymatic hydrolysis and microbial fermentation are two important bioprocessing methods. Cellulase and pectinase can degrade cellulose and pectin components in plant cell walls, disrupting cell wall structure and promoting the release of intracellular active ingredients. Microbial fermentation technology can alter the component structure of Polygonatum sibiricum through microbial metabolism, generating novel active substances, and effectively releasing and transforming functional components such as Polygonatum sibiricum polysaccharides and saponins through enzymatic hydrolysis. Furthermore, Polygonatum sibiricum polysaccharides have high glucose and fructose residue content. For individuals with high blood sugar, if the polysaccharides are degraded into free monosaccharides in vivo or during processing, it may pose a potential risk of blood sugar fluctuations. Therefore, when preparing Polygonatum sibiricum oligosaccharides, simultaneously reducing the content of free glucose and fructose in the fermentation products would be safer and more suitable for individuals with high blood sugar and high cholesterol. Studies have shown that probiotics such as Lactobacillus plantarum can secrete various enzymes (such as β-glucanase, α-galactosidase, and isomerase) during fermentation, which can degrade large-molecule polysaccharides in Polygonatum sibiricum into small-molecule oligosaccharides and alter the structure of the oligosaccharides. Therefore, this invention provides a method for preparing fermented Polygonatum extract rich in galactomannic acid oligosaccharides, a product thereof, and its application.

[0031] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0032] The fresh Polygonatum used in this embodiment of the invention comes from Funiu Mountain in Henan Province and is Polygonatum sibiricum (also known as chicken head Polygonatum). Polygonatum sibiricum ).

[0033] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0034] Example 1: Preparation of Polygonatum hydrolysate Fresh Polygonatum rhizome was washed, dried, and then pulverized in a grinder to obtain Polygonatum rhizome powder. The powder was then passed through a 60-200 mesh sieve. 100 g of Polygonatum rhizome powder was accurately weighed and mixed with an appropriate amount of distilled water to achieve a material-to-liquid ratio of 1:5 to 1:20 (g / g). The mixture was stirred thoroughly, and then cellulase and pectinase were added. The amount of cellulase added was 0.1% to 1.0% of the mass of the Polygonatum rhizome powder, and the amount of pectinase added was 0.05% to 0.5% of the mass of the Polygonatum rhizome powder. The mixture was then enzymatically hydrolyzed in a water bath at 40-60℃ for 1-4 hours, with continuous stirring. After hydrolysis, the hydrolysate was heated in a 95℃ water bath for 10 minutes to inactivate the enzymes, yielding a Polygonatum rhizome hydrolysate. The total sugar content of the hydrolysate was determined and converted to the total sugar content per gram of Polygonatum rhizome powder. The release rate of Polygonatum rhizome polysaccharides was then calculated. Total sugar content: 0.2 mL of the above Polygonatum hydrolysate, 0.4 mL of 6% phenol solution, and 3 mL of concentrated sulfuric acid were mixed thoroughly and left at room temperature for 30 min. The absorbance was measured at 490 nm. Standard curves were plotted using glucose solutions of different concentrations (30, 60, 90, 120, 160, and 200 µg / mL) as standards. The results are shown in Table 1. The size of the Polygonatum powder, the material-to-liquid ratio, the amount of cellulase and pectinase added, the hydrolysis temperature, and the hydrolysis time all affect the release rate of Polygonatum polysaccharides. Based on the polysaccharide release rate, the following conditions were selected for preparing the Polygonatum hydrolysate: 100-mesh Polygonatum powder, a material-to-liquid ratio of 1:20, a cellulase addition of 1.0%, a pectinase addition of 0.5%, a hydrolysis temperature of 50℃, and a hydrolysis time of 2 h. Under these conditions, the polysaccharide release rate was 75.47%. Under the same treatment conditions, the polysaccharide release rate of the enzyme-added group was approximately 30% higher than that of the enzyme-free group.

[0035] Table 1 shows the total sugar content of Polygonatum hydrolysate under different treatment conditions in Example 1.

[0036] Example 2 Preparation of Polygonatum sibiricum fermentation broth According to the optimal conditions selected in Example 1 (100-mesh Polygonatum powder, material-to-liquid ratio of 1:20, cellulase addition of 1.0%, pectinase addition of 0.5%, hydrolysis temperature of 50℃, and hydrolysis time of 2 h), Polygonatum hydrolysate was prepared and sterilized in a high-temperature and high-pressure autoclave (115℃, 0.1 MPa) for 30 min. Lactobacillus plantarum YZ11 (CGMCC No. 39764) and Lactobacillus rhamnosus ZL73 (CGMCC No. 39765) were activated with MRS broth medium and cultured statically at 37℃ for 24 h. Compound probiotic solutions were prepared according to different volume ratios. After the sterilized hydrolysate cooled, probiotics (Lactobacillus plantarum CGMCC No. 39764, OD...) were inoculated. 600The OD values ​​of the two strains were 1.0–1.2 (Lactobacillus rhamnosus CGMCC No. 39765, and a mixture of both). 600 All values ​​were 1.0–1.2, with a single-strain inoculation rate of 5% (V / V) and a mixed inoculation rate of 1%–10% (V / V). *Lactobacillus plantarum* comprised 30%–70% of the volume, with the remainder being *Lactobacillus rhamnosus*. Fermentation was carried out at 30–37℃ for 12–72 h, and the pH and total sugar content of the fermentation broth were recorded under different conditions. The enzymatic hydrolysate, after sterilization and cooling, was not inoculated (enzymatic hydrolysis group) and served as a control. As shown in Table 2, the ratio of the two probiotic strains, the inoculation rate, fermentation temperature, and fermentation time all affected the microbial conversion of the active components of *Polygonatum sibiricum*. With prolonged fermentation time, the pH showed a decreasing trend, indicating that the probiotics could utilize the sugars in *Polygonatum sibiricum* to produce acid and lower the pH of the fermentation broth. However, with prolonged fermentation time, the total sugar content showed a fluctuating trend of first decreasing and then increasing. In the initial stage of fermentation, probiotics rapidly consume the fermentable sugars in the Polygonatum hydrolysate, leading to a decrease in total sugar content. As fermentation progresses, extracellular enzymes secreted by probiotics (such as β-glucanase and α-galactosidase) continuously degrade the macromolecular polysaccharides in Polygonatum into oligosaccharides. Some of the released sugars are not completely consumed, thus the total sugar content rebounds. The fluctuation in total sugar content indicates that while probiotics utilize sugars for growth, they also continuously release new sugar components.

[0037] Table 2 shows the pH value and total sugar content of the Polygonatum odoratum fermentation broth under different treatment conditions in Example 2.

[0038] Example 3: Determination of molecular weight and monosaccharide composition of galactomannic acid oligosaccharides in Polygonatum sibiricum fermentation broth The Polygonatum sibiricum fermentation broth prepared in Example 2 was centrifuged at 4℃ and 8000 rpm for 15 min to remove insoluble impurities and bacterial cells. The supernatant was collected and dialyzed with a 1000 Da dialysis bag for 24 h to remove small molecule metabolites. The monosaccharide composition and molecular weight distribution of Polygonatum sibiricum polysaccharides in the fermentation broth were determined. A water extract of Polygonatum sibiricum of equal concentration was prepared with distilled water (without enzymatic hydrolysis and probiotic fermentation). An enzymatic hydrolysate was sterilized, cooled, and not inoculated (enzymatic hydrolysis group) as a control.

[0039] Monosaccharide composition determination method: 1 mL of fermentation broth was placed in an ampoule, and 1 mL of 4 mol / L trifluoroacetic acid (TFA) was added. The ampoule was sealed with an alcohol burner and hydrolyzed at 110 °C for 6 h. After cooling to room temperature, 3 times the volume of methanol was added, and TFA was removed using a rotary evaporator. This process was repeated three times. A 1 mol / L mixed monosaccharide standard solution was prepared, and the hydrolyzed product was subjected to PMP derivatization. The derivatization method was as follows: 100 µL of the mixed monosaccharide standard solution and the hydrolyzed product were mixed with 210 µL of 0.3 mol / L NaOH solution. 200 µL of 0.5 mol / L PMP methanol solution was added, and the mixture was thoroughly mixed. The mixture was then placed in a 70 °C oven for 60 min. After the reaction, the mixture was cooled to room temperature, and 210 µL of 0.3 mol / L HCl was added to neutralize excess alkali. Add 3 mL of dichloromethane to the mixture, shake, and let stand until it separates into layers. Discard the lower layer and repeat this extraction process three times. Filter the upper layer through a 0.22 µm microporous membrane and perform HPLC analysis. HPLC column: Agilent ZORBAX 300 XDB-C18; HPLC conditions: 0.05 mol / L KH₂PO₄ phosphate buffer (pH 6.7) / CH₃CN (83:17, V:V) as mobile phase; column temperature: 35 ℃; flow rate: 1 mL / min; injection volume: 20 µL; detection wavelength: 245 nm. The monosaccharide composition of the fermentation broth is shown in Table 3. The original extract (without enzymatic hydrolysis and microbial fermentation, corresponding to the "control" in the table, i.e., the above-mentioned Polygonatum odoratum aqueous extract) contained glucose, fructose, mannose, galacturonic acid, and galactose. The monosaccharide composition remained essentially unchanged after enzymatic hydrolysis. After single-strain fermentation with *Lactobacillus plantarum* (group 13), the glucose and fructose contents decreased significantly, indicating that *Lactobacillus plantarum* can secrete glucosidase and fructanase to degrade and utilize glucose and fructose, while the mannose and galactose contents increased significantly. In contrast, after single-strain fermentation with *Lactobacillus rhamnosus* (group 14), glucose was undetectable, while the galactose and galacturonic acid contents increased significantly. After co-fermentation with two strains under different conditions, glucose and fructose were undetectable, and the polysaccharide component contained only mannose, galactose, and galacturonic acid, in a ratio of 0.43–1.54: 0.88–2.83: 1. These results indicate that co-fermentation with *Lactobacillus plantarum* and *Lactobacillus rhamnosus* can completely consume the glucose and fructose residues in *Polygonatum sibiricum* polysaccharides or use isomerases to convert them into the remaining monosaccharides, transforming the original glucose and fructose-rich *Polygonatum sibiricum* polysaccharides into polysaccharides containing only mannose, galactose, and galacturonic acid. This has important safety implications for people with hyperglycemia.

[0040] The molecular weight determination conditions were as follows: the supernatant was filtered through a 0.22 µm microporous membrane and then analyzed by HPLC. The HPLC column was a Shodex OHpak SB-803 HQ, the mobile phase was 0.2 mol / L NaNO3 solution and 0.01 mol / L NaH2PO4 solution, the column temperature was 30 ℃, the flow rate was 0.8 mL / min, and the injection volume was 20 µL. The molecular weight distribution of the fermentation broth is shown in Table 4. In the original extract (without enzymatic hydrolysis and microbial fermentation), the proportion of components with a molecular weight >10000 Da was greater than 98%. After enzymatic hydrolysis and microbial transformation, the molecular weight decreased significantly, and the proportion of components with a molecular weight of 1000-10000 Da was 62.36%~79.26%. Among them, group 12 had the lowest average molecular weight. Figure 1 As shown, group 12 includes three components in the 1000-10000 Da range: 5843, 3363, and 1401 Da.

[0041] Table 3 shows the monosaccharide composition of the Polygonatum odoratum fermentation broth under different treatment conditions in Example 3.

[0042] Table 4 shows the molecular weight distribution of the Polygonatum sibiricum fermentation broth under different treatment conditions in Example 3.

[0043] Example 4: Physicochemical Properties and Composition Analysis of Polygonatum Fermentation Broth (1) Determination of saponin content Preparation of water-saturated n-butanol: Take an appropriate amount of n-butanol into a stoppered conical flask or reagent bottle, add excess pure water (such as 1 / 3 to 1 / 2 of the volume of n-butanol), shake vigorously for 2-3 minutes, and then let stand overnight. When using, simply take the supernatant.

[0044] Take 1.5 mL of fermentation supernatant, add 1.5 mL of water-saturated n-butanol, vortex for 2 min, centrifuge at 3000 g for 5 min, and collect the n-butanol layer (upper layer); repeat the extraction twice, combine the n-butanol layers, evaporate to dryness, and redissolve in 1 mL of methanol. Take 0.3 mL of the sample redissolved in methanol, evaporate the solvent in a 60°C water bath, add 0.20 mL of 5% vanillin-glacial acetic acid solution and 0.8 mL of pure perchloric acid, shake well, heat in a 60°C water bath for 15 min, cool in ice water for 5 min, add 5 mL of glacial acetic acid, shake well, and let stand for 15 min. Using a reagent blank as a control, measure its absorbance at a wavelength of 550 nm. Preparation of the standard curve: A 500 µg / mL diosgenin standard solution was prepared using methanol. Accurately pipette 0, 0.10, 0.20, 0.30, 0.40, 0.50, and 0.60 mL of the standard solution into 10 mL stoppered test tubes. The solvent was evaporated in a 60°C water bath. All other procedures were performed in the same manner as the standard group. The results are shown in Table 5. Enzymatic hydrolysis was beneficial for releasing some saponins, increasing the content by 12.97% compared to the control group. The fermentation group significantly increased the saponin content, with the highest increase reaching 68.93%.

[0045] Table 5 shows the saponin content and increase rate of Polygonatum odoratum fermentation broth under different treatment conditions in Example 4.

[0046] (2) Determination of total phenol content Take 0.1 mL of fermentation supernatant from different groups, add 0.1 mL of 1.0 mol / L Folin-Ciocalteu reagent, shake well and let stand for 6 min. Then add 0.4 mL of 10% sodium carbonate solution and 0.4 mL of pure water to each group, shake well, and let stand in a 40 ℃ water bath in the dark for 60 min. After cooling, measure the absorbance at 760 nm. Use gallic acid solutions of different concentrations as standards to plot standard curves. The results are shown in Table 6. The fermentation groups significantly increased the total phenol content, with the highest increase rate reaching 71.05%.

[0047] Table 6 shows the total phenol content and improvement rate of Polygonatum sibiricum fermentation broth under different treatment conditions in Example 4.

[0048] (3) Determination of total flavonoid content Take 0.2 mL of fermentation supernatant from different groups, add 0.1 mL of 5% sodium nitrite solution, shake well and let stand for 6 min, add 0.1 mL of 10% aluminum nitrate solution, shake well and let stand for 6 min, add 0.2 mL of 10% NaOH solution, and measure the absorbance at 510 nm after 15 min. Use rutin solutions of different concentrations as standards to plot standard curves. The results are shown in Table 7. The fermentation groups significantly increased the total flavonoid content, with the highest increase rate reaching 55.54%.

[0049] Table 7 shows the total flavonoid content and enhancement rate of Polygonatum sibiricum fermentation broth under different treatment conditions in Example 4.

[0050] As shown in Tables 5, 6, and 7, compared to the original Polygonatum extract, enzymatic hydrolysis promoted the release of a small amount of saponins, but had no significant effect on the promotion of total phenols and total flavonoids. Under the same inoculum size, fermentation temperature, and fermentation time, single-strain fermentation and dual-strain combined fermentation showed significant differences in their effects on the promotion of saponins, total phenols, and total flavonoids. Dual-strain combined fermentation was more effective in increasing the content of saponins, total phenols, and total flavonoids in the fermentation broth. Among these, the contents of all three components were highest when using 70% Lactobacillus plantarum and 30% Lactobacillus rhamnosus.

[0051] (4) Analysis of free amino acid composition Take 2 mL of fermentation supernatant, add 2 mL of 5% sulfosalicylic acid solution, centrifuge at 10000 rpm and 4 ℃ for 10 min, filter using a 0.22 μm microporous membrane, and determine the composition and content of free amino acids using a Hitachi L-8900 fully automated amino acid analyzer. As shown in Table 8, the fermentation group contained 24-25 kinds of free amino acids, including the 9 essential amino acids required by the human body. After fermentation, the total amount of sweet amino acids decreased by about 21%-30% compared with the control group, mainly due to the significant decrease in serine, threonine, and glycine, while alanine increased slightly. In addition, γ-aminobutyric acid increased significantly after fermentation, by about 14%-25% compared with the control group, and arginine increased by 7%-18%. Based on the above component analysis results, the conditions of group 12 were selected as the optimal conditions for preparing Polygonatum sibiricum fermentation broth for subsequent activity evaluation.

[0052] Table 8 shows the composition and content of free amino acids in the Polygonatum odoratum fermentation broth under different treatment conditions in Example 4.

[0053] Example 5: The ameliorative effect of fermented Polygonatum extract on a fatty liver animal model. (1) Establishment of NAFLD mouse model Forty-eight six-week-old SPF-grade male C57BL / 6J mice were randomly divided into two groups: a control group (NC, n=8) and a high-fat diet model group (HFD, n=40). Mice in the NC group were fed a normal diet, while mice in the HFD group were fed a high-fat diet. Body weight changes were recorded weekly. Eight weeks after modeling, two mice from each group were randomly selected for serum biochemical markers and liver pathological examination. Significantly elevated blood lipid levels and fat accumulation in more than one-third of the hepatocytes confirmed the successful establishment of the NAFLD model. Mice in the HFD group were randomly divided into 5 groups: model group (HFD, high-fat diet + 10 mL / kg PBS), pioglitazone group (PIG, high-fat diet + 10 mg / kg pioglitazone), unfermented Polygonatum extract group (PR, high-fat diet + 10 mL / kg), fermented Polygonatum extract group (FPR, high-fat diet + 10 mL / kg), and nine-times-steamed and nine-times-dried Polygonatum extract group (NPR, high-fat diet + 10 mL / kg). They were administered the extracts daily by gavage for 4 weeks, and changes in body weight and blood glucose were recorded. Results are as follows: Figure 2 As shown, compared with the model group, the treatment groups significantly reduced the weight gain rate of HFD mice. After 4 weeks of intervention, the PIG group decreased by 19.11%, the PR group by 25.72%, the FPR group by 35.34%, and the NPR group by 28.00%. The FPR group's weight was closest to that of the NC group. Furthermore, after 3 weeks of intervention, the blood glucose level in the FPR group returned to normal, while the other intervention groups remained slightly higher than the NC group after 4 weeks of intervention.

[0054] (2) Liver index measurement At the end of the experiment, liver tissue was collected from all mice, rinsed with physiological saline, dried with kitchen paper, and weighed.

[0055] Liver index (%) = Liver weight (g) / Body weight (g) × 100 The results are as follows Figure 3 As shown, all treatment groups significantly reduced the liver index of mice in the HFD group (P<0.05), but there was no significant difference among the intervention groups (PIG, PR, FPR, NPR) (P>0.05).

[0056] (3) Serum marker detection At the end of the experiment, mice were anesthetized with ether, and blood was collected from the posterior ocular venous plexus. Whole blood was allowed to stand at room temperature for 2-4 hours, then centrifuged at 3,000 r / min for 10 minutes at 4°C to collect serum. Serum biochemical parameters, including triglycerides, total cholesterol, low-density lipoprotein (LDL), and high-density lipoprotein (HDL), were measured using kits provided by Nanjing Jiancheng. Decreased TC, TG, LDL-C, and HDL-C are the main characteristics of lipid metabolism disorders. Figure 4As shown, compared with the NC group, the HFD group had significantly higher levels of TC, TG, and LDL-C, and significantly lower levels of HDL-C (P<0.001). The intervention group significantly improved lipid metabolism abnormalities, restoring TC, TG, LDL-C, and HDL-C to normal levels. Specifically, the FPR group had significantly lower TG and LDL-C levels than the PR and NPR groups, and significantly higher HDL-C levels than the PR and NPR groups (P<0.05), indicating that the fermented Polygonatum extract of this invention can effectively reduce hepatic lipid accumulation.

[0057] (4) Liver histopathology in NAFLD mice Mouse livers were collected and fixed in 4% paraformaldehyde fixative for at least 24 hours. After fixation, the tissues were sequentially dehydrated with graded ethanol, then cleared and embedded in paraffin. Tissue sections were prepared using a paraffin microtome, dewaxed, rehydrated, and stained with hematoxylin and eosin (HE). After staining, morphological changes were observed and images were acquired under an optical microscope. The dissected mouse liver is shown below. Figure 5 As shown, the HFD group had enlarged livers with significant fat accumulation, while the intervention group showed significant improvement. The FPR group's liver morphology and color were closest to the NC group. HE staining was as follows. Figure 6 As shown, the sinusoidal structure of the NC group was clear, and the hepatic cords were arranged radially and orderly. In contrast, the sinusoidal structure of the HFD group was blurred, and the hepatocytes showed obvious fatty vacuolar degeneration, disordered arrangement of hepatic cords, and inflammatory cell infiltration. After treatment in the intervention group, the fatty degeneration of hepatocytes was significantly reduced, and the hepatocyte structure tended to be normal. No obvious fatty vacuoles were observed in the hepatocytes of the FPR group, while a small number of fatty vacuoles were observed in the PIG, PR, and NPR groups. This indicates that the fermented Polygonatum sibiricum broth can improve fatty liver in mice, and the effect is better than that of unfermented Polygonatum sibiricum and nine-steamed and nine-dried Polygonatum sibiricum.

[0058] Example 6: Dosage Form Application of Fermented Polygonatum Extract (1) Preparation of fermented Polygonatum sibiricum oral liquid Take 1000 mL of fermented Polygonatum sibiricum liquid prepared under the optimal conditions in Example 2, add 0-30 g xylitol, 0-0.5 g steviol glycosides, 0-20 g erythritol, 0-2.0 g citric acid, 0-30 g fructooligosaccharides, 0-5 g pectin, and excipients such as jujube powder and wolfberry powder, stir evenly, homogenize under high pressure (pressure 20-40 MPa, temperature 55-65℃), degas under vacuum (vacuum degree -0.06 to -0.08 MPa), fill and seal, and then sterilize by pasteurization (70-80℃ for 10-20 min). After cooling, the fermented Polygonatum sibiricum oral liquid is obtained.

[0059] (2) Preparation of freeze-dried powder of fermented Polygonatum sibiricum The fermented Polygonatum sibiricum broth prepared under the optimal conditions in Example 2 was mixed with maltodextrin or soluble starch (accounting for 10%–30% of the solid content of the fermentation broth) as an excipient, stirred evenly, pre-frozen at -20°C for 12–24 h, and then placed in a freeze dryer and dried at a vacuum of 10–50 Pa and a cold trap temperature of -50°C for 24–48 h to obtain the freeze-dried Polygonatum sibiricum powder. The obtained freeze-dried powder can be further mixed with food- or pharmaceutically acceptable excipients to prepare various dosage forms. Tablets: Take 100 parts of lyophilized fermented Polygonatum powder, add 20-40 parts of microcrystalline cellulose, 5-15 parts of sodium carboxymethyl starch, and 0.5-2 parts of magnesium stearate, mix evenly and compress into tablets to obtain fermented Polygonatum tablets.

[0060] Capsules: Take 100 parts of fermented Polygonatum lyophilized powder, add 10-30 parts of pregelatinized starch and 0.5-2 parts of silicon dioxide, mix evenly, and fill into empty capsules to obtain fermented Polygonatum lyophilized capsules.

[0061] Granules: Take 100 parts of freeze-dried fermented Polygonatum powder, add 20-50 parts of dextrin and 10-30 parts of sucrose powder, use an appropriate amount of ethanol as a wetting agent to make a soft material, granulate it through a 10-20 mesh sieve, dry it at 50-60℃, and granulate it to obtain fermented Polygonatum granules.

[0062] Solid beverage: Take 100 parts of fermented Polygonatum lyophilized powder, add 20-50 parts of fructooligosaccharides, 10-30 parts of erythritol, 1-5 parts of citric acid, and 5-20 parts of fruit powder, mix well, and you will get fermented Polygonatum lyophilized solid beverage.

[0063] The food- or pharmaceutically acceptable excipients include, but are not limited to: xylitol, steviol glycosides, erythritol, fructooligosaccharides, maltodextrin, dextrin, microcrystalline cellulose, sodium carboxymethyl starch, magnesium stearate, pregelatinized starch, silicon dioxide, sucrose powder, citric acid, pectin, jujube, goji berries, kudzu root, yam, apple powder, peach powder, etc.

[0064] (3) Application of other dosage forms Based on the above preparation method, those skilled in the art can further prepare the fermented Polygonatum sibiricum liquid prepared in Example 2 or the freeze-dried powder prepared therefrom into food forms such as beverages, jellies, gummies, and meal replacement powders, or into health food or pharmaceutical dosage forms such as oral liquids, granules, tablets, and capsules, all of which fall within the protection scope of this invention.

[0065] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A fermented polygonatum extract rich in galactomannic acid oligosaccharides, characterized in that, The total sugar content in the fermented Polygonatum sibiricum extract is 30.56–39.59 mg / mL, of which galactomannic acid oligosaccharides with a molecular weight of 1000–10000 Da account for 62.36%–79.26% of the total sugar mass. The monosaccharide composition of the galactomannic acid oligosaccharides includes mannose, galactose, and galacturonic acid, and the molar ratio of mannose, galactose, and galacturonic acid is 0.43–1.54 : 0.88–2.83 :

1. The pH of the fermented Polygonatum sibiricum extract was 3.04–5.23, and the saponin content was 152.84–180.37 μg / mL. The total phenol content in the fermented Polygonatum sibiricum extract was 30.25–45.62 μg / mL; The total flavonoid content in the fermented Polygonatum extract was 70.26–102.86 μg / mL.

2. A type of Lactobacillus plantarum ( Lactiplantibacillus plantarum ), characterized in that, The Lactobacillus plantarum YZ11 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 39764 and deposit date of July 20, 2026.

3. A type of Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ), characterized in that, The Lactobacillus rhamnosus ZL73 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 39765 and deposit date of July 20, 2026.

4. The use of *Lactobacillus plantarum* as described in claim 2 and / or *Lactobacillus rhamnosus* as described in claim 3 in the fermentation preparation of oligosaccharides.

5. A method for preparing fermented polygonatum extract rich in galactomannic acid oligosaccharides as described in claim 1, characterized in that, Includes the following steps: Mix Polygonatum powder with water, add cellulase and pectinase for enzymatic hydrolysis to obtain Polygonatum hydrolysate; The enzymatic hydrolysate of Polygonatum was sterilized, cooled, and then inoculated with compound probiotics for fermentation to obtain the fermentation product. The fermentation product is subjected to solid-liquid separation, and the supernatant is collected to obtain fermented Polygonatum sibiricum liquid rich in galactomannic acid oligosaccharides. The compound probiotic is a mixture of Lactobacillus plantarum as described in claim 2 and Lactobacillus rhamnosus as described in claim 3.

6. The preparation method according to claim 5, characterized in that, The particle size of the Polygonatum odoratum powder is 60-200 mesh; the mass ratio of the Polygonatum odoratum powder to water is 1:5-1:20; the enzymatic hydrolysis temperature is 40-60℃, and the enzymatic hydrolysis time is 1-4 h; the amount of cellulase added is 0.1%-1.0% of the mass of the Polygonatum odoratum powder, and the amount of pectinase added is 0.05%-0.5% of the mass of the Polygonatum odoratum powder.

7. The preparation method according to claim 5, characterized in that, The compound probiotic contains 30%–70% Lactobacillus plantarum by volume, with the remainder being Lactobacillus rhamnosus; the inoculation amount of the compound probiotic is 1%–10% (V / V); the fermentation temperature is 30–37℃, and the fermentation time is 12–72 h.

8. The use of the fermented Polygonatum extract as described in claim 1 in the preparation of products for improving fatty liver and / or metabolic diseases.

9. The application according to claim 8, characterized in that, The fatty liver and / or metabolic diseases mentioned include reducing fasting blood glucose, reducing liver triglyceride levels, reducing serum total cholesterol and / or low-density lipoprotein cholesterol levels, increasing serum high-density lipoprotein cholesterol levels, alleviating hepatic steatosis, and improving non-alcoholic fatty liver disease; the products mentioned include functional foods, health foods, pharmaceuticals, or special medical foods.

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