Preparation method of modified stropharia rugoso-annulata crude polysaccharide and application of modified stropharia rugoso-annulata crude polysaccharide in lactobacillus fermentation of rhizoma polygonati

Abstract

The invention provides a preparation method of modified stropharia rugosoannulata crude polysaccharide and application of the modified stropharia rugosoannulata crude polysaccharide in lactobacillus fermentation of rhizoma polygonati, and belongs to the technical field of bioengineering. The method comprises the following steps: carrying out irradiation treatment on crude stropharia rugoso-annulata polysaccharide, and then carrying out ultrahigh pressure treatment to obtain the modified crude stropharia rugoso-annulata polysaccharide. In order to solve the problem that the proliferation of lactic acid bacteria is inhibited in the fermentation process of rhizoma polygonati, the growth of the lactic acid bacteria is promoted by adding the modified stropharia rugoso-annulata crude polysaccharide. The method can effectively reduce the inhibition effect of the polygonatum sibiricum primary pulp on lactic acid bacteria and improve the fermentation efficiency. After the fermentation broth is applied to polygonatum sibiricum fermentation, the content of irritant components is reduced, the taste and flavor of primary pulp are improved, the content of saponins, total phenols and total flavonoids is remarkably increased, and meanwhile the content of cholesterol is reduced. In addition, the inhibition capability and the antioxidant activity of the fermented polygonatum sibiricum primary pulp on alpha-glucosidase are enhanced, and the comprehensive quality of the product is remarkably improved.

Description

A method for preparing modified crude polysaccharide from *Stropharia macrocarpa* and its application in lactic acid bacteria fermentation of *Polygonatum sibiricum*. Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to a method for preparing modified Pleurotus ostreatus crude polysaccharide and its application in lactic acid bacteria fermentation of Polygonatum sibiricum. Background Technology

[0002] Polygonatum is a food and medicine homology substance, rich in polysaccharides, saponins, flavonoids and other active ingredients, exhibiting multiple pharmacological activities such as anti-oxidation, hypoglycemia, hypolipidemia, anti-inflammation and anti-tumor effects. Polygonatum has the effects of "tonifying qi and nourishing yin, strengthening the spleen and moistening the lungs, and benefiting the kidneys," and is widely used in functional foods, medicinal cuisine and health products, with broad application prospects. Although the medicinal value of Polygonatum has always been highly regarded, the calcium oxalate, mucilage and other components contained in its raw medicinal material can easily cause significant irritation to the oral and pharyngeal mucosa, and direct consumption often causes discomfort such as numbness of the mouth and tongue, and burning sensation in the throat.

[0003] Traditionally, a "nine-steaming and nine-processing" technique is used to alleviate this problem. While this technique effectively reduces tongue numbing and irritation, it suffers from a long processing cycle, high energy consumption, and the risk of loss of heat-sensitive active ingredients during repeated steaming and drying. Microbial fermentation technology, as an important pathway in modern Chinese medicine processing, utilizes bacterial enzyme systems to biotransform medicinal components, effectively achieving "detoxification and enhanced efficacy." It also breaks down large molecules into more easily absorbed smaller molecules, significantly improving bioavailability. However, due to the pharmacological components of Polygonatum sibiricum, it has a certain inhibitory effect on lactic acid bacteria, hindering its microbial fermentation. Summary of the Invention

[0004] In view of this, the present invention provides a method for preparing modified Stropharia macrocarpa crude polysaccharide. The modified Stropharia macrocarpa crude polysaccharide prepared by the method can significantly reduce the inhibitory effect of Polygonatum on the proliferation of lactic acid bacteria and accelerate the growth of lactic acid bacteria. Adding it to the Polygonatum fermentation system can effectively improve the numbing taste of Polygonatum and enhance its functional quality.

[0005] To achieve the above objectives, the present invention provides the following technical solution: The present invention provides a method for preparing modified *Stropharia macrocarpa* crude polysaccharide, comprising: irradiating the crude polysaccharide of *Stropharia macrocarpa*, and subjecting the irradiated crude polysaccharide of *Stropharia macrocarpa* to ultra-high pressure treatment to obtain modified crude polysaccharide of *Stropharia macrocarpa*; wherein the irradiation dose is 5~15 kGy; the ultra-high pressure treatment pressure is 300~600 MPa, and the ultra-high pressure treatment time is 20~40 min.

[0006] Preferably, the radioactive source for the irradiation treatment is a Co60 isotope.

[0007] This invention provides a modified crude polysaccharide of *Agaricus bisporus* prepared by the aforementioned preparation method.

[0008] This invention provides an application of the modified *Stropharia macrocarpa* crude polysaccharide in lactic acid bacteria fermentation of *Polygonatum sibiricum*.

[0009] Preferably, the lactic acid bacteria include Streptococcus thermophilus and / or Lactobacillus delbrueckii subsp. bulgaricus.

[0010] This invention provides a method for preparing Polygonatum sibiricum fermented raw pulp, comprising the following steps: fermenting Polygonatum sibiricum raw pulp under the action of lactic acid bacteria and the modified Pleurotus ostreatus crude polysaccharide to obtain Polygonatum sibiricum fermented raw pulp.

[0011] Preferably, the Polygonatum slurry comprises Polygonatum and water, and the mass ratio of Polygonatum to water in the Polygonatum slurry is 1:(5~20); in the fermentation system, the initial concentration of the lactic acid bacteria is 6.5~8.5 1g CFU / mL, and the initial concentration of the modified Pleurotus ostreatus crude polysaccharide is 1~10 mg / mL.

[0012] Preferably, the lactic acid bacteria include Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus; the ratio of viable Streptococcus thermophilus to Lactobacillus delbrueckii subsp. bulgaricus is 1:3.

[0013] Preferably, the fermentation temperature is 35~39℃; the fermentation time is 12~96 h.

[0014] Preferably, the fermentation process further includes homogenization.

[0015] Compared with existing technologies, this invention has the following advantages: This invention provides a method for preparing modified *Stropharia macrocarpa* crude polysaccharide, comprising: irradiating the crude polysaccharide with irradiation, and subjecting the irradiated crude polysaccharide to ultra-high pressure treatment to obtain modified *Stropharia macrocarpa* crude polysaccharide; the irradiation dose is 5-15 kGy; the ultra-high pressure treatment pressure is 300-600 MPa, and the ultra-high pressure treatment time is 20-40 min. This invention modifies *Stropharia macrocarpa* crude polysaccharide through a combination of irradiation and ultra-high pressure treatment, which not only effectively reduces the molecular weight of the polysaccharide but also improves its biological activity. The modified *Stropharia macrocarpa* crude polysaccharide prepared by this method can significantly reduce the inhibitory effect of *Polygonatum sibiricum* raw material on the proliferation of lactic acid bacteria and accelerate the growth of lactic acid bacteria. Adding it to the *Polygonatum sibiricum* fermentation system can effectively improve the numbing sensation of *Polygonatum sibiricum* and simultaneously enhance its functional quality.

[0016] This invention provides an application of the modified *Scutellaria baicalensis* crude polysaccharide in the fermentation of *Polygonatum sibiricum* using lactic acid bacteria. Addressing key industrial challenges in the modern processing of *Polygonatum sibiricum*, a resource used for both food and medicine, this invention proposes for the first time a processing strategy of fermenting *Polygonatum sibiricum* pulp with lactic acid bacteria and adding polysaccharides. By adding *Scutellaria baicalensis* polysaccharides, the antibacterial effect of the *Polygonatum sibiricum* pulp is effectively alleviated, and the proliferation of lactic acid bacteria is promoted, effectively solving the technical bottleneck in improving the taste and function of the initial *Polygonatum sibiricum* pulp. This invention utilizes the metabolic activity of lactic acid bacteria to significantly reduce the numbing sensation on the tongue of the *Polygonatum sibiricum* pulp, balancing the sweet and sour flavor, thereby improving the overall sensory quality. Simultaneously, the fermentation process also promotes the accumulation of active ingredients such as saponins, total phenols, and total flavonoids in the *Polygonatum sibiricum* pulp, reduces cholesterol content, and enhances the inhibitory ability of the *Polygonatum sibiricum* pulp against α-glucosidase and its antioxidant properties. This process is rationally designed, with no exogenous components added throughout, significantly improving the functional characteristics of the *Polygonatum sibiricum* pulp while optimizing its taste, achieving a comprehensive upgrade in the sensory quality and functional value of the fermented *Polygonatum sibiricum* pulp. Attached Figure Description

[0017] Figure 1 is a sensory comparison radar chart of Polygonatum fermentation pulp; Figure 2 is a comparison chart of the inhibitory ability of Polygonatum fermentation pulp on α-glucosidase; Figure 3 is a comparison chart of the antioxidant capacity (ABTS) of Polygonatum fermentation pulp; Figure 4 is a comparison chart of the molecular weight of polysaccharides in Polygonatum fermentation pulp of Examples 1-8. Detailed Implementation

[0018] This invention provides a method for preparing modified *Stropharia macrocarpa* crude polysaccharide, comprising: irradiating the crude polysaccharide with irradiation, and subjecting the irradiated crude polysaccharide to ultra-high pressure treatment to obtain modified *Stropharia macrocarpa* crude polysaccharide; wherein the irradiation dose is 5-15 kGy; the ultra-high pressure treatment pressure is 300-600 MPa, and the ultra-high pressure treatment time is 20-40 min.

[0019] In this invention, the crude polysaccharide from *Stropharia macrocarpa* can be prepared in-house or directly derived from existing *Stropharia macrocarpa* crude polysaccharides. When prepared in-house, the crude polysaccharide is preferably extracted from *Stropharia macrocarpa*, which may include open-capped, commercially worthless *Stropharia macrocarpa* (products directly eliminated at the production site and not circulated in the market). *Stropharia macrocarpa* polysaccharide has a positive promoting effect on the proliferation of probiotics. This invention innovatively employs irradiation combined with ultra-high pressure technology to modify this type of *Stropharia macrocarpa* polysaccharide and adds it to the fermentation system of *Polygonatum sibiricum* pulp, which can significantly promote the fermentation and proliferation of lactic acid bacteria. Examples of this invention demonstrate that, compared to *Pleurotus ostreatus*, fermenting *Polygonatum sibiricum* with modified crude polysaccharide prepared from *Stropharia macrocarpa* yields better sensory scores, physicochemical indicators, and functional indicators. This invention does not impose any special limitations on the extraction method of the crude polysaccharide from *Stropharia macrocarpa*; conventional crude polysaccharide extraction methods in the art are acceptable. As an optional implementation, the extraction method of crude polysaccharide from *Stropharia macrocarpa* includes drying, pulverizing, ultrasonically extracting, concentrating, precipitating with alcohol, and filtering to obtain crude polysaccharide. The drying temperature is preferably 50-60℃, more preferably 55℃; the particle size of the pulverized material is preferably 100-150 μm; the ultrasonic extraction power is preferably 200-400 W, more preferably 250-350 W, and most preferably 300 W; the ultrasonic extraction time is preferably 1-2 h, more preferably 1.5 h; the material-to-liquid ratio of the ultrasonic extraction is preferably 1:10-30 (w / v), more preferably 1:15-25, and most preferably 1:20. The concentration method is vacuum concentration at 50℃. The concentrated liquid is then precipitated with alcohol at four times its volume and filtered to obtain crude polysaccharide from *Stropharia macrocarpa*. The crude polysaccharide is preferably further dried, and the moisture content after drying is preferably 10%-20%, more preferably 12%-18%, and most preferably 15%.

[0020] After obtaining the crude polysaccharide from *Stropharia macrocarpa*, this invention subjectes the crude polysaccharide to irradiation treatment. The preferred irradiation dose is 6-12 kGy, but can be 7, 8, 9, 10, or 11 kGy. The preferred radioactive source for the irradiation treatment is Co60 isotope.

[0021] After irradiation treatment, the present invention preferably prepares a solution of the irradiated *Stropharia macrocarpa* crude polysaccharide with water, and then subjectes the solution to ultra-high pressure treatment to obtain modified *Stropharia macrocarpa* crude polysaccharide. The concentration of *Stropharia macrocarpa* crude polysaccharide in the solution is preferably 0.1~0.2 g / mL, more preferably 0.15 g / mL. Preparing the solution results in a more pronounced ultra-high pressure effect and facilitates the addition of *Stropharia macrocarpa* polysaccharide in a completely dissolved state. The solvent of the solution in the present invention preferably includes water. The mass percentage of the irradiated *Stropharia macrocarpa* crude polysaccharide in the solution is preferably 10%~20%, more preferably 13%~18%, and most preferably 16.67%. The pressure of the ultra-high pressure treatment is preferably 350~550 MPa, but can be 400, 450, or 500 MPa. The ultra-high pressure treatment time is preferably 25~35 min, more preferably 30 min. This invention further modifies the crude polysaccharide of *Stropharia masticata* (a type of mushroom) by ultra-high pressure treatment after irradiation. This specific combination of irradiation and ultra-high pressure treatment effectively reduces the molecular weight of the polysaccharide, optimizes its molecular structure and bioactivity, and enriches the modified *Stropharia masticata* crude polysaccharide with prebiotics. Through repeated experiments, this invention demonstrates that adding the modified polysaccharide to the *Polygonatum sibiricum* fermentation system significantly reduces the inhibitory effect of *Polygonatum sibiricum* raw pulp on lactic acid bacteria proliferation and accelerates lactic acid bacteria growth, thereby effectively improving the sensory score, physicochemical indicators, and functional indicators of the *Polygonatum sibiricum* fermentation raw pulp.

[0022] This invention provides a modified crude polysaccharide of *Agaricus macrocarpa* prepared by the aforementioned method.

[0023] The modified *Stropharia spp.* crude polysaccharide of this invention preferably has a molecular weight distribution range of 5000~20000 Da, more preferably 8000~18000 Da, and even more preferably 8237~17372 Da. This modified *Stropharia spp.* crude polysaccharide can effectively promote the proliferation of lactic acid bacteria and is rich in prebiotics and other active ingredients, which can improve the flavor and taste of *Polygonatum sibiricum* pulp and increase the content of its functional active ingredients. Embodiments of this invention demonstrate that adding the modified *Stropharia spp.* crude polysaccharide to the *Polygonatum sibiricum* fermentation system not only completely removes the unpleasant numbing sensation of *Polygonatum sibiricum* (the numbing sensation is mainly caused by calcium oxalate), but also achieves a harmonious and palatable sweet and sour ratio. Furthermore, it significantly improves several functional indicators: the content of active ingredients such as saponins, total phenols, and total flavonoids increases significantly, while the cholesterol content decreases; simultaneously, the α-glucosidase inhibition capacity and antioxidant capacity are also enhanced, achieving a comprehensive upgrade in the sensory quality and functional value of the *Polygonatum sibiricum* fermentation pulp.

[0024] In view of this, the present invention provides an application of the modified Pleurotus ostreatus crude polysaccharide in lactic acid bacteria fermentation of Polygonatum sibiricum.

[0025] In this invention, the lactic acid bacteria preferably include *Streptococcus thermophilus* and / or *Lactobacillus delbrueckii* subsp. bulgaricus. The *Polygonatum* is preferably a dried stem tuber obtained through a single steaming and processing method. This invention does not specifically limit the single steaming and processing method; any conventional single steaming and processing method for *Polygonatum* in the art can be used. The preferred varieties of *Polygonatum* include *Polygonatum sibiricum* and / or *Polygonatum multiflorum*. This invention utilizes the modified *Scutellaria baicalensis* crude polysaccharide for lactic acid bacteria fermentation of *Polygonatum*, which can improve at least one of the following properties of *Polygonatum*: reducing calcium oxalate content, destroying the calcium oxalate form, weakening the numbing sensation of *Polygonatum* raw pulp, reducing cholesterol content, increasing saponin content, increasing total phenolic content, increasing total flavonoid content, improving α-glucosidase inhibition ability, and antioxidant capacity.

[0026] This invention provides a method for preparing Polygonatum sibiricum fermented raw pulp, comprising the following steps: fermenting Polygonatum sibiricum raw pulp under the action of lactic acid bacteria and the modified Pleurotus ostreatus crude polysaccharide to obtain Polygonatum sibiricum fermented raw pulp.

[0027] This invention preferably involves pulping Polygonatum sibiricum to obtain Polygonatum sibiricum pulp. The preferred Polygonatum sibiricum is the same as described above and will not be repeated here. This invention does not impose any special limitation on the pulping method; any conventional Polygonatum sibiricum pulping method in the art is acceptable. As an optional embodiment, Polygonatum sibiricum can be soaked in water, rehydrated, and then homogenized to obtain Polygonatum sibiricum pulp. The preferred mass ratio of Polygonatum sibiricum to water is 1:5~20, more preferably 1:7~15, even more preferably 1:8~13, and most preferably 1:10. The preferred soaking and rehydration time is 5~10 h, more preferably 6~8 h, and most preferably 7 h. The Polygonatum sibiricum pulp prepared by this method ensures suitable viscosity and fluidity, which is beneficial for subsequent commercial processing.

[0028] After obtaining the Polygonatum sibiricum pulp, the present invention ferments the Polygonatum sibiricum pulp under the action of lactic acid bacteria and the modified Pleurotus ostreatus crude polysaccharide to obtain Polygonatum sibiricum fermented pulp. The Polygonatum sibiricum pulp includes Polygonatum sibiricum and water, and is obtained by soaking dried Polygonatum sibiricum slices, rehydrating them, and then homogenizing them; the mass ratio of Polygonatum sibiricum to water in the Polygonatum sibiricum pulp is 1:(5~20), more preferably 1:(8~15), and most preferably 1:10; in the fermentation system of the present invention, the initial concentration of the lactic acid bacteria is 6.5~8.5 1 g CFU / mL, more preferably 7.5 1 g CFU / mL, and the initial concentration of the modified Pleurotus ostreatus crude polysaccharide is preferably 1~10 mg / mL, more preferably 3~6 mg / mL, and most preferably 4.5 mg / mL. In the embodiments of the present invention, the added concentration of the modified Pleurotus ostreatus crude polysaccharide is 0.15 g / mL, and the added amount is 2%, 3%, 4%, or 5% of the weight of the Polygonatum sibiricum pulp. The concentration of the added lactic acid bacteria is 8.5 lg CFU / mL, and the amount added is 6%, 8%, 10%, or 12% of the weight of the Polygonatum sibiricum pulp. The lactic acid bacteria preferably include Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus; the ratio of viable bacteria of Streptococcus thermophilus to Lactobacillus delbrueckii is preferably 1:3. This ratio is beneficial for promoting the conversion and release of active ingredients in Polygonatum sibiricum, and for optimizing the taste and enhancing the function of the Polygonatum sibiricum pulp. This invention does not impose any special limitations on the source of the Streptococcus thermophilus and Lactobacillus delbrueckii; they can be purchased through conventional commercial channels. In this invention, the fermentation temperature is preferably 35-39℃, more preferably 36-38℃, and most preferably 37℃; the fermentation time is preferably 12-96 h, more preferably 24-72 h, and even more preferably 36-48 h. After fermentation, homogenization is preferably also performed. The homogenization pressure is preferably 15–30 MPa, more preferably 15–25 MPa, and most preferably 20 MPa. The homogenization cycle is preferably 3–8 cycles, more preferably 3–5 cycles, and even more preferably 4 cycles. The homogenization process improves the physical stability and texture uniformity of the Polygonatum fermentation pulp. After homogenization, the Polygonatum fermentation pulp is preferably packaged in aluminum foil bags for sterilization at 121°C for 30 minutes. This sterilization process enhances the commercial applicability of the product, ensures its commercial storage and distribution, and provides technical support for its market promotion.

[0029] This invention reduces the inhibitory effect of Polygonatum sibiricum extract on lactic acid bacteria proliferation by adding modified Pleurotus ostreatus polysaccharide, effectively promoting lactic acid bacteria growth. Simultaneously, through the metabolic activity of lactic acid bacteria, it alleviates the numbing sensation on the tongue, balances the sweet and sour flavor, improves the smoothness of the texture, and enhances the overall sensory quality of the product. Furthermore, the fermentation process promotes the conversion and release of active ingredients in Polygonatum sibiricum, thus overcoming the technical bottlenecks in optimizing the taste and enhancing the functionality of the initial Polygonatum sibiricum extract. The fermented Polygonatum sibiricum prepared using the method of this invention has a balanced sweet and sour taste and excellent palatability. Compared with the initial Polygonatum sibiricum without the addition of modified polysaccharides and lactic acid bacteria (Comparative Example 1), it is comprehensively optimized: the saponin content is increased by 16.51%~36.61%, the total phenol content is increased by 80.00%~135.00%, the total flavonoid content is increased by 51.49%~133.89%, and the cholesterol content is reduced by 17.61%~49.66%; the α-glucosidase inhibition ability is increased by 15.15%~48.36%, the antioxidant capacity (ABTS free radical scavenging ability) is increased by 55.17%~108.28%, and the product stability is improved by 18.82%~22.10%.

[0030] To further illustrate the present invention, the solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0031] Experimental Materials: 1. Preparation of Polygonatum sibiricum pulp: Polygonatum sibiricum stems were steamed thoroughly for about 5 hours, then dried at 55°C until the moisture content was about 15%, resulting in Polygonatum sibiricum stems that had undergone one steaming and one drying process. The dried Polygonatum sibiricum stems were sliced, and then 10 times their weight of water were added for rehydration. After homogenization, Polygonatum sibiricum pulp was obtained. In this embodiment of the invention, three types of Polygonatum sibiricum pulp were prepared based on different slice thicknesses and rehydration times.

[0032] Polygonatum slurry 1: slice thickness 5 mm, rehydration time 8 h; Polygonatum slurry 2: slice thickness 8 mm, rehydration time 5 h; Polygonatum slurry 3: slice thickness 10 mm, rehydration time 10 h.

[0033] 2. Preparation of crude polysaccharide from *Stropharia macrocarpa*: *Stropharia macrocarpa* mushrooms with open caps and no commercial value were selected. The mushrooms were dried, pulverized, subjected to ultrasonic thermal extraction, concentrated, precipitated with alcohol, and filtered to obtain *Stropharia macrocarpa* polysaccharide. Specifically, conventional polysaccharide extraction methods were used for crude polysaccharide extraction: Fresh *Stropharia macrocarpa* mushrooms were dried at 55℃, pulverized, and passed through an 80-mesh sieve. An appropriate amount of mushroom powder was added to distilled water at a material-to-liquid ratio of 1:20, and ultrasonic extraction was performed at 70℃ and 300W for 1.5 hours. After centrifugation, the supernatant was collected, concentrated under reduced pressure at 50℃, and finally precipitated with four times its volume of ethanol. The mixture was then filtered to obtain crude polysaccharide from *Stropharia macrocarpa*. The crude polysaccharide was further dried, with a final water content of 15%.

[0034] 3. Preparation of seed bacterial suspension: ① Activation of bacterial strain: Activation of bacterial strain: On a clean bench, take 1 g each of Streptococcus thermophilus and Lactobacillus bulgaricus (both purchased from Shaanxi Yunqi Biotechnology Co., Ltd., catalog numbers YQ20250213 and YQ20250215 respectively), transfer them to 10 mL of liquid MRS medium, gently shake to completely dissolve the bacterial powder, and incubate at 37 ℃ and 180 r / min for 2 h.

[0035] ② Preparation of seed bacterial suspension: On a clean bench, 1 mL of activated bacterial suspension was placed in 50 mL of liquid MRS medium and incubated at 37°C until OD500 reached. 600 At approximately 1.0, *Streptococcus thermophilus* seed suspension and *Lactobacillus bulgaricus* seed suspension were prepared. The concentration of the bacterial suspension was adjusted to 8.5 lg CFU / mL for subsequent inoculation and fermentation of *Polygonatum sibiricum* syrup.

[0036] Liquid MRS medium: containing 10 g / L peptone, 10 g / L beef extract, 10 g / L glucose, 5 g / L yeast extract, 3.02 g / L sodium acetate, 1.16 g / L dipotassium hydrogen phosphate, 0.05 g / L magnesium sulfate, 2 g / L triammonium citrate, 0.03 g / L manganese sulfate, and 1 mL Tween 80, with a pH of 6.0–6.4, sterilized at 121°C for 30 min to obtain liquid MRS medium.

[0037] Example 1: A method for preparing Polygonatum sibiricum fermented raw pulp, the steps are as follows: 1. Preparation of modified Pleurotus ostreatus crude polysaccharide solution: Irradiation combined with ultra-high pressure modification of Pleurotus ostreatus crude polysaccharide: First, Pleurotus ostreatus crude polysaccharide is treated with a radioactive source Co60 isotope, the irradiation dose is 8 kGy. 1. Irradiation of crude polysaccharide: Add 5 times its weight of water to redissolve the crude polysaccharide, then subject it to ultra-high pressure treatment at 400 MPa for 30 min. Adjust the concentration with water to obtain a modified *Scutellaria baicalensis* crude polysaccharide solution with a concentration of 0.15 g / mL. 2. Fermentation of *Polygonatum sibiricum* raw pulp: Add 3% of the modified *Scutellaria baicalensis* crude polysaccharide solution to the *Polygonatum sibiricum* raw pulp by weight, and add seed culture suspension at an inoculum amount of 8% of the *Polygonatum sibiricum* raw pulp. Ferment at 37℃ for 48 h. The viable cell ratio of *Streptococcus thermophilus* and *Lactobacillus bulgaricus* in the seed culture suspension is 1:3. 3. Homogenization and sterilization: After the fermentation of *Polygonatum sibiricum* raw pulp is completed, subsequent homogenization treatment is performed at 20 MPa for 4 cycles. The pulp is then canned in aluminum foil bags and sterilized at 121℃ for 30 min to obtain *Polygonatum sibiricum* fermented raw pulp.

[0038] Example 2: A method for preparing Polygonatum sibiricum fermented pulp, comprising the following steps: 1. Preparation of modified Pleurotus ostreatus crude polysaccharide solution: The difference from Example 1 is that the irradiation dose is 6 kGy and the ultra-high pressure treatment is 300 MPa; 2. Fermentation of Polygonatum sibiricum pulp: 2% of modified Pleurotus ostreatus crude polysaccharide solution is added to Polygonatum sibiricum pulp, and 6% of seed culture suspension is added as an inoculum. Fermentation is carried out at 37°C for 36 h; the ratio of viable bacteria of Streptococcus thermophilus and Lactobacillus bulgaricus in the seed culture suspension is 1:3; 3. Homogenization and sterilization: After the fermentation of Polygonatum sibiricum pulp is completed, subsequent homogenization treatment is carried out, with 5 cycles of treatment at 20 MPa. The pulp is then bottled in aluminum foil bags and sterilized at 121°C for 30 min to obtain Polygonatum sibiricum fermented pulp.

[0039] Example 3: A method for preparing Polygonatum sibiricum fermented raw pulp, comprising the following steps: 1. Preparation of modified Pleurotus ostreatus crude polysaccharide solution: The difference from Example 1 is that the irradiation dose is 6 kGy; 2. Fermentation of Polygonatum sibiricum raw pulp: 2% of modified Pleurotus ostreatus crude polysaccharide solution is added to Polygonatum sibiricum raw pulp by weight, and seed culture suspension is added by inoculation amount of 12% of Polygonatum sibiricum raw pulp. Fermentation is carried out at 37℃ for 36 h; the ratio of viable bacteria of Streptococcus thermophilus and Lactobacillus bulgaricus in the seed culture suspension is 1:3; 3. Homogenization and sterilization: After the Polygonatum sibiricum raw pulp fermentation is completed, subsequent homogenization treatment is carried out, with 4 cycles of treatment at 25 MPa, and it is bottled in aluminum foil bags and sterilized at 121℃ for 30 min to obtain Polygonatum sibiricum fermented raw pulp.

[0040] Example 4: A method for preparing Polygonatum sibiricum fermented pulp, comprising the following steps: 1. Preparation of modified Pleurotus ostreatus crude polysaccharide solution: The difference from Example 1 is that the irradiation dose is 9 kGy and the ultra-high pressure treatment is 500 MPa; 2. Fermentation of Polygonatum sibiricum pulp: 3% of modified Pleurotus ostreatus crude polysaccharide solution is added to Polygonatum sibiricum pulp, and 10% of seed culture suspension is added as an inoculum. Fermentation is carried out at 37°C for 36 h; the ratio of viable bacteria of Streptococcus thermophilus and Lactobacillus bulgaricus in the seed culture suspension is 1:3; 3. Homogenization and sterilization: After the fermentation of Polygonatum sibiricum pulp is completed, subsequent homogenization treatment is carried out, with 4 cycles of treatment at 15 MPa. The pulp is then bottled in aluminum foil bags and sterilized at 121°C for 30 min to obtain Polygonatum sibiricum fermented pulp.

[0041] Example 5: A method for preparing Polygonatum sibiricum fermented pulp, comprising the following steps: 1. Preparation of modified Pleurotus ostreatus crude polysaccharide solution: The difference from Example 1 is that the irradiation dose is 12 kGy and the ultra-high pressure treatment is 600 MPa; 2. Fermentation of Polygonatum sibiricum pulp: 5% of modified Pleurotus ostreatus crude polysaccharide solution is added to Polygonatum sibiricum pulp, and 10% of seed culture suspension is added as an inoculum. Fermentation is carried out at 37°C for 12 h. The ratio of viable bacteria of Streptococcus thermophilus and Lactobacillus bulgaricus in the seed culture suspension is 1:3; 3. Homogenization and sterilization: After the fermentation of Polygonatum sibiricum pulp is completed, subsequent homogenization treatment is carried out, with 4 cycles of treatment at 15 MPa. The pulp is then bottled in aluminum foil bags and sterilized at 121°C for 30 min to obtain Polygonatum sibiricum fermented pulp.

[0042] Example 6: A method for preparing Polygonatum sibiricum fermented pulp, comprising the following steps: 1. Preparation of modified Pleurotus ostreatus crude polysaccharide solution: The difference from Example 1 is that the irradiation dose is 12 kGy and the ultra-high pressure treatment is 500 MPa; 2. Fermentation of Polygonatum sibiricum pulp: 4% of modified Pleurotus ostreatus crude polysaccharide solution is added to Polygonatum sibiricum pulp, and 12% of seed culture suspension is added as an inoculum. Fermentation is carried out at 37°C for 72 h. The ratio of viable bacteria of Streptococcus thermophilus and Lactobacillus bulgaricus in the seed culture suspension is 1:3; 3. Homogenization and sterilization: After the fermentation of Polygonatum sibiricum pulp is completed, subsequent homogenization treatment is carried out, with 5 cycles of treatment at 15 MPa. The pulp is then bottled in aluminum foil bags and sterilized at 121°C for 30 min to obtain Polygonatum sibiricum fermented pulp.

[0043] Example 7: A method for preparing Polygonatum sibiricum fermented pulp, comprising the following steps: 1. Preparation of modified Pleurotus ostreatus crude polysaccharide solution: The difference from Example 1 is that the irradiation dose is 10 kGy and the ultra-high pressure treatment is 600 MPa; 2. Fermentation of Polygonatum sibiricum pulp: 5% of modified Pleurotus ostreatus crude polysaccharide solution is added to Polygonatum sibiricum pulp, and 10% of seed culture suspension is added as an inoculum. Fermentation is carried out at 37°C for 36 h. The ratio of viable bacteria of Streptococcus thermophilus and Lactobacillus bulgaricus in the seed culture suspension is 1:3; 3. Homogenization and sterilization: After the fermentation of Polygonatum sibiricum pulp is completed, subsequent homogenization treatment is carried out, with 3 cycles of treatment at 15 MPa. The pulp is then bottled in aluminum foil bags and sterilized at 121°C for 30 min to obtain Polygonatum sibiricum fermented pulp.

[0044] Example 8: A method for preparing Polygonatum sibiricum fermented pulp, comprising the following steps: 1. Preparation of modified Pleurotus ostreatus crude polysaccharide solution: The difference from Example 1 is that the irradiation dose is 7 kGy and the ultra-high pressure treatment is 300 MPa; 2. Fermentation of Polygonatum sibiricum pulp: 4% of modified Pleurotus ostreatus crude polysaccharide solution is added to Polygonatum sibiricum pulp, and 6% of seed culture suspension is added as an inoculum. Fermentation is carried out at 37°C for 24 h. The ratio of viable bacteria of Streptococcus thermophilus and Lactobacillus bulgaricus in the seed culture suspension is 1:3; 3. Homogenization and sterilization: After the fermentation of Polygonatum sibiricum pulp is completed, subsequent homogenization treatment is carried out, with 4 cycles of treatment at 20 MPa. The pulp is then bottled in aluminum foil bags and sterilized at 121°C for 30 min to obtain Polygonatum sibiricum fermented pulp.

[0045] The preparation methods of comparative examples 1 to 8 of Polygonatum sibiricum fermented raw pulp are shown in Table 1.

[0046] Table 1 Comparative settings of Polygonatum sibiricum fermentation raw pulp and preparation methods

[0047] Test Example 1: Sensory quality, physicochemical indicators, functional indicators, and stability of the Polygonatum sibiricum fermented raw pulp from the Example and Comparative examples were determined. The specific test indicators are as follows.

[0048] 1. Sensory quality: Multi-dimensional sensory evaluation is adopted, covering odor, color, taste, texture, sweetness and sourness and acceptability. The sensory evaluation standards of Polygonatum fermentation pulp are shown in Table 2.

[0049] Table 2 Sensory Evaluation Criteria for Polygonatum Fermentation Mash

[0050] 2. Physicochemical indicators: including titratable acid, pH value, soluble solids, and the content of active ingredients such as saponins, total phenols and total flavonoids, as well as cholesterol content.

[0051] Titratable acids shall be tested according to the method of GB / T 12293-1990, and soluble solids shall be tested according to the method of GB / T 10788-1989.

[0052] Saponin content detection: The sample was extracted with ethanol, evaporated to dryness, and tested using diosgenin as a standard with vanillin-glacial acetic acid solution for color development.

[0053] Total phenol content detection: After alcohol extraction, the sample was tested using the Folin-Ciocalteu colorimetric method with gallic acid as the standard.

[0054] Total flavonoid content detection: After alcohol extraction, the sample was tested using the sodium nitrite-aluminum nitrate method with catechin as the standard.

[0055] Cholesterol content detection: The detection method is o-phthalaldehyde solution-concentrated sulfuric acid reaction.

[0056] Calcium oxalate content detection: Soluble oxalic acid was removed by washing with water, and the raw pulp of Polygonatum sibiricum was acid-hydrolyzed. The oxalic acid content in the raw pulp of Polygonatum sibiricum was detected by liquid chromatography.

[0057] 3. Functional indicators: mainly α-glucosidase inhibition capacity and antioxidant capacity.

[0058] The inhibitory effect of Polygonatum odoratum extract on α-glucosidase was evaluated by colorimetric analysis using an enzyme-linked immunosorbent assay (ELISA) reader.

[0059] Antioxidant capacity was measured using an ABTS kit and expressed as Trolox antioxidant capacity equivalents.

[0060] 4. Stability test: Take a certain amount of sample, centrifuge (2000 rpm, 10 min), and calculate the percentage of the total volume of the separated water layer.

[0061] All the above experiments were repeated three times, and the average value of the results was taken.

[0062] 5. The sensory indicators (odor, color, taste, texture, sweetness and sourness, acceptability and total score) of Polygonatum sibiricum pulp are shown in Table 3, and the corresponding flavor radar chart is shown in Figure 1.

[0063] The physicochemical and functional indicators (titerable acid, pH value, soluble solids, lactic acid bacteria count, saponins, total phenols, total flavonoids, calcium oxalate, cholesterol content and stability) of Polygonatum sibiricum raw pulp are shown in Table 4.

[0064] Table 3 Sensory evaluation results of Polygonatum sibiricum fermented raw pulp

[0065] Table 4 Physicochemical and Functional Indicators of Polygonatum Fermentation Proceedings

[0066] The results show that the preparation method of Polygonatum sibiricum fermented pulp provided in this embodiment of the invention significantly reduces the calcium oxalate content in the Polygonatum sibiricum pulp (Table 4), improves the flavor and taste of the pulp (Figure 1), and increases the content of its functional active ingredients, thereby comprehensively enhancing the functional characteristics of the product (Figures 2 and 3). In addition, the homogenization treatment used in the process effectively improves the stability of the pulp (Table 4), which provides a key guarantee for extending the shelf life of the product and realizing commercial production.

[0067] Compared to Comparative Example 1, the saponin content, total phenol content, and total flavonoid content of the Polygonatum fermented raw pulp prepared in Examples 1-8 increased by 16.51%-36.61%, 80.00%-135.00%, and 51.49%-133.89%, while cholesterol content decreased by 25.36%-43.52% and calcium oxalate content decreased by 83.42%-85.52%. Furthermore, the Polygonatum raw pulp showed increased α-glucosidase inhibition by 15.15%-48.36%, increased antioxidant capacity (ABTS free radical scavenging capacity) by 55.17%-108.28%, and improved stability by 18.82%-22.10%. It is noteworthy that while Comparative Example 2 (without polysaccharide addition) and Comparative Example 3 (with only Streptococcus thermophilus addition) showed limited improvements in active ingredients and efficacy, their flavor and taste scores were significantly lower. This may be because the substrate without added polysaccharides in Comparative Example 2 was not conducive to the growth of lactic acid bacteria, while the single-strain fermentation in Comparative Example 3 was insufficient to form rich flavor compounds. Comparative Example 5, the unhomogenized pulp, had the worst pulp stability. Comparative Example 6 added unmodified (i.e., untreated by irradiation and ultra-high pressure) crude polysaccharide from *Scutellaria baicalensis* during the fermentation process. Compared to Comparative Example 1, this group promoted lactic acid bacteria proliferation, increased the content of functional components, and improved functional quality, but all results were inferior to the examples. This further verifies the positive impact of polysaccharide modification on *Scutellaria baicalensis* fermentation. Comparative Example 7 added modified *Pleurotus ostreatus* polysaccharide during fermentation, but its fermentation effect was still far inferior to Example 1 using *Scutellaria baicalensis* polysaccharide, which may be related to the latter's superior prebiotic properties. Comparative Example 8 directly inoculated *Lactobacillus plantarum* into the *Scutellaria baicalensis* pulp for fermentation, but the lactic acid bacteria failed to proliferate effectively in the system, resulting in a low colony count and insufficient accumulation of active ingredients. The improvement in functional quality was also inferior to Examples 1-8.

[0068] As shown in Figure 4, the molecular weight distribution of the polysaccharide from *Schefflera heptaphylla* treated with irradiation and ultra-high pressure ranged from 8237 to 17372 Da. The results indicate that higher irradiation doses and high-pressure treatment (Examples 1 and 4-7) can further reduce the molecular weight of the polysaccharide (8287-10579 Da). It is generally believed that lower molecular weight polysaccharides are more readily utilized by lactic acid bacteria, thereby promoting their proliferation. However, the viable counts of lactic acid bacteria in the embodiments of this invention were ranked as follows: Example 4 > Example 1 > Example 2 > Example 5 > Example 6 > Example 8 > Example 7. It is noteworthy that although the polysaccharide molecular weights of Examples 6 and 5 were low (8287 Da and 8846 Da, respectively), their lactic acid bacteria proliferation effects were not optimal. This may be related to the effect of excessively high irradiation doses on the polysaccharide structure or biological activity; the specific mechanism requires further investigation. In the experiment, various lactic acid bacteria fermentation combinations were screened, and it was ultimately determined that a 1:3 mixture of *Streptococcus thermophilus* and *Lactobacillus bulgaricus* was most suitable for the fermentation of *Polygonatum sibiricum* extract.

[0069] Based on the above results, Example 4 determined the optimal fermentation process parameters: A crude polysaccharide solution of *Scutellaria baicalensis* obtained by irradiation with 9 kGy combined with ultra-high pressure treatment at 500 MPa was added to the initial *Polygonatum sibiricum* slurry at a dosage of 3%, followed by inoculation with a mixed bacterial suspension (*Streptococcus thermophilus*: *Lactobacillus bulgaricus* = 1:3) at a dosage of 10%, and fermented at 37°C for 36 h. After homogenization, the fermentation product exhibited the best overall quality in terms of flavor, content of functional components, efficacy, and stability.

[0070] Mechanism analysis shows that the addition of modified polysaccharides from *Stropharia macrocarpa* effectively promotes the growth of lactic acid bacteria, accelerates acid production, and lowers pH, while also partially consuming soluble solids in the system. However, if *Streptococcus thermophilus* (as in Comparative Example 3) is used alone, its vigorous metabolism, while lowering pH, can easily lead to excessive acidity and flavor imbalance in the product. Therefore, this invention employs a modified polysaccharide addition combined with a synergistic fermentation strategy of *Streptococcus thermophilus* and *Lactobacillus bulgaricus*. This promotes the growth of lactic acid bacteria while avoiding the metabolic defects of single-strain bacteria. Through synergistic effects, it not only enhances the content of functional components and product efficacy but also significantly improves the overall flavor and taste, providing a stable and efficient solution for the industrialization of *Polygonatum sibiricum* fermented pulp. In summary, this invention, through reasonable process design, achieves simultaneous improvement in the taste and functional properties of *Polygonatum sibiricum* pulp.

[0071] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing modified crude polysaccharide from *Stropharia macrocarpa*, characterized in that, include: The crude polysaccharide of *Stropharia macrocarpa* was irradiated, and the irradiated crude polysaccharide was subjected to ultra-high pressure treatment to obtain modified crude polysaccharide of *Stropharia macrocarpa*. The irradiation dose is 5~15 kGy; the ultra-high pressure treatment pressure is 300~600 MPa, and the ultra-high pressure treatment time is 20~40 min.

2. The preparation method according to claim 1, characterized in that, The radioactive source used for the irradiation treatment was a Co60 isotope.

3. A modified crude polysaccharide of *Agaricus macrocarpa* prepared by the preparation method described in claim 1 or 2.

4. The application of the modified Pleurotus ostreatus crude polysaccharide as described in claim 3 in the fermentation of Polygonatum sibiricum by lactic acid bacteria.

5. The application according to claim 4, characterized in that, The lactic acid bacteria include Streptococcus thermophilus and / or Lactobacillus delbrueckii subsp. bulgaricus.

6. A method for preparing Polygonatum sibiricum fermented raw pulp, characterized in that, Includes the following steps: The raw material of Polygonatum odoratum was fermented under the action of lactic acid bacteria and the modified Pleurotus ostreatus crude polysaccharide as described in claim 3 to obtain Polygonatum odoratum fermented raw material.

7. The preparation method according to claim 6, characterized in that, The Polygonatum slurry comprises Polygonatum and water, and the mass ratio of Polygonatum to water in the Polygonatum slurry is 1:(5~20); in the fermentation system, the initial concentration of the lactic acid bacteria is 6.5~8.5 1g CFU / mL, and the initial concentration of the modified Pleurotus ostreatus crude polysaccharide is 1~10 mg / mL.

8. The preparation method according to claim 6, characterized in that, The lactic acid bacteria include Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus; the ratio of viable Streptococcus thermophilus to Lactobacillus delbrueckii subsp. bulgaricus is 1:

3.

9. The preparation method according to claim 6, characterized in that, The fermentation temperature is 35~39℃; the fermentation time is 12~96 h.

10. The preparation method according to any one of claims 6 to 9, characterized in that, After fermentation, homogenization is also performed.

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