Method for preparing ganoderma polysaccharide rich in mannan-galactan structure by using aureobacterium bacteria GS-7 and ganoderma lucidum YJ0724 and application thereof

Abstract

The application discloses a method for preparing ganoderma polysaccharide rich in mannan-galactan structure by using a bacterium of the genus chryseobacterium GS-7 and ganoderma lucidum YJ0724 and application thereof, and relates to the technical field of microbial fermentation, and comprises the following steps: preparing a seed solution of the bacterium of the genus chryseobacterium GS-7 and a seed solution of the ganoderma lucidum YJ0724; a. inoculating the seed solution of the bacterium of the genus chryseobacterium GS-7 and the seed solution of the ganoderma lucidum YJ0724 into a fermentation medium at the same time for co-culturing to obtain a fermentation liquor containing ganoderma polysaccharide rich in mannan-galactan structure. The obtained ganoderma polysaccharide rich in mannan-galactan structure can improve immunoregulatory activity.

Description

Technical Field

[0001] This application relates to the field of microbial fermentation technology, and in particular to a method for preparing Ganoderma lucidum polysaccharides rich in mannogalactan structure from Chlorella vulgaris GS-7 and Ganoderma lucidum YJ0724, and its application. Background Technology

[0002] Ganoderma lucidum ( Ganoderma lucidum Ganoderma lucidum, commonly known as Lingzhi, belongs to the Basidiomycota, Agaricomycetes, Apophyceales, Ganodermataceae, and Ganoderma genus. It is a famous medicinal fungus in my country with over 2000 years of history, possessing effects such as "tonifying qi, calming the mind, and strengthening the heart," and has extremely high application value in nutrition, health care, and medicine. Modern medical research has also proven that Ganoderma lucidum has varying degrees of efficacy in the treatment and adjuvant therapy of diseases such as chronic bronchitis, coronary heart disease, hepatitis, hyperlipidemia, diabetes, and cancer. Ganoderma lucidum is rich in various bioactive components, with its main active ingredients being Ganoderma lucidum polysaccharides and triterpenoids. Ganoderma lucidum polysaccharides, as its most abundant component, have received considerable attention in recent years. Ganoderma lucidum polysaccharides (GLP) are mainly composed of (1→3), (1→6)-α / β-glucan, glycoproteins, and water-soluble heteropolysaccharides. Ganoderma lucidum (GLP) exerts its anti-cancer effects by inhibiting tumor growth and metastasis, enhancing patients' immune function, and through multiple mechanisms including anti-proliferation, pro-apoptosis, anti-metastasis, anti-angiogenesis, anti-inflammation, anti-oxidation, and immunomodulation. Currently, the market application of Ganoderma lucidum mainly focuses on four aspects: fermentation broth, mycelium, fruiting bodies, and spores, with fruiting bodies and spores being the most prevalent. However, wild Ganoderma lucidum faces harsh growing conditions and severely insufficient yield; artificial cultivation suffers from unstable yields, significant quality variations, long production cycles, large land areas required, and susceptibility to environmental conditions.

[0003] Therefore, developing technologies that offer short production cycles, high yields, stable quality, and industrial-scale production capabilities is crucial. Current research indicates that high yields of Ganoderma lucidum polysaccharides can be obtained through deep liquid fermentation technology, with the mycelium containing even higher levels of major nutrients such as protein, fat, sugar, and ash than the fruiting body. Studies also show significant differences in medicinal efficacy between mycelium, fruiting body, and spore powder.

[0004] Currently, deep fermentation of Ganoderma lucidum mainly focuses on single-strain culture under standard laboratory conditions. In fact, regardless of whether in natural or artificial cultivation, macrofungi, besides being influenced by soil and environment, also form competitive, parasitic, or symbiotic relationships with other microorganisms. Their interactions with other microorganisms in the growth environment are crucial for the activation of their metabolites. In contrast, co-culture can simulate the symbiotic relationships between microorganisms in nature, influencing fungal morphology and developmental patterns through interspecific interactions, activating silent genes / gene clusters to synthesize new metabolites or release extracellular enzymes. Co-culture not only activates the biosynthesis of various secondary metabolites but is also an important means of revealing fungal interactions and the functions of new genes.

[0005] Co-culture is completely different from simply providing a culture medium for signaling molecules. It mainly promotes gene transcription and translation in microorganisms and activates new biosynthetic pathways by simulating the competitive relationships in natural ecosystems.

[0006] Therefore, co-culture technology has been widely used in the discovery of natural products from microbial sources, but there are no examples of applying this technology to the discovery of functional new polysaccharides in large edible and medicinal fungi.

[0007] The information disclosed in the background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art. Summary of the Invention

[0008] This application addresses the aforementioned technical problems by providing a method for preparing Ganoderma lucidum polysaccharides rich in mannogalactan structure using Chlorella vulgaris GS-7 and Ganoderma lucidum YJ0724, and its application. The resulting Ganoderma lucidum polysaccharides rich in mannogalactan structure can enhance immunomodulatory activity.

[0009] This application provides a method for preparing Ganoderma lucidum polysaccharides rich in mannogalactan structure from Chlorella vulgaris GS-7 and Ganoderma lucidum YJ0724, including the following steps:

[0010] Prepare seed culture of *Aureobacterium* GS-7 and seed culture of *Ganoderma lucidum* YJ0724;

[0011] a. The seed culture of Aureobacterium GS-7 and the seed culture of Ganoderma lucidum YJ0724 were simultaneously inoculated into the fermentation medium and co-cultured to obtain a fermentation broth containing Ganoderma lucidum polysaccharides with a mannogalactan structure.

[0012] Alternatively, b) after filtering and sterilizing the fermentation broth of *Aureobacterium* GS-7, it is simultaneously inoculated with *Ganoderma lucidum* seed liquid YJ0724 into a fermentation medium for co-culture to obtain a fermentation broth containing *Ganoderma lucidum* polysaccharides rich in mannogalactan structure.

[0013] Taxonomic nomenclature of *Chlorella* spp. GS-7 Chryseobacterium piperi GS-7; Preservation number CCTCC NO: M 20251587; Classification and nomenclature of Ganoderma lucidum YJ0724 Ganoderma lucidum YJ0724; Accession number CCTCC NO: M 20251588;

[0014] Ganoderma lucidum polysaccharide GG-1c, rich in mannan-galactan structure, with the molecular formula C 194 H 152 O 304 ;

[0015] The structural formula of GG-1c is: Preferably, in method a, the inoculation amount of Ganoderma lucidum YJ0724 seed culture is at least 1% of the total amount of PIB medium, and the inoculation amount of Chlorella spp. GS-7 seed culture is at least 10% of the total amount of PIB medium.

[0016] Preferably, in method b, the inoculum of Chlorella spp. GS-7 filtrate is at least 10% of the total amount of PIB medium, and the inoculum of Ganoderma lucidum seed liquid YJ0724 is at least 10% of the total amount of PIB medium.

[0017] Preferably, the culture medium used for co-culture is PIB medium; the co-culture conditions after inoculation are 20~35℃ and 100~250rpm for 10~20 days.

[0018] Preferably, the fermentation broth containing Ganoderma lucidum polysaccharides with a mannogalactan structure is filtered, centrifuged, fractionated and precipitated, and subjected to DEAE anion exchange chromatography. The eluent is purified by ultrafiltration membrane with a molecular weight cutoff of 3.5 kDa, separated by gel size exclusion chromatography, and the components with similar chromatographic behavior are combined to obtain Ganoderma lucidum polysaccharides with a mannogalactan structure.

[0019] Preferably, the obtained Ganoderma lucidum polysaccharide rich in mannogalactan structure is purified by Sephadex G-50 gel chromatography column, the eluent is collected, the salt is removed, and it is freeze-dried to obtain Ganoderma lucidum polysaccharide GG-1c.

[0020] Preferably, during Sephadex G-50 gel chromatography purification, the mobile phase is 0.1M NaCl, and the mobile phase flow rate is 0.18 mL / min; a 3.5 kDa dialysis bag is used for salt removal.

[0021] Another aspect of this application provides the application of the Ganoderma lucidum polysaccharide rich in mannogalactan structure obtained by the above-mentioned method of preparing Ganoderma lucidum polysaccharide rich in mannogalactan structure from Chlorella vulgaris GS-7 and Ganoderma lucidum YJ0724 in the preparation of preparations to enhance immune activity, wherein the Ganoderma lucidum polysaccharide rich in mannogalactan structure is Ganoderma lucidum polysaccharide GG-1c.

[0022] This substance can be used to prepare preparations that enhance immune activity. These preparations can be solid preparations such as tablets, capsules, granules, pills, powders, etc.; liquid preparations such as oral liquids, syrups, mixtures, lotions, drops, liniments, infusions, etc.; semi-solid preparations such as ointments, creams, gels, pastes, eye ointments, etc.; and gaseous and spray preparations.

[0023] Preferably, the Mn value of Ganoderma lucidum polysaccharide GG-1c is 10.14 kDa and the Mw value is 14.31 kDa.

[0024] Preferably, the Ganoderma lucidum polysaccharide GG-1c is composed of 45.80% mannose, 39.82% galactose, 12.24% fructose, and 2.14% glucose.

[0025] The beneficial effects that this application can produce include:

[0026] 1) This application provides a method for preparing Ganoderma lucidum polysaccharides rich in mannogalactan structure using *Chlorella vulgaris* GS-7 and *Ganoderma lucidum* YJ0724. This method can isolate and purify a uniform Ganoderma lucidum polysaccharide GG-1c rich in mannogalactan structure. The fermentation broth and bacterial cells obtained by this method show significantly increased Man content and significantly decreased Glc content. Co-culture may promote the conversion of Glc to Man by regulating carbohydrate metabolism pathways. Co-culture also increases the diversity of other monosaccharides, such as Gal content, which increases to 15.74% in the co-culture system. At the same time, the enrichment effect of Man in the fermentation broth of the co-culture system is significant. Co-culture can also promote the synthesis of polysaccharides with larger molecular weights, exhibiting a moderate molecular weight distribution. The provided co-culture system can obtain high-purity polysaccharide GG-1c, which has a narrow molecular weight distribution, stable chromatographic behavior, and low impurity content.

[0027] 2) The application of the Ganoderma lucidum polysaccharide rich in mannogalactan structure provided in this application, specifically the homogeneous polysaccharide GG-1c obtained from the fermentation broth of the co-culture system of Ganoderma lucidum YJ0724 and GS-7, shows a Man / Gal ratio close to 1:1, indicating that GG-1c has a more regular Man-Gal alternation or segmental arrangement structure. GG-1c exhibits significant immunomodulatory activity, selectively enhancing the release of IL-6 and IL-1β and NO production, while also showing a concentration-dependent regulatory effect on TNF-α. The promoting effect on NO and IL-6 is most significant.

[0028] A bacterium of the genus *Citrus* GS-7 was deposited on July 11, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, 430072, China. The accession number is (027) 68754052. The classification and nomenclature are as follows: Chryseobacterium piperi GS-7;

[0029] Ganoderma lucidum YJ0724 was deposited on July 11, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, 430072, China. Telephone: (027) 68754052, Accession No.: CCTCC NO: M 20251588. Ganoderma lucidum YJ0724; Attached Figure Description

[0030] Figure 1 Colony morphology A and phylogenetic tree B of the *Aureobacter* strain GS-7 in Example 1 provided in this application.

[0031] Figure 2 For the analysis of monosaccharide composition in the fermentation broth and mycelium obtained in each treatment of Example 2 provided in this application, A is the HPLC analysis chromatogram of monosaccharide composition of polysaccharides in the fermentation broth of different treatment groups; B is the HPLC analysis chromatogram of monosaccharide composition of polysaccharides in mycelium of each treatment group; C is the high-performance gel permeation chromatography analysis of polysaccharides in the fermentation broth of each treatment group; D is the relative content distribution of different monosaccharides in the polysaccharide structure of fermentation broth and mycelium of each treatment group; STD group is the standard of different monosaccharides; GL-FB, GL-Str, GS7-FB, GS7-Str, CC-Ⅰ-FB, CC-Ⅱ-Str respectively represent red... The fermentation broth of Ganoderma lucidum YJ0724, the mycelium of Ganoderma lucidum YJ0724, the fermentation broth of Chlorella vulgaris GS-7, the bacterial cells of Chlorella vulgaris GS-7, and fermentation broth and mycelium samples obtained by two different co-culture methods are shown in the figure. STD in the figure represents 9 monosaccharide standards: D-mannose (Man), L-rhamnose (Rha), D-glucuronic acid (GlcA), D-galacturonic acid (GalA), D-glucose (Glc), D-galactose (Gal), L-arabinose (Ara), fucose (Fuc), and N-acetylglucosamine (GlcNAc).

[0032] Figure 3 The physicochemical properties of GG-1 and GG-1c obtained in Example 3 of this application are analyzed as follows: A is the HPGPC analysis of GG-1 and GG-1c, with the inset showing the molecular weight distribution curve; B is the UV spectrum analysis of GG-1c; C is the HPLC analysis of the monosaccharide composition of GG-1 and GG-1c; and D is the FT-IR spectrum analysis of GG-1c.

[0033] Figure 4The polysaccharide methylation results of GG-1c obtained in Example 3 of this application are shown in Figure A. A is the GC-MS total ion chromatogram of partially methylated sugar alcohol acetyl esters of GG-1c. B is the mass spectrum of each partially methylated sugar alcohol acetyl ester: B1 is the mass spectrum after T-Fucp glycosidic bond methylation; B2 is the mass spectrum after T-Glcp glycosidic bond methylation; B3 is the mass spectrum after T-Manp glycosidic bond methylation; B4 is the mass spectrum after 1,2-Manp glycosidic bond methylation; B5 is the mass spectrum after 1,6-Manp glycosidic bond methylation; B6 is the mass spectrum after 1,6-Galp glycosidic bond methylation; B7 is the mass spectrum after 1,2,6-Manp glycosidic bond methylation; and B8 is the mass spectrum after 1,2,6-Galp glycosidic bond methylation.

[0034] Figure 5 1D / 2D NMR analysis and structure of GG-1c obtained in Example 3 of this application; A is ¹H-NMR spectrum; B is ¹³C-NMR spectrum; C is ¹H-¹HCOSY spectrum; D is ¹H-¹³CHSQC spectrum; E is HSQC-TOCSY spectrum; F is ¹H-¹³CHMBC spectrum.

[0035] Figure 6 The effect of GG-1c obtained in Example 3 of this application on RAW264.7 cells. A represents the relative NO release rate; B represents the TNF-α expression level; C represents the IL-6 expression level; D represents the IL-1β expression level; all data in the figure are expressed as mean ± standard deviation (n=3). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, compared with the NC group (negative control group).

[0036] Figure 7 The GG-1c structure provided in this application. Detailed Implementation

[0037] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments, but this does not limit the present invention in any way. Any modifications or improvements made based on the teachings of the present invention shall fall within the protection scope of the present invention.

[0038] Unless otherwise specified, all materials and instruments used in the following examples were obtained commercially; all detection methods used are existing methods. Unless otherwise specified, all culture media, solvents, and solutions used in the following examples were prepared using formulas known in the art. TSA liquid culture medium: 15.0 g / L casein digest, 5.0 g / L soybean peptone, 5.0 g / L sodium chloride, 1 g / L glucose, prepared to a volume of 1 L, sterilized at 121°C for 20 minutes. Solid culture medium was prepared by adding 15.0 g / L agar to the TSA liquid culture medium, with all other conditions remaining the same. ISP II liquid culture medium: 4.0 g / L yeast extract, 10.0 g / L malt extract, 4.0 g / L glucose, prepared to a volume of 1 L, sterilized at 121°C for 20 minutes. ISP II solid culture medium was prepared by adding 15.0 g / L agar to the ISP II liquid culture medium, with all other conditions remaining the same. PDB medium: 200 g / L peeled potatoes, 20 g / L glucose, distilled water to a final volume of 1 L, pH: natural (approximately 5.6), sterilize at 121°C for 20 minutes. PDA is prepared by adding 15.0 g / L agar to PDB, with all other conditions the same. PIB medium: After preparing ISP II liquid medium and PDB medium, mix them in a 1:1 volume ratio and sterilize at 121°C for 20 minutes.

[0039] Example 1: Chlorobacterium spp. C. piperi GS-7 Isolation, Purification and Identification

[0040] Using 10mL centrifuge tubes sterilized at 121℃ for 20 min, appropriate amounts of soil samples from the growing area of ​​Ganoderma lucidum were collected in Jinning District, Kunming City, Yunnan Province. The samples were quickly placed in an incubator with ice packs, and bacterial isolation was performed as soon as possible. The soil samples were serially diluted with PBS buffer solution, and samples from each dilution were spread onto a cloth. The serial dilution concentration was 10... -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 10 -8 10 -9 1 mL of soil sample was added to 9 mL of PBS buffer solution, serially diluted, and then plated onto TSA and ISP II solid agar plates. The plates were incubated statically for 24 h at 25 °C. Single colonies were then selected from the isolation plates for streak purification. Single colonies from the TSA and ISP II agar plates, after being incubated statically at 25 °C for 24 h using a bamboo stick sterilized at 121 °C for 20 min, were used to obtain strain GS-7, which is *Chlorobacterium*. C. piperi GS-7 colonies on ISP II solid medium are round ( Figure 1(A) Smooth edges, flat colony surface, and orange-yellow color. DNA was extracted from GS-7, and then 16S rRNA was amplified using 27F / 1492R primers. The PCR product was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The obtained 16S rRNA sequence is (No. 1):

[0041] The strain was compared with the NCBI database, and the resulting phylogenetic tree is shown below. Figure 1 As shown in Figure B, this strain is Chryseobacterium piperi .

[0042] Colonies of Ganoderma lucidum strain YJ0724 were collected on July 24, 2024, in Jinning District, Kunming City, Yunnan Province. DNA was extracted from YJ0724 using existing microbial isolation and culture methods, and then 16S rRNA was amplified using ITS1 / ITS4 primers. The PCR product was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The obtained 16S rRNA sequence is (No. 2):

[0043] The strain was compared with the NCBI database and identified as [the strain name]. Ganoderma lucidum .

[0044] The obtained strain is a genus of *Cyclocarya*. Chryseobacterium piperi GS-7 and Ganoderma lucidum Ganoderma lucidum YJ0724 is deposited at the China Center for Type Culture Collection. The taxonomic nomenclature of *Chlorella* bacteria GS-7 is as follows. Chryseobacterium piperi GS-7; Preservation number CCTCC NO: M 20251587; Classification and nomenclature of Ganoderma lucidum YJ0724 Ganoderma lucidum YJ0724; Accession number CCTCC NO: M 20251588.

[0045] Example 2: Construction of a co-culture system of *Cryptobacter* strain GS-7 and *Ganoderma lucidum* strain YJ0724

[0046] First, a single colony of Ganoderma lucidum strain YJ0724 was inoculated onto PDA agar plates and incubated statically at 25°C for 24 hours. Then, 0.5cm × 0.5cm samples were cut from the resulting PDA plates. 2 The colony blocks were inoculated into PDB medium (20 colony blocks were inoculated into each 500mL Erlenmeyer flask containing 200mL of medium), and cultured at 25℃ and 150rpm for 7 days to obtain the seed liquid of Ganoderma lucidum strain YJ0724 for later use.

[0047] The preserved GS-7 strain was streaked and activated on ISP II solid medium (25℃, 24h). Single colonies were picked and transferred to ISP II liquid medium (100mL medium per 250mL Erlenmeyer flask). The culture was carried out at 25℃ and 150rpm for 24h to obtain the GS-7 seed culture for later use.

[0048] The following procedures were followed to obtain the GS-7 pure culture group, the GL pure culture control group, and the co-culture treatment groups CC-I and CC-II, respectively:

[0049] Treatment group CC-I: GS-7 seed culture and Ganoderma lucidum YJ0724 seed culture were co-inoculated into PIB medium at 1% and 10% of the total PIB medium volume, respectively (200 mL of medium per 500 mL Erlenmeyer flask). The fermentation broth of the treatment group CC-I co-culture system was obtained by culturing at 25℃ and 150 rpm for 14 days. The fermentation broth was centrifuged at 4000 rpm for 15 min, and the supernatant (CC-IFB) and cell precipitate (CC-I Str) were collected.

[0050] Treatment group CC-II: The filtrate of GS-7 seed culture after sterilization by filtration through a 0.22-micron aqueous filter and Ganoderma lucidum YJ0724 seed culture were inoculated into PIB medium at 10% of the total PIB medium (200 mL of medium per 500 mL Erlenmeyer flask). The fermentation broth of the treatment group CC-II co-culture system was obtained by culturing at 25℃ and 150 rpm for 14 days. The fermentation broth was centrifuged at 4000 rpm for 15 min, and the supernatant (CC-II FB) and cell precipitate (CC-II Str) were collected.

[0051] GS-7 pure culture control group: GS-7 seed culture was inoculated into PIB medium at 1% of the total amount of PIB medium (200 mL of medium per 500 mL Erlenmeyer flask). The fermentation broth of the GS-7 pure culture control group was obtained by culturing at 25℃ and 150 rpm for 14 days. The fermentation broth was centrifuged at 4000 rpm for 15 min, and the supernatant (GS7-FB) and cell precipitate (GS7-Str) were collected.

[0052] GL pure culture control group: The seed culture of Ganoderma lucidum YJ0724 was inoculated into PIB medium at 10% of the total amount of PIB medium (200 mL of medium per 500 mL Erlenmeyer flask). The fermentation broth of Ganoderma lucidum YJ0724 pure culture control group was obtained by culturing at 25℃ and 150 rpm for 14 days. The fermentation broth was centrifuged at 4000 rpm for 15 min, and the supernatant (GL-FB) and cell precipitate (GL-Str) were collected.

[0053] Example 3: Extraction, separation and purification of polysaccharides

[0054] The fermentation broth, after 14 days of co-cultivation, was filtered through a filter cloth to separate the mycelium and filtrate. The filtrate was centrifuged (8000 rpm, 15 min), and the supernatant was sequentially fractionated using 20%, 40%, 60%, and 80% ethanol solutions.

[0055] The supernatant of the fermentation broth was centrifuged and mixed with a 20% ethanol solution and allowed to stand until no more precipitate formed. The mixture was then filtered to obtain 20g of precipitate GG-20.

[0056] The supernatant of the fermentation broth was centrifuged and mixed with a 40% ethanol solution, and then allowed to stand until no more precipitate formed. After filtration, 0.64 g of precipitate GG-40 was obtained.

[0057] The supernatant of the fermentation broth was centrifuged and mixed with a 60% ethanol solution, and then allowed to stand until no more precipitate formed. After filtration, 1.8g of precipitate GG-60 was obtained.

[0058] The supernatant of the fermentation broth was centrifuged and mixed with an 80% ethanol solution, and then allowed to stand until no more precipitate was formed. After filtration, 2.5g of precipitate GG-80 was obtained; precipitates GG-20 and GG-40 were discarded.

[0059] DEAE anion exchange chromatography: Precipitates GG-80 and GG-60 were dissolved in deionized water and then loaded onto a DEAE-52 cellulose anion exchange column. Neutral polysaccharide fractions were obtained by elution with 0.5M NaCl. The resulting eluates were combined and then used for subsequent steps.

[0060] The resulting eluent was purified by ultrafiltration membrane with a molecular weight cutoff of 3.5 kDa to obtain a precipitate. The precipitate was concentrated to obtain 600 mg of crude polysaccharide, of which 200 mg was used for subsequent purification.

[0061] Size exclusion chromatography (GPC) separation: 200 mg of crude polysaccharide was loaded onto a Sephadex G-100 column. The eluent was collected and fractionated. The eluent was analyzed by HPGPC and fractionated again. Fractions with similar chromatographic behavior were combined to obtain GG-1. GG-1 was further purified by GPC using a Sephadex G-50 column with 0.1 M NaCl as the mobile phase and a flow rate of 0.18 mL / min. After collecting the eluent, the salts were removed using a 3.5 kDa dialysis bag, and the product was lyophilized to obtain the target polysaccharide fraction GG-1c.

[0062] Similar chromatographic behavior refers to the phenomenon where samples with the same retention time are grouped together when eluted with different eluents for HPGPC analysis.

[0063] Example 4: Structural characterization of polysaccharide component GG-1c

[0064] (1) Determination of molecular weight: GG-1c degree and molecular weight were analyzed by high performance gel permeation chromatography (HPGPC) combined with differential refractive index detector (RID).

[0065] The analytical conditions were as follows: sample concentration 2 mg / mL, flow rate 0.5 mL / min, column temperature 25 °C, and separation was performed using a Shodex SB-804 HQ analytical column (7.8 × 300 mm) coupled with an OHpak SB-G guard column. The molecular weight of GG-1c (1 mg / mL) was determined by gel permeation chromatography (GPC) equipped with a Wyatt Technology Optilab T-rEX differential detector and a DAWN HELEOS II multi-angle laser light scattering (MALS) detector.

[0066] The separation system consisted of Ohpak SB-805 HQ (300×8 mm) and SB-803 HQ (300×8 mm) gel columns connected in series, with a column temperature of 45℃ and an injection volume of 100 μL. The mobile phase was a 0.1 M NaNO3 solution containing 0.02% NaN3, with isocratic elution for 75 min at a flow rate of 0.6 mL / min.

[0067] (2) Monosaccharide composition analysis: The monosaccharide composition was determined by pre-column derivatization using PMP (1-phenyl-3-methyl-5-pyrazolone). The specific steps were as follows: 1.0 mg of sample was added to 2.0 mL of 4 M trifluoroacetic acid (TFA), hydrolyzed in an oil bath at 110 °C for 4 hours, and then dried under vacuum. The residue was reconstituted with 200 μL of deionized water, followed by the addition of 200 μL of 0.6 M NaOH and 400 μL of 0.5 M PMP-methanol solution. The derivatization reaction was carried out at 70 °C for 1 hour, and then neutralized with 400 μL of 0.3 M HCl. After chloroform extraction, the aqueous phase was centrifuged and filtered through a 0.22 μm filter membrane before HPLC analysis. Chromatographic conditions: Hadesil C18-Bio column (5 μm, 4.6 × 250 mm), column temperature 25 °C, detection wavelength 250 nm, flow rate 1.0 mL / min, acetonitrile gradient elution program (17%-20%, 60 min).

[0068] (3) Fourier transform infrared spectroscopy and ultraviolet spectroscopy analysis: Fourier transform infrared spectroscopy (FT-IR) was used to analyze the characteristic functional groups of the isolated and purified polysaccharides. The specific steps were as follows: 1.0 mg of GG-1c sample was mixed with dry potassium bromide (KBr) at a ratio of 1:40 (w / w), pressed into a 1 mm thick sheet, and the transmission spectrum was scanned in the wavenumber range of 4000–400 cm⁻¹ using a Bruker TENSOR 27 infrared spectrometer.

[0069] The characteristic absorbance of proteins (280 nm) and nucleic acids (260 nm) in GG-1c was detected using a Shimadzu UV-2401PC UV-Vis spectrophotometer. The instrument performed a full-band scan in the wavelength range of 200–600 nm to accurately quantify the presence of the aforementioned biomolecules in the sample.

[0070] (4) Methylation analysis: The polysaccharide was first carbonylated and reduced. The specific steps were as follows: 3.0 mg GG-1c was mixed with 0.6 mL of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 0.5 g / mL) and 0.2 mL of 2-morpholinoethanesulfonic acid (MES, 0.2 M). The pH was adjusted to 4.75 with 0.1 M HCl and the reaction was allowed to proceed for 3 hours. 1.0 mL of imidazole (4.0 M) was added and the mixture was shaken and mixed. The mixture was then divided into two portions: one portion was treated with 0.8 mL of sodium borodeuteride (NaBD4, 70 mg / mL) in an ice bath for 8 hours and labeled as GG-1cI; the other portion was treated with 0.8 mL of sodium borohydride (NaBH4, 70 mg / mL) in the same manner and labeled as GG-1cII. After the reaction was terminated, the product was purified by dialysis using a 3.5 kDa filter and lyophilized to obtain the reduced polysaccharide. The reduced polysaccharide was then methylated as follows: 100 μg of sample was dissolved in 250 μL NaOH / DMSO (30 mg / mL), and 60 μL iodomethane was added. The mixture was magnetically stirred at room temperature for 0.5 hours (repeated 3 times). After the reaction was terminated, the sample was extracted with chloroform and the organic phase was dried under nitrogen. The sample was hydrolyzed with 0.3 mL trifluoroacetic acid (TFA, 2.0 M) at 110 °C for 2 hours. The hydrolysis product was then reduced with 250 μL ammonia (2.0 M) and 5 mg NaBD4 at 50 °C for 1 hour, and the reaction was terminated with 125 μL acetic acid. The reduction product was acetylated with pyridine (70 μL) and acetic anhydride (250 μL) at 110 °C for 1 hour. The final product was concentrated to 60 μL for GC-MS analysis.

[0071] GC-MS analysis was performed using an Agilent 7890A gas chromatograph-5957C mass spectrometer (DB-5MS column). Helium was used as the carrier gas (flow rate 1.5 mL / min, split ratio 5:1). The temperature program was: initial 100℃, held for 5 minutes, then increased to 250℃ at 5℃ / min. Electron impact ionization (EI) was used in full scan mode for a total run time of 40 minutes. Finally, partial acid hydrolysis and ABEE derivatization were performed. The specific steps were as follows: 1.5 mg GG-1c was added to 2.0 mL of 0.5 M LTA and hydrolyzed at 65℃ for 30 minutes. After redissolving the hydrolysate, 80 μL of acetic acid, 7.1 mg of sodium cyanoborohydride, and 4.0 mg of ABEE were added, and derivatization was performed at 65℃ for 2 hours.

[0072] After extraction with diethyl ether, the aqueous phase of the reaction solution was analyzed by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) in positive ion mode.

[0073] (5) Nuclear magnetic resonance analysis: The structure of GG-1c was analyzed using a Bruker AVANCE 800 MHz nuclear magnetic resonance spectrometer (Bruker, Germany). The specific method was as follows: 40 mg of sample was dissolved in 0.5 mL L2O (containing sodium tetramethylsilanepropionate (TSP) as an internal standard, with the δC / δH chemical shift reference point at 0 ppm), and the following spectra were collected at 25 °C: ¹H NMR, ¹³C NMR and two-dimensional spectra (COSY, TCOSY, HSQC, HSQC-TOCSY, HSQC-COSY, HMBC, ROESY).

[0074] Results Analysis

[0075] 1) HPLC results analysis: The HPLC results of each treatment fermentation broth are as follows: Figure 2 HPLC results of mycelium in treatment A are as follows: Figure 2 Figure B indicates that under the pure culture conditions of Ganoderma lucidum in Example 2, Glc was the main component in the GL-FB fermentation broth (57.61%), while the Man content was relatively low (20.67%). The samples obtained from fermentation of strain GS7 alone showed significant increases in Man content in both the fermentation broth and mycelium of strains GS-7 and YJ0724 (CC-I, CC-II), reaching 35.83% (fermentation broth) and 33.87% (mycelium), respectively, after co-culturing GS-7 with Ganoderma lucidum YJ0724. Simultaneously, the Glc content decreased significantly, dropping to 25.18% (fermentation broth) and 26.79% (mycelium), respectively.

[0076] This significant change in composition suggests that co-cultivation may promote the conversion of Glc to Man by regulating carbohydrate metabolic pathways. Furthermore, co-cultivation also increased the diversity of other monosaccharides; for example, the Gal content increased to 15.74% in the CC-II system, higher than the 9.76% under pure Ganoderma lucidum culture conditions.

[0077] 2) Analysis of mycelial polysaccharides in the two co-culture systems yielded the following results: Figure 2 B, Figure 2 The results (D) indicate that Glc remains a monosaccharide component in the mycelium, and its content remains at a high level even under co-culture conditions (GL-Str: 72.36%, CC-I-Str: 63.60%, CC-II-Str: 55.99%).

[0078] Although the Man content in the CC-I-Str and CC-II-Str systems was higher than that in the GL group (8.10%), reaching 12.88% and 17.65% respectively, their relative contents were significantly lower than the accumulation levels in the fermentation broth (FB) of the corresponding treatment groups (CC-I: 35.83%, CC-II: 33.87%). This result indicates that Man-containing Ganoderma lucidum polysaccharides are mainly enriched in the fermentation broth of the co-culture system rather than in the bacterial cells, providing important experimental evidence for the subsequent isolation and purification of Man-rich polysaccharides.

[0079] Further through the Figure 2 Analysis of the molecular weight distribution of polysaccharides in FB (fiber fibroblasts) from different treatment groups revealed significant differences. The GL-FB polysaccharide had a molecular weight distribution (Mw) of 5.2 kDa, a molecular weight distribution (Mn) of 2.2 kDa, and a molecular weight index (PDI) of 2.34, indicating a relatively uniform molecular weight distribution. The GS7-FB polysaccharide showed a narrower molecular weight distribution with Mw and Mn decreasing to 3.5 kDa and 2.1 kDa, respectively, and a PDI of 1.69. In the CC-I system, the Mw and Mn of the polysaccharide increased to 9.0 kDa and 2.9 kDa, respectively, and the PDI increased to 3.07, indicating that co-cultivation promoted the synthesis of larger molecular weight polysaccharides. In the CC-II system, a distinct bimodal distribution of polysaccharides was observed: the major component (97.46%) had Mw and Mn of 6.4 kDa and 2.6 kDa, respectively, and a PDI of 2.49; while the minor component (2.54%) showed a significantly increased molecular weight, with a Mw reaching 324.5 kDa.

[0080] Comparison of the two co-culture systems revealed significant differences in polysaccharide composition and molecular weight distribution. The CC-I system exhibited higher Man accumulation (35.83%) and a moderate molecular weight distribution (Mw 9.0 kDa, PDI 3.07) in FB, with higher Man content and relatively uniform molecular weight distribution in Ganoderma lucidum polysaccharides. While the CC-II system also promoted Man accumulation (33.87%), its polysaccharide molecular weight distribution showed a bimodal phenomenon, indicating a more complex synthetic regulation mode. Considering the uniformity of Man content, polysaccharide molecular weight distribution, and process operability, the CC-I system was chosen as the focus of subsequent research.

[0081] 3) The molecular weight determination results of GG-1 and GG-1c were obtained by analyzing the HPGPC and standard dextran calibration curves. Figure 3In step A), it was found that GG-1 exhibited a significant secondary peak within the retention time range of 11–16 min, indicating that the polysaccharide still contained polysaccharide subfractions with different molecular weights. In contrast, GG-1c showed a single, well-symmetrical main peak at 19.7 min, confirming that further purification by Sephadex G-100 size exclusion chromatography significantly improved the homogeneity of the polysaccharide. Quantitative analysis showed that the Mn and Mw values ​​of GG-1 were 6.71 kDa and 19.13 kDa, respectively, with a PDI of 2.85, indicating that this component had a wide molecular weight distribution and low homogeneity. In contrast, the Mn and Mw values ​​of GG-1c polysaccharide were 10.14 kDa and 14.31 kDa, respectively, and its PDI value was significantly reduced to 1.41, approaching the characteristics of an ideal monodisperse system (PDI=1), further confirming that GG-1c has high homogeneity. Figure 3 As can be observed from the molecular weight distribution in the illustration of A, the cumulative molecular weight distribution curve of GG-1c shows a steeper slope and a narrower distribution range, while GG-1 exhibits a broad distribution characteristic, which is consistent with the calculated PDI value.

[0082] UV analysis results ( Figure 3 As shown in Figure B), GG-1c exhibits almost no significant absorption in the 200–400 nm wavelength range, with only extremely weak absorption at 260 nm and 280 nm, and absorbance values ​​far below 0.2. This indicates that the sample contains virtually no impurities such as nucleic acids (260 nm) and proteins / peptides (280 nm). This lack of characteristic absorption peaks is consistent with typical high-purity polysaccharides, providing further spectroscopic evidence for the high homogeneity of GG-1c and confirming the effectiveness of the purification method employed.

[0083] The results of HPGPC and UV analysis show that the separation and purification strategy adopted in this study successfully purified the multi-component mixture GG-1 into a highly homogeneous GG-1c polysaccharide with a narrow molecular weight distribution, stable chromatographic behavior, and low impurity content.

[0084] The HPLC results of the monosaccharide composition of GG-1 and GG-1c are as follows: Figure 3 GG-1C is composed of seven monosaccharides: Man (50.10%), Gal (29.07%), and Fuc (9.94%), with small amounts of Glc (6.83%), GlcNAc (1.12%), Xyl (1.66%), and Ara (1.29%). GG-1C exhibits a more homogeneous sugar composition, consisting of four monosaccharides: Man (45.80%), Gal (39.82%), Fuc (12.24%), and Glc (2.14%). GlcNAc, Xyl, and Ara are no longer detected, indicating that the purification process effectively removed these non-major sugar components.

[0085] The Man / Gal ratio of GG-1 decreased from 1.72:1 to 1.15:1 in GG-1c. The ratio of GG-1c is close to 1:1, indicating that GG-1c may have a more regular Man-Gal alternation or segmental arrangement structure.

[0086] Compared to GG-1c, GG-1a shows that the Fuc content is relatively stable, indicating that the Fuc-rich domains are preferentially retained during purification. This retention may be closely related to the conformational stability and biological activity of the polysaccharide.

[0087] b. The significant decrease in Glc content from 6.83% to 2.14% indicates that the GG-1c purification process effectively removed non-major structural components, and Glc may be derived from heteropolysaccharides in the co-culture system.

[0088] c. In GG-1c, Man (mannose) and Gal (galactose) together account for 85.62% of the total sugar molar content. Combined with the characteristic fucose modification, GG-1c exhibits typical mannogalactan characteristics.

[0089] 4) The FT-IR spectrum of GG-1c is as follows: Figure 3 As shown in Figure D, the strong broad peak at 3423.9 cm⁻¹ in the spectrum is attributed to the stretching vibration of the hydroxyl group (OH) in the polysaccharide molecule. The moderate intensity absorption in the 2928.1 cm⁻¹ region corresponds to the CH stretching vibration. In the high-frequency region, the moderate intensity absorption band at 1650.3 cm⁻¹ can be attributed to the HOH bending vibration of bound water. In the fingerprint region (1300-400 cm⁻¹), the characteristic absorption peaks of the polysaccharide are more pronounced: the absorptions at 1383.3 cm⁻¹ and 1357.5 cm⁻¹ represent the CH bending vibration; the absorption at 1250.2 cm⁻¹ may be related to the CO stretching vibration; 1135.5 cm⁻¹ belongs to the COC stretching vibration of the pyran ring; and the strong absorption peaks at 1068.1 cm⁻¹ and 1033.3 cm⁻¹ are the characteristic absorptions of the C-OH stretching vibration and the COC on the ring.

[0090] Characteristic fingerprint region absorption peak analysis revealed that the absorption at 916.6 cm⁻¹ indicates the presence of α-configuration glycosidic bonds, while the signal at 814.8 cm⁻¹ is particularly correlated with α-configuration mannosyl residues. These spectral features, combined with the high proportions of Man and Gal detected in monosaccharide composition analysis, suggest that the glycosidic bonds in GG-1c are predominantly α-configuration, consistent with typical mannogalactan structural characteristics. Other absorptions at 974.2 cm⁻¹, 870.5 cm⁻¹, 650.7 cm⁻¹, and 578.7 cm⁻¹ further corroborate the pyran ring structure and its deformed vibrational modes; the positions and intensities of these peaks are highly consistent with the characteristic absorptions of α-configuration mannogalactans reported in the literature.

[0091] Based on the combined results of infrared spectroscopy and monosaccharide composition analysis, GG-1c can be identified as a mannogalactan with α-glycosidic bonds as the main linkage.

[0092] 5) GG-1c methylation results are as follows Figure 4 China A and Figure 4 As shown in B1~B8 and Table 1:

[0093] Table 1: Methylation analysis results of GG-1c

[0094]

[0095] Methylation analysis of GG-1c revealed eight glycosidic bond types, including T-Fuc p T-Glc p T-Man p 1,2-Man p 1,6-Man p 1,6-Gal p 1,2,6-Man p and 1,2,6-Gal p The results are shown in Table 1. Figure 4 China A Figure 4 As shown in Figures B1 to B8, the molar percentages of each component after molecular weight correction are 4.31%, 8.47%, 22.98%, 12.45%, 2.63%, 32.73%, 5.86%, and 10.56%, respectively.

[0096] Monosaccharide composition analysis and methylation results indicate that GG-1c has a highly branched structural feature. Theoretically, each branch point is connected to nearly two terminal units on average, forming a complex spatial structure similar to a "tree".

[0097] (6) GG-1c NMR results are as follows Figure 5 As shown in A~F and Table 2:

[0098] Table 2: GG-1c 1 H and 13 C chemical shift (δ, ppm)

[0099]

[0100] according to Figure 5 Analysis of A~F and Table 2 shows that the molecular formula of GG-1c is C 194 H 152 O 304 , structural formula as Figure 7 As shown.

[0101] Example 5: Evaluation of Immunological Activity

[0102] RAW264.7 cells were cultured in DMEM medium containing 10% fetal bovine serum (Biological Industries, Germany) at 37°C and 5% CO2. Cells were sputtered at a rate of 1.5 × 10⁻⁶ cells / year. 5 Inoculate 96-well plates at a density of 1 cell / well and treat with 25-800 μg / mL LGLP-1b (3 replicates per group). A positive control (1 μg / mL LLPS) and a blank control (complete culture medium) are also included.

[0103] After 24 hours of treatment, MTT reagent (Promega, USA) was added to each well, and the cells were cultured overnight. Absorbance was measured at 490 nm using a microplate reader (Thermo Fisher Scientific, USA). Cell phagocytic activity was then assessed using the following procedure: cells (1.5 × 10⁻⁶ cells / well). 5 (each well) was treated with GG-1c aqueous solution at concentrations of 50, 100, and 200 μg / mL for 24 hours, with a positive control (1 μg / mL LPS) and a blank control.

[0104] Add 100 μL of 0.1% neutral red solution to each well and continue culturing for 1.5 hours. Discard the supernatant, wash three times with PBS, add 150 μL of lysis buffer (ethanol:acetic acid:water = 1:1:2) to each well, incubate at 37°C for 2 hours, and measure the absorbance at 570 nm.

[0105] Finally, NO and cytokine levels were detected. Specifically, cell supernatant was collected, and NO was detected at 540 nm using the Griess reagent method. IL-6, IL-1β, and TNF-α levels were measured by ELISA (detection wavelength 450 nm). All experiments were performed in triplicate.

[0106] The results of GG-1c immune activity evaluation are as follows: Figure 6As shown in Figures A through D, the experimental results indicate that GG-1c exhibits selective and concentration-dependent regulatory effects on different inflammatory factors.

[0107] GG-1c significantly increased IL-6 release levels at all concentrations (4.2, 4.5, 4.2 pg / mL), an increase of approximately 82.61–95.65% compared to the negative control, and even higher than the LPS stimulation group. GG-1c also significantly increased IL-1β levels at all concentrations (22.5–23.5 pg / mL), an increase of approximately 114.29–123.81% compared to the negative control, comparable to the LPS group, indicating that GG-1c can enhance IL-1β release even at low concentrations.

[0108] TNF-α expression levels increased with increasing GG-1c concentration (57.4, 59.5, 64.3 pg / mL), representing increases of 26.71%, 31.35%, and 41.94% compared to the negative control (NC), respectively, showing a clear concentration-dependent characteristic.

[0109] GG-1c significantly promoted NO production, with relative NO release rates of 57.8%, 90.3%, and 88.6% at the three concentrations (with LPS as 100%), which were 49.35%, 133.33%, and 129.97% higher than the negative control.

[0110] The results showed that GG-1c had the most significant promoting effect on NO and IL-6 at a concentration of 100 μg / mL. The homogeneous polysaccharide GG-1c obtained from the co-culture system of Ganoderma lucidum YJ0724 and GS-7 exhibited significant immunomodulatory activity, selectively enhancing the release of IL-6 and IL-1β and NO production, while also showing a concentration-dependent regulatory effect on TNF-α.

[0111] Example 6: Immune-enhancing agents:

[0112] The Ganoderma lucidum polysaccharide GG-1c obtained in Example 3 was mixed with pure water to prepare an oral liquid.

[0113] Example 7

[0114] The difference from the CC-I group in Example 2 is that the co-culture conditions are 20°C and 100 rpm for 10 days.

[0115] Example 8

[0116] The difference from Example 2CC-II group is that the co-culture conditions are 35°C and 250 rpm for 20 days.

[0117] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing Ganoderma lucidum polysaccharides rich in mannogalactan structure from *Aureobacillus* GS-7 and *Ganoderma lucidum* YJ0724, characterized in that... Includes the following steps: Prepare seed culture of *Aureobacterium* GS-7 and seed culture of *Ganoderma lucidum* YJ0724; a. The seed culture of Aureobacterium GS-7 and the seed culture of Ganoderma lucidum YJ0724 were simultaneously inoculated into the fermentation medium and co-cultured to obtain a fermentation broth containing Ganoderma lucidum polysaccharides with a mannogalactan structure. Taxonomic designation of the bacterium Chryseobacterium sp. GS-7 Chryseobacterium piperi GS-7; Accession No. CCTCC NO: M 20251587; Taxonomic designation of the bacterium Ganoderma lucidum YJ0724 Ganoderma lucidum YJ0724; Accession No. CCTCC NO: M 20251588; Ganoderma polysaccharide GG-1c rich in mannan-galactan structure, molecular formula is C 194 H 152 O 304 ; GG-1c has the structure: .

2. The method of claim 1, wherein, In method a, the inoculum amount of Ganoderma lucidum YJ0724 is at least 1% of the total amount of PIB medium, and the inoculum amount of Chlorella spp. GS-7 is at least 10% of the total amount of PIB medium.

3. The method of claim 1, wherein, The culture medium used for co-culture was PIB medium; after inoculation, the co-culture conditions were 20-35℃ and 100-250rpm for 10-20 days.

4. The method of claim 1, wherein, The fermentation broth containing Ganoderma lucidum polysaccharides with a mannogalactan structure was filtered, centrifuged, fractionated, precipitated, and subjected to DEAE anion exchange chromatography. The eluent was purified by ultrafiltration membrane with a molecular weight cutoff of 3.5 kDa and separated by gel size exclusion chromatography. Components with similar chromatographic behavior were combined to obtain Ganoderma lucidum polysaccharides with a mannogalactan structure.

5. The method of claim 4, wherein, The obtained Ganoderma lucidum polysaccharide rich in mannan-galactan structure was purified by Sephadex G-50 gel chromatography column, and the eluent was collected, the salt was removed, and then freeze-dried.

6. The method of claim 5, wherein, During Sephadex G-50 gel chromatography purification, the mobile phase was 0.1M NaCl, and the flow rate was 0.18 mL / min. Salt removal was performed using a 3.5 kDa dialysis bag to obtain Ganoderma lucidum polysaccharide GG-1c.

7. The application of the Ganoderma lucidum polysaccharide rich in mannogalactan structure obtained by the method for preparing Ganoderma lucidum polysaccharide rich in mannogalactan structure from *Aureobacterium* GS-7 and *Ganoderma lucidum* YJ0724 as described in any one of claims 1 to 6 in the preparation of immune-enhancing agents, characterized in that... The Ganoderma lucidum polysaccharide rich in mannan-galactomannan structure is GG-1c.

8. Use according to claim 7, characterized in that, The Mn value of Ganoderma lucidum polysaccharide GG-1c is 10.14 kDa, the Mw value is 14.31 kDa, and the Man / Gal ratio is 1.15:

1.

9. Use according to claim 7, characterized in that, Ganoderma lucidum polysaccharide GG-1c is composed of 45.80% mannose, 39.82% galactose, 12.24% fructose, and 2.14% glucose.

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