Priestia aryabhattai and microbial preparation and application thereof

Abstract

The application discloses a Priestia aryabhattai strain and a microbial preparation and application thereof. The preservation unit of the Priestia aryabhattai strain is a Guangdong Microbial Culture Collection Center, and the preservation number is GDMCC 68502. The microbial preparation comprises the Priestia aryabhattai strain or at least one of a bacterial suspension, a fermentation liquid, a fermentation supernatant, a bacterial body, a spore or a metabolite prepared from the Priestia aryabhattai strain. The Priestia aryabhattai strain and the microbial preparation provided by the application can significantly improve the content of polysaccharide in ganoderma lucidum mycelium, the effect is stable, and the application prospect is good in ganoderma lucidum cultivation and ganoderma lucidum polysaccharide product production.

Description

Technical Field

[0001] This invention relates to the fields of microbial technology and cultivation technology of edible and medicinal fungi, specifically to a strain of Priestia aryabhattai and its microbial preparations and applications. Background Technology

[0002] Ganoderma lucidum polysaccharides are the main active components of Ganoderma lucidum, and their content directly determines its medicinal and commercial value. Existing yield-increasing methods, such as strain selection, culture optimization, or chemical inducers, suffer from long cycles, limited effectiveness, or safety issues. Although *Priestia aryabhattai* is widely used in agricultural growth promotion and environmental remediation, it has not yet been found to be used to regulate Ganoderma lucidum polysaccharide synthesis. Summary of the Invention

[0003] The present invention aims to provide a strain of Priestia aryabhattai and its microbial preparations and applications.

[0004] To achieve the above objectives, the first aspect of the present invention provides:

[0005] A strain of *Priestia aryabhattai*, deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC 68502.

[0006] Preferably, the 16S rDNA sequence of the *Bacillus argentea* is shown in SEQ ID NO:1.

[0007] The second aspect of the present invention provides:

[0008] A microbial preparation comprising the aforementioned Bacillus argentea, or comprising at least one of the following: a bacterial suspension, fermentation broth, fermentation supernatant, bacterial cells, spores, or metabolites prepared from the aforementioned Bacillus argentea.

[0009] Preferably, the effective viable count of *Bacillus argentea* in the microbial preparation is not less than 1 × 10⁻⁶. 6 CFU / mL or CFU / g.

[0010] The third aspect of the present invention is provided as follows:

[0011] The application of the aforementioned Bacillus aureus or the aforementioned microbial preparation in increasing the polysaccharide content of Ganoderma lucidum.

[0012] The fourth aspect of the present invention is provided as follows:

[0013] The application of the aforementioned Bacillus aureus or the aforementioned microbial preparation in Ganoderma lucidum cultivation.

[0014] The fifth aspect of the present invention provides:

[0015] The application of the aforementioned Bacillus aureus, or the aforementioned microbial preparation, in the preparation of products with increased Ganoderma lucidum polysaccharide content.

[0016] The sixth aspect of the present invention provides:

[0017] A method for increasing the polysaccharide content of Ganoderma lucidum includes the step of co-culturing the aforementioned Bacillus aureus or the aforementioned microbial preparation with Ganoderma lucidum.

[0018] Preferably, the Ganoderma is a fungus belonging to the genus Ganoderma.

[0019] Preferably, the Ganoderma fungus is selected from at least one of Ganoderma lucidum, Ganoderma sinense, Ganoderma tsugae, and Ganoderma leucocontextum.

[0020] The present invention has the following beneficial effects:

[0021] 1. This invention is the first to discover that *Bacillus argentea* can significantly increase the polysaccharide content of *Ganoderma lucidum*. The strain can act as a biological inducer, effectively activating the metabolic pathways of *Ganoderma lucidum* growth and significantly promoting polysaccharide synthesis. Plate co-culture experiments showed that, compared with the control group not inoculated with *Bacillus argentea*, the polysaccharide content of *Ganoderma lucidum* treated with *Bacillus argentea* of this invention was significantly increased, reaching nearly twice that of the control group, demonstrating a significant and stable yield increase.

[0022] 2. Using naturally derived Bacillus oryzae probiotics as an induction method to replace traditional chemical inducers (such as methyl jasmonate, salicylic acid, etc.) avoids potential toxicity, residues and environmental safety issues.

[0023] 3. The Bacillus argentea strain can be made into liquid or solid microbial preparations and directly added to Ganoderma lucidum cultivation material or fermentation medium without changing the existing cultivation or fermentation process. It has strong compatibility, low cost, and is suitable for large-scale promotion and application.

[0024] 4. The Bacillus argentea itself has functions such as producing plant hormones and solubilizing phosphorus and potassium. It can not only increase the polysaccharide content, but also promote the growth of Ganoderma lucidum mycelium, shorten the fruiting cycle or enhance the resistance to stress, thereby improving the dual benefits of Ganoderma lucidum yield and quality. Attached Figure Description

[0025] Figure 1This invention relates to a phylogenetic tree of Bacillus argentea Soil25HZ-CG-01-02-B12 constructed based on the 16S rDNA sequence.

[0026] Figure 2 This is a comparison chart of the content of Ganoderma lucidum polysaccharides under different treatment conditions according to the present invention. Detailed Implementation

[0027] To enable those skilled in the art to more clearly understand the technical solutions of the present invention, the technical solutions will be clearly and completely described below in conjunction with specific embodiments. The described embodiments are only a part of the embodiments of the present invention, and not all of them. Specific conditions are not specified in the following embodiments; they are generally implemented under conventional conditions or according to the conditions recommended by the manufacturer.

[0028] Reishi mushroom is an important medicinal fungus, containing various bioactive components such as polysaccharides, triterpenes, sterols, and nucleosides in its fruiting body and mycelium. Among these, reishi polysaccharides are one of the most important active components, possessing multiple biological activities including immunomodulation, antitumor, antioxidant, hypoglycemic, and hepatoprotective effects, and are widely used in health foods, pharmaceuticals, and functional foods. The content of reishi polysaccharides is a crucial indicator for evaluating the quality and efficacy of reishi and its products; therefore, increasing the yield and content of reishi polysaccharides has always been a core concern of the reishi industry.

[0029] Currently, methods to increase Ganoderma lucidum polysaccharide yield mainly include the selection and breeding of superior strains, optimization of cultivation substrates and culture medium formulations, control of fermentation and cultivation conditions, and the addition of exogenous substances such as elicitors to stimulate polysaccharide synthesis. However, strain selection has a long cycle and limited efficiency; the improvement in physicochemical conditions often faces significant bottlenecks; and chemical elicitors raise concerns about cost, residues, and safety. Therefore, developing green, safe, efficient, and easy-to-implement methods to increase Ganoderma lucidum polysaccharide yield has significant application value.

[0030] Interactions are common among microorganisms. Some beneficial microorganisms can act as growth promoters or biological inducers, influencing fungal growth, development, and secondary metabolism, thereby regulating the accumulation of target active ingredients. *Priestiaaryabhattai* is a group of Gram-positive, spore-forming bacteria widely found in soil and other environments. Existing research and patent literature report that this bacterium has multiple functions, including promoting plant growth, potassium solubilization, phosphorus solubilization, nitrogen fixation, plant hormone production, antagonism of pathogens, degradation of environmental pollutants, and tolerance to abiotic stress. It has been applied in agricultural microbial fertilizers, biological control, and environmental remediation.

[0031] However, current applications of *Bacillus argentea* mainly focus on promoting growth in higher plants, biological control, and environmental remediation. There are no reports of its application in Ganoderma lucidum cultivation, particularly in increasing its polysaccharide content. In other words, a technical solution for using *Bacillus argentea* to regulate the accumulation of active ingredients in this medicinal fungus and increase its polysaccharide content is currently lacking in the field.

[0032] Therefore, there is an urgent need to provide a strain that can safely and effectively increase the polysaccharide content of Ganoderma lucidum and corresponding application methods.

[0033] Therefore, the present invention aims to provide a strain of *Priestia aryabhattai*, which is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC 68502.

[0034] The *Priestia aryabhattai* strain described in this invention was isolated from soil samples in our laboratory. Its original name was Soil25HZ-CG-01-02-B12, and it was deposited on June 24, 2026, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, China, with accession number GDMCC 68502. *Priestia aryabhattai* Soil25HZ-CG-01-02-B12 was isolated from field soil samples from Chenggu County, Hanzhong City, Shaanxi Province. It was identified as *Priestia aryabhattai* through morphological observation, physiological and biochemical assays, and 16S rDNA sequence analysis.

[0035] More specifically, the 16S rDNA sequence of the aforementioned Bacillus auricula is shown in SEQ ID NO:1:

[0036] CGGCTAGCCTCCTTACGGTTACTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACG

[0037] GGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAG

[0038] CGATTCCAGCTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGAATGGTTTTATG

[0039] GGATTGGCTTGACCTCGCGGTCTTGCAGCCCTTTGTACCATCCATTGTAGCACGTGTGTA

[0040] GCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCAC

[0041] CGGCAGTCACCTTAGAGTGCCCAACTAAATGCTGGCAACTAAGATCAAGGGTTGCGCTCG

[0042] TTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTC

[0043] ACTCTGTCCCCCGAAGGGGAACGCTCTATCTCTAGAGTTGTCAGAGGATGTCAAGACCTG

[0044] GTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCC

[0045] GTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGT

[0046] TAGCTGCAGCACTAAAGGGCGGAAACCCTCTAACACTTAGCACTCATCGTTTACGGCGTG

[0047] GACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCGCCTCAGCGTCAGTTAC

[0048] AGACCAAAAAGCCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTA

[0049] CACGTGGAATTCCGCTTTTCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCTCCA

[0050] CGGTTGAGCCGTGGGCTTTCACATCAGACTTAAGAAACCGCCTGCGCGCGCTTTACGCCC

[0051] AATAATTCCGGATAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGC

[0052] CGTGGCTTTCTGGTTAGGTACCGTCAAGGTACGAGCAGTTACTCTCGTACTTGTTCTTCC

[0053] CTAACAACAGAGTTTTACGACCCGAAAGCCTTCATCACTCACGCGGCGTTGCTCCGTCAG

[0054] ACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTC

[0055] TCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTATGCATCGTTGCCTTGGTGAGC

[0056] CGTTACCTCACCAACTAGCTAATGCACCGCGGGCCCATCTGTAAGTGATAGCCGAAACCA

[0057] TCTTTCAATCATCTCCCATGAAGGAGAAGATCCTATCCGGTATTAGCTTCGGTTTCCCGA

[0058] AGTTATCCCAGTCTTACAGGCAGGTTGCCCACGTGTTACTCACCCGTCCGCCGCTAACGT

[0059] CATAGAAGCAAGCTTCTAATCAGTTCGCTCGACTTGCATGTATTAGGCACGCCGCCAGCG

[0060] TTCATCCTGAG

[0061] This invention marks the first discovery that *Priestia aryabhattai* GDMCC 68502 can serve as a highly efficient biological inducer, significantly activating the growth and metabolic pathways of *Ganoderma lucidum* and promoting polysaccharide synthesis. Plate co-culture experiments showed that its polysaccharide content could reach nearly twice that of the control group. This strain originates from the natural environment, replacing traditional chemical inducers; it is non-toxic, residue-free, and environmentally friendly. Its formulation can be directly added to the cultivation substrate or fermentation system without altering existing processes, making it simple to operate, low-cost, and easy to scale up. Furthermore, this strain also possesses functions such as producing plant hormones and solubilizing phosphorus and potassium. While increasing polysaccharide content, it also promotes mycelial growth, shortens the fruiting cycle, and enhances stress resistance, achieving a dual improvement in both the yield and quality of *Ganoderma lucidum*.

[0062] The second aspect of the invention provides:

[0063] A microbial preparation comprising the aforementioned *Priestia aryabhattai* GDMCC 68502, or at least one of the following: a bacterial suspension, fermentation broth, fermentation supernatant, bacterial cells, spores, or metabolites prepared from the aforementioned *Priestia aryabhattai* GDMCC 68502.

[0064] As a preferred embodiment, the microbial preparation can be in the form of liquid, powder, granules or wettable powder, etc. The preparation can be applied to the Ganoderma lucidum cultivation substrate or fermentation culture medium by spraying, mixing, root irrigation or direct addition to the liquid fermentation system, etc., to effectively promote the biosynthesis and accumulation of Ganoderma lucidum polysaccharides without changing the existing Ganoderma lucidum cultivation or fermentation process.

[0065] Based on the aforementioned microbial preparations, the live bacteria or spores can colonize in the cultivation environment and continuously release signaling molecules, activating the growth and metabolic pathways of Ganoderma lucidum. The fermentation broth and bacterial suspension contain both live bacteria and secretions, making them suitable for various inoculation methods. Even after removing the bacteria, the fermentation supernatant retains significant activity, indicating that its effective components are water-soluble metabolites secreted by the strain (such as organic acids, small molecule peptides, or unknown signaling substances), which can be directly sensed and utilized by Ganoderma lucidum. Spores, due to their high stability, facilitate preparation storage and transportation, making them suitable for solid dosage forms. Furthermore, the metabolites themselves can be used to develop biostimulants without live bacteria to meet specific production needs. Experiments show that all of the above methods can effectively increase the polysaccharide content of Ganoderma lucidum.

[0066] In a preferred embodiment, the effective viable count of *Bacillus aeruginosa* in the microbial preparation is not less than 1 × 10⁻⁶. 6 CFU / mL or CFU / g.

[0067] In the preferred embodiment described above, the effective viable count of *Priestiaaryabhattai* (GDMCC 68502) in the microbial preparation is not less than 1 × 10⁻⁶. 6 The concentration threshold is CFU / mL (liquid dosage form) or CFU / g (solid dosage form), which is the minimum effective dose determined by the inventors through inventive effort: when the concentration of live bacteria is below this level, it is difficult to establish effective microecological interactions or continuously release sufficient bioactive signaling molecules in the Ganoderma lucidum cultivation substrate or fermentation system, resulting in a significant weakening or even failure of the induction effect on the Ganoderma lucidum polysaccharide synthesis pathway; while maintaining a concentration of 1×10 6 At concentrations above CFU, the strain can be ensured to rapidly colonize and survive stably after inoculation, and to fully secrete growth-promoting substances (such as plant hormones, organic acids, and unknown inducing factors), thereby effectively activating the expression of genes related to Ganoderma lucidum growth and metabolism, and significantly increasing polysaccharide production. Simultaneously, this concentration balances formulation stability and production costs. For example, while ensuring biological activity, it avoids problems such as nutrient depletion, accumulation of metabolic waste, and rapid inactivation of bacteria during storage caused by excessively high live cell density, thus helping to maintain the stability and efficacy consistency of the formulation throughout its shelf life. Furthermore, it avoids resource waste due to excessively high bacterial counts while ensuring reliable induction performance under different cultivation environments.

[0068] In a preferred embodiment, the microbial preparation further includes carriers, protectants, or adjuvants acceptable in the field of microbial preparations.

[0069] In the preferred embodiments described above, the microbial preparation may further include carriers, protectants, or adjuvants acceptable in the field of microbial preparations to improve its stability, storability, ease of application, and bioactivity. The carrier may be selected from diatomaceous earth, bacterial bran, biochar, wheat bran, corn cob powder, or sterile water, etc., to adsorb or suspend the bacterial cells, facilitating the preparation of powders, granules, or liquids, and aiding in the uniform dispersion and colonization of the strains in the Ganoderma lucidum cultivation substrate. Protectants may include trehalose, skim milk powder, polyvinyl alcohol (PVA), xanthan gum, or ascorbic acid, etc., which can protect cell membrane structure, maintain spore viability, and significantly improve the survival rate of the preparation under high temperature, dryness, or long-term storage conditions during freeze-drying or drying. Adjuvants may include surfactants (such as Tween-80), pH buffers (such as phosphate buffer), or nutritional supplements (such as yeast extract or peptone), to improve the wettability of the bacterial suspension, stabilize the system pH, and promote bacterial recovery and metabolic activity. The above-mentioned components can be added in the post-fermentation treatment stage according to conventional processes, such as resuspending the cells in a buffer solution containing a protectant after centrifugation, or mixing with a solid carrier for granulation and drying, thereby synergistically enhancing the overall performance of the formulation and its field application effect.

[0070] It is understood that the carriers, protective agents or adjuvants used include, but are not limited to, the types listed above, and any other substances commonly used or equivalent in the field of microbial preparation technology may be applied.

[0071] The third aspect of the present invention provides:

[0072] The application of the aforementioned *Priestia aryabhattai* GDMCC 68502, or the aforementioned microbial preparation, in increasing the polysaccharide content of *Ganoderma lucidum*.

[0073] Specifically, the aforementioned *Priestia aryabhattai* GDMCC 68502, or microbial preparations made with it as an active ingredient, can be used to increase the polysaccharide content of *Ganoderma lucidum*.

[0074] As a preferred embodiment, the application includes at least one of the following:

[0075] 1. In Ganoderma lucidum soil covering cultivation, the microbial preparation is applied to the soil environment of the cultivation substrate (such as a culture medium composed of sawdust, cottonseed hulls, bran, etc.) by spraying or mixing with the substrate for inoculation.

[0076] 2. In the process of producing Ganoderma lucidum mycelium or polysaccharide through liquid submerged fermentation, this preparation is introduced as a biological inducer at the initial stage of fermentation;

[0077] 3. In-situ regulation can also be achieved during the development stage of Ganoderma lucidum fruiting bodies through root irrigation or surface atomization treatment.

[0078] The above applications can effectively activate the growth and metabolic pathways of Ganoderma lucidum, significantly increasing the accumulation of intracellular and extracellular polysaccharides. It should be emphasized that the applications described in this invention are not limited to the above embodiments. Any technical pathway that utilizes this strain or its preparations to directly or indirectly promote the synthesis and enrichment of Ganoderma lucidum polysaccharides falls within the scope of protection of this invention.

[0079] The fourth aspect of the present invention provides:

[0080] The application of the aforementioned *Priestia aryabhattai* GDMCC 68502, or the aforementioned microbial preparation, in the cultivation of *Ganoderma lucidum*.

[0081] Specifically, the aforementioned *Priestia aryabhattai* GDMCC 68502, or microbial preparations made with it as an active ingredient, can be widely used in the cultivation of Ganoderma lucidum to promote mycelial growth, shorten the fruiting cycle, enhance stress resistance, and increase the content of active ingredients such as polysaccharides in the fruiting body.

[0082] As a preferred embodiment, the application includes at least one of the following:

[0083] 1. Before inoculating Ganoderma lucidum spawn after sterilizing the culture medium, spray the microbial preparation evenly or mix it into the cultivation substrate (such as a mixture of sawdust, cottonseed hulls, corn cobs, wheat bran, etc.) for pretreatment.

[0084] 2. During the mycelial growth stage of Ganoderma lucidum, the preparation can be introduced through surface inoculation with soil or local spot application to form a beneficial microecology;

[0085] 3. Apply regularly during the primordia differentiation and fruiting body growth period by means of root irrigation, spraying or atomized fumigation to continuously regulate the rhizosphere or substrate microenvironment.

[0086] 4. In addition, this preparation can also be used in Ganoderma lucidum liquid culture propagation systems or in various modes such as industrialized bag cultivation, bottle cultivation, and log cultivation.

[0087] It should be noted that the application of this invention in Ganoderma lucidum cultivation is not limited to the specific methods described above. Any technical solution that uses Bacillus argentea or its preparations to directly or indirectly improve the growth and development of Ganoderma lucidum, the accumulation of metabolites, or the cultivation efficiency should be considered as an equivalent implementation of this invention and included in the scope of protection.

[0088] The fifth aspect of the present invention provides:

[0089] The application of the aforementioned *Priestia aryabhattai* GDMCC 68502, or the aforementioned microbial preparation, in the preparation of products with increased Ganoderma lucidum polysaccharide content.

[0090] Specifically, the aforementioned *Priestia aryabhattai* GDMCC 68502, or microbial preparations made with it as an active ingredient, can be used to prepare functional products aimed at increasing the polysaccharide content of *Ganoderma lucidum*.

[0091] As a preferred embodiment, the application includes: in the processing of products such as Ganoderma lucidum fruiting bodies, Ganoderma lucidum spore powder, broken-cell wall spore powder, freeze-dried Ganoderma lucidum mycelium powder, or Ganoderma lucidum extracts, by co-fermenting, post-ripening, or biotransformation of the microbial preparation with the Ganoderma lucidum raw materials, activating the endogenous metabolic enzyme system of Ganoderma lucidum, promoting the synthesis, enrichment, or structural optimization of polysaccharides, thereby obtaining high-value functional products with significantly increased polysaccharide content. For example, the microbial agent can be used for secondary fermentation of broken-cell wall Ganoderma lucidum spore powder, or for biological pretreatment before the preparation of Ganoderma lucidum whole herb powder. It can also be introduced into the raw material preparation stage of health foods such as Ganoderma lucidum oral liquids, capsules, and tablets to enhance their polysaccharide efficacy. It should be noted that the application of this invention is not limited to the specific product forms mentioned above. All functional products such as foods, health foods, pharmaceuticals, or cosmetics that are based on Ganoderma lucidum or its derivatives and achieve increased polysaccharide content through the introduction of the aforementioned Bacillus aureus or its preparations are within the scope of protection of this invention.

[0092] The sixth aspect of the present invention provides:

[0093] A method for increasing the polysaccharide content of Ganoderma lucidum includes the step of co-culturing the aforementioned Bacillus aureus or the aforementioned microbial preparation with Ganoderma lucidum.

[0094] As a preferred embodiment, the method may include the following steps: First, Bacillus aureus is activated under sterile conditions and prepared into a bacterial suspension or solid inoculum of suitable concentration. Then, it is inoculated into a solid cultivation substrate (such as sawdust-wheat bran mixture) that has been inoculated with Ganoderma lucidum strains at a certain ratio, or the inoculum is added to the Ganoderma lucidum liquid fermentation system at the early stage of growth. The two are then co-cultured for 7–10 days under controlled temperature and humidity (such as 25–30°C, relative humidity 85%–95%) and aeration conditions. During this process, Bacillus aureus regulates the metabolic pathways of Ganoderma lucidum by secreting metabolically active substances (such as organic acids, enzymes, or signaling molecules), promoting the synthesis and accumulation of polysaccharides such as β-glucan. After co-culture, the fruiting bodies, mycelia, or fermentation broth of Ganoderma lucidum are harvested, and polysaccharides are extracted by conventional processes such as water extraction and alcohol precipitation to obtain a product with a polysaccharide content significantly higher than that of the control group. This method is not only simple to operate and environmentally friendly, but also applicable to various Ganoderma lucidum production modes such as bag cultivation, bottle cultivation, log cultivation and liquid fermentation, and has good prospects for industrial application.

[0095] In a preferred embodiment, the co-culture temperature is 25–30°C, the culture time is 7–10 days, and / or the inoculum size of Bacillus aureus is 0.5%.

[0096] In the preferred embodiment described above, the co-culture temperature is set at 25–30°C, which takes into account the optimal growth temperature of Ganoderma lucidum mycelium (usually 25–28°C) and Priestia lactea. The optimal range for the metabolic activity of Ganoderma lucidum (28–30℃) is the equilibrium point after this range. Within this range, the two can achieve good symbiotic compatibility and metabolic interaction, avoiding the inhibition of Ganoderma lucidum growth by high temperature while ensuring that Bacillus can effectively secrete growth-promoting or inducing factors. The culture time of 7–10 days corresponds to the initial stage of Ganoderma lucidum metabolism to the stage of rapid polysaccharide accumulation. At this time, the introduction of microbial stimulation can significantly upregulate the expression of key enzymes in polysaccharide synthesis (such as UDP-glucose pyrophosphorylase and β-1,3-glucan synthase). Too short a time will result in insufficient induction, while too long a time may lead to contamination by other microorganisms or substrate depletion. The inoculum amount of 0.5% has been verified by gradient testing (0.1%–2.0%) to provide sufficient active cells to exert bioregulatory effects without inhibiting the normal metabolism of Ganoderma lucidum due to excessive proliferation competing for nutrients or changing the pH of the substrate microenvironment. Thus, the optimal balance is achieved between safety, economy and efficacy.

[0097] In a preferred embodiment, the Ganoderma is a fungus belonging to the genus Ganoderma.

[0098] The aforementioned Ganoderma refers to fungi belonging to the genus Ganoderma, which belongs to the phylum Basidiomycota, class Agaricales, order Polyporaceales, and family Ganodermataceae. It is a group of large fungi with important medicinal and health-promoting value, including Ganoderma lucidum, Ganoderma sinense, and Ganoderma lucidum. Representative species include Ganoderma lucidum (Ganoderma tsugae) and Ganoderma lucidum (Ganoderma contextum), which are widely used in traditional Chinese medicine and the development of modern functional foods and pharmaceuticals. Ganoderma fungi are rich in various bioactive components such as polysaccharides, triterpenes, proteins, amino acids, and trace elements. In particular, their β-glucan-type polysaccharides have been proven to have significant immunomodulatory, antitumor, antioxidant, and anti-fatigue pharmacological effects, and are one of the core indicators for evaluating the quality and efficacy of Ganoderma lucidum. This invention selects Ganoderma fungi as the co-culture object not only because of their mature cultivation system and clear metabolic regulation potential, but also because their polysaccharide synthesis pathway is highly responsive to beneficial microorganisms and their metabolites. This facilitates the targeted increase of polysaccharide content by introducing Bacillus aureus, thereby obtaining high-value-added Ganoderma lucidum products.

[0099] In a preferred embodiment, the Ganoderma fungus is selected from at least one of Ganoderma lucidum, Ganoderma sinense, Ganoderma tsugae, and Ganoderma maleucocontextum.

[0100] In the preferred embodiments described above, the Ganoderma fungus is selected from at least one of Ganoderma lucidum, Ganoderma sinense, Ganoderma tsugae, and Ganoderma maleucocontextum. The selection was based on the comprehensive advantages of the four species in terms of medicinal value, polysaccharide content, and responsiveness to microbial co-culture: *Ganoderma lucidum* is a legally recognized medicinal Ganoderma listed in the Chinese Pharmacopoeia, with clear quality standards and a broad industrial application base; its polysaccharide components have been studied most extensively. *Ganoderma sinense*, also a pharmacopoeia species, is traditionally used for "calming the mind and invigorating qi"; its fruiting body polysaccharide structure is unique, exhibiting significant immune activity, and it is maturely cultivated in southern regions. *Ganoderma tsugae* is mainly distributed in the isothermal coniferous forests of Changbai Mountain in my country; its fruiting body has a high polysaccharide content (up to 50% or more), is rich in triterpenes and phenolic acids, and exhibits significant immunomodulatory and antioxidant activities; it responds well to the microbial environment and demonstrates strong metabolic potential in co-culture systems. *Ganoderma leucocontextum*, as a high-polysaccharide resource-type Ganoderma that has attracted much attention in recent years, has white flesh, a short growth cycle, and has shown high sensitivity to beneficial microbial stimulation in previous studies, demonstrating outstanding polysaccharide synthesis potential. Therefore, selecting the above four Ganoderma species not only ensures the stability and reproducibility of the technical solution of this invention, but also takes into account the application needs under different regions and product positioning, which is conducive to the efficient and targeted development of high polysaccharide Ganoderma products.

[0101] Example 1: Isolation, identification and preservation of Bacillus aureus Soil25HZ-CG-01-02-B12

[0102] 1.1 Isolation and purification of strains

[0103] Prepare field soil samples from Chenggu County, Hanzhong City, Shaanxi Province, China. Place a sterile 50mL centrifuge tube on an analytical balance, peel it, and use a sterile spatula to take 0.5g of soil sample into the centrifuge tube. In a laminar flow hood, add 50mL of dd H2O to the centrifuge tube containing the soil sample and shake well for about 10 seconds. Vortex thoroughly for 15 seconds. Using the above soil suspension as the original concentration of bacterial solution, dilute it 10 times and 100 times in a laminar flow hood. Transfer 1mL of the original solution and 9mL of dd H2O to a new sterile centrifuge tube to prepare a 10-fold diluted bacterial solution. Then transfer 1mL of the 10-fold diluted bacterial solution and 9mL of dd H2O to a new sterile centrifuge tube to prepare a 100-fold diluted bacterial solution. Three gradients of bacterial suspension were added to LB solid medium, and spread using disposable spreaders. Five replicates were performed for each gradient. After spreading, the plates were sealed and incubated at 25°C in a biochemical incubator. Single colonies were observed every 12 hours. Once colonies appeared, their morphology was observed. Thirty morphologically distinct single colonies, not in contact with other colonies, were selected in a clean bench and added to eight-tube strips containing 50 μL of LB liquid medium. These were then incubated at 37°C in a shaker for 1 hour. After shaker incubation, 20 μL of bacterial suspension was pipetteed into 30 LB solid medium strips and streaked. The plates were then sealed and incubated at 25°C in a biochemical incubator. Single colony growth was observed every 12 hours. Once single colonies appeared, the transfer and incubation process was repeated three times for purification. The resulting pure culture strain was designated Soil25HZ-CG-01-02-B12.

[0104] 1.2 Molecular biological identification

[0105] Genomic DNA was extracted from strain Soil25HZ-CG-01-02-B12 and amplified by PCR using universal primers for bacterial 16S rDNA [27F: AGA GTT TGA TCC TGG CTC AG / 1492R: GGT TAC CTT GTT ACG ACT T]. The amplified product was sequenced to obtain the 16S rDNA sequence (as shown in SEQ ID NO:1, 1451 bp in length). BLAST alignment of this sequence in the GenBank database showed a similarity of over 99% with the type strain of *Priestia aryabhattai*. A phylogenetic tree was constructed based on the 16S rDNA sequence (e.g., ...). Figure 1(Note: A phylogenetic tree was constructed using 16S sequences of closely related taxa of the genus Bacillus and the outgroup Escherichia coli.) The results showed that strain Soil25HZ-CG-01-02-B12 clustered with Priestia aryabhattai, thus confirming that strain Soil25HZ-CG-01-02-B12 is Priestia aryabhattai.

[0106] 1.3 Preservation of bacterial strains

[0107] The strain Soil25HZ-CG-01-02-B12 was deposited on June 24, 2026 at the Guangdong Provincial Center for Microbial Culture Collection, located at No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, China, with accession number GDMCC 68502.

[0108] Example 2: Effect of Bacillus argentea Soil25HZ-CG-01-02-B12 on Ganoderma lucidum polysaccharide content (plate co-culture experiment)

[0109] 2.1 Test Materials

[0110] Ganoderma lucidum strain: Guangzhi No. 3, a Ganoderma lucidum strain selected and bred by the research group; Bacillus aureus: strain Soil25HZ-CG-01-02-B12 obtained in Example 1; culture medium: potato agarose medium (PDA).

[0111] "Guangzhi No. 3" is a high-yield, high-activity new variety that was isolated from high-quality wild Ganoderma lucidum collected from a nature reserve in Anhui Province by the Institute of Microbiology, Guangdong Academy of Sciences in 2014. It was developed through years of compound breeding and domestication screening and was approved by the Guangdong Provincial Crop Variety Approval Committee in 2026 (the first officially approved edible fungus variety in Guangdong Province).

[0112] 2.2 Experimental Design and Co-culture Methods

[0113] The activated Ganoderma lucidum spawn was inoculated into the center of a PDA plate. The experiment included a control group and a treatment group: the control group was inoculated with Ganoderma lucidum only; the treatment group was inoculated with Bacillus oryzae Soil25HZ-CG-01-02-B12 (50 μL) before inoculating with Ganoderma lucidum, and then covered with a sterile celluloid film (pre-trimmed to the plate size and autoclaved). Each treatment was replicated three times. The plates were incubated at 25°C in the dark for 10 days.

[0114] 2.3 Determination of Ganoderma lucidum polysaccharide content

[0115] After cultivation, Ganoderma lucidum mycelium from the control group and the treatment group were collected. Ganoderma lucidum polysaccharides were extracted through drying, pulverizing, hot water extraction, alcohol precipitation, and deproteinization. The polysaccharide content was determined by phenol-sulfuric acid method with glucose as standard, and the polysaccharide content of each treatment group was calculated.

[0116] 2.4 Test Results

[0117] The results are shown in Table 1 below. Figure 2 (The control group CK consisted of Ganoderma lucidum cultured alone (average mycelial polysaccharide content was 0.846%), and the treatment group T consisted of Ganoderma lucidum inoculated with *Bacillus argentea* of the present invention (average mycelial polysaccharide content was 1.640%).) Compared with the control group, the polysaccharide content of Ganoderma lucidum inoculated with *Bacillus argentea* Soil25HZ-CG-01-02-B12 of the present invention was significantly increased, reaching nearly twice that of the control, with a statistically significant difference (P<0.001). This indicates that *Bacillus argentea* of the present invention can significantly promote the accumulation of Ganoderma lucidum polysaccharides.

[0118] Table 1. Ganoderma lucidum polysaccharide content under different treatments

[0119]

[0120] Example 3: Preparation of microbial inoculum containing Bacillus oryzae Soil25HZ-CG-01-02-B12

[0121] The strain Soil25HZ-CG-01-02-B12 was inoculated into LB liquid medium and cultured at 37℃ with shaking at 250 rpm for 12 h until the effective viable count reached 1×10⁻⁶. 6 The fermentation broth was obtained by dispersing CFU / mL. The fermentation broth can be used directly as a liquid inoculum, or it can be processed into a solid (powder / granule) inoculum through processes such as freeze drying.

[0122] Example 4: Effect of microbial inoculum containing Bacillus oryzae Soil25HZ-CG-01-02-B12 on the content of Ganoderma lucidum polysaccharides.

[0123] 3.1 Test Materials

[0124] Ganoderma lucidum strain: Guangzhi No. 3; microbial agent: the microbial agent containing Bacillus argentea Soil25HZ-CG-01-02-B12 as described in Example 3; culture medium: PDA medium.

[0125] 3.2 Experimental Design and Co-culture Methods

[0126] The activated Ganoderma lucidum strain No. 3 was inoculated into the center of a PDA plate. The experiment included a control group and a treatment group: the control group was inoculated with Ganoderma lucidum only; the treatment group was sprayed with 50 μL of the above-mentioned microbial agent (equivalent to 0.5% inoculum volume) evenly before inoculation with Ganoderma lucidum, and then covered with a sterile celluloid film. Each treatment was replicated three times and incubated at 25°C in the dark for 10 days.

[0127] 3.3 Determination of Ganoderma lucidum polysaccharide content

[0128] After cultivation, the content of Ganoderma lucidum polysaccharides was extracted and determined according to the method described in Example 2.3.

[0129] 3.4 Test Results

[0130] The results are shown in Table 2. Compared with the control group, the polysaccharide content in Ganoderma lucidum mycelium treated with the microbial agent of this invention was significantly increased, reaching 1.578%, which was 1.852 times that of the control group (0.852%), and the difference was extremely significant (P<0.001). This result indicates that the microbial agent prepared by Bacillus aspergillus Soil25HZ-CG-01-02-B12 can significantly promote the accumulation of Ganoderma lucidum polysaccharides, with stable effects and practical application value.

[0131] Table 2. Ganoderma lucidum polysaccharide content under different treatments

[0132]

[0133] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A strain of *Priestia aryabhattai*, characterized in that, The depository of the aforementioned Bacillus aureus is the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC 68502.

2. The *Bacillus argentea* according to claim 1, wherein the 16S rDNA sequence is shown in SEQ ID NO:

1.

3. A microbial preparation, characterized in that, The microbial preparation includes Bacillus aureus as described in claim 1 or 2, or includes at least one of the following: bacterial suspension, fermentation broth, fermentation supernatant, bacterial cells, spores, or metabolites prepared from Bacillus aureus as described in claim 1 or 2.

4. The microbial preparation according to claim 3, characterized in that, The effective viable count of Bacillus aureus in the microbial preparation is not less than 1×10⁻⁶. 6 CFU / mL or CFU / g.

5. The use of Bacillus auricularis as described in claim 1 or 2, or the microbial preparation as described in claim 3 or 4, in increasing the content of Ganoderma lucidum polysaccharides.

6. The use of Bacillus auricula-judae as described in claim 1 or 2, or the microbial preparation as described in claim 3 or 4, in the cultivation of Ganoderma lucidum.

7. The use of Bacillus auricula-judae as described in claim 1 or 2, or the microbial preparation as described in claim 3 or 4, in the preparation of products with increased Ganoderma lucidum polysaccharide content.

8. A method for increasing the polysaccharide content of Ganoderma lucidum, characterized in that, The method includes the step of co-culturing Ganoderma lucidum with Bacillus argentis as described in claim 1 or 2, or with the microbial preparation as described in claim 3 or 4.

9. The method according to claim 8, characterized in that, The Ganoderma mentioned is a fungus belonging to the genus Ganoderma.

10. The method according to claim 9, characterized in that, The Ganoderma fungi mentioned are selected from at least one of Ganoderma lucidum, Ganoderma sinense, Ganoderma tsugae, and Ganoderma leucocontextum.

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