Preparation method of small molecule ganoderma lucidum embryo easily absorbed

Abstract

This invention provides a method for manufacturing easily absorbed Ganoderma lucidum embryos. The method involves screening high-expression strains using Q-PCR technology to obtain superior Ganoderma lucidum mother cultures. These mother cultures are inoculated into a spore-forming semi-solid culture medium and cultured in two stages to obtain a high-concentration Ganoderma lucidum spore culture. The spore culture is then transferred to a liquid submersible culture tank and cultured in two stages with the addition of a chitin-containing bio-inducer to induce spore growth and obtain Ganoderma lucidum embryo liquid. Further enzymatic processing using stepwise pH regulation yields easily absorbed Ganoderma lucidum embryo liquid. Finally, the liquid is bottled and sterilized to produce a Ganoderma lucidum embryo beverage. This product is rich in bioactive components such as small-molecule polysaccharides, ganoderic acids, and small peptides. This invention solves the problems of Ganoderma lucidum embryo formation and absorption. Animal experiments have shown an absorption rate of over 94%, and the product possesses functions such as immune protection and anti-tumor activity, antibacterial and anti-inflammatory effects, liver and kidney protection, lung function improvement, and intestinal regulation.

Description

Technical Field

[0001] This invention belongs to the fields of bioengineering and biocatalysis. More specifically, this invention relates to a method for efficiently producing Ganoderma lucidum active products using metabolic regulation, obtaining easily absorbed small-molecule functional components through biocatalytic cell wall disruption and degradation, and evaluating efficacy through animal experiments. This invention also provides a method for screening Ganoderma lucidum strains using RT-PCR technology. Background Technology

[0002] Reishi mushroom has been a precious medicinal fungus in my country since ancient times, known as the "immortal herb" and considered a national treasure. The first Chinese pharmacopoeia, the *Shennong Bencao Jing*, written over 2000 years ago, records the medicinal effects of higher fungi; Li Shizhen's *Compendium of Materia Medica* from the Ming Dynasty also lists the medicinal effects of reishi. It can be used to treat liver disease, chronic hepatitis, nephritis, hypertension, hyperlipidemia, neurasthenia, insomnia, forgetfulness, tracheitis and bronchitis, enteritis, arteriosclerosis, leukemia, diabetes, and physical weakness caused by prolonged illness. In recent years, its unique longevity-promoting effects have been demonstrated in anti-cancer and anti-tumor research.

[0003] Currently, most Ganoderma lucidum products are produced using artificially cultivated fruiting bodies as raw materials, either by extracting or collecting spores for processing into various products. The main problems with this method include: First, the high degree of lignification in the raw materials makes cell wall breaking difficult, resulting in poor absorption rates. Second, although Ganoderma lucidum is a fungus, it differs from other filamentous fungi; it does not form spores during the mycelial growth stage, but only after the fruiting body matures. Therefore, there are no research reports on how to produce spores in the later stages of mycelial growth. Third, the cultivation process is difficult to control, thus the yield of the active ingredients Ganoderma lucidum polysaccharides and Ganoderma lucidum acids cannot be targeted and increased.

[0004] Reishi mushrooms are higher fungi, and their wild growth history includes the process from spores to mycelium to fruiting bodies. The stage of spore germination into mycelium has long been overlooked; this invention defines it as the embryonic stage. During the spore stage, various nutrients in Reishi mushrooms are tightly stored within the cell wall. However, during the embryonic stage, small molecule nutrients such as polysaccharides, polypeptides, and ganoderic acids are formed and released from the cell wall. Products developed using Reishi embryos from this stage show significantly improved absorption rates. Therefore, how to break free from dependence on natural spores, directly and efficiently produce spores at the mycelial stage and simultaneously germinate them into embryos, while also directionally increasing the content of active ingredients, has become a pressing technical challenge in this field.

[0005] To overcome the aforementioned challenges, those skilled in the art have explored various technical approaches. Regarding enzymatic hydrolysis, patent CN200410019093.9 discloses a method for preparing Ganoderma lucidum polysaccharides through enzymatic hydrolysis. This method utilizes the mycelium's own enzyme system combined with exogenous enzymes such as cellulase and β-glucanase for enzymatic hydrolysis, resulting in 70%-90% of the polysaccharides having a molecular weight of 6000-50000. While this technology utilizes the mycelium's own enzyme system, it still requires the addition of multiple exogenous enzymes, making the process complex and posing a risk of exogenous enzyme residue. In contrast, this invention employs a stepwise pH-controlled enzymatic hydrolysis technique (first adjusting the pH to 3.5-5.0 and then incubating, followed by adjusting the pH to 7.5-9.0 and incubating again), relying entirely on the Ganoderma lucidum germ's own enzyme system to achieve highly efficient enzymatic hydrolysis without the need for any exogenous enzymes. This simplifies the process while improving product safety.

[0006] Regarding spore germination, the published patent CN200510036103.4 discloses a method for germinating Ganoderma lucidum spores, which involves soaking Ganoderma lucidum spores in activated water until the germ pores are exposed. This method only achieves the initial germination of spores, cannot be scaled up for mass production, and lacks precise control over the germination process. To address this deficiency, this invention employs a two-stage precise culture in a liquid submersible (stage 0-72 hours: ventilation ratio 1:0.75-1:1, temperature 25℃-33℃, pH 3.5-5.0; stage 72-96 hours: ventilation ratio 1:0.1-1:0.3, temperature 10℃-15℃, pH 5.5-7.0), and adds a chitin-containing bio-inducer to fully germinate the spores and grow them into embryos, thus achieving mass production.

[0007] Regarding metabolic regulation, patent CN201710148624.1 discloses a liquid culture medium for Ganoderma lucidum with high yields of polysaccharides and ganoderic acids, and its preparation method, which adds 5.4-7.2 mM calcium chloride to increase the content of polysaccharides and ganoderic acids. In this technology, calcium and magnesium salts are added as micronutrients at concentrations only in the millimolecular range. However, this invention has found that increasing the calcium salt concentration to 0.9-2.0 mol / L and the magnesium salt concentration to 0.5-1.0 mol / L (2-3 orders of magnitude higher), combined with 1-90 mg / L ozone and an acidic environment of pH 3.5-4.5, the synergistic effect of these three elements can efficiently induce mycelium to produce spores, while significantly increasing the yield of ganoderic acids and polysaccharides. This high-concentration synergistic culture strategy overcomes the technical prejudice held by those skilled in the art that high concentrations of metal ions inhibit fungal growth.

[0008] Furthermore, regarding strain selection, the published patent CN201910120979.9 discloses a high-yielding Ganoderma lucidum polysaccharide strain RWHBW-1 and its applications. This strain obtains high-yielding Ganoderma lucidum polysaccharide through mutagenesis; however, this method has a long screening cycle and uncontrollable mutation direction. In contrast, this invention uses HMGR as the target gene and employs Q-PCR to detect its mRNA expression level for targeted screening, achieving quantitative and efficient strain selection.

[0009] In summary, none of the existing technologies disclose the core technical solution of this invention: directly inducing mycelium to produce spores on a semi-solid culture medium through the synergistic stress of high-concentration calcium and magnesium salts, ozone, and low pH; then simultaneously germinating the spores into embryos through two-stage deep liquid culture; and finally obtaining small-molecule active ingredients by combining stepwise pH-controlled self-enzymatic hydrolysis technology. The technical solution of this invention achieves a dual technological leap from "spore collection" to "direct mycelial sporulation" and from "external enzyme cell wall disruption" to "self-enzymatic hydrolysis," resulting in a product absorption rate significantly superior to existing Ganoderma lucidum products. Summary of the Invention

[0010] Purpose of the invention: The purpose of this invention is to provide a method for manufacturing small-molecule, easily absorbed Ganoderma lucidum germ, thereby solving the absorption problem of current Ganoderma lucidum products and producing a small-molecule, easily absorbed Ganoderma lucidum germ product.

[0011] Technical Solution: To achieve the above objectives, this invention adopts the following technical approach: First, addressing the technical challenge of Ganoderma lucidum mycelium's inability to directly produce spores under natural conditions, this invention successfully induces mycelium to directly produce spores on a semi-solid culture medium by optimizing nutrient factors (high concentration of calcium and magnesium salts) and non-nutrient factors (ozone, low pH, segmented temperature control) during the mycelial growth stage, thus eliminating dependence on natural spores. Second, addressing the problem of low ganoderic acid yield, this invention uses glutaryl-CoA reductase (HMGR) as a target gene and employs Q-PCR technology to screen for high-expression strains. HMGR is a key rate-limiting enzyme in the ganoderic acid biosynthesis pathway, and its expression level is positively correlated with ganoderic acid yield; using this as a screening indicator enables targeted breeding of high-yielding strains. Third, this invention utilizes the control of combinations of nutrient and non-nutrient factors as one of the metabolic regulation strategies to achieve high yields of Ganoderma lucidum polysaccharides and ganoderic acids.

[0012] Based on the above technical concept, the present invention specifically adopts the following technical solution: In a first aspect, the present invention discloses a method for manufacturing Ganoderma lucidum germ with easily absorbed small molecules, comprising the following steps: Step (a). Select Ganoderma lucidum strains to obtain active Ganoderma lucidum mother strains; Step (b). Prepare a semi-solid culture medium for spore formation and use it for mother culture; Step (c). The Ganoderma lucidum mother culture obtained in step (a) is inoculated into the spore-forming semi-solid culture medium prepared in step (b) and cultured under specific environmental conditions to obtain Ganoderma lucidum spore culture. Step (d). The Ganoderma lucidum spore culture obtained in step (c) is transferred to a liquid deep culture tank to grow buds and obtain Ganoderma lucidum embryo solution; Step (e). The Ganoderma lucidum germ liquid described in step (d) is subjected to enzymatic treatment to obtain a small molecule Ganoderma lucidum germ liquid that is easily absorbed. Step (f). The small-molecule, easily absorbed Ganoderma lucidum germ liquid obtained in step (e) is bottled and sterilized to obtain Ganoderma lucidum germ beverage.

[0013] In some specific embodiments, the method for selecting the Ganoderma lucidum strain in step (a) includes: using the HMGR gene as the target gene, detecting its mRNA expression level by Q-PCR, and selecting the strains with the highest expression levels as production strains.

[0014] In some specific embodiments, the spore-forming semi-solid culture medium in step (b) comprises: 0.5%-5% carbon source, 0.05%-3% nitrogen source, 0.05%-0.75% gelling agent, 0.9-2.0 mol / L calcium salt, and 0.5-1.0 mol / L magnesium salt, and the pH of the spore-forming semi-solid culture medium is 3.5-4.5.

[0015] In some specific implementations, the specific environmental conditions in step (c) are as follows: Phase 1, days 1-10: Temperature 15℃-35℃, humidity 55%-95%, light intensity 10lx-100lx; The second stage, 1-7 days: temperature 1℃-15℃, humidity 30%-50%, light intensity 200lx-400lx, and ozone 1mg / L-90mg / L added; Ultimately, the spore concentration in the obtained Ganoderma lucidum spore culture was 5 × 10⁻⁶. 9 / g-5×10 10 / g.

[0016] In some specific embodiments, the culture medium in the liquid submersible in step (d) comprises: 0.5%-5% carbon source, 0.05%-3% nitrogen source, 0.5-0.75 mol / L calcium salt, and 0.15-0.35 mol / L magnesium salt, and the pH of the culture medium is controlled at 3.5-7.0, and a two-stage culture is carried out.

[0017] In some specific implementations, the conditions for the two-stage cultivation are as follows: Phase 1, 0-72 hours: ventilation ratio 1:0.75-1:1, temperature 25℃-33℃, pH 3.5-5.0; The second stage, 72-96 hours: ventilation ratio 1:0.1-1:0.3, temperature 10℃-15℃, pH 5.5-7.0, and addition of 0.5%-3% of a chitin-containing biological inducer, wherein the biological inducer is selected from at least one of cicada pupa powder, chitin, and edible fungus mycelium powder.

[0018] In some specific embodiments, the enzymatic treatment in step (e) is a stepwise pH-controlled enzymatic treatment, specifically as follows: First, adjust the pH of the Ganoderma lucidum germ solution to 3.5-5.0, raise the temperature to 45℃-65℃, and keep it warm for 5-30 hours; Then, adjust the pH to 7.5-9.0, raise the temperature to 45℃-65℃, and keep it at that temperature for 5-30 hours; Furthermore, the enzymatic treatment does not involve the addition of exogenous enzymes, but relies entirely on the enzyme system of the Ganoderma lucidum germ itself.

[0019] In some specific embodiments, the Ganoderma lucidum germ solution obtained in step (e) contains: The polysaccharide content is 1.5g / 100g-3.0g / 100g. The content of ganoderic acid is 0.5g / 100g-1.75g / 100g. The polysaccharides have a molecular weight of 1kDa-50kDa, with small molecule polysaccharides having a molecular weight of less than 50kDa accounting for more than 85%.

[0020] In some specific embodiments, step (f) involves sterilizing the germ solution in a jar at 100°C-135°C for 5 seconds to 60 minutes.

[0021] Secondly, this invention discloses a small-molecule, easily absorbed Ganoderma lucidum germ product, which is manufactured by the method of manufacturing the small-molecule, easily absorbed Ganoderma lucidum germ. The product has immune-anti-tumor, antibacterial and anti-inflammatory, liver-protecting and kidney-strengthening, lung-improving and intestinal-regulating effects, and its absorption rate in animal experiments reaches over 94%.

[0022] Compared with the prior art, the present invention has the following beneficial effects: First, the method of the present invention enables direct sporulation of mycelium, eliminating the dependence on natural spores.

[0023] This invention utilizes the synergistic effect of three key elements—high concentrations of calcium and magnesium salts (0.9-2.0 mol / L calcium salt and 0.5-1.0 mol / L magnesium salt), ozone (1-90 mg / L), and low pH (3.5-4.5)—to successfully induce the direct production of spores from Ganoderma lucidum mycelium on a semi-solid culture medium. This technological breakthrough eliminates the reliance on fruiting body cultivation for Ganoderma lucidum spore production, enabling industrialized and large-scale production.

[0024] Second, the method of the present invention enables spores to germinate into embryos simultaneously without the need for additional cell wall breaking treatment.

[0025] This invention utilizes a two-stage precise culture process in a deep liquid culture tank, allowing the cell walls of spores to naturally soften and rupture during germination, thus releasing active ingredients naturally. This technical solution eliminates the need for a dedicated cell wall disruption process, simplifies the workflow, and reduces production costs.

[0026] Third, the method of the present invention achieves self-enzymatic hydrolysis without the addition of exogenous enzymes, and the product has high safety.

[0027] This invention utilizes a stepwise pH-controlled enzymatic hydrolysis technique (first adjusting the pH to 3.5-5.0 and then incubating, followed by adjusting the pH to 7.5-9.0 and incubating again), relying entirely on the Ganoderma lucidum embryo's own enzyme system to achieve highly efficient enzymatic hydrolysis.

[0028] Fourth, the product of this invention has a small molecular weight, high absorption rate, and significantly improved bioavailability. The Ganoderma lucidum germ extract obtained by this invention contains polysaccharides with a molecular weight of 1-50 kDa, with small molecule polysaccharides accounting for over 85%, and ganoderic acid content ranging from 0.5g / 100g to 1.75g / 100g. Animal experiments show that the absorption rate of the product of this invention reaches 94%, while the absorption rates of commercially available Ganoderma lucidum powder and Ganoderma lucidum spore powder are only 32% and 35%, respectively. This significant increase in absorption rate means a substantial increase in the amount of active ingredients entering the bloodstream at the same dosage, thereby improving the product's efficacy and cost-effectiveness.

[0029] Fifth, the product of this invention has multiple health care functions. Animal experiments have shown that the product of this invention has the following health care functions: (1) immune and anti-tumor effects; (2) enhancing the phagocytic function of macrophages; (3) protecting immune organs; and (4) antibacterial and anti-inflammatory effects.

[0030] Sixth, the process of this invention is simple, low-cost, and suitable for industrial production. Compared with the prior art, this invention has the following process advantages: (1) It uses spores as inoculation raw materials and can be produced on a large scale through fermentation engineering, without being limited by season or region; (2) It does not require additional cell wall breaking process or the addition of exogenous enzymes, simplifying the process flow; (3) Liquid deep fermentation and stepwise pH-controlled enzymatic reaction are both completed in conventional equipment, with low equipment investment and easy scale-up production. Therefore, this invention has good industrial application prospects and economic benefits. Attached Figure Description

[0031] Figure 1 Schematic flow chart of the manufacturing method of small molecule easily absorbable Ganoderma lucidum germ Figure 2 Schematic diagram of the morphology of Ganoderma lucidum spores in semi-solid medium of the present invention Figure 3 Schematic diagram of the morphology of Ganoderma lucidum mycelium in semi-solid medium of the present invention Specific embodiments

[0032] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions or according to the conditions recommended by the manufacturers.

[0033] Unless otherwise specified, percentages and parts are calculated by weight.

[0034] Example 1: Screening of high-yield Ganoderma lucidum strains Collect Ganoderma lucidum fruit bodies from the wild, obtain pure strains using aseptic isolation techniques in the laboratory, culture on PDA slants, collect mycelia to extract RNA, and detect the HMGR expression levels of each strain by Q-PCR. Different varieties such as Qingzhi, Chizhi, Zizhi, Huangzhi, Baizhi, and Heizhi are selected respectively. Some of the screening results are shown in Table 1 below, and the high-yield strain DBS200010-Chi (Chizhi) is obtained.

[0035] Table 1 Screening of high-yield Ganoderma lucidum strains by Q-PCR Note: Qing (Qingzhi), Huang (Huangzhi), Chi (Chizhi), Zi (Zizhi), Bai (Baizhi), Hei (Heizhi).

[0036] As can be seen from Table 1, the HMGR expression level of the DBS200010-Chi strain is the highest (9.7), and its ganoderic acid content is also the highest (1.12 g / 100 g), indicating that the HMGR expression level is positively correlated with the ganoderic acid yield. Therefore, this strain is selected as the production strain for subsequent experiments.

[0037] Example 2: Preparation of sporulation semi-solid medium Weigh 0.2% carboxymethyl cellulose, 0.5% agar powder, 1% glucose, 0.15% yeast powder, 10% calcium chloride (about 0.9 mol / l), and 5% magnesium chloride (about 0.53 mol / l) into potato juice, adjust the pH to 4.5, and sterilize at 121 °C under high-pressure steam for 25 minutes. After cooling, it is the sporulation semi-solid medium (Semi-S).

[0038] Simultaneously, a semi-solid culture medium with calcium chloride and magnesium chloride removed and a solid culture medium (S) containing calcium chloride and magnesium chloride were prepared as controls.

[0039] Example 3: Cultivation of Ganoderma lucidum spores The high-yielding Ganoderma lucidum strain DBS200010-Chi selected in Example 1 was inoculated into the semi-solid sporulation medium prepared in Example 2. It was first cultured at 27°C, 75% humidity, and 50 lx light intensity for 7 days. Then, it was cultured at 12°C, 30% humidity, 200 lx light intensity, and 60 mg / L ozone concentration for 5 days. Comparative experiments were conducted simultaneously, and the results are shown in Table 2. Morphological observations are shown in... Figure 2 and Figure 3 .

[0040] Table 2 Effects of different culture conditions on Ganoderma lucidum spore formation The culture conditions for each item in the table above are as follows: 1- Semi-solid sporulation medium, 2- Solid medium (control), 3- Semi-solid medium without 10% calcium chloride and 5% magnesium chloride (control), 4- Semi-solid medium containing 9% calcium chloride and 4% magnesium chloride (control), 5- Culture with ozone, 6- Culture without ozone (control), 7- Constant temperature culture at 30℃. Spore yield "++" indicates high yield, "-" indicates not detected.

[0041] Table 2 shows that Ganoderma lucidum mycelium can only produce spores efficiently under the combined conditions of semi-solid culture medium, high concentration of calcium and magnesium salts (10% calcium chloride + 5% magnesium chloride), ozone (60 mg / L), and segmented temperature culture (conditions 1 and 5). This indicates that a semi-solid culture medium combined with high concentration of calcium and magnesium salts, ozone, and segmented temperature culture are necessary conditions for Ganoderma lucidum mycelium to produce spores.

[0042] Example 4: Obtaining Ganoderma lucidum embryos Culture medium composition: 3.5% glucose, 1% fructose, 0.1% corn flour, 0.5% wheat bran, 1% soybean flour, 5% calcium chloride (approximately 0.45 mol / L), 2% magnesium chloride (approximately 0.21 mol / L); pH of the culture medium was 4.5. The medium was autoclaved at 121°C for 45 minutes and then cooled before inoculating with spores obtained in Example 3 at a 10% inoculum.

[0043] The training is divided into the following two stages: Phase 1: 0-72 hours, with ventilation controlled at 1:1, temperature 28℃, pH 4.5, and constant temperature and pH controlled culture.

[0044] Second stage: 72-96 hours, with ventilation controlled at 1:0.3, temperature at 15℃, pH at 6.5, and constant temperature and pH controlled culture, during which 2% cicada pupa powder is added as a chitin-containing biological inducer.

[0045] Four control groups were set up: one without cicada pupa powder, one without calcium chloride or magnesium chloride, and one without two-stage culture (temperature and pH control). The experimental results are shown in Table 3.

[0046] Table 3. Effects of different culture conditions on the formation of effective components in Ganoderma lucidum. The culture conditions for each item in the table above are as follows: 1- Culture conditions of this patent, 2- No calcium chloride or magnesium chloride added to the culture medium (control), 3- No cicada pupa powder added during the second stage of culture (control), 4- No second stage of culture (control), @ Determination method under Ganoderma lucidum in the National Pharmacopoeia is adopted, * Determination method of GB / T34799-2017 is adopted, # refers to neutral protease, the activity determination adopts the method in GB / T23527.1-2023, & Dextranase activity determination adopts the method specified in GB / T23874-2021.

[0047] As shown in Table 3, under the cultivation conditions of this invention (Condition 1), the content of ganoderic acid (1.72 g / 100 g), polysaccharide content (2.85%), and the activities of the three enzymes were significantly higher than those in the control groups. This indicates that the combined effects of high concentrations of calcium and magnesium salts, chitin-containing biological inducers, and two-stage cultivation are key to improving the content of effective components and enzyme activities in Ganoderma lucidum.

[0048] Example 5: Preparation of small molecule Ganoderma lucidum germ solution (stepwise pH-controlled enzymatic reaction) The patented process involves adjusting the pH of the Ganoderma lucidum germ solution obtained in Example 4 to 4.5, heating it to 45°C, and holding it therefore for 20 hours; then adjusting the pH to 7.5, heating it to 55°C, and holding it therefore for 15 hours.

[0049] Control 1: pH not adjusted, directly incubated at 50℃ for 35 hours.

[0050] Control 2: First, adjust the pH to 7.5, then incubate at 55℃ for 15 hours, then adjust the pH to 4.5, cool to 45℃, and incubate for 20 hours.

[0051] The experimental results are shown in Table 4.

[0052] Table 4. Effects of different treatment processes on the formation of effective components in Ganoderma lucidum. Wherein: @ uses the determination method under Ganoderma lucidum in the National Pharmacopoeia, * uses the Ubbelohde viscometer method, and the molecular weight is less than 50KD.

[0053] As shown in Table 4, the process of this invention (stepwise pH control with acid followed by alkali) yielded the highest content of ganoderic acid (1.75 g / 100 g) and polysaccharide (2.97%), with small molecule polysaccharides accounting for 87.3%, while almost no small molecule polysaccharides were detected in controls 1 and 2. This indicates that the specific pH control sequence of acid followed by alkali is key to obtaining a high proportion of small molecule polysaccharides, and that no exogenous enzymes were added during the entire enzymatic process, relying entirely on the enzyme system of the Ganoderma lucidum germ itself.

[0054] Comparative Example 1: Effects of different conditions on sporulation and active ingredients in Ganoderma lucidum To demonstrate the synergistic effect of the three elements of high-concentration calcium and magnesium salts, ozone, and segmented temperature culture in this invention, the following control experiment was set up.

[0055] (1) Experimental design Based on the sporulation semi-solid culture medium of Example 2, and keeping other culture conditions (inoculum size, culture period, etc.) consistent, the following treatment groups were set up: Group A (Complete conditions of this invention): Semi-solid spore-forming medium (containing 10% calcium chloride + 5% magnesium chloride) + ozone (60 mg / L) + segmented temperature culture (first stage: 27℃, 7 days; second stage: 12℃, 5 days); Group B (calcium and magnesium salt deficient): Semi-solid spore-forming medium without calcium chloride and magnesium chloride + ozone (60 mg / L) + segmented temperature culture; Group C (ozone-deficient): Semi-solid spore-forming medium (containing 10% calcium chloride + 5% magnesium chloride) in ozone-free + segmented temperature culture; Group D (lacking segmented temperature): Semi-solid spore-forming medium (containing 10% calcium chloride + 5% magnesium chloride) + ozone (60 mg / L) + constant temperature 30℃ culture (unsegmented); Group E (low concentration of calcium and magnesium salts): semi-solid spore-forming medium containing low concentrations of calcium chloride (1%, approximately 0.09 mol / L) and magnesium chloride (0.5%, approximately 0.05 mol / L) + ozone (60 mg / L) + segmented temperature culture; Group F (single-factor control): the average of the experimental results under three single-factor conditions (high concentration of calcium and magnesium salt alone, ozone alone, and segmented temperature alone).

[0056] (2) Measurement indicators After cultivation, the spore concentration and ganoderic acid content of each group were measured. Spore concentration was determined using a hemocytometer; ganoderic acid content was determined using the method described in the Ganoderma section of the Chinese Pharmacopoeia.

[0057] (3) Experimental results The experimental results are shown in Table 1, the control example.

[0058] Comparative Example Table 1: Effects of Different Stress Conditions on Ganoderma lucidum Sporulation and Active Components Note: High concentration refers to 10% calcium chloride (approximately 0.9 mol / L) + 5% magnesium chloride (approximately 0.53 mol / L); low concentration refers to 1% calcium chloride (approximately 0.09 mol / L) + 0.5% magnesium chloride (approximately 0.05 mol / L); "+" indicates that the condition was applied, "-" indicates that the condition was not applied; spore concentration "<1×10 6 "" indicates that no sporulation occurred; group F is the average of three single-factor conditions (high concentration of calcium and magnesium salts alone, ozone alone, and segmented temperature alone).

[0059] (4) Results Analysis As can be seen from the comparison table 1: First, all three elements are indispensable. Only the complete conditions of this invention (Group A) can achieve efficient sporulation (4.8 × 10⁻⁶). 9 The spore concentration was high (1.72 g / 100 g) and high in ganoderic acid (1.72 g / 100 g); the spore concentration of groups lacking any element (groups B, C, and D) was below the detection limit (<1×10⁻⁶ g / g). 6 The content of ganoderic acid was also significantly reduced ( / g). This indicates that there is a synergistic effect among high concentrations of calcium and magnesium salts, ozone, and segmented temperature cultivation, and all three must be present simultaneously to achieve the purpose of the invention.

[0060] Second, there is a critical effect of calcium and magnesium salt concentration. Low concentrations of calcium and magnesium salts (group E) can produce a small number of spores (5.2 × 10⁻⁶). 7 However, its spore concentration is less than 1 / 90 of that of the present invention (Group A), and its ganoderic acid content is also much lower than that of Group A. This indicates that the high concentration (0.9 mol / L) selected in the present invention is a necessary condition for achieving efficient spore production; below this concentration, the same effect cannot be achieved.

[0061] Third, single factors are completely ineffective. The single-factor control group (Group F) failed to induce sporulation, further demonstrating that the technical effect of this invention stems from the synergistic combination of the three elements, rather than the simple superposition of a single factor.

[0062] In summary, this comparative example demonstrates that the three essential elements of high-concentration calcium and magnesium salts, ozone, and segmented temperature cultivation must coexist to efficiently induce sporulation of Ganoderma lucidum mycelium; there is a critical effect of calcium and magnesium salt concentration, and the high concentration (0.9 mol / L) selected in this invention is a necessary condition for achieving efficient sporulation.

[0063] Comparative Example 2: Effects of high concentrations of calcium and magnesium salts on sporulation and active ingredients in Ganoderma lucidum To verify the feasibility of the calcium salt concentration range of 0.9-2.0 mol / L and magnesium salt concentration of 0.5-1.0 mol / L in this invention, and to demonstrate the promoting effect of high concentration calcium and magnesium salts (1.5-2.0 mol / L) on sporulation and active ingredients of Ganoderma lucidum, the following supplementary experiments were conducted.

[0064] (1) Experimental design Based on the sporulation semi-solid culture medium of Example 2, and keeping other culture conditions consistent (ozone 60 mg / L, segmented temperature culture), the following calcium and magnesium salt concentration gradient treatment groups were set up: Group 1 (concentration in the embodiments of the present invention): 10% calcium chloride (approximately 0.9 mol / L) + 5% magnesium chloride (approximately 0.53 mol / L); Group 2 (medium to high concentration): 15% calcium chloride (approximately 1.35 mol / L) + 7% magnesium chloride (approximately 0.74 mol / L); Group 3 (high concentration): 18% calcium chloride (approximately 1.62 mol / L) + 9% magnesium chloride (approximately 0.95 mol / L); Group 4 (high concentration): 22% calcium chloride (approximately 1.98 mol / L) + 10% magnesium chloride (approximately 1.05 mol / L); Group 5 (overly high concentration control): 25% calcium chloride (approximately 2.25 mol / L) + 12% magnesium chloride (approximately 1.26 mol / L); Three parallel experiments were set up for each group, and the average value was taken as the final result. After the culture was completed, the spore concentration, ganoderic acid content and polysaccharide content were measured respectively.

[0065] (2) Measurement method a. Spore concentration: determined by hemocytometer method; b. Ganoderma lucidum acid content: determined by the method under Ganoderma lucidum in the Chinese Pharmacopoeia; c. Polysaccharide content: determined by phenol-sulfuric acid method.

[0066] (3) Experimental results The experimental results are shown in Table 2 of the control examples.

[0067] Comparative Example Table 2: Effects of different concentrations of calcium and magnesium salts on sporulation and active ingredients of Ganoderma lucidum (4) Results Analysis As can be seen from the comparison table 2: First, the concentration range of this invention is fully achievable. Within the calcium and magnesium salt concentration range of Groups 1 to 4 (calcium salt 0.9-1.98 mol / L, magnesium salt 0.53-1.05 mol / L), the spore concentration all reached 4.5 × 10⁻⁶. 9The content of ganoderic acid is 1.68g / 100g-1.89g / 100g, and the polysaccharide content is 2.78%-3.12%, both of which are at a relatively high level. This indicates that the calcium salt concentration range (0.9-2.0mol / L) and magnesium salt concentration range (0.5-1.0mol / L) defined in this invention are completely achievable and effective ranges.

[0068] Second, appropriately increasing the calcium and magnesium salt concentration yielded better results. When the calcium and magnesium salt concentration was increased from 0.9 mol / L (Group 1) to 1.35 mol / L (Group 2), the spore concentration increased to 5.6 × 10⁻⁶. 9 The content of ganoderic acid was increased to 1.89 g / 100g, and the polysaccharide content was increased to 3.12%, with all indicators reaching optimal values. This indicates that, within the scope of this invention, appropriately increasing the concentration of calcium and magnesium salts can further promote sporulation and the accumulation of active ingredients.

[0069] Third, the effect decreased when the concentration exceeded 2.0 mol / L. When the calcium and magnesium salt concentration exceeded 2.0 mol / L (group 5, 2.25 mol / L), the spore concentration (2.1 × 10⁻⁶) decreased. 9 The content of ganoderic acid (1.21g / 100g) and polysaccharide (1.96%) all decreased significantly, indicating that excessively high concentrations would exert excessive stress on the Ganoderma lucidum mycelium, inhibiting its growth and sporulation.

[0070] In summary, this comparative example demonstrates that the calcium salt concentration range (0.9-2.0 mol / L) and magnesium salt concentration range (0.5-1.0 mol / L) defined in this invention are both achievable, and efficient sporulation (spore concentration ≥ 4.5 × 10⁻⁶) can be obtained within these ranges. 9 The concentration of ganoderic acid ≥ 1.68 g / 100 g and polysaccharide ≥ 2.78% is high; the effect decreases when the concentration exceeds 2.0 mol / L.

[0071] Example 6: Ganoderma lucidum germ absorption rate and immune antitumor experiment 6.1 Experimental Materials Drugs: The original Ganoderma lucidum embryo samples prepared in Example 5 of this invention were divided into three dosage groups (low, medium, and high), denoted as: Ganoderma lucidum embryo a, Ganoderma lucidum embryo b, and Ganoderma lucidum embryo c; Positive control group: cyclophosphamide, Shanghai Hualian Pharmaceutical Co., Ltd.; commercially available Ganoderma lucidum spore powder; commercially available Ganoderma lucidum powder; Negative control: self-made physiological saline.

[0072] Animals and tumor spectrum: Kunming mice, half male and half female, weighing 18-22 grams, were provided by Shanghai Slack Animal House.

[0073] 6.2 Methods 6.2.1 Digestion and Absorption Experiment of Ganoderma lucidum Embryo The content of Ganoderma lucidum triterpenoids in feces is measured daily, and the formula for calculating the digestibility and absorption rate is as follows: Digestibility and absorption rate (%) = (Ganoderic acid content in the sample before gavage - Ganoderic acid content in the feces) / Ganoderic acid content in the sample before gavage × 100% 6.2.2 Antitumor effect on mouse S180 As per standard procedure, S180 tumor masses were aseptically scraped from mice, minced, and homogenized into single cells using a glass homogenizer. The cells were diluted 1:3 with physiological saline to adjust the cell count, and 0.2 ml was injected into the right axilla of each mouse. Twenty-four hours later, the mice were randomly divided into 13 groups of six mice each, including a physiological saline control group and a cyclophosphamide positive control group. Samples were further divided into high, medium, and low dose groups. Administration continued for 10 days (by gavage). On day 11, the mice were sacrificed, the tumor masses were collected, and the tumor weight was measured to calculate the inhibition rate.

[0074] 6.2.3 Effects on phagocytic function of mouse peritoneal macrophages Mice were grouped, administered the drug at the same dosage and via the same route as above. After 10 consecutive days of administration, 48 hours before sacrifice, each mouse was intraperitoneally injected with 1 ml of 2% soluble starch. Two hours after the last administration, each mouse was intraperitoneally injected with 0.5 ml of 5% chicken erythrocyte (CRBC) suspension. Four hours after injection, the animals were sacrificed, and peritoneal macrophages (MΦ) were flushed out with Hank's solution, placed on a glass slide, and incubated at 37°C for 30 minutes. The free cells were then rinsed off with physiological saline and dried. Giemsa-Wright staining was performed, and the phagocytic rate of CRBCs by 100 MΦ cells was calculated.

[0075] 6.2.4 Effects on white blood cell count and immune organ weight in mice The mice were grouped and administered the drugs as described above, and were given the drugs continuously for 10 days. Twenty-four hours after the last administration, blood was collected from the tail vein to count the white blood cells. The animals were then sacrificed, and the thymus and spleen were weighed.

[0076] 6.3 Results and Discussion 6.3.1 Determination of the digestibility and absorption rate of Ganoderma lucidum germ The experimental data are shown in Table 5. The absorption rate of Ganoderma lucidum germ reached 94%, which was significantly higher than that of Ganoderma lucidum whole powder (32%) and Ganoderma lucidum spore powder (35%), indicating that the product of this invention has excellent digestibility and absorption.

[0077] Table 5. Experimental results of absorption rates of Ganoderma lucidum germ, Ganoderma lucidum powder, and Ganoderma lucidum spore powder. 6.3.2 Antitumor effect of Ganoderma lucidum germ on S180 tumors in mice The results are shown in Table 6 below. As can be seen from Table 6, the original Ganoderma lucidum embryo samples of the present invention have significant antitumor effects (P<0.01 or P<0.05). The tumor inhibition rates of the medium and high dose groups were 40.9% and 58.8%, respectively, showing a dose-dependent effect. The high dose group (58.8%) was close to the positive control cyclophosphamide (67.1%), but without the toxic side effects of chemical drugs.

[0078] Table 6. Antitumor effect of Ganoderma lucidum germ on S180 tumors in mice (n=6) 6.3.3 Effect of Ganoderma lucidum germ on phagocytic function of mouse peritoneal macrophages The results are shown in Table 7 below. Table 7 shows that cyclophosphamide inhibits the phagocytic function of macrophages (MΦ); Ganoderma lucidum whole powder and spore powder do not significantly improve the phagocytic function of MΦ. However, the Ganoderma lucidum embryo sample of this invention can significantly improve the phagocytic rate of mouse peritoneal macrophages, with the high-dose group reaching 80.5%, which is significantly higher than the control group (43.1%).

[0079] Table 7 Effects of Ganoderma lucidum germ on peritoneal phagocytic and bactericidal function of mice (n=6) (X mean ± SD) 6.3.4 Effects of Ganoderma lucidum germ on white blood cell count and immune organ weight in mice As shown in Table 8 below, cyclophosphamide reduced the white blood cell count in mice by 67.4%, and caused atrophy of the thymus and spleen by 35.7% and 48.6%, respectively, with the spleen atrophy being more pronounced. The improvement effects of Ganoderma lucidum whole powder and spore powder were not significant. However, the Ganoderma lucidum embryo sample of this invention significantly increased the white blood cell count and the weight of immune organs in mice; the white blood cell count in the high-dose group increased from 9.5 × 10⁻⁶ in the control group. 9 / L increased to 22.1×10 9 / L, thymus weight increased from 39.8 mg / 10g to 58.7 mg / 10g, and spleen weight increased from 36.8 mg / 10g to 61.8 mg / 10g.

[0080] Example 7: Experiment on the inhibition of pathogenic bacteria by Ganoderma lucidum embryo Test strains: Staphylococcus aureus (SA), Escherichia coli (EL), and Candida albicans (CA).

[0081] Experimental method: The antibacterial efficacy test method was adopted according to the 2015 edition of the Chinese Pharmacopoeia. Test bacterial concentration: 5 × 10⁻⁶ 8 cfu / ml.

[0082] The experimental results are shown in Table 9 below. The Ganoderma lucidum germ samples of the present invention have a significant inhibitory effect on the three pathogenic bacteria, with inhibition rates of 94.1% (Staphylococcus aureus), 92.6% (Escherichia coli) and 79.6% (Candida albicans) after 24 hours.

[0083] Table 9. Antibacterial effect of ganoderic acid (concentration of tested bacterial solution: 5×10⁻⁶) 8 cfu / ml) In summary, the present invention has the following advantages: I. The method of the present invention enables direct sporulation of mycelium, eliminating the dependence on natural spores.

[0084] In existing technologies, the acquisition of Ganoderma lucidum spores relies on the collection of fruiting bodies after natural maturation, which is time-consuming, costly, and lacks quality control. This invention utilizes the synergistic effect of three key elements—high concentrations of calcium and magnesium salts (0.9-2.0 mol / L calcium salt, 0.5-1.0 mol / L magnesium salt), ozone (1-90 mg / L), and low pH (3.5-4.5)—to successfully induce the direct production of spores from Ganoderma lucidum mycelium on a semi-solid culture medium.

[0085] As shown in Example 3, under the culture conditions of the present invention, the spore concentration reached 5 × 10⁻⁶. 9 / g-5×10 10 / g, while the control groups without added calcium and magnesium salts or ozone failed to produce spores. Example 1 further demonstrates that the absence of any one factor leads to sporulation failure (spore concentrations in groups B, C, and D were all below 1×10⁻⁶). 6 / g), the spore concentration of low-concentration calcium-magnesium salts (Group E) is only about 1 / 90 (5.2 × 10⁻⁶) of that under intact conditions. 7 The single factor (group F) alone could not induce sporulation. This technological breakthrough has made Ganoderma lucidum spore production no longer dependent on fruiting body cultivation, realizing industrialized and large-scale production.

[0086] Second, the method of the present invention enables spores to germinate into embryos simultaneously without the need for additional cell wall breaking treatment.

[0087] In existing technologies, Ganoderma lucidum spores have tough chitin-containing cell walls, requiring mechanical or enzymatic disruption to release active ingredients. This disruption process not only increases costs but may also damage heat-sensitive active substances. This invention utilizes a two-stage precise culture process in a deep liquid culture tank, allowing the cell walls to soften and rupture naturally during spore germination, thus releasing the active ingredients naturally.

[0088] As shown in Example 4, under the two-stage cultivation conditions of the present invention (first stage: ventilation rate 1:0.75-1:1, temperature 25-33℃, pH 3.5-5.0; second stage: ventilation rate 1:0.1-1:0.3, temperature 10-15℃, pH 5.5-7.0, with the addition of cicada pupa powder and other biological inducers), the ganoderic acid content reached 1.72g / 100g, and the polysaccharide content reached 2.85%, significantly higher than the control group without the addition of inducers (0.48g / 100g and 1.79%) or without two-stage cultivation (0.39g / 100g and 1.13%). This technical solution eliminates the need for a dedicated cell wall breaking process, simplifies the process flow, and reduces production costs.

[0089] Third, the method of the present invention achieves self-enzymatic hydrolysis without the addition of exogenous enzymes, and the product has high safety.

[0090] In existing technologies, the enzymatic hydrolysis preparation of Ganoderma lucidum polysaccharides usually requires the addition of exogenous enzymes such as cellulase and β-glucanase, which not only increases costs but also poses safety risks due to residual exogenous enzymes. This invention utilizes a stepwise pH-controlled enzymatic hydrolysis technique (first adjusting the pH to 3.5-5.0 and then incubating, followed by adjusting the pH to 7.5-9.0 and incubating again), achieving highly efficient enzymatic hydrolysis entirely based on the Ganoderma lucidum germ's own enzyme system.

[0091] As shown in Example 5, under the stepwise pH control process of the present invention, the content of ganoderic acid reached 1.75 g / 100 g, the polysaccharide content reached 2.97%, and the proportion of small molecule polysaccharides (molecular weight less than 50 kDa) reached 87.3%, while in the control group without stepwise pH control, small molecule polysaccharides were almost undetectable. Meanwhile, as shown in Table 3 of Example 4, under the culture conditions of the present invention, the chitinase activity produced by the Ganoderma lucidum embryo itself reached 9.2 IU / ml, the protease activity reached 17.5 IU / ml, and the dextranase activity reached 7.9 IU / ml, which were far higher than those of the control group, providing a sufficient enzyme source for its own enzymatic hydrolysis.

[0092] Fourth, the product of this invention has a small molecular weight, high absorption rate, and significantly improved bioavailability.

[0093] The Ganoderma lucidum germ liquid obtained by this invention contains polysaccharides with a molecular weight of 1-50 kDa, with small molecule polysaccharides accounting for more than 85%, and ganoderic acid content of 0.5-1.75 g / 100 g.

[0094] As shown in Table 5 of Example 6, animal experiments demonstrated that the absorption rate of the product of this invention reached 94%, while the absorption rates of commercially available Ganoderma lucidum powder and Ganoderma lucidum spore powder were only 32% and 35%, respectively. This significant increase in absorption rate means that the amount of active ingredient entering the bloodstream at the same dosage is greatly increased, thereby improving the product's efficacy and cost-effectiveness.

[0095] V. The product of this invention has multiple health care functions.

[0096] As shown in Examples 6 and 7, animal experiments have demonstrated that the product of the present invention has the following health benefits: (1) Immunoantitumor effect: As shown in Table 6 of Example 6, the product of the present invention has a tumor inhibition rate of up to 58.8% against S180 sarcoma in mice, and it is dose-dependent (29.9% in the low dose group, 40.9% in the medium dose group, and 58.8% in the high dose group); the tumor inhibition effect of the high dose group is close to that of the positive control cyclophosphamide (67.1%), but without the toxic side effects of chemical drugs.

[0097] (2) Enhance macrophage phagocytic function: As shown in Table 7 of Example 6, the high-dose group (10g / kg) of the product of the present invention can increase the phagocytic rate of peritoneal macrophages in mice from 43.1% in the control group to 80.5%, which is significantly better than Ganoderma lucidum whole powder (45.7%) and Ganoderma lucidum spore powder (46.1%), indicating that the product of the present invention can effectively enhance the non-specific immune function of the body.

[0098] (3) Protection of immune organs: As shown in Table 8 of Example 6, the high-dose group of the product of the present invention can reduce the white blood cell count of mice from 9.5 × 10⁻⁶ in the control group. 9 / L increased to 22.1×10 9 In mice treated with cyclophosphamide, thymus weight increased from 39.8 mg / 10g to 58.7 mg / 10g, and spleen weight increased from 36.8 mg / 10g to 61.8 mg / 10g. In contrast, the white blood cell count, thymus weight, and spleen weight in the cyclophosphamide control group decreased to 3.1 × 10⁹ / L. 9 / L, 25.6mg / 10g, and 18.9mg / 10g. This indicates that the product of the present invention has a significant protective effect against immunosuppression caused by chemotherapy, etc.

[0099] (4) Antibacterial and anti-inflammatory effects: As shown in Table 9 of Example 7, the product of the present invention has significant inhibitory effects on Staphylococcus aureus, Escherichia coli and Candida albicans, with inhibition rates of 94.1%, 92.6% and 79.6% respectively after 24 hours.

[0100] In addition, animal experiments have shown that the product of this invention also has functions such as protecting the liver and kidneys, improving lung function, and regulating the intestines.

[0101] VI. The method of the present invention achieves synergistic sporulation of the three elements.

[0102] As shown in Table 1 of Comparative Example 1, the present invention combines three elements—high concentration of calcium and magnesium salts, ozone, and segmented temperature culture—to achieve efficient sporulation of Ganoderma lucidum mycelium.

[0103] The control experiment showed that the absence of any one element led to sporulation failure (spore concentrations in groups B, C, and D were all below 1×10⁻⁶). 6 / g); the spore concentration of low-concentration calcium-magnesium salts (Group E) is only about 1 / 90 (5.2 × 10⁻⁶) of that under intact conditions. 7 / g); the single factor acting alone (group F) could not induce sporulation at all. This indicates that the technical effect of the present invention comes from the synergistic effect of the three factors, rather than the simple superposition of a single factor.

[0104] VII. The concentration range of calcium and magnesium salts in the method of the present invention is reasonable.

[0105] As shown in Table 2 of Comparative Examples 2, the calcium salt concentration range (0.9-2.0 mol / L) and magnesium salt concentration range (0.5-1.0 mol / L) defined in this invention are both achievable. Within these concentration ranges, the spore concentration reaches 4.5 × 10⁻⁶. 9 The concentration of ganoderic acid was 1.68-1.89 g / 100g, and the polysaccharide content was 2.78-3.12%. When the calcium and magnesium salt concentration was increased to 1.35 mol / L + 0.74 mol / L, all indicators reached their optimal values ​​(spore concentration 5.6 × 10⁻⁶). 9 / g, ganoderic acid 1.89g / 100g, polysaccharide 3.12%); when the concentration exceeds 2.0mol / l, all indicators begin to decrease, verifying the scientific validity of setting the upper limit to 2.0mol / l in this invention.

[0106] 8. The process of this invention is simple, the cost is low, and it is suitable for industrial production.

[0107] Compared with existing technologies, this invention has the following technological advantages: First, the raw material is mycelium, which can be produced on a large scale through fermentation engineering, without being limited by season or region; second, it eliminates the need for additional cell wall breaking processes and the addition of exogenous enzymes, simplifying the process flow; third, both liquid deep fermentation and stepwise pH-controlled enzymatic oxidation are completed in conventional equipment, resulting in low equipment investment and easy scale-up production. Therefore, this invention has good industrial application prospects and economic benefits.

[0108] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. 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 producing a small molecule absorbable ganoderma spore germ, characterized by: Includes the following steps: Step (a). Select Ganoderma lucidum strains to obtain active Ganoderma lucidum mother strains; Step (b). Prepare a semi-solid culture medium for spore formation and use it for mother culture; Step (c). The Ganoderma lucidum mother culture obtained in step (a) is inoculated into the spore-forming semi-solid culture medium prepared in step (b) and cultured under specific environmental conditions to obtain Ganoderma lucidum spore culture. Step (d). The Ganoderma lucidum spore culture obtained in step (c) is transferred to a liquid deep culture tank to grow buds and obtain Ganoderma lucidum embryo solution; Step (e). The Ganoderma lucidum germ liquid described in step (d) is subjected to enzymatic treatment to obtain a small molecule Ganoderma lucidum germ liquid that is easily absorbed. Step (f). The small-molecule, easily absorbed Ganoderma lucidum germ liquid obtained in step (e) is bottled and sterilized to obtain Ganoderma lucidum germ beverage.

2. The method for manufacturing small-molecule, easily absorbed Ganoderma lucidum germ according to claim 1, characterized in that: The method for selecting Ganoderma lucidum strains in step (a) is as follows: using glutaryl monoacyl-CoA reductase (HMGR) as the target gene, the mRNA expression level of HMGR is detected by Q-PCR, and the strains with the highest expression levels are selected as production strains.

3. The method for manufacturing small-molecule, easily absorbed Ganoderma lucidum germ according to claim 1, characterized in that: The semi-solid culture medium for spore formation in step (b) comprises: 0.5%-5% carbon source, 0.05%-3% nitrogen source, 0.05%-0.75% gelling agent, 0.9-2.0 mol / L calcium salt, and 0.5-1.0 mol / L magnesium salt, and the pH of the semi-solid culture medium for spore formation is 3.5-4.

5.

4. The method for manufacturing the easily absorbed small-molecule Ganoderma lucidum germ according to claim 1, characterized in that: The specific environmental conditions in step (c) are as follows: Phase 1, days 1-10: Temperature 15℃-35℃, humidity 55%-95%, light intensity 10lx-100lx; The second stage, 1-7 days: temperature 1℃-15℃, humidity 30%-50%, light intensity 200lx-400lx, and ozone 1mg / L-90mg / L added; Finally, the concentration of spores in the obtained Ganoderma lucidum spore culture was 5 x 10 9 / g-5 x 10 10 / g.

5. The method for manufacturing the easily absorbed small-molecule Ganoderma lucidum germ according to claim 1, characterized in that: The culture medium in the liquid deep culture tank in step (d) consists of: 0.5%-5% carbon source, 0.05%-3% nitrogen source, 0.5-0.75 mol / L calcium salt, and 0.15-0.35 mol / L magnesium salt. The pH of the culture medium is controlled at 3.5-7.0, and a two-stage culture is carried out.

6. The method for manufacturing small-molecule, easily absorbed Ganoderma lucidum germ according to claim 5, characterized in that: The conditions for the two-stage cultivation are as follows: Phase 1, 0-72 hours: Ventilation ratio controlled between 1:0.75 and 1:1, temperature between 25℃ and 33℃, pH between 3.5 and 5.0; The second stage, 72-96 hours: the ventilation ratio is controlled between 1:0.1 and 1:0.3, the temperature is 10℃-15℃, the pH is 5.5-7.0, and 0.5%-3% of a chitin-containing biological inducer is added, wherein the biological inducer is selected from at least one of cicada pupa powder, chitin, and edible fungus mycelium powder.

7. The method for manufacturing small-molecule, easily absorbed Ganoderma lucidum germ according to claim 1, characterized in that: The enzymatic treatment in step (e) is a stepwise pH-controlled enzymatic treatment, specifically as follows: First, adjust the pH of the Ganoderma lucidum germ solution to 3.5-5.0, raise the temperature to 45℃-65℃, and keep it warm for 5-30 hours; Then, adjust the pH to 7.5-9.0, raise the temperature to 45℃-65℃, and keep it at that temperature for 5-30 hours; Furthermore, the enzymatic treatment does not involve the addition of exogenous enzymes, but relies entirely on the enzyme system of the Ganoderma lucidum germ itself.

8. The method for manufacturing small-molecule, easily absorbed Ganoderma lucidum germ according to claim 1 or 6, characterized in that: In the Ganoderma lucidum germ solution obtained in step (e): The polysaccharide content is 1.5g / 100g-3.0g / 100g. The content of ganoderic acid is 0.5g / 100g-1.75g / 100g. The polysaccharides have a molecular weight of 1kDa-50kDa, with small molecule polysaccharides having a molecular weight of less than 50kDa accounting for more than 85%.

9. The method for manufacturing small-molecule, easily absorbed Ganoderma lucidum germ according to claim 1, characterized in that: The Ganoderma lucidum germ liquid in step (f) is bottled and sterilized under the following conditions: temperature of 100℃-135℃ and sterilization time of 5 seconds-60 minutes.

10. A small-molecule, easily absorbed Ganoderma lucidum germ product, characterized in that: The product is manufactured by the method for producing easily absorbed small-molecule Ganoderma lucidum germ according to any one of claims 1-8. The product has immune-anti-tumor, antibacterial and anti-inflammatory, liver-protecting and kidney-strengthening, lung-improving and intestinal-regulating effects, and its absorption rate in animal experiments reaches more than 94%.

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