A chrysanthemum morifolium fermented composition and a preparation method thereof

Abstract

The application belongs to the field of pharmaceutical preparations, and particularly discloses a ferulae and ganoderma lucidum fermentation composition and a preparation method thereof. The ferulae and ganoderma lucidum fermentation composition is obtained by adding enzymes to ferment substrates of ferulae and ganoderma lucidum powder. The application uses the ferulae and ganoderma lucidum powder as the substrates, and obtains the fermentation composition through three-stage enzyme fermentation. The test shows that the fermentation composition has high contents of active components of the ferulae and ganoderma lucidum, and has rich aroma. The fermentation composition can be used as traditional Chinese medicinal materials to improve the efficacy, and can also be used as excellent spices to improve the quality of life, and has practical popularization and application value.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical preparations, specifically relating to an agarwood and Ganoderma lucidum fermentation composition and its preparation method. Background Technology

[0002] Agarwood is a resinous wood formed from the resin of the Aquilaria sinensis tree (a member of the Thymelaeaceae family) after it is infected by fungi and undergoes a long and complex physiological and biochemical reaction. Its formation process typically takes decades or even longer, making it scarce and highly valuable. In Traditional Chinese Medicine (TCM), agarwood is used to treat chest and abdominal distension, stomach cold with vomiting, and kidney deficiency with shortness of breath. Furthermore, agarwood has a unique and long-lasting aroma, possessing both a deep woody note and a sweet and cool quality, holding an important place in traditional incense culture and considered the "king of fragrances." It is precisely this dual value—both medicinal and aromatic—that makes agarwood highly regarded in both ancient and modern TCM and health preservation fields.

[0003] Reishi mushroom, the fruiting body of a fungus in the Polyporaceae family, is traditionally hailed as the "immortal herb" and is a top-grade tonic in traditional Chinese medicine. It is neutral in nature and sweet in taste, possessing the effects of tonifying qi and calming the mind, relieving cough and asthma, and is suitable for symptoms such as weakness, restlessness, insomnia, and cough. Modern scientific research has further confirmed that reishi is rich in various bioactive components such as reishi polysaccharides, triterpenoids, and adenosine, showing broad application potential in areas such as immune regulation, anti-tumor activity, anti-oxidation, and liver protection. Therefore, reishi not only holds a place in the traditional Chinese medicine system but has also become an important raw material in the modern functional food and health product field.

[0004] Although medicinal and food products with agarwood and Ganoderma lucidum as the main raw materials are currently used in traditional Chinese medicine and modern health products, aiming to synergistically exert the dual effects of regulating qi and tonifying the body, there are no research reports to date on combining agarwood and Ganoderma lucidum for fermentation to simultaneously increase the content of their active ingredients and improve the aroma quality. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides an agarwood and Ganoderma lucidum fermentation composition, which is made by naturally fermenting agarwood powder and Ganoderma lucidum powder with the addition of enzymes. The enzymes are cellulase, β-glucanase, chitinase, lignin peroxidase, and neutral protease; The cellulase, β-glucanase, and chitinase were added to the substrate at the start of fermentation; lignin peroxidase was added to the substrate 2 days after fermentation; and neutral protease was added to the substrate 2 days and 4 days after fermentation, respectively.

[0006] Furthermore, the substrate is added with water before the enzyme is added; the mass ratio of water to substrate is 100:40~60.

[0007] Furthermore, the mass ratio of water to substrate is 100:52.

[0008] Further, the mass percentage of cellulase, β-glucanase or chitinase to substrate is 0.5%-1.5%; the mass percentage of lignin peroxidase to substrate is 0.2%-0.8%; and the mass percentage of neutral protease to substrate is 0.2%-1.0%.

[0009] Furthermore, the mass percentage of cellulase, β-glucanase, or chitinase to substrate is 0.5%; the mass percentage of lignin peroxidase to substrate is 0.2%; the mass percentage of neutral protease added after 2 days of fermentation to substrate is 0.2%; and the mass percentage of neutral protease added after 4 days of fermentation to substrate is 0.3%.

[0010] Furthermore, the fermentation temperature is 30-40°C, the humidity is 60-70%, and the time is 6-8 days.

[0011] Furthermore, the fermentation temperature is 30°C, the humidity is 60%, and the time is 6 days.

[0012] Furthermore, the mass ratio of agarwood powder to Ganoderma lucidum powder is 5~15:2.

[0013] Furthermore, the mass ratio of agarwood powder to Ganoderma lucidum powder is 8:2.

[0014] This invention also provides a method for preparing an agarwood and Ganoderma lucidum fermentation composition, comprising the following steps: Mix agarwood powder and Ganoderma lucidum powder, add water to the mixture and mix well, then spray with enzyme solution, stir evenly and let it ferment naturally to obtain the product; The enzyme solutions are respectively cellulase, β-glucanase, chitinase, lignin peroxidase and neutral protease; The cellulase, β-glucanase, and chitinase were sprayed into the mixed substrate at the start of fermentation; lignin peroxidase was sprayed into the mixed substrate 2 days after fermentation; and neutral protease was sprayed into the mixed substrate 2 days and 4 days after fermentation, respectively.

[0015] Furthermore, the mass ratio of water to the mixed substrate is 100:40~60.

[0016] Furthermore, the mass ratio of water to substrate is 100:52.

[0017] Furthermore, the fermentation temperature is 30-40°C, the humidity is 60-70%, and the time is 6-8 days.

[0018] Furthermore, the fermentation temperature is 30°C, the humidity is 60%, and the time is 6 days.

[0019] Further, the mass percentage of cellulase, β-glucanase or chitinase to the mixed substrate is 0.5%-1.5%; the mass percentage of lignin peroxidase to the mixed substrate is 0.2%-0.8%; and the mass percentage of neutral protease to the mixed substrate is 0.2%-1.0%.

[0020] Furthermore, the neutral protease is sprayed into the mixed substrate at a mass percentage of 0.2% - 0.8% after 2 days of fermentation, and at a mass percentage of 0.3% - 1.0% after 4 days of fermentation.

[0021] Furthermore, the cellulase, β-glucanase, and chitinase are each sprayed into the mixed substrate at a mass percentage of 0.5% at the start of fermentation; lignin peroxidase is sprayed into the mixed substrate at a mass percentage of 0.2% after 2 days of fermentation; and neutral protease is sprayed into the mixed substrate at a mass percentage of 0.2% after 2 days of fermentation and at a mass percentage of 0.3% after 4 days of fermentation.

[0022] Furthermore, the mass ratio of agarwood powder to Ganoderma lucidum powder is 5~15:2.

[0023] Furthermore, the mass ratio of agarwood powder to Ganoderma lucidum powder is 8:2.

[0024] Furthermore, both the agarwood powder and the Ganoderma lucidum powder are powders that have passed through an 80-mesh sieve.

[0025] Furthermore, the enzyme solution is prepared by diluting the enzyme with an acetate-sodium acetate buffer solution at pH 3.6–5.6; the enzyme concentration in the enzyme solution is 1000~1500 U / mL.

[0026] Furthermore, the enzyme solution is prepared by diluting the enzyme with a pH 5 acetate-sodium acetate buffer solution.

[0027] The natural fermentation described in this invention refers to a fermentation process that does not involve artificial inoculation with specific strains, but relies on the raw materials themselves and naturally occurring microorganisms in the environment to carry out the fermentation.

[0028] The Ganoderma powder described in this invention is Ganoderma lucidum fruiting body powder.

[0029] The agarwood powder described in this invention is the powder of the resin-containing wood of Aquilaria sinensis (Lour.) Gilg, a plant of the Thymelaeaceae family.

[0030] This invention relates to an agarwood and Ganoderma lucidum fermentation composition. Using a specific ratio of Ganoderma lucidum and agarwood as substrates, it employs a three-stage enzymatic fermentation process, adding specific enzymes at different fermentation stages for precise catalysis. This process not only significantly increases the content of active ingredients in the fermentation composition but also effectively enriches the characteristic components of agarwood and Ganoderma lucidum, resulting in a richer, more complex, and more complex aroma, and superior overall quality. This fermentation composition can be used as a traditional Chinese medicine to enhance efficacy and as a high-grade fragrance to improve quality of life, thus possessing broad prospects for practical application.

[0031] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0032] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0033] Figure 1 This is a schematic diagram of the three-stage enzymatic fermentation process of agarwood-Ganoderma lucidum dual substrates according to the present invention.

[0034] Figure 2 Endpoint comparison of active ingredients in fermentation compositions with different agarwood and Ganoderma lucidum ratios.

[0035] Figure 3 Heatmaps showing the normalized performance and overall score for each formulation group.

[0036] Figure 4 To supplement the normalized comparison chart of quality and marker components. Detailed Implementation

[0037] The raw materials, reagents, and equipment used in the specific embodiments of this invention were all purchased commercially.

[0038] Example 1: Preparation of Agarwood and Ganoderma Fermentation Composition 1. Raw material preparation: (1) Take agarwood powder and Ganoderma lucidum powder respectively, pass them through an 80-mesh sieve, and use them as substrate raw materials for fermentation; (2) Take cellulase, β-glucanase, chitinase, lignin peroxidase and neutral protease respectively, and dilute them with pH 5 acetate-sodium acetate buffer to prepare enzyme solutions of 1000~1500 U / mL.

[0039] 2. Preparation of Fermentation Composition Take the substrate raw materials, agarwood powder and Ganoderma lucidum powder, mix them in a ratio of 8:2, add water at a ratio of 100:52 (mass ratio of water to substrate), mix thoroughly, and then ferment at a temperature of 30℃ and a humidity of 60%. During fermentation, at the beginning of fermentation, cellulase solution, β-glucanase solution, and chitinase solution were sprayed and stirred evenly, so that the mass percentage of the three enzymes and the mixed substrate was 0.5% each. After 2 days of fermentation, lignin peroxidase solution and neutral protease solution were sprayed and stirred evenly, so that the mass percentage of the two enzymes and the mixed substrate was 0.2% each. After 4 days of fermentation, neutral protease solution was sprayed and stirred evenly, so that the mass percentage of the enzymes and the mixture was 0.3% each. Fermentation continued until the 6th day, and the agarwood and Ganoderma lucidum fermentation composition was obtained.

[0040] Example 2 Preparation of Agarwood and Ganoderma Fermentation Composition 1. Raw material preparation: (1) Take agarwood powder and Ganoderma lucidum powder respectively, pass them through an 80-mesh sieve, and use them as substrate raw materials for fermentation; (2) Take cellulase, β-glucanase, chitinase, lignin peroxidase and neutral protease respectively, and dilute them with pH 5 acetate-sodium acetate buffer to prepare enzyme solutions of 1000~1500 U / mL.

[0041] 2. Preparation of Fermentation Composition Take agarwood powder and Ganoderma lucidum powder as the substrate materials, mix them in a ratio of 5:2, add water at a ratio of 100:50 (water to substrate mass ratio) and mix thoroughly. Then ferment at a temperature of 40℃ and a humidity of 70%. During fermentation, at the beginning of fermentation, cellulase solution, β-glucanase solution, and chitinase solution were sprayed and stirred evenly, so that the mass percentage of the three enzymes and the mixed substrate was 1.5%. After 2 days of fermentation, lignin peroxidase solution and neutral protease solution were sprayed and stirred evenly, so that the mass percentage of the two enzymes and the mixed substrate was 0.8%. After 4 days of fermentation, neutral protease solution was sprayed and stirred evenly, so that the mass percentage of the enzymes and the mixture was 1.0%. Fermentation continued until the 8th day, and the agarwood and Ganoderma lucidum fermentation composition was obtained.

[0042] Example 3 Preparation of Agarwood and Ganoderma Fermentation Composition 1. Raw material preparation: (1) Take agarwood powder and Ganoderma lucidum powder respectively, pass them through an 80-mesh sieve, and use them as substrate raw materials for fermentation; (2) Take cellulase, β-glucanase, chitinase, lignin peroxidase and neutral protease respectively, and dilute them with pH 5 acetate-sodium acetate buffer to prepare enzyme solutions of 1000~1500 U / mL.

[0043] 2. Preparation of Fermentation Composition Take the substrate raw materials, agarwood powder and Ganoderma lucidum powder, mix them in a ratio of 15:2, add water in a ratio of 100:60 (mass ratio of water to substrate), mix thoroughly, and then ferment at a temperature of 40℃ and a humidity of 70%. During fermentation, at the beginning of fermentation, cellulase solution, β-glucanase solution, and chitinase solution were sprayed and stirred evenly, so that the mass percentage of the three enzymes and the mixed substrate was 0.8% each. After 2 days of fermentation, lignin peroxidase solution and neutral protease solution were sprayed and stirred evenly, so that the mass percentage of the two enzymes and the mixed substrate was 0.6% each. After 4 days of fermentation, neutral protease solution was sprayed and stirred evenly, so that the mass percentage of the enzymes and the mixture was 0.5% each. Fermentation continued until the 8th day, and the agarwood and Ganoderma lucidum fermentation composition was obtained.

[0044] Example 4: Preparation of Agarwood and Ganoderma Fermentation Composition 1. Raw material preparation: (1) Take agarwood powder and Ganoderma lucidum powder respectively, pass them through an 80-mesh sieve, and use them as substrate raw materials for fermentation; (2) Take cellulase, β-glucanase, chitinase, lignin peroxidase and neutral protease respectively, and dilute them with pH 5 acetate-sodium acetate buffer to prepare enzyme solutions of 1000~1500 U / mL.

[0045] 2. Preparation of Fermentation Composition Take agarwood powder and Ganoderma lucidum powder as the substrate materials, mix them in a ratio of 8:2, add water at a ratio of 100:40 (water to substrate mass ratio) and mix thoroughly. Then ferment at a temperature of 35℃ and a humidity of 65%. During fermentation, at the beginning of fermentation, cellulase solution, β-glucanase solution, and chitinase solution were sprayed and stirred evenly, so that the mass percentage of the three enzymes and the mixed substrate was 1.5%. After 2 days of fermentation, lignin peroxidase solution and neutral protease solution were sprayed and stirred evenly, so that the mass percentage of the two enzymes and the mixed substrate was 0.8%. After 4 days of fermentation, neutral protease solution was sprayed and stirred evenly, so that the mass percentage of the enzymes and the mixture was 1.0%. Fermentation continued until the 7th day, and the agarwood and Ganoderma lucidum fermentation composition was obtained.

[0046] Example 5: Preparation of Agarwood and Ganoderma Fermentation Composition 1. Raw material preparation: (1) Take agarwood powder and Ganoderma lucidum powder respectively, pass them through an 80-mesh sieve, and use them as substrate raw materials for fermentation; (2) Take cellulase, β-glucanase, chitinase, lignin peroxidase and neutral protease respectively, and dilute them with pH 5 acetate-sodium acetate buffer to prepare enzyme solutions of 1000~1500 U / mL.

[0047] 2. Preparation of Fermentation Composition Take agarwood powder and Ganoderma lucidum powder as the substrate materials, mix them in a ratio of 8:2, add water at a ratio of 100:40 (water to substrate mass ratio) and mix thoroughly. Then ferment at a temperature of 35℃ and a humidity of 65%. During fermentation, at the beginning of fermentation, cellulase solution, β-glucanase solution, and chitinase solution were sprayed and stirred evenly, so that the mass percentage of the three enzymes and the mixed substrate was 1% each. After 2 days of fermentation, lignin peroxidase solution and neutral protease solution were sprayed and stirred evenly, so that the mass percentage of the two enzymes and the mixed substrate was 0.5% each. After 4 days of fermentation, neutral protease solution was sprayed and stirred evenly, so that the mass percentage of the enzymes and the mixture was 0.7% each. Fermentation continued until the 7th day, and the agarwood and Ganoderma lucidum fermentation composition was obtained.

[0048] The following experimental examples illustrate the beneficial effects of the present invention.

[0049] Experimental Example 1: Study on the Influence of Substrate Ratio of Agarwood and Ganoderma Lucidum on Enzymatic Fermentation Process Previous research on the enzymatic fermentation of agarwood revealed that adding different enzymes at three different stages of natural fermentation, using agarwood as a substrate, can significantly improve its quality. Given that both agarwood and Ganoderma lucidum are scarce traditional Chinese medicinal materials and possess the effects of strengthening the body and consolidating the foundation, they are commonly used in clinical practice as raw materials for health preservation. Based on this, our team innovatively constructed a three-stage enzymatic fermentation system using agarwood and Ganoderma lucidum as common substrates, aiming to organically integrate their characteristic components during fermentation. Through systematic exploration, the three-stage enzyme addition strategy was finally established as follows: cellulase solution, β-glucanase solution, and chitinase solution are added at the beginning of fermentation; lignin peroxidase solution and neutral protease solution are added after 2 days of fermentation; and neutral protease solution is added again after 4 days of fermentation. Figure 1 Furthermore, it was found that the ratio of agarwood to Ganoderma lucidum in the substrate had a significant impact on the quality of the final fermentation composition. This is further illustrated below through a comparative experiment of substrate ratios: 1. Materials and Instruments 1.1 Raw materials: 80-mesh agarwood powder (passed through an 80-mesh standard sieve, and the sieve residue is collected), 80-mesh Ganoderma lucidum powder (passed through an 80-mesh standard sieve, and the sieve residue is collected).

[0050] 1.2 Enzyme preparations: cellulase (purchased from Shanghai Maclean Biotechnology Co., Ltd., enzyme activity ≥200,000 U / g), β-glucanase (purchased from Shanghai Maclean Biotechnology Co., Ltd., enzyme activity ≥30,000 U / g), chitinase (purchased from Shanghai Maclean Biotechnology Co., Ltd., enzyme activity ≥5,000 U / g), lignin peroxidase (purchased from Shanghai Maclean Biotechnology Co., Ltd., enzyme activity ≥100 U / g), neutral protease (purchased from BASF Biotechnology Co., Ltd., Hefei, enzyme activity ≥50,000 U / g).

[0051] 1.3 Main instruments: Electronic balance (FA2004N, Shanghai Precision Scientific Instruments Co., Ltd.), constant temperature and humidity fermentation incubator (HWS-250, Shanghai Yiheng Scientific Instruments Co., Ltd.), high performance liquid chromatograph (Agilent 1260, Agilent Technologies, USA), ultraviolet-visible spectrophotometer (UV-2600, Shimadzu Corporation, Japan), high speed refrigerated centrifuge (TGL-16M, Hunan Xiangyi Laboratory Instrument Development Co., Ltd.).

[0052] 2 methods According to the experimental groups in Table 1, the main active ingredients, total polysaccharides, total triterpenes, and total phenols of the fermentation end products with different agarwood and Ganoderma lucidum ratios as substrates were detected. The aroma score and comprehensive evaluation were also carried out, as well as the determination of the core indicators of traditional Chinese medicine quality control, ash content, agarwood marker components linalool and isolinalool, and Ganoderma lucidum marker component ganoderic acid A.

[0053] 2.1 Preparation of Fermentation End Product Samples According to the grouping, agarwood powder and Ganoderma lucidum powder were mixed in different mass ratios, and then water was added at a ratio of 100:52 (water to substrate mass ratio) and thoroughly mixed. Fermentation was carried out at 30℃ and 60% humidity for 6 days. At the beginning of fermentation, cellulase, β-glucanase, and chitinase solutions were added, each at 0.5% of the mixed substrate mass. After 2 days of fermentation, lignin peroxidase and neutral protease solutions were added, each at 0.2% of the mixed substrate mass. After 4 days of fermentation, neutral protease solution was added again, at 0.3% of the mixed substrate mass. The enzyme solutions were prepared by diluting the enzymes with pH 5 acetate-sodium acetate buffer to a concentration of 1000–1500 U / mL. Three parallel samples were set up for each group.

[0054] 2.2 Detection Indicators ①The ash content was determined by constant weight ashing at 550-600℃.

[0055] ②Agaricone, isoagaricone, and ganoderic acid A were determined by HPLC external standard method; Preparation of test solution: Accurately weigh 0.50 g of fermentation sample powder, add 25 mL of methanol, extract by sonication for 30 min, cool and make up the weight loss with methanol, centrifuge at 10000 r / min for 10 min, take the supernatant and filter it through a 0.22 μm microporous membrane for HPLC analysis; Chromatographic conditions: C18 column (4.6 mm × 250 mm, 5 μm), column temperature 30℃, flow rate 1.0 mL / min, injection volume 10 μL. For the detection of agaritol and isogaritol, the mobile phase was acetonitrile-water, with gradient elution: 0–10 min, acetonitrile 22%; 10–25 min, acetonitrile 22%→32%; 25–35 min, acetonitrile 32%, detection wavelength 252 nm. For the detection of ganoderic acid A, the mobile phase was acetonitrile-0.1% phosphoric acid aqueous solution, with gradient elution: 0–15 min, acetonitrile 28%→32%; 15–30 min, acetonitrile 32%→38%; 30–40 min, acetonitrile 38%, detection wavelength 252 nm. A standard curve was prepared using agaric tetraol, isogaric tetraol, and ganoderic acid A as reference standards, and the content of each component in the sample was calculated using the external standard method.

[0056] ③ Total polysaccharide content: determined by the sulfuric acid-anthrone colorimetric method; Accurately weigh 0.20 g of sample, add 20 mL of distilled water, extract in a 90℃ water bath for 2 h, cool, make up to volume and centrifuge, take 1.0 mL of supernatant, add 5.0 mL of anthrone-sulfuric acid colorimetric solution, react in a boiling water bath for 10 min, cool to room temperature and measure absorbance at 625 nm, use glucose as a reference to plot a standard curve, and calculate the total polysaccharide content.

[0057] ④ Total triterpenes: The total triterpenes were determined by the vanillin-glacial acetic acid colorimetric method (referring to the method under Ganoderma lucidum in Part I of the 2020 edition of the Chinese Pharmacopoeia), with oleanolic acid as the reference standard. The absorbance was measured at a wavelength of 548 nm, and the total triterpenes content was calculated.

[0058] ⑤ Total phenols: The Folin-Ciocalteu test was used. Specifically, 0.2 g of sample was accurately weighed, 30 mL of 70% ethanol was added, and the mixture was extracted in an 80℃ water bath for 40 min. The extract was filtered and the volume was adjusted to 50 mL. 1 mL of the extract was accurately measured, 0.5 mL of Folin-Ciocalteu reagent was added, and the mixture was shaken well. 2 mL of 10% sodium carbonate solution was added, and the volume was adjusted to 10 mL with water. The mixture was allowed to stand at room temperature for 60 min, and the absorbance was measured at 765 nm. A standard curve was plotted using gallic acid as a reference, and the total phenol content was calculated.

[0059] ⑥ Aroma Evaluation: Five professional aroma evaluators with over five years of industry experience from agarwood companies were invited to conduct a blind sensory evaluation of the fermentation products. Evaluation indicators included aroma intensity, aroma harmony, and longevity. Each indicator was scored out of 10 points, for a total of 30 points. The average score from the five evaluators was taken as the final aroma score. The evaluation criteria are as follows: Aroma intensity (10 points): 8~10 points (rich and mellow aroma), 5~7 points (moderate aroma), 1~4 points (light aroma or off-putting smell); Fragrance Harmony (10 points): 8~10 points (harmonious and unified fragrance, no abruptness), 5~7 points (basic harmony of fragrance), 1~4 points (chaotic fragrance, abruptness). Fragrance duration (10 minutes): 8~10 minutes (fragrance duration ≥8h), 5~7 minutes (fragrance duration 4~8h), 1~4 minutes (fragrance duration <4h).

[0060] Table 1. Experimental grouping and the ratio of agarwood to Ganoderma lucidum. 3 Results 3.1 Main active ingredients Table 2. Results of active ingredients at the endpoint for each formulation group Note: p The values ​​are obtained by comparing each proportion group with the invention group.

[0061] From Table 2 and Figure 2 It is evident that the content of major active ingredients in the fermentation end product varies with the ratio of the substrates agarwood and Ganoderma lucidum. The greatest impact is on total polysaccharides; the invention group showed significant, highly significant, or highly highly significant differences compared to all control groups except control group 6. The second most significant impact is on total phenols; the invention group showed significant differences compared to control groups 7-8, and highly significant differences compared to control group 6. The impact on total triterpenes was relatively small; the invention group only showed significant differences compared to control groups 6 and 7. Overall, the invention group achieved optimal levels in total polysaccharides, total triterpenes, and total phenols, indicating that it can effectively achieve synergistic transformation of both types of substrates.

[0062] 3.2 Aroma Score and Overall Score Aroma scores were assigned to each group of fermentation end products using an aroma evaluation method, with a score of 30 points. The scores were then weighted and summed according to the following weights: total polysaccharides 0.30, total triterpenes 0.25, total phenols 0.20, and aroma score 0.25. The final comprehensive score for each group of fermentation products was then calculated and used as the evaluation basis for optimizing substrate formulation.

[0063] Table 3 Fragrance and Overall Performance From Table 3 and Figure 3 The results show that the invention group scored 27.8 points in aroma, the highest among all groups, and its overall score also ranked first. The invention group achieved a good balance between aroma and release of functional components in the ratio of agarwood and Ganoderma lucidum, thus gaining significant advantages in both taste and efficacy.

[0064] 3.3 Quality and Marker Components Table 4. Detection results of ash content and marker components in each formulation group Note: p The values ​​are obtained by comparing each proportion group with the invention group.

[0065] From Table 4 and Figure 4 It is evident that the invention group demonstrated superior ash control, with its ash content significantly lower than that of all control groups except Control 1 and Control 3, indicating that the group effectively reduced inorganic impurities during fermentation. Simultaneously, regarding the enrichment of medicinal substances, the content of agarwood marker components in the invention group was significantly higher than that of all control groups except Control 1, while the content of Ganoderma lucidum marker components was significantly higher than all control groups. In particular, compared to Control 2, which used Ganoderma lucidum as the sole substrate for fermentation, the invention group achieved a significant increase in Ganoderma lucidum marker components through a three-stage fermentation process using agarwood-Ganoderma lucidum dual substrates. This result fully demonstrates that agarwood has a positive contribution to the production of Ganoderma lucidum active ingredients. Overall, the fermentation end product of the invention group exhibits comprehensive advantages in both purity and medicinal substance enrichment.

[0066] Experimental Example 2: Study on Fermentation Process Parameters of Agarwood and Ganoderma Lucidum Based on the previous enzymatic hydrolysis and fermentation process of agarwood monomers, the fermentation parameters were further optimized to obtain a three-stage enzyme addition process suitable for dual-substrate fermentation of agarwood and Ganoderma lucidum, so as to improve the quality of the fermentation composition of agarwood and Ganoderma lucidum.

[0067] 1. Method Based on a substrate ratio of agarwood powder to Ganoderma lucidum powder of 8:2, the total polysaccharide, total triterpenoid, and total phenol content, as well as aroma score, were used as evaluation indicators. The comprehensive score was calculated using the method described in "3.2, Aroma Score and Comprehensive Score" of Experiment 1. Key process parameters such as moisture content, enzyme level, fermentation temperature, relative humidity, and fermentation time were progressively screened. During the screening process, all other conditions for preparing the fermentation product samples remained consistent with Experiment 1, only the factors under investigation were changed; the detection methods for each indicator were also the same as in Experiment 1 to ensure data comparability and facilitate process optimization.

[0068] 2 Results (1) Moisture content screening The study examined three levels of water-to-substrate mass ratios: 100:40, 100:52, and 100:60. The results are shown in Table 5.

[0069] Table 5. Screening results based on moisture content The results showed that the material was too dry under the 100:60 condition, which was not conducive to the synchronous opening of the agarwood cellulose structure and the Ganoderma lucidum cell wall; the material was prone to clumping under the 100:40 condition, which reduced the aeration and made the aroma coordination and repeatability worse in the later stage; the fermentation was most uniform under the 100:52 condition, and the three indicators showed the best overall performance. Therefore, the optimal mass ratio of water to mixed substrate was 100:52.

[0070] (2) Enzyme level screening Three enzyme levels were investigated: low, medium, and high. The low level consisted of 0.5% each of cellulase, β-glucanase, and chitinase at the start of fermentation; 0.2% each of lignin peroxidase and neutral protease after 2 days of fermentation; and 0.3% of neutral protease after 4 days of fermentation. The medium level consisted of 0.8% cellulase, 0.8% β-glucanase, 0.8% chitinase, 0.6% lignin peroxidase, and 0.6% and 0.5% neutral protease added in two separate additions. The high level consisted of 1.5% cellulase, 1.5% β-glucanase, 1.5% chitinase, 0.8% lignin peroxidase, and 0.8% and 1.0% neutral protease added in two separate additions. The results are shown in Table 6.

[0071] Table 6. Screening results for enzyme addition levels The results showed that the low-level group had advantages in maintaining the aroma of agarwood, releasing the functional components of Ganoderma lucidum, and comprehensively evaluating the three measured indicators. Although the high-level group could enhance cell wall breaking in the early stage, it was prone to aroma distortion and decreased stability in the later stage. Therefore, the addition amounts of each enzyme were determined as follows: cellulase 0.5%–1.5%, β-glucanase 0.5%–1.5%, chitinase 0.5%–1.5%, lignin peroxidase 0.2%–0.8%, neutral protease 0.2%–0.8% added after 2 days of fermentation, and neutral protease 0.3%–1.0% added after 4 days of fermentation. The optimal values ​​were found in the low-level group, with cellulase, β-glucanase, and chitinase each at 0.5% at the beginning, lignin peroxidase and neutral protease each at 0.2% after 2 days of fermentation, and neutral protease at 0.3% after 4 days of fermentation.

[0072] (3) Screening of fermentation temperature The temperature was examined at three levels: 30℃, 35℃, and 40℃. The results are shown in Table 7. Table 7. Results of Fermentation Temperature Screening The results showed that the aroma of agarwood was well preserved and the transformation of ganoderic acids was stable at 30℃, with the best overall performance across the three indicators. The target product could also be formed at 35℃, but the overall score was slightly lower. Although 40℃ could accelerate the initial reaction, the purity of the aroma decreased in the later stage. Therefore, the fermentation temperature range was determined to be 30-40℃, with 30℃ being the optimal range.

[0073] (4) Relative humidity screening The study examined three levels: 60%, 65%, and 70%. The results are shown in Table 8. Table 8 Relative Humidity Screening Results The results showed that 60%–70% relative humidity was suitable for two-substrate three-stage enzyme fermentation, with 60% humidity providing stable substrate conditions and good reproducibility. While higher humidity could maintain the conversion, it increased moisture fluctuations, which was detrimental to scale-up control. Therefore, the relative humidity range was determined to be 60%–70%, with 60% being optimal.

[0074] (5) Screening of fermentation time The study examined three levels: 6 days, 7 days, and 8 days. The results are shown in Table 9. Table 9. Results of Fermentation Time Screening The results showed that by day 6 of fermentation, the release of agarwood aroma and Ganoderma lucidum active ingredients had reached a good balance; extending the fermentation to 7-8 days resulted in limited gains in some indicators, and the overall improvement in color and quality was not significant in the later stages. Therefore, the fermentation time range was determined to be 6-8 days, with 6 days being the optimal time.

[0075] The results show that: before fermentation, water was added to the substrate until the water-to-substrate mass ratio was 100:52, and then fermented for 6 days at 30℃ and 60% relative humidity; at the beginning of fermentation, 0.5% each of cellulase, β-glucanase, and chitinase were added; after 2 days of fermentation, 0.2% lignin peroxidase was added, and 0.2% neutral protease was added for the first time; after 4 days of fermentation, 0.3% neutral protease was added for the second time. This process can balance the preservation of the characteristic aroma of agarwood and the release of functional components of Ganoderma lucidum, and is suitable for obtaining stable compound fermentation products.

[0076] In summary, this invention uses Ganoderma lucidum and agarwood in a specific ratio as substrates and employs a three-stage enzymatic fermentation process. This not only significantly increases the content of active ingredients in the fermentation composition but also effectively enriches the characteristic components of agarwood and Ganoderma lucidum, resulting in a richer, more complex aroma and superior overall quality. The fermentation composition of this invention can be used as a traditional Chinese medicine to enhance efficacy and as a high-grade fragrance to improve quality of life, thus possessing broad prospects for practical application.

Claims

1. A fermented composition of agarwood and Ganoderma lucidum, characterized in that: It is made by naturally fermenting agarwood powder and Ganoderma lucidum powder with added enzymes. The enzymes are cellulase, β-glucanase, chitinase, lignin peroxidase, and neutral protease; The cellulase, β-glucanase, and chitinase were added to the substrate at the start of fermentation; lignin peroxidase was added to the substrate 2 days after fermentation; and neutral protease was added to the substrate 2 days and 4 days after fermentation, respectively. The mass ratio of agarwood powder to Ganoderma lucidum powder is 5~15:2; The fermentation temperature is 30~40°C, the humidity is 60~70%, and the time is 6~8 days.

2. The agarwood and Ganoderma lucidum fermentation composition according to claim 1, characterized in that: The substrate is added with water before the enzyme is added; the mass ratio of water to substrate is 100:40~60. The mass percentages of cellulase, β-glucanase, and chitinase relative to the substrate are all 0.5%-1.5%; the mass percentage of lignin peroxidase relative to the substrate is 0.2%-0.8%; and the mass percentage of neutral protease relative to the substrate is 0.2%-1.0%.

3. The agarwood and Ganoderma lucidum fermentation composition according to claim 1, characterized in that: The mass ratio of agarwood powder to Ganoderma lucidum powder is 8:

2.

4. A method for preparing an agarwood and Ganoderma lucidum fermentation composition, characterized in that: Includes the following steps: Mix agarwood powder and Ganoderma lucidum powder, add water to the mixture and mix well, then spray with enzyme solution, stir evenly and let it ferment naturally to obtain the product; The enzyme solutions are respectively cellulase, β-glucanase, chitinase, lignin peroxidase and neutral protease; The cellulase, β-glucanase, and chitinase were sprayed into the mixed substrate at the start of fermentation; lignin peroxidase was sprayed into the mixed substrate 2 days after fermentation; and neutral protease was sprayed into the mixed substrate 2 days and 4 days after fermentation, respectively. The mass ratio of agarwood to Ganoderma lucidum powder is 5~15:2; The fermentation temperature is 30~40°C, the humidity is 60~70%, and the time is 6~8 days.

5. The preparation method according to claim 4, characterized in that: The mass ratio of water to the mixed substrate is 100:40~60.

6. The preparation method according to claim 4, characterized in that: The mass percentages of cellulase, β-glucanase, and chitinase in the mixed substrate are all 0.5%-1.5%; the mass percentage of lignin peroxidase in the mixed substrate is 0.2%-0.8%; and the mass percentage of neutral protease in the mixed substrate is 0.2%-1.0%.

7. The preparation method according to claim 4, characterized in that: The neutral protease was sprayed into the mixed substrate at a mass percentage of 0.2%-0.8% after 2 days of fermentation, and at a mass percentage of 0.3%-1.0% after 4 days of fermentation.

8. The preparation method according to claim 4, characterized in that: The mass ratio of agarwood powder to Ganoderma lucidum powder is 8:

2.

9. The preparation method according to claim 4, characterized in that: The enzyme solution is prepared by diluting the enzyme with an acetate-sodium acetate buffer solution at pH 3.6–5.6; the enzyme concentration in the enzyme solution is 1000~1500 U / mL.

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