Method for extracting ganoderan through microwave-assisted green chemistry-enzyme method and application

Abstract

The invention discloses a method for extracting ganoderma lucidum polysaccharide by a microwave-assisted green chemical-enzyme method and application, which comprises the following steps: taking ganoderma lucidum sporocarp powder, adding cellulase and water, uniformly mixing, then adding 30% hydrogen peroxide, uniformly mixing, adjusting the pH value to 3-7, and carrying out microwave-assisted extraction for 40-120 minutes under the conditions that the power is 400-800W and the temperature is 50-90 DEG C; and centrifuging, taking supernate, concentrating to 30-50% of the original volume, and freeze-drying to obtain the ganoderma lucidum polysaccharide. The method is high in ganoderma lucidum polysaccharide extraction efficiency and more complete in ganoderma lucidum polysaccharide activity retention. The ganoderma lucidum sporocarp polysaccharide product is free of peculiar smell and good in sensory property, the SOD activity in C.elegans under the hydrogen peroxide stress state can be improved by 33.76%, the CAT activity can be improved by 229.36%, and the MDA content can be reduced by 85.65%.

Description

(1) Technical field

[0001] The present invention relates to a method for extracting ganoderma lucidum polysaccharide by microwave-assisted green chemical-enzymatic method and its application. (2) Background technology

[0002] Ganoderma lucidum is a fungus belonging to the phylum Basidiomycetes, class Agaricomycetes, family Polyporaceae, and genus Ganoderma. It contains a variety of active ingredients, including polysaccharides, triterpenes, proteins, alkaloids, and ergosterol, which are effective in relieving symptoms such as palpitations, fatigue, and hypertension. Ganoderma polysaccharides are a class of components in Ganoderma with significant biological activity, exhibiting anti-tumor, immune-regulating, antibacterial, and blood sugar-lowering properties.

[0003] The fruiting body of Ganoderma lucidum consists of a cap (cap) and a stipe. The cap usually develops on one side of the stipe's top, while the stipe is located to the side of the cap and is purple-red or brown-red in color. Its texture is fleshy when young, and becomes corky when mature and dry, which makes the cell wall of the Ganoderma lucidum fruiting body exceptionally tough. The exceptionally tough cell wall structure of the fruiting body of Ganoderma lucidum, a Basidiomycetes, serves as a strong protective layer and barrier. Whether it can be broken directly affects the dissolution and structure of Ganoderma lucidum polysaccharides. Polysaccharide activity is closely related to its structure. Once the structure of the polysaccharide is destroyed, its biological activity will be significantly reduced or even lost. Therefore, it is extremely important to effectively break the cell wall structure of the Ganoderma lucidum fruiting body without destroying the integrity of the "active fragments" of Ganoderma lucidum polysaccharides, and to use an extraction method that has high extraction yield, is pollution-free, and has low equipment requirements.

[0004] Ganoderma lucidum, a member of the phylum Basidiomycetes, has a typical fungal cell wall structure. The chitin within its structure is composed of β-1→4-linked glucosamine and acetylglucosamine. Adjacent chitin chains are hydrogen-bonded to form antiparallel microfibrils exceeding 1 μm in length. On the outer side of the chitin, β-1→3-glucan forms the most abundant polymer in the cell wall. Three glucan chains form a triple helix, linked together by hydrogen bonds. β-1→3-glucan is also linked to β-1→6-glucan, forming a highly branched, elastic polymer network. The structural proteins on the outer side of the cell wall are glycoproteins containing N- and O-linked carbohydrates. These proteins typically include mannose-rich mannoproteins and other glycoproteins containing mannose and galactose residues. Glycoproteins in the cell wall are linked to the plasma membrane via glycophosphatidylinositol (GPI) anchors and cross-linked to chitin microfibrils and glucans. Transmission electron microscopy reveals that the fungal cell wall appears as discrete layers, with multiple interwoven components within each layer.

[0005] Common extraction methods for Ganoderma lucidum polysaccharides include hot water extraction, reflux extraction, ultrasonic-assisted extraction, enzymatic extraction, and combined extraction. Hot water extraction and reflux extraction are simple to operate but time-consuming, resulting in incomplete extraction, low yield, and easy polysaccharide degradation. Ultrasonic / microwave-assisted extraction is more efficient, but the high temperatures generated by prolonged ultrasound / microwave exposure can damage the components and require high equipment. Enzymatic extraction exploits the differences in the cell wall-breaking sites of different enzymes to attack the cell wall layer of the fungal cell wall. Simultaneously, changes in the osmotic pressure of the buffer solution rupture the cell membrane and release the cytoplasm. Due to the complex structure of the fungal cell wall, it is difficult for a single enzyme to effectively break it down. Combined cell wall-breaking methods combine two or more methods, incorporating the characteristics of each extraction method to significantly improve the polysaccharide extraction yield.

[0006] Hydrogen peroxide is a clean, green, and efficient oxidant and a typical environmentally friendly agent. It is inexpensive, pollution-free, requires minimal equipment, and consumes little energy. It easily decomposes into water and oxygen, leaving no residue during the entire unit operation process. Research on hydrogen peroxide in areas such as lignin removal, dietary fiber modification, and the degradation of natural macromolecular polysaccharides continues to attract attention. In particular, the literature on polysaccharide degradation has increased year by year, with hydrogen peroxide found to enhance the bioactivity of degraded polysaccharides. However, studies on hydrogen peroxide in polysaccharide degradation or dietary fiber modification have all employed alkaline conditions, and few studies have examined its combined use with enzymatic methods for the extraction of fungal polysaccharides. Hydrogen peroxide-enzyme synergistic extraction of Ganoderma lucidum polysaccharides is a composite extraction method that combines chemical and enzymatic hydrolysis. Its advantages lie in extraction efficiency, polysaccharide activity retention, and environmental friendliness.

[0007] Ganoderma lucidum is a precious edible and medicinal fungus, and its polysaccharides have extremely high medicinal value. Taking advantage of the extremely tough cell wall structure of Ganoderma lucidum fruiting bodies, which is difficult to destroy under conventional physical or chemical conditions, a microwave-assisted green chemistry-bioenzyme synergistic method was developed. The extraction yield of Ganoderma lucidum polysaccharides was used as the response value, supplemented by a BBD design method to optimize extraction conditions. Scanning electron microscopy was used to observe the surface characteristics of the cell wall through the green chemistry-bioenzyme complex. This method was used to obtain Ganoderma lucidum polysaccharides with high yield and strong bioactivity (improving the lifespan of Caenorhabditis elegans under oxidative stress), which can realize new applications in Ganoderma lucidum processing. (3) Summary of the invention

[0008] The purpose of the present invention is to provide a method and application for extracting Ganoderma lucidum polysaccharides by microwave-assisted green chemistry-enzyme method, which utilizes microwave-assisted green chemistry-biological enzymes to collaboratively break the cell walls of Ganoderma lucidum fruiting bodies, extract, and decolorize Ganoderma lucidum polysaccharides. Ganoderma lucidum fruiting bodies are used as raw materials to obtain Ganoderma lucidum polysaccharides with high yield and strong biological activity (such as improving the lifespan of Caenorhabditis elegans under oxidative stress and other anti-oxidative stress capabilities), effectively solving the problems of low extraction rate and biological activity of Ganoderma lucidum polysaccharides and complex extraction process in existing methods.

[0009] The technical solution adopted in the present invention is:

[0010] In a first aspect, the present invention provides a method for extracting Ganoderma lucidum polysaccharides using a microwave-assisted green chemical-enzymatic method. The method comprises the following steps: taking Ganoderma lucidum fruiting body powder, adding cellulase and water to mix, then adding 30% hydrogen peroxide to mix, adjusting the pH to 3-7, using a microwave chemical reactor, and performing microwave-assisted extraction for 40-120 minutes at a power of 400-800W and 50-90°C; centrifuging (preferably at 8000 rpm for 20 minutes), taking the supernatant and concentrating it to 30-50% of the original volume to obtain a Ganoderma lucidum polysaccharide liquid, and freeze-drying to obtain Ganoderma lucidum polysaccharide.

[0011] Furthermore, the Ganoderma lucidum fruiting body powder is obtained by drying the Ganoderma lucidum fruiting body in an oven at 50° C. until the mass moisture content is less than 8% (preferably 7.5%), crushing the Ganoderma lucidum fruiting body powder, and passing the powder through a 20-100 mesh sieve (preferably 80-100 mesh sieve).

[0012] Furthermore, the amount of the cellulase added is 0.9-2.7%, preferably 1.8-2.4% (more preferably 2%) based on the weight of the Ganoderma lucidum fruiting body powder. The enzymatic activity of the cellulase is preferably 10,000 U / g.

[0013] Furthermore, water and 30% hydrogen peroxide constitute the extractant, the volume dosage of the extractant is 50-150 mL / g (preferably 70-150 mL / g, more preferably 135 mL / g) based on the weight of the Ganoderma lucidum fruiting body powder; the mass concentration of hydrogen peroxide in the extractant is 1.5-4.5% (preferably 3-3.6%).

[0014] Furthermore, the pH is preferably 4-6, more preferably 5-5.5.

[0015] Furthermore, it is preferred to extract at a power of 500-800 W and a temperature of 50-70° C. for 90-120 min, and more preferably at a power of 600 W and a temperature of 50° C. for 105 min.

[0016] Furthermore, the freeze-drying conditions were an initial temperature of -30°C and a vacuum degree of 80Pa.

[0017] In a second aspect, the present invention provides a Ganoderma lucidum polysaccharide prepared by the method.

[0018] In a third aspect, the present invention provides a use of the Ganoderma lucidum polysaccharide in preparing a drug for extending the lifespan of Caenorhabditis elegans under oxidative stress.

[0019] Furthermore, the Caenorhabditis elegans is the wild strain N2 of Caenorhabditis elegans.

[0020] Compared with the existing methods, the beneficial effects of the present invention are mainly reflected in:

[0021] 1. The extraction efficiency of Ganoderma lucidum polysaccharides is high, and the polysaccharide yield is improved. The hydroxyl free radicals generated by hydrogen peroxide under the action of microwaves are a strong oxidant that can destroy the tough and thick structure of Ganoderma lucidum cell walls, increase cell permeability, and thus promote the release of polysaccharides. Combined with biological enzymatic hydrolysis (cellulase), the polysaccharide-protein complex in the Ganoderma lucidum cell wall can be further decomposed to release more polysaccharides. Cellulase and hydrogen peroxide work together, and the effects of the two may be complementary. That is, after cellulase decomposes the cell wall structure, hydrogen peroxide can penetrate more easily. At the same time, hydrogen peroxide destroys the cell membrane lipids through oxidation, making it easier for cellulase to act. The two work together to accelerate the complete disintegration of the cell structure. The advantage of synergistic extraction lies in dynamic complementarity and maximum efficiency. Through the synchronous action of enzymes and hydrogen peroxide, the efficient disintegration of cell structure is achieved, while reducing the loss of target objects.

[0022] 2. The activity of Ganoderma lucidum polysaccharides is more completely retained. The traditional high-temperature water extraction method may cause thermal degradation or structural damage of polysaccharides, while the hydrogen peroxide-enzyme synergistic method reduces the damage to the tertiary helical structure of polysaccharides under low temperature conditions and retains its biological activity. Combining the BBD design method with the cell wall breaking morphology of the SEM scanning electron microscope, the controllable fragmentation effect of the cell wall is fully guaranteed, forming a loose and porous state, which is not only conducive to the full dissolution of the polysaccharides in the wall, but also conducive to the preparation of active polysaccharides with a narrow molecular weight distribution, the integrity of the polysaccharide "active fragments" is not destroyed during the wall breaking process, and the yield is high. At the same time, it has many advantages such as no impurities are introduced in the entire operation process, no pollution, low equipment requirements and low energy consumption.

[0023] 3. The process is environmentally friendly and safe. Compared to traditional alkaline and acid extraction methods, hydrogen peroxide decomposes into water and oxygen after the reaction, leaving no residual toxicity and better meeting the requirements of green chemistry. Furthermore, the mild conditions of the enzymatic step prevent corrosion of equipment by strong acids and bases, making it suitable for industrial production.

[0024] 4. Wide range of applications and better impurity removal. Traditional water extraction methods easily dissolve impurities such as proteins and pigments, requiring additional steps (such as the Sevag method) to remove impurities. However, hydrogen peroxide pretreatment can oxidatively decompose some proteins and pigments. Combined with enzymatic hydrolysis, it can specifically degrade cell wall components, reducing the difficulty of subsequent purification and achieving a one-step process of cell wall destruction, extraction, and decolorization.

[0025] 5. The microwave-assisted green chemistry-enzyme synergistic extraction of Ganoderma lucidum fruiting body polysaccharides was odorless and exhibited excellent sensory properties. The extraction yield and effect on the average lifespan of C. elegans were 11.98 times and 1.18 times higher, respectively, than those obtained with traditional hot water extraction; 9.75 times and 1.16 times higher, respectively, than those obtained with microwave-assisted hot water extraction; 5.21 times and 1.11 times higher, respectively, than those obtained with hydrogen peroxide extraction; 7.23 times and 1.22 times higher, respectively, than those obtained with microwave-assisted hydrogen peroxide extraction; 6.82 times and 1.11 times higher, respectively, than those obtained with microwave-assisted cellulase extraction; 2.33 times and 1.21 times higher, respectively, than those obtained with microwave-assisted cellulase followed by hydrogen peroxide extraction; and 3.44 times and 1.15 times higher, respectively, than those obtained with microwave-assisted hydrogen peroxide followed by cellulase extraction. Compared with the negative control group, Ganoderma lucidum polysaccharide (0.5 mg / mL) can increase the SOD activity of C. elegans by 33.76%, CAT activity by 229.36%, and reduce the MDA content by 85.65% under hydrogen peroxide stress, and the differences are significant compared with the negative control group (p<0.05). (IV) Description of the accompanying drawings

[0026] Figure 1 , glucose standard curve.

[0027] Figure 2 , Actual photos of Ganoderma lucidum fruiting bodies after crushing without sieve (A) and after sieve through 20 mesh sieve (B).

[0028] Figure 3 , a bar chart showing the effects of different single factors (microwave power, microwave temperature, microwave time, material-liquid ratio and hydrogen peroxide concentration) on the extraction yield of Ganoderma lucidum polysaccharides.

[0029] Figure 4 , response surface diagram of the effect of microwave-assisted hydrogen peroxide extraction of Ganoderma lucidum polysaccharides on the polysaccharide extraction yield.

[0030] Figure 5 , bar chart showing the effects of different single factors (extraction time, enzyme addition, pH, temperature) on the extraction yield of Ganoderma lucidum polysaccharides.

[0031] Figure 6 , response surface diagram of the effect of microwave-assisted cellulase extraction of Ganoderma lucidum polysaccharides on the polysaccharide extraction yield.

[0032] Figure 7 , bar chart showing the effect of microwave-assisted hydrogen peroxide followed by enzyme extraction on the extraction yield of Ganoderma lucidum polysaccharides.

[0033] Figure 8 , bar chart showing the effect of microwave-assisted enzyme extraction followed by hydrogen peroxide extraction on the extraction yield of Ganoderma lucidum polysaccharides.

[0034] Figure 9, a bar chart showing the effects of different single factors (time, enzyme addition, pH, temperature) on the extraction rate of Ganoderma lucidum polysaccharides.

[0035] Figure 10 , response surface diagram of the effect of microwave-assisted extraction with cellulase followed by hydrogen peroxide on the extraction yield of Ganoderma lucidum polysaccharides.

[0036] Figure 11 , a bar chart showing the effects of different single factors (extraction time, enzyme addition amount, material-liquid ratio, temperature and hydrogen peroxide concentration) on the extraction yield of Ganoderma lucidum polysaccharides.

[0037] Figure 12 , response surface diagram of the effect of microwave-assisted enzyme and hydrogen peroxide synergistic extraction of Ganoderma lucidum polysaccharides on the polysaccharide extraction yield.

[0038] Figure 13 , bar graph showing the effects of Ganoderma lucidum powder with different particle sizes on the extraction yield of Ganoderma lucidum polysaccharides by microwave-assisted enzyme and hydrogen peroxide synergistic extraction.

[0039] Figure 14 , electron microscopic images of the surface characteristics of Ganoderma lucidum cell wall; A represents Ganoderma lucidum fruiting body powder; B represents hot water extraction; C represents hydrogen peroxide extraction; D represents cellulase-hydrogen peroxide synergy; ×5000.

[0040] Figure 15 , bar graph showing the effects of microwave-assisted hydrogen peroxide-extracted Ganoderma lucidum polysaccharides on the average lifespan of C. elegans.

[0041] Figure 16 , bar graph showing the effects of microwave-assisted cellulase-extracted Ganoderma lucidum polysaccharides on the average lifespan of C.elegans.

[0042] Figure 17 , bar graph showing the effect of microwave-assisted hydrogen peroxide followed by cellulase extraction of Ganoderma lucidum polysaccharides on the average lifespan of C. elegans.

[0043] Figure 18 , bar graph showing the effect of microwave-assisted extraction of Ganoderma lucidum polysaccharides using cellulase followed by hydrogen peroxide on the average lifespan of C.elegans.

[0044] Figure 19 , bar graph showing the effects of microwave-assisted cellulase-hydrogen peroxide synergistic extraction of Ganoderma lucidum polysaccharides on the average lifespan of C. elegans.

[0045] Figure 20 , bar graph showing the effects of microwave-assisted cellulase-hydrogen peroxide synergistic extraction of Ganoderma lucidum polysaccharides on the activities of SOD (left) and CAT (right) in C. elegans.

[0046] Figure 21, bar graph showing the effects of microwave-assisted cellulase-hydrogen peroxide co-extraction of Ganoderma lucidum polysaccharides on MDA levels in C. elegans. (V) Specific implementation methods

[0047] The present invention is further described below with reference to specific embodiments, but the protection scope of the present invention is not limited thereto:

[0048] Example 1

[0049] 1 Materials and reagents

[0050] Ganoderma lucidum fruiting body (provided by Zhejiang Baixing Food Co., Ltd.).

[0051] Phenol, concentrated sulfuric acid, hydrochloric acid, hydrogen peroxide, and deionized water were used. Cellulase (activity 10,000 U / g) was purchased from Shanghai MacLean Biochemical Technology Co., Ltd. Superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) assay kits were purchased from Nanjing Jiancheng Bioengineering Institute.

[0052] Caenorhabditis elegans N2 wild strain and Escherichia coli OP50 were kindly provided by the College of Animal Science, Zhejiang University.

[0053] 2 Experimental instruments

[0054] JP-250A-2 high-speed multifunctional pulverizer, Shanghai Jiupin Industry and Trade Co., Ltd.; XH-MC-1 microwave chemical reactor, Beijing Xianghu Technology Development Co., Ltd.; TG16-WS desktop high-speed centrifuge, Hunan Xiangyi Centrifuge Instrument Co., Ltd.; UV-visible spectrophotometer i3, Haineng Instrument; SHZ-D(III) circulating water multi-purpose vacuum pump, Zhengzhou Kelu Instrument Equipment Co., Ltd.; VS-840K-U clean bench, Suzhou Antai Air Technology Co., Ltd.; intelligent biochemical incubator, Ningbo Haishu Saifu Experimental Instrument Factory; itachi SU8010 field emission scanning electron microscope (SEM); Keyence RE-52 rotary evaporator, Shanghai Yarong Biochemical Instrument Factory; DHG-9240A electric constant temperature blast drying oven, Shanghai Precision Laboratory Equipment Co., Ltd.

[0055] 3 Experimental methods

[0056] 3.1 Ganoderma lucidum fruiting body powder

[0057] The fruiting bodies of Ganoderma lucidum were placed in an oven at 50℃ and dried until the water content was 7.5%. Figure 2 Before use, crush the fruiting bodies of Ganoderma lucidum to a fluffy state and pass through a 20-mesh sieve to obtain Ganoderma lucidum fruiting body powder ( Figure 2Middle B).

[0058] 3.2 Optimization of microwave-assisted hydrogen peroxide extraction process of Ganoderma lucidum polysaccharides

[0059] (1) Single factor experiment on Ganoderma lucidum polysaccharide extraction:

[0060] A. Microwave power: Weigh 1 g of the Ganoderma lucidum fruiting body powder prepared by the method in 3.1, add 5 mL of 30% hydrogen peroxide and 95 mL of water as the extractant, i.e., the concentration of hydrogen peroxide in the extractant is 1.5%, and the liquid-to-solid ratio of the extractant to the Ganoderma lucidum fruiting body powder is 100:1 (mL / g). Use a microwave chemical reactor to extract for 30 min at microwave powers of 400, 500, 600, 700, and 800 W, respectively, at 80°C. After extraction, centrifuge at 8000 rpm for 20 min. Record the volume of the supernatant, take 1 mL of the supernatant, and detect the polysaccharide content in the supernatant by the phenol-sulfuric acid method and convert it into the polysaccharide extraction yield.

[0061] B. Microwave temperature: Based on A, the microwave power is fixed at 600W, and the temperature is changed to 60-100℃ (60, 70, 80, 90, 100℃). Other operations are the same.

[0062] C Microwave time: Based on A, the microwave power is fixed at 600W, and the time is changed to 10-50min (10, 20, 30, 40, 50min). Other operations are the same.

[0063] D Liquid-to-material ratio: Based on A, the microwave power was fixed at 600 W, and the liquid-to-material ratio was changed to 40-130 mL / g (40, 60, 80, 90, 100, 110, 120, 130 mL / g). Other operations were the same.

[0064] E. Hydrogen peroxide concentration: Based on A, the microwave power was fixed at 600 W, and the hydrogen peroxide concentration in the extractant was changed to 0.6%-3.0% (0.6, 0.9, 1.2, 1.5, 1.8, 2.1, 2.4, 2.7, 3.0%), and other operations were the same.

[0065] Polysaccharide content detection method: refer to GB / T 15672-2009.

[0066] Polysaccharide extraction rate (%) = [c*V*F*10 -6 / M]*100.

[0067] Wherein, c is the concentration of the polysaccharide sample to be tested obtained according to the glucose standard curve (μg / mL); V is the volume of the supernatant of the extracted polysaccharide sample (mL); F is the dilution factor; M is the mass of the Ganoderma lucidum fruiting body powder (g).

[0068] Glucose standard curve see Figure 1, the standard equation is y = 4.4873x-0.0039 (R 2 =0.9935).

[0069] (2) Response surface optimization:

[0070] On the basis of single factor, the polysaccharide extraction rate was used as the response value. According to the principle of central composite test, the liquid-to-solid ratio, time and hydrogen peroxide concentration were used as variables. The three-factor three-level response surface analysis experiment was designed using Design-Expert 8.0.6 software to carry out Box Benhnken Design response surface analysis.

[0071] 3.3 Optimization of microwave-assisted enzymatic extraction conditions for Ganoderma lucidum polysaccharides

[0072] (1) Single factor experiment on microwave-assisted enzymatic extraction of Ganoderma lucidum polysaccharides

[0073] A. Amount of enzyme added: Place 1 g of the Ganoderma lucidum fruiting body powder prepared by the method in 3.1 in a three-necked flask, and add 0.006, 0.009, 0.012, 0.015, 0.018, and 0.021 g of cellulase, respectively (the amount of cellulase added is 0.6-2.1% based on the mass of the Ganoderma lucidum fruiting body powder), add 100 mL of water and mix well, adjust the pH to 5.0, use a microwave chemical reactor, power 600 W, 60°C, after enzymatic extraction for 30 min, centrifuge at 8000 rpm for 20 min, record the volume of the supernatant, take 1 mL of the supernatant, use the phenol-sulfuric acid method to detect the polysaccharide content in the supernatant and convert it into the polysaccharide extraction yield.

[0074] B Temperature: Based on A, the enzyme amount was selected as 0.015 g, the temperature was changed to 50-90°C (50, 60, 70, 80, 90°C), and other operations were the same.

[0075] C time: Based on A, the enzyme amount was selected as 0.015 g, and the enzymatic hydrolysis time was changed to 10-50 min (10, 20, 30, 40, 50 min). Other operations were the same.

[0076] D pH: Based on A, the enzyme amount was selected as 0.015 g and the pH was changed to 3.0-7.0 (3.0, 4.0, 5.0, 6.0, 7.0). Other operations were the same.

[0077] (2) Response surface optimization. Based on the single factor, the extraction rate of Ganoderma lucidum polysaccharide was used as the response value. According to the principle of central composite test, the hydrolysis temperature, enzyme dosage and hydrolysis time were used as variables. A three-factor three-level response surface analysis experiment was designed using Design-Expert 8.0.6 software to perform Box Benhnken Design response surface optimization.

[0078] 3.4 Effects of microwave-assisted extraction methods of enzyme followed by hydrogen peroxide and hydrogen peroxide followed by enzyme on the yield of Ganoderma lucidum polysaccharides

[0079] Hydrogen peroxide followed by enzymatic extraction: Weigh 1 g of the Ganoderma lucidum fruiting body powder prepared by the method in 3.1, add 7.7 mL of 30% hydrogen peroxide and 102.3 mL of water as the extractant (the mass concentration of hydrogen peroxide in the extractant is 2.1%), use a microwave chemical reactor at a power of 600 W and 70°C for microwave extraction for 50 min, then add 0.009, 0.012, 0.015, 0.018, and 0.021 g of cellulase, respectively, adjust the pH to 5.0, and perform microwave enzymatic extraction at a power of 600 W and 60°C for 20 min. Centrifuge at 8000 rpm for 20 min, record the volume of the supernatant, take 1 mL of the supernatant, and use the phenol-sulfuric acid method to detect the polysaccharide content in the supernatant and convert it into the polysaccharide extraction yield.

[0080] Extraction by enzyme followed by hydrogen peroxide: Weigh 1 g of the Ganoderma lucidum fruiting body powder prepared by the method in 3.1, add 0.009, 0.012, 0.015, 0.018, and 0.021 g of cellulase, respectively, add 102.3 mL of water and mix well, adjust the pH to 5.0, and use a microwave chemical reactor to perform enzymatic extraction at a power of 600 W and 60°C for 20 min. Then, add 7.7 mL of 30% hydrogen peroxide (the mass concentration of hydrogen peroxide in 110 mL of extractant is 2.1%). Further, extract at a microwave power of 600 W and 70°C for 50 min, centrifuge at 8000 rpm for 20 min, record the volume of the supernatant, take 1 mL of the supernatant, and detect the polysaccharide content in the supernatant by the phenol-sulfuric acid method and convert it into the polysaccharide extraction yield.

[0081] The extraction method with high yield of Ganoderma lucidum polysaccharides was further optimized using single factor and response surface design for the extraction process parameters.

[0082] 3.5 Microwave-assisted enzyme-first followed by hydrogen peroxide single factor and response surface optimization

[0083] (1) Single factor experiment of microwave-assisted extraction of Ganoderma lucidum polysaccharide using enzyme followed by hydrogen peroxide

[0084] A. Amount of enzyme added: 1 g of the Ganoderma lucidum fruiting body powder prepared by the method of 3.1 was placed in a three-necked flask, and 0.009, 0.012, 0.015, 0.018, and 0.021 g of cellulase were added respectively (the amount of cellulase added was 0.9-2.1% based on the mass of the Ganoderma lucidum fruiting body powder). After adding 102.3 mL of water and mixing, the pH was adjusted to 5.0. After enzymatic extraction for 30 min at 600 W and 60° C. in a microwave chemical reactor, 7.7 mL of 30% hydrogen peroxide (mass concentration of hydrogen peroxide in 110 mL of extractant was 2.1%) was added. After further extraction at 600 W and 70° C. in a microwave power of 50 min, the mixture was centrifuged at 8000 rpm for 20 min. The volume of the supernatant was recorded, and 1 mL of the supernatant was taken. The polysaccharide content in the supernatant was detected by the phenol-sulfuric acid method and converted into the polysaccharide extraction yield.

[0085] B temperature: Based on A, the enzyme amount was selected as 0.015 g, and the enzymatic hydrolysis temperature was changed to 50-90°C (50, 60, 70, 80, 90°C). Other operations were the same.

[0086] C time: Based on A, the enzyme amount was selected as 0.015 g, and the enzymatic hydrolysis time was changed to 10-50 min (10, 20, 30, 40, 50 min). Other operations were the same.

[0087] D pH: Based on A, the enzyme amount was selected as 0.015 g and the pH was changed to 3.0-7.0 (3.0, 4.0, 5.0, 6.0, 7.0). Other operations were the same.

[0088] (2) Response surface optimization. Based on the single factor, the extraction rate of Ganoderma lucidum polysaccharide was used as the response value. According to the principle of central composite test, the hydrolysis temperature, enzyme dosage and hydrolysis time were used as variables. A three-factor three-level response surface analysis experiment was designed using Design-Expert 8.0.6 software to perform Box Benhnken Design response surface optimization.

[0089] 3.6 Optimization of microwave-assisted hydrogen peroxide and enzyme-assisted extraction conditions for Ganoderma lucidum polysaccharides

[0090] (1) Single factor experiment on microwave-assisted hydrogen peroxide and enzyme synergistic extraction of Ganoderma lucidum polysaccharides

[0091] A. Amount of enzyme added: 1 g of Ganoderma lucidum fruiting body powder prepared by the method of 3.1 was placed in a three-necked flask, and 0.009, 0.012, 0.015, 0.018, 0.021, 0.025, and 0.027 g of cellulase were added respectively. After adding 90 mL of water and mixing, the pH was adjusted to 5.5. 10 mL of 30% hydrogen peroxide (the mass concentration of hydrogen peroxide in 100 mL of extractant is 3%) was added. Using a microwave chemical reactor, extraction was carried out at a power of 600 W and 60°C for 60 min. After centrifugation at 8000 rpm for 20 min, the volume of the supernatant was recorded, 1 mL of the supernatant was taken, and the polysaccharide content in the supernatant was detected by the phenol-sulfuric acid method and converted into the polysaccharide extraction yield.

[0092] B. Liquid-to-solid ratio: 1 g of the Ganoderma lucidum fruiting body powder prepared by the method of 3.1 was placed in a three-necked flask, 0.015 g of cellulase was added, and 30% hydrogen peroxide and water were added as extractants, i.e., (7 mL hydrogen peroxide + 63 mL water), (9 mL hydrogen peroxide + 81 mL water), (11 mL hydrogen peroxide + 99 mL water), (13 mL hydrogen peroxide + 117 mL water), (15 mL hydrogen peroxide + 135 mL water) were added respectively, so that the mass concentration of hydrogen peroxide added to the extractant was 1. The extraction concentration was 3.0%, the liquid-to-material ratio of the extractant to Ganoderma lucidum fruiting body powder was 70:1, 90:1, 110:1, 130:1, and 150:1 (mL / g), the pH was adjusted to 5.5, and a microwave chemical reactor was used. After extraction for 60 min at a power of 600 W and 60°C, the mixture was centrifuged at 8000 rpm for 20 min, the supernatant volume was recorded, and 1 mL of the supernatant was taken. The polysaccharide content in the supernatant was detected by the phenol-sulfuric acid method and converted into the polysaccharide extraction yield.

[0093] C temperature: Place 1 g of Ganoderma lucidum fruiting body powder prepared by the method in 3.1 in a three-necked flask, add 0.015 g of cellulase, add 90 mL of water and mix well, add 10 mL of 30% hydrogen peroxide, adjust the pH to 5.5, use a microwave chemical reactor, power 600 W, temperatures 50, 60, 70, 80, and 90 ° C, extract for 60 min, centrifuge at 8000 rpm for 20 min, record the volume of the supernatant, take 1 mL of the supernatant, use the phenol-sulfuric acid method to detect the polysaccharide content in the supernatant and convert it into the polysaccharide extraction yield.

[0094] D time: Place 1 g of Ganoderma lucidum fruiting body powder prepared by the method of 3.1 in a three-necked flask, add 0.015 g of cellulase, add 90 mL of water and mix well, adjust the pH to 5.5, add 10 mL of 30% hydrogen peroxide, use a microwave chemical reactor, power 600 W, 60 ° C conditions, extraction for 40, 60, 80, 100, and 120 min, respectively, after centrifugation at 8000 rpm for 20 min, record the volume of the supernatant, take 1 mL of the supernatant, use the phenol-sulfuric acid method to detect the polysaccharide content in the supernatant and convert it into the polysaccharide extraction yield.

[0095] E. Hydrogen peroxide concentration: 1 g of Ganoderma lucidum fruiting body powder prepared by the method of 3.1 was placed in a three-necked flask, 0.015 g of cellulase was added, and 30% hydrogen peroxide and water were added as extractants respectively (95 mL water with 5 mL 30% hydrogen peroxide), (94 mL water with 6 mL 30% hydrogen peroxide), (93 mL water with 7 mL 30% hydrogen peroxide), (92 mL water with 8 mL 30% hydrogen peroxide), (91 mL water with 9 mL 30% hydrogen peroxide), (90 mL water with 10 mL 30% hydrogen peroxide), (89 mL water with 11 mL 30% hydrogen peroxide), (88 mL water with 12 mL 30% hydrogen peroxide), (87 mL water with 13 mL 30% hydrogen peroxide), (86 mL water with 14 mL 30% hydrogen peroxide), (85 mL water with 15 mL 30% hydrogen peroxide), the pH was adjusted to 5.5, so that the mass concentration of hydrogen peroxide in the extractant was 1.5, 1.8, 2.1, 2.4, 2.7, 3.0, 3.3, 3.6, 3.9, 4.2, and 4.5%, respectively. A microwave chemical reactor was used, and extraction was carried out at a power of 600 W and 60°C for 60 min. The samples were then centrifuged at 8000 rpm for 20 min, and the supernatant volume was recorded. 1 mL of the supernatant was taken, and the polysaccharide content in the supernatant was detected by the phenol-sulfuric acid method and converted into the polysaccharide extraction yield.

[0096] (2) Response surface optimization. Based on the single factor, the extraction rate of Ganoderma lucidum polysaccharide was used as the response value. According to the principle of central composite test, the hydrolysis temperature, enzyme dosage and hydrolysis time were used as variables. A three-factor three-level response surface analysis experiment was designed using Design-Expert 8.0.6 software to perform Box Benhnken Design response surface optimization.

[0097] 3.7 Effect of different particle sizes of Ganoderma lucidum powder on the extraction yield of Ganoderma lucidum polysaccharides

[0098] 1.0 g of Ganoderma lucidum fruiting body powder of different particle sizes (>40, 40-60, 60-80, 80-100, 100-150, <150) was weighed, 0.020 g of cellulase was added, 121.5 mL of water was added, and then 13.5 mL of 30% hydrogen peroxide (the concentration of hydrogen peroxide in the extractant was 3.0%) was added and mixed. The pH was adjusted to 5.5. A microwave chemical reactor was used, and extraction was performed at 600 W power and 50°C for 105 min according to the extraction parameters optimized in 3.6. The mixture was centrifuged at 8000 rpm for 20 min. The supernatant was collected and tested according to the method in 3.2 to calculate the extraction yield of Ganoderma lucidum polysaccharides of different particle sizes.

[0099] 3.8 Optimization of Enzyme-Hydrogen Peroxide Synergistic Extraction Conditions for Ganoderma Lucidum Polysaccharides

[0100] 1 g of the Ganoderma lucidum fruiting body powder prepared by the method in 3.1 was placed in a three-necked flask, 0.02 g of cellulase was added, 126 mL of water was added and mixed, 9 mL of 30% hydrogen peroxide (3% by mass concentration in the extractant) was added, the pH was adjusted to 5.5, and extraction was performed using a microwave chemical reactor at 600 W power and 50°C for 105 min. The extract was then centrifuged at 8000 rpm for 20 min. The volume of the supernatant was recorded, and 1 mL of the supernatant was collected. The polysaccharide content in the supernatant was determined using the phenol-sulfuric acid method and converted to yield. The supernatant was concentrated to 50% of its original volume to obtain a Ganoderma lucidum polysaccharide solution, which was then freeze-dried (initial temperature: -30°C, vacuum: 80 Pa) to obtain Ganoderma lucidum polysaccharide.

[0101] 3.9 Scanning electron microscopy (SEM) observation of the microstructure of Ganoderma lucidum fruiting bodies and the residue after polysaccharide extraction using enzyme-hydrogen peroxide method

[0102] 0.5 mg of Ganoderma lucidum fruiting body powder and Ganoderma lucidum polysaccharide extracted according to the optimized conditions in 3.8 were respectively taken, coated on MC1000 ion sputtering instrument (icn sputter), and observed using German ZEISS GeminiSEM 300 scanning electron microscope (SEM).

[0103] 3.10 In vivo bioactivity index detection

[0104] Preparation of Caenorhabditis elegans growth medium (NGM medium): 1000 mL of NGM medium contains 2.5 g of peptone, 3 g of NaCl, 17 g of agar, 25 mL of PBS buffer (pH 6.0, 1 M), and 975 mL of deionized water. After sterilization, add 1 mL of a 0.22 μm-filtered cholesterol solution (5 mg / mL), 1 mL of a 1 M MgSO₄ solution, and 1 mL of a 1 M CaCl₂ solution.

[0105] 1000 mL of LB liquid medium contained 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl, which was adjusted to neutrality with 5 M NaOH, and the solvent was distilled water.

[0106] Preparation of 1000 mL of M9 buffer: 15.12 g Na2HPO4·12H2O, 3 g KH2PO4, 5 g NaCl, 0.25 g MgSO4·7H2O, the solvent is deionized water.

[0107] Lysis buffer: 0.5 mL deionized water, 0.3 mL NaClO, 0.2 mL 10 mol / L NaOH aqueous solution, prepared and used immediately.

[0108] Nematode culture: E. coli OP50 was inoculated into LB liquid medium and cultured overnight at 37°C in a shaker. The culture was then stored at 4°C until ready for use. Wild-type C. elegans strain N2 was inoculated onto NGM medium coated with E. coli OP50 and well-grown bacteria. After 72 hours of incubation at 20°C, a large number of adults and larvae were visible on the medium.

[0109] Synchronizing nematodes: Select a plate with well-grown nematodes and pipette 1.5 mL of M9 buffer onto the plate. Wash the plate several times until most nematodes are removed. Pipette 1 mL of the nematode suspension into a 1.5 mL EP tube and centrifuge at 3000 rpm for 1 minute. Discard the supernatant and reconstitute the pellet with 0.25 mL of deionized water. Then, add 0.15 mL of lysis buffer, vortex for 5 minutes, centrifuge at 4000 rpm for 1 minute, discard the supernatant, and reconstitute with 1 mL of M9 buffer. Vortex and wash, then centrifuge at 4000 rpm for 1 minute. Repeat the washing process until the sodium hypochlorite smell disappears. Place the lysed eggs onto NGM medium uncoated with E. coli OP50 and incubate in a 20°C incubator for 16 hours to obtain L1 nematodes. Wash the nematodes with M9 buffer, place them onto NGM medium coated with E. coli OP50, and incubate in a 20°C incubator for 48 hours to obtain synchronized L4 nematodes.

[0110] C.elegans survival rate experiment: NGM culture medium was divided into negative control group, positive control group and Ganoderma lucidum polysaccharide sample group, with 3 parallels in each group. The negative control group was coated with 1 mL of M9 buffer, the positive control group was coated with 1 mL of M9 buffer containing 0.5 mg / mL Vc at a final concentration, and the Ganoderma lucidum polysaccharide sample groups were coated with 1 mL of M9 buffer containing 4, 2, 1, 0.5, 0.25, 0.125 mg / mL Ganoderma lucidum polysaccharide at final concentrations. The Ganoderma lucidum polysaccharide was prepared according to the optimized parameters of 3.2 microwave-assisted hydrogen peroxide, 3.3 microwave-assisted cellulase, 3.4 microwave-assisted hydrogen peroxide first and then cellulase, 3.5 microwave-assisted cellulase first and then hydrogen peroxide, and 3.6 microwave-assisted cellulase-hydrogen peroxide synergistic extraction. The Ganoderma lucidum polysaccharide was prepared according to the method of 3.8. 150 μL of E. coli OP50 (concentration at 10 9 CFU / mL). Synchronized L4 nematodes were placed on plates for each group, with 60 C. elegans in triplicate per group. The nematodes were then incubated at 20°C for 48 h. The nematodes in each group were then transferred to a 96-well plate, and 200 μL of M9 buffer containing 4 mM hydrogen peroxide was added to each well. The plates were then incubated at 20°C. The number of C. elegans deaths was recorded every 40 min, and the survival rate of C. elegans at different time points was calculated.

[0111] According to the kit instructions provided by Nanjing Jiancheng Research Institute, the activities of superoxide dismutase (SOD) and catalase (CAT) in C. elegans, as well as the level of malondialdehyde (MDA), the terminal product of lipid peroxidation in C. elegans, were detected.

[0112] 3.11 Data Processing

[0113] The experimental data were expressed as Mean ± SD, and the significance difference was analyzed using one-way analysis of variance (ANOVA) test and LSD multiple comparison analysis in IBM SPSS Statistics 25 software. The difference was considered significant when p < 0.05.

[0114] 4 Experimental results

[0115] 4.1 Single factor experiment and response surface optimization of Ganoderma lucidum polysaccharide extraction

[0116] 4.1.1 Single-factor experiment and response surface optimization of microwave-assisted hydrogen peroxide extraction of Ganoderma lucidum polysaccharides

[0117] The effects of microwave power, microwave temperature, microwave time, material-liquid ratio and hydrogen peroxide concentration on the extraction yield of Ganoderma lucidum polysaccharides are shown in Figure 3 .

[0118] like Figure 3 As shown in the figure, when the microwave power increased from 400W to 800W, the extraction yield of Ganoderma lucidum polysaccharides gradually increased and then gradually decreased, but there were no significant differences between the levels. Therefore, no further response surface optimization was required and the temperature was set at 600W. When the temperature was gradually increased from 60°C to 90°C, the polysaccharide extraction yield continued to increase. When it was further increased to 100°C, the yield showed a downward trend. There were no significant differences between the various temperature levels from 70°C to 100°C. Considering the feasibility of practical operation, the temperature was set at 70°C and no further response surface optimization was required.

[0119] Based on the other three single-factor experiments, a three-factor, three-level response surface analysis experiment was designed using Design-Expert.V8.0.6 software according to the central composite experimental principle. The response surface experiment factor levels are shown in Table 1, and the experimental design and results are shown in Table 2.

[0120] Table 1 Experimental design factor levels for microwave-assisted hydrogen peroxide extraction of Ganoderma lucidum polysaccharides

[0121] Coding level A: Material-liquid ratio (mL / g) B: Hydrogen peroxide concentration (%) C: Processing time (min) -1 90 1.5 10 0 110 2.1 30 1 130 2.7 50

[0122] Table 2 Experimental design and results of microwave-assisted hydrogen peroxide extraction of Ganoderma lucidum polysaccharides

[0123]

[0124] The experimental data in Table 2 were subjected to multiple regression fitting to obtain the regression equation with polysaccharide yield (Y) as the response value:

[0125] Y=4.64+0.088A+0.263B+0.285C+0.174AB-0.005AC-0.226BC-1.35A 2 -0.538B 2 -0.047C 2

[0126] The regression equation was subjected to variance analysis, and the results are shown in Table 3.

[0127] Table 3 Regression model analysis of microwave-assisted hydrogen peroxide extraction yield of Ganoderma lucidum polysaccharides

[0128]

[0129]

[0130] Equation correlation coefficient R 2 =0.9845, indicating that the model has good correlation; the adjusted correlation coefficient R 2 adj =0.9645, indicating that the equation can explain 96.45% of the response value changes and has a good degree of fit; the coefficient of variation is 4.20%, indicating that the model has high credibility; a precision value greater than 4.0 is considered reasonable, and the precision of this experiment is 18.59, indicating that it is an appropriate signal; therefore, the equation can be used to infer the test results.

[0131] The results of variance analysis show that the first-order terms B and C have a very significant impact on the response value; the second-order term A 2 、B 2 The effect on the response value curve effect is extremely significant; the interaction term BC has a very significant effect on the response value surface effect, and the interaction terms AB and AC have no significant effect on the response value surface effect, indicating that there is an obvious synergistic effect between hydrogen peroxide concentration and microwave time, while there is no synergistic effect between the solid-liquid ratio and microwave time, or between the solid-liquid ratio and hydrogen peroxide concentration; from the size of the F value, it can be inferred that the order of the influence of the three factors on the extraction yield is: C>B>A, that is, microwave time>hydrogen peroxide concentration>solid-liquid ratio.

[0132] Based on the regression equation, the response surface and contour map of the interaction factors are drawn as follows: Figure 4 .

[0133] A large slope of the response surface plot indicates that the factor has a large impact on the response value, and dense elliptical contour lines indicate a large interactive effect between the two factors, while a gentle slope and circular contour lines indicate the opposite. Figure 4In the figure, the contour lines of the two groups of interaction effects of the three factors approach an ellipse, indicating that there is an interaction between the two.

[0134] The optimal conditions optimized using the model were: a liquid-to-solid ratio of 110.6664 mL / g, a microwave time of 49.9999 min, and a hydrogen peroxide concentration of 2.12405%. Under these conditions, the optimal adjustment was: a liquid-to-solid ratio of 110 mL / g, a microwave time of 50 min, and a hydrogen peroxide concentration of 2.1%. To further verify the validity and accuracy of the model with the actual situation, three parallel experiments were conducted using the extraction conditions optimized by the response surface analysis. The yield of Ganoderma lucidum polysaccharides reached 4.92% ± 0.02%, which is consistent with the predicted value. This demonstrates the reliability of the response surface analysis method and its good fit with the actual situation, thus verifying the validity of the regression equation.

[0135] 4.1.2 Single-factor experiment and response surface optimization of microwave-assisted cellulase extraction of Ganoderma lucidum polysaccharides

[0136] Effects of extraction time, enzyme addition, pH, and temperature on the extraction yield of Ganoderma lucidum polysaccharides are shown in Figure 5 .

[0137] Depend on Figure 5 As can be seen, as the extraction time increased from 10 to 20 minutes, the yield of Ganoderma lucidum polysaccharides showed a significant upward trend. When the extraction time increased beyond 20 minutes, the yield of Ganoderma lucidum polysaccharides showed a significant downward trend. At 20 minutes, the yield of Ganoderma lucidum polysaccharides was significantly higher than at other extraction times. Therefore, 20 minutes was used as the center point in subsequent optimization experiments.

[0138] The extraction rate of Ganoderma lucidum polysaccharides showed a significant upward trend with increasing enzyme addition. When the enzyme addition reached 1.5%, the extraction rate of Ganoderma lucidum polysaccharides showed a significant downward trend with further increase of enzyme addition. Therefore, the enzyme addition rate of 1.5% was used as the center point in subsequent optimization experiments.

[0139] The extraction yield of Ganoderma lucidum polysaccharides showed a significant upward trend with increasing pH, reaching its highest yield at pH 5.0. Further increases in pH resulted in a significant downward trend in the extraction yield. Therefore, a pH of 5.0 was used as the central point in subsequent optimization experiments.

[0140] The extraction yield of Ganoderma lucidum polysaccharides increased with increasing temperature, reaching its highest yield at 60°C. As the temperature continued to rise, the extraction yield showed a downward and upward trend, suggesting that the optimal enzyme temperature is around 60°C, and increasing the temperature deviates from the optimal enzyme temperature. The upward trend observed at 90°C may be due to the fact that the temperature increase accelerated the dissolution of the polysaccharide, but this effect was not as significant as that of the enzyme. Therefore, 60°C was selected as the central point for subsequent optimization experiments.

[0141] Based on the analysis of the single-factor experimental results and the principle of central composite experimental design, Design-Expert 8.0.6 software was used. The response surface experiment factor levels are shown in Table 4, and the experimental design and results are shown in Table 5.

[0142] Table 4 Experimental design factor levels for microwave-assisted cellulase extraction of Ganoderma lucidum polysaccharides

[0143] Coding level A: Processing time (min) B: Temperature (℃) C: Enzyme addition amount (%) D:pH -1 10 50 1.2 4.0 0 20 60 1.5 5.0 1 30 70 1.8 6.0

[0144] Table 5 Experimental design and results of microwave-assisted cellulase extraction of Ganoderma lucidum polysaccharides

[0145]

[0146] The experimental data in Table 5 were subjected to multiple regression fitting to obtain the regression equation with polysaccharide yield (Y) as the response value:

[0147] Y=5.37+0.257A+0.0017B+0.098C+0.78D-0.814AB+0.069AC-0.013AD-0.0125BC-1.60BD-0.0795CD-1.30A 2 -1.03B 2 -0.227C 2 -0.960D 2

[0148] The regression equation was subjected to variance analysis, and the results are shown in Table 6.

[0149] Table 6 Regression model analysis of the extraction rate of Ganoderma lucidum polysaccharides by microwave-assisted cellulase extraction

[0150]

[0151] Equation correlation coefficient R 2 =0.9121, indicating that the model has good correlation; the adjusted correlation coefficient R 2 adj=0.8243, indicating that the equation can explain 82.43% of the response value changes and has a good degree of fit; the coefficient of variation is 13.22%, indicating that the model has high credibility; a precision value greater than 4.0 is considered reasonable, and the precision of this experiment is 12.7841, indicating that it is an appropriate signal; therefore, the equation can be used to infer the test results.

[0152] The results of variance analysis show that the first-order term D has a very significant effect on the response value; the second-order term A 2 、B 2 、D 2 The response curve was significantly affected, while the interaction terms AB and BD had a significant impact on the response surface, indicating a significant synergistic effect between treatment time and temperature, and between temperature and pH. The F values ​​indicate that the order of influence of the four factors on extraction yield is: D > A > C > B, i.e., pH > treatment time > enzyme dosage > temperature.

[0153] The response surface of the interaction factors based on the regression equation is as follows Figure 6 .

[0154] A steep slope in the response surface plot indicates a significant influence of the factor on the response value, while densely packed elliptical contour lines indicate a significant interaction between two factors. A gentle slope and circular contour lines indicate the opposite. The optimal response surface optimization conditions were: a treatment time of 18.4045 min, an extraction temperature of 54.4633°C, an enzyme dosage of 1.73984%, and a pH of 5.62285. Under these conditions, a polysaccharide yield of 5.4677% was achieved. Based on actual laboratory conditions, the optimal conditions were adjusted to: a treatment time of 18 min, an extraction temperature of 55°C, an enzyme dosage of 1.7%, and a pH of 5.6. To further validate the model's accuracy and effectiveness, three parallel experiments were conducted using the extraction conditions optimized by the response surface analysis. The resulting polysaccharide yield reached 5.215% ± 0.021%, consistent with the predicted value. This demonstrates the reliability of the response surface analysis method and its good fit with the actual situation, thus verifying the validity of the regression equation.

[0155] 4.1.3 Effects of microwave-assisted hydrogen peroxide followed by enzyme extraction and enzyme followed by hydrogen peroxide extraction on the yield of Ganoderma lucidum polysaccharides

[0156] 3.4 Effect of microwave-assisted hydrogen peroxide followed by enzyme extraction on the yield of Ganoderma lucidum polysaccharides Figure 7 The effect of microwave-assisted enzyme extraction followed by hydrogen peroxide extraction on the yield of Ganoderma lucidum polysaccharides is shown in Figure 8 . Figure 7 and Figure 8Both methods showed an initial increase and then decrease in the extraction rate of Ganoderma lucidum polysaccharides with increasing cellulase addition, with the highest extraction rate occurring at a cellulase addition of 1.5%. However, at the same enzyme addition of 1.5%, the enzyme-first, hydrogen peroxide-second extraction method was superior to hydrogen peroxide-first, enzyme-second extraction. This is hypothesized to be due to the following factors: the former method is more efficient in disrupting the cell wall; enzymatic pretreatment enhances cell wall permeability; cellulase specifically decomposes cellulose, chitin, and other components of the Ganoderma lucidum cell wall, disrupting the cell wall structure and facilitating the release of intracellular polysaccharides; and this pretreatment provides a more open channel for subsequent hydrogen peroxide penetration, thereby improving polysaccharide dissolution efficiency. The following sections further optimized the parameters for the microwave-assisted enzyme-first, hydrogen peroxide-second extraction method.

[0157] 4.1.4 Microwave-assisted single-factor experiment and response surface optimization of Ganoderma lucidum polysaccharide extraction using enzyme followed by hydrogen peroxide

[0158] According to the results and analysis of 4.1.3, in the microwave-assisted enzyme-first followed by hydrogen peroxide extraction method, enzymatic pretreatment is the key to improving the extraction yield of Ganoderma lucidum polysaccharides. Therefore, a single-factor experiment was conducted on the time of enzymatic extraction, enzyme addition, pH and enzymatic hydrolysis temperature in the microwave-assisted enzyme-first followed by hydrogen peroxide method. The hydrogen peroxide extraction after enzyme treatment referred to the optimal process parameters of microwave-assisted hydrogen peroxide extraction optimized in 4.1.1, that is, the extraction time was continued for 50 minutes under the conditions of a hydrogen peroxide concentration of 2.1% and 70°C. The effects of the time of enzymatic extraction, enzyme addition, pH and enzymatic hydrolysis temperature on the extraction yield of Ganoderma lucidum polysaccharides in the microwave-assisted enzyme-first followed by hydrogen peroxide method can be seen in Figure 9 .

[0159] Depend on Figure 9 As can be seen, as the extraction time increased from 10 to 20 minutes, the yield of Ganoderma lucidum polysaccharides showed a significant upward trend. When the extraction time increased beyond 20 minutes, the yield showed a significant decrease, followed by an upward and downward trend. At 20 minutes, the yield of Ganoderma lucidum polysaccharides was significantly higher than at other extraction times. Therefore, 20 minutes was used as the central point in subsequent optimization experiments.

[0160] The extraction yield of Ganoderma lucidum polysaccharides showed a significant upward trend with increasing enzyme addition, reaching its peak at 1.5%. Further increases in enzyme addition led to a significant decrease in the yield. Therefore, 1.5% enzyme addition was used as the baseline for subsequent optimization experiments.

[0161] The extraction yield of Ganoderma lucidum polysaccharides showed a significant upward trend with increasing pH, reaching its highest yield at pH 5.0. Further increases in pH resulted in a significant downward trend in the extraction yield. Therefore, a pH of 5.0 was used as the central point in subsequent optimization experiments.

[0162] The extraction yield of Ganoderma lucidum polysaccharides increased with increasing temperature, reaching its highest yield at 60°C. As the temperature continued to increase, the extraction yield of Ganoderma lucidum polysaccharides showed a downward and upward trend, but it was still not as high as the yield at 60°C. Therefore, 60°C was selected as the central point for subsequent optimization experiments.

[0163] Based on the analysis of the single-factor experimental results and the principle of central composite experimental design, Design-Expert 8.0.6 software was used. The response surface experiment factor levels are shown in Table 7, and the experimental design and results are shown in Table 8.

[0164] Table 7 Experimental design factor levels of microwave-assisted extraction of Ganoderma lucidum polysaccharides using cellulase followed by hydrogen peroxide

[0165] Coding level A: Processing time (min) B: Temperature (℃) C: Enzyme addition amount (%) D:pH -1 10 50 1.2 4.0 0 20 60 1.5 5.0 1 30 70 1.8 6.0

[0166] Table 8 Experimental design and results of microwave-assisted extraction of Ganoderma lucidum polysaccharides using cellulase followed by hydrogen peroxide

[0167]

[0168]

[0169] The experimental data in Table 8 were subjected to multiple regression fitting to obtain the regression equation with polysaccharide yield (Y) as the response value:

[0170] Y=13.94+0.6676A+0.522B+0.3104C+2.30D-2.21AB+0.3699AC+0.0564AD-0.1127BC-3.96BD-0.231CD-3.13A 2 -2.60B 2 -0.03C 2 -2.40D 2

[0171] The regression equation was subjected to variance analysis, and the results are shown in Table 9.

[0172] Table 9 Regression model analysis of the extraction yield of Ganoderma lucidum polysaccharide by microwave-assisted extraction of cellulase followed by hydrogen peroxide

[0173]

[0174]

[0175] Equation correlation coefficient R 2 =0.9589, indicating that the model has good correlation; the adjusted correlation coefficient R 2 adj=0.9178, indicating that the equation can explain 91.78% of the response value changes and has a good degree of fit; the coefficient of variation is 9.19%, indicating that the model has high credibility; a precision value greater than 4.0 is considered reasonable, and the precision of this experiment is 19.1119, indicating that it is an appropriate signal; therefore, the equation can be used to infer the test results.

[0176] The results of variance analysis show that the first-order terms A and D among the four factors have a very significant impact on the response value; the second-order terms A 2 、B 2 、D 2 The response curve was significantly affected, while the interaction terms AB and BD significantly affected the response surface, indicating a significant synergistic effect between treatment time and temperature, and between temperature and pH. The F values ​​indicate that the order of influence of the four factors on extraction yield is: D > A > B > C, i.e., pH > treatment time > temperature > enzyme dosage.

[0177] The response surface of the interaction factors based on the regression equation is as follows Figure 10 .

[0178] A large slope of the response surface plot indicates that the factor has a large impact on the response value, and dense elliptical contour lines indicate a large interactive effect between the two factors, while a gentle slope and circular contour lines indicate the opposite. Figure 10 In the figure, the interaction effect contours of the two groups AB and BD approach ellipses, indicating that there is an interaction between the two.

[0179] The optimal conditions for response surface analysis were: a treatment time of 23.072 minutes, an extraction temperature of 56.765°C, an enzyme dosage of 1.8%, and a pH of 5.547. Under these conditions, the polysaccharide dissolution rate reached 15.948%. Based on actual laboratory conditions, the optimal conditions were adjusted to: a treatment time of 23 minutes, an extraction temperature of 57°C, an enzyme dosage of 1.8%, and a pH of 5.5. To further verify the validity and accuracy of the model with the actual conditions, three parallel experiments were conducted using the extraction conditions optimized by the response surface analysis. The yield of Ganoderma lucidum polysaccharides reached 15.26% ± 0.33%, which is consistent with the predicted value. This demonstrates the reliability of the response surface analysis method and its good fit with the actual conditions, thus validating the validity of the regression equation.

[0180] 4.1.5 Single-factor experiment and response surface optimization of microwave-assisted enzyme and hydrogen peroxide synergistic extraction of Ganoderma lucidum polysaccharides

[0181] The effects of extraction time, enzyme addition, material-liquid ratio, temperature and hydrogen peroxide concentration on the extraction yield of Ganoderma lucidum polysaccharides are shown in Figure 11 .

[0182] like Figure 11As shown in the figure, as the extraction time increased from 40 to 100 minutes, the extraction yield of Ganoderma lucidum polysaccharides showed a significant upward trend. When the extraction time increased to more than 100 minutes, the extraction yield showed a significant downward trend. At an extraction time of 100 minutes, the extraction yield of Ganoderma lucidum polysaccharides was significantly higher than the yields at other extraction times. Therefore, 100 minutes was used as the center point in subsequent optimization experiments.

[0183] The extraction yield of Ganoderma lucidum polysaccharides showed a significant increase, then decrease, then increase, then decrease again with increasing enzyme addition (from 0.9% to 2.7%), reaching its highest yield when the enzyme addition reached 2.1%. Therefore, 2.1% enzyme addition was used as the central point in subsequent optimization experiments.

[0184] As the liquid-to-solid ratio increased from 70 mL / g to 110 mL / g, the extraction yield of Ganoderma lucidum polysaccharides continued to increase significantly. The highest extraction yield was achieved at 130 mL / g, but there was no difference compared to the yield at 110 mL / g. As the liquid-to-solid ratio continued to increase, the extraction yield showed a downward trend. Therefore, a liquid-to-solid ratio of 110 mL / g was used as the center point in subsequent optimization experiments.

[0185] As the temperature increased from 50°C to 70°C, the extraction yield of Ganoderma lucidum polysaccharides showed a downward trend, but there was no significant difference between the various levels. At 80°C, the extraction yield of Ganoderma lucidum polysaccharides showed a significant downward and upward trend, with no inflection point in the entire temperature range. The extraction yield at 90°C was similar to that at temperatures between 50°C and 70°C. For energy conservation purposes, 50°C was selected for subsequent experiments without temperature optimization.

[0186] As the hydrogen peroxide concentration increased from 1.5% to 2.7%, the extraction yield of Ganoderma lucidum polysaccharides fluctuated, but no differences were observed between levels. When the hydrogen peroxide concentration increased from 2.7% to 3.0%, the extraction yield of Ganoderma lucidum polysaccharides showed a significant upward trend. When the hydrogen peroxide concentration continued to increase from 3.0% to 4.5%, the extraction yield of Ganoderma lucidum polysaccharides initially increased and then decreased, but no differences were observed between levels. The extraction yield of polysaccharides did not show an inflection point across the entire hydrogen peroxide concentration range, so no optimization of the hydrogen peroxide concentration was required for subsequent experiments; 3.0% was selected.

[0187] Based on the analysis of the single-factor experimental results and the principle of central composite experimental design, a three-factor, three-level response surface analysis experiment was designed using Design-Expert 8.0.6 software. The response surface experiment factor levels are shown in Table 10, and the experimental design and results are shown in Table 11.

[0188] Table 10 Experimental design factor levels for microwave-assisted enzyme and hydrogen peroxide synergistic extraction of Ganoderma lucidum polysaccharides

[0189] Coding level A: Extraction time (min) B: Enzyme addition amount (%) C: Material-liquid ratio (mL / g) -1 90 1.8 70 0 105 2.1 110 1 120 2.4 150

[0190] Table 11 Experimental design and results of microwave-assisted enzyme and hydrogen peroxide synergistic extraction of Ganoderma lucidum polysaccharides

[0191]

[0192] The experimental data in Table 11 were subjected to multiple regression fitting to obtain the regression equation with polysaccharide yield (Y) as the response value:

[0193] Y=18.55+0.2526A+0.4189B+3.35C+0.6482AB-1.08AC-1.49BC-3.01A 2 -2.32B 2 -2.17C 2

[0194] The regression equation was subjected to variance analysis, and the results are shown in Table 12.

[0195] Table 12 Regression model analysis of the extraction yield of Ganoderma lucidum polysaccharides by microwave-assisted enzyme and hydrogen peroxide synergistic extraction

[0196]

[0197]

[0198] Equation correlation coefficient R 2 =0.9953, indicating that the model has good correlation; the adjusted correlation coefficient R 2 adj =0.9891, indicating that the equation can explain 98.91% of the response value changes and has a good degree of fit; the coefficient of variation is 2.44%, indicating that the model has high credibility; a precision value greater than 4.0 is considered reasonable, and the precision of this experiment is 35.1004, indicating that it is an appropriate signal; therefore, the equation can be used to infer the test results.

[0199] The results of variance analysis show that the first-order terms B and C have a very significant impact on the response value; the second-order term A 2 、B 2 、C 2 The effect on the response value curve is extremely significant; the interaction terms AB, AC, and BC have extremely significant effects on the response value surface effect, indicating that there is an obvious synergistic effect between the extraction time, enzyme addition amount and material-liquid ratio; from the size of the F value, it can be inferred that the order of the influence of the three factors on the extraction yield is: C>B>A, that is, material-liquid ratio>enzyme addition amount>time.

[0200] Based on the regression equation, the response surface and contour map of the interaction factors are drawn as follows: Figure 12 .

[0201] A large slope of the response surface plot indicates that the factor has a large impact on the response value, and dense elliptical contour lines indicate a large interactive effect between the two factors, while a gentle slope and circular contour lines indicate the opposite. Figure 12 In the figure, the contour lines of the two groups of interaction effects of the three factors AB, AC and BC approach ellipses, indicating that there is an interaction between the two.

[0202] The optimal conditions optimized using the model were: extraction time of 104.913 minutes, enzyme addition of 2.00% (mass fraction), and liquid-to-solid ratio of 136.313 mL / g. Under these conditions, the polysaccharide dissolution rate reached 19.831%. Based on actual laboratory conditions, the optimal conditions were adjusted to: extraction time of 105 minutes, enzyme addition of 2.0% (mass fraction), and liquid-to-solid ratio of 135 mL / g. To further verify the validity and accuracy of the model with the actual situation, three parallel experiments were conducted using the extraction conditions optimized by response surface analysis. The extraction yield of Ganoderma lucidum polysaccharides reached 19.56% ± 0.11%, which is consistent with the predicted value. This demonstrates that the response surface analysis method is reliable and fits the actual situation well, thus verifying the validity of the regression equation.

[0203] 4.1.6 Effect of different particle sizes of Ganoderma lucidum powder on the extraction yield of Ganoderma lucidum polysaccharides

[0204] Effects of different particle sizes of Ganoderma lucidum powder on the extraction rate of Ganoderma lucidum polysaccharides Figure 13 As the particle size of Ganoderma lucidum powder continued to decrease, the extraction rate of Ganoderma lucidum polysaccharides showed a trend of continuous significant increase and then significant decrease. When the particle size of Ganoderma lucidum powder was between 80-100 mesh, the extraction rate of Ganoderma lucidum polysaccharides was 35.59% ± 0.578%, which was significantly higher than that of other particle sizes.

[0205] In summary, among the methods of microwave-assisted hydrogen peroxide, microwave-assisted cellulase, microwave-assisted cellulase first and then hydrogen peroxide, microwave-assisted hydrogen peroxide first and then cellulase, and microwave-assisted cellulase-hydrogen peroxide synergistic extraction of Ganoderma lucidum polysaccharide, the microwave-assisted cellulase-hydrogen peroxide synergistic extraction effect is best when the particle size of Ganoderma lucidum powder is 80-100 mesh.

[0206] 4.2 Scanning electron microscopy (SEM) observation of the cell wall and surface characteristics of Ganoderma lucidum polysaccharides

[0207] SEM images of Ganoderma lucidum polysaccharides obtained under the optimal extraction conditions of 3.1 Ganoderma lucidum fruiting body powder raw material, hot water in Comparative Example 1, hydrogen peroxide in Comparative Example 3, and microwave-assisted cellulase-hydrogen peroxide synergistic extraction in 3.8 are shown in FIG. Figure 14 .Depend on Figure 14It can be seen that the cell wall and mycelium of the Ganoderma lucidum fruiting body are basically intact, with few cracks and holes. The surface of the sample treated with hot water has a flocculent structure and is slightly cracked. The cell wall of the sample treated with hydrogen peroxide is more damaged, with rods appearing on the surface and a clear porous structure. The surface of the sample treated with microwave-assisted cellulase-hydrogen peroxide is loose, with more prominent rods and a clear porous structure. It can be seen that different treatment methods have a great influence on the microstructure of the cell wall of Ganoderma lucidum fruiting body, and it is speculated that they also have a great influence on the microstructure of the polysaccharides in the cell wall. The yield, molecular structure and biological activity of polysaccharides in the cell wall strongly depend on the cell wall breaking treatment method used, suggesting that the biological activity of polysaccharides obtained by several cell wall breaking methods also varies.

[0208] 4.3 Effects of Ganoderma lucidum polysaccharides on the lifespan of C. elegans under oxidative stress

[0209] Ganoderma lucidum polysaccharides were prepared according to the optimized parameters of microwave-assisted hydrogen peroxide, microwave-assisted cellulase, microwave-assisted cellulase followed by hydrogen peroxide, microwave-assisted hydrogen peroxide followed by cellulase, and microwave-assisted cellulase-hydrogen peroxide synergistic extraction in method 4.1. The effects of these on the average lifespan of C. elegans under hydrogen peroxide-induced oxidative stress are shown in Figure 4. Figures 15-19 , Vc was selected as the positive control.

[0210] Hydrogen peroxide can diffuse in cells and tissues. In living systems, hydrogen peroxide can react with metal ions to produce strong OH free radicals, which can induce intracellular oxidative stress reactions in nematodes. The maximum survival time (Tmax) of nematodes in the negative control (M9) group under hydrogen peroxide stress was 240 min, and the positive control group Vc could extend the Tmax of nematodes to more than 360 min. After hydrogen peroxide stress treatment, all concentrations (0.125-4.0 mg / mL) of Ganoderma lucidum polysaccharides extracted by five methods (microwave-assisted hydrogen peroxide, microwave-assisted cellulase, microwave-assisted cellulase first and then hydrogen peroxide, microwave-assisted hydrogen peroxide first and then cellulase, and microwave-assisted cellulase-hydrogen peroxide synergistically) can shift the growth curve of nematodes under hydrogen peroxide stress to the right, and the Tmax of Ganoderma lucidum polysaccharides extracted by the five methods at the optimal concentration reaches 320 min. Figures 15-19The average survival time of nematodes in the negative control group was less than 130 minutes, while the average survival time of nematodes in the positive control group was around 200 minutes. Compared with the negative control group, Ganoderma lucidum polysaccharides extracted using all five methods prolonged the average survival time of nematodes under stress at all concentrations tested. Among them, medium- and high-concentration Ganoderma lucidum polysaccharides (0.5-4.0 mg / mL) significantly prolonged the average lifespan of nematodes (p < 0.05), while low concentrations (0.125-0.25 mg / mL) had a minimal effect on nematode survival (p > 0.05). When the concentration of Ganoderma lucidum polysaccharide was 0.5 mg / mL, the polysaccharide extracted by microwave-assisted cellulase-hydrogen peroxide synergistic method could extend the average lifespan of C. elegans to 210.83 min ± 15.14 min, which was 55.57%, 11.42%, 20.50% and 14.58% higher than the polysaccharide extracted by microwave-assisted hydrogen peroxide (172.00 min ± 10.58 min), microwave-assisted cellulase (189.42 min ± 8.46 min), microwave-assisted cellulase followed by hydrogen peroxide (174.96 min ± 11.31 min) and microwave-assisted hydrogen peroxide followed by cellulase (184.00 min ± 10.58 min), respectively.

[0211] 4.4 Effects of Ganoderma lucidum polysaccharides on antioxidants and antioxidant enzymes in C. elegans under oxidative stress

[0212] Based on the results in Section 4.3, Ganoderma lucidum polysaccharides prepared by all five methods were able to extend the average lifespan of C. elegans under oxidative stress. This indicates that Ganoderma lucidum polysaccharides extracted using either hydrogen peroxide, cellulase, or both can enhance the lifespan of C. elegans under oxidative stress, and that none of the five methods affected the function of Ganoderma lucidum polysaccharides. The following section further investigated the bioactivity of Ganoderma lucidum polysaccharides extracted using microwave-assisted cellulase-hydrogen peroxide co-extraction, by measuring lipid peroxide levels and antioxidant enzyme activity in C. elegans under oxidative stress.

[0213] (1) Effects of Ganoderma lucidum polysaccharides on antioxidant enzymes

[0214] Superoxide dismutase (SOD) is an enzyme containing a metal cofactor that catalyzes the dismutation of superoxide anions to produce hydrogen peroxide and oxygen. Catalase (CAT), a peroxisome marker enzyme, is widely expressed in mammalian tissues. Its primary function is to catalyze the decomposition of hydrogen peroxide into oxygen and water.

[0215] Effects of Ganoderma lucidum polysaccharides on SOD activity in C.elegans Figure 20(Left panel) Compared with the negative control group (M9), Ganoderma lucidum polysaccharide concentrations of 0.25, 0.5, and 2 mg / mL significantly increased SOD activity in C. elegans (p<0.05), increasing by 47.06%, 33.76%, and 134.14%, respectively. This suggests that a certain concentration of Ganoderma lucidum polysaccharide can significantly enhance SOD activity in C. elegans under oxidative stress.

[0216] Effects of Ganoderma lucidum polysaccharides on CAT activity in C.elegans Figure 20 (Right figure) All different concentrations of Ganoderma lucidum polysaccharide (0.125mg / mL to 2.0mg / mL) significantly increased CAT activity in C. elegans (p<0.05). There was no significant difference between the 0.125mg / mL and 0.5mg / mL concentrations compared to the positive control group (p>0.05). At a concentration of 1.0mg / mL, the effect on CAT activity was significantly superior to that of the positive control group (p<0.05). Compared to the negative control group, when the concentration of Ganoderma lucidum polysaccharide increased from 0.125mg / mL to 2.0mg / mL, CAT activity in C. elegans increased by 163.30%, 116.74%, 229.36%, 320.64%, and 73.39%, respectively.

[0217] (2) Effects of Ganoderma lucidum polysaccharides on malondialdehyde, the terminal product of lipid peroxides in C. elegans

[0218] Malondialdehyde (MDA) is a product of the reaction between free radicals and lipid oxidation, which can produce cytotoxicity and aggravate cell membrane damage. Therefore, MDA levels are often used as an indicator in anti-aging research. Figure 21 As shown in the results, except for the high concentration of 2.0 mg / mL, other concentrations of Ganoderma lucidum polysaccharide significantly reduced the MDA content in C. elegans (p < 0.05). Compared with the negative control group, the MDA content in C. elegans was reduced by 42.51%, 93.43%, 85.65% and 39.58% after treatment with 0.125, 0.25, 0.5 and 1.0 mg / mL of Ganoderma lucidum polysaccharide, respectively. The high concentration of Ganoderma lucidum polysaccharide reduced the MDA content by 7.14%, but there was no difference compared with the negative control group. It is speculated that this may be due to the high permeability caused by the high concentration of Ganoderma lucidum polysaccharide.

[0219] Example 2

[0220] 1.0 g of Ganoderma lucidum fruiting body powder (passed through an 80-100 mesh sieve) was added with 0.020 g of cellulase, 121.5 mL of water, and 13.5 mL of 30% hydrogen peroxide (the concentration of hydrogen peroxide in the extractant was 3.0%). The mixture was then mixed and the pH was adjusted to 5.5. The extract was extracted using a microwave chemical reactor at 600 W power and 50°C for 105 minutes. Centrifugation was performed at 8000 rpm for 20 minutes, and the supernatant was concentrated to a volume of 50 mL to obtain a Ganoderma lucidum polysaccharide solution. The solution was freeze-dried (initial temperature: -30°C, vacuum: 80 Pa) to obtain 0.3559 g of Ganoderma lucidum polysaccharide. Three replicates were set up.

[0221] Using the detection method of 3.2 in Example 1, the average extraction yield of Ganoderma lucidum polysaccharide was 20.30%; using the detection method of 3.10 in Example 1, Ganoderma lucidum polysaccharide (0.5 mg / mL) could extend the average lifespan of C. elegans under hydrogen peroxide stress to 210.83 min±15.14 min, an increase of 88.24% compared with the negative control group (average lifespan 112.0 min±12.0 min); at the same time, the SOD activity in C. elegans under hydrogen peroxide stress was increased by 33.76%, the CAT activity was increased by 229.36%, and the MDA content was reduced by 85.65%, and the differences were significant compared with the negative control group (p<0.05).

[0222] Comparative Example 1: Hot Water Extraction

[0223] 1.0 g of Ganoderma lucidum fruiting body powder (passed through an 80-100 mesh sieve) was added to 135 mL of water and extracted in a shaking water bath at 70°C for 105 min. The mixture was centrifuged at 8000 rpm for 20 min, and the supernatant was concentrated to a volume of 50 mL to obtain a Ganoderma lucidum polysaccharide solution. This solution was freeze-dried (initial temperature: -30°C, vacuum: 80 Pa) to obtain 0.029 g of Ganoderma lucidum polysaccharide. The polysaccharide detection method and in vivo bioactivity analysis were the same as in Example 1. The results are shown in Table 13.

[0224] Table 13 Yields of Ganoderma lucidum polysaccharides extracted by different treatments and their in vitro and in vivo bioactivity indicators

[0225]

[0226] As shown in Table 13, the extraction process of Ganoderma lucidum polysaccharide has a great influence on the extraction yield of polysaccharide and the average lifespan of C.elegans under stress state. Microwave-assisted cellulase-hydrogen peroxide synergistic extraction can significantly improve the extraction yield of Ganoderma lucidum polysaccharide and the average lifespan of C.elegans under stress state. The extraction yield and the average lifespan of C.elegans under oxidative stress state are increased by 11.98 times and 1.18 times respectively compared with Ganoderma lucidum polysaccharide extracted by hot water.

[0227] Comparative Example 2: Microwave-assisted hot water extraction

[0228] 1.0 g of Ganoderma lucidum fruiting body powder (passed through an 80-100 mesh sieve) was added to 135 mL of water and extracted using a microwave chemical reactor at 600 W power and 70°C for 105 min. The extract was centrifuged at 8000 rpm for 20 min, and the supernatant was concentrated to a volume of 50 mL to obtain a Ganoderma lucidum polysaccharide solution. This solution was freeze-dried (initial temperature: -30°C, vacuum: 80 Pa) to obtain 0.036 g of Ganoderma lucidum polysaccharide. The polysaccharide detection method and in vivo bioactivity analysis were the same as in Example 1. The results are shown in Table 14.

[0229] Table 14 Yields of Ganoderma lucidum polysaccharides extracted by different treatments and their in vitro and in vivo bioactivity indicators

[0230]

[0231] As shown in Table 14, the extraction process of Ganoderma lucidum polysaccharide has a great influence on the extraction yield of polysaccharide and the average lifespan of C.elegans under stress state. Microwave-assisted cellulase-hydrogen peroxide synergistic extraction can significantly improve the extraction yield of Ganoderma lucidum polysaccharide and the average lifespan of C.elegans under stress state. Among them, the extraction yield and the average lifespan of C.elegans under oxidative stress state are increased by 9.75 times and 1.16 times respectively compared with the Ganoderma lucidum polysaccharide extracted by microwave-assisted hot water.

[0232] Comparative Example 3: Hydrogen Peroxide Extraction

[0233] 1.0 g of Ganoderma lucidum fruiting body powder (passed through an 80-100 mesh sieve) was added to 121.5 mL of water, followed by 13.5 mL of 30% hydrogen peroxide, and mixed thoroughly. The mixture was extracted in a shaking water bath at 70°C for 105 min. The mixture was centrifuged at 8000 rpm for 20 min, and the supernatant was concentrated to a volume of 50 mL to obtain a Ganoderma lucidum polysaccharide solution. This solution was freeze-dried (initial temperature: -30°C, vacuum: 80 Pa) to obtain 0.068 g of Ganoderma lucidum polysaccharide. The polysaccharide detection method and in vivo bioactivity analysis were the same as in Example 1. The results are shown in Table 15.

[0234] Table 15 Yields of Ganoderma lucidum polysaccharides extracted by different treatments and their in vitro and in vivo bioactivity indicators

[0235]

[0236] As shown in Table 15, the extraction process of Ganoderma lucidum polysaccharide has a great influence on the extraction yield of polysaccharide and the average lifespan of C.elegans under stress state. Microwave-assisted cellulase-hydrogen peroxide synergistic extraction can significantly improve the extraction yield of Ganoderma lucidum polysaccharide and the average lifespan of C.elegans under stress state. Among them, the extraction yield and the average lifespan of C.elegans under oxidative stress state are increased by 5.21 times and 1.11 times respectively compared with Ganoderma lucidum polysaccharide extracted with hydrogen peroxide.

[0237] Example 3

[0238] Accurately weigh 1.0g of Ganoderma lucidum fruiting body powder (passed through an 80-100 mesh sieve), add 0.018g of cellulase, 132mL of water, and then 18mL of 30% hydrogen peroxide (the concentration of hydrogen peroxide in the system is 3.6%). Mix thoroughly, adjust the pH to 6.0, and extract using a microwave chemical reactor at 700W power and 70°C for 120 minutes. Centrifuge at 8000rpm for 20 minutes, and concentrate the supernatant to 50mL to obtain a Ganoderma lucidum polysaccharide solution. Freeze-dry (initial temperature -30°C, vacuum 80Pa) to obtain 0.329g of Ganoderma lucidum polysaccharide. Three replicates were set up. The average polysaccharide extraction yield was 19.3%, and the polysaccharide was shown to extend the average lifespan of C. elegans under oxidative stress to 197.23 minutes.

[0239] Example 4

[0240] Accurately weigh 1.0g of Ganoderma lucidum fruiting body powder (passed through an 80-100 mesh sieve), add 0.024g of cellulase, 63mL of water, and then 7mL of 30% hydrogen peroxide (the concentration of hydrogen peroxide in the system is 3.0%). Mix thoroughly and extract using a microwave chemical reactor at 600W power, pH 4.0, and 60°C for 90 minutes. Centrifuge at 8000rpm for 20 minutes, and concentrate the supernatant to 35mL to obtain a Ganoderma lucidum polysaccharide solution. Freeze-drying (initial temperature -30°C, vacuum 80Pa) yielded 0.311g of Ganoderma lucidum polysaccharide. Three replicates were set up. The average polysaccharide extraction yield was 18.5%, and the polysaccharide was shown to extend the average lifespan of C. elegans under oxidative stress to 191.16 minutes.

[0241] Example 5

[0242] Accurately weigh 1.0g of Ganoderma lucidum fruiting body powder (passed through an 80-100 mesh sieve), add 0.021g of cellulase, 89mL of water, and 11mL of 30% hydrogen peroxide (the concentration of hydrogen peroxide in the system is 3.3%). Mix thoroughly, adjust the pH to 5.0, and extract using a microwave chemical reactor at 500W power and 50°C for 105 minutes. Centrifuge at 8000rpm for 20 minutes, and concentrate the supernatant to 50mL to obtain a Ganoderma lucidum polysaccharide solution. Freeze-dry (initial temperature -30°C, vacuum 80Pa) to obtain 0.321g of Ganoderma lucidum polysaccharide. Three replicates were set up. The average polysaccharide extraction yield was 19.1%, and the polysaccharide was shown to extend the average lifespan of C. elegans under oxidative stress to 188.60 minutes.

[0243] Example 6

[0244] Accurately weigh 1.0g of Ganoderma lucidum fruiting body powder (passed through an 80-100 mesh sieve), add 0.020g of cellulase, 99mL of water, and then 11mL of 30% hydrogen peroxide (the concentration of hydrogen peroxide in the system is 3.0%). Mix thoroughly, adjust the pH to 5.5, and extract using a microwave chemical reactor at 800W power and 60°C for 120 minutes. Centrifuge at 8000rpm for 20 minutes, and concentrate the supernatant to 50mL to obtain a Ganoderma lucidum polysaccharide solution. Freeze-dry (initial temperature -30°C, vacuum 80Pa) to obtain 0.317g of Ganoderma lucidum polysaccharide. Three replicates were set up. The average polysaccharide extraction yield was 18.7%, and the polysaccharide was shown to extend the average lifespan of C. elegans under oxidative stress to 193.52 minutes.

Claims

1. A method for extracting Ganoderma lucidum polysaccharides by microwave-assisted green chemistry-enzymatic method, characterized in that: The method comprises the following steps: taking ganoderma lucidum fruiting body powder, adding cellulase and water, mixing evenly, then adding 30% hydrogen peroxide, mixing evenly, adjusting the pH to 3-7, and performing microwave-assisted extraction at a power of 400-800W and a temperature of 50-90°C for 40-120 minutes; The supernatant was concentrated to 30-50% of the original volume and freeze-dried to obtain Ganoderma lucidum polysaccharide.

2. The method according to claim 1, wherein Ganoderma lucidum fruiting body powder is obtained by drying Ganoderma lucidum fruiting body in a 50°C oven until the water content is less than 8%, crushing the powder, and passing the powder through a 20-100 mesh sieve.

3. The method according to claim 1, wherein The amount of cellulase added is 0.9-2.7% based on the weight of the Ganoderma lucidum fruiting body powder.

4. The method according to claim 1 or 3, wherein: The enzymatic activity of the cellulase is 10,000 U / g.

5. The method according to claim 1, wherein Water and 30% hydrogen peroxide constitute the extractant, the volume dosage of the extractant is 50-150 mL / g based on the weight of the ganoderma lucidum fruiting body powder; the mass concentration of the hydrogen peroxide in the extractant is 1.5-4.5%.

6. The method according to claim 1, wherein pH is 4-6.

7. The method according to claim 1, wherein Extraction was carried out at a power of 500-800W and a temperature of 50-70°C for 90-120 minutes.

8. The method according to claim 1, wherein The freeze-drying conditions were an initial temperature of -30°C and a vacuum degree of 80 Pa.

9. Ganoderma lucidum polysaccharide prepared by the method according to claim 1.

10. Use of the Ganoderma lucidum polysaccharide according to claim 9 in preparing a drug for extending the lifespan of Caenorhabditis elegans under oxidative stress.

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