Preparation method of ganoderma lucidum bud polysaccharide through ultrasonic extraction

Abstract

The invention discloses a preparation method for ultrasonically extracting ganoderma lucidum bud water-soluble polysaccharide, relates to the field of extraction of effective components of natural medicines, and adopts a single factor response surface method to optimize an extraction process and evaluate the antioxidant activity of the ganoderma lucidum bud water-soluble polysaccharide. The extraction rate, the antioxidant activity and the energy consumption are taken as evaluation indexes, comprehensive evaluation is performed by adopting an entropy weight method, and the optimal extraction process after optimization is as follows: the extraction power is 280W, the liquid-material ratio is 38: 1 (mL: g), the extraction time is 27.5 min, and the extraction temperature is 47 DEG C; the optimal polysaccharide extraction rate of a water extract is 2.22 + / -0.02%, the ABTS clearance rate is 52.37 + / -1.27%, the energy consumption is 0.292 + / -0.0001 KW / h, the comprehensive score is 43.77 + / -1.02 score, and the ganoderma lucidum bud polysaccharide has good antioxidant activity, which proves that the ganoderma lucidum bud polysaccharide obtained by the extraction process is high in extraction rate and good in activity.

Description

Technical Field

[0001] The present invention relates to the field of extraction of effective components of natural medicines, and particularly relates to a process for extracting ganoderma lucidum bud polysaccharides using an ultrasonic method. Background Art

[0002] Ganoderma lucidum belongs to the Basidiomycetes, Hymenomycetes, Aphyllophytes, Ganoderma, and Ganoderma genus. It is listed in the Compendium of Materia Medica and possesses extremely high nutritional and health benefits. Ganoderma contains a variety of bioactive ingredients, such as nucleotides, triterpenes, polysaccharides, glycoproteins, and alkaloids. Among these numerous active ingredients, Ganoderma polysaccharides are a hot topic, exhibiting multiple biological activities such as antioxidant, anti-tumor, and immunomodulatory activities, playing an important role in maintaining human health. Ganoderma buds are the young branches of the Ganoderma fruiting body during the bud stage, before the cap has formed on the stipe. They are sweet and neutral in nature, primarily composed of an elongated stalk and an undifferentiated cap. Compared to the fruiting body, they are slightly more immature. Traditional Chinese Medicine often uses the fruiting body, while ignoring the young buds. Around May each year, to ensure the quality of Ganoderma lucidum, growers prune off excess branches. These buds, valued at 10%-12% of the total Ganoderma lucidum output, are often discarded as waste due to their rapid deterioration and unpleasant odor. While these buds contain active ingredients similar to those in Ganoderma lucidum fruiting bodies, their specific effects are unclear. Therefore, further research and development are necessary to inject new vitality into the Ganoderma lucidum industry.

[0003] There are many methods for extracting Ganoderma lucidum polysaccharides. Different methods are suitable for different raw material characteristics and application scenarios, and each has its own advantages and disadvantages. Currently, the main methods include hot water extraction, enzyme extraction, and ultrasonic extraction. Multiple methods can also be combined, such as ultrasonic-enzyme-assisted extraction, ultrasonic-microwave combination, water-enzyme combination, and ultrasonic-pulse extraction. In addition, deep eutectic solvent extraction, high-pressure extraction, extrusion processing, rapid solvent extraction, steam explosion and other technologies are also gradually being applied to the extraction of Ganoderma lucidum polysaccharides.

[0004] The traditional hot water extraction process is relatively mature and simple to operate, but it has problems such as unstable extraction efficiency and large losses. Li Bo et al. extracted Ganoderma lucidum fruiting body polysaccharides under the conditions of 90℃ hot water extraction for 90 minutes, a liquid-to-solid ratio of 20:1, and two extractions, with an extraction rate of 2.67% (Li Bo, Zhang Yaru, Zhou Yali, et al. Study on the extraction process of triterpenes and polysaccharides from Ganoderma lucidum [J]. Edible Fungi, 2022, 44(02): 54-57.). Li Xiaohan et al. used water extraction and alcohol precipitation to extract polysaccharides from the fruiting body of Ganoderma lucidum. The optimal extraction conditions were extraction temperature of 92.4℃, extraction time of 5h 5min, liquid-to-solid ratio of 21:1 (mL:g), and the yield was 2.967%. After separation and purification, GLP-B-1 was obtained. When its mass concentration was 0.7mg / mL, its scavenging rate for ABTS free radicals was the highest, which was 57.529%. When its mass concentration was 0.8mg / mL, its scavenging rate for hydroxyl free radicals was the highest, which was 5.116% (Li Xiaohan, Bian Wenjuan, Qiao Xueying, et al. Optimization of the extraction process of Ganoderma lucidum polysaccharides by response surface methodology and its antioxidant activity [J]. Chinese Journal of Pharmaceutical Sciences, 2025, 60(09): 949-955.). Enzyme extraction can get rid of the restrictions of cell membrane and cell wall, allowing the intracellular polysaccharides to be fully released, thereby increasing the polysaccharide yield. Luo Tongyan et al. optimized the enzymatic hydrolysis process of water-soluble sugars from the cell wall of Ganoderma lucidum spore powder and found that the optimal extraction process was 2% of lytic enzyme, 5h of enzymatic hydrolysis time, 10:1 (mL:g) liquid-to-solid ratio, 40℃, and pH 6.5. Under these conditions, the content of water-soluble sugars from the cell wall of Ganoderma lucidum spore powder reached 17.86% (Luo Tongyan, Tang Qingjiu, Liang Rui, et al. Optimization of the process and molecular weight distribution of water-soluble sugars from the cell wall of Ganoderma lucidum spore powder by enzymatic extraction [J]. Journal of Edible Fungi, 2024, 31(02):76-84.). Ding Xiaoxiao et al. used a composite enzyme method to extract polysaccharides from Ganoderma lucidum fruiting bodies. The results showed that the composite enzyme ratio was: 3.5% cellulase, 4.0% hemicellulase, and 3.0% papain; the optimal enzymatic hydrolysis extraction conditions were: pH value, temperature, and time of enzymatic hydrolysis treatment were 5.70, 50℃, and 81min, respectively. Under these conditions, the extraction rate of Ganoderma lucidum polysaccharides was 3.73%. (Ding Xiaoxiao, Li Fengwei, Shang Yueling, et al. Optimization of the complex enzyme extraction process of Ganoderma lucidum polysaccharides [J]. Food Research and Development, 2020, 41(05): 34-39+53.). Ultrasonic extraction is also widely used in Ganoderma lucidum polysaccharides. Compared with the hot extraction method, it has the characteristics of high extraction efficiency and short extraction time. Zhang Lingyu et al. used ultrasonic extraction of Ganoderma lucidum spore powder polysaccharides and optimized it through orthogonal experiments. The optimal extraction process parameters were liquid-to-solid ratio of 20:1 (mL:g), extraction temperature of 35℃, extraction time of 1.5h, and extraction power of 450W. Under these conditions, the extraction rate of Ganoderma lucidum spore powder polysaccharides was 4.99% (Zhang Lingyu, Zhong Bao. Optimization of ultrasonic-assisted extraction process of Ganoderma lucidum spore powder polysaccharides [J]. Agricultural Products Processing, 2020, (20): 40-42.).Sun Jiaze et al. optimized the extraction process of polysaccharides from Ganoderma lucidum stipes using ultrasound-enzyme-assisted extraction. They found that the optimal extraction conditions were 30 min of ultrasound time, 50 °C of ultrasound temperature, 1:25 (g:mL), and 500 U / g of enzyme addition. The polysaccharide yield was 1.57%. When the concentration reached 4.0 mg / mL, the scavenging rate of polysaccharides on hydroxyl radicals was 39.60% (Sun Jiaze, Sun Ao, Wu Tong, et al. Optimization of the extraction process of polysaccharides and triterpenes from Ganoderma lucidum stipes and their anti-tumor and antioxidant activities [J]. Food Research and Development, 2025, 46(07): 102-109.). Wang Qingbo used the extraction rate of Ganoderma lucidum water extract as an influencing factor and optimized the ultrasonic extraction process using the response surface methodology. The results showed that the optimal conditions were extraction temperature of 51.63°C, ultrasonic time of 4.11h, ultrasonic power of 286.32W, and solid-liquid ratio of 1:31.89g / mL. The maximum extraction rate under these conditions was 10.2796%. After separation and purification, the main antioxidant active substance CBG3 was obtained. When the sample mass concentration reached the experimental maximum concentration value of 12mg / mL, the hydroxyl radical scavenging rate was 85.72% (Wang Qingbo. Research on extraction and anti-inflammatory and antioxidant effects of microorganisms and polysaccharides in Ganoderma lucidum [D]. Changchun University of Technology, 2022). Li Caolong et al. proposed the optimal conditions for ultrasonic extraction of Ganoderma lucidum fruiting body polysaccharides based on response surface optimization: ultrasonic power 400W, ultrasonic time 40min, and liquid-to-solid ratio 15:1 (mL:g). Under these optimal process conditions, the polysaccharide extraction rate was 3.22%, which was higher than that of hot water extraction (Li Caolong, Wu Xirou, Wang Xiaoshuang, et al. Optimization of Ganoderma lucidum fruiting body polysaccharide extraction process and its improvement effect on asthma mice [J]. China Brewing, 2024, 43(11): 119-124.).

[0005] The entropy weight method is an objective weighting method based on the amount of information. It determines the weight of each evaluation index by measuring the degree of change in the value of each evaluation index. The greater the difference in the index, the more information it contains, the greater the entropy weight, and the more significant the impact on the evaluation result. This method is highly objective and avoids the bias of subjective weighting. It is based on the information entropy theory and has rigorous mathematical theory. At present, there are many literatures that use the response surface method (RSM) alone to optimize the extraction process, but there are few studies that explicitly mention the combination of the entropy weight method. In recent years, there has been a slow growth trend. The combined application of the entropy weight method and the response surface method in the extraction of active ingredients of natural medicines has gradually attracted attention. Luo Jinchen et al. used the response surface method combined with the entropy weight method to optimize the multi-component extraction process of Ganoderma lucidum spore powder. Under the optimal conditions, the extraction rates of total polysaccharides and total triterpenes were 4.27% and 1.10%, respectively (Luo Jinchen, Ma Xinyu, Hu Yang, et al. Optimization of the extraction process of Ganoderma lucidum spore powder [J]. Chemical Engineer, 2025, 39(06): 6-12.).

[0006] Although various extraction technologies are currently available, most of the response values ​​are based on a single extraction rate, ignoring its antioxidant capacity and the energy consumption generated by instrument use. Energy conservation and environmental protection are not considered, the promotion of sustainable energy utilization is neglected, and there is a lack of systematic comprehensive evaluation of multiple indicators. To address the above problems, the present invention, based on existing conventional technologies, uses extraction rate, antioxidant activity, and energy consumption as response values. Through single-factor preliminary screening, entropy weight method and response surface method are used for comprehensive evaluation to obtain the optimal extraction process parameters, achieving green development of extraction and energy consumption. Summary of the Invention

[0007] The present invention provides a process optimization method for ultrasonically extracting polysaccharides from Ganoderma lucidum buds, which specifically comprises the following steps:

[0008] 1. Pretreatment of Ganoderma lucidum bud materials

[0009] Select fresh Ganoderma lucidum buds that are undamaged, free of pests and diseases, and uniform in size, rinse the surface dirt with tap water, drain the water, dry them at 50°C to constant weight, and grind them into Ganoderma lucidum bud powder.

[0010] Soak the Ganoderma lucidum bud powder and ethanol at a ratio of 1:10 (g:mL) for 24 hours to remove fat and fat-soluble pigments. After degreasing, place the recovered Ganoderma lucidum bud powder in a cool place to air-dry for later use.

[0011] 2. Single-factor experiment

[0012] Taking dried Ganoderma lucidum bud powder as the extraction raw material, the effects of extraction power, extraction time, liquid-to-material ratio and extraction temperature on the polysaccharide extraction rate, antioxidant activity and energy consumption of Ganoderma lucidum bud aqueous extract were investigated. The entropy weight method was used to determine the weights and a comprehensive score was performed. The results showed that the optimal extraction power was 280W, the optimal extraction time was 30min, the optimal liquid-to-material ratio was 1:40 and the optimal extraction temperature was 50℃.

[0013] 3. Response surface analysis and determination of the optimal extraction process

[0014] Based on the results of single-factor experiments, with extraction power, extraction time, liquid-to-solid ratio and extraction temperature as independent variables, a four-factor three-level response surface experimental plan was designed using Design-Expert software and the experimental results were analyzed. The optimal extraction process conditions for Ganoderma lucidum bud polysaccharides by ultrasonic extraction were obtained: extraction power of 280W, liquid-to-solid ratio of 38:1 (mL:g), extraction time of 27.5min, and extraction temperature of 47℃; the optimal polysaccharide extraction rate was 2.22±0.02%, the ABTS clearance rate was 52.37±1.27%, the energy consumption was 0.292±0.001KW / h, and the comprehensive score was 43.77±1.02 points.

[0015] Compared with the prior art, the present invention has the following significant advantages:

[0016] 1. Break through the limitations of traditional single indicators and build a scientific and comprehensive extraction process optimization system

[0017] Traditional natural medicine extraction process optimization typically focuses solely on extraction yield, ignoring the activity of the active ingredients and the environmental friendliness of the extraction process. This can lead to biased optimization results in practical applications. This innovative multi-index comprehensive evaluation system incorporates extraction yield, antioxidant activity, and energy consumption into a unified analytical framework. Through normalization, this system comprehensively reflects the overall effectiveness of the extraction process, avoiding one-sided optimization strategies such as "high extraction yield but low activity" or "high cost but low benefit."

[0018] 2. Data-driven objective decision-making to improve process reliability

[0019] At present, there are many documents that use the single factor-response surface method (RSM) to optimize the extraction process, which relies on manually setting the weights of multiple indicators. However, there are few studies that explicitly mention the combination of entropy weight method. The present invention adopts the response surface method combined with the entropy weight method to optimize the extraction process. The entropy value is used to clarify the influence of each indicator on the process. The data is determined by the degree of variation of the data itself to avoid human interference. The extraction rate and antioxidant activity are used as positive indicators, and the scores of the two are added together; energy consumption is used as a negative indicator, and the score is taken as a negative value to obtain a comprehensive score, establish an RSM mathematical model, and predict the optimal process parameters.

[0020] 3. Green and efficient extraction, enabling sustainable development

[0021] The present invention optimizes the optimal process conditions: ultrasonic power of 280W, liquid-to-solid ratio of 38:1 (mL:g), time of 27.5min, and temperature of 47°C, to obtain a polysaccharide extraction rate of 2.22±0.02% in the Ganoderma lucidum bud water extract, which is superior to a hot reflux method (1.51±0.01%) and a microwave method (1.79±0.02%) under the same conditions. In addition, the present invention does not require an organic solvent, incorporates energy consumption into an evaluation system, complies with the principles of green chemistry, and achieves a win-win situation of environmental protection, green development, and active ingredient extraction. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Trend chart of the effect of ultrasound single factor on the comprehensive score of Ganoderma lucidum bud polysaccharide extraction

[0023] Figure 2 3D response surface diagram of Ganoderma lucidum bud polysaccharide extracted by ultrasonic method DETAILED DESCRIPTION

[0024] The present invention is described in detail below with reference to diagrams and specific implementation operations.

[0025] Example 1: Pretreatment of Ganoderma lucidum bud material

[0026] Select fresh Ganoderma lucidum buds that are undamaged, free of pests and diseases, and of uniform size. Rinse the surface dirt with tap water and drain the water. Place them flat in an oven and dry them at 50°C to constant weight. Transfer them to a Chinese herbal medicine grinder to make uniform powder.

[0027] Soak 50 g of Ganoderma lucidum bud powder in 500 mL of ethanol for 24 hours to remove fat and fat-soluble pigments. After defatting, spread the recovered Ganoderma lucidum bud powder on a tray and place it in a cool place to air-dry for later use.

[0028] Example 2: Evaluation indicators and methods

[0029] Polysaccharide extraction rate determination method: phenol-sulfuric acid method. Using anhydrous glucose standard as a reference, accurately weigh and prepare anhydrous glucose solutions of varying concentrations. Measure the absorbance at 490 nm using a UV spectrophotometer. Plot a standard curve with the concentration of the anhydrous glucose solution (mg / mL) as the abscissa and the absorbance as the ordinate. The standard curve equation is y = 7.1105x + 0.1141, r = 0.9995, and exhibits good linearity in the range of 0-0.1 mg / mL.

[0030] Accurately weigh 1g of Ganoderma lucidum bud powder, place it in a conical flask, and perform ultrasonic extraction under the corresponding extraction conditions. After the extraction is completed, filter and wash the filter residue with a small amount of ultrapure water twice. Combine the filtrate, dilute to 100mL, and shake well. Take 1mL of Ganoderma lucidum bud polysaccharide sample solution, dilute it 6 times, accurately draw the diluent, 5% phenol solution, and concentrated sulfuric acid in a ratio of 1:1:5 and add them to the test tube in sequence and shake well. Let it stand for 30 minutes, measure the absorbance at 490nm, and calculate the polysaccharide content by the regression equation of the glucose standard curve. Calculate the extraction rate according to formula (1):

[0031] Polysaccharide extraction rate (%) = N × C × V / m × 100% (1) Where:

[0032] N——dilution multiple;

[0033] C——measured concentration (mg / mL);

[0034] V——extraction volume (mL);

[0035] m——dry mass of raw material (mg).

[0036] ABTS free radical scavenging rate assay: Use the ABTS free radical scavenging assay. Mix 7.4 mM / L ABTS solution with 2.6 mM / L K₂S₂O₄ solution in a 1:1 ratio. Incubate the mixture in the dark for 12-16 hours to obtain an ABTS free radical stock solution, which should be stored in the dark. Dilute with purified water to an absorbance of 0.70 ± 0.02 (734 nm) before use.

[0037] Accurately weigh 1g of Ganoderma lucidum bud powder, place it in a conical flask, and perform ultrasonic extraction under the corresponding extraction conditions. After the extraction is completed, filter and wash the filter residue with a small amount of ultrapure water twice. Combine the filtrate, dilute to 100mL, shake well, take 1mL of Ganoderma lucidum bud polysaccharide sample solution, dilute 3 times, and obtain a sample solution. This solution is the ABTS determination solution of the present invention. Sample 0.5mL + ABTS working solution 4.5mL, shake well, react in the dark at room temperature for 10min, and measure the absorbance at a wavelength of 734nm. Calculate the clearance rate according to formula (2):

[0038]

[0039] Where:

[0040] A0——water + ABTS working fluid;

[0041] A1——sample+water;

[0042] A2——Sample + ABTS working solution.

[0043] Energy consumption was measured using a power monitor (Deli Power Monitor DL333501C). Select a power monitor suitable for the voltage, current, and power requirements of the ultrasonic equipment. With the equipment powered off, connect the monitor to the equipment's power supply, ensuring proper and secure connections between the circuit and the equipment. Following the instructions in the user manual, reset the monitor to zero, start the ultrasonic equipment (GT SONIC-P20 Ultrasonic Cleaner), and allow it to operate. The monitor will automatically record accumulated energy consumption.

[0044] Example 3: Single factor experiment

[0045] The following four factors were selected for investigation: extraction power, extraction time, liquid-to-solid ratio, and extraction temperature. Five levels were selected for each factor. Extraction rate, antioxidant activity, and energy consumption were used as investigation indicators. Each experiment changed one of the factors and fixed the other experimental conditions. A single-factor experiment was conducted, and the entropy weight method was used to determine the weights for comprehensive scoring.

[0046] (1) Effect of extraction power on Ganoderma lucidum bud polysaccharides

[0047] Accurately weigh 1.0g of Ganoderma lucidum bud powder, fix the extraction time to 30min, and the liquid-to-solid ratio to 40:1

[0048] Extraction was performed at 50°C using an ultrasonic instrument with extraction powers set at 200, 240, 280, 320, and 360 W. The residue and filtrate were separated by vacuum filtration. The filtrate was then diluted to 100 mL and diluted 6-fold to determine the extraction yield and 3-fold to determine the antioxidant activity. Energy consumption was measured using a power monitor. The entropy weight method was used to determine the weights for comprehensive scoring and to preliminarily screen for the optimal extraction power for polysaccharide extraction from Ganoderma lucidum bud aqueous extracts. The results showed that the optimal extraction power was 280 W.

[0049] Table 1 Results of single factor experiment on extraction power

[0050]

[0051]

[0052] (2) Effect of extraction time on Ganoderma lucidum bud polysaccharides

[0053] The extraction power was fixed at 280W, and the extraction time was set to 10, 20, 30, 40, and 50 min, respectively. The other experimental conditions were the same as those in Example 3 (1). The results showed that the optimal extraction time was 30 min.

[0054] Table 2 Results of single factor experiment on extraction time

[0055]

[0056] (3) Effect of liquid-to-solid ratio on Ganoderma lucidum bud polysaccharides

[0057] The extraction power was fixed at 280W, the extraction time was 30min, and the liquid-to-solid ratio was set at 20:1, 30:1, 40:1, and 50:1 (mL:g). The rest was the same as in Example 3 (1). The results showed that the optimal liquid-to-solid ratio was 40:1.

[0058] Table 3 Results of single factor experiment on liquid-to-solid ratio

[0059]

[0060] (4) Effect of extraction temperature on Ganoderma lucidum bud polysaccharides

[0061] The extraction power was fixed at 280W, and the extraction temperatures were set at 30, 40, 50, 60, and 70°C, respectively. The rest was the same as in Example 3 (1). The results showed that the optimal extraction temperature was 50°C.

[0062] Table 4 Results of single factor experiment on extraction temperature

[0063]

[0064] The changing trend of each influencing factor on the comprehensive score is as follows Figure 1 As shown in the figure, the optimal levels of each parameter in the single-factor experiment are: extraction power 280W, extraction time 30min, liquid-to-solid ratio 40:1, and extraction temperature 50℃.

[0065] The entropy weight method was used to construct a matrix using single-factor experimental data. The extraction rate and ABTS clearance rate were set as positive indicators, and energy consumption was set as a negative indicator. The weights of each indicator were obtained, and the comprehensive score was (polysaccharide extraction rate score × 41.0%) + (ABTS clearance rate × 28.1%) - (energy consumption score × 30.9%).

[0066] Example 4: Response surface analysis and determination of the optimal extraction process

[0067] Based on the optimal conditions obtained from the single-factor experiment screening, three adjacent level conditions containing the optimal process were selected. The comprehensive score was used as the response value. Using the Design-Expert software, a four-factor three-level Box-Behnken experimental design was established as shown in Table 5:

[0068] Table 5 Box-Behnken central combination experimental factors and levels

[0069]

[0070] Ultrasonic power (A), liquid-to-solid ratio (B), ultrasonic time (C), and ultrasonic temperature (D) were optimized for a total of 29 experiments, with the overall score serving as the response value. The response surface Box-Behnken central combination experimental factor and level design and the results are shown in Table 6.

[0071] Table 6 Response surface design and experimental results

[0072]

[0073] The results of the response surface optimization experiment are shown in Table 6. The data were processed using Design Expert 12.0 software. The regression model of the comprehensive score of Ganoderma lucidum bud polysaccharide was highly significant (P < 0.0001), and the lack of fit term was not significant (P > 0.05), indicating that the model was effective. The regression model formula was obtained as follows: comprehensive score Y = 44.76-3.02×A-0.5867×B-1.97×C-3.28×D+0.6125×AB+0.4575×AC-2.03×AD-0.0300×BC-1.11×BD+0.2150×CD-4.58×A2-1.64×B2-4.38×C2-4.78×D2(R 2=0.9228, P<0.05). In order to verify the feasibility of the quadratic multinomial regression equation for the extraction process conditions of Ganoderma lucidum bud polysaccharide, the regression model was subjected to variance analysis and significance test. The results are shown in Table 7. Among the four indicators examined, A, C, and D are highly significant items. Among the interaction indicators, AD is a significant item. Among the quadratic indicators of the independent variable, A 2 、C 2 、D 2 is a highly significant term, B 2 The order of the influence of various factors on the extraction of Ganoderma lucidum bud polysaccharide is: extraction power = extraction temperature > extraction time > liquid-to-solid ratio.

[0074] Table 7 Model ANOVA

[0075]

[0076] The response surface diagram can intuitively reflect the interaction between two factors and their impact on the comprehensive score, while the contour map can clearly show the intensity of the interaction. Figure 2 The influence of the interaction between various factors on the comprehensive score. Figure 2 It can be seen that the interaction response surface diagram of ultrasonic power (A) and extraction temperature (D) is the steepest, the surface change is the most significant, and its contour map is elliptical, indicating that the interaction between A and D has the greatest impact on the comprehensive score of Ganoderma lucidum bud polysaccharide. Combined with the contour map, it can be seen that the interaction between A and D has a significantly higher impact on the comprehensive score than the interaction between A and liquid-to-solid ratio (B), A and ultrasonic time (C), and B and D, B and C, and C and D. Among them, Figure 2 The contour plot of the interaction between B and C is closest to a circle, indicating that the interaction between B and C has the least impact on the comprehensive evaluation. According to the data in Table 7, the P value for the AD interaction is the lowest (0.0083), while the P value for the BC interaction is the highest (0.9645), indicating that the AD interaction has the most significant impact on the sensory evaluation, while the BC interaction has a relatively weaker impact. Therefore, the degree of influence of each interaction on the comprehensive score of Ganoderma lucidum bud polysaccharides is AD > BD > AB > AC > CD > BC.

[0077] Optimal condition verification experiment: Based on the above experimental data, the Box-Behnken experimental design was used in Design-Expert software. The optimal extraction conditions of Ganoderma lucidum bud polysaccharides predicted by the optimization analysis model were as follows: ultrasonic power 268.37W, liquid-to-solid ratio 38.61:1 (mL:g), ultrasonic time 27.53min, extraction temperature 47.29℃, and the theoretical value of the comprehensive score was 45.92. According to the actual experimental conditions, in order to facilitate the experimental operation and the influence of the equipment's own accuracy, three parallel experiments were carried out under the conditions of ultrasonic power 280W, liquid-to-solid ratio 38:1 (mL / g), ultrasonic time 27.5min, and extraction temperature 47℃. The polysaccharide extraction rate was 2.22±0.02%, the ABTS clearance rate was 52.37±1.27%, the energy consumption was 0.292±0.001KW / h, and the comprehensive score reached 43.77±1.02 points, which was close to the theoretical value. The response surface method optimization model fitted the actual situation well, and the model was reliable.

[0078] Example 5: Comparison of process parameters

[0079] Ultrasonic method: extraction was carried out according to the optimal conditions of Example 4: extraction power of 280 W, liquid-to-solid ratio of 38:1 (mL:g), extraction time of 27.5 min, and extraction temperature of 47°C;

[0080] Hot reflux method: liquid-to-solid ratio 40:1 (mL:g), reflux time 30 min, extraction temperature 50°C;

[0081] Microwave method: microwave power 280 W, liquid-to-solid ratio 40:1 (mL:g), extraction time 30 min, extraction temperature 50 °C;

[0082] Literature 1: Extraction power 400W, liquid-to-solid ratio 15:1 (mL:g), ultrasonic time 40min, extraction at room temperature (Li Caolong, Wu Xirou, Wang Xiaoshuang, et al. Optimization of Ganoderma lucidum fruiting body polysaccharide extraction process and its improvement effect on asthma mice [J]. China Brewing, 2024, 43(11): 119-124.);

[0083] Reference 2: Extraction power 286.32W, liquid-to-solid ratio 31.89:1 (mL:g), extraction time 4.11h, extraction temperature 51.63℃, (Wang Qingbo. Research on extraction and anti-inflammatory and antioxidant properties of microorganisms and polysaccharides from Ganoderma lucidum [D]. Changchun University of Technology, 2022.).

[0084] The polysaccharide extraction yield, ABTS antioxidant activity, and energy consumption of Ganoderma lucidum bud aqueous extracts were measured and scored. The results are shown in Table 8. The ultrasonic method received the highest score, demonstrating superior extraction yield and ABTS antioxidant activity compared to other extraction methods and references, while also significantly lower energy consumption than Reference 2. Although the reflux method had the lowest energy consumption, its extraction yield was not high, resulting in the lowest overall score, indicating that energy-saving extraction cannot meet the requirements for obtaining active ingredients. While the microwave method offered moderate extraction yields and energy consumption, its ABTS antioxidant activity was low, indicating that the ultrasonic method offers a better balance between efficient and energy-saving extraction and retention of active ingredients.

[0085] Reference 1 used high-power extraction, but the low liquid-to-solid ratio and temperature limited its overall score. Reference 2's prolonged extraction significantly increased energy consumption, resulting in a low extraction rate and the lowest ABTS free radical scavenging rate, affecting its overall score. This validates the synergistic effect of liquid-to-solid ratio and extraction time on extraction effectiveness. This patented method optimizes extraction power to 280W, liquid-to-solid ratio to 38:1 (mL:g), extraction time to 27.5min, and extraction temperature to 47°C, while ensuring low-energy and high-efficiency extraction. Through comprehensive evaluation across multiple indicators and aspects, it achieves a balance among polysaccharide extraction rate, antioxidant activity, and energy consumption. Compared to other extraction methods, this overall technical solution has significant advantages (P < 0.001).

[0086] Table 8 Comparison of comprehensive scores of different extraction methods under the same conditions

[0087]

[0088] Note: ABTS antioxidant activity (%) refers to the same volume of test solution extracted by different extraction methods but with the same dilution treatment. * indicates p < 0.05 (significant difference), ** indicates p < 0.01 (extremely significant difference), *** indicates

[0089] p<0.001 (extremely significant difference)

[0090] In summary, the present invention introduces ultrasonic-assisted extraction technology on the basis of traditional water extraction process, optimizes the ultrasonic extraction process of Ganoderma lucidum bud polysaccharides by multiple objectives, and innovatively adopts the entropy weight method to construct a comprehensive scoring model with polysaccharide extraction rate (41.0%), ABTS antioxidant activity (28.1%) and energy consumption (30.9%) as weight indicators to achieve a balance between efficient extraction and low energy consumption. On the basis of single factor experiments, the entropy weight method and response surface method (Box-Behnken design) are used to synergistically optimize the extraction power of 280W, liquid-to-solid ratio of 38:1 (mL:g), extraction time of 27.5min, and extraction temperature of 47°C. Experiments have shown that this process promotes the release of active ingredients through ultrasonic cavitation effect and mechanical action, with a comprehensive score of 64.97±1.01, which is better than the hot reflux method 53.13±0.74 and the microwave method 56.56±1.01.

Claims

1. A process for extracting water-soluble polysaccharides from Ganoderma lucidum buds using an ultrasonic method, characterized in that: Ganoderma lucidum buds were dried in an oven to constant weight, defatted and processed into Ganoderma lucidum bud powder. Ultrasonic extraction was performed and the energy consumption was recorded. Vacuum filtration was performed and the aqueous extract was collected for comprehensive evaluation.

2. According to claim 1, it is characterized in that Take fresh Ganoderma lucidum buds that are undamaged, free of pests and diseases, and uniform in size, wash away the dirt on the surface, drain the water, dry them at 50°C to constant weight, and grind them into Ganoderma lucidum bud powder.

3. As claimed in claim 1, it is characterized in that Soak the Ganoderma lucidum bud powder and ethanol at a ratio of 1:10 (g:mL) for 24 hours to remove fat and fat-soluble pigments. After degreasing, place the recovered Ganoderma lucidum bud powder in a cool place to air-dry for later use.

4. The method according to claim 1, wherein: Taking extraction rate, antioxidant activity and energy consumption as indicators, the entropy weight method was used to determine the weights and conduct a comprehensive evaluation. The single factor-response surface methodology was used to investigate the comprehensive effects of four factors, namely, extraction power, extraction temperature, extraction time and liquid-to-solid ratio, on the three indicators.

5. As claimed in claim 4, it is characterized in that The evaluation weights of Ganoderma lucidum bud polysaccharides were determined by the entropy weight method, with the extraction rate accounting for 41.0%, the antioxidant activity accounting for 28.1%, and the energy consumption accounting for 30.9%. The comprehensive score = (polysaccharide extraction rate / maximum polysaccharide extraction rate × 41.0%) + (antioxidant activity / maximum antioxidant activity × 28.1%) - (energy consumption / maximum energy consumption × 30.9%).

6. As claimed in claim 4, it is characterized in that The optimal extraction process conditions are: extraction power of 280W, liquid-to-solid ratio of 38:1 (mL:g), extraction time of 27.5min, and extraction temperature of 47℃; the optimal polysaccharide extraction rate is 2.22±0.02%, the ABTS clearance rate is 52.37±1.27%, the energy consumption is 0.292±0.0001KW / h, and the comprehensive score is 43.77±1.02 points.

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