Experimental Response Surface Research Articles

Synergistic Fermentation of Pichia kluyveri and Saccharomyces cerevisiae Integrated with Two-Step Sugar-Supplement for Preparing High-Alcohol Kiwifruit Wine

Wild yeast suitable for kiwifruit wine fermentation was isolated and purified, and the fermentation process was optimized to increase the alcohol content of the kiwifruit wine. Pichia kluyveri was isolated from kiwifruit pulp by lineation separating, screened by morphological characteristics in Wallerstein Laboratory Nutrient Agar (WL) medium and microscope observation, and further identified by 26S rDNA D1/D2 domain sequence analysis. Taking alcohol content and sensory evaluation as two indexes, the fermentation condition for kiwifruit wine was optimized by single factor and response surface experiment. The optimal fermentation conditions were optimized as follows: the fermentation temperature was at 24 °C, the initial pH was 3.8, the sugar dosage in second step was 8% (w/w), and the inoculating quantity of Pichia kluyveri and Saccharomyces cerevisiae was 0.15 g/L at equal proportion. Under these optimal conditions, the maximum estimated alcohol content was 15.6 vol%, and the kiwifruit wine was light green in color with strong kiwifruit aroma and mellow taste.

Design and Experimentation of Rice Seedling Throwing Apparatus Mounted on Unmanned Aerial Vehicle

In order to further exploit the production advantages of rice throwing, this paper proposes a systematically designed throwing device suitable for integration with unmanned aerial vehicles (UAVs). The device primarily comprises a seedling carrying and connection system, a seedling pushing mechanism, and a seedling guiding device. The operational principles and workflow of the device are initially elucidated. Subsequently, an analysis of factors influencing rice throwing effectiveness is conducted, with throwing height, working speed, and the bottom diameter of the seedling guide tube identified as key factors. Seedling spacing uniformity and seedling uprightness are designated as performance indicators. A three-factor, three-level response surface experiment is conducted, yielding regression models for the experimental indicators. Through an analysis of the response surface, the optimal parameter combination is determined to be a throwing height of 142.79 cm, a working speed of 55.38 r/min, and a bottom diameter of the seedling guide tube of 43.51 mm. At these parameters, the model predicts a seedling spacing uniformity of 88.43% and a seedling uprightness of 88.12%. Field experiments validate the accuracy of the optimized model results. Experimental data indicate that under the optimal operational parameters calculated by the regression model, the seedling spacing uniformity is 86.7%, and the seedling uprightness is 84.2%. The experimental results meet the design requirements, providing valuable insights for UAV rice-throwing operations.

Optimization of Fermentation Culture Medium for Sanghuangporus alpinus Using Response-Surface Methodology

The newly identified Sanghuangporus alpinus species of the Sanghuang mushroom genus has been found to possess significant medical benefits. However, the current artificial cultivation technology has not reached the requisite maturity. The response-surface methodology (RSM) was used to optimize the Sanghuangporus alpinus culture medium formulation and evaluate the functional activity of S. alpinus exopolysaccharides. First, a single-factor experiment was conducted to screen for optimal carbon and nitrogen sources for S. alpinus. Then, using Box–Behnken’s central composite design, a response-surface experiment was conducted to determine optimal culture parameters. Finally, the rationality of those parameters was assessed in a shaking flask experiment. The optimal culture parameters, determined through regression analysis, were 20.20 ± 0.17 g/L fructose (carbon source), 7.29 ± 0.10 g/L yeast extract (nitrogen source), and 0.99 ± 0.01 g/L dandelion. With optimization, the S. alpinus yield increased to 12.79 ± 1.41 g/L, twice that obtained from the initial culture medium. The S. alpinus exopolysaccharide exhibited an excellent antioxidant capacity, with the strongest scavenging effect noted on ABTS free radicals (lowest half-inhibitory concentration: 0.039 mg/mL). Additionally, this exopolysaccharide effectively inhibited various cancer cells, exhibiting the strongest activity against human glioma cells U251 (half-inhibitory concentration: 0.91 mg/mL). The RSM used to optimize the fermentation culture parameters of S. alpinus significantly increased the mycelial biomass. The improvement of Sanghuangporus alpinus yield through liquid fermentation and optimizing the fermentation medium could fill the existing gap in the cultivation of Sanghuangporus alpinus, as well as provide valuable data for the large-scale production of S. alpinus.

Utilizing experimental design and desirability function in optimizing RP-HPLC method for simultaneous determination of some skeletal muscle relaxants and analgesics

An experimental design and response surface methodologies using Plackett–Burman and Box-Behnken designs were applied for selecting and optimizing the most appropriate parameters which significantly affect the separation and quantitative estimation of five skeletal muscle relaxants and four analgesic drugs (baclofen, methocarbamol, dantrolene sodium, orphenadrine citrate, cyclobenzaprine hydrochloride, ketoprofen, etoricoxib, ibuprofen, and mefenamic acid) with a relatively short duration of analysis in a single run. For the separation of the nine drugs, an INERTSIL ODS-V3-5 µm C18 column (250 × 4.6 mm I.D.) was used with the optimum mobile phase conditions (45.15 mM ammonium acetate buffer pH 5.56 adjusted with acetic acid, acetonitrile, and methanol in a ratio of 30.5:29.5:40, v/v/v with a flow rate of 1.5 mL/min) and UV-detection at 220 nm. The optimized method was successfully subjected to the validation steps as described in ICH guidelines for linearity, precision, accuracy, robustness, and sensitivity. The optimized and validated method was effectively applied to determine the content of the studied drugs in their pharmaceutical preparations and to expand its applicability to the counterfeit estimation of etoricoxib in different brands of tablet dosage forms.

Using Response Surface Methodology Approach to Modeling and Optimization of the Combustion Process in Cement Kiln

AbstractIn this study, this process has been modeled and optimized using the design of experiment technique and response surface methodology (RSM). The RSM was employed to optimize the combustion process in the cement kiln. Kiln feed (X1 = 160–190 t h−1), fan speed (X2 = 840–870 rpm), kiln rotational speed (X3 = 2.8–3.4 rpm), kiln fuel rate (X4 = 3800–4150 L h−1), calciner fuel rate (X5 = 5700–6200 L h−1), pressure of kiln inlet (X6 = 98–102 Pa), and limestone saturation factor (LSF) (X7 = 94–95.8 %) as independent variables were investigated. The carbon monoxide and the kiln body temperature in RSM were considered as response variables. The absolute average error in the model predictions for the combustion process in the cement kiln, for the kiln body's temperature, is about 1.6 % and for carbon monoxide is about 15.5 %.

Numerical analysis and process optimization of electron beam melting for superalloys

This study aims to determine the optimal parameters for the electron beam melting process by employing a modified transient three-dimensional thermal model. The validity of the thermal model is confirmed through experimental results. Moreover, the proposed method establishes mathematical relationships between the resulting molten pool temperature and process parameters. A broader range of process parameters for a new Ni–Co-based superalloy was achieved by investigating the effects of scanning velocities (10–100 mm/s), scanning radius (20–60 mm), and melting mass (1500–3100 g) on temperature at various electron beam power levels (10–30 kW). Regression models were established between scanning velocities-temperature, and scanning radius-temperature under different electron beam power levels. These models laid the foundation for real-time automatic control of molten pool temperature. Significantly, orthogonal experimental design and response surface method are employed to conduct a multi-objective optimization based on sixteen experiments and three parameters. The results of multi-objective optimization indicate that with a smelting mass of 1.5 kg, an electron beam power of 11 kW, a scanning radius of 60 mm, and a scanning velocity of 75 mm/s, the energy consumption is 34.95 kW h, and the theoretical mass loss of Ni element is 0.002 kg. The desirability is 91.2 %.

Virtual parameter calibration of pod pepper seeds based on discrete element simulation

In order to achieve numerical optimization of the pod pepper seed sowing device, the contact parameters of pod pepper seeds were calibrated, with the angle of repose used as the response value. A set of discrete element method (DEM) models of pod pepper seeds was developed to simulate the formation of seed repose angles using reverse engineering reconstruction techniques. An eight-factor, three-level response surface experiment based on the Box-Behnken central combination test method was performed to study the effects of various factors on the angle of repose of seeds. The angle of repose obtained from physical experiments with a value of 27.56° was taken as the target value. The optimal combination of parameters is obtained as follows: seed Poisson's ratio of 0.22, seed shear modulus of 15.47 MPa, seed-to-seed static friction coefficient of 0.25, seed-to-seed rolling friction coefficient of 0.67, seed-to-seed collision recovery coefficient of 0.64, seed-to-steel-plate static friction coefficient of 0.55, seed-to-steel-plate rolling friction coefficient of 0.45, and seed-to-steel plate collision recovery coefficient of 0.34. A two-sample t-test of the angle of repose obtained by the cylinder lifting method and the pumping plate method against the target value yielded P > 0.05, indicating the reliability of the simulation experiments.

Experimental Study and Response Surface Methodology Optimization of Electro-Fenton Process Reactive Orange 16 Dye Treatment

Experimental Study and Response Surface Methodology Optimization of Electro-Fenton Process Reactive Orange 16 Dye Treatment

Magnetic biochar catalyst from reed straw and electric furnace dust for biodiesel production and life cycle assessment

A novel magnetic carbon-based catalyst was prepared from solid waste (reed straw and electric furnace dust) by coprecipitation and high temperature pyrolysis with excellent application potential. Single factor and response surface experiments were used to optimize the catalyst preparation process (mass ratio of reed straw/electric furnace dust of 5:1 at 500 °C in 4 h) and biodiesel production, respectively, with results of 99.89 wt% biodiesel yield in the first cycle (93.61 wt% after 11 cycles) obtained at 74.15 °C in 4.16 h with a 14.06:1 M ratio of methanol/oil and 7.75 wt% catalyst. The life cycle assessment of magnetic carbon-based catalysts for biodiesel production suggested that the soybean oil extraction stage had the greatest impact on the environment, mainly from the use of soybeans and electricity. Solid waste as raw material to prepare catalysts for biomass energy conversion was proven to be a good strategy for energy conservation and emission reduction. This study provides guidance sustainable biomass energy conversion with low cost, low energy consumption, and minimal negative environmental impact.

Production of Mannooligosaccharides from Açaí Seed by Immobilized β-Mannanase

In this work, an enzyme cocktail with β-mannanase as the main activity was immobilized on epoxy resin foams filled with fibers from annatto capsules. The catalytic system was characterized by SEM, FTIR, and a mechanical crush resistance test. The behavior of the pH and temperature for the hydrolysis of the locust bean gum were also studied. With the same substrate and with respect to the free enzyme, the immobilized enzyme showed an activity retention of 79.61%. Its operational stability in ten reuse cycles did not show any statistically significant loss of activity. This catalytic system was used to study the preferential release of MOS of two to five degrees of polymerization from mannan present in dried and ground açaí seeds, which were not subjected to any other pretreatment. Using an experimental response surface design, the predicted quadratic models for the M2–M5 MOS content were obtained and they fit well with the experimental data, predicting a production range between 0.435 and 20 g/L of MOS (M2–M5). In addition, the production reached about 12 g/L under the optimized conditions. These results indicate that the used foamed epoxy resin supports and immobilization methodology are suitable for catalyzing the hydrolysis of mannan from açaí seeds.

Optimization of Ultrasound‐Assisted Deep Eutectic Solvent Extraction and Bioactivities of Polysaccharide from Porphyra haitanensis

AbstractIn this paper, ultrasound‐assisted method and deep eutectic solvent (DES) are used to extract polysaccharides from Porphyra haitanensis. The single‐factor experiment is carried out first, and then the response surface experiment is designed to obtain the optimal process conditions. The optimal extraction conditions are the molar ratio of choline chloride to oxalate of 1:1, the water content of DES of 60%, the solid–liquid ratio of 1:60 g mL−1, the time of 120 min, the power of 200 W, and the temperature of 80 °C, with the extraction yield of 4.42%. And the yield of P. haitanensis polysaccharides (PHPs) is 3.14% and 3.31% by water immersion and ultrasonic water extraction, respectively, indicating that the DES extraction method is superior to these two methods, and the optimization effect is good. The extracted polysaccharides are tested for biological activity after separation and purification on DEAE and Sephadex G‐75 columns. The results of α‐glucosidase inhibitory activity and α‐amylase inhibitory activity show that PHPs have certain antioxidant activity. 1,1‐Diphenyl‐3‐nitrophenylhydrazine (DPPH) and 2,2‐diazo‐di‐3‐ethylbenzothiazolin‐6‐sulfonic acid (ABTS) free radical scavenging assays show that PHPs have certain antiglycemic activity.

Enhancing H2O2 production with a self-breathing gas diffusion electrode fabricated via ultrasonic impregnation and vacuum filtration

In this study, an electro-Fenton gas diffusion electrode featuring air self-respiration was fabricated by incorporating carbon nanotubes, carbon nitride, and polytetrafluoroethylene onto graphite felt. The fabrication process involved a synergistic approach of ultrasonic impregnation and vacuum filtration, establishing a gas–liquid-solid tri-phase interface both on the surface and within the electrode. This preparation method facilitates autonomous air intake, diffusing it to the tri-phase reaction interface, thereby eliminating the necessity for aeration and reducing operational costs. The optimal electrode preparation conditions were determined through Box-Behnken Design response surface experiments, leading to the enhancement of H2O2 production conditions. Under the optimized conditions, H2O2 accumulation reached 45.83 mg L−1 cm−2h−1, surpassing the performance achieved with conventional ultrasonic impregnation and vacuum filtration methods. Furthermore, the performance of self-breathing and the impact factors are explored. Finally, the electrode’s efficacy was demonstrated through the degradation of phenol and bisphenol A with concentrations of 100 mg L−1 exhibiting degradation rates of 92 % and 95 %, respectively, within 60 min.

Optimization of preparation conditions for Salsola laricifolia protoplasts using response surface methodology and artificial neural network modeling

BackgroundSalsola laricifolia is a typical C3–C4 typical desert plant, belonging to the family Amaranthaceae. An efficient single-cell system is crucial to study the gene function of this plant. In this study, we optimized the experimental conditions by using Box-Behnken experimental design and Response Surface Methodology (RSM)-Artificial Neural Network (ANN) model based on the previous studies.ResultsAmong the 17 experiment groups designed by Box-Behnken experimental design, the maximum yield (1.566 × 106/100 mg) and the maximum number of viable cells (1.367 × 106/100 mg) were obtained in group 12, and the maximum viability (90.81%) was obtained in group 5. Based on these results, both the RSM and ANN models were employed for evaluating the impact of experimental factors. By RSM model, cellulase R-10 content was the most influential factor on protoplast yield, followed by macerozyme R-10 content and mannitol concentration. For protoplast viability, the macerozyme R-10 content had the highest influence, followed by cellulase R-10 content and mannitol concentration. The RSM model performed better than the ANN model in predicting yield and viability. However, the ANN model showed significant improvement in predicting the number of viable cells. After comprehensive evaluation of the protoplast yield, the viability and number of viable cells, the optimal results was predicted by ANN yield model and tested. The amount of protoplast yield was 1.550 × 106/100 mg, with viability of 90.65% and the number of viable cells of 1.405 × 106/100 mg. The corresponding conditions were 1.98% cellulase R-10, 1.00% macerozyme R-10, and 0.50 mol L−1 mannitol. Using the obtained protoplasts, the reference genes (18SrRNA, β-actin and EF1-α) were screened for expression, and transformed with PEG-mediated pBI121-SaNADP-ME2-GFP plasmid vector. There was no significant difference in the expression of β-actin and EF1-α before and after treatment, suggesting that they can be used as internal reference genes in protoplast experiments. And SaNADP-ME2 localized in chloroplasts.ConclusionThe current study validated and evaluated the effectiveness and results of RSM and ANN in optimizing the conditions for protoplast preparation using S. laricifolia as materials. These two methods can be used independently of experimental materials, making them suitable for isolating protoplasts from other plant materials. The selection of the number of viable cells as an evaluation index for protoplast experiments is based on its ability to consider both protoplast yield and viability. The findings of this study provide an efficient single-cell system for future genetic experiments in S. laricifolia and can serve as a reference method for preparing protoplasts from other materials.

花生籽粒物理参数测定与离散元参数标定

This study measured the intrinsic and contact parameters through physical experiments to improve the accuracy of discrete element simulation analysis of peanut seeds. Discrete element models for five different peanut seed filling ball numbers were established. The simulation parameters were calibrated through a combination of physical and simulation experiments. Firstly, the Plackett-Burman test was used to screen the significance of simulation parameters. Then, the steepest climbing test was conducted to determine the optimal range of significance parameters using the relative error be-tween the simulated and the physical experimental as the evaluation index. Finally, a response surface experiment with three factors and three levels was conducted using the repose angle as the response value. The static and rolling friction coefficients among peanut seeds were set as 0.43 and 0.50 separately, and the rolling coefficient between peanut seeds and steel plate was set as 0.12. During verification experiments, the simulated repose angle was 25.18°, with a relative error of 2.42% compared to the physical repose angle, further verifying the reliability of the simulation model. The re-search group used different numbers of filling balls with optimal parameters in the repose angle experiment. Then they evaluate the simulation time and the error value of repose angle between the simulated and physical experiment. The optimal number of filled balls is the Sphere 1178. The research results indicate that discrete element model of peanut seeds and calibration parameters are reliable. Based on the results of this research, an intelligent peanut precision sowing machine can be developed.

Optimizing the quality of bran-fortified stewing noodles using extruded wheat bran and improvers

Aimed to improve the tensile properties, color, and sensory quality of bran-fortified stewing noodles using bran extrusion and improvers. A single-factor experiment and Box–Behnken design response surface experiment were designed, and the multi-index comprehensive weighted scoring method was applied. The single-factor experiment indicated high-quality bran-fortified stewing noodles when wheat gluten, glucose oxidase, and guar gum contents were 10%, 40 mg/kg, and 0.3%, respectively. The response surface experiment showed that the comprehensive weighted score in the response surface optimal group was maximum (86.33 points) when wheat gluten, glucose oxidase, and guar gum contents were 5%, 40 mg/kg, and 0.2%, respectively. The verified model experiment showed that the average comprehensive weighted score was 89.31±0.49 points, indicating that the model was feasible to improve the quality of bran-fortified stewing noodles. The optimal formula involved wheat gluten, glucose oxidase, and guar gum contents of 6.67%, 28 mg/kg, and 0.22%, respectively. Bran extrusion and improvers can improve the quality of bran-fortified stewing noodles. The findings may serve as a reference for the quality improvement and industrial production of fiber-rich stewing noodles.

Influence of coal cowl parameters on the coal loading process of thin coal seam shearer drum

The low loading rate of the thin coal seam shearer drum is a severe obstacle to the efficient mining of thin seam resources, and the auxiliary drum loading through the cowl is an effective measure to alleviate this situation. However, the working mechanism of the coal cowl still remains unclear. In this paper, with the help of the discrete element method and the modeling experiment method, the effects of coal cowl’s offset distance, tilt angle and wrap angle on the coal loading rate under different loading modes of the drum are investigated; and the significance of various factors and their interactions to the drum coal loading rate is explored by designing response surface experiments. The findings show that a monotonous negative correlation between the offset distance of the coal cowl and the coal loading rate is identified, and that a smaller offset distance can effectively improve the coal loading rate of the drum. The conveying torque is significantly increased, easily inducing the drum choking, coal recycling coal over-crushing. Along with the increasing tilt angle, the rate of ejection loading decreases monotonically, and the rate of pushing loading increases first and then decreases. Coal loading rate is weakly affected by changes in coal cowl’s wrap angle. The results of response surface analysis reveal that the most significant factors affecting the drum’s coal loading rate are tilt angle and offset distance in ejection and pushing loading modes, respectively. The conclusions drawn here offer implications for improving the coal loading performance of the thin coal seam shearer drum, as well as certain guidance on the optimal design of coal cowl parameters.

Performance and emission characteristics of a diesel engine fuelled with Mesua ferrea biodiesel with chromium oxide (Cr2O3) nanoparticles: Experimental approach and response surface methodology

This study investigates the effects of using nano-fuelled diesel engines, including Mesua ferrea biodiesel and chromium oxide (Cr2O3) nanoparticles. The RSM model was created to make accurate predictions of the operating parameters. The amount of Cr2O3 nanoparticles was set at 60, 80, and 100 mg/L and an equal amount of surfactant and dispersant was added to the nanoparticles. The test was carried out under different compression ratios and load conditions. The parameters evaluated were the engine loads (3, 6, 9, and 12 kgf), fuel samples, and compression ratios (16.5:1, 17.5:1, and 18.5:1) as inputs and the parameters BTE, BSFC, CO, UHC, NOx, and smoke opacity as outputs. The BD20 + Cr2O3 80 mg/L + DSP 80 mg/L fuel sample resulted in a significant improvement in BTE by 16.58 % and a reduction in BSFC by 0.58 % compared to the other samples, while CO concentration decreased by 31.85 %, UHC concentration by 22.23 %, NOx concentration by 6.16 % and smoke opacity by 62.61 % at CR 18.5:1 and full load. The correlation coefficient (R2), which was determined using response surface methods and artificial neural networks, was between 0.96 and 0.98 for all output parameters. The observed range of values demonstrates a strong correlation between the experimental data and the predicted results.

The Effects of Processing Parameters during the Wire Arc Additive Manufacturing of 308L Stainless Steel on the Formation of a Thin-Walled Structure.

Wire arc additive manufacturing (WAAM) excels in producing medium to large components with a high deposition rate. Process optimization is crucial for uniform, defect-free components. This research employs orthogonal experimental design and response surface methodology (RSM) to control TIG WAAM-ed 308L stainless steel components. Varied parameters, including tungsten electrode angle, welding current, and speed, target weld bead attributes. Analysis of variance (ANOVA) evaluates multi-processing parameter influence on weld bead formation. Comparison with experimental results confirms accurate modeling of the relationship between parameters and bead attributes. The study optimizes process parameters and swing to enhance dimensional accuracy in single-layer and multi-layer components, improving precision, quality, and accuracy in thin-walled structures.

Analysis and optimization of effect factors of refrigeration performance of portable micro-refrigerator

The objective of this study was to ascertain an efficient portable cold container, considering the constrained space, low airflow velocity, and uneven heat distribution of a micro-refrigerator, which lead to in insufficient heat dissipation and low refrigeration efficiency. A thermoelectric micro-refrigerator model with a volume of 9083.8 cm3 was established under forced convection heat transfer conditions. Through single-factor and response surface experiments, the impact of thermoelectric elements configuration on refrigeration efficiency was identified. These elements include the input current of the Thermo Electric Cooler (TEC), the derating factor (actual power) of the cooling fan, and the height of the heat sink fin.The results indicate that optimizing the refrigeration conditions significantly enhances the refrigeration efficiency of the micro-refrigerator. At an ambient temperature of 25℃, with a fan derating factor of 0.77 (corresponding to an actual power of 0.8218 W), a heat sink fin height of 59.15 mm, and a TEC input current of 5.09A, under stable conditions,the cold side temperature can reach −11.3 ℃. The hot side temperature is 38.2 ℃, consistent with the response surface experiment result. Numerical simulation under these conditions reveals a cold side temperature of −12.4 ℃ and a hot side temperature of 41.9 ℃, showing a 9.7 % difference from the experimental results. The simulation data further demonstrate that the vertical arrangement of the cooling fan and heat sink can achieve a cold side temperature of −12.9 ℃, effectively maintaining the hot side temperature below 40 ℃. The thermoelectric system investigated in this paper holds promising potential for applications in portable fresh-keeping and refrigeration.

Biphasic Fermentation of Trapa bispinosa Shells by Ganoderma sinense and Characterization of Its Polysaccharides and Alcoholic Extract and Analysis of Their Bioactivity.

Trapa bispinosa shells (TBs) and its flesh (TBf) have been recognized for their medicinal properties, including antioxidant, antitumor, and immunomodulatory effects. Despite these benefits, TBs are often discarded as waste material, and their applications remain to be further explored. In this study, we optimized the solid-state fermentation process of Ganoderma sinense (GS) with TBs using a response surface experiment methodology to obtain the fermented production with the highest water extract rate and DPPH free radical scavenging activity. We prepared and characterized pre-fermentation purified polysaccharides (P1) and post-fermentation purified polysaccharides (P2). Alcoholic extracts before (AE1) and after (AE2) fermentation were analyzed for active components such as polyphenols and flavonoids using UPLC-QTOF-MS/MS (ultra-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry). Mouse macrophages (RAW 264.7) were employed to compare the immune-stimulating ability of polysaccharides and the antioxidant activity of AE1 and AE2. Optimal fermentation conditions comprised a duration of 2 days, a temperature of 14 °C, and a humidity of 77%. The peak water extract yield and DPPH free radical scavenging rate of the water extract from TBs fermented by GS were observed under these conditions. The enhanced activity may be attributed to changes in the polysaccharide structure and the components of the alcoholic extract. The P2 treatment group indicated more secretion of RAW 264.7 cells of NO, iNOS, IL-2, IL-10, and TNF-α than P1, which shows that the polysaccharides demonstrated increased immune-stimulating ability, with their effect linked to the NF-кB pathway. Moreover, the results of the AE2 treatment group indicated that secretion of RAW 264.7 cells of T-AOC and T-SOD increased and MDA decreased, which shows that the alcoholic extract demonstrated enhanced antioxidant activity, with its effect linked to the Nrf2/Keap1-ARE pathway. Biphasic fermentation of Trapa bispinosa shells by Ganoderma sinense could change the composition and structure of the polysaccharides and the composition of the alcoholic extract, which could increase the products' immunomodulatory and antioxidant activity.