{"paper_id":"11488906-c01d-4f4b-bc4c-80be69aec96b","body_text":"Sequential Enzymatic and Ultrasonic Extraction of Lentinula edodes Mushroom Proteins Leading to Enhanced Yield and Significant Immunoactivity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Sequential Enzymatic and Ultrasonic Extraction of Lentinula edodes Mushroom Proteins Leading to Enhanced Yield and Significant Immunoactivity Zi Chen ZHAO, Yan Yu ZHU, Fang Ting GU, Lin Xi HUANG, Xuwei LIU, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6257446/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 12 You are reading this latest preprint version Abstract Lentinula edodes is a widely consumed edible fungus and a rich source of proteins with both nutritional and medicinal value. This study discovered an effective process to extract proteins from L. edodes mushroom by comparison of water extraction (WE), enzyme-assisted extraction (EAE), ultrasound-assisted extraction (UAE), and various combinations of EAE with UAE. The two-step and sequential scheme by EAE and then UAE, designated EUE resulted in the highest protein yield compared with EAE after UAE (UEE) and simultaneous EAE and UAE (SEUE). The soluble protein yield by EUE (9.4%) was nearly three times that by UEE (3.6%) and 1.4 times and around two times higher than by UAE (6.9%) and EAE (4.9%), respectively. Compared with those by other extraction methods, the protein fraction by EUE had the highest protein content (56.0%) and β-sheet content (55.8%) and exhibited the strongest in vitro immunostimulatory activity. Through statistically designed experiments and response surface methodology, EUE conditions were optimized as enzyme 0.28% (w/v), ultrasound amplitude 62%, and (NH 4 ) 2 SO 4 saturation 69%, achieving 9.7% protein yield and 58.4% protein content. The distribution of protein molecular weight (MW) was below 10 kDa and between 25-75 kDa. The protein fraction contained nutritional amino acids and significant immunostimulatory activities in vitro . EUE has shown promising potential for efficient extraction of proteins from mushrooms in the food industry. Lentinula edodes Enzyme- and ultrasound-assisted extraction Proteins Amino acid profile Nutrition value Immunoactivity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Lentinula edodes , is one of the more popular edible mushrooms and is especially favored in the Asia region for its appealing aroma, plus notable nutritional, and medicinal properties. Among the most abundant and also beneficial components are polysaccharides (PS) and proteins (Wasser, 2004 ; Du et al., 2024 ). The PS derived from L. edodes have been extensively reported in the literature on their notable antitumor, and immunomodulatory effects and many other bioactivities (Wang et al., 2022 ). Proteins represent another important component of L. edodes are gaining attention for their medicinal potential (Wong et al., 2010 ; Das & Prakash, 2021 ). It exhibits abundant essential amino acids (EAA) (Yu et al., 2023 ). which is beneficial for human health. Moreover, protein also exhibit fascinating biological functionalities, including antitumor, antioxidant, and antimicrobial activities (Ngai & Ng, 2003 ; Gao et al., 2023 ). Our recent research have shown that the protein-rich fractions from L. edodes has stronger immunostimulatory activity compared to the PS-rich extracts (Zhao et al., 2024 ) and also showed high nutritional value of AA (Zhao et al., 2024 ). Extraction is a crucial step for acquiring the desired proteins from mushrooms and other food and medicinal materials. Extraction with water is the most common method for extracting proteins from mushrooms (Jing et al., 2021 ) or with aqueous solutions containing salt (NaCl) or surfactants (SDS, Triton100) for higher protein solubility (Chatterjee et al., 2012 ). Alkaline or acid extraction may be more effective for the proteins with high solubility in the respective pH (Du et al., 2018 ; Gerliani et al., 2019 ). After the extraction process, an organic solvent, typically ethanol or methanol, is usually used to precipitate and isolate the proteins from prolamin, urea, phenol, and other denaturing agents (Chatterjee et al., 2012 ; Capellini et al., 2017 ; Chen et al., 2019 ; Bose et al., 2019 ). However, the aforementioned extraction and separation methods are rather tedious and time-consuming, and not environmentally friendly. EAE and UAE are two of the most widely used methods for improving the water extraction of natural substances from diverse origins (Cannavacciuolo et al., 2024 ; Goktayoglu et al., 2023 ). It can be operated at a lower temperature than hot-water extraction. EAE and UAE are particularly favorable for heat-sensitive components such as proteins. EAE and UAE have also been considered efficient in enhancing protein extraction from various mushrooms (Ahmed et al. 2024 ). It is well established that high-intensity ultrasound enhances water extraction mainly through acoustic cavitation in water which produces strong shear forces, causing cell wall disruption and promoting the release of cellular components (Wu, 2019 ). A recent study has shown that UAE with intensive ultrasound enhances protein extraction from L. edodes mushroom to some extent (Wang et al., 2023 ). However, the strong shear forces generated by high-intensive ultrasound may also induce adverse effects on the integrity and quality of protein extracts (Higuera-Barraza et al., 2016 ). In contrast, EAE utilizes specific enzymes like cellulases to selectively degrade cellulose and other glycan components of the cell walls, keeping the proteins intact (Culter, 2008 ). The complex structure of the fungal cell wall mainly contains proteins, glucan, chitin, as well as cellulose (Rivillas-Acevedo and Soriano-García 2006 ), which compounds within cells can be further enhanced by enzymes (Zhao et al., 2016 ). The enzymatic hydrolysis can make the cellular structure more permeable and easier to disrupt, so that some proteins can be released. Zhang et al. ( 2015 ) assessed the use of cellulase for protein extraction from Pleurotus eryngii mushroom and increased the protein yield under specific extraction conditions. Additionally, another study found that extraction sequentially by UAE and EAE with cellulase can also enhance the protein yield from Pleurotus eryngii (Xu et al., 2020 ). However, while these studies have demonstrated the potential of hybrid approaches, the treatment sequence (enzymes before or ultrasound after) remains underexplored. Critically, no reported study has been found in the literature on EAE or its combination with UAE of proteins from L. edodes , excepted for a few reported studies employing either ultrasound or enzyme methods individually (Wang et al., 2023 ; Prandi et al., 2023 ). Based on the above background, we hypothesize that the proper combination of EAE and UAE may ultimately enhance the protein extraction through a biochemical mechanism (by enzyme) and a physical or mechanical mechanism (by ultrasound). This study aimed to identify and optimize the most efficient protein extraction scheme and conditions based on a combination of EAE and UAE. Initially, several extraction methods were compared based on the protein yield and content, including WE, UAE, and EAE, and the synergy that depends on the order of sequential combinations of EAE and UAE under various conditions was thoroughly evaluated. The two-step sequential extraction by EAE and UAE (EUE) was identified as the most efficient method and was further optimized by statistical experimental design as well as response surface methodology (RSM). Furthermore, the characteristics and immunostimulatory functions of the protein obtained following RSM analysis were evaluated. 2. Materials and methods 2.1 Chemical and biochemical reagents The ammonium sulfate ((NH 4 ) 2 SO 4 ) used was sourced from AnalaR Normapur, Vienna, Austria. Cellulase, bovine serum albumin (BSA), penicillin, lipopolysaccharide (LPS), streptomycin, sulphanilamide, phosphoric acid, as well as anthrone were sourced from Sigma-Aldrich in St. Louis, MO, USA. Potassium persulphate as well as sodium nitrite were obtained from BDH in Poole, England. Fetal bovine serum and Dulbecco's modified Eagle medium were acquired from Thermo Fisher Scientific in Waltham, MA, in the United States. Sodium chloride (NaCl) was purchased from Macklin (Shanghai, China). All remaining chemicals were sourced from reliable vendors in analytical reagent (AR) grade or higher. 2.2 Extraction of proteins with different methods The dried mushroom bodies were pulverized into a powder using an electric mill. Then, it was sifted via mesh sieves (850 µm) to varying mean particle sizes. Mushroom powder was then defatted using 95% ethanol and dried at approximately 50°C until the weight remained constant. To identify the best effective extraction method, several methods were compared initially including WE, UAE, EAE, and various combinations of EAE and UAE as described in detail below. 2.2.1 Water extraction (WE) A preliminary test was performed with the conventional WE extraction method using alkaline and acid precipitation, resulting in a very low protein content (3.5 ± 0.7%). As a result, the optimized WE method incorporating (NH 4 ) 2 SO 4 and isoelectric precipitation was chosen. The WE procedure and conditions were selected based on the literature (Yang et al., 2023 ). The dried defatted mushroom powder (3 g) was dispersed into an aqueous solution of 1% (w/v) NaCl at 1:30 (w/v) and macerated for 30 minutes (in plastic centrifuge tubes). To lower the pH of the mixture to 10, 1 M NaOH was put into the samples. Then, the mixture was agitated for three hours at 50°C and later was centrifuged for thirty minutes at 4000 rpm at 4°C. The supernatant was collected for protein precipitation as follows. Firstly, 1M HCl was added to the liquid to adjust the pH to 4, and then (NH 4 ) 2 SO 4 was added to 70% saturation, was left to stand overnight (4°C). The liquid was centrifuged at 9000 rpm for twenty minutes and the resulting pellet was gathered and dissolved in water. The liquid was centrifuged again (4000 rpm, 30 minutes) to attain a solid-free solution of crude protein. To remove salts, the protein solution was dialyzed in distilled water for 48 h by a 3.5 kDa molecular weight (MW) cutoff membrane. The crude protein fraction was then obtained by freeze-drying. The protein yield (%) was represented by the mass percentage of freeze-dried soluble protein in the original mushroom powder. Lowry method was applied to the calculation of the total protein yield, and the Anthrone test, as outlined in earlier studies, was applied to measure the total sugar (or carbohydrate) amount. 2.2.2 Ultrasound-assisted extraction (UAE) The dried defatted mushroom powder (3 g) was pre-treated using the same method as outlined in the WE method, followed by UAE. UAE was conducted using an ultrasonic processor (20 kHz), the Sonics & Materials Inc. Model VCX-130 from Newton, USA, providing a maximum output power of 130 W, following established procedures with some adjustments. In brief, a 12 mm-diameter ultrasonic horn was submerged into the liquid samples. Ice was employed during the UAE process to prevent overheating. The US amplitude was maintained at 60% (0.87 W/mL), and the ultrasonic process was conducted for 40 min. After UAE, the solid-liquid mixture was spun at 4000 rpm for thirty minutes. The collected liquid underwent protein fraction isolation and partial purification, following the WE method as detailed earlier. 2.2.3 Enzyme-assisted extraction (EAE) Cellulase was chosen as the extracting enzyme for its well-known effectiveness in enhancing extraction of mushrooms and other organisms (Fernandes, 2018 ). The enzyme-assisted extraction procedure was adapted from Xu et al. ( 2020 ). As for the WE method, the dried defatted mushroom powder (3 g) was macerated for thirty minutes as well as the pH of the liquid was adapted to 4.5 via adding 1 M HCl (the optimal pH for the enzyme). Cellulase was gradually introduced into the liquid at a concentration of 0.3% (w/v), and EAE was carried out at 45°C for 1 h. The extraction process was stopped by briefly heating the sample solution at 100°C. Afterwards, the blend was cooled to ambient temperature. The solution pH was increased to 10 by 1 M NaOH. The sample was centrifuged for 30 minutes at 4000 rpm. The collected supernatant was utilized for protein isolation following the previously outlined procedure. 2.2.4 Combined EAE and UAE extraction Three schemes of combined EAE and UAE extraction were initially tested including EUE (EAE followed by UAE), UEE (UAE followed by EAE), and SEUE (simultaneous use of EAE and UAE). Table S1 shows the specific procedures and conditions for the combined extraction schemes. With all the extraction schemes, 3 grams of defatted mushroom powder were treated according to the procedure outlined for the WE method, and the supernatants obtained from these three schemes were applied for protein isolation and purification as for the WE method. 2.3 Optimization of EUE extraction Preliminary experiment results showed that the two-step sequential EUE method was the most effective in order to separate and extract the protein from the L. edodes ( Figure S1 ). Therefore, the following extraction experiments were all conducted in two separate steps, enzyme using cellulase during the enzyme treatment. The major process factors, including enzyme content, enzyme treatment time, UAE treatment time, US amplitude percentage, and (NH 4 ) 2 SO 4 saturation concentration, were evaluated for their effects on protein extraction yield. Figure 1 presents a flowchart for the EUE extraction scheme including all major experimental steps starting from the raw mushroom including the major steps EAE, and UAE, and the possible extraction mechanisms. Cellulase enzyme, within the concentration range of 0.2-1.0% (w/v), was gradually introduced into the mushroom extract solution (pH 4.5) within a plastic centrifuge tube. The mixture was then extracted at 45°C for 30 to 120 minutes. The EAE was ceased by heating at 100°C. Once the sample cooled, its pH was increased to 10 by 1 M NaOH. The treatment was further extended with UAE by adjusting the ultrasound amplitude to 20–100% (0.29–1.44 W/mL) for a duration of 20–60 minutes. After centrifuging for thirty minutes at 4000 rpm, the liquid of samples was collected for protein precipitation by adding (NH 4 ) 2 SO 4 to reach 20–100% saturation and allowing it to precipitate overnight at 4°C. The following procedure was performed for partial purification and recovery of the protein fraction as for the WE method. 2.4 Statistical experiment design for EUE optimization by RSM According to the results of the optimization of the EUE experiments mentioned above, a 3-central point definitive screening experimental design was selected. RSM was used to improve the protein extraction conditions utilizing a 3 3 , three-factor, three-level factorial Box-Behnken design (BBD). This design created seven experimental runs, including 5 replicates at the central points and 12 factorial points. The choice of experimental factors and their respective levels was guided by the prior experiments (Table 1 ), with enzyme concentration (% w/v, X 1 ), ultrasound amplitude (US) (%, X 2 ), and (NH 4 ) 2 SO 4 concentration (%, X 3 ) serving as independent variables. The protein yield and content were chosen as the two response values. The response surface analysis was performed based on the BBD response surface design. The RSM was designed by the Design-Expert 11 software program (Stat-Ease, Inc., Minneapolis, USA). Analysis of Variance (ANOVA) was conducted to perform the statistical analysis, used to determine the optimal conditions for EUE extraction. Table 1 Factors and levels used in the optimization of EUE. Factor Coded factor levels -1 0 1 X 1 Enzyme amount (% w/v) 0.1 0.3 0.5 X 2 US amplitude (%) 40 60 80 X 3 (NH 4 ) 2 SO 4 saturation (%) 50 70 90 2.5 Analysis of isolated protein fractions 2.5.1 Protein secondary structure analysis The secondary structures of the WE, UAE, EAE, EUE, and optimized EUE protein samples were analyzed using Circular Dichroism (CD) spectroscopy. The experiments were conducted at room temperature with a Jasco J-1500 CD spectropolarimeter from Japan. The identification of protein extract samples was in accordance with prior published research (Zhao et al., 2024 ). 2.5.2 Composition, molecular weight, and FT-IR spectroscopy analysis The MW of optimized EUE-extracted protein samples was examined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, following the procedures outlined in prior documentation (Cheung et al., 2012 ). The optimized EUE protein samples were crushed into KBr pellets and subjected to Fourier transform-infrared (FT-IR) spectroscopy over the wavenumber range of 500–4000 cm − 1 (Nexus 670 FT-IR spectrometer) (Thermo Nicolet Co., Cambridge, UK). Protein samples composition of AA was assessed in accordance with the description provided in a prior report. Prior to analysis using ultra-performance liquid chromatography-electrospray ionization triple quadrupole mass spectrometry (UPLC-ESI-TQMS) (Agilent Technologies, Santa Clara, CA, USA) coupled with a CORTECS™ UPLC C18 (1.6 µm, 2.1 x 150 mm column) as detailed by Gray et al. ( 2017 ) as well as Guba et al. ( 2022 ), protein samples underwent a 30-second treatment with nitrogen followed by hydrolysis with 12M HCl at 110 ℃ for 24 hours. Following this, the samples were diluted 60-fold to attain a final concentration of 0.1 M. The diluted samples were derivatized using the Waters Kairos AA kit (Waters, Milford, MA, USA). 2.6 Immunoactivity assay in vitro As previously reported, RAW 264.7 cell culture was used to evaluate the immunomodulatory activities of protein fractions. According to the previous study (Zhao et al., 2024 ), the concentration of all protein fractions was set at 4 µg/mL, and LPS (200 ng/mL) was chosen as a positive control. Experiments including macrophage cell proliferation, nitric oxide (NO) generation, and phagocytic activity were initially conducted on protein extract samples from WE, EAE, UAE, and EUE to pinpoint the optimized protein exhibiting both high yield and biological activities. 2.6.1 Analysis of cell viability and nitric oxide (NO) generation RAW264.7 cell culture was prepared as previously described (Zhao et al., 2024 ), and then seeded into 100 µL of 96-well plates (5 × 10 4 cells/mL). The cells in the logarithmic growth phase were treated with protein samples and LPS for 24 hours. Cell viability was determined using MTS as previously described. The supernatants of the cell culture medium underwent treatment with Griess reagent to quantify NO. Absorbance readings were taken by utilizing a microplate reader at 540 nm. Subsequently, the absorbance measurement was calibrated against NO concentrations using sodium nitrite. 2.6.2 Neutral red uptake analysis The neutral red uptake method was applied to assess the phagocytic potential of cells. Cells were cultured and seeded as described in section 2.6.1 . After protein and LPS treatment for 24 hours, the supernatant was removed, and the subsequent experiments were conducted as in previous study. 2.6.3 ROS production The amount of reactive oxygen species (ROS) was assessed by the fluorescent probe 2’,7’-Dichlorodihydrofluorescein diacetate (DCFH-DA) from Sigma as described by Jayasinghe et al. ( 2023 ) with slight adjustments. In summary, each well of a 96-well black plate was seeded and incubated according to the procedure outlined in section 2.6.1 . After 24 hours of cultivation, 100 µL of EUE-extracted protein (0.2 µg/mL) was further added to each well. Following a one-hour incubation at 37 ℃, cells were treated with DCFH-DA (10 µM) and further cultured for 23 h at 37 ℃. Fluorescence intensity was measured with an excitation wavelength of 485 nm and an emission wavelength of 527 nm. 2.7 Statistical Data Analysis The results are presented as the mean ± standard deviation (SD) from three independent replicates. Statistical analyses were performed in Prism 9 using a student’s T-test or one-way ANOVA followed by a Tukey post-hoc test. A p -value of less than 0.05 was considered statistically significant. 3. Results and discussion 3.1 Comparison of protein yield and content from various extraction methods Table 2 is a summary of the crude protein yields by various extraction methods. When the UAE process was applied alone, the protein content was the lowest compared to the other methods. Compared to UAE, protein extraction using the WE extraction method with a NaCl solution and precipitation with (NH 4 ) 2 SO 4 led to higher yields and content. This may be caused by the ability of salt ions to inhibit electrostatic protein-protein interactions, consequently improving the extractability. Cellulase is a highly effective biocatalyst for degrading carbohydrate fiber and cell walls, so as to facilitate protein extraction. In the single-step enzyme-assisted extraction EAE process, the protein yield was 4.9% and the protein content was 50% (Table 2 ) . In comparison, the two-step EUE process resulted in a higher protein yield and content. Two alternative process schemes were compared with the EUE, UAE followed by EAE (UEE) and simultaneous enzymatic and SEUE (concurrent application of EAE and UAE), both resulting in lower protein yield and content ( Figure S1 ). The lower extraction efficiency of UEE than EUE suggests that the mechanical effect of ultrasound is more effective when the cell walls are partially disrupted by the enzyme cellulase, but less effective when the cell walls are intact. In the UEE method (enzyme extraction after ultrasound extraction), intact cell walls likely resist disruption due to their dense, unmodified structure. In contrast, the EUE method employs enzymatic pretreatment to weaken the cell wall, making it more susceptible to cavitation-induced fracture during ultrasound treatment. Specifically, cellulase breaks down cell walls by hydrolyzing β-1,4-glycosidic bonds in cellulose, degrading the crystalline cellulose network and hemicellulose matrix. This enzymatic action reduces cell wall rigidity and compromises its mechanical integrity (Nakazawa et al., 2024 ; Zhang et al., 2018 ). Enzymatic pretreatment fragments the cell wall structure, enhancing permeability and weakening its integrity (Fig. 1 B), which reduces the energy required for ultrasound to breach the compromised cellulose-hemicellulose network, thereby maximizing physical disruption efficiency and facilitating the release of intracellular proteins for higher extraction yields. This concurs with previous findings by Turker and Isleroglu ( 2024 ) that enzyme pretreatment followed by ultrasonication was effective for protein extraction. As for the SEUE scheme, the lower efficiency may be explained by the fact that the two require different optimal conditions for effective protein extraction. Consequently, the EUE scheme achieved a higher protein yield due to a greater synergy between enzyme and ultrasound. Meanwhile, the EUE extraction method significantly enhances extraction efficiency and reduces extraction time, particularly when compared to the modified traditional WE method. Additionally, the protein extracted by the EUE method exhibited stronger immunostimulatory activities than those by the other three methods (Fig. 2 ). This is likely attributable to the higher protein content achieved through the EUE method compared to other extraction techniques, potentially offering more accessible epitopes for immune recognition (Zhao et al., 2024 ). Besides, the EUE method demonstrated the highest β-sheet content (Table 2 ), which contributes to creating a stable and rigid framework. This structural feature helps maintain the conformation of the epitope, thereby enhancing its accessibility for antibody binding (Rodrigues et al., 2019 ). Meanwhile, its high α-helix content contributes to overall structural stability without introducing excessive rigidity, potentially balancing stability with the flexibility necessary for optimal epitope exposure. Despite its low β-turn content (2.7%), the high proportion of β-sheets compensates by preserving structural integrity and ensuring epitope accessibility. In contrast, EAE exhibited the weakest activity, which can be attributed to its low β-turn content (Table 2 ). This deficiency may result in protein misfolding, reduced flexibility, and stability issues, potentially hindering epitope exposure and effective binding to immune receptors (Marcelino & Gierasch, 2008 ). Although the WE method did not exhibit a high β-sheet structure, it possessed a high random coil content (Table 2 ), which may enhance flexibility and improve epitope exposure, thereby facilitating recognition by immune cells (Fan et al., 2024 ). Regarding UAE, its β-turn content was higher than that of EAE (Table 2 ), which may aid in maintaining local conformational stability and facilitate the correct positioning of epitopes. In summary, the EUE method was chosen for optimization to achieve maximum protein yield because it can produce a high yield with substantial protein content and strong immunostimulatory activity. Table 2 Protein yields, contents, and secondary structures by various separation methods. Protein yield (%) a Total protein content (%) Total sugar content (%) WE 6.5 ± 1.1 47.3 ± 1.0 43.1 ± 2.5 UAE 6.9 ± 1.5 40.9 ± 5.8 51.4 ± 4.4 EAE 4.9 ± 1.3 50.2 ± 2.7 45.8 ± 3.7 EUE 9.4 ± 1.4 56.0 ± 2.2 38.7 ± 2.0 Protein secondary structure Random coil% α-Helix% β-Sheet% β-Turn% WE 40.2 ± 3.4 20.9 ± 1.7 20.6 ± 1.6 18.3 ± 1.4 UAE 33.3 ± 0.3 13.9 ± 1.0 38.5 ± 4.0 14.3 ± 0.2 EAE 29.5 ± 3.3 19.9 ± 2.1 43.2 ± 1.9 7.4 ± 1.1 EUE 15.2 ± 1.3 26.3 ± 3.5 55.8 ± 3.9 2.7 ± 0.3 a. Equal to the mass of freeze-dried soluble protein divided by the mass of original mushroom powder. 3.2 Effects of EUE conditions on protein yield Figure 3 A demonstrates the impact of enzyme concentration ranging from 0.2 to 0.8 (w/v) on the protein yield. All other conditions held constant (EAE 60 minutes, UAE 40 minutes, US amplitude 60%, and (NH 4 ) 2 SO 4 saturation 70%. As the amount of cellulase increased, both protein yield and content initially rose, then declined, and eventually stabilized. The lowest protein yield was achieved at 0.2%, which shows a significant difference compared to the yield at 0.3%. A 0.3% cellulase addition resulted in the highest protein yield and content. At certain liquid-to-material ratios, the protein extraction efficiency increases with cellulase levels rise. However, as the amount of cellulase added increases, it leads to a weakening of the diffusive effect of the mushroom powder and a decrease in the diffusive effect of cellulase, resulting in a decrease in the protein extraction rate (Xu et al., 2020 ). Therefore, 0.3% was chosen as the central enzyme concentration for the RSM analysis. Figure 3 B indicates the effect of enzyme treatment time (30–120 minutes). Although there was insignificant ( p > 0.05) between the various time periods, the protein output showed a rising and then declining pattern as treatment time rose, while the protein content appeared a significant difference ( p < 0.05). The protein extraction rate increased with time (30–60 minutes), reaching its maximum at 60 minutes, and then declining trend. With a longer treatment time, cellulase further hydrolyzes the broken cell walls of L. edodes , resulting in increased protein solubilization and extraction rate. However, the protein content decreased due probably to protein aggregation, which can negatively impact the extraction rate. The protein yield was little impacted by changing the UAE time from 20 to 60 minutes, as seen in Fig. 3 C ( p > 0.05) while obviously affected on soluble protein content when the other conditions remained constant. As illustrated in Fig. 3 C, within the interval of 20 to 40 minutes of ultrasound treatment, the protein content increases with the extension of ultrasound time, reaching its maximum at 40 minutes. With a further increase in ultrasound treatment time, the protein content starts to decrease, which may be protein denaturation and subsequently lowering the extraction rate. Protein production varies with US amplitude, as shown in Fig. 3 D. At a US amplitude of 60%, the maximum protein content was observed at 56%. Protein content enhanced with the rise in US amplitude from 20%-60% but started to decline with a higher amplitude. Despite the decrease in protein extraction rate mentioned earlier, the crude protein yield continued to increase. This can be attributed to the release of other components such as PS, and polyphenols along with the protein as the US amplitude increased. These other components, although not affected by the destructive effects of ultrasound, could increase the crude protein yield. In order to attain a high protein content, a central point of 60% US amplitude was chosen for the response surface methodology (RSM) experiments. Figure 3 E shows the effect of (NH 4 ) 2 SO 4 saturation from 20–100% on protein extraction yield. With the rise of (NH 4 ) 2 SO 4 amount, protein content and yield were both increased, probably attributed to the stronger salting-out effect. No significant difference was seen in the crude protein yield between 70% as well as 100% saturation of (NH 4 ) 2 SO 4 ( p > 0.05). A central point of 70% (NH 4 ) 2 SO 4 saturation was selected for the RSM experiments. 3.3 EUE extraction conditions optimized using RSM 3.3.1 Model Fitting and Statistical Evaluation RSM analysis was used to determine the optimal EUE protein extraction conditions from L. edodes. The effects of enzyme additional amount, US amplitude, and (NH 4 ) 2 SO 4 saturation on the protein yield and content were tested using a BBD. The results of all 17 experimental runs are presented in Table 3 . Regression analysis was performed by fitting a response surface model to all the responses, from which the multiple regression equation was derived. These equations represent the empirical correlation between the responses and the independent variables, as displayed: $$\\:{Y}_{1}=-29.01+18.99{X}_{1}+\\:0.47{X}_{2}+0.59{X}_{3}+0.20{X}_{1}{X\\:}_{2}-0.01{X}_{1}{X}_{3}-0.003{X}_{2}{X}_{3}-53.24{X}_{1}^{2}-0.002{X}_{2}^{3}-0.003{X}_{3}^{2}$$ 3.1 $$\\:{Y}_{2}=-68.35+175.93{X}_{1}-\\:0.10{X}_{2}+3.11{X}_{3}-0.02\\:{X}_{1}{X}_{2}-0.90{X}_{1}{X}_{3}\\:-0.01{X}_{2}{X}_{3}-207.06{X}_{1}^{2}+0.003{X}_{2}^{2}-0.02{X}_{3}^{2}$$ 3.2 In the following regression equations, Y 1 and Y 2 are the responses, representing protein yield and its content, respectively. X 1 - X 3 denotes the actual values of the independent variables. The F -test was used to assess the significant impact of X 1 - X 3 on the Y 1 and Y 2 . US amplitude ( X 2 ) and (NH 4 ) 2 SO 4 saturation ( X 3 ) were found to significantly influence both protein yield and content ( p < 0.05). In contrast, enzyme amount ( X 1 ) as well as the interactive effects of US amplitude & (NH 4 ) 2 SO 4 saturation ( X 2 : X 3 ) significantly affected protein content ( p < 0.05). However, enzyme amount ( X 1 ) did not significantly affect protein yield. Besides, the model was determined to be significant for both responses ( Y 1 and Y 2 ) (Table 4 ). Lack-of-fit value showing no significant difference, as evidenced by p -values of 0.16 and 0.32 for model equations 3.1 and 3.2 , respectively. This demonstrates that the model is reliable for predicting protein yield and content. The adequacy of this model was confirmed via the coefficient of determination (R 2 ), with values of 0.944 ( Y 1 ) and 0.924 for ( Y 2 ), respectively. A higher R² value indicates a strong fit between the empirical models and the experimental data. Three-dimensional response surface plots were generated in order to explore the interaction effects of X 1 -X 3 on Y 1 and Y 2 (Fig. 4 ). One variable was kept constant while the other two were changed in these graphs. Multiple solutions were generated via this approach, with the most desirable solution, exhibiting a desirability value of 0.736, being presented in this study, which is close to 1. The contour plots (Fig. 5 ) show that the optimum conditions for the protein with high content and high yield by the EUE extraction were 0.28% (w/v) enzyme, 62% US amplitude and 69% (NH 4 ) 2 SO 4 saturation, predicting a maximum protein yield of 9.7% and content of 57.1%. The optimal conditions were validated through a verification experiment. The verification experiment yielded a protein yield of 9.7 ± 1.6% and a content of 58.4 ± 1.3%, which closely matched the model-predicted value. The findings also indicated that the response models accurately represented the optimization targets. In general, the accuracy was satisfactory, and the response surface models were effective for predicting responses. Table 3 RSM experimental design (3 3 BBD) and the resulting protein yield and content outcomes. Run Independent variables Response Y 1 Response Y 2 X 1 X 2 X 3 Experiment Predicted Experiment Predicted 1 0.5 60 90 5.0 ± 0.5 5.1 39.3 ± 1.7 38.4 2 0.3 60 70 9.0 ± 1.4 9.5 55.4 ± 1.2 57.5 3 0.5 60 50 6.3 ± 0.5 6.6 43.6 ± 2.9 41.8 4 0.3 60 70 9.7 ± 1.9 9.5 58.3 ± 2.9 57.5 5 0.3 40 90 5.9 ± 1.1 6.3 63.5 ± 0.8 62.9 6 0.1 60 50 7.3 ± 0.1 7.1 38.8 ± 1.3 39.8 7 0.1 80 70 7.3 ± 0.7 7.9 48.9 ± 0.9 47.4 8 0.1 40 70 6.1 ± 1.6 6.0 60.0 ± 1.9 58.7 9 0.3 80 90 7.5 ± 1.7 7.3 45.6 ± 1.3 45.3 10 0.3 60 70 9.4 ± 1.3 9.5 57.2 ± 1.1 57.5 11 0.3 40 50 5.1 ± 0.7 5.3 52.5 ± 0.8 52.9 12 0.3 60 70 10.0 ± 1.9 9.5 60.3 ± 1.2 57.5 13 0.3 60 70 9.4 ± 1.3 9.5 56.4 ± 1.4 57.5 14 0.5 40 70 4.3 ± 0.5 3.8 52.2 ± 1.3 53.7 15 0.5 80 70 8.8 ± 0.4 8.9 40.7 ± 1.9 42.0 16 0.1 60 90 6.0 ± 0.02 5.7 48.9 ± 1.5 50.8 17 0.3 80 50 11.7 ± 0.5 11.3 47.0 ± 1.2 47.6 Table 4 ANOVA of fitted quadratic models of two responses. Source Y 1 : protein yield (%) Y 2 : protein content (%) F p F p Model 31.05 < 0.0001 22.63 0.0002 X 1 -enzyme amount 2.55 0.1542 11.74 0.0110 X 2 -US amplitude 99.06 < 0.0001 57.36 0.0001 X 3 -(NH 4 ) 2 SO 4 saturation 17.83 0.0039 6.39 0.0393 X 1 X 2 10.49 0.0143 0.0080 0.9311 X 1 X 3 0.0126 0.9139 11.39 0.0118 X 2 X 3 25.72 0.0014 8.38 0.0231 X 1 ² 77.77 < 0.0001 63.01 < 0.001 X 2 ² 9.39 0.0182 1.32 0.2876 X 3 ² 26.12 0.0014 39.68 0.0004 Lack of Fit 2.95 0.1613 1.62 0.3192 R 2 = 0.976 R 2 adj = 0.944 C.V.% = 6.54 R 2 = 0.967 R 2 adj = 0.924 C.V.% = 4.19 3.4 Structure characteristics of EUE-extracted proteins In the CD spectrum ( Figure S2 ), the protein structure was characterized by random coil 10.0 ± 1.4%, α-helix 29.3 ± 2.6%, β-sheet 56.7 ± 6.2%, and β-turn 4.0 ± 0.6%. A previous study identified the β-sheet as the predominant structure in proteins extracted from the stipe of L. edodes mushrooms, which is consistent with our findings (Hu, 2019 ). In Figure S3 , the EUE-extracted protein under optimized conditions exhibits characteristic peaks of amide I (1638 cm − 1 ) and amide III (1242 cm − 1 ). Amide I and amide III bands result from the N-H and C-N stretching vibrations, as well as N-H bending vibrations. The absorption band at 3411 cm − 1 is associated with the stretching vibration of O–H in the molecular structure, probably due to the presence of PS. This aligns with our findings, as the EUE-extracted protein can also acquire PS, as shown in Table 2 . The anti-symmetric stretching vibration of alkanes (-C-H-) is responsible for the band at 2919 cm − 1 . The absorption bands at 1547 cm − 1 as well as 1080 cm − 1 arise from the in-plane deformation vibration of NH 2 and the in-plane bending vibration of NH, respectively, in the amide II region. From the SDS-PAGE analysis ( Figure S4 ), it was found that most protein bands were < 10 kDa. Protein bands appeared at 25, 34 and 43–75 kDa. 3.5 Composition of proteins extracted through EUE Table 5 presents the AA profiles of partially purified proteins extracted by EUE. As shown in Table 5 A, the ratio of the sum of Glu and Asp to the sum of Lys, Arg, as well as His was 1.22. Thus, the EUE-extracted protein sample was acidic proteins. The protein nutritional value is primarily determined by the type, content, quantity, as well as composition of EAA. The protein extracted via EUE comprised EAA and non-essential amino acids (NEAA) with a total amount of 437.0 mg/g, which exceeded the amount of protein in other edible fungi such as oyster mushrooms and enoki mushrooms. The content of EEA constitutes over 40% of the total amino acids (TAA) content. EAA/NEAA value was close to 80%, in line with the recommendations proposed by FAO/WHO ideal protein condition. Furthermore, the value of isoleucine and valine exceeds the range required by FAO/WHO for adults (Wang et al., 2023 ), indicating that L. edodes can be used as a good nutritional protein source for adults. As stated by the AA scoring model recommended by FAO/WHO and the AA profile of egg protein, the amino acid score (AAS) as well as chemical score (CS) values for EUE protein are presented in Table 5 B. When AAS was used as the standard, isoleucine content was the highest, 2.08 times of the standard. The first limiting AA was Met + Cys, while Lys was the second limiting AA. The highest isoleucine content was 1.41 times the standard when CS was used as the standard. Met + Cys was also recognized as first limiting AA. Therefore, the main limiting AA in the L. edodes protein were Met + Cys and lysine. Met + Cy was also identified as the first limiting AA based on the RC value. Lysine was the second limiting AA. This result aligns with a previous study that identified Met + Cys as the first limiting AA in the fungus L. edodes . It was also agreed with our previous findings, which showed that Met + Cys is lacking in L. edodes . The RC value of Leu was close to 1, indicating that the composition ratio of this AA in L. edodes protein was close to that in the model spectrum. Val was a relative deficiency, and Ile and Thr were relatively abundance. Mushroom protein should be combined with other proteins to optimize its nutritional value based on the protein complementation theory. The nutritional quality of the protein improves as its SRC value increases. In this study, SRC value of EUE-extracted protein was 69.8%, which was comparable to that of soy protein, milk (72.60%), milk powder (67.31%), and walnuts (62.65%). The protein extracted from L. edodes mushrooms using the EUE method possesses high nutritional value and offers potential for development and utilization. Table 5 Amino acids in EUE-extracted protein samples. (A) Amino acid composition and contents (mg/g crude protein) EAA Content NEAA Content Histidine (His) 9.8 ± 2.6 Alanine (Ala) 26.3 ± 1.6 Isoleucine (Ile) 33.3 ± 1.9 Asparagine (Arg) 2.3 ± 0.04 Leucine (Leu) 39.2 ± 2.6 Aspartic acid (Asp) 36.3 ± 2.7 Lysine (Lys) 23.2 ± 0.9 Arginine (Arg) 22.9 ± 1.5 Methionine (Met) 9.1 ± 1.0 Glutamic acid (Glu) 31.7 ± 2.2 Phenylalanine (Phe) 23.2 ± 1.5 Glycine (Gly) 23.0 ± 0.9 Threonine (Thr) 25.9 ± 1.4 Serine (Ser) 27.2 ± 2.0 Valine (Val) 25.0 ± 1.4 Tyrosine (Tyr) 17.6 ± 1.6 Cysteine (Cys) 2.1 ± 0.3 Glutamine (Glu) 38.4 ± 5.6 Proline 20.5 ± 1.4 TAA EAA NEAA EAA/TAA (%) EAA/NEAA (%) 437.0 ± 33.1 188.7 ± 13.3 248.3 ± 19.8 43.2 ± 0.2 76.0 ± 0.7 (B) Amino acid and chemical scores of protein samples AAS (%) CS (%) RAA RC Ile 83.3 ± 4.8 61.7 ± 3.5 0.83 ± 0.05 1.47 ± 0.01 Leu 56.0 ± 3.7 45.6 ± 3.0 0.56 ± 0.04 0.99 ± 0.00 Lys 42.2 ± 1.6 33.1 ± 1.3 0.42 ± 0.02 0.75 ± 0.02 Met + Cys 32.0 ± 3.7 19.6 ± 2.3 0.32 ± 0.04 0.56 ± 0.03 Phe + Tyr 68.0 ± 5.2 43.9 ± 3.3 0.68 ± 0.05 1.21 ± 0.01 Thr 64.8 ± 3.5 55.1 ± 3.0 0.65 ± 0.03 1.14 ± 0.01 Val 50.0 ± 2.8 37.9 ± 2.1 0.50 ± 0.03 0.88 ± 0.01 SRC 69.8 ± 0.6% Note: AAS = content of a specific EAA in the protein sample divided by its content in the FAO/WHO model protein ( Table S2 ) (FAO/WHO, 1973 ; FAO/WHO, 2007 ); CS = content of an EAA in protein sample divided by its content in whole egg protein ( Table S2 ); RAA (AA ratio) = content of an AA in protein divided by the its content in the FAO/WHO pattern spectrum; RC (ratio coefficient) = RAA value divided by the average RAA; SRC (score of ratio coefficient) = the standard deviation coefficient of RC (Zhao et al., 2024 ). 3.6 Immunomodulatory activities At a concentration of 4 µg/mL, the EUE-extracted protein demonstrated no toxicity to RAW 264.7 cells (Fig. 6 A). NO is essential in various physiological functions, particularly in host defense. NO production serves as a reliable measure of the immunocompetence of RAW 264.7 cells. As displayed in Fig. 6 B, EUE-extracted protein's NO production was more significant than the control group ( p < 0.05). NO production level was around five times higher than that of the control group. Furthermore, phagocytosis is also one of the essential functions of macrophages. Neutral red phagocytosis was employed in this investigation as a marker of immune response activity, and the outcomes were consistent with the NO generation findings. The protein group's neutral red uptake was almost 1.5 times more than the control group's ( p < 0.05) (Fig. 6 C &D ). Moreover, as defensive elements and signaling molecules in immunological pathways, ROS can be produced by macrophage cells during phagocytosis. As anticipated, the EUE protein fractions stimulated ROS production in macrophage cells similar to pinocytic activity (Fig. 6 E). Overall, the results confirmed that the EUE-extracted protein rich fraction exhibits in vitro immunostimulatory activity. 4. Conclusions The present study has demonstrated that the two-step, sequential enzyme- followed by ultrasound-assisted (EUE) was the effective process scheme for L. edodes mushroom protein extraction with the highest protein yield and protein content, and immunostimulatory activity in comparison with the one-step process including WE, EAE, UAE. The EUE method also achieved a higher protein yield or content compared to both the simultaneous combination of EAE and UAE, as well as the two-step process of ultrasound followed by enzyme-assisted extraction (UEE). This outcome also suggests that the process scheme is a significant factor in achieving the synergistic or cooperative effect of different means and mechanisms of extraction. Moreover, enzyme concentration, ultrasound power, and ammonium salt saturation were the major process factors for the EUE process. The protein fraction showed a high nutritional value and significant in vitro immunostimulatory activity. The results and findings from the present study may provide new ideas and a theoretical basis for the combined EUE extraction scheme in the extraction of proteins from L. edodes and other mushrooms. However, its efficacy and feasibility for industrial application is still subject to pilot-scale trials and process economy analysis. It is also meaningful to conduct a more detailed investigation into the protein structures and their interactions with PS in relation to immunostimulatory activities by different extraction methods. For fundamental understanding and rational application, further research is needed to elucidate the mechanisms underlying the synergistic effects of enzyme and ultrasound interactions at the molecular level. Declarations Acknowledgments Special thanks to So-Pu Kin for his support of LC-MS experiments and analysis. Author Contributions ZC Zhao: Methodology, Investigation, Writing-original draft; YY Zhu: Methodology; FT Gu: Methodology; LX Huang: Methodology; XW Liu: Methodology; JY Wu: Funding, Project administration, Supervision, Writing - review & editing. Data Availability The datasets produced or analyzed in this study can be obtained from the corresponding author upon request. Funding was provided by The Hong Kong Polytechnic University (Ri-Food Project CD59). Competing Interests: The authors declare no competing interests. References Ahmed, T., Suzauddula, M., Akter, K., Hossen, M., & Islam, M. N. (2024). Green Technology for Fungal Protein Extraction—A Review. Separations , 11 (6), 186. https://doi.org/10.3390/separations11060186 Bose, U., Broadbent, J. A., Byrne, K., Hasan, S., Howitt, C. A., & Colgrave, M. L. (2019). 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-6257446\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":436749193,\"identity\":\"24d8a1a9-4807-4095-a472-d2fb90341df8\",\"order_by\":0,\"name\":\"Zi Chen ZHAO\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Hong Kong Polytechnic University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Zi\",\"middleName\":\"Chen\",\"lastName\":\"ZHAO\",\"suffix\":\"\"},{\"id\":436749194,\"identity\":\"badadb32-7d38-45b5-9a71-41395debb28f\",\"order_by\":1,\"name\":\"Yan Yu ZHU\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Hong Kong Polytechnic University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Yan\",\"middleName\":\"Yu\",\"lastName\":\"ZHU\",\"suffix\":\"\"},{\"id\":436749195,\"identity\":\"5ef24729-1286-463e-9c29-e142a3fa58de\",\"order_by\":2,\"name\":\"Fang Ting GU\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Hong Kong Polytechnic University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Fang\",\"middleName\":\"Ting\",\"lastName\":\"GU\",\"suffix\":\"\"},{\"id\":436749196,\"identity\":\"59356977-ae5c-4c40-9367-48c7cb61baab\",\"order_by\":3,\"name\":\"Lin Xi HUANG\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Hong Kong Polytechnic University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Lin\",\"middleName\":\"Xi\",\"lastName\":\"HUANG\",\"suffix\":\"\"},{\"id\":436749197,\"identity\":\"dc19b06a-f2e0-4d44-ac81-2b8d8a3bde8f\",\"order_by\":4,\"name\":\"Xuwei LIU\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Hong Kong Polytechnic University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Xuwei\",\"middleName\":\"\",\"lastName\":\"LIU\",\"suffix\":\"\"},{\"id\":436749198,\"identity\":\"dbf8d96c-b7d6-4b1a-a644-48f3aedd3553\",\"order_by\":5,\"name\":\"Jian Yong WU\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABA0lEQVRIiWNgGAWjYBACxmZk3gcGBhkwg4eBIUGCGC2MM8CKCWhBAcw8xGhhbmd+9oCxzSZP3oHHTNp2hw2PwY0Exgdv2xjyJBtwOYzN3ICxLa3Y8ABQS+6ZNJAWZsO5bQzF0rj9Yib9t+1w4sYG3m3SuW2HQVrYpHnbGBLn4dTC/k2CEabFEqKF/Td+LTxmYC3zGYBaGKG2MIO0zMatpUyC4Vxa4gZm/s+WvUC/SJ552Cw555xE4kwc3jfsP75NgqHMJnF+e1vijZ87bOT4jicf/PAGKDLjAA4tIKMY2RgYDA6DGECewgEQyYA7IuXB5B8gowGqRR6He0bBKBgFo2DkAgCMDlW6BZEO8AAAAABJRU5ErkJggg==\",\"orcid\":\"\",\"institution\":\"Hong Kong Polytechnic University\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Jian\",\"middleName\":\"Yong\",\"lastName\":\"WU\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2025-03-19 03:23:16\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-6257446/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-6257446/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":79902813,\"identity\":\"2a4d64cc-d226-4d82-859a-3b0fd6a45293\",\"added_by\":\"auto\",\"created_at\":\"2025-04-04 10:28:01\",\"extension\":\"jpg\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":174972,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eProcess flow diagram (A) and the possible mechanisms (B) for enzyme- and ultrasound-assisted extraction of proteins from mushroom.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Picture1.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6257446/v1/4c254e9a6f7adc2b9fe5c359.jpg\"},{\"id\":79902811,\"identity\":\"0c400ee1-d84b-43a4-a842-6deb01268257\",\"added_by\":\"auto\",\"created_at\":\"2025-04-04 10:28:01\",\"extension\":\"jpg\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":35732,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eImmunoregulating activities of isolated fractions on the (A) cell viability, (B) NO release of macrophages, and (C) phagocytic activity measured by neutral red uptake. **, *** and ****: statistically significant differences from the control group at \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.01 and \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.001, \\u003cem\\u003ep\\u003c/em\\u003e\\u0026lt; 0.0001 respectively.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Picture2.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6257446/v1/908f434ad7ec4e40b6542c97.jpg\"},{\"id\":79902843,\"identity\":\"e3706c5d-1ec4-435b-9ac1-60d2a2104ff3\",\"added_by\":\"auto\",\"created_at\":\"2025-04-04 10:28:04\",\"extension\":\"jpg\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":74042,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eEffect of (A) cellulase addition amount, (B) enzyme treatment time, (C) UAE treatment time, (D) US amplitude, (E) (NH\\u003csub\\u003e4\\u003c/sub\\u003e)\\u003csub\\u003e2\\u003c/sub\\u003eSO\\u003csub\\u003e4 \\u003c/sub\\u003esaturation concentration on protein extraction yield and content of \\u003cem\\u003eL. edodes\\u003c/em\\u003e.\\u0026nbsp;(Common conditions: enzyme concentration 0.3%, enzyme treatment time 60 min, ultrasound treatment time 40 min, US amplitude 60% and (NH\\u003csub\\u003e4\\u003c/sub\\u003e)\\u003csub\\u003e2\\u003c/sub\\u003eSO\\u003csub\\u003e4\\u003c/sub\\u003e 70% saturation; Different letters a, b…f indicating insignificant difference).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Picture3.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6257446/v1/3b94d02dab2b90ad0a5e8b31.jpg\"},{\"id\":79902829,\"identity\":\"23083c9c-358d-4dd9-a11a-b4843c20dbc1\",\"added_by\":\"auto\",\"created_at\":\"2025-04-04 10:28:02\",\"extension\":\"jpg\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":89957,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eResponse surface plots of Protein yield (\\u003cem\\u003eY\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e1\\u003c/em\\u003e\\u003c/sub\\u003e) versus two experimental variables: (A) enzyme amount (\\u003cem\\u003eX\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e1\\u003c/em\\u003e\\u003c/sub\\u003e); and ultrasound amplitude (\\u003cem\\u003eX\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e2\\u003c/em\\u003e\\u003c/sub\\u003e); (B) enzyme amount (\\u003cem\\u003eX\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e1\\u003c/em\\u003e\\u003c/sub\\u003e) and the (NH\\u003csub\\u003e4\\u003c/sub\\u003e)\\u003csub\\u003e2\\u003c/sub\\u003eSO\\u003csub\\u003e4\\u003c/sub\\u003e saturation (\\u003cem\\u003eX\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e3\\u003c/em\\u003e\\u003c/sub\\u003e); (C) US amplitude power (\\u003cem\\u003eX\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e2\\u003c/em\\u003e\\u003c/sub\\u003e) and (NH\\u003csub\\u003e4\\u003c/sub\\u003e)\\u003csub\\u003e2\\u003c/sub\\u003eSO\\u003csub\\u003e4\\u003c/sub\\u003e saturation (\\u003cem\\u003eX\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e3\\u003c/em\\u003e\\u003c/sub\\u003e); Protein content (\\u003cem\\u003eY\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e2\\u003c/em\\u003e\\u003c/sub\\u003e) versus two experimental variables: (D) enzyme amount (\\u003cem\\u003eX\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e1\\u003c/em\\u003e\\u003c/sub\\u003e); and US amplitude power (\\u003cem\\u003eX\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e2\\u003c/em\\u003e\\u003c/sub\\u003e); (E) enzyme amount (\\u003cem\\u003eX\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e1\\u003c/em\\u003e\\u003c/sub\\u003e) and the (NH\\u003csub\\u003e4\\u003c/sub\\u003e)\\u003csub\\u003e2\\u003c/sub\\u003eSO\\u003csub\\u003e4\\u003c/sub\\u003e saturation (\\u003cem\\u003eX\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e3\\u003c/em\\u003e\\u003c/sub\\u003e); (F) US amplitude power (\\u003cem\\u003eX\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e2\\u003c/em\\u003e\\u003c/sub\\u003e) and (NH\\u003csub\\u003e4\\u003c/sub\\u003e)\\u003csub\\u003e2\\u003c/sub\\u003eSO\\u003csub\\u003e4\\u003c/sub\\u003e saturation (\\u003cem\\u003eX\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e3\\u003c/em\\u003e\\u003c/sub\\u003e).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Picture4.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6257446/v1/82a41591ea9575614b59acee.jpg\"},{\"id\":79902830,\"identity\":\"415ad6d4-2fee-4e2b-bd4c-f606389ef7cc\",\"added_by\":\"auto\",\"created_at\":\"2025-04-04 10:28:02\",\"extension\":\"jpg\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":119896,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eContour plots with the desirable response goals to determine the optimum protein yield and content at an enzyme additional amount of 0.28%.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Picture5.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6257446/v1/714186a29694f175e7410956.jpg\"},{\"id\":79902823,\"identity\":\"7c27a921-c6ee-4a88-95f5-343baf0c213a\",\"added_by\":\"auto\",\"created_at\":\"2025-04-04 10:28:02\",\"extension\":\"jpg\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":49100,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eImmunoregulating activities of EUE-extracted protein on the (A) cell viability, (B) NO release of macrophages, (C, D) phagocytic activity measured by neutral red uptake, and (E) ROS expression. \\u0026nbsp;Data are expressed as the means ± SD (n = 3). The error bar represents the standard deviation. *, ** and ****: statistically significant differences from the control group at \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05 and \\u003cem\\u003ep\\u003c/em\\u003e\\u0026lt; 0.01, \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.0001 respectively.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Picture6.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6257446/v1/4d1d6d1199613104f4fb6798.jpg\"},{\"id\":79905051,\"identity\":\"f1d15c16-c40c-4867-bac2-2767dca18882\",\"added_by\":\"auto\",\"created_at\":\"2025-04-04 10:52:03\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":2090081,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6257446/v1/c59bb4c1-08a7-4941-ad89-f4309308d196.pdf\"},{\"id\":79902814,\"identity\":\"ccdf5d63-43b6-4e55-b1f2-95b916d0787b\",\"added_by\":\"auto\",\"created_at\":\"2025-04-04 10:28:01\",\"extension\":\"docx\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":1227693,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Supplementarymaterials.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6257446/v1/2d7ce6b79af9de136a1b7b05.docx\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Sequential Enzymatic and Ultrasonic Extraction of Lentinula edodes Mushroom Proteins Leading to Enhanced Yield and Significant Immunoactivity\",\"fulltext\":[{\"header\":\"1. Introduction\",\"content\":\"\\u003cp\\u003e \\u003cem\\u003eLentinula edodes\\u003c/em\\u003e, is one of the more popular edible mushrooms and is especially favored in the Asia region for its appealing aroma, plus notable nutritional, and medicinal properties. Among the most abundant and also beneficial components are polysaccharides (PS) and proteins (Wasser, \\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e2004\\u003c/span\\u003e; Du et al., \\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e). The PS derived from \\u003cem\\u003eL. edodes\\u003c/em\\u003e have been extensively reported in the literature on their notable antitumor, and immunomodulatory effects and many other bioactivities (Wang et al., \\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e). Proteins represent another important component of \\u003cem\\u003eL. edodes\\u003c/em\\u003e are gaining attention for their medicinal potential (Wong et al., \\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e2010\\u003c/span\\u003e; Das \\u0026amp; Prakash, \\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e). It exhibits abundant essential amino acids (EAA) (Yu et al., \\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e). which is beneficial for human health. Moreover, protein also exhibit fascinating biological functionalities, including antitumor, antioxidant, and antimicrobial activities (Ngai \\u0026amp; Ng, \\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e2003\\u003c/span\\u003e; Gao et al., \\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e). Our recent research have shown that the protein-rich fractions from \\u003cem\\u003eL. edodes\\u003c/em\\u003e has stronger immunostimulatory activity compared to the PS-rich extracts (Zhao et al., \\u003cspan citationid=\\\"CR43\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e) and also showed high nutritional value of AA (Zhao et al., \\u003cspan citationid=\\\"CR43\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eExtraction is a crucial step for acquiring the desired proteins from mushrooms and other food and medicinal materials. Extraction with water is the most common method for extracting proteins from mushrooms (Jing et al., \\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e) or with aqueous solutions containing salt (NaCl) or surfactants (SDS, Triton100) for higher protein solubility (Chatterjee et al., \\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e2012\\u003c/span\\u003e). Alkaline or acid extraction may be more effective for the proteins with high solubility in the respective pH (Du et al., \\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e; Gerliani et al., \\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e). After the extraction process, an organic solvent, typically ethanol or methanol, is usually used to precipitate and isolate the proteins from prolamin, urea, phenol, and other denaturing agents (Chatterjee et al., \\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e2012\\u003c/span\\u003e; Capellini et al., \\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e; Chen et al., \\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e; Bose et al., \\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e). However, the aforementioned extraction and separation methods are rather tedious and time-consuming, and not environmentally friendly.\\u003c/p\\u003e \\u003cp\\u003eEAE and UAE are two of the most widely used methods for improving the water extraction of natural substances from diverse origins (Cannavacciuolo et al., \\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e; Goktayoglu et al., \\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e). It can be operated at a lower temperature than hot-water extraction. EAE and UAE are particularly favorable for heat-sensitive components such as proteins. EAE and UAE have also been considered efficient in enhancing protein extraction from various mushrooms (Ahmed et al. \\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e). It is well established that high-intensity ultrasound enhances water extraction mainly through acoustic cavitation in water which produces strong shear forces, causing cell wall disruption and promoting the release of cellular components (Wu, \\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e). A recent study has shown that UAE with intensive ultrasound enhances protein extraction from \\u003cem\\u003eL. edodes\\u003c/em\\u003e mushroom to some extent (Wang et al., \\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e). However, the strong shear forces generated by high-intensive ultrasound may also induce adverse effects on the integrity and quality of protein extracts (Higuera-Barraza et al., \\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e2016\\u003c/span\\u003e). In contrast, EAE utilizes specific enzymes like cellulases to selectively degrade cellulose and other glycan components of the cell walls, keeping the proteins intact (Culter, \\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e2008\\u003c/span\\u003e). The complex structure of the fungal cell wall mainly contains proteins, glucan, chitin, as well as cellulose (Rivillas-Acevedo and Soriano-Garc\\u0026iacute;a \\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e2006\\u003c/span\\u003e), which compounds within cells can be further enhanced by enzymes (Zhao et al., \\u003cspan citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e2016\\u003c/span\\u003e). The enzymatic hydrolysis can make the cellular structure more permeable and easier to disrupt, so that some proteins can be released. Zhang et al. (\\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e2015\\u003c/span\\u003e) assessed the use of cellulase for protein extraction from \\u003cem\\u003ePleurotus eryngii\\u003c/em\\u003e mushroom and increased the protein yield under specific extraction conditions. Additionally, another study found that extraction sequentially by UAE and EAE with cellulase can also enhance the protein yield from \\u003cem\\u003ePleurotus eryngii\\u003c/em\\u003e (Xu et al., \\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e). However, while these studies have demonstrated the potential of hybrid approaches, the treatment sequence (enzymes before or ultrasound after) remains underexplored. Critically, no reported study has been found in the literature on EAE or its combination with UAE of proteins from \\u003cem\\u003eL. edodes\\u003c/em\\u003e, excepted for a few reported studies employing either ultrasound or enzyme methods individually (Wang et al., \\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e; Prandi et al., \\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eBased on the above background, we hypothesize that the proper combination of EAE and UAE may ultimately enhance the protein extraction through a biochemical mechanism (by enzyme) and a physical or mechanical mechanism (by ultrasound). This study aimed to identify and optimize the most efficient protein extraction scheme and conditions based on a combination of EAE and UAE. Initially, several extraction methods were compared based on the protein yield and content, including WE, UAE, and EAE, and the synergy that depends on the order of sequential combinations of EAE and UAE under various conditions was thoroughly evaluated. The two-step sequential extraction by EAE and UAE (EUE) was identified as the most efficient method and was further optimized by statistical experimental design as well as response surface methodology (RSM). Furthermore, the characteristics and immunostimulatory functions of the protein obtained following RSM analysis were evaluated.\\u003c/p\\u003e\"},{\"header\":\"2. Materials and methods\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.1 Chemical and biochemical reagents\\u003c/h2\\u003e \\u003cp\\u003eThe ammonium sulfate ((NH\\u003csub\\u003e4\\u003c/sub\\u003e)\\u003csub\\u003e2\\u003c/sub\\u003eSO\\u003csub\\u003e4\\u003c/sub\\u003e) used was sourced from AnalaR Normapur, Vienna, Austria. Cellulase, bovine serum albumin (BSA), penicillin, lipopolysaccharide (LPS), streptomycin, sulphanilamide, phosphoric acid, as well as anthrone were sourced from Sigma-Aldrich in St. Louis, MO, USA. Potassium persulphate as well as sodium nitrite were obtained from BDH in Poole, England. Fetal bovine serum and Dulbecco's modified Eagle medium were acquired from Thermo Fisher Scientific in Waltham, MA, in the United States. Sodium chloride (NaCl) was purchased from Macklin (Shanghai, China). All remaining chemicals were sourced from reliable vendors in analytical reagent (AR) grade or higher.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec4\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.2 Extraction of proteins with different methods\\u003c/h2\\u003e \\u003cp\\u003eThe dried mushroom bodies were pulverized into a powder using an electric mill. Then, it was sifted via mesh sieves (850 \\u0026micro;m) to varying mean particle sizes. Mushroom powder was then defatted using 95% ethanol and dried at approximately 50\\u0026deg;C until the weight remained constant. To identify the best effective extraction method, several methods were compared initially including WE, UAE, EAE, and various combinations of EAE and UAE as described in detail below.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec5\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.2.1 Water extraction (WE)\\u003c/h2\\u003e \\u003cp\\u003eA preliminary test was performed with the conventional WE extraction method using alkaline and acid precipitation, resulting in a very low protein content (3.5\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.7%). As a result, the optimized WE method incorporating (NH\\u003csub\\u003e4\\u003c/sub\\u003e)\\u003csub\\u003e2\\u003c/sub\\u003eSO\\u003csub\\u003e4\\u003c/sub\\u003e and isoelectric precipitation was chosen. The WE procedure and conditions were selected based on the literature (Yang et al., \\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e). The dried defatted mushroom powder (3 g) was dispersed into an aqueous solution of 1% (w/v) NaCl at 1:30 (w/v) and macerated for 30 minutes (in plastic centrifuge tubes). To lower the pH of the mixture to 10, 1 M NaOH was put into the samples. Then, the mixture was agitated for three hours at 50\\u0026deg;C and later was centrifuged for thirty minutes at 4000 rpm at 4\\u0026deg;C. The supernatant was collected for protein precipitation as follows. Firstly, 1M HCl was added to the liquid to adjust the pH to 4, and then (NH\\u003csub\\u003e4\\u003c/sub\\u003e)\\u003csub\\u003e2\\u003c/sub\\u003eSO\\u003csub\\u003e4\\u003c/sub\\u003e was added to 70% saturation, was left to stand overnight (4\\u0026deg;C). The liquid was centrifuged at 9000 rpm for twenty minutes and the resulting pellet was gathered and dissolved in water. The liquid was centrifuged again (4000 rpm, 30 minutes) to attain a solid-free solution of crude protein. To remove salts, the protein solution was dialyzed in distilled water for 48 h by a 3.5 kDa molecular weight (MW) cutoff membrane. The crude protein fraction was then obtained by freeze-drying.\\u003c/p\\u003e \\u003cp\\u003eThe protein yield (%) was represented by the mass percentage of freeze-dried soluble protein in the original mushroom powder. Lowry method was applied to the calculation of the total protein yield, and the Anthrone test, as outlined in earlier studies, was applied to measure the total sugar (or carbohydrate) amount.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec6\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.2.2 Ultrasound-assisted extraction (UAE)\\u003c/h2\\u003e \\u003cp\\u003eThe dried defatted mushroom powder (3 g) was pre-treated using the same method as outlined in the WE method, followed by UAE. UAE was conducted using an ultrasonic processor (20 kHz), the Sonics \\u0026amp; Materials Inc. Model VCX-130 from Newton, USA, providing a maximum output power of 130 W, following established procedures with some adjustments. In brief, a 12 mm-diameter ultrasonic horn was submerged into the liquid samples. Ice was employed during the UAE process to prevent overheating. The US amplitude was maintained at 60% (0.87 W/mL), and the ultrasonic process was conducted for 40 min. After UAE, the solid-liquid mixture was spun at 4000 rpm for thirty minutes. The collected liquid underwent protein fraction isolation and partial purification, following the WE method as detailed earlier.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec7\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.2.3 Enzyme-assisted extraction (EAE)\\u003c/h2\\u003e \\u003cp\\u003eCellulase was chosen as the extracting enzyme for its well-known effectiveness in enhancing extraction of mushrooms and other organisms (Fernandes, \\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e). The enzyme-assisted extraction procedure was adapted from Xu et al. (\\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e). As for the WE method, the dried defatted mushroom powder (3 g) was macerated for thirty minutes as well as the pH of the liquid was adapted to 4.5 via adding 1 M HCl (the optimal pH for the enzyme). Cellulase was gradually introduced into the liquid at a concentration of 0.3% (w/v), and EAE was carried out at 45\\u0026deg;C for 1 h. The extraction process was stopped by briefly heating the sample solution at 100\\u0026deg;C. Afterwards, the blend was cooled to ambient temperature. The solution pH was increased to 10 by 1 M NaOH. The sample was centrifuged for 30 minutes at 4000 rpm. The collected supernatant was utilized for protein isolation following the previously outlined procedure.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec8\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.2.4 Combined EAE and UAE extraction\\u003c/h2\\u003e \\u003cp\\u003eThree schemes of combined EAE and UAE extraction were initially tested including EUE (EAE followed by UAE), UEE (UAE followed by EAE), and SEUE (simultaneous use of EAE and UAE). \\u003cb\\u003eTable S1\\u003c/b\\u003e shows the specific procedures and conditions for the combined extraction schemes. With all the extraction schemes, 3 grams of defatted mushroom powder were treated according to the procedure outlined for the WE method, and the supernatants obtained from these three schemes were applied for protein isolation and purification as for the WE method.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec9\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.3 Optimization of EUE extraction\\u003c/h2\\u003e \\u003cp\\u003ePreliminary experiment results showed that the two-step sequential EUE method was the most effective in order to separate and extract the protein from the \\u003cem\\u003eL. edodes\\u003c/em\\u003e (\\u003cb\\u003eFigure S1\\u003c/b\\u003e). Therefore, the following extraction experiments were all conducted in two separate steps, enzyme using cellulase during the enzyme treatment. The major process factors, including enzyme content, enzyme treatment time, UAE treatment time, US amplitude percentage, and (NH\\u003csub\\u003e4\\u003c/sub\\u003e)\\u003csub\\u003e2\\u003c/sub\\u003eSO\\u003csub\\u003e4\\u003c/sub\\u003e saturation concentration, were evaluated for their effects on protein extraction yield. Figure\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e presents a flowchart for the EUE extraction scheme including all major experimental steps starting from the raw mushroom including the major steps EAE, and UAE, and the possible extraction mechanisms.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eCellulase enzyme, within the concentration range of 0.2-1.0% (w/v), was gradually introduced into the mushroom extract solution (pH 4.5) within a plastic centrifuge tube. The mixture was then extracted at 45\\u0026deg;C for 30 to 120 minutes. The EAE was ceased by heating at 100\\u0026deg;C. Once the sample cooled, its pH was increased to 10 by 1 M NaOH. The treatment was further extended with UAE by adjusting the ultrasound amplitude to 20\\u0026ndash;100% (0.29\\u0026ndash;1.44 W/mL) for a duration of 20\\u0026ndash;60 minutes. After centrifuging for thirty minutes at 4000 rpm, the liquid of samples was collected for protein precipitation by adding (NH\\u003csub\\u003e4\\u003c/sub\\u003e)\\u003csub\\u003e2\\u003c/sub\\u003eSO\\u003csub\\u003e4\\u003c/sub\\u003e to reach 20\\u0026ndash;100% saturation and allowing it to precipitate overnight at 4\\u0026deg;C. The following procedure was performed for partial purification and recovery of the protein fraction as for the WE method.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec10\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.4 Statistical experiment design for EUE optimization by RSM\\u003c/h2\\u003e \\u003cp\\u003eAccording to the results of the optimization of the EUE experiments mentioned above, a 3-central point definitive screening experimental design was selected. RSM was used to improve the protein extraction conditions utilizing a 3\\u003csup\\u003e3\\u003c/sup\\u003e, three-factor, three-level factorial Box-Behnken design (BBD). This design created seven experimental runs, including 5 replicates at the central points and 12 factorial points. The choice of experimental factors and their respective levels was guided by the prior experiments (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e), with enzyme concentration (% w/v, \\u003cem\\u003eX\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e1\\u003c/em\\u003e\\u003c/sub\\u003e), ultrasound amplitude (US) (%, \\u003cem\\u003eX\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e2\\u003c/em\\u003e\\u003c/sub\\u003e), and (NH\\u003csub\\u003e4\\u003c/sub\\u003e)\\u003csub\\u003e2\\u003c/sub\\u003eSO\\u003csub\\u003e4\\u003c/sub\\u003e concentration (%, \\u003cem\\u003eX\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e3\\u003c/em\\u003e\\u003c/sub\\u003e) serving as independent variables. The protein yield and content were chosen as the two response values. The response surface analysis was performed based on the BBD response surface design. The RSM was designed by the Design-Expert 11 software program (Stat-Ease, Inc., Minneapolis, USA). Analysis of Variance (ANOVA) was conducted to perform the statistical analysis, used to determine the optimal conditions for EUE extraction.\\u003c/p\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab1\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 1\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003eFactors and levels used in the optimization of EUE.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"4\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e \\u003cp\\u003eFactor\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colspan=\\\"3\\\" nameend=\\\"c4\\\" namest=\\\"c2\\\"\\u003e \\u003cp\\u003eCoded factor levels\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e-1\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e1\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eX\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e1\\u003c/em\\u003e\\u003c/sub\\u003e Enzyme amount (% w/v)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e0.1\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e0.3\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e0.5\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eX\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e2\\u003c/em\\u003e\\u003c/sub\\u003e US amplitude (%)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e40\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e60\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e80\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eX\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e3\\u003c/em\\u003e\\u003c/sub\\u003e (NH\\u003csub\\u003e4\\u003c/sub\\u003e)\\u003csub\\u003e2\\u003c/sub\\u003eSO\\u003csub\\u003e4\\u003c/sub\\u003e saturation (%)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e50\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e70\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e90\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.5 Analysis of isolated protein fractions\\u003c/h2\\u003e \\u003cdiv id=\\\"Sec12\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.5.1 Protein secondary structure analysis\\u003c/h2\\u003e \\u003cp\\u003eThe secondary structures of the WE, UAE, EAE, EUE, and optimized EUE protein samples were analyzed using Circular Dichroism (CD) spectroscopy. The experiments were conducted at room temperature with a Jasco J-1500 CD spectropolarimeter from Japan. The identification of protein extract samples was in accordance with prior published research (Zhao et al., \\u003cspan citationid=\\\"CR43\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec13\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.5.2 Composition, molecular weight, and FT-IR spectroscopy analysis\\u003c/h2\\u003e \\u003cp\\u003eThe MW of optimized EUE-extracted protein samples was examined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, following the procedures outlined in prior documentation (Cheung et al., \\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e2012\\u003c/span\\u003e). The optimized EUE protein samples were crushed into KBr pellets and subjected to Fourier transform-infrared (FT-IR) spectroscopy over the wavenumber range of 500\\u0026ndash;4000 cm\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e (Nexus 670 FT-IR spectrometer) (Thermo Nicolet Co., Cambridge, UK). Protein samples composition of AA was assessed in accordance with the description provided in a prior report. Prior to analysis using ultra-performance liquid chromatography-electrospray ionization triple quadrupole mass spectrometry (UPLC-ESI-TQMS) (Agilent Technologies, Santa Clara, CA, USA) coupled with a CORTECS\\u0026trade; UPLC C18 (1.6 \\u0026micro;m, 2.1 x 150 mm column) as detailed by Gray et al. (\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e) as well as Guba et al. (\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e), protein samples underwent a 30-second treatment with nitrogen followed by hydrolysis with 12M HCl at 110 ℃ for 24 hours. Following this, the samples were diluted 60-fold to attain a final concentration of 0.1 M. The diluted samples were derivatized using the Waters Kairos AA kit (Waters, Milford, MA, USA).\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec14\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.6 Immunoactivity assay \\u003cem\\u003ein vitro\\u003c/em\\u003e\\u003c/h2\\u003e \\u003cp\\u003eAs previously reported, RAW 264.7 cell culture was used to evaluate the immunomodulatory activities of protein fractions. According to the previous study (Zhao et al., \\u003cspan citationid=\\\"CR43\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e), the concentration of all protein fractions was set at 4 \\u0026micro;g/mL, and LPS (200 ng/mL) was chosen as a positive control. Experiments including macrophage cell proliferation, nitric oxide (NO) generation, and phagocytic activity were initially conducted on protein extract samples from WE, EAE, UAE, and EUE to pinpoint the optimized protein exhibiting both high yield and biological activities.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec15\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.6.1 Analysis of cell viability and nitric oxide (NO) generation\\u003c/h2\\u003e \\u003cp\\u003eRAW264.7 cell culture was prepared as previously described (Zhao et al., \\u003cspan citationid=\\\"CR43\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e), and then seeded into 100 \\u0026micro;L of 96-well plates (5 \\u0026times; 10\\u003csup\\u003e4\\u003c/sup\\u003e cells/mL). The cells in the logarithmic growth phase were treated with protein samples and LPS for 24 hours. Cell viability was determined using MTS as previously described. The supernatants of the cell culture medium underwent treatment with Griess reagent to quantify NO. Absorbance readings were taken by utilizing a microplate reader at 540 nm. Subsequently, the absorbance measurement was calibrated against NO concentrations using sodium nitrite.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec16\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.6.2 Neutral red uptake analysis\\u003c/h2\\u003e \\u003cp\\u003eThe neutral red uptake method was applied to assess the phagocytic potential of cells. Cells were cultured and seeded as described in section \\u003cspan refid=\\\"Sec15\\\" class=\\\"InternalRef\\\"\\u003e2.6.1\\u003c/span\\u003e. After protein and LPS treatment for 24 hours, the supernatant was removed, and the subsequent experiments were conducted as in previous study.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec17\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.6.3 ROS production\\u003c/h2\\u003e \\u003cp\\u003eThe amount of reactive oxygen species (ROS) was assessed by the fluorescent probe 2\\u0026rsquo;,7\\u0026rsquo;-Dichlorodihydrofluorescein diacetate (DCFH-DA) from Sigma as described by Jayasinghe et al. (\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e) with slight adjustments. In summary, each well of a 96-well black plate was seeded and incubated according to the procedure outlined in section \\u003cspan refid=\\\"Sec15\\\" class=\\\"InternalRef\\\"\\u003e2.6.1\\u003c/span\\u003e. After 24 hours of cultivation, 100 \\u0026micro;L of EUE-extracted protein (0.2 \\u0026micro;g/mL) was further added to each well. Following a one-hour incubation at 37 ℃, cells were treated with DCFH-DA (10 \\u0026micro;M) and further cultured for 23 h at 37 ℃. Fluorescence intensity was measured with an excitation wavelength of 485 nm and an emission wavelength of 527 nm.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec18\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.7 Statistical Data Analysis\\u003c/h2\\u003e \\u003cp\\u003eThe results are presented as the mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;standard deviation (SD) from three independent replicates. Statistical analyses were performed in Prism 9 using a student\\u0026rsquo;s T-test or one-way ANOVA followed by a Tukey post-hoc test. A \\u003cem\\u003ep\\u003c/em\\u003e-value of less than 0.05 was considered statistically significant.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"3. Results and discussion\",\"content\":\"\\u003cdiv id=\\\"Sec20\\\" class=\\\"Section2\\\"\\u003e\\n \\u003ch2\\u003e3.1 Comparison of protein yield and content from various extraction methods\\u003c/h2\\u003e\\n \\u003cp\\u003eTable \\u003cspan class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e is a summary of the crude protein yields by various extraction methods. When the UAE process was applied alone, the protein content was the lowest compared to the other methods. Compared to UAE, protein extraction using the WE extraction method with a NaCl solution and precipitation with (NH\\u003csub\\u003e4\\u003c/sub\\u003e)\\u003csub\\u003e2\\u003c/sub\\u003eSO\\u003csub\\u003e4\\u003c/sub\\u003e led to higher yields and content. This may be caused by the ability of salt ions to inhibit electrostatic protein-protein interactions, consequently improving the extractability. Cellulase is a highly effective biocatalyst for degrading carbohydrate fiber and cell walls, so as to facilitate protein extraction. In the single-step enzyme-assisted extraction EAE process, the protein yield was 4.9% and the protein content was 50% (Table \\u003cspan class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e\\u003cstrong\\u003e)\\u003c/strong\\u003e. In comparison, the two-step EUE process resulted in a higher protein yield and content.\\u003c/p\\u003e\\n \\u003cp\\u003eTwo alternative process schemes were compared with the EUE, UAE followed by EAE (UEE) and simultaneous enzymatic and SEUE (concurrent application of EAE and UAE), both resulting in lower protein yield and content (\\u003cstrong\\u003eFigure S1\\u003c/strong\\u003e). The lower extraction efficiency of UEE than EUE suggests that the mechanical effect of ultrasound is more effective when the cell walls are partially disrupted by the enzyme cellulase, but less effective when the cell walls are intact. In the UEE method (enzyme extraction after ultrasound extraction), intact cell walls likely resist disruption due to their dense, unmodified structure. In contrast, the EUE method employs enzymatic pretreatment to weaken the cell wall, making it more susceptible to cavitation-induced fracture during ultrasound treatment. Specifically, cellulase breaks down cell walls by hydrolyzing \\u0026beta;-1,4-glycosidic bonds in cellulose, degrading the crystalline cellulose network and hemicellulose matrix. This enzymatic action reduces cell wall rigidity and compromises its mechanical integrity (Nakazawa et al., \\u003cspan class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e; Zhang et al., \\u003cspan class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e). Enzymatic pretreatment fragments the cell wall structure, enhancing permeability and weakening its integrity (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eB), which reduces the energy required for ultrasound to breach the compromised cellulose-hemicellulose network, thereby maximizing physical disruption efficiency and facilitating the release of intracellular proteins for higher extraction yields. This concurs with previous findings by Turker and Isleroglu (\\u003cspan class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e) that enzyme pretreatment followed by ultrasonication was effective for protein extraction. As for the SEUE scheme, the lower efficiency may be explained by the fact that the two require different optimal conditions for effective protein extraction. Consequently, the EUE scheme achieved a higher protein yield due to a greater synergy between enzyme and ultrasound. Meanwhile, the EUE extraction method significantly enhances extraction efficiency and reduces extraction time, particularly when compared to the modified traditional WE method.\\u003c/p\\u003e\\n \\u003cp\\u003eAdditionally, the protein extracted by the EUE method exhibited stronger immunostimulatory activities than those by the other three methods (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e). This is likely attributable to the higher protein content achieved through the EUE method compared to other extraction techniques, potentially offering more accessible epitopes for immune recognition (Zhao et al., \\u003cspan class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e). Besides, the EUE method demonstrated the highest \\u0026beta;-sheet content (Table \\u003cspan class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e), which contributes to creating a stable and rigid framework. This structural feature helps maintain the conformation of the epitope, thereby enhancing its accessibility for antibody binding (Rodrigues et al., \\u003cspan class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e). Meanwhile, its high \\u0026alpha;-helix content contributes to overall structural stability without introducing excessive rigidity, potentially balancing stability with the flexibility necessary for optimal epitope exposure. Despite its low \\u0026beta;-turn content (2.7%), the high proportion of \\u0026beta;-sheets compensates by preserving structural integrity and ensuring epitope accessibility. In contrast, EAE exhibited the weakest activity, which can be attributed to its low \\u0026beta;-turn content (Table \\u003cspan class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e). This deficiency may result in protein misfolding, reduced flexibility, and stability issues, potentially hindering epitope exposure and effective binding to immune receptors (Marcelino \\u0026amp; Gierasch, \\u003cspan class=\\\"CitationRef\\\"\\u003e2008\\u003c/span\\u003e). Although the WE method did not exhibit a high \\u0026beta;-sheet structure, it possessed a high random coil content (Table \\u003cspan class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e), which may enhance flexibility and improve epitope exposure, thereby facilitating recognition by immune cells (Fan et al., \\u003cspan class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e). Regarding UAE, its \\u0026beta;-turn content was higher than that of EAE (Table\\u0026nbsp;\\u003cspan class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e), which may aid in maintaining local conformational stability and facilitate the correct positioning of epitopes. In summary, the EUE method was chosen for optimization to achieve maximum protein yield because it can produce a high yield with substantial protein content and strong immunostimulatory activity.\\u003c/p\\u003e\\n \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u0026nbsp;\\u003ctable id=\\\"Tab2\\\" border=\\\"1\\\"\\u003e\\n \\u003ccaption language=\\\"En\\\"\\u003e\\n \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 2\\u003c/div\\u003e\\n \\u003cdiv class=\\\"CaptionContent\\\"\\u003e\\n \\u003cp\\u003eProtein yields, contents, and secondary structures by various separation methods.\\u003c/p\\u003e\\n \\u003c/div\\u003e\\n \\u003c/caption\\u003e\\n \\u003ccolgroup cols=\\\"10\\\"\\u003e\\u003c/colgroup\\u003e\\n \\u003cthead\\u003e\\n \\u003ctr\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\u0026nbsp;\\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\" colspan=\\\"3\\\"\\u003e\\n \\u003cp\\u003eProtein yield (%) \\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003eTotal protein content (%)\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\" colspan=\\\"3\\\"\\u003e\\n \\u003cp\\u003eTotal sugar content (%)\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\" colspan=\\\"1\\\"\\u003e\\u0026nbsp;\\u003c/th\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/thead\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eWE\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"3\\\"\\u003e\\n \\u003cp\\u003e6.5\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003e47.3\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.0\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"3\\\"\\u003e\\n \\u003cp\\u003e43.1\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2.5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"1\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eUAE\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"3\\\"\\u003e\\n \\u003cp\\u003e6.9\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003e40.9\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;5.8\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"3\\\"\\u003e\\n \\u003cp\\u003e51.4\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;4.4\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"1\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eEAE\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"3\\\"\\u003e\\n \\u003cp\\u003e4.9\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.3\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003e50.2\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2.7\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"3\\\"\\u003e\\n \\u003cp\\u003e45.8\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;3.7\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"1\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eEUE\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"3\\\"\\u003e\\n \\u003cp\\u003e9.4\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.4\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003e56.0\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2.2\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"3\\\"\\u003e\\n \\u003cp\\u003e38.7\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2.0\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"1\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"8\\\"\\u003e\\n \\u003cp\\u003eProtein secondary structure\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eRandom coil%\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003e\\u0026alpha;-Helix%\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003e\\u0026beta;-Sheet%\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"3\\\"\\u003e\\n \\u003cp\\u003e\\u0026beta;-Turn%\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003eWE\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e40.2\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;3.4\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003e20.9\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.7\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003e20.6\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.6\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"3\\\"\\u003e\\n \\u003cp\\u003e18.3\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.4\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003eUAE\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e33.3\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.3\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003e13.9\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.0\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003e38.5\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;4.0\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"3\\\"\\u003e\\n \\u003cp\\u003e14.3\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.2\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003eEAE\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e29.5\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;3.3\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003e19.9\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2.1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003e43.2\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.9\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"3\\\"\\u003e\\n \\u003cp\\u003e7.4\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003eEUE\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e15.2\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.3\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003e26.3\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;3.5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003e55.8\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;3.9\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"3\\\"\\u003e\\n \\u003cp\\u003e2.7\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.3\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n \\u003c/table\\u003e\\n \\u003c/div\\u003e\\n \\u003cp\\u003ea. Equal to the mass of freeze-dried soluble protein divided by the mass of original mushroom powder.\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003cdiv id=\\\"Sec21\\\" class=\\\"Section2\\\"\\u003e\\n \\u003ch2\\u003e3.2 Effects of EUE conditions on protein yield\\u003c/h2\\u003e\\n \\u003cp\\u003eFigure \\u003cspan class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA demonstrates the impact of enzyme concentration ranging from 0.2 to 0.8 (w/v) on the protein yield. All other conditions held constant (EAE 60 minutes, UAE 40 minutes, US amplitude 60%, and (NH\\u003csub\\u003e4\\u003c/sub\\u003e)\\u003csub\\u003e2\\u003c/sub\\u003eSO\\u003csub\\u003e4\\u003c/sub\\u003e saturation 70%. As the amount of cellulase increased, both protein yield and content initially rose, then declined, and eventually stabilized. The lowest protein yield was achieved at 0.2%, which shows a significant difference compared to the yield at 0.3%. A 0.3% cellulase addition resulted in the highest protein yield and content. At certain liquid-to-material ratios, the protein extraction efficiency increases with cellulase levels rise. However, as the amount of cellulase added increases, it leads to a weakening of the diffusive effect of the mushroom powder and a decrease in the diffusive effect of cellulase, resulting in a decrease in the protein extraction rate (Xu et al., \\u003cspan class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e). Therefore, 0.3% was chosen as the central enzyme concentration for the RSM analysis.\\u003c/p\\u003e\\n \\u003cp\\u003eFigure \\u003cspan class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eB indicates the effect of enzyme treatment time (30\\u0026ndash;120 minutes). Although there was insignificant (\\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;\\u0026gt;\\u0026thinsp;0.05) between the various time periods, the protein output showed a rising and then declining pattern as treatment time rose, while the protein content appeared a significant difference (\\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05). The protein extraction rate increased with time (30\\u0026ndash;60 minutes), reaching its maximum at 60 minutes, and then declining trend. With a longer treatment time, cellulase further hydrolyzes the broken cell walls of \\u003cem\\u003eL. edodes\\u003c/em\\u003e, resulting in increased protein solubilization and extraction rate. However, the protein content decreased due probably to protein aggregation, which can negatively impact the extraction rate.\\u003c/p\\u003e\\n \\u003cp\\u003eThe protein yield was little impacted by changing the UAE time from 20 to 60 minutes, as seen in Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eC (\\u003cem\\u003ep\\u0026thinsp;\\u0026gt;\\u003c/em\\u003e\\u0026thinsp;0.05) while obviously affected on soluble protein content when the other conditions remained constant. As illustrated in Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eC, within the interval of 20 to 40 minutes of ultrasound treatment, the protein content increases with the extension of ultrasound time, reaching its maximum at 40 minutes. With a further increase in ultrasound treatment time, the protein content starts to decrease, which may be protein denaturation and subsequently lowering the extraction rate.\\u003c/p\\u003e\\n \\u003cp\\u003eProtein production varies with US amplitude, as shown in Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eD. At a US amplitude of 60%, the maximum protein content was observed at 56%. Protein content enhanced with the rise in US amplitude from 20%-60% but started to decline with a higher amplitude. Despite the decrease in protein extraction rate mentioned earlier, the crude protein yield continued to increase. This can be attributed to the release of other components such as PS, and polyphenols along with the protein as the US amplitude increased. These other components, although not affected by the destructive effects of ultrasound, could increase the crude protein yield. In order to attain a high protein content, a central point of 60% US amplitude was chosen for the response surface methodology (RSM) experiments.\\u003c/p\\u003e\\n \\u003cp\\u003eFigure \\u003cspan class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eE shows the effect of (NH\\u003csub\\u003e4\\u003c/sub\\u003e)\\u003csub\\u003e2\\u003c/sub\\u003eSO\\u003csub\\u003e4\\u003c/sub\\u003e saturation from 20\\u0026ndash;100% on protein extraction yield. With the rise of (NH\\u003csub\\u003e4\\u003c/sub\\u003e)\\u003csub\\u003e2\\u003c/sub\\u003eSO\\u003csub\\u003e4\\u003c/sub\\u003e amount, protein content and yield were both increased, probably attributed to the stronger salting-out effect. No significant difference was seen in the crude protein yield between 70% as well as 100% saturation of (NH\\u003csub\\u003e4\\u003c/sub\\u003e)\\u003csub\\u003e2\\u003c/sub\\u003eSO\\u003csub\\u003e4\\u003c/sub\\u003e (\\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;\\u0026gt;\\u0026thinsp;0.05). A central point of 70% (NH\\u003csub\\u003e4\\u003c/sub\\u003e)\\u003csub\\u003e2\\u003c/sub\\u003eSO\\u003csub\\u003e4\\u003c/sub\\u003e saturation was selected for the RSM experiments.\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e3.3 EUE extraction conditions optimized using RSM\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cdiv id=\\\"Sec22\\\" class=\\\"Section3\\\"\\u003e\\n \\u003ch2\\u003e3.3.1 Model Fitting and Statistical Evaluation\\u003c/h2\\u003e\\n \\u003cp\\u003eRSM analysis was used to determine the optimal EUE protein extraction conditions from \\u003cem\\u003eL. edodes.\\u003c/em\\u003e The effects of enzyme additional amount, US amplitude, and (NH\\u003csub\\u003e4\\u003c/sub\\u003e)\\u003csub\\u003e2\\u003c/sub\\u003eSO\\u003csub\\u003e4\\u003c/sub\\u003e saturation on the protein yield and content were tested using a BBD. The results of all 17 experimental runs are presented in Table \\u003cspan class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e. Regression analysis was performed by fitting a response surface model to all the responses, from which the multiple regression equation was derived. These equations represent the empirical correlation between the responses and the independent variables, as displayed:\\u003c/p\\u003e\\n \\u003cdiv id=\\\"Equ1\\\" class=\\\"Equation\\\"\\u003e\\n \\u003cdiv class=\\\"mathdisplay\\\" id=\\\"FileID_Equ1\\\" name=\\\"EquationSource\\\"\\u003e$$\\\\:{Y}_{1}=-29.01+18.99{X}_{1}+\\\\:0.47{X}_{2}+0.59{X}_{3}+0.20{X}_{1}{X\\\\:}_{2}-0.01{X}_{1}{X}_{3}-0.003{X}_{2}{X}_{3}-53.24{X}_{1}^{2}-0.002{X}_{2}^{3}-0.003{X}_{3}^{2}$$\\u003c/div\\u003e\\n \\u003cdiv class=\\\"EquationNumber\\\"\\u003e3.1\\u003c/div\\u003e\\n \\u003c/div\\u003e\\n \\u003cdiv id=\\\"Equ2\\\" class=\\\"Equation\\\"\\u003e\\n \\u003cdiv class=\\\"mathdisplay\\\" id=\\\"FileID_Equ2\\\" name=\\\"EquationSource\\\"\\u003e$$\\\\:{Y}_{2}=-68.35+175.93{X}_{1}-\\\\:0.10{X}_{2}+3.11{X}_{3}-0.02\\\\:{X}_{1}{X}_{2}-0.90{X}_{1}{X}_{3}\\\\:-0.01{X}_{2}{X}_{3}-207.06{X}_{1}^{2}+0.003{X}_{2}^{2}-0.02{X}_{3}^{2}$$\\u003c/div\\u003e\\n \\u003cdiv class=\\\"EquationNumber\\\"\\u003e3.2\\u003c/div\\u003e\\n \\u003c/div\\u003e\\n \\u003cp\\u003eIn the following regression equations, \\u003cem\\u003eY\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e1\\u003c/em\\u003e\\u003c/sub\\u003e and \\u003cem\\u003eY\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e2\\u003c/em\\u003e\\u003c/sub\\u003e are the responses, representing protein yield and its content, respectively. \\u003cem\\u003eX\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e1\\u003c/em\\u003e\\u003c/sub\\u003e-\\u003cem\\u003eX\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e3\\u003c/em\\u003e\\u003c/sub\\u003e denotes the actual values of the independent variables. The \\u003cem\\u003eF\\u003c/em\\u003e-test was used to assess the significant impact of \\u003cem\\u003eX\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e1\\u003c/em\\u003e\\u003c/sub\\u003e-\\u003cem\\u003eX\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e3\\u003c/em\\u003e\\u003c/sub\\u003e on the \\u003cem\\u003eY\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e1\\u003c/em\\u003e\\u003c/sub\\u003e and \\u003cem\\u003eY\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e2\\u003c/em\\u003e\\u003c/sub\\u003e. US amplitude (\\u003cem\\u003eX\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e2\\u003c/em\\u003e\\u003c/sub\\u003e) and (NH\\u003csub\\u003e4\\u003c/sub\\u003e)\\u003csub\\u003e2\\u003c/sub\\u003eSO\\u003csub\\u003e4\\u003c/sub\\u003e saturation (\\u003cem\\u003eX\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e3\\u003c/em\\u003e\\u003c/sub\\u003e) were found to significantly influence both protein yield and content (\\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05). In contrast, enzyme amount (\\u003cem\\u003eX\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e1\\u003c/em\\u003e\\u003c/sub\\u003e) as well as the interactive effects of US amplitude \\u0026amp; (NH\\u003csub\\u003e4\\u003c/sub\\u003e)\\u003csub\\u003e2\\u003c/sub\\u003eSO\\u003csub\\u003e4\\u003c/sub\\u003e saturation (\\u003cem\\u003eX\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e2\\u003c/em\\u003e\\u003c/sub\\u003e : \\u003cem\\u003eX\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e3\\u003c/em\\u003e\\u003c/sub\\u003e) significantly affected protein content (\\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05). However, enzyme amount (\\u003cem\\u003eX\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e1\\u003c/em\\u003e\\u003c/sub\\u003e) did not significantly affect protein yield. Besides, the model was determined to be significant for both responses (\\u003cem\\u003eY\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e1\\u003c/em\\u003e\\u003c/sub\\u003e and \\u003cem\\u003eY\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e2\\u003c/em\\u003e\\u003c/sub\\u003e) (Table \\u003cspan class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e). Lack-of-fit value showing no significant difference, as evidenced by \\u003cem\\u003ep\\u003c/em\\u003e-values of 0.16 and 0.32 for model equations \\u003cspan class=\\\"InternalRef\\\"\\u003e3.1\\u003c/span\\u003e and \\u003cspan class=\\\"InternalRef\\\"\\u003e3.2\\u003c/span\\u003e, respectively. This demonstrates that the model is reliable for predicting protein yield and content. The adequacy of this model was confirmed via the coefficient of determination (R\\u003csup\\u003e2\\u003c/sup\\u003e), with values of 0.944 (\\u003cem\\u003eY\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e1\\u003c/em\\u003e\\u003c/sub\\u003e) and 0.924 for (\\u003cem\\u003eY\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e2\\u003c/em\\u003e\\u003c/sub\\u003e), respectively. A higher R\\u0026sup2; value indicates a strong fit between the empirical models and the experimental data. Three-dimensional response surface plots were generated in order to explore the interaction effects of \\u003cem\\u003eX\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e1\\u003c/em\\u003e\\u003c/sub\\u003e\\u003cem\\u003e-X\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e3\\u003c/em\\u003e\\u003c/sub\\u003e on \\u003cem\\u003eY\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e1\\u003c/em\\u003e\\u003c/sub\\u003e and \\u003cem\\u003eY\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e2\\u003c/em\\u003e\\u003c/sub\\u003e (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e). One variable was kept constant while the other two were changed in these graphs. Multiple solutions were generated via this approach, with the most desirable solution, exhibiting a desirability value of 0.736, being presented in this study, which is close to 1. The contour plots (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e) show that the optimum conditions for the protein with high content and high yield by the EUE extraction were 0.28% (w/v) enzyme, 62% US amplitude and 69% (NH\\u003csub\\u003e4\\u003c/sub\\u003e)\\u003csub\\u003e2\\u003c/sub\\u003eSO\\u003csub\\u003e4\\u003c/sub\\u003e saturation, predicting a maximum protein yield of 9.7% and content of 57.1%. The optimal conditions were validated through a verification experiment. The verification experiment yielded a protein yield of 9.7\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.6% and a content of 58.4\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.3%, which closely matched the model-predicted value. The findings also indicated that the response models accurately represented the optimization targets. In general, the accuracy was satisfactory, and the response surface models were effective for predicting responses.\\u003c/p\\u003e\\n \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u0026nbsp;\\u003ctable id=\\\"Tab3\\\" border=\\\"1\\\"\\u003e\\n \\u003ccaption language=\\\"En\\\"\\u003e\\n \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 3\\u003c/div\\u003e\\n \\u003cdiv class=\\\"CaptionContent\\\"\\u003e\\n \\u003cp\\u003eRSM experimental design (3\\u003csup\\u003e3\\u003c/sup\\u003e BBD) and the resulting protein yield and content outcomes.\\u003c/p\\u003e\\n \\u003c/div\\u003e\\n \\u003c/caption\\u003e\\n \\u003ccolgroup cols=\\\"9\\\"\\u003e\\u003c/colgroup\\u003e\\n \\u003cthead\\u003e\\n \\u003ctr\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eRun\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\" colspan=\\\"4\\\"\\u003e\\n \\u003cp\\u003eIndependent variables\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003eResponse \\u003cem\\u003eY\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e1\\u003c/em\\u003e\\u003c/sub\\u003e\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003eResponse \\u003cem\\u003eY\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e2\\u003c/em\\u003e\\u003c/sub\\u003e\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/thead\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003eX\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e1\\u003c/em\\u003e\\u003c/sub\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003eX\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e2\\u003c/em\\u003e\\u003c/sub\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003eX\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e3\\u003c/em\\u003e\\u003c/sub\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eExperiment\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003ePredicted\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eExperiment\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003ePredicted\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003e1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e0.5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e60\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e90\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e5.0\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e5.1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e39.3\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.7\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e38.4\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003e2\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e0.3\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e60\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e70\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e9.0\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.4\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e9.5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e55.4\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.2\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e57.5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003e3\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e0.5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e60\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e50\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e6.3\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e6.6\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e43.6\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2.9\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e41.8\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003e4\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e0.3\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e60\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e70\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e9.7\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.9\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e9.5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e58.3\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2.9\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e57.5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003e5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e0.3\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e40\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e90\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e5.9\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e6.3\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e63.5\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.8\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e62.9\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003e6\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e0.1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e60\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e50\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e7.3\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e7.1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e38.8\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.3\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e39.8\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003e7\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e0.1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e80\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e70\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e7.3\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.7\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e7.9\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e48.9\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.9\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e47.4\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003e8\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e0.1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e40\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e70\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e6.1\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.6\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e6.0\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e60.0\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.9\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e58.7\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003e9\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e0.3\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e80\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e90\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e7.5\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.7\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e7.3\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e45.6\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.3\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e45.3\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003e10\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e0.3\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e60\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e70\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e9.4\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.3\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e9.5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e57.2\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e57.5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003e11\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e0.3\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e40\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e50\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e5.1\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.7\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e5.3\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e52.5\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.8\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e52.9\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003e12\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e0.3\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e60\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e70\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e10.0\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.9\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e9.5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e60.3\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.2\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e57.5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003e13\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e0.3\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e60\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e70\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e9.4\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.3\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e9.5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e56.4\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.4\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e57.5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003e14\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e0.5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e40\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e70\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e4.3\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e3.8\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e52.2\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.3\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e53.7\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003e15\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e0.5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e80\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e70\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e8.8\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.4\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e8.9\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e40.7\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.9\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e42.0\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003e16\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e0.1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e60\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e90\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e6.0\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.02\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e5.7\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e48.9\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e50.8\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003e17\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e0.3\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e80\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e50\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e11.7\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e11.3\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e47.0\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.2\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e47.6\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n \\u003c/table\\u003e\\n \\u003c/div\\u003e\\n \\u003cdiv class=\\\"gridtable\\\"\\u003e\\n \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\"\\u003e\\u003cbr\\u003e\\u003c/div\\u003e\\u0026nbsp;\\u003ctable id=\\\"Tab4\\\" border=\\\"1\\\"\\u003e\\n \\u003ccaption language=\\\"En\\\"\\u003e\\n \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 4\\u003c/div\\u003e\\n \\u003cdiv class=\\\"CaptionContent\\\"\\u003e\\n \\u003cp\\u003eANOVA of fitted quadratic models of two responses.\\u003c/p\\u003e\\n \\u003c/div\\u003e\\n \\u003c/caption\\u003e\\n \\u003ccolgroup cols=\\\"5\\\"\\u003e\\u003c/colgroup\\u003e\\n \\u003cthead\\u003e\\n \\u003ctr\\u003e\\n \\u003cth align=\\\"left\\\" rowspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003eSource\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003eY\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e1\\u003c/em\\u003e\\u003c/sub\\u003e: protein yield (%)\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003eY\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e2\\u003c/em\\u003e\\u003c/sub\\u003e: protein content (%)\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003eF\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003ep\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003eF\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003ep\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/thead\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eModel\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e31.05\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e\\u0026lt;\\u0026thinsp;0.0001\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e22.63\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e0.0002\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003eX\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e1\\u003c/em\\u003e\\u003c/sub\\u003e-enzyme amount\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e2.55\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e0.1542\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e11.74\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e0.0110\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003eX\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e2\\u003c/em\\u003e\\u003c/sub\\u003e-US amplitude\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e99.06\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e\\u0026lt;\\u0026thinsp;0.0001\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e57.36\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e0.0001\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003eX\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e3\\u003c/em\\u003e\\u003c/sub\\u003e-(NH\\u003csub\\u003e4\\u003c/sub\\u003e)\\u003csub\\u003e2\\u003c/sub\\u003eSO\\u003csub\\u003e4\\u003c/sub\\u003e saturation\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e17.83\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e0.0039\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e6.39\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e0.0393\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003eX\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e1\\u003c/em\\u003e\\u003c/sub\\u003e\\u003cem\\u003eX\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e2\\u003c/em\\u003e\\u003c/sub\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e10.49\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e0.0143\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e0.0080\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e0.9311\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003eX\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e1\\u003c/em\\u003e\\u003c/sub\\u003e\\u003cem\\u003eX\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e3\\u003c/em\\u003e\\u003c/sub\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e0.0126\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e0.9139\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e11.39\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e0.0118\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003eX\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e2\\u003c/em\\u003e\\u003c/sub\\u003e\\u003cem\\u003eX\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e3\\u003c/em\\u003e\\u003c/sub\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e25.72\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e0.0014\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e8.38\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e0.0231\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003eX\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e1\\u003c/em\\u003e\\u003c/sub\\u003e\\u003cem\\u003e\\u0026sup2;\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e77.77\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e\\u0026lt;\\u0026thinsp;0.0001\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e63.01\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e\\u0026lt;\\u0026thinsp;0.001\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003eX\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e2\\u003c/em\\u003e\\u003c/sub\\u003e\\u003cem\\u003e\\u0026sup2;\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e9.39\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e0.0182\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e1.32\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e0.2876\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003eX\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e3\\u003c/em\\u003e\\u003c/sub\\u003e\\u003cem\\u003e\\u0026sup2;\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e26.12\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e0.0014\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e39.68\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e0.0004\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eLack of Fit\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e2.95\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e0.1613\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e1.62\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e0.3192\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eR\\u003csup\\u003e2\\u003c/sup\\u003e\\u0026thinsp;=\\u0026thinsp;0.976\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eR\\u003csup\\u003e2\\u003c/sup\\u003e\\u003csub\\u003eadj\\u003c/sub\\u003e\\u0026thinsp;=\\u0026thinsp;0.944\\u003c/p\\u003e\\n \\u003cp\\u003eC.V.% = 6.54\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eR\\u003csup\\u003e2\\u003c/sup\\u003e\\u0026thinsp;=\\u0026thinsp;0.967\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eR\\u003csup\\u003e2\\u003c/sup\\u003e\\u003csub\\u003eadj\\u003c/sub\\u003e\\u0026thinsp;=\\u0026thinsp;0.924\\u003c/p\\u003e\\n \\u003cp\\u003eC.V.% = 4.19\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n \\u003c/table\\u003e\\n \\u003c/div\\u003e\\n \\u003c/div\\u003e\\n\\u003c/div\\u003e\\n\\u003cdiv id=\\\"Sec23\\\" class=\\\"Section2\\\"\\u003e\\n \\u003ch2\\u003e3.4 Structure characteristics of EUE-extracted proteins\\u003c/h2\\u003e\\n \\u003cp\\u003eIn the CD spectrum (\\u003cstrong\\u003eFigure S2\\u003c/strong\\u003e), the protein structure was characterized by random coil 10.0\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.4%, \\u0026alpha;-helix 29.3\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2.6%, \\u0026beta;-sheet 56.7\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;6.2%, and \\u0026beta;-turn 4.0\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.6%. A previous study identified the \\u0026beta;-sheet as the predominant structure in proteins extracted from the stipe of \\u003cem\\u003eL. edodes\\u003c/em\\u003e mushrooms, which is consistent with our findings (Hu, \\u003cspan class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e). In \\u003cstrong\\u003eFigure S3\\u003c/strong\\u003e, the EUE-extracted protein under optimized conditions exhibits characteristic peaks of amide I (1638 cm\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e) and amide III (1242 cm\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e). Amide I and amide III bands result from the N-H and C-N stretching vibrations, as well as N-H bending vibrations. The absorption band at 3411 cm\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e is associated with the stretching vibration of O\\u0026ndash;H in the molecular structure, probably due to the presence of PS. This aligns with our findings, as the EUE-extracted protein can also acquire PS, as shown in Table \\u003cspan class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e. The anti-symmetric stretching vibration of alkanes (-C-H-) is responsible for the band at 2919 cm\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e. The absorption bands at 1547 cm\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e as well as 1080 cm\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e arise from the in-plane deformation vibration of NH\\u003csub\\u003e2\\u003c/sub\\u003e and the in-plane bending vibration of NH, respectively, in the amide II region. From the SDS-PAGE analysis (\\u003cstrong\\u003eFigure S4\\u003c/strong\\u003e), it was found that most protein bands were \\u0026lt;\\u0026thinsp;10 kDa. Protein bands appeared at 25, 34 and 43\\u0026ndash;75 kDa.\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003cdiv id=\\\"Sec24\\\" class=\\\"Section2\\\"\\u003e\\n \\u003ch2\\u003e3.5 Composition of proteins extracted through EUE\\u003c/h2\\u003e\\n \\u003cp\\u003eTable \\u003cspan class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e presents the AA profiles of partially purified proteins extracted by EUE. As shown in Table \\u003cspan class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eA, the ratio of the sum of Glu and Asp to the sum of Lys, Arg, as well as His was 1.22. Thus, the EUE-extracted protein sample was acidic proteins. The protein nutritional value is primarily determined by the type, content, quantity, as well as composition of EAA. The protein extracted via EUE comprised EAA and non-essential amino acids (NEAA) with a total amount of 437.0 mg/g, which exceeded the amount of protein in other edible fungi such as oyster mushrooms and enoki mushrooms. The content of EEA constitutes over 40% of the total amino acids (TAA) content. EAA/NEAA value was close to 80%, in line with the recommendations proposed by FAO/WHO ideal protein condition. Furthermore, the value of isoleucine and valine exceeds the range required by FAO/WHO for adults (Wang et al., \\u003cspan class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e), indicating that \\u003cem\\u003eL. edodes\\u003c/em\\u003e can be used as a good nutritional protein source for adults.\\u003c/p\\u003e\\n \\u003cp\\u003eAs stated by the AA scoring model recommended by FAO/WHO and the AA profile of egg protein, the amino acid score (AAS) as well as chemical score (CS) values for EUE protein are presented in Table \\u003cspan class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eB. When AAS was used as the standard, isoleucine content was the highest, 2.08 times of the standard. The first limiting AA was Met\\u0026thinsp;+\\u0026thinsp;Cys, while Lys was the second limiting AA. The highest isoleucine content was 1.41 times the standard when CS was used as the standard. Met\\u0026thinsp;+\\u0026thinsp;Cys was also recognized as first limiting AA. Therefore, the main limiting AA in the \\u003cem\\u003eL. edodes\\u003c/em\\u003e protein were Met\\u0026thinsp;+\\u0026thinsp;Cys and lysine. Met\\u0026thinsp;+\\u0026thinsp;Cy was also identified as the first limiting AA based on the RC value. Lysine was the second limiting AA. This result aligns with a previous study that identified Met\\u0026thinsp;+\\u0026thinsp;Cys as the first limiting AA in the fungus \\u003cem\\u003eL. edodes\\u003c/em\\u003e. It was also agreed with our previous findings, which showed that Met\\u0026thinsp;+\\u0026thinsp;Cys is lacking in \\u003cem\\u003eL. edodes\\u003c/em\\u003e. The RC value of Leu was close to 1, indicating that the composition ratio of this AA in \\u003cem\\u003eL. edodes\\u003c/em\\u003e protein was close to that in the model spectrum. Val was a relative deficiency, and Ile and Thr were relatively abundance. Mushroom protein should be combined with other proteins to optimize its nutritional value based on the protein complementation theory. The nutritional quality of the protein improves as its SRC value increases. In this study, SRC value of EUE-extracted protein was 69.8%, which was comparable to that of soy protein, milk (72.60%), milk powder (67.31%), and walnuts (62.65%). The protein extracted from\\u0026nbsp;\\u003cem\\u003eL. edodes\\u003c/em\\u003e mushrooms using the EUE method possesses high nutritional value and offers potential for development and utilization.\\u003c/p\\u003e\\n \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u0026nbsp;\\u003ctable id=\\\"Tab7\\\" border=\\\"1\\\"\\u003e\\n \\u003ccaption language=\\\"En\\\"\\u003e\\n \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 5\\u003c/div\\u003e\\n \\u003cdiv class=\\\"CaptionContent\\\"\\u003e\\n \\u003cp\\u003eAmino acids in EUE-extracted protein samples.\\u003c/p\\u003e\\n \\u003c/div\\u003e\\n \\u003c/caption\\u003e\\n \\u003ccolgroup cols=\\\"7\\\"\\u003e\\u003c/colgroup\\u003e\\n \\u003cthead\\u003e\\n \\u003ctr\\u003e\\n \\u003cth align=\\\"left\\\" colspan=\\\"7\\\"\\u003e\\n \\u003cp\\u003e(A) Amino acid composition and contents (mg/g crude protein)\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/thead\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eEAA\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eContent\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"3\\\"\\u003e\\n \\u003cp\\u003eNEAA\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003eContent\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eHistidine (His)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e9.8\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2.6\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"3\\\"\\u003e\\n \\u003cp\\u003eAlanine (Ala)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003e26.3\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.6\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eIsoleucine (Ile)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e33.3\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.9\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"3\\\"\\u003e\\n \\u003cp\\u003eAsparagine (Arg)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003e2.3\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.04\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eLeucine (Leu)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e39.2\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2.6\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"3\\\"\\u003e\\n \\u003cp\\u003eAspartic acid (Asp)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003e36.3\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2.7\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eLysine (Lys)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e23.2\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.9\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"3\\\"\\u003e\\n \\u003cp\\u003eArginine (Arg)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003e22.9\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eMethionine (Met)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e9.1\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.0\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"3\\\"\\u003e\\n \\u003cp\\u003eGlutamic acid (Glu)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003e31.7\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2.2\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003ePhenylalanine (Phe)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e23.2\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"3\\\"\\u003e\\n \\u003cp\\u003eGlycine (Gly)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003e23.0\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.9\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eThreonine (Thr)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e25.9\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.4\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"3\\\"\\u003e\\n \\u003cp\\u003eSerine (Ser)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003e27.2\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2.0\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eValine (Val)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e25.0\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.4\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"3\\\"\\u003e\\n \\u003cp\\u003eTyrosine (Tyr)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003e17.6\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.6\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"3\\\"\\u003e\\n \\u003cp\\u003eCysteine (Cys)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003e2.1\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.3\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"3\\\"\\u003e\\n \\u003cp\\u003eGlutamine (Glu)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003e38.4\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;5.6\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"3\\\"\\u003e\\n \\u003cp\\u003eProline\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003e20.5\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.4\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eTAA\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eEAA\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eNEAA\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"3\\\"\\u003e\\n \\u003cp\\u003eEAA/TAA (%)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eEAA/NEAA (%)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e437.0\\u0026nbsp;\\u0026plusmn; 33.1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e188.7\\u0026nbsp;\\u0026plusmn; 13.3\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003e248.3\\u0026nbsp;\\u0026plusmn; 19.8\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003e43.2 \\u0026plusmn; 0.2\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e76.0\\u0026nbsp;\\u0026plusmn; 0.7\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"7\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e(B) Amino acid and chemical scores of protein samples\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u0026nbsp;\\u003ctable id=\\\"Taba\\\" border=\\\"1\\\"\\u003e\\n \\u003ccolgroup cols=\\\"5\\\"\\u003e\\u003c/colgroup\\u003e\\n \\u003cthead\\u003e\\n \\u003ctr\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\u0026nbsp;\\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eAAS (%)\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eCS (%)\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eRAA\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eRC\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/thead\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eIle\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e83.3\\u0026nbsp;\\u0026plusmn; 4.8\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e61.7\\u0026nbsp;\\u0026plusmn; 3.5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e0.83\\u0026nbsp;\\u0026plusmn; 0.05\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e1.47\\u0026nbsp;\\u0026plusmn; 0.01\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eLeu\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e56.0\\u0026nbsp;\\u0026plusmn; 3.7\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e45.6\\u0026nbsp;\\u0026plusmn; 3.0\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e0.56\\u0026nbsp;\\u0026plusmn; 0.04\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e0.99\\u0026nbsp;\\u0026plusmn; 0.00\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eLys\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e42.2\\u0026nbsp;\\u0026plusmn; 1.6\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e33.1\\u0026nbsp;\\u0026plusmn; 1.3\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e0.42\\u0026nbsp;\\u0026plusmn; 0.02\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e0.75\\u0026nbsp;\\u0026plusmn; 0.02\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eMet\\u0026thinsp;+\\u0026thinsp;Cys\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e32.0\\u0026nbsp;\\u0026plusmn; 3.7\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e19.6\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2.3\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e0.32\\u0026nbsp;\\u0026plusmn; 0.04\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e0.56\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.03\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003ePhe\\u0026thinsp;+\\u0026thinsp;Tyr\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e68.0\\u0026nbsp;\\u0026plusmn; 5.2\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e43.9\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;3.3\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e0.68\\u0026nbsp;\\u0026plusmn; 0.05\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e1.21\\u0026nbsp;\\u0026plusmn; 0.01\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eThr\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e64.8\\u0026nbsp;\\u0026plusmn; 3.5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e55.1\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;3.0\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e0.65\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.03\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e1.14\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.01\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eVal\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e50.0\\u0026nbsp;\\u0026plusmn; 2.8\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e37.9\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2.1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e0.50\\u0026nbsp;\\u0026plusmn; 0.03\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e0.88\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.01\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eSRC\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e69.8\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.6%\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n \\u003c/table\\u003e\\n \\u003c/div\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n \\u003ctfoot\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"7\\\"\\u003eNote: AAS\\u0026thinsp;=\\u0026thinsp;content of a specific EAA in the protein sample divided by its content in the FAO/WHO model protein (\\u003cstrong\\u003eTable S2\\u003c/strong\\u003e) (FAO/WHO, \\u003cspan class=\\\"CitationRef\\\"\\u003e1973\\u003c/span\\u003e; FAO/WHO, \\u003cspan class=\\\"CitationRef\\\"\\u003e2007\\u003c/span\\u003e); CS\\u0026thinsp;=\\u0026thinsp;content of an EAA in protein sample divided by its content in whole egg protein (\\u003cstrong\\u003eTable S2\\u003c/strong\\u003e); RAA (AA ratio)\\u0026thinsp;=\\u0026thinsp;content of an AA in protein divided by the its content in the FAO/WHO pattern spectrum; RC (ratio coefficient)\\u0026thinsp;=\\u0026thinsp;RAA value divided by the average RAA; SRC (score of ratio coefficient)\\u0026thinsp;=\\u0026thinsp;the standard deviation coefficient of RC (Zhao et al., \\u003cspan class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e).\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tfoot\\u003e\\n \\u003c/table\\u003e\\n \\u003c/div\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e3.6 Immunomodulatory activities\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003eAt a concentration of 4 \\u0026micro;g/mL, the EUE-extracted protein demonstrated no toxicity to RAW 264.7 cells (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eA). NO is essential in various physiological functions, particularly in host defense. NO production serves as a reliable measure of the immunocompetence of RAW 264.7 cells. As displayed in Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eB, EUE-extracted protein\\u0026apos;s NO production was more significant than the control group (\\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05). NO production level was around five times higher than that of the control group. Furthermore, phagocytosis is also one of the essential functions of macrophages. Neutral red phagocytosis was employed in this investigation as a marker of immune response activity, and the outcomes were consistent with the NO generation findings. The protein group\\u0026apos;s neutral red uptake was almost 1.5 times more than the control group\\u0026apos;s (\\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05) (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eC \\u003cstrong\\u003e\\u0026amp;D\\u003c/strong\\u003e). Moreover, as defensive elements and signaling molecules in immunological pathways, ROS can be produced by macrophage cells during phagocytosis. As anticipated, the EUE protein fractions stimulated ROS production in macrophage cells similar to pinocytic activity (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eE). Overall, the results confirmed that the EUE-extracted protein rich fraction exhibits in \\u003cem\\u003evitro\\u003c/em\\u003e immunostimulatory activity.\\u003c/p\\u003e\\n\\u003c/div\\u003e\"},{\"header\":\"4. Conclusions\",\"content\":\"\\u003cp\\u003eThe present study has demonstrated that the two-step, sequential enzyme- followed by ultrasound-assisted (EUE) was the effective process scheme for \\u003cem\\u003eL. edodes\\u003c/em\\u003e mushroom protein extraction with the highest protein yield and protein content, and immunostimulatory activity in comparison with the one-step process including WE, EAE, UAE. The EUE method also achieved a higher protein yield or content compared to both the simultaneous combination of EAE and UAE, as well as the two-step process of ultrasound followed by enzyme-assisted extraction (UEE). This outcome also suggests that the process scheme is a significant factor in achieving the synergistic or cooperative effect of different means and mechanisms of extraction. Moreover, enzyme concentration, ultrasound power, and ammonium salt saturation were the major process factors for the EUE process. The protein fraction showed a high nutritional value and significant \\u003cem\\u003ein vitro\\u003c/em\\u003e immunostimulatory activity. The results and findings from the present study may provide new ideas and a theoretical basis for the combined EUE extraction scheme in the extraction of proteins from \\u003cem\\u003eL. edodes\\u003c/em\\u003e and other mushrooms. However, its efficacy and feasibility for industrial application is still subject to pilot-scale trials and process economy analysis. It is also meaningful to conduct a more detailed investigation into the protein structures and their interactions with PS in relation to immunostimulatory activities by different extraction methods. For fundamental understanding and rational application, further research is needed to elucidate the mechanisms underlying the synergistic effects of enzyme and ultrasound interactions at the molecular level.\\u003c/p\\u003e \"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgments\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eSpecial thanks to So-Pu Kin for his support of LC-MS experiments and analysis.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthor Contributions\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eZC Zhao: Methodology, Investigation, Writing-original draft; YY Zhu: Methodology; FT Gu: Methodology; LX Huang: Methodology; XW Liu: Methodology; JY Wu: Funding, Project\\u003c/p\\u003e\\n\\u003cp\\u003eadministration, Supervision, Writing - review \\u0026amp; editing.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eData Availability\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe datasets produced or analyzed in this study can be obtained from the corresponding author upon request.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFunding\\u0026nbsp;\\u003c/strong\\u003ewas provided by The Hong Kong Polytechnic University (Ri-Food Project CD59).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCompeting Interests:\\u003c/strong\\u003e The authors declare no competing interests.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eAhmed, T., Suzauddula, M., Akter, K., Hossen, M., \\u0026amp; Islam, M. 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Extraction, purification and anti-proliferative activities of polysaccharides from \\u003cem\\u003eLentinus edodes\\u003c/em\\u003e. \\u003cem\\u003eInternational Journal of Biological Macromolecules\\u003c/em\\u003e, \\u003cem\\u003e93\\u003c/em\\u003e, 136\\u0026ndash;144. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1016/j.ijbiomac.2016.05.100\\u003c/span\\u003e\\u003cspan address=\\\"10.1016/j.ijbiomac.2016.05.100\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eZhao, Z. C., Gu, F. T., Li, J. H., Zhu, Y. Y., Huang, L. X., \\u0026amp; Wu, J. Y. (2024). Fractionation, characterization, and assessment of nutritional and immunostimulatory protein-rich polysaccharide-protein complexes isolated from \\u003cem\\u003eLentinula edodes\\u003c/em\\u003e mushroom. \\u003cem\\u003eInternational Journal of Biological Macromolecules\\u003c/em\\u003e, \\u003cem\\u003e280\\u003c/em\\u003e, 136082. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1016/j.ijbiomac.2024.136082\\u003c/span\\u003e\\u003cspan address=\\\"10.1016/j.ijbiomac.2024.136082\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eZhao, Z. C., Huang, L. X., Dong, X. L., \\u0026amp; Wu, J. Y. (2024). Evaluation of Three-Phase Partitioning for Efficient and Simultaneous Isolation of Immunomodulatory Polysaccharides and Proteins from \\u003cem\\u003eLentinula Edodes\\u003c/em\\u003e Mushroom. \\u003cem\\u003eFood and Bioprocess Technology\\u003c/em\\u003e, \\u003cem\\u003e17\\u003c/em\\u003e(8), 2277\\u0026ndash;2291. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1007/s11947-023-03262-3\\u003c/span\\u003e\\u003cspan address=\\\"10.1007/s11947-023-03262-3\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":true,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"food-and-bioprocess-technology\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"\",\"sideBox\":\"Learn more about [Food and Bioprocess Technology](https://www.springer.com/journal/11947)\",\"snPcode\":\"11947\",\"submissionUrl\":\"https://submission.nature.com/new-submission/11947/3\",\"title\":\"Food and Bioprocess Technology\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"Springer Hybrid\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false},\"keywords\":\"Lentinula edodes, Enzyme- and ultrasound-assisted extraction, Proteins, Amino acid profile, Nutrition value, Immunoactivity\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-6257446/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-6257446/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003e\\u003cem\\u003eLentinula edodes \\u003c/em\\u003eis a widely consumed edible fungus\\u003cem\\u003e \\u003c/em\\u003eand a rich source of proteins with both nutritional and medicinal value. This study discovered an effective process to extract proteins from\\u003cem\\u003e L. edodes\\u003c/em\\u003e mushroom by comparison of water extraction (WE), enzyme-assisted extraction (EAE), ultrasound-assisted extraction (UAE), and various combinations of EAE with UAE. The two-step and sequential scheme by EAE and then UAE, designated EUE resulted in the highest protein yield compared with EAE after UAE (UEE) and simultaneous EAE and UAE (SEUE). The soluble protein yield by EUE (9.4%) was nearly three times that by UEE (3.6%) and 1.4 times and around two times higher than by UAE (6.9%) and EAE (4.9%), respectively. Compared with those by other extraction methods, the protein fraction by EUE had the highest protein content (56.0%) and β-sheet content (55.8%) and exhibited the strongest \\u003cem\\u003ein vitro \\u003c/em\\u003eimmunostimulatory activity. Through statistically designed experiments and response surface methodology, EUE conditions were optimized as enzyme 0.28% (w/v), ultrasound amplitude 62%, and (NH\\u003csub\\u003e4\\u003c/sub\\u003e)\\u003csub\\u003e2\\u003c/sub\\u003eSO\\u003csub\\u003e4\\u003c/sub\\u003e saturation 69%, achieving 9.7% protein yield and 58.4% protein content. The distribution of protein molecular weight (MW) was below 10 kDa and between 25-75 kDa. The protein fraction contained nutritional amino acids and significant immunostimulatory activities \\u003cem\\u003ein vitro\\u003c/em\\u003e. EUE has shown promising potential for efficient extraction of proteins from mushrooms in the food industry.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Sequential Enzymatic and Ultrasonic Extraction of Lentinula edodes Mushroom Proteins Leading to Enhanced Yield and Significant Immunoactivity\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-04-04 10:27:56\",\"doi\":\"10.21203/rs.3.rs-6257446/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"decision\",\"content\":\"Revision requested\",\"date\":\"2025-05-12T14:39:15+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2025-04-24T16:12:17+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2025-03-31T09:16:47+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"206921928497629364122506047854476391529\",\"date\":\"2025-03-29T11:25:55+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"231678357495625533602522964094306971597\",\"date\":\"2025-03-28T08:18:59+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"228356944765999067741684466317071720611\",\"date\":\"2025-03-27T22:31:18+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"338089100232034558588856075862438931643\",\"date\":\"2025-03-27T18:22:51+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"302827795306971822371498531893611447294\",\"date\":\"2025-03-26T06:09:12+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2025-03-25T20:54:36+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2025-03-20T11:33:37+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"checksComplete\",\"content\":\"\",\"date\":\"2025-03-20T01:52:31+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Food and Bioprocess Technology\",\"date\":\"2025-03-19T03:18:47+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"food-and-bioprocess-technology\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"\",\"sideBox\":\"Learn more about [Food and Bioprocess Technology](https://www.springer.com/journal/11947)\",\"snPcode\":\"11947\",\"submissionUrl\":\"https://submission.nature.com/new-submission/11947/3\",\"title\":\"Food and Bioprocess Technology\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"Springer Hybrid\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false}}],\"origin\":\"\",\"ownerIdentity\":\"4b0669d4-dd0f-4513-9aa0-b4d37a11d379\",\"owner\":[],\"postedDate\":\"April 4th, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"under-review\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2025-08-20T09:23:51+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2025-04-04 10:27:56\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-6257446\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-6257446\",\"identity\":\"rs-6257446\",\"version\":[\"v1\"]},\"buildId\":\"8U1c8b4HqxoKbykW_rLl7\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}