Statistical optimization of fermentation conditions for metabolites production by Lentinus edodes and investigation of anticancer activity

Statistical optimization of fermentation conditions for metabolites production by Lentinus edodes and investigation of anticancer activity | 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 Article Statistical optimization of fermentation conditions for metabolites production by Lentinus edodes and investigation of anticancer activity Samaneh Zarea Garizi, Hamid Reza Samadlouie, Ahmad Rajaei, Amir Salek Farrokhi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6748184/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The investigation was carried out to increase polysaccharide yield utilizing statistical strategies, starting with submerge and shifting to solid-state fermentation, with the main aim of promoting the anticancer properties of Lentinus edodes (shiitake) fruit and exploring the rheological properties of polysaccharide. The Plackett–Burman (P.B) screening indicated that wheat starch, meat peptone, and vitamin B6 had a more pronounced impacted on polysaccharide production than the other substrates. The optimal substrates for polysaccharide production, identified using response surface methodology (RSM) after screening, was revealed to contain 48.56 g/l wheat starch, 15 g/l meat peptone, and 300 µg B6 vitamins. Shiitake polysaccharide solutions exhibited shear-thinning behavior, enhanced viscosity, and gel-like properties at higher concentrations. Shiitake polysaccharide also demonstrated superior emulsifying capacity for oil-in-water emulsions, suggesting its potential as an effective stabilizer in food formulations. The fruit body of shiitake produced under optimal polysaccharide conditions had the highest diversity in flavonoids and phenolic acids and significant anticancer activity against MDA-MB-231 breast cancer cell. More to the point, the nanoparticle induced aggravation of human breast cancer cell death were evident with IC50 levels of 182 μg/mL for fruit moss and 138 μg/mL for biomass cultivated in submerge condition containing MgO2 NPs. Biological sciences/Biotechnology Biological sciences/Microbiology Lentinus edode Response surface methodology nanoparticle polysaccharide rheological properties anticancer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Medical mushrooms, which serve as primary producers of bioactive components and biological macromolecule with remarkable health-promoting effects, represent a significant area of interest in the fields of biotechnology and pharmacology 1 , 2 . Among the diverse array of medical mushrooms, shiitake has garnered substantial attention due to its ability to synthesize valuable biological macromolecule such as polysaccharides, particularly β-glucans, ergosterol, and phenolic compounds 3 , possessing the potential to address various health conditions, including diabetes, cancer, the flu, heart disease, high blood pressure, and weakness 4 . Over the past two decades, numerous researchers have endeavored to investigate the flush of mushrooms and secondary metabolites of medical mushrooms when cultivated under various formulated deciduous woods 5 , 6 . Even with its effectiveness, this fermentation method has distinct drawbacks, one of which is its sole reliance on the culture medium for secondary metabolite production and the long period of cultivation required to harvest the fruit when it is at optimal stage of maturity 7 . The solid-state fermentation of shiitake is significantly influenced by the choice of key substrates utilized in formulated media 8 . Among these substrates, starch from rice bran and wheat bran plays a vital role in enhancing the production of secondary metabolites by Shiitake 9 , 10 . However, the formulation of solid-state media for mushroom cultivation heavily relies on the experience and expertise of cultivators, as no statistical analyses have been conducted to determine the most effective substrate for promoting shiitake growth and metabolite production 11 . The absence of precise measurements of biomass and metabolites during the solid-state fermentation prior to the fruiting process has impeded the optimization efforts 12 . More importantly, the cultivation of Shiitake mushrooms requires a considerable amount of time for the development of fully mature fruiting bodies, which is an essential aspect of their growth process 13 . The entire cycle, from the initial mycelium stage to the final maturity of the fruiting bodies, can span several months. Significantly, previous research comparing submerged and solid-state fermentation has indicated that submerged fermentation offers several advantages over solid-state fermentation. These advantages include the ease of controlling the physical and chemical conditions of the media, relatively lower spore contamination, and cost-effectiveness as a fermentation method 6 . The consensus among biotechnology experts is that the availability of macro elements, including carbon, nitrogen, and mineral substrates, nanoparticles and vitamins greatly influences the proliferation of mushrooms and the synthesis of secondary metabolites in submerged fermentation 6 , 14 . A substantial and expanding body of literature has been dedicated to identifying the suitable statistical methods for optimizing the physicochemical conditions of the medium to enhance metabolite productions 15 . Among various statistical techniques, RSM has been widely employed worldwide to mathematically determine the optimal conditions for key substrates that impact the desired responses 6 . Prior to optimization, screening methods such as P.B designs are commonly used to identify key substrates that have a significant positive impact on the responses 16 . It is worth noting that there is a scarcity of studies investigating the effects of titanium dioxide (TiO2 NPs), magnesium oxide nanoparticles (MgO2 NPs), and vitamin B6 on the production of secondary metabolites under submerged conditions. Therefore, this study aims to provide recent updates on the utilization of these substrates in formulated media. Additionally, the phenolic components present in various parts of shiitake, including the fruiting body, moss, stem, and shell, have not been thoroughly characterized. Hence, the objective of this research is to optimize the production of shiitake's polysaccharides and dry weight biomass (DWB) through submerged fermentation. Subsequently, the optimal conditions determined will be applied to solid-state fermentation, and the total phenol, and phenolic component profiles of the mushroom fruiting body will be characterized. Finally, the anticancer activities of the extracts obtained from different parts of shiitake fruiting body and biomass of optimized submerge fermentation conditions were examined. Results and discussion Screening of key substrates in polysaccharide production under P.B design Extensive investigations have been done to analyze how substrates used in submerge fermentation affect the production of polysaccharides and biomass from Shiitake 17 – 19 .So far, however, the consequences of different macro and micro substrates on CSP and DWB using suitable statistical methods are yet to be determined. Therefore through the use of the P.B design as a screening approach and subsequent RSM optimization of process parameters, the shiitake fermentation medium was perfected to maximize DWB and CSP yield. The use of the P.B method proved to be a promising approach for examining diverse macro and micro substrates and identifying their significant influences 20 . Throughout the screening, a range of substrates (glucose, glucose syrup, glycerol, starch, B6 and B12 vitamin, casein peptone, meat peptone yeast extract, TiO2 NPs, MgO2 NPs) were utilized to explore their influence on the production of CSP and DWB, and to identify significant factors in metabolites production compared to other variables (table-1). Table 1 P.B design matrix with CSP (percentage of DWB) and DWB (percentage of medium) production. Glucose syrup (G.S), Glucose (G), Glycerol (Gly), casein peptone (CP), meat peptone (MP), yeast extract (YE), Magnesium oxide nanoparticles (Mgo), Titanium dioxide nanoparticles (Tio2) , Test order G.S (g/l) G (g/l) Gly (g/l) Starch (g/l) C.P (g/l) M.P (g/l) YE (g/l) Mgo (g/l) Tio 2 (g/l) B 12 µl/l B 6 µl/l DWB CSP 1 15 5 15 15 2 2 2 0.1 0.03 300 300 0.375 5.6 2 15 15 5 5 4 2 4 0.1 0.03 100 300 0.26 6.25 3 5 5 15 5 4 2 4 0.1 0.1 300 100 0.352 1.1 4 15 15 15 5 2 4 2 0.1 0.1 100 100 0.562 5.3 5 5 15 15 15 2 2 4 0.03 0.1 100 300 0.36 3.7 6 15 5 5 5 2 4 4 0.03 0.1 300 300 0.316 7.1 7 5 15 15 5 4 4 2 0.03 0.03 300 300 0.32 6.5 8 10 10 10 10 3 3 3 0.065 0.065 200 200 0.503 0.99 9 15 5 15 15 4 4 4 0.03 0.03 100 100 0.644 4.9 10 15 15 5 15 4 2 2 0.03 0.1 300 100 0.497 2.01 11 5 15 5 15 2 4 4 0.1 0.03 300 100 0.592 2.3 12 10 10 10 10 3 3 3 0.065 0.065 200 200 0.461 1.9 13 5 5 5 15 4 4 2 0.1 0.1 100 300 0.45 4.4 14 5 5 5 5 2 2 2 0.03 0.03 100 100 0.347 0.86 The statistical analysis of the data was conducted using Design-Expert software (version 6.0.10), and the square root method was employed to develop the linear equation. The coefficient of determination (R 2 ) for DWB and CSP was found to be 0.91 for both, indicating a high level of agreement between the data and the regression model. The p-value was utilized to assess the significance of each variable. To determine the linear effects of the variables on the responses, a significance level of 5% was chosen. Subsequently, the results obtained from the P.B design were further examined through analysis of variance (ANOVA) (Table 2 – 3 ). Table 2 ANOVA for P.B. Model Analysis of variance of DWB. Glucose syrup (G.S) Meat peptone(M.P) Source Sum of Squares df Mean Square F Value p-value Prob > F Model 3.617818 3 1.205939 31.42607 < 0.0001 significant G S 0.963965 1 0.963965 25.12036 0.0007 M. P 0.832221 1 0.832221 21.68719 0.0012 B 6 1.821631 1 1.821631 47.47064 < 0.0001 Curvature 1.026895 1 1.026895 26.76029 0.0006 significant Residual 0.345365 9 0.038374 Lack of Fit 0.27186 8 0.033983 0.462319 0.8204 not significant Pure Error 0.073504 1 0.073504 Cor Total 4.990078 13 In terms of biomass production, the ANOVA analysis showed a significant association with glucose syrup, meat peptone, and vitamin B6, while starch, meat peptone, and vitamin B6 were also found to have a significant influence on CSP production (p F Model 0.157745 3 0.052582 40.16591 < 0.0001 significant WS 0.04826 1 0.04826 36.86477 0.0002 M.P 0.040021 1 0.040021 30.57093 0.0004 B 6 0.069464 1 0.069464 53.06202 < 0.0001 Curvature 0.005984 1 0.005984 4.571268 0.0612 not significant Residual 0.011782 9 0.001309 Lack of Fit 0.0109 8 0.001363 1.544785 0.5557 not significant Pure Error 0.000882 1 0.000882 Cor Total 0.175511 13 B 6 proved to be the most important source for DWB and CSP production, as confirmed by its higher F value in Table 2 – 3 compared to the carbon and nitrogen source. In terms of promoting biomass and CSP yield, glucose syrup and starch emerged as the most effective carbon sources respectively, alongside meat peptone Optimization of selected substrates on CSP production using RSM The implementation of central composite rotatable design (CCRD) in optimization is a commonly employed and successful strategy in experimental planning 19 . Through P.B design analysis, it was observed that wheat starch (W.S), meat peptone (M.P) and vitamin B6 are essential substrates for increasing CSP production (Table 4 ). Table 4 Results of FCCCD using two variables indicating observed results. Run WS MP B6 CSP DWB g/l 1 10 5 300 5.57 4.4 2 30 10 400 6.4 4.3 3 63.63586 10 400 4.25 6.05714 4 50 5 300 6.15 5.64286 5 30 1.591036 400 6.88 4.67143 6 50 15 500 6.64 4.52857 7 30 10 568.1793 7.1 6.4 8 30 10 400 6.3 4.44286 9 30 10 231.8207 6.46 7.1 10 30 18.40896 400 6.05 3.7 11 10 15 300 2.7 4.8 12 10 15 500 4 3.57143 13 50 15 300 6.98 6.78571 14 50 5 500 5 7 15 10 5 500 6.5 6.47143 16 1 10 400 1.5 4.2 In addition to these variables, 5 glucose, 10 glycerol and 2 yeast extract extract were used as a basal media. Design-Expert software (version 6.0.10) was utilized for the statistical analysis of the data. The least squares method was employed to formulate the second equation. WS, M.P, and vitamin B6 were considered as variable factors. The significance of these factors was evaluated at a confidence level of 5% to determine their effects on CSP production. The analysis of variance (ANOVA) results indicated that the model terms, as well as the linear and interactive effects of W.S, M.P, and vitamin B6, were found to be statistically significant. The "Prob > F" values were less than 0.05 (Table 5 ). Table 5 ANOVA for Response Surface Quadratic. Model Analysis of variance table Source Sum of Squares df Mean Square F Value p-value Prob > F Model 39.77114 9 4.419016 153.3863 < 0.0001 significant A-S.W 8.266116 1 8.266116 286.9211 < 0.0001 B-MP 1.351311 1 1.351311 46.90468 0.0005 C-B6 0.241572 1 0.241572 8.385098 0.0275 AB 7.6832 1 7.6832 266.6878 < 0.0001 AC 1.7298 1 1.7298 60.04224 0.0002 BC 0.17405 1 0.17405 6.041364 0.0493 A^2 13.31115 1 13.31115 462.0367 < 0.0001 B^2 0.046138 1 0.046138 1.601478 0.2526 C^2 0.306665 1 0.306665 10.64449 0.0172 Residual 0.172858 6 0.02881 Lack of Fit 0.167858 5 0.033572 6.714333 0.2846 not significant Pure Error 0.005 1 0.005 Cor Total 39.944 15 R 2 index used as an adequacy of model 21 was 98. The F-value of W.S was higher than that of the protein and vitamin sources. This suggests that the W.S had a stronger impact on CSP production compared to the protein and vitamin sources. Consequently, it can be inferred that the carbon source played a more quantitatively significant role in influencing polysaccharide production than the protein source. The lack of fit of the final model was non-significant. This indicates that the model used to analyze the data provides a good fit to the observed data. Analysis of variance (ANOVA) was applied to fit a second order polynomial equation. The fitted equation of CSP production over the W.S, .P and B6 vitamin was shown as where, Y was the CSP and A, B and C were W.S, M.P and B 6 vitamin respectively. p = 7.09 + 0.21 × A -0.53×B-9.20E-003 × C + 9.8E-003 × A × B -2.32E-004× A ×C + 2.95E-004× B ×C-2.3E-003× A 2 + 2.82E-003× B 2 + 1.82E-005×C2 3.3 Verification of optimum condition There was no considerable difference between the predicted values and the actual values of the CSP. The comparison of the experimental values (actual values) with the predicted values, as shown in Fig. 1 , supports the adequacy of the RSM model. As the experimental values closely align with the predicted values, it suggests that the model is reliable in capturing the effects of the W.S, M.P and B6 vitamin on CSP production. CPS production The 3-dimensional plot of the relationship between the quantity of W.S, M.P, and CSP production revealed that the highest CSP production was achieved at the highest levels of W.S and M.P (Fig. 2 ). The three-dimensional graph depicting the effect of vitamin and W.S on CSP production revealed that increasing the W.S concentration to above 40 g/L resulted in an increase in CSP production. On the flip side, changes in vitamin levels did not greatly contribute to the increase. However, the lowest B6 vitamin level did show a slight rise in CSP accumulation (Fig. 3 ) At the maximum levels of M.P and the minimum level of vitamin B6, the highest amount of CSP was produced. However, an excessive amount of M.P and a moderate level of vitamin B6 hindered the production of CSP. The most precise method for stimulating the production of CSP was found to be through the use of high levels of W.S and M.P, as observed (Fig. 4 ). Validation of the optimized culture conditions Under the optimal conditions, which involved the utilization of 48.56 g/l of W.S, 15 g/l of M.P, and 300 µg of B6 vitamins, the model predicted that the CSP content would peak at 7.025 percent. In order to assess the accuracy of the model equation, the predicted optimal conditions were implemented in an experimental setting. Under these experimental conditions, the measured CSP was determined to be 7.5 ± 0.2 g/L, which slightly exceeded the predicted maximum value. The results demonstrate that the model equation offered a dependable estimation of the optimal conditions for CSP. The attained CSP in the experimental validation closely aligned with the predicted value, confirming the suitability and accuracy of the model. The optimization of conditions using RSM led to a 6 percent rise in intracellular polysaccharides production in Shiitake strain LeS 22 . The specific selection of substrates, statistical methods, and substrate concentrations in the RSM approach resulted in a notable increase in CSP content in shiitake. Solid state formulation toward shiitake fruiting To prepare the solid-state medium, the optimal conditions for polysaccharide production, as determined through the RSM, were employed in the formulation of the solid-state medium. During the initial test, the formulated media, which consisted of beech wood sawdust and chips in equal amounts, enriched with 48.56 g/l of W.S, 15 g/l of M.P, and 300 µg of vitamin B6, experienced contamination with heat-resistant spores, This contamination occurred despite the media being subjected to sterilization at 120 ºC for 2.5 hours. The presence of heat-resistant spores in the formulated media posed a challenge to achieving a sterile environment for the desired fermentation process. To address this issue and reduce the resistance of the heat-resistant spores prior to the sterilization process, all substrates used in the formulation were soaked in a 50 ppm NaOCl solution for 6 hours. For the cultivation of shiitake mushrooms, an optimal environmental condition was established following the recommendations outlined in the book "Stamets, 2011." After a period of three months, all the fruit bodies were harvested, resulting in the highest CSP recorded at 12.5 ± 0.8% of the dry biomass. The study findings indicated a substantial enhancement in CSP through solid-state fermentation in comparison to submerged fermentation. Specifically, the CSP exhibited an increase of over 66%, rising from 7.5–12.5% of the dry biomass. Compared to submerged fermentation, solid-state fermentation has different records of successful applications in the production of secondary metabolites 6 . In addition to CSP, the study also examined the total phenolic content and phenolic profiles of the moss, shell, and stem of the shiitake fruiting body. The analysis of phenolic components and their profiles aimed to gain insights into the composition and distribution of phenolic compounds across different parts of the fruiting body. Total phenolic content and phenolic profiles The study investigated the impact of five different nanoparticles—specifically titanium dioxide, magnesium oxide, ferric oxide, zinc oxide, and silicon nanoparticles—on the DWB and phenolic content of shiitake (Table 6 ). The mushrooms were cultivated in medium comprising (g/l) 48.56 W.S, 15 M.P, and 300 µg of B6 along with 5 glucose, 10 glycerol and 2 yeast extract, at a temperature of 18°C. Magnesium oxide and iron oxide nanoparticles exhibited a stimulatory effect on the growth of shiitake, representing a 45% and 13 percent enhancement compared to the control. In contrast, the utilization of silicon oxide nanoparticles had a detrimental effect on the growth of the shiitake mushrooms. In terms of phenolic production, the application of silicon oxide and magnesium oxide nanoparticles led to a 20% and 4% increase, respectively, compared to the control sample. However, when considering the biomass content, the sample treated with magnesium oxide nanoparticles exhibited a higher total phenol content compared to the sample treated with silicon oxide nanoparticles. In the remaining treated samples, the amount of phenolic compounds was lower than that of the control sample. Table 6 the effect of various nanoparticles on biomass and phenolic content of shiitake nanoparticle (µg/ml)nanoparticle concentration DWB Phenolic (mg GAE /g) 1 sample 0 0.66 ± 0.01 c 15.58 ± 0.2 c 2 ZnO 500 0.56 ± 0.03 d 13.48 ± 0.1 d 3 Fe 3 O 4 500 0.75 ± 0.05 b 13.79 ± 0.3 d 4 TiO 2 500 0.67 ± 0.04 c 15.53 ± 0.5 c 5 MgO 500 0.96 ± 0.03 a 16.5 ± 0.2 b 6 SiO 2 500 0.41 ± 0.02 e 17.23 ± 0.3 a It was previously established that phenolic compounds make up about 4.79 to 8 mg GAE/g of the dry weight of biomass in submerge fermentation 23 , a quantity that is naturally lower than that found in shiitake fruit 24 , 25 . The selection of the most suitable medium designed in this research was recognized as a key factor in enhancing the production of phenolic components in shiitake mushrooms, surpassing the total phenolic values documented in previous studies. What remains unclear is the variation in phenolic profiles among different parts of shiitake mushrooms. The phenolic profiles of shiitake mushrooms were analyzed using an Agilent 6410 QqQ equipped with an Electrospray Ionization interface (ESI), and the results were presented in Table 6 Table 6 Phenolic acids and flavonoids extracted from moss, shell, stem and biomass of shiitake Phenolic Compounds Polyphenol Classes moss Shell Stem Biomass with Mgo Malic acid Phenolic acid + + + + Protocatechuic acid Phenolic acid + + + + Gallic acid Phenolic acid - - - - Sinapic acid Phenolic acid + + + + Homogentisic acid Phenolic acid + - + + Sulfosalicylic acid Phenolic acid + + + + Gentisic acid Phenolic acid + + + + p-Hydroxybenzoic acid Phenolic acid + - + + Chlorogenic acid Phenolic acid + + + + Vanillic acid Phenolic acid + - + + Caffeic acid Phenolic acid + + + + Syringic acid Phenolic acid + + + + Cinnamic acid Phenolic acid + + + - p-Coumaric acid Phenolic acid + + + + Ferulic acid Phenolic acid + + + + Veratric acid Phenolic acid + + + + Salicylic acid Phenolic acid + - + - Benzoic acid Phenolic acid + + + + o-Coumaric acid Phenolic acid + + + + Abscisic acid Phenolic acid + + + + 3,4-Dihydroxybenzoic acid Phenolic acid + + + + Coumaric acid Phenolic acid + + + + Kaempferol Flavonoid + + + + Isorhamnetin Flavonoid + + + + Catechin Flavonoid + + + + Rutin Flavonoid + + + + Naringin Flavonoid + + + + Myricetin Flavonoid + + + + Quercetin Flavonoid + + + + Naringenin Flavonoid + + - - Hesperetin Flavonoid + + + + Formononetin Flavonoid + + + + Biochanin A Flavonoid + + + + Epicatechin Flavonoid + + + + Quercetin-3-D-galactoside Flavonoid + + + + Resveratrol Stilbene + + + + Pyrogallol Other polyphenols + - + + Vanillin Other polyphenols + - + + Syringaldehyde Other polyphenols + + + + 3,4-Dihydroxybenzaldehyde Other polyphenols + - + + Among the different parts of the shiitake fruiting bodies, the moss and stem exhibited the highest diversity of phenolic components, specifically 21 phenolic acids. The biomass obtained through submerged fermentation supplemented with magnesium oxide nanoparticles contained 19 phenolic compounds, showing slightly lower diversity compared to the moss and stem parts of the shiitake fruiting bodies. The mushroom skin showed the lowest diversity of phenolic compounds, with only 17 phenolic acids identified. The moss of the fruit did not contain gallic acid. In contrast, five phenolic compounds—gallic acid, homogentisic acid, p-hydroxybenzoic acid, vanillic acid, and salicylic acid—were not detected in the mushroom fruit shell. Similarly, in the mushroom biomass, three phenolic compounds—gallic acid, cinnamic acid, and salicylic acid—were not detected. All 13 listed flavonoids were found in the moss and shell of shiitake mushrooms. Additionally, 12 flavonoids were detected in the stem and biomass of the mushrooms. The only flavonoid that was not found in the stem of the fruit and biomass was naringenin. The recent research revealed the detection of phenolic compounds including gallicacid, chlorogenic acid, syringic acid, rutin, p-coumaric acid, ferulic acid, 2-hydroxy cinnamic acid, caffeic acid, protocatechuic acid, quercetin, abscisic acid, and trans-cinnamic acid 26 , 27 , which were also identified in the present study.Analysis of shiitake usingboth base and acid hydrolysisrevealed the presence of phenolic compounds including hesperidin, luteolin, and cinnamic acid 27 , but two of these compounds, excluding cinnamicacid,were not found in the presentshiitake fruit analysis. Rheological properties Flow behavior In order to investigate the rheological behavior and emulsifying properties of CSP, pectin was selected among several commercial polysaccharides. Preliminary experiments revealed that the rheological behavior of pectin solution closely resembled that of CSP at similar concentrations, unlike other commercial polysaccharides (specific results not provided). Figure 5 demonstrates that the viscosity of CSP solutions at 1% concentration and pectin solutions at 1% and 2% concentrations did not exhibit significant changes with increasing shear rate. This suggests that the viscosity of both 1% and 2% pectin solutions, as well as 1% shiitake polysaccharide solution, remained independent of shear rate and displayed nearly Newtonian behavior. However, the viscosity of the 2% shiitake polysaccharide solution notably decreased with increasing shear rate, indicating a shear-thinning behavior, characteristic of pseudoplastic fluids. The shear-thinning behavior in polysaccharide solutions occurs due to the alignment of polymer molecular chains in the flow field, which leads to a decrease in viscosity. Additionally, the results demonstrated that an increase in solution concentration, for both CSP and pectin, resulted in higher viscosity. This increase in viscosity can be attributed to the augmented effective volume of the dispersed phase 28 . Moreover, Fig. 6 revealed that at very low shear rates, the 2% CSP solution exhibited the highest viscosity. However, overall, the 2% pectin solution displayed the highest viscosity, while the 1% CSP solution had the lowest viscosity. This observation indicates that CSP solutions generally exhibit lower thickening properties compared to pectin solutions. Furthermore, at a shear rate higher than 80 [1/s], the viscosity of the 2% CSP solution approached that of the 1% pectin solution. The findings regarding the shear-thinning behavior of the 2% CSP solution align with the reports by Xu et al. in 2016. Xu et al. investigated the viscosity of CSP solutions with different concentrations and reported varying rates of viscosity change with different concentrations. Solutions with higher concentrations exhibited a faster decrease in viscosity. They also noted that a 0.8% CSP solution showed Newtonian behavior at low shear rates. Their research indicated that more concentrated CSP solutions had a diluting effect with higher shear rates 29 . Xu et al. in 2008 stated that lentinan, a polysaccharide is derived from the shiitake, displays shear-thinning behavior. However, this behavior occurs at very low shear rates 30 . These findings are congruent with the results obtained in this study. The obtained results are also consistent with the information provided by Kumar and colleagues in 2023. They reported that lentinan polysaccharide solutions exhibit Newtonian flow behavior in dilute solutions and shear-thinning behavior in semi-dilute solutions at 25°C 31 . The high viscosity at low shear rates and the pseudoplastic behavior of CSP make them suitable stabilizers in various food formulations such as mayonnaise, salad dressing, mustard sauce, and vegetable soup 32 . Viscoelastic properties As shown in Fig. 6 , the 2% CSP solution, like the 2% pectin solution, in the low frequency domain, the storage modulus (G') is greater than the loss modulus(G"),which indicates the dominance of elastic behavior over viscous behavior and indicates their gel-like structure. On the other hand, in the high frequency domain, the G"is greater than the G', which shows the gel structure is formed by weak intermolecular forces. Nevertheless in all frequencies tested on 1% pectin and 1% CSP solutions, the G"is higher than the G', which indicated the dominance of viscous behavior in these samples. Also, the values of G" and G' in different frequencies of 1% pectin and 1% CSP samples are almost equal. In general, the G" and G'in 2% pectin and 2% CSP samples were higher than those with 1% concentration, which indicates that intermolecular forces formed at higher concentrations are stronger. The results of this research largely corroborate the findings reported by Xu et al. in 2016. Xu et al. reported that, except for the 0.8% CSP sample, the G' exceeded the G" at all frequencies tested. However, in the 0.8% CSP sample, these two moduli intersected, resembling the characteristics of concentrated solutions, viscoelastic fluids, and entangled systems. In their investigations, the 1% CSP solution exhibited a slightly higher G'compared to the G", with both moduli appearing almost parallel to each other across all frequencies, indicating limited gel formation. They suggested that increasing the concentration of CSP solutions promotes gel formation due to enhanced entanglement between polymer molecular chains at higher concentrations. These entanglements contribute to gel formation 29 . Emulsifying properties The present study investigated the stability of different oil-in-water emulsions containing 1% and 2% CSP and pectin over a one-week storage period. In general, the stability of emulsions containing CSP was higher compared to emulsions containing pectin. To expedite the estimation of creaming, the prepared emulsions were subjected to centrifugation. This method also revealed that the highest degree of phase separation was observed in 1% and 2% pectin emulsions. In order to quantitatively assess emulsion instability, the creaming index was calculated for various emulsions after one week of storage. As depicted in Fig. 7 A, the highest creaming index was observed in the emulsions with1% and 2% pectin, while the creaming index for the emulsionwith 2% CSP remained zero after one week. Furthermore, the emulsifying capacity of different emulsions was determined. Figure 7 B indicates that emulsions containing pectin exhibited the lowest emulsifying capacity, whereas the emulsioncontaining2% CSP displayed the highest emulsifying capacity, reaching 100%. Swelling power reflects the ability of polymers to absorb and retain water within their structure. The results indicated that pectin possessed higher swelling power than CSP (Fig. 7 C). This finding is in accordance with a study by Tu et al. in 2022, where they measured the swelling power of CSP by incorporating shiitake powder into sorghum flour. The reported swelling power value of CSP (4.74) coincides with the results obtained in our investigation. Additionally, the oil absorption capacity of CSP was determined (Fig. 7 C) as part of this study. The findings revealed that pectin exhibited lower oil absorption capacity compared to CSP, suggesting a higher hydrophilicity of pectin polysaccharide compared to CSP 33 . Based on the results obtained regarding the emulsifying properties, it can be concluded that CSP possesses superior emulsifying properties compared to pectin polysaccharide. Moreover, the 2% concentration of CSP exhibited enhanced emulsifying properties. Overall, CSP demonstrates the potential to enhance viscosity and stabilize emulsions, which may prove valuable in the context of Pickering emulsions. Cytotoxicity of shiitake against human breast cancer cell line MDA - MB - 231 The therapeutic benefits of shiitake mushroom extracts in treating and preventing diseases are well-established, with polysaccharides and phenolic components being highlighted as the most powerful anticancer agents among the bioactive components 34 – 36 .The investigation into the cytotoxic effects of six distinct shiitake mushroom extracts on MDA-MB-231 breast cancer cell lines demonstrated that all extracts led to significant cell death at a concentration of 500 µg/l. Following a 48-hour incubation period, the IC50 levels were evaluated for each extract to determine the concentration required to inhibit 50% of cell growth. The IC50 values obtained for the extracts were as follows: Extracted polysaccharide (225 µg), Fruit moss extract (182 µg), Fruit stem extract (194 µg), Fruit shell extract (244 µg), Mycelium extract of the control sample (202 µg), and Mycelium extract cultivated in MgO-enriched culture medium (138 µg) (Fig. 8 ). In a study which set out to determine anticancer properties of shiitake mushroom, 37 found that IC50 of the aqueous extract of shiitake QF808-1 strain against lung cancers was 198.5 µg/mL. Out of all the parts of the shiitake mushroom fruiting bodies, it was the extract from the moss that showed the strongest inhibitory effect on the growth of breast cancer cells. Of greater significance is the fact that the polysaccharide extracted from shiitake mushrooms, functioning as a biological macromolecule, displayed anti-cancer properties. Out of all the mycelium extracts, the extract derived from the biomass grown in an MgO-enriched medium showed the strongest inhibitory effect in comparison to the rest. In terms of inhibitory power, the mycelium extract from the MgO-enriched culture medium outperformed the rest of the samples tested. The findings suggest that shiitake mushroom extracts, particularly those derived from the moss and mycelium cultivated in an MgO-enriched culture medium, have the considerable potential to trigger cytotoxic effects on MDA-MB-231breast cancer cell lines. These discoveries indicate that these extracts have the potential to serve as effective therapeutic treatments for breast cancer. Conclusion The effects of the primary substrates and their concentrations on polysaccharide production were assessed utilizing P.B design as a screening method before employing RSM. The observation of the highest polysaccharide content in the W.S substrate suggests that the biosynthesis of polysaccharides in shiitake mushrooms is stimulated by this substrate, potentially through facilitating the polymerization process of the consumed substrate. Shiitake polysaccharide demonstrated notable rheological properties, exhibiting shear-thinning behavior and enhanced viscosity at higher concentrations. Shiitake polysaccharide solutions also showed superior emulsifying capacity compared to pectin, making them promising candidates for stabilizing oil-in-water emulsions. These findings suggest that Shiitake polysaccharide could be an effective stabilizer in various food formulations, particularly for improving viscosity and emulsion stability. The moss from shiitake fruit bodies, rich in a diverse array of flavonoids and phenolic acids, exhibited the most significant cytotoxicity against the MDA-MB-231 cell line, with the lowest IC50 compared to various fruit parts extracts. Notably, the utilization of nanoparticles not only enhanced phenolic production but also bolstered the biomass's anticancer properties, rivaling those of the shiitake fruit itself. In conclusion, the application of nanoparticles spurred the synthesis of secondary metabolites, resulting in submerged-cultivated biomass with elevated levels of bioactive compounds and more potent anti-cancer properties compared to solid-state-cultivated shiitake fruit which require significant time and expense for production. Material methods Shiitake (strain no. M3714) was obtained from the Mycelia company, Deinze, Belgium. Almost all materials were purchased from Merck Company. Concentrations were obtained under low pressure in a rotary evaporator (HeidolphLaborota 4000 efficient rotary evaporator, Germany). The extracts were dried by vacuum freeze-drying (Christ Alpha 1–2 freeze-dryer). TiO2 NPs and MgO2 NPs were attained from United States Research Nanomaterials Inc. with a purity of ± 99% and an average size of 15 and 20nm, respectively Microorganisms, inoculums, and cultivation conditions The strain of mushrooms was initially cultivated on potato dextrose agar (PDA) medium. To preserve the strain for long-term use, it was stored at a low temperature of 4°C. For the fermentation process, 250mL Erlenmeyer flasks were utilized, each containing 50mL of a formulated medium to ensure adequate oxygen supply for the growth of the mushroom and production of metabolites. The formulated medium consisted of 30g/L glucose and 10g/L yeast extract, supplemented with micro mineral elements. This medium was used during a 10-day fermentation period to facilitate the development of inoculums with maximum cell viability and concentration. 5 percent of a fresh seed culture was added to the fermentation media. This addition served two purposes: First, it helped improve the growth efficiency of the fungi, promoting their proliferation. Second, it acted as a stimulus for the production of secondary metabolites, which are bioactive compounds of interest 6 Fermentation Media In the study, various substrates (glucose, glucose syrup, glycerol, wheat starch, B6 and B12 vitamin, casein peptone, meat peptone yeast extract, TiO2 NPs, MgO2 NPs) were evaluated to assess their impact on the production of polysaccharides and DWB. The medium was supplemented with MgSO4·7H2O (1g/L), CaCl2 (0.2g/L), KH2PO4 (1.5g/L), and CaCO3 (1.5g/L) as constant factors. A rotary shaker set at 185 RPM and a temperature of 18°C were chosen as the constant conditions for the culture. In an innovative approach to enhance the production of secondary metabolites, the study investigated the effects of titanium dioxide nanoparticles (TiO2 NPs), magnesium oxide nanoparticles (MgO2 NPs), vitamin pyridoxine (B 6 ) and B 12 on the growth and formation of secondary metabolites of shiitake in a submerge conditions were examined 38 Isolation and purification of polysaccharide To extract polysaccharides from the shiitake biomass, the following procedure was employed: The shiitake biomass was separated from the formulated medium by centrifugation at 12,000g for 5 minutes. The separated biomass was washed twice with distilled water to remove any impurities. The washed biomass was then dried and crushed to facilitate the extraction of polysaccharides. The dried and crushed biomass was heated three times in a boiling 96% ethanol solution, each time for 3 hours. After the heating process, the polysaccharides were extracted from the dry biomass by treating it three times with distilled water at a temperature of 70°C for 3 hours each time. The resulting solution containing the polysaccharides was centrifuged at 3,000rpm.To remove proteins, the solution was deproteinated using the sevag method, which involved adding a sevag reagent (a mixture of 1-butanol and chloroform in a ratio of 1:4, v/v) to the solution 39 .The sevag reagent was then removed, and the remaining solution was concentrated using a vacuum rotary evaporator to remove approximately two-thirds of the water phase (supernatant).The concentrated polysaccharide solution was dialyzed against deionized water for 48 hours to remove any remaining impurities and salts. The water-soluble polysaccharides were precipitated by adding 96% ethanol maintained at 4°C for 24 hours. The precipitated polysaccharides were collected by centrifugation. The collected polysaccharides were further processed using a vacuum freeze-dryer to obtain the crude shiitake polysaccharides (CSP) in a dry form 40 . Preparation of Shiitake solid state medium for fruit production Beech wood sawdust and chips were combined in equal amounts to generate a basic substrate. This substrate provides a suitable environment for the growth of shiitake. The beech wood substrate was detoxified by treating it with an alkaline solution, typically sodium hydroxide (NaOH). This detoxification process helps eliminate contaminants and enhance the growth of shiitake. The substrate was formulated by adding starch and meat peptone. These components serve as a nutrient source for the mushroom, promoting its growth and fruiting. Since the beech wood substrate may naturally contain spore contamination, the mixed substrates were soaked in the 50 ppm NaOCl solution for 6 hours 41 . After soaking, the mixed substrates were sterilized by subjecting them to a temperature of 121°C for 2.5 hours. Spawn of shiitake were added to the sterilized medium. The medium with the added spawn was incubated under appropriate conditions, following the guidelines specified by 42 . These conditions typically involve maintaining specific temperature, humidity, and light conditions to support the growth and development of shiitake. The fruit bodies can then be harvested after three months for further processing. Extraction of shiitake dry matter, total phenolic content, and analysis of phenolic compounds In the study, dried and finely crushed moss, stem, and shell of shiitake were subjected to extraction using 90% ethanol. The extraction was carried out at 25°C for 72 hours. After the extraction, the extracts were centrifuged and concentrated using a vacuum rotary evaporator 6 . To measure the total phenolic contents (TPCs) in the extracts, the Folin–Ciocalteu reagent and gallic acid (GAE) standard method were used. The GAE standard, dissolved in absolute ethanol, was mixed with the Folin–Ciocalteu reagent. After 10 minutes, a sodium carbonate solution was added to the mixture, and it was maintained at ambient temperature for 30 minutes. The absorbance of the mixed solution was measured at 765 nm using a UV–Vis spectrophotometer 43 . The TPC was reported as milligrams of GAE per gram of Shiitake dry tissue 44 . Additionally, the phenolic components extracted from shiitake dry biomass were analyzed to determine their phenolic profiles. The analysis was performed using an Agilent 6410 QqQ mass spectrometer equipped with an Electrospray Ionization (ESI) interface. The phenolic components' structure was examined under specific settings, including drying gas temperature (nitrogen): 350°C, drying gas flow rate: 9L/min, nebulizer pressure: 40 psig, Injection Volume: 25µL, skimmer: 60V, fragmentor voltage: 220V, capillary voltage: 3,500V, and scan range of m/z: 50–1,000 6 . Rheological properties Hydrocolloid solutions were prepared using shiitake and pectin polysaccharides at concentrations of 1% and 2% (w/v). Pectin solutions were acidified with citric acid, and 0.15% (w/v) calcium chloride was added. These solutions were then heated to 90°C for 2 hours. Rheological properties, including flow behavior and viscoelastic characteristics, were analyzed using a rheometer(Anton PaarPhysica MCR 302, Austria) at 25°C. Viscosity measurements were conducted with concentric cylinders, while viscoelastic properties were assessed using cone and plate geometries. All samples were allowed a rest period of 600 seconds before testing. To determine the linear viscoelastic range, a strain sweep test (strain range: 0.1 to 100%, frequency: 1 rad/s) was conducted. Subsequently, dynamic tests were performed within the linear viscoelastic region, and storage (G') and loss (G") moduli were plotted against frequency. Creaming index, emulsifying capacity, swelling index and fat binding capacity Sunflower oil-in-water emulsions (10% oil ratio) were prepared to determine the creaming index and emulsifying capacity. Emulsions were formulated using 8 ml of 1% and 2% pectin gum and shiitake polysaccharide solutions mixed with 2 ml of sunflower oil. These mixtures were homogenized using a high-shear mixer at 12,000 rpm for 3 minutes. The creaming index was calculated after two weeks of storage at room temperature using the Eq. (1): Creaming-index = \(\:\frac{SH}{TH}\times\:100\) (1) where SH was the height of the serum layer and TH was the height of the whole emulsion. Also, the emulsifying capacity was calculated according to Eq. (2). Emulsion capacity (%) = \(\:\frac{{e}_{\nu\:}}{{t}_{\nu\:}}\times\:100\) (2) wheree ν was the emulsion volume and t ν was the total volume. Swelling power of the shiitake and pectin polysaccharides was measured following the method described by 45 . Briefly, 0.3 g of polysaccharide samples were immersed in 10 ml of distilled water and heated in a boiling water bath for 10 minutes. After cooling, the samples were centrifuged at 1700 rpm for 4 minutes. The swelling power was calculated using the Eq. (3): Swelling power = \(\:\frac{wet\:precipitate\:weight}{dry\:weight}\) (3) Fat binding capacity was also evaluated using the method by 45 . A 0.2 g sample of shiitake and pectin polysaccharides was mixed with 10 ml of sunflower oil. The mixtures were held at room temperature for 1 hour, with vortexing every 15 minutes. Afterward, the samples were centrifuged at 1600 rpm for 15 minutes, the supernatant was removed, and the residue was weighed 45 . The amount of absorbed oil was calculated using the Eq. (4): Fat binding capacity = \(\:\frac{Final\:weight\:of\:dry\:samples}{Initial\:weight\:of\:dry\:samples}\) (4) Cell culture and viability analysis The breast cancer cell line MDA-MB-231 was obtained from the National Cell Bank of the Institute Pasteur of Iran. The cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 1% penicillin/streptomycin and 10% heat-inactivated fetal bovine serum. The culture was maintained at 37°C with 5% CO2 36 . To ensure high cellular viability, the MDA-MB-231 cells were subcultured using trypsin when they reached near confluency. Approximately 1 million cells were reseeded into a fresh T-75 flask for further experiments. To determine the cell viability of MDA-MB-231 cells and the half maximal inhibitory concentration (IC50) values of the stem, shell, and moss extracts of Shiitake against MDA-MB231 cells, the MTT assay was employed. For this assay, 10,000 cells per well were seeded in a 96-well plate and exposed to different concentrations of shiitake extracts for 48 hours at 37°C. Serial dilutions of the Shiitake extracts at concentrations of 0, 62.5, 125, 250, 500, and 1,000 µg/mL were prepared and added to the wells containing the cells. The cytotoxic effects of the extracts on the MDA-MB-231 cancer cell line were evaluated using the MTT assay, following the protocol described in a previous research study conducted by 46 . Statistical method In the study, a P.B design was employed to identify the key substrates that had a significant influence on DWB and polysaccharide. After identifying the key substrates, a RSM approach was used to optimize their quantities. The Central Composite Design (CCD), which is a common form of RSM, was implemented using the statistical package Design-Expert, version 7.0.0, developed by Stat-Ease, Inc., Minneapolis, MN, United States ( https://design-expert-7.software.informer.com/ ). The significance of the models, substrates, and their interactions on the responses was verified using analysis of variance (ANOVA). The means and standard deviations (SD) of the experimental data were determined using Microsoft Excel software (presumably referring to Excel version 2023, https://www.microsoft.com/en-us/microsoft-365/excel ). To compare the differences among the means at a significance level of 0.05, Duncan's multiple range tests was performed using SPSS 16.0 software ( https://spss.software.informer.com/16.0/ ). Statements & Declarations Competing interests The authors declare no competing interests.. Funding The authors declare that no funds, grants, or other support were received during the preparation of this manuscript Author Contribution S.Z.G: Data curation, Formal analysis, Investigation, Project administration, Resources, Software, Writing – review & editing. H.R.S: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Software, Supervision, Validation, Visualization, Writing – review & editing.A.R.:Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Software, Supervision, Validation, Visualization, Writing – review & editing. A.S.F : Data curation, Investigation, Methodology, Project administration, Resources, Writing – review & editing. Data Availability All data generated or analysed during this study are included in this article References Thomas, L. & Mago, P. 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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-6748184","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":471141859,"identity":"b29fa2fc-c3ef-4e1e-8975-862835a90eb8","order_by":0,"name":"Samaneh Zarea Garizi","email":"","orcid":"","institution":"Shahrood University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Samaneh","middleName":"Zarea","lastName":"Garizi","suffix":""},{"id":471141861,"identity":"89b2da5d-c920-465c-a6b8-af3a05fb9d18","order_by":1,"name":"Hamid Reza 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Technology","correspondingAuthor":false,"prefix":"","firstName":"Ahmad","middleName":"","lastName":"Rajaei","suffix":""},{"id":471141864,"identity":"b6654deb-ed8d-4258-a3e0-0fae4a92f51a","order_by":3,"name":"Amir Salek Farrokhi","email":"","orcid":"","institution":"Pasteur Institute of Iran","correspondingAuthor":false,"prefix":"","firstName":"Amir","middleName":"Salek","lastName":"Farrokhi","suffix":""}],"badges":[],"createdAt":"2025-05-26 07:39:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6748184/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6748184/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84728870,"identity":"11fe1b46-0fdd-4ef0-b254-44069754d9dd","added_by":"auto","created_at":"2025-06-16 16:25:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":15472,"visible":true,"origin":"","legend":"\u003cp\u003eThe curve of predicted values against actual values of the response\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6748184/v1/fb6ea95de35d716051a947c3.png"},{"id":84728095,"identity":"b9747d1d-9208-43ee-92cc-1d36a84a3396","added_by":"auto","created_at":"2025-06-16 16:17:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":29486,"visible":true,"origin":"","legend":"\u003cp\u003eResponse surface curve for CSPproduction by shiitake as a function of W.S and M.P\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6748184/v1/b0d41cee4b364bca04e7b9b2.png"},{"id":84728097,"identity":"d796a43a-0cd4-47db-897e-31df667af55a","added_by":"auto","created_at":"2025-06-16 16:17:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":26125,"visible":true,"origin":"","legend":"\u003cp\u003eResponse surface curve for CSP production by as a function of W.S and B6 vitamin\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6748184/v1/1d895f60bbe8ad6793719f48.png"},{"id":84728099,"identity":"cd383850-21ee-48eb-b985-450fbf2860d4","added_by":"auto","created_at":"2025-06-16 16:17:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":37868,"visible":true,"origin":"","legend":"\u003cp\u003eResponse surface curve for CSP production by as a function of B\u003csub\u003e6\u003c/sub\u003e vitamin and meat peptone\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6748184/v1/c87ea1af47d1178b9968c7f6.png"},{"id":84728102,"identity":"09cf6179-3471-48f4-8d57-836c24c3a1ce","added_by":"auto","created_at":"2025-06-16 16:17:53","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":95829,"visible":true,"origin":"","legend":"\u003cp\u003eViscosity of 1% and 2% pectin and CSP solutions.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6748184/v1/9049cb2b06bf93b484923f86.png"},{"id":84728101,"identity":"89f8c8b7-0393-4043-a20e-73a0326b306f","added_by":"auto","created_at":"2025-06-16 16:17:53","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":70657,"visible":true,"origin":"","legend":"\u003cp\u003eFrequency sweep of 1% and 2% pectin and CSP solutions\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6748184/v1/0596e778758529df04d12a76.png"},{"id":84728871,"identity":"0b643f51-c863-4807-8889-11442f9844f4","added_by":"auto","created_at":"2025-06-16 16:25:53","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":23551,"visible":true,"origin":"","legend":"\u003cp\u003eCreaming index (A); emulsifying capacity (B); swelling power and fat binding capacity (C)\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6748184/v1/f5b16bc2a2c465fc6790ade9.png"},{"id":84729795,"identity":"f97ad6c2-a12c-4c25-80ac-66747dad29d6","added_by":"auto","created_at":"2025-06-16 16:41:53","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":139358,"visible":true,"origin":"","legend":"\u003cp\u003eCytotoxic impact of shiitake on MDA-MB-231 cells. Each bar indicated the mean ± SD of three experiments. fruit moss (a), extracted polysaccharide (b), fruit stem (c), shiitake mycelium (d), Mycelium cultivated in MgO-enriched culture medium (e) and Fruit shell (f).\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6748184/v1/08b6186c2d2503c2611528d7.png"},{"id":87465962,"identity":"d6fc9a59-7bae-4dbc-9cb4-6601eba2852f","added_by":"auto","created_at":"2025-07-24 07:23:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2050329,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6748184/v1/59b72edb-0988-4a6c-8b1b-3de7cf1e4512.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Statistical optimization of fermentation conditions for metabolites production by Lentinus edodes and investigation of anticancer activity","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMedical mushrooms, which serve as primary producers of bioactive components and biological macromolecule with remarkable health-promoting effects, represent a significant area of interest in the fields of biotechnology and pharmacology \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Among the diverse array of medical mushrooms, shiitake has garnered substantial attention due to its ability to synthesize valuable biological macromolecule such as polysaccharides, particularly β-glucans, ergosterol, and phenolic compounds \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, possessing the potential to address various health conditions, including diabetes, cancer, the flu, heart disease, high blood pressure, and weakness \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Over the past two decades, numerous researchers have endeavored to investigate the flush of mushrooms and secondary metabolites of medical mushrooms when cultivated under various formulated deciduous woods \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Even with its effectiveness, this fermentation method has distinct drawbacks, one of which is its sole reliance on the culture medium for secondary metabolite production and the long period of cultivation required to harvest the fruit when it is at optimal stage of maturity \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. The solid-state fermentation of shiitake is significantly influenced by the choice of key substrates utilized in formulated media \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Among these substrates, starch from rice bran and wheat bran plays a vital role in enhancing the production of secondary metabolites by Shiitake \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. However, the formulation of solid-state media for mushroom cultivation heavily relies on the experience and expertise of cultivators, as no statistical analyses have been conducted to determine the most effective substrate for promoting shiitake growth and metabolite production \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. The absence of precise measurements of biomass and metabolites during the solid-state fermentation prior to the fruiting process has impeded the optimization efforts \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. More importantly, the cultivation of Shiitake mushrooms requires a considerable amount of time for the development of fully mature fruiting bodies, which is an essential aspect of their growth process \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. The entire cycle, from the initial mycelium stage to the final maturity of the fruiting bodies, can span several months. Significantly, previous research comparing submerged and solid-state fermentation has indicated that submerged fermentation offers several advantages over solid-state fermentation. These advantages include the ease of controlling the physical and chemical conditions of the media, relatively lower spore contamination, and cost-effectiveness as a fermentation method \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. The consensus among biotechnology experts is that the availability of macro elements, including carbon, nitrogen, and mineral substrates, nanoparticles and vitamins greatly influences the proliferation of mushrooms and the synthesis of secondary metabolites in submerged fermentation\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. A substantial and expanding body of literature has been dedicated to identifying the suitable statistical methods for optimizing the physicochemical conditions of the medium to enhance metabolite productions \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Among various statistical techniques, RSM has been widely employed worldwide to mathematically determine the optimal conditions for key substrates that impact the desired responses\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Prior to optimization, screening methods such as P.B designs are commonly used to identify key substrates that have a significant positive impact on the responses\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. It is worth noting that there is a scarcity of studies investigating the effects of titanium dioxide (TiO2 NPs), magnesium oxide nanoparticles (MgO2 NPs), and vitamin B6 on the production of secondary metabolites under submerged conditions. Therefore, this study aims to provide recent updates on the utilization of these substrates in formulated media. Additionally, the phenolic components present in various parts of shiitake, including the fruiting body, moss, stem, and shell, have not been thoroughly characterized. Hence, the objective of this research is to optimize the production of shiitake's polysaccharides and dry weight biomass (DWB) through submerged fermentation. Subsequently, the optimal conditions determined will be applied to solid-state fermentation, and the total phenol, and phenolic component profiles of the mushroom fruiting body will be characterized. Finally, the anticancer activities of the extracts obtained from different parts of shiitake fruiting body and biomass of optimized submerge fermentation conditions were examined.\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eScreening of key substrates in polysaccharide production under P.B design\u003c/h2\u003e \u003cp\u003eExtensive investigations have been done to analyze how substrates used in submerge fermentation affect the production of polysaccharides and biomass from Shiitake \u003csup\u003e\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.So far, however, the consequences of different macro and micro substrates on CSP and DWB using suitable statistical methods are yet to be determined. Therefore through the use of the P.B design as a screening approach and subsequent RSM optimization of process parameters, the shiitake fermentation medium was perfected to maximize DWB and CSP yield. The use of the P.B method proved to be a promising approach for examining diverse macro and micro substrates and identifying their significant influences\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Throughout the screening, a range of substrates (glucose, glucose syrup, glycerol, starch, B6 and B12 vitamin, casein peptone, meat peptone yeast extract, TiO2 NPs, MgO2 NPs) were utilized to explore their influence on the production of CSP and DWB, and to identify significant factors in metabolites production compared to other variables (table-1).\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\u003eP.B design matrix with CSP (percentage of DWB) and DWB (percentage of medium) production. Glucose syrup (G.S), Glucose (G), Glycerol (Gly), casein peptone (CP), meat peptone (MP), yeast extract (YE), Magnesium oxide nanoparticles (Mgo), Titanium dioxide nanoparticles (Tio2) ,\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"14\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c14\" colnum=\"14\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTest order\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG.S (g/l)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eG\u003c/p\u003e \u003cp\u003e(g/l)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGly\u003c/p\u003e \u003cp\u003e(g/l)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eStarch\u003c/p\u003e \u003cp\u003e(g/l)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eC.P\u003c/p\u003e \u003cp\u003e(g/l)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eM.P\u003c/p\u003e \u003cp\u003e(g/l)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eYE\u003c/p\u003e \u003cp\u003e(g/l)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eMgo\u003c/p\u003e \u003cp\u003e(g/l)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eTio\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e(g/l)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003eB\u003csub\u003e12\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e\u0026micro;l/l\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\"\u003e \u003cp\u003eB\u003csub\u003e6\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e\u0026micro;l/l\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c13\"\u003e \u003cp\u003eDWB\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c14\"\u003e \u003cp\u003eCSP\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.375\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e5.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e6.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.352\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e1.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.562\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e5.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e3.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.316\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e7.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e6.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.065\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.065\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.503\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e0.99\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.644\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e4.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.497\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e2.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.592\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e2.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.065\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.065\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.461\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e1.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e4.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.347\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e0.86\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe statistical analysis of the data was conducted using Design-Expert software (version 6.0.10), and the square root method was employed to develop the linear equation. The coefficient of determination (R\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e) for DWB and CSP was found to be 0.91 for both, indicating a high level of agreement between the data and the regression model. The p-value was utilized to assess the significance of each variable. To determine the linear effects of the variables on the responses, a significance level of 5% was chosen. Subsequently, the results obtained from the P.B design were further examined through analysis of variance (ANOVA) (Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eANOVA for P.B. Model Analysis of variance of DWB. Glucose syrup (G.S) Meat peptone(M.P)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSource\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSum of\u003c/p\u003e \u003cp\u003eSquares\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003edf\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMean\u003c/p\u003e \u003cp\u003eSquare\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eF\u003c/p\u003e \u003cp\u003eValue\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003cp\u003eProb\u0026thinsp;\u0026gt;\u0026thinsp;F\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eModel\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.617818\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.205939\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e31.42607\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003esignificant\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eG S\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.963965\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.963965\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e25.12036\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.0007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM. P\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.832221\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.832221\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e21.68719\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.0012\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB\u003csub\u003e6\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.821631\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.821631\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e47.47064\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCurvature\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.026895\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.026895\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e26.76029\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.0006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003esignificant\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eResidual\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.345365\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.038374\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLack of Fit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.27186\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.033983\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.462319\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.8204\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003enot significant\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePure Error\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.073504\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.073504\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCor Total\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.990078\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIn terms of biomass production, the ANOVA analysis showed a significant association with glucose syrup, meat peptone, and vitamin B6, while starch, meat peptone, and vitamin B6 were also found to have a significant influence on CSP production (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eANOVA for P.B. Model Analysis of variance of CSP. Wheat starch (WS)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSource\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSum of\u003c/p\u003e \u003cp\u003eSquares\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003edf\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMean\u003c/p\u003e \u003cp\u003eSquare\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eF\u003c/p\u003e \u003cp\u003eValue\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003cp\u003eProb\u0026thinsp;\u0026gt;\u0026thinsp;F\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eModel\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.157745\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.052582\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e40.16591\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003esignificant\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.04826\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.04826\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e36.86477\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.0002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM.P\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.040021\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.040021\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e30.57093\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.0004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB\u003csub\u003e6\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.069464\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.069464\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e53.06202\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCurvature\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.005984\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.005984\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.571268\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.0612\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003enot significant\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eResidual\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.011782\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.001309\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLack of Fit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.0109\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.001363\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.544785\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.5557\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003enot significant\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePure Error\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.000882\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.000882\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCor Total\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.175511\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eB\u003csub\u003e6\u003c/sub\u003e proved to be the most important source for DWB and CSP production, as confirmed by its higher F value in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e compared to the carbon and nitrogen source. In terms of promoting biomass and CSP yield, glucose syrup and starch emerged as the most effective carbon sources respectively, alongside meat peptone\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eOptimization of selected substrates on CSP production using RSM\u003c/h3\u003e\n\u003cp\u003eThe implementation of central composite rotatable design (CCRD) in optimization is a commonly employed and successful strategy in experimental planning \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Through P.B design analysis, it was observed that wheat starch (W.S), meat peptone (M.P) and vitamin B6 are essential substrates for increasing CSP production (Table \u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eResults of FCCCD using two variables indicating observed results.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\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 \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRun\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eB6\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCSP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eDWB g/l\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e63.63586\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.05714\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\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\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.64286\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.591036\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.67143\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\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\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.52857\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e568.1793\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.44286\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e231.8207\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18.40896\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.57143\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13\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\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.78571\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14\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\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.47143\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIn addition to these variables, 5 glucose, 10 glycerol and 2 yeast extract extract were used as a basal media. Design-Expert software (version 6.0.10) was utilized for the statistical analysis of the data. The least squares method was employed to formulate the second equation. WS, M.P, and vitamin B6 were considered as variable factors. The significance of these factors was evaluated at a confidence level of 5% to determine their effects on CSP production. The analysis of variance (ANOVA) results indicated that the model terms, as well as the linear and interactive effects of W.S, M.P, and vitamin B6, were found to be statistically significant. The \"Prob\u0026thinsp;\u0026gt;\u0026thinsp;F\" values were less than 0.05 (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eANOVA for Response Surface Quadratic. Model Analysis of variance table\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSource\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSum of\u003c/p\u003e \u003cp\u003eSquares\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003edf\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMean\u003c/p\u003e \u003cp\u003eSquare\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eF\u003c/p\u003e \u003cp\u003eValue\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003cp\u003eProb\u0026thinsp;\u0026gt;\u0026thinsp;F\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eModel\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e39.77114\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4.419016\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e153.3863\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003esignificant\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA-S.W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e8.266116\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8.266116\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e286.9211\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB-MP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.351311\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.351311\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e46.90468\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.0005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC-B6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.241572\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.241572\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8.385098\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.0275\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7.6832\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7.6832\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e266.6878\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.7298\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.7298\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e60.04224\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.0002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.17405\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.17405\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.041364\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.0493\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA^2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e13.31115\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e13.31115\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e462.0367\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB^2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.046138\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.046138\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.601478\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.2526\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC^2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.306665\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.306665\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e10.64449\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.0172\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eResidual\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.172858\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.02881\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLack of Fit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.167858\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.033572\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.714333\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.2846\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003enot significant\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePure Error\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCor Total\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e39.944\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eR\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003eindex used as an adequacy of model \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e was 98. The F-value of W.S was higher than that of the protein and vitamin sources. This suggests that the W.S had a stronger impact on CSP production compared to the protein and vitamin sources. Consequently, it can be inferred that the carbon source played a more quantitatively significant role in influencing polysaccharide production than the protein source. The lack of fit of the final model was non-significant. This indicates that the model used to analyze the data provides a good fit to the observed data.\u003c/p\u003e \u003cp\u003eAnalysis of variance (ANOVA) was applied to fit a second order polynomial equation. The fitted equation of CSP production over the W.S, .P and B6 vitamin was shown as where, Y was the CSP and A, B and C were W.S, M.P and B\u003csub\u003e6\u003c/sub\u003e vitamin respectively.\u003c/p\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;7.09\u0026thinsp;+\u0026thinsp;0.21 \u0026times; A -0.53\u0026times;B-9.20E-003 \u0026times; C\u0026thinsp;+\u0026thinsp;9.8E-003 \u0026times; A \u0026times; B -2.32E-004\u0026times; A \u0026times;C\u0026thinsp;+\u0026thinsp;2.95E-004\u0026times; B \u0026times;C-2.3E-003\u0026times; A 2\u0026thinsp;+\u0026thinsp;2.82E-003\u0026times; B 2\u0026thinsp;+\u0026thinsp;1.82E-005\u0026times;C2\u003c/p\u003e \u003cp\u003e3.3 Verification of optimum condition\u003c/p\u003e \u003cp\u003eThere was no considerable difference between the predicted values and the actual values of the CSP. The comparison of the experimental values (actual values) with the predicted values, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, supports the adequacy of the RSM model. As the experimental values closely align with the predicted values, it suggests that the model is reliable in capturing the effects of the W.S, M.P and B6 vitamin on CSP production.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eCPS production\u003c/h3\u003e\n\u003cp\u003eThe 3-dimensional plot of the relationship between the quantity of W.S, M.P, and CSP production revealed that the highest CSP production was achieved at the highest levels of W.S and M.P (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe three-dimensional graph depicting the effect of vitamin and W.S on CSP production revealed that increasing the W.S concentration to above 40 g/L resulted in an increase in CSP production. On the flip side, changes in vitamin levels did not greatly contribute to the increase. However, the lowest B6 vitamin level did show a slight rise in CSP accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAt the maximum levels of M.P and the minimum level of vitamin B6, the highest amount of CSP was produced. However, an excessive amount of M.P and a moderate level of vitamin B6 hindered the production of CSP. The most precise method for stimulating the production of CSP was found to be through the use of high levels of W.S and M.P, as observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eValidation of the optimized culture conditions\u003c/h3\u003e\n\u003cp\u003eUnder the optimal conditions, which involved the utilization of 48.56 g/l of W.S, 15 g/l of M.P, and 300 \u0026micro;g of B6 vitamins, the model predicted that the CSP content would peak at 7.025 percent. In order to assess the accuracy of the model equation, the predicted optimal conditions were implemented in an experimental setting. Under these experimental conditions, the measured CSP was determined to be 7.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 g/L, which slightly exceeded the predicted maximum value. The results demonstrate that the model equation offered a dependable estimation of the optimal conditions for CSP. The attained CSP in the experimental validation closely aligned with the predicted value, confirming the suitability and accuracy of the model. The optimization of conditions using RSM led to a 6 percent rise in intracellular polysaccharides production in \u003cem\u003eShiitake\u003c/em\u003e strain LeS\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. The specific selection of substrates, statistical methods, and substrate concentrations in the RSM approach resulted in a notable increase in CSP content in shiitake.\u003c/p\u003e\n\u003ch3\u003eSolid state formulation toward shiitake fruiting\u003c/h3\u003e\n\u003cp\u003eTo prepare the solid-state medium, the optimal conditions for polysaccharide production, as determined through the RSM, were employed in the formulation of the solid-state medium. During the initial test, the formulated media, which consisted of beech wood sawdust and chips in equal amounts, enriched with 48.56 g/l of W.S, 15 g/l of M.P, and 300 \u0026micro;g of vitamin B6, experienced contamination with heat-resistant spores, This contamination occurred despite the media being subjected to sterilization at 120 \u0026ordm;C for 2.5 hours. The presence of heat-resistant spores in the formulated media posed a challenge to achieving a sterile environment for the desired fermentation process. To address this issue and reduce the resistance of the heat-resistant spores prior to the sterilization process, all substrates used in the formulation were soaked in a 50 ppm NaOCl solution for 6 hours. For the cultivation of shiitake mushrooms, an optimal environmental condition was established following the recommendations outlined in the book \"Stamets, 2011.\" After a period of three months, all the fruit bodies were harvested, resulting in the highest CSP recorded at 12.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8% of the dry biomass. The study findings indicated a substantial enhancement in CSP through solid-state fermentation in comparison to submerged fermentation. Specifically, the CSP exhibited an increase of over 66%, rising from 7.5\u0026ndash;12.5% of the dry biomass. Compared to submerged fermentation, solid-state fermentation has different records of successful applications in the production of secondary metabolites\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. In addition to CSP, the study also examined the total phenolic content and phenolic profiles of the moss, shell, and stem of the shiitake fruiting body. The analysis of phenolic components and their profiles aimed to gain insights into the composition and distribution of phenolic compounds across different parts of the fruiting body.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eTotal phenolic content and phenolic profiles\u003c/h2\u003e \u003cp\u003eThe study investigated the impact of five different nanoparticles\u0026mdash;specifically titanium dioxide, magnesium oxide, ferric oxide, zinc oxide, and silicon nanoparticles\u0026mdash;on the DWB and phenolic content of shiitake (Table \u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The mushrooms were cultivated in medium comprising (g/l) 48.56 W.S, 15 M.P, and 300 \u0026micro;g of B6 along with 5 glucose, 10 glycerol and 2 yeast extract, at a temperature of 18\u0026deg;C. Magnesium oxide and iron oxide nanoparticles exhibited a stimulatory effect on the growth of shiitake, representing a 45% and 13 percent enhancement compared to the control. In contrast, the utilization of silicon oxide nanoparticles had a detrimental effect on the growth of the shiitake mushrooms. In terms of phenolic production, the application of silicon oxide and magnesium oxide nanoparticles led to a 20% and 4% increase, respectively, compared to the control sample. However, when considering the biomass content, the sample treated with magnesium oxide nanoparticles exhibited a higher total phenol content compared to the sample treated with silicon oxide nanoparticles. In the remaining treated samples, the amount of phenolic compounds was lower than that of the control sample.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ethe effect of various nanoparticles on biomass and phenolic content of shiitake\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\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=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003enanoparticle\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(\u0026micro;g/ml)nanoparticle concentration\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDWB\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePhenolic (mg GAE /g)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003esample\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e15.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZnO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e13.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e13.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTiO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e15.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMgO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e16.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSiO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e17.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIt was previously established that phenolic compounds make up about 4.79 to 8 mg GAE/g of the dry weight of biomass in submerge fermentation \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, a quantity that is naturally lower than that found in shiitake fruit \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. The selection of the most suitable medium designed in this research was recognized as a key factor in enhancing the production of phenolic components in shiitake mushrooms, surpassing the total phenolic values documented in previous studies. What remains unclear is the variation in phenolic profiles among different parts of shiitake mushrooms. The phenolic profiles of shiitake mushrooms were analyzed using an Agilent 6410 QqQ equipped with an Electrospray Ionization interface (ESI), and the results were presented in Table\u0026nbsp;\u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e6\u003c/span\u003e\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab7\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePhenolic acids and flavonoids extracted from moss, shell, stem and biomass of shiitake\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\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 \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePhenolic Compounds\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePolyphenol Classes\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003emoss\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eShell\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eStem\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eBiomass\u003c/p\u003e \u003cp\u003ewith Mgo\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMalic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePhenolic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProtocatechuic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePhenolic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGallic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePhenolic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSinapic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePhenolic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHomogentisic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePhenolic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSulfosalicylic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePhenolic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGentisic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePhenolic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ep-Hydroxybenzoic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePhenolic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChlorogenic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePhenolic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVanillic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePhenolic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCaffeic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePhenolic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSyringic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePhenolic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCinnamic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePhenolic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ep-Coumaric acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePhenolic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFerulic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePhenolic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVeratric acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePhenolic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSalicylic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePhenolic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBenzoic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePhenolic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eo-Coumaric acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePhenolic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAbscisic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePhenolic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3,4-Dihydroxybenzoic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePhenolic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCoumaric acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePhenolic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKaempferol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFlavonoid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIsorhamnetin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFlavonoid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCatechin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFlavonoid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRutin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFlavonoid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNaringin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFlavonoid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMyricetin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFlavonoid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQuercetin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFlavonoid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNaringenin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFlavonoid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHesperetin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFlavonoid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFormononetin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFlavonoid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBiochanin A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFlavonoid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEpicatechin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFlavonoid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQuercetin-3-D-galactoside\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFlavonoid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eResveratrol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eStilbene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePyrogallol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOther polyphenols\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVanillin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOther polyphenols\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSyringaldehyde\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOther polyphenols\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3,4-Dihydroxybenzaldehyde\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOther polyphenols\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAmong the different parts of the shiitake fruiting bodies, the moss and stem exhibited the highest diversity of phenolic components, specifically 21 phenolic acids. The biomass obtained through submerged fermentation supplemented with magnesium oxide nanoparticles contained 19 phenolic compounds, showing slightly lower diversity compared to the moss and stem parts of the shiitake fruiting bodies. The mushroom skin showed the lowest diversity of phenolic compounds, with only 17 phenolic acids identified. The moss of the fruit did not contain gallic acid. In contrast, five phenolic compounds\u0026mdash;gallic acid, homogentisic acid, p-hydroxybenzoic acid, vanillic acid, and salicylic acid\u0026mdash;were not detected in the mushroom fruit shell. Similarly, in the mushroom biomass, three phenolic compounds\u0026mdash;gallic acid, cinnamic acid, and salicylic acid\u0026mdash;were not detected. All 13 listed flavonoids were found in the moss and shell of shiitake mushrooms. Additionally, 12 flavonoids were detected in the stem and biomass of the mushrooms. The only flavonoid that was not found in the stem of the fruit and biomass was naringenin. The recent research revealed the detection of phenolic compounds including gallicacid, chlorogenic acid, syringic acid, rutin, p-coumaric acid, ferulic acid, 2-hydroxy cinnamic acid, caffeic acid, protocatechuic acid, quercetin, abscisic acid, and trans-cinnamic acid\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, which were also identified in the present study.Analysis of shiitake usingboth base and acid hydrolysisrevealed the presence of phenolic compounds including hesperidin, luteolin, and cinnamic acid\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, but two of these compounds, excluding cinnamicacid,were not found in the presentshiitake fruit analysis.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRheological properties\u003c/h3\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eFlow behavior\u003c/h2\u003e \u003cp\u003eIn order to investigate the rheological behavior and emulsifying properties of CSP, pectin was selected among several commercial polysaccharides. Preliminary experiments revealed that the rheological behavior of pectin solution closely resembled that of CSP at similar concentrations, unlike other commercial polysaccharides (specific results not provided). Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e demonstrates that the viscosity of CSP solutions at 1% concentration and pectin solutions at 1% and 2% concentrations did not exhibit significant changes with increasing shear rate. This suggests that the viscosity of both 1% and 2% pectin solutions, as well as 1% shiitake polysaccharide solution, remained independent of shear rate and displayed nearly Newtonian behavior. However, the viscosity of the 2% shiitake polysaccharide solution notably decreased with increasing shear rate, indicating a shear-thinning behavior, characteristic of pseudoplastic fluids. The shear-thinning behavior in polysaccharide solutions occurs due to the alignment of polymer molecular chains in the flow field, which leads to a decrease in viscosity. Additionally, the results demonstrated that an increase in solution concentration, for both CSP and pectin, resulted in higher viscosity. This increase in viscosity can be attributed to the augmented effective volume of the dispersed phase\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMoreover, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e revealed that at very low shear rates, the 2% CSP solution exhibited the highest viscosity. However, overall, the 2% pectin solution displayed the highest viscosity, while the 1% CSP solution had the lowest viscosity. This observation indicates that CSP solutions generally exhibit lower thickening properties compared to pectin solutions. Furthermore, at a shear rate higher than 80 [1/s], the viscosity of the 2% CSP solution approached that of the 1% pectin solution.\u003c/p\u003e \u003cp\u003eThe findings regarding the shear-thinning behavior of the 2% CSP solution align with the reports by Xu et al. in 2016. Xu et al. investigated the viscosity of CSP solutions with different concentrations and reported varying rates of viscosity change with different concentrations. Solutions with higher concentrations exhibited a faster decrease in viscosity. They also noted that a 0.8% CSP solution showed Newtonian behavior at low shear rates. Their research indicated that more concentrated CSP solutions had a diluting effect with higher shear rates\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Xu et al. in 2008 stated that lentinan, a polysaccharide is derived from the shiitake, displays shear-thinning behavior. However, this behavior occurs at very low shear rates\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. These findings are congruent with the results obtained in this study. The obtained results are also consistent with the information provided by Kumar and colleagues in 2023. They reported that lentinan polysaccharide solutions exhibit Newtonian flow behavior in dilute solutions and shear-thinning behavior in semi-dilute solutions at 25\u0026deg;C\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. The high viscosity at low shear rates and the pseudoplastic behavior of CSP make them suitable stabilizers in various food formulations such as mayonnaise, salad dressing, mustard sauce, and vegetable soup\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eViscoelastic properties\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, the 2% CSP solution, like the 2% pectin solution, in the low frequency domain, the storage modulus (G') is greater than the loss modulus(G\"),which indicates the dominance of elastic behavior over viscous behavior and indicates their gel-like structure. On the other hand, in the high frequency domain, the G\"is greater than the G', which shows the gel structure is formed by weak intermolecular forces. Nevertheless in all frequencies tested on 1% pectin and 1% CSP solutions, the G\"is higher than the G', which indicated the dominance of viscous behavior in these samples. Also, the values of G\" and G' in different frequencies of 1% pectin and 1% CSP samples are almost equal. In general, the G\" and G'in 2% pectin and 2% CSP samples were higher than those with 1% concentration, which indicates that intermolecular forces formed at higher concentrations are stronger.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe results of this research largely corroborate the findings reported by Xu et al. in 2016. Xu et al. reported that, except for the 0.8% CSP sample, the G' exceeded the G\" at all frequencies tested. However, in the 0.8% CSP sample, these two moduli intersected, resembling the characteristics of concentrated solutions, viscoelastic fluids, and entangled systems. In their investigations, the 1% CSP solution exhibited a slightly higher G'compared to the G\", with both moduli appearing almost parallel to each other across all frequencies, indicating limited gel formation. They suggested that increasing the concentration of CSP solutions promotes gel formation due to enhanced entanglement between polymer molecular chains at higher concentrations. These entanglements contribute to gel formation \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eEmulsifying properties\u003c/h2\u003e \u003cp\u003eThe present study investigated the stability of different oil-in-water emulsions containing 1% and 2% CSP and pectin over a one-week storage period. In general, the stability of emulsions containing CSP was higher compared to emulsions containing pectin. To expedite the estimation of creaming, the prepared emulsions were subjected to centrifugation. This method also revealed that the highest degree of phase separation was observed in 1% and 2% pectin emulsions. In order to quantitatively assess emulsion instability, the creaming index was calculated for various emulsions after one week of storage. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, the highest creaming index was observed in the emulsions with1% and 2% pectin, while the creaming index for the emulsionwith 2% CSP remained zero after one week. Furthermore, the emulsifying capacity of different emulsions was determined. Figure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB indicates that emulsions containing pectin exhibited the lowest emulsifying capacity, whereas the emulsioncontaining2% CSP displayed the highest emulsifying capacity, reaching 100%.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSwelling power reflects the ability of polymers to absorb and retain water within their structure. The results indicated that pectin possessed higher swelling power than CSP (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). This finding is in accordance with a study by Tu et al. in 2022, where they measured the swelling power of CSP by incorporating shiitake powder into sorghum flour. The reported swelling power value of CSP (4.74) coincides with the results obtained in our investigation. Additionally, the oil absorption capacity of CSP was determined (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC) as part of this study. The findings revealed that pectin exhibited lower oil absorption capacity compared to CSP, suggesting a higher hydrophilicity of pectin polysaccharide compared to CSP \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Based on the results obtained regarding the emulsifying properties, it can be concluded that CSP possesses superior emulsifying properties compared to pectin polysaccharide. Moreover, the 2% concentration of CSP exhibited enhanced emulsifying properties. Overall, CSP demonstrates the potential to enhance viscosity and stabilize emulsions, which may prove valuable in the context of Pickering emulsions.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCytotoxicity of shiitake against human breast cancer cell line MDA\u003c/b\u003e \u003cb\u003e-\u003c/b\u003e \u003cb\u003eMB\u003c/b\u003e \u003cb\u003e-\u003c/b\u003e \u003cb\u003e231\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe therapeutic benefits of shiitake mushroom extracts in treating and preventing diseases are well-established, with polysaccharides and phenolic components being highlighted as the most powerful anticancer agents among the bioactive components \u003csup\u003e\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e.The investigation into the cytotoxic effects of six distinct shiitake mushroom extracts on MDA-MB-231 breast cancer cell lines demonstrated that all extracts led to significant cell death at a concentration of 500 \u0026micro;g/l. Following a 48-hour incubation period, the IC50 levels were evaluated for each extract to determine the concentration required to inhibit 50% of cell growth. The IC50 values obtained for the extracts were as follows: Extracted polysaccharide (225 \u0026micro;g), Fruit moss extract (182 \u0026micro;g), Fruit stem extract (194 \u0026micro;g), Fruit shell extract (244 \u0026micro;g), Mycelium extract of the control sample (202 \u0026micro;g), and Mycelium extract cultivated in MgO-enriched culture medium (138 \u0026micro;g) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn a study which set out to determine anticancer properties of shiitake mushroom, \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e found that IC50 of the aqueous extract of shiitake QF808-1 strain against lung cancers was 198.5 \u0026micro;g/mL. Out of all the parts of the shiitake mushroom fruiting bodies, it was the extract from the moss that showed the strongest inhibitory effect on the growth of breast cancer cells. Of greater significance is the fact that the polysaccharide extracted from shiitake mushrooms, functioning as a biological macromolecule, displayed anti-cancer properties. Out of all the mycelium extracts, the extract derived from the biomass grown in an MgO-enriched medium showed the strongest inhibitory effect in comparison to the rest. In terms of inhibitory power, the mycelium extract from the MgO-enriched culture medium outperformed the rest of the samples tested. The findings suggest that shiitake mushroom extracts, particularly those derived from the moss and mycelium cultivated in an MgO-enriched culture medium, have the considerable potential to trigger cytotoxic effects on MDA-MB-231breast cancer cell lines. These discoveries indicate that these extracts have the potential to serve as effective therapeutic treatments for breast cancer.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe effects of the primary substrates and their concentrations on polysaccharide production were assessed utilizing P.B design as a screening method before employing RSM. The observation of the highest polysaccharide content in the W.S substrate suggests that the biosynthesis of polysaccharides in shiitake mushrooms is stimulated by this substrate, potentially through facilitating the polymerization process of the consumed substrate. Shiitake polysaccharide demonstrated notable rheological properties, exhibiting shear-thinning behavior and enhanced viscosity at higher concentrations. Shiitake polysaccharide solutions also showed superior emulsifying capacity compared to pectin, making them promising candidates for stabilizing oil-in-water emulsions. These findings suggest that Shiitake polysaccharide could be an effective stabilizer in various food formulations, particularly for improving viscosity and emulsion stability. The moss from shiitake fruit bodies, rich in a diverse array of flavonoids and phenolic acids, exhibited the most significant cytotoxicity against the MDA-MB-231 cell line, with the lowest IC50 compared to various fruit parts extracts. Notably, the utilization of nanoparticles not only enhanced phenolic production but also bolstered the biomass's anticancer properties, rivaling those of the shiitake fruit itself. In conclusion, the application of nanoparticles spurred the synthesis of secondary metabolites, resulting in submerged-cultivated biomass with elevated levels of bioactive compounds and more potent anti-cancer properties compared to solid-state-cultivated shiitake fruit which require significant time and expense for production.\u003c/p\u003e"},{"header":"Material methods","content":"\u003cp\u003eShiitake (strain no. M3714) was obtained from the Mycelia company, Deinze, Belgium. Almost all materials were purchased from Merck Company. Concentrations were obtained under low pressure in a rotary evaporator (HeidolphLaborota 4000 efficient rotary evaporator, Germany). The extracts were dried by vacuum freeze-drying (Christ Alpha 1\u0026ndash;2 freeze-dryer). TiO2 NPs and MgO2 NPs were attained from United States Research Nanomaterials Inc. with a purity of \u0026plusmn;\u0026thinsp;99% and an average size of 15 and 20nm, respectively\u003c/p\u003e\n\u003ch2\u003eMicroorganisms, inoculums, and cultivation conditions\u003c/h2\u003e\n\u003cp\u003eThe strain of mushrooms was initially cultivated on potato dextrose agar (PDA) medium. To preserve the strain for long-term use, it was stored at a low temperature of 4\u0026deg;C. For the fermentation process, 250mL Erlenmeyer flasks were utilized, each containing 50mL of a formulated medium to ensure adequate oxygen supply for the growth of the mushroom and production of metabolites. The formulated medium consisted of 30g/L glucose and 10g/L yeast extract, supplemented with micro mineral elements. This medium was used during a 10-day fermentation period to facilitate the development of inoculums with maximum cell viability and concentration. 5 percent of a fresh seed culture was added to the fermentation media. This addition served two purposes: First, it helped improve the growth efficiency of the fungi, promoting their proliferation. Second, it acted as a stimulus for the production of secondary metabolites, which are bioactive compounds of interest \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\n\u003ch2\u003eFermentation Media\u003c/h2\u003e\n\u003cp\u003eIn the study, various substrates (glucose, glucose syrup, glycerol, wheat starch, B6 and B12 vitamin, casein peptone, meat peptone yeast extract, TiO2 NPs, MgO2 NPs) were evaluated to assess their impact on the production of polysaccharides and DWB. The medium was supplemented with MgSO4\u0026middot;7H2O (1g/L), CaCl2 (0.2g/L), KH2PO4 (1.5g/L), and CaCO3 (1.5g/L) as constant factors. A rotary shaker set at 185 RPM and a temperature of 18\u0026deg;C were chosen as the constant conditions for the culture. In an innovative approach to enhance the production of secondary metabolites, the study investigated the effects of titanium dioxide nanoparticles (TiO2 NPs), magnesium oxide nanoparticles (MgO2 NPs), vitamin pyridoxine (B\u003csub\u003e6\u003c/sub\u003e) and B\u003csub\u003e12\u003c/sub\u003e on the growth and formation of secondary metabolites of shiitake in a submerge conditions were examined\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\n\u003ch2\u003eIsolation and purification of polysaccharide\u003c/h2\u003e\n\u003cp\u003eTo extract polysaccharides from the shiitake biomass, the following procedure was employed: The shiitake biomass was separated from the formulated medium by centrifugation at 12,000g for 5 minutes. The separated biomass was washed twice with distilled water to remove any impurities. The washed biomass was then dried and crushed to facilitate the extraction of polysaccharides. The dried and crushed biomass was heated three times in a boiling 96% ethanol solution, each time for 3 hours. After the heating process, the polysaccharides were extracted from the dry biomass by treating it three times with distilled water at a temperature of 70\u0026deg;C for 3 hours each time. The resulting solution containing the polysaccharides was centrifuged at 3,000rpm.To remove proteins, the solution was deproteinated using the sevag method, which involved adding a sevag reagent (a mixture of 1-butanol and chloroform in a ratio of 1:4, v/v) to the solution\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e.The sevag reagent was then removed, and the remaining solution was concentrated using a vacuum rotary evaporator to remove approximately two-thirds of the water phase (supernatant).The concentrated polysaccharide solution was dialyzed against deionized water for 48 hours to remove any remaining impurities and salts. The water-soluble polysaccharides were precipitated by adding 96% ethanol maintained at 4\u0026deg;C for 24 hours. The precipitated polysaccharides were collected by centrifugation. The collected polysaccharides were further processed using a vacuum freeze-dryer to obtain the crude shiitake polysaccharides (CSP) in a dry form \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003ch2\u003ePreparation of Shiitake solid state medium for fruit production\u003c/h2\u003e\n\u003cp\u003eBeech wood sawdust and chips were combined in equal amounts to generate a basic substrate. This substrate provides a suitable environment for the growth of shiitake. The beech wood substrate was detoxified by treating it with an alkaline solution, typically sodium hydroxide (NaOH). This detoxification process helps eliminate contaminants and enhance the growth of shiitake. The substrate was formulated by adding starch and meat peptone. These components serve as a nutrient source for the mushroom, promoting its growth and fruiting. Since the beech wood substrate may naturally contain spore contamination, the mixed substrates were soaked in the 50 ppm NaOCl solution for 6 hours \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. After soaking, the mixed substrates were sterilized by subjecting them to a temperature of 121\u0026deg;C for 2.5 hours. Spawn of shiitake were added to the sterilized medium. The medium with the added spawn was incubated under appropriate conditions, following the guidelines specified by \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. These conditions typically involve maintaining specific temperature, humidity, and light conditions to support the growth and development of shiitake. The fruit bodies can then be harvested after three months for further processing.\u003c/p\u003e\n\u003ch2\u003eExtraction of shiitake dry matter, total phenolic content, and analysis of phenolic compounds\u003c/h2\u003e\n\u003cp\u003eIn the study, dried and finely crushed moss, stem, and shell of shiitake were subjected to extraction using 90% ethanol. The extraction was carried out at 25\u0026deg;C for 72 hours. After the extraction, the extracts were centrifuged and concentrated using a vacuum rotary evaporator\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. To measure the total phenolic contents (TPCs) in the extracts, the Folin\u0026ndash;Ciocalteu reagent and gallic acid (GAE) standard method were used. The GAE standard, dissolved in absolute ethanol, was mixed with the Folin\u0026ndash;Ciocalteu reagent. After 10 minutes, a sodium carbonate solution was added to the mixture, and it was maintained at ambient temperature for 30 minutes. The absorbance of the mixed solution was measured at 765 nm using a UV\u0026ndash;Vis spectrophotometer\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. The TPC was reported as milligrams of GAE per gram of Shiitake dry tissue\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Additionally, the phenolic components extracted from shiitake dry biomass were analyzed to determine their phenolic profiles. The analysis was performed using an Agilent 6410 QqQ mass spectrometer equipped with an Electrospray Ionization (ESI) interface. The phenolic components\u0026apos; structure was examined under specific settings, including drying gas temperature (nitrogen): 350\u0026deg;C, drying gas flow rate: 9L/min, nebulizer pressure: 40 psig, Injection Volume: 25\u0026micro;L, skimmer: 60V, fragmentor voltage: 220V, capillary voltage: 3,500V, and scan range of m/z: 50\u0026ndash;1,000\u003csup\u003e6\u003c/sup\u003e.\u003c/p\u003e\n\u003ch2\u003eRheological properties\u003c/h2\u003e\n\u003cp\u003eHydrocolloid solutions were prepared using shiitake and pectin polysaccharides at concentrations of 1% and 2% (w/v). Pectin solutions were acidified with citric acid, and 0.15% (w/v) calcium chloride was added. These solutions were then heated to 90\u0026deg;C for 2 hours. Rheological properties, including flow behavior and viscoelastic characteristics, were analyzed using a rheometer(Anton PaarPhysica MCR 302, Austria) at 25\u0026deg;C. Viscosity measurements were conducted with concentric cylinders, while viscoelastic properties were assessed using cone and plate geometries. All samples were allowed a rest period of 600 seconds before testing. To determine the linear viscoelastic range, a strain sweep test (strain range: 0.1 to 100%, frequency: 1 rad/s) was conducted. Subsequently, dynamic tests were performed within the linear viscoelastic region, and storage (G\u0026apos;) and loss (G\u0026quot;) moduli were plotted against frequency.\u003c/p\u003e\n\u003ch2\u003eCreaming index, emulsifying capacity, swelling index and fat binding capacity\u003c/h2\u003e\n\u003cp\u003eSunflower oil-in-water emulsions (10% oil ratio) were prepared to determine the creaming index and emulsifying capacity. Emulsions were formulated using 8 ml of 1% and 2% pectin gum and shiitake polysaccharide solutions mixed with 2 ml of sunflower oil. These mixtures were homogenized using a high-shear mixer at 12,000 rpm for 3 minutes. The creaming index was calculated after two weeks of storage at room temperature using the Eq.\u0026nbsp;(1):\u003c/p\u003e\n\u003cp\u003eCreaming-index = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{SH}{TH}\\times\\:100\\)\u003c/span\u003e\u003c/span\u003e (1)\u003c/p\u003e\n\u003cp\u003ewhere SH was the height of the serum layer and TH was the height of the whole emulsion.\u003c/p\u003e\n\u003cp\u003eAlso, the emulsifying capacity was calculated according to Eq.\u0026nbsp;(2).\u003c/p\u003e\n\u003cp\u003eEmulsion capacity (%) =\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{{e}_{\\nu\\:}}{{t}_{\\nu\\:}}\\times\\:100\\)\u003c/span\u003e\u003c/span\u003e (2)\u003c/p\u003e\n\u003cp\u003ewheree\u003csub\u003e\u0026nu;\u003c/sub\u003e was the emulsion volume and t\u003csub\u003e\u0026nu;\u003c/sub\u003e was the total volume.\u003c/p\u003e\n\u003cp\u003eSwelling power of the shiitake and pectin polysaccharides was measured following the method described by \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Briefly, 0.3 g of polysaccharide samples were immersed in 10 ml of distilled water and heated in a boiling water bath for 10 minutes. After cooling, the samples were centrifuged at 1700 rpm for 4 minutes. The swelling power was calculated using the Eq.\u0026nbsp;(3):\u003c/p\u003e\n\u003cp\u003eSwelling power = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{wet\\:precipitate\\:weight}{dry\\:weight}\\)\u003c/span\u003e\u003c/span\u003e (3)\u003c/p\u003e\n\u003cp\u003eFat binding capacity was also evaluated using the method by\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. A 0.2 g sample of shiitake and pectin polysaccharides was mixed with 10 ml of sunflower oil. The mixtures were held at room temperature for 1 hour, with vortexing every 15 minutes. Afterward, the samples were centrifuged at 1600 rpm for 15 minutes, the supernatant was removed, and the residue was weighed\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. The amount of absorbed oil was calculated using the Eq.\u0026nbsp;(4):\u003c/p\u003e\n\u003cp\u003eFat binding capacity = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{Final\\:weight\\:of\\:dry\\:samples}{Initial\\:weight\\:of\\:dry\\:samples}\\)\u003c/span\u003e\u003c/span\u003e (4)\u003c/p\u003e\n\u003ch2\u003eCell culture and viability analysis\u003c/h2\u003e\n\u003cp\u003eThe breast cancer cell line MDA-MB-231 was obtained from the National Cell Bank of the Institute Pasteur of Iran. The cells were cultured in Dulbecco\u0026apos;s Modified Eagle Medium (DMEM) supplemented with 1% penicillin/streptomycin and 10% heat-inactivated fetal bovine serum. The culture was maintained at 37\u0026deg;C with 5% CO2\u003csup\u003e36\u003c/sup\u003e. To ensure high cellular viability, the MDA-MB-231 cells were subcultured using trypsin when they reached near confluency. Approximately 1 million cells were reseeded into a fresh T-75 flask for further experiments. To determine the cell viability of MDA-MB-231 cells and the half maximal inhibitory concentration (IC50) values of the stem, shell, and moss extracts of Shiitake against MDA-MB231 cells, the MTT assay was employed. For this assay, 10,000 cells per well were seeded in a 96-well plate and exposed to different concentrations of shiitake extracts for 48 hours at 37\u0026deg;C. Serial dilutions of the Shiitake extracts at concentrations of 0, 62.5, 125, 250, 500, and 1,000 \u0026micro;g/mL were prepared and added to the wells containing the cells. The cytotoxic effects of the extracts on the MDA-MB-231 cancer cell line were evaluated using the MTT assay, following the protocol described in a previous research study conducted by \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003ch2\u003eStatistical method\u003c/h2\u003e\n\u003cp\u003eIn the study, a P.B design was employed to identify the key substrates that had a significant influence on DWB and polysaccharide. After identifying the key substrates, a RSM approach was used to optimize their quantities. The Central Composite Design (CCD), which is a common form of RSM, was implemented using the statistical package Design-Expert, version 7.0.0, developed by Stat-Ease, Inc., Minneapolis, MN, United States (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://design-expert-7.software.informer.com/\u003c/span\u003e\u003c/span\u003e). The significance of the models, substrates, and their interactions on the responses was verified using analysis of variance (ANOVA). The means and standard deviations (SD) of the experimental data were determined using Microsoft Excel software (presumably referring to Excel version 2023, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.microsoft.com/en-us/microsoft-365/excel\u003c/span\u003e\u003c/span\u003e). To compare the differences among the means at a significance level of 0.05, Duncan\u0026apos;s multiple range tests was performed using SPSS 16.0 software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://spss.software.informer.com/16.0/\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e"},{"header":"Statements \u0026 Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests..\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThe authors declare that no funds, grants, or other support were received during the preparation of this manuscript\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eS.Z.G: Data curation, Formal analysis, Investigation, Project administration, Resources, Software, Writing \u0026ndash; review \u0026amp; editing. H.R.S: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Software, Supervision, Validation, Visualization, Writing \u0026ndash; review \u0026amp; editing.A.R.:Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Software, Supervision, Validation, Visualization, Writing \u0026ndash; review \u0026amp; editing. A.S.F : Data curation, Investigation, Methodology, Project administration, Resources, Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data generated or analysed during this study are included in this article\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eThomas, L. \u0026amp; Mago, P. Unearthing the therapeutic benefits of culinary-medicinal mushrooms for humans: Emerging sustainable bioresources of 21st century. \u003cem\u003eJ. 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Avenanthramide-C reduces the viability of MDA-MB-231 breast cancer cells through an apoptotic mechanism. \u003cem\u003eCancer Cell Int.\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, 1\u0026ndash;12 (2017).\u003cspan dir=\"RTL\"\u003e\u003c/span\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Lentinus edode, Response surface methodology, nanoparticle, polysaccharide, rheological properties, anticancer","lastPublishedDoi":"10.21203/rs.3.rs-6748184/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6748184/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe investigation was carried out to increase polysaccharide yield utilizing statistical strategies, starting with submerge and shifting to solid-state fermentation, with the main aim of promoting the anticancer properties of \u003cem\u003eLentinus edodes \u003c/em\u003e(shiitake) fruit and exploring the rheological properties of polysaccharide. The Plackett–Burman (P.B) screening indicated that wheat starch, meat peptone, and vitamin B6 had a more pronounced impacted on polysaccharide production than the other substrates. The optimal substrates for polysaccharide production, identified using response surface methodology (RSM) after screening, was revealed to contain 48.56 g/l wheat starch, 15 g/l meat peptone, and 300 µg B6 vitamins. Shiitake polysaccharide solutions exhibited shear-thinning behavior, enhanced viscosity, and gel-like properties at higher concentrations. Shiitake polysaccharide also demonstrated superior emulsifying capacity for oil-in-water emulsions, suggesting its potential as an effective stabilizer in food formulations. The fruit body of shiitake produced under optimal polysaccharide conditions had the highest diversity in flavonoids and phenolic acids and significant anticancer activity against MDA-MB-231 breast cancer cell. More to the point, the nanoparticle induced aggravation of human breast cancer cell death were evident with IC50 levels of 182 μg/mL for fruit moss and 138 μg/mL for biomass cultivated in submerge condition containing MgO2 NPs.\u003c/p\u003e","manuscriptTitle":"Statistical optimization of fermentation conditions for metabolites production by Lentinus edodes and investigation of anticancer activity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-16 16:17:49","doi":"10.21203/rs.3.rs-6748184/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"86471b10-8dde-421f-9a16-ae03078c9fc7","owner":[],"postedDate":"June 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":50037855,"name":"Biological sciences/Biotechnology"},{"id":50037856,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2025-07-24T07:23:12+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-16 16:17:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6748184","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6748184","identity":"rs-6748184","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}