Optimization of polysaccharide conditions and analysis of antioxidant capacity in the co-culture of Sanghuangporus vaninii and Pleurotus sapidus

Measurement of DPPH free radical scavenging capacity

For measuring DPPH free radical scavenging capacity, the methods described by Zeng et al. (2012) were followed, with slight adjustments. Samples were accurately weighed and diluted with distilled water to create solutions of varying concentrations. DPPH was weighed precisely and mixed with absolute ethanol to create a 0.1 mmol/L DPPH solution. Subsequently, 2 mL of a sample solution at a specific concentration was mixed with 2 mL of the DPPH solution, thoroughly mixed, and incubated in the dark at 30 °C for 30 min. The absorbance at 517 nm, denoted as A₁, was then measured. The absorbance when distilled water was used instead of the DPPH solution was recorded as A₂, and the absorbance when distilled water was used in place of the sample solution was recorded as A₀. The DPPH free radical scavenging rate was calculated using the following formula:

$$\mathrm{D}\mathrm{P}\mathrm{P}\mathrm{H}\mathrm{f}\mathrm{r}\mathrm{e}\mathrm{e}\mathrm{r}\mathrm{a}\mathrm{d}\mathrm{i}\mathrm{c}\mathrm{a}\mathrm{l}\mathrm{s}\mathrm{c}\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{i}\mathrm{n}\mathrm{g}(\mathrm{\%})=\left(1-{\displaystyle \frac{A_1-A_2}{A_0}}\right)\times 100$$

Measurement of ABTS free radical scavenging capacity

For measuring ABTS free radical scavenging capacity, the methods of Jo, Cho & Chun (2021) were followed, with slight modifications. Equal amounts of 7 mmol/L ABTS solution and 2.45 mmol/L K₂O₈S₂ solution were mixed evenly and left to stand in the dark at room temperature for 12 h to obtain the ABTS mother solution. The ABTS mother solution was then diluted with distilled water until its absorbance at 734 nm was 0.7 ± 0.02, recorded as A₀, to obtain the ABTS working solution. Sample solutions of different concentrations (1 mL each) were mixed evenly with 4 mL of ABTS working solution, placed in the dark at 30 °C for 6 min, and the absorbance at 734 nm was measured and recorded as A. The ABTS free radical scavenging rate was calculated using the following formula:

$$\mathrm{A}\mathrm{B}\mathrm{T}\mathrm{S}\mathrm{f}\mathrm{r}\mathrm{e}\mathrm{e}\mathrm{r}\mathrm{a}\mathrm{d}\mathrm{i}\mathrm{c}\mathrm{a}\mathrm{l}\mathrm{s}\mathrm{c}\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{i}\mathrm{n}\mathrm{g}(\mathrm{\%})=\left(1-{\displaystyle \frac{A}{A_0}}\right)\times 100$$

Glucose concentration screening

According to the results (Fig. 3A), the IPS content showed a gradual increase within the glucose concentration range of 10–30 g/L. At a glucose concentration of 30 g/L, the IPS content reached its highest value of 57.9784 ± 0.4536 mg/g. However, within the 30–70 g/L glucose concentration range, the IPS content decreased. Conversely, the biomass consistently increased from 0.6604 ± 0.0100 g/100 mL to 1.0310 ± 0.0455 g/100 mL within the 10–70 g/L glucose concentration range. Based on the selection criterion of IPS content, the optimal glucose concentration was determined to be 30 g/L.

Yeast extract concentration screening

The results indicate (Fig. 3B) that both the IPS content and biomass increased as the yeast extract concentration increased in the 2–8 g/L range. At a yeast extract concentration of 8 g/L, both the IPS content and biomass reached their maximum values, measuring 59.4230 ± 0.4821 mg/g and 1.1008 ± 0.0902 g/100 mL, respectively. However, when the yeast extract concentration exceeded 8 g/L and ranged from 8–20 g/L, the IPS content showed a decreasing trend, while the biomass remained relatively stable. Therefore, the optimal yeast extract concentration was determined to be 8 g/L.

MgSO₄·7H₂O concentration screening

The results indicate (Fig. 3C) that there was an increasing trend in IPS content as MgSO₄·7H₂O concentration increased in the 0.2–2 g/L range. The IPS content reached its highest value of 62.0891 ± 0.4404 mg/g when the concentration of MgSO₄·7H₂O was 2 g/L, after which it decreased. Similarly, the biomass showed an increasing trend as MgSO₄·7H₂O concentration increased in the 0.2–2.5 g/L range. The biomass reached its peak value of 0.8379 ± 0.0079 g/100 mL when the concentration of MgSO₄·7H₂O was 2.5 g/L and then remained stable. Based on the selection criterion of IPS content, the optimal concentration of MgSO₄·7H₂O was determined to be 2 g/L.

KH₂PO₄ concentration screening

As KH₂PO₄ concentration increased from 0.5 to 1.5 g/L of KH₂PO₄, both IPS content and biomass showed an increasing trend. However, at a KH₂PO₄ concentration of 1.5 g/L, both the IPS content and biomass reached their maximum values, measuring 57.6404 ± 0.5523 mg/g and 0.7956 ± 0.0053 g/100 mL, respectively. As KH₂PO₄ concentration increased from 1.5 to 3.5 g/L, IPS content decreased, while biomass initially decreased and then increased (Fig. 3D). Therefore, the optimal concentration of KH₂PO₄ was determined to be 1.5 g/L.

Initial pH screening

The concentration of IPS showed an increasing trend across the pH range of 3–9. Initially, the IPS concentration was relatively low within the pH range of 3–4. However, it rapidly increased as the pH increased from 4 to 5, followed by a gradual increase within the pH range of 5–9. The highest IPS content of 61.7806 ± 0.5062 mg/g was observed at pH 9, after which it started to decrease. Conversely, biomass showed an upward trajectory within the pH range of 3–6, reaching its highest value of 0.7904 ± 0.0104 g/100 mL at pH 6 before starting to decline (Fig. 3E). Based on the evaluation of IPS content, the optimal pH was determined to be nine.

Sanghuangporus vaninii inoculum ratio screening

Within the 20–80% inoculation ratio range of S. vaninii, the IPS content showed an overall ascending-descending-ascending-descending trend. At a 30% and 70% inoculation ratio, the IPS content reached peak values of 65.2716 ± 0.3909 mg/g and 65.5855 ± 0.4270 mg/g, respectively, with no significant difference between these two peaks (Fig. 3F). Conversely, biomass consistently decreased. Due to the faster mycelial growth rate of P. sapidus compared to S. vaninii, the biomass increased as the proportion of P. sapidus inoculation increased. Therefore, to establish a more comprehensive co-culture state of the two strains and maximize their interaction, the optimal inoculation ratio for S. vaninii was determined to be 70%.

Liquid loading volume screening

The IPS content showed an increasing trend as the liquid volume increased in the 50–100 mL range. At a liquid volume of 100 mL, the IPS content reached its peak at 60.7687 ± 0.3657 mg/g, and then decreased as liquid volume increased. Similarly, the biomass increased as the liquid volume increased from 50 to 150 mL, reaching its highest value of 0.8736 ± 0.0064 g/100 mL before declining (Fig. 3G). Based on the evaluation criterion of IPS content, the optimal liquid volume was determined to be 100 mL.

Inoculum volume screening

IPS content showed an ascending-descending-ascending-descending trend as inoculum volume increased from 5% to 35%. At inoculum volumes of 10% and 20%, the IPS content reached peak values of 61.8494 ± 0.4158 mg/g and 60.8487 ± 0.4427 mg/g, respectively, with a significant difference between these two peaks. Conversely, biomass increased as inoculum volume increased from 5% to 15%, reaching its highest point at 15% with a value of 0.8378 ± 0.008 g/100 mL, after which it started to decline (Fig. 3H). Based on the evaluation criterion of IPS content, the optimal inoculum volume was determined to be 10%.

The optimal culture conditions were determined through Single-factor experiments, with the following parameters: glucose concentration of 30 g/L, yeast extract concentration of 8 g/L, MgSO₄·7H₂O concentration of 2 g/L, KH₂PO₄ concentration of 1.5 g/L, pH of 9, S. vaninii inoculum ratio of 70%, Liquid loading volume 100 mL, and inoculum volume of 10%.

PB design

In this study, a PB design strategy was used to systematically investigate the effects of different elements on IPS content in the co-culture and identify which factors significantly affected IPS content. The experimental design and interpretation of results were conducted using Design Expert 13 software. The specific methodologies and outcomes of the experiments are presented in Table 5.

A variance analysis was performed on the experimental data, and the results are shown in Table 6. The high values of the model confirmation coefficient R² and the adjusted coefficient Adj R² indicate that the model fits well and the results are highly reliable. Among the elements studied, X₂, X₆, and X₈ had significant influences on the response variable. The extent of their influence was ranked as X₂ > X₆ > X₈. Therefore, future experiments will focus on examining the effects of these three elements on the response variable to determine the optimal culture conditions.

RSM experimental design and results

This investigation used the Box-Behnken central composite design strategy with adjustable variables including yeast extract concentration, S. vaninii inoculum ratio, and liquid loading volume. A three-factor, three-level experimental design was implemented to refine the culture conditions, with IPS content as the response parameter. The results are presented in Table 7. The generated IPS content was evaluated and fitted using Design Expert 13 software.

The regression equation for IPS content (Y) was calculated as:

$$\begin{array}{rl}Y=& -234.42+17.72A+1.62B+3.67C-0.02AB-0.06AC+0.03BC\\ & -0.86A^2-0.03B^2-0.03C^2.\end{array}$$

The variance analysis of the RSM analysis model is shown in Table 8. The default term had a p-value of 0.0845, which exceeds 0.05, indicating the model’s soundness. The determination coefficient R² and adjusted coefficient Adj R² were 0.9970 and 0.9932, respectively, demonstrating an excellent correlation between the theoretical and actual values of IPS content. The F-values of factors A, B, and C were 581.67, 49.24, and 181.67, respectively, indicating the precedence of their effects on IPS content as A > C > B. Factors A, B, C, and the interaction terms AC, BC, A², B², and C² exhibited statistically significant effects on IPS content (p < 0.05).

RSM analysis

The RSM analysis results demonstrated the interaction effects between yeast extract concentration, S. vaninii inoculum ratio, and liquid loading volume on IPS content (Fig. 4). The contour plot visually represents the transition from the blue region to the yellow area, with a steeper gradient indicating more significant results. The surfaces of yeast extract concentration, S. vaninii inoculum ratio, and liquid loading volume exhibited a high degree of curvature, suggesting a highly significant impact of these three factors on IPS content.

Design-Expert 13 software was used to optimize the cultivation conditions for IPS content, and the optimal culture conditions were determined to be a yeast extract concentration of 6.12 g/L, S. vaninii inoculum ratio of 68.87%, and a liquid loading volume of 105.55 mL. Under these conditions, the projected IPS content was calculated to be 69.4559 mg/g. However, due to practical constraints, the yeast extract concentration was adjusted to 6 g/L, the S. vaninii inoculum ratio was recalibrated to 69%, and the liquid loading volume was adjusted to 106 mL.

Regression model validation

The results obtained from the optimization design experiments of the RSM showed that the IPS content was 69.4559 mg/g under optimal cultivation conditions, which was 16.19% higher than the IPS content under the pre-optimized culture conditions. Furthermore, when the optimum conditions determined by the RSM optimization method were applied in the actual culture, the IPS content reached 69.9626 mg/g, with a deviation of only 0.73% from the projected value (Table 9). These findings highlight the accuracy and reliability of the projected results obtained through RSM optimization.

Analysis of DPPH free radical scavenging capacity

The scavenging rate of DPPH radicals by S. vaninii, P. sapidus, and the S. vaninii and P. sapidus co-culture increased with the higher mass concentration of IPS. At a mass concentration of 0.02 mg/mL, the scavenging rates were 3.71%, 6.26%, and 7.20% for S. vaninii, P. sapidus, and the S. vaninii and P. sapidus co-culture, respectively. As the mass concentration increased to 0.4 mg/mL, the scavenging rates for S. vaninii and the S. vaninii and P. sapidus co-culture rapidly rose to 87.10% and 80.23%, stabilizing around 87% and 81%, respectively. When the mass concentration reached 0.6 mg/mL, the P. sapidus scavenging rate peaked at 84.99% and remained stable at around 85% (Fig. 5A). The mass concentration of antioxidants (IC₅₀) at a scavenging rate of 50% was used as the evaluation criterion to assess the antioxidant performance and free radical scavenging ability of antioxidants. A lower IC₅₀ value indicates a higher ability of antioxidants to scavenge free radicals (Tang et al., 2010). The IC₅₀ values of IPS from S. vaninii, P. sapidus, and the S. vaninii and P. sapidus co-culture were calculated as 0.1875, 0.2581, and 0.1687 mg/mL, respectively. This suggests that IPS from S. vaninii and P. sapidus co-cultures exhibit a stronger ability to scavenge DPPH free radicals compared to pure cultures of S. vaninii and P. sapidus.

Analysis of ABTS free radical scavenging capacity

The scavenging rate of ABTS free radicals by S. vaninii, P. sapidus, and the S. vaninii and P. sapidus co-culture increased as the mass concentration of IPS increased. At a mass concentration of 0.02 mg/mL, the scavenging rates were 25.62%, 24.86%, and 25.19% for S. vaninii, P. sapidus, and the S. vaninii and P. sapidus co-culture, respectively. As the mass concentration of IPS increased to 0.6 mg/mL, the scavenging rates rapidly rose to 97%, 95.05%, and 97.86% for S. vaninii, P. sapidus, and the S. vaninii and P. sapidus co-culture. With further increases in mass concentration, the clearance remained around 98% (Fig. 5B). The IC₅₀ of IPS on ABTS radical scavenging for S. vaninii, P. sapidus, and the S. vaninii and P. sapidus co-culture were calculated to be 0.1397, 0.1515, and 0.1136 mg/mL, respectively. This indicates that the IPS of S. vaninii and P. sapidus co-cultures exhibit a stronger scavenging ability of ABTS radicals compared to the IPS of S. vaninii and P. sapidus pure cultures.