Effect of the addition of different concentrations of LRM anthocyanin extract on ABSC fermentation
To determine the effect of LRM anthocyanins on ABSC growth and metabolism, different concentrations (0.00, 0.03, 0.06, 0.12 mg/mL) of LRM anthocyanins extract were added to the ABSC fermentation medium, and biomass and EPS contents (mg/mL) were used as parameters to determine the optimal concentration leading to improvement in ABSC fermentation. After 5 days of fermentation, biomass and EPS content initially increased and then decreased with the addition of increasing concentrations of LMR anthocyanins (Fig. 1). Specifically, 0.06 mg/mL of anthocyanins significantly increased biomass and EPS content (P < 0.05). Compared with the control group (fermentation medium without the addition of anthocyanins), biomass increased by 1.05-fold (47.32 ± 0.82 mg/mL), and EPS content showed a twofold increase (13.67 ± 0.25 mg/mL). Hence, the addition of 0.06 mg/mL anthocyanins had an effective influence on the production of biomass and EPS during ABSC fermentation. To further study the effect of anthocyanin extract and its main components, i.e., proanthocyanidin and petunia anthocyanin, on ABSC fermentation, aliquots of anthocyanin crude extract, proanthocyanidin, and petunia anthocyanin extracts (0.06 mg/mL) were, respectively, added to the ABSC fermentation medium, and the effect on biomass and EPS content was investigated.
Effect of the addition of different types of anthocyanins on ABSC biomass and EPS synthesis
ABSC fermentation was carried out in a liquid medium containing different fractions obtained from LMR anthocyanin crude extract (0.06 mg/mL) to determine which fraction was most effective in promoting ABSC fermentation. Samples were obtained every 24 h for the determination of biomass and EPS contents and ABSC growth status. Biomass in all three experimental groups was significantly higher compared with the control (P < 0.05) (Fig. 3). Among added extracts, LMR anthocyanin had the greatest effect on promoting effective biomass accumulation (P < 0.05) (Fig. 2), followed by proanthocyanidins and petunia anthocyanin. ABSC grew well for 72 h, after which mycelia entered the decay phase, accompanied by increasing viscosity of fermentation broth and mycelial autolysis with gradual decrease in biomass. Therefore, the addition of anthocyanins to the ABSC fermentation medium promotes the accumulation of biomass produced by ABSC, and the degree of biomass accumulation is likely determined by the nature of the anthocyanin molecule added to the fermentation medium.
EPS content stimulated by the addition of anthocyanin extracts in the ABSC fermentation medium was higher compared with the control (P < 0.05), and peaked after 72 h of fermentation. As shown in Fig. 3, in the early stage of fermentation (24–72 h), exogenous addition of anthocyanins promoted significantly EPS accumulation. In the late stages of fermentation (96–120 h), with mycelia decay and catabolism prevailing over anabolism, a decrease in EPS accumulation was observed. Additionally, proanthocyanidins were the most effective compound to induce EPS synthesis, followed by petunia anthocyanin.
Hence, the addition of different types of anthocyanins into the ABSC fermentation medium led to remarkable changes in mycelial biomass and EPS production, as well as in mycelial growth. At 72 h of fermentation, biomass and EPS production peaked, resulting in mycelial biomass and EPS yield 3.27 and 2.11 higher, respectively, compared to the control group. Therefore, anthocyanins extracted from LRM can promote ABSC biomass accumulation and EPS production. This promoting effect is presumably related to the nature of the active component (procyanidins or petunia anthocyanin) and might be caused by a synergistic effect among the bioactive components.
Effect of different types of anthocyanins on ABSC hyphal morphology
Mycelium growth is closely related to hyphae morphology and structure. To investigate the effect of the addition of the LRM anthocyanin extract and its major components (procyanidins and petunia anthocyanins) on ABSC, fermentation was conducted for 5 days in a medium containing different anthocyanins. Mycelial morphology investigated by SEM revealed a different degree of change in ABSC hyphae structures after the addition of LRM anthocyanin extract and its major components to the fermentation medium. Mycelial density after stimulation by LRM anthocyanins and major components was altered, with mycelium showing more branches under stimulation by LRM anthocyanins. The surface of the mycelium in the control group was smooth and intact after 72 h of fermentation (Fig. 4a). In contrast, ABSC mycelium in medium containing LRM anthocyanins showed evident folds and slight indentations (Fig. 4b). Therefore, we speculated that the addition of LRM anthocyanins led to an increase in cell membrane fluidity and alterations in permeability, which was conducive to greater exchange of nutrients and elimination of metabolites, thus accelerating ABSC growth and synthesis of polysaccharides.
Effect of LRM anthocyanins on the activity of enzymes involved in EPS synthesis
PGM and PGI are enzymes known to be involved in determining the composition of EPS monosaccharides in Ganoderma lucidum . Therefore, the effect of LRM anthocyanins extract on the activity of these enzymes was investigated. PGI activity in ABSC is shown in Fig. 5. PGI activity after the addition of LRM anthocyanins increased initially and then decreased, reaching a peak at 48 h. PGI activity in the medium containing LRM anthocyanins was significantly different from that in the control group (P < 0.05), with an increase in PGI conversion rate by 349.9%. Interestingly, PGI activity in the medium containing proanthocyanidin or petunia anthocyanin extracts was 100% and 88% compared with the control group, respectively, which indicates a less-pronounced effect of such fractions compared to the LRM anthocyanin crude extract. Moreover, PGM activity in medium with LRM anthocyanin crude extract, proanthocyanidin, and petunia anthocyanin extracts group were 171.6, 150.0, and 120.0% (P < 0.05), respectively, compared with the control group.
Moreover, PMI activity during ABSC fermentation in medium with added LRM anthocyanin crude extract, proanthocyanidin, and petunia anthocyanin extracts was 20.3%, 14.8%, and 7.5% higher than in the control group (P < 0.05), respectively. Therefore, it can be inferred that the addition of LMR anthocyanin crude extract, and to a less-pronounced degree of proanthocyanidins and petunia anthocyanin extracts, had a remarkable effect in enhancing PGI, PGM, and PMI activity in ABSC during fermentation.
Collectively, LMR anthocyanin crude extract has been shown to increase the activity of key enzymes involved in the synthesis of EPS, thus positively affecting EPS yield. Moreover, LRM anthocyanins showed a more pronounced effect than proanthocyanidins and petunia anthocyanins in promoting the activity of a key enzyme involved in EPS synthesis. Overall, LRM anthocyanins contributed to promoting growth and EPS accumulation during liquid fermentation of ABSC, and this strategy can be applied to accelerate the fermentation process.
Transcriptomic and functional analysis of ABSC by RNA-Seq
To further explore the mechanism underlying the LRM anthocyanin-mediated effect on EPS synthesis in ABSC, RNA-Seq analysis was conducted to evaluate the transcriptomic response of ABSC in medium containing LRM anthocyanins. Transcriptomic data revealed a total of 56,115 transcripts; the average transcript size was 3450.28 bp, average GC content was 53.73%, and Q30 score was between 96.14 and 96.16% (Table 1). A total of 349 genes were differentially expressed, among which 93 genes were up-regulated and 256 genes were down-regulated (log2 Fold Change ≥ 2, False discovery rate (FDR) < 0.05) (Fig. 6). Gene ontology (GO) enrichment analysis was used to assign and quantify transcripts according to their presumed function in the cell (Fig. 7). GO analysis indicated 22 subgroups in cellular components, 17 subgroups in molecular functions, and 26 subgroups in biological processes. Differentially expressed genes (DEGs) in the cellular components category mainly included cell, cell part, organelle, and cell membrane. Within the molecular function category, the highest number of transcripts of DEGs were assigned to binding and catalytic activity. In addition, a high number of transcripts were included in biological processes, cell processes, metabolic processes, and response to stimulus categories.
Levels of PGI and PGM in ABSC grown in the presence of LRM anthocyanins extract varied compared with the control group (Fig. 5), but no significant differences in the expression of genes involved in key metabolic pathways were observed (Fig. 8). Findings discussed earlier indicated that activity of PGI and PGM was greater in treated groups than in the control group. Therefore, LRM anthocyanins extracts alter the activity of PGI and PGM at the protein level, but do not induce changes at the transcription sites of polysaccharide-related genes.