Evaluating the Application Potential of a Recombinant Ganoderma Protein as Bioactive Ingredients in Cosmetics.

The aim of this study was to evaluate the application potential of a recombinant fungal immunomodulatory protein from Ganoderma lucidum (rFIP-glu). First, a recombinant plasmid pPIC9K::FIP-glu-His was transferred into Pichia pastoris for the production of protein. The protein was then to assess its free radical scavenging abilities and the effect on the viability of both human immortalized keratinocytes (HaCaT cells) and mouse B16-F10 melanoma cells (B16 cells) in vitro, followed by the effect on the melanin synthesis of B16 cells. The results of SDS-PAGE and western blot showed that rFIP-glu was successfully expressed. Furtherly, a bioactivity assay in vitro indicated that the scavenging rate of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals reached 84.5% at 6.0 mg/mL (p ≤ 0.0001) of rFIP-glu, showing strong antioxidant activity. Subsequently, a safety evaluation demonstrated that rFIP-glu promoted the proliferation of HaCaT cells, with the cell viability reaching 124.3% at 48 µg/mL (p ≤ 0.01), regarding the cell viability of B16 cells after exposure to rFIP-glu (48 µg/mL) significantly inhibited, to 80.7% (p ≤ 0.01). Besides, rFIP-glu inhibited the melanin synthesis of B16 cells in a dose-dependent manner from 100-1000 µg/mL, and rFIP-glu at 500 µg/mL (p ≤ 0.01) exhibited the highest intracellular melanin amount reduction of 16.8%. Furthermore, a mechanism analysis showed that rFIP-glu inhibited tyrosinase (TYR) activity by up-regulating the expression of the microphthalmia-associated transcription factor (MITF) and down-regulating the gene expression of TYR and tyrosinase-related protein-1 (TYRP-1), thus inhibiting melanin synthesis. The data implied that rFIP-glu had significant antioxidant activity and whitening potency. It should be used as raw materials for cosmeceutical applications.

N-Glycosylated Ganoderma lucidum immunomodulatory protein improved anti-inflammatory activity via inhibition of the p38 MAPK pathway.

The global health emergency generated by coronavirus disease-2019 has prompted the search for immunomodulatory agents. There are many potential natural products for drug discovery and development to tackle this disease. One of these candidates is the Ganoderma lucidum fungal immunomodulatory protein (FIP-glu). In the present study, we clarify the influences of N-linked glycans on the improvement of anti-inflammatory activity and the potential mechanisms of action. Four proteins, including FIP-glu (WT) and its mutants N31S, T36N and N31S/T36N, were successfully expressed in P. pastoris, of which T36N and N31S/T36N were glycoproteins. After treatment with peptide-N-glycosidase F, the results of SDS-PAGE and Western blot showed that the glycan moiety was removed completely, indicating that the glycan moiety was N-linked. This was also demonstrated by UPLC-qTOF-MS. The cytotoxicity assay showed that N-linked glycans decreased the cytotoxicity of WT; while, the RT-qPCR assay showed that N-glycosylated WT regulated the mRNA expression of IL-6 and TGF-ß1. The Western blot results showed that N-glycosylated WT reduced the phosphorylation level of p38 MAPK. In conclusion, our findings revealed a novel mechanism by which N-glycosylation of FIP-glu improved its anti-inflammatory activity through the regulation of the expression of inflammatory cytokines in RAW264.7 via inhibition of p38 MAPK phosphorylation. It was proved that N-glycosylation significantly improved the functional properties of FIP-glu, providing theoretical and technical support for expanding the application of FIPs in the food and pharmaceutical industries.

Statistical Optimization of Polysaccharides Production by the Lingzhi or Reishi Medicinal Mushroom Ganoderma lucidum (Agaricomycetes) in Solid-State Fermentation Using Highland Barley Grains.

Ganoderma lucidum is a widely used medicinal mushroom in traditional Chinese medicine that creates a diverse set of bioactive compounds. Highland barley, a typical nutrition-balanced crop, is not convenient for direct consumption but its nutritional characteristics meet the modern requirements of health food. In the present study, barley grains were used as substrates on solid-state fermentation (SSF) of G. lucidum. Statistical optimization of media composition was employed for enhancing the production of polysaccharides. Peptone, medlar, and KH2PO4 were identified as the most important components for producing polysaccharide. Based on the one-factor-at-a-time experimentation, a quadratic regression model with the polysaccharide contents as the response value was established by central composite design (CCD). The results showed that the predicted variables were 2.38% peptone, 1.14% medlar, and 0.25% KH2PO4 for the maximum yield of predicted polysaccharides of 11.64% in the fermented substrate. The experimental polysaccharides obtained using the predicted optimum media composition constituted 11.61% of the fermented substrate, which validates the high degree of accuracy of optimization for enhanced production of polysaccharides by SSF. This study suggested that in the process of barley grains fermentation by G. lucidum for polysaccharides production, the optimized SSF substrate consists of 71.4% barley grains, 2.38% peptone, 1.14% medlar, 0.25% KH2PO4, 2.5% glucose, 0.25% MgSO4 · 7H2O, and 1% CaCO3. According to this study, the strain G. lucidum 00679 showed a good fermentation property by optimizing the media. It might be a candidate industrial strain for further process optimization and scale-up study.

Optimization of the fermentation parameters for the production of Ganoderma lucidum immunomodulatory protein by Pichia pastoris.

In order to obtain a better fermentation parameter for the production of recombinant Ganoderma lucidum immunomodulatory protein (rFIP-glu), an engineered Pichia pastoris GS115 was investigated on the fermentation time, temperature, methanol concentration and initial pH of media, while immunomodulatory activities of the rFIP-glu was confirmed. L9(33) orthogonal experiment were firstly employed to optimize various fermentation parameters in the shake-flask level. The optimized fermentation parameters were subsequently verified in a 5 L fermenter. Biological activities including cell viability and tumor necrosis factor-alpha (TNF-α) mRNA of the rFIP-glu were evaluated on murine macrophage RAW264.7 cells. The results showed that the yield of rFIP-glu was up to 368.71 µg/ml in the shake-flask, and 613.47 µg/ml in the 5 L fermenter, when the Pichia pastoris was incubated in basic media with the methanol concentration 1.0% and initial pH 6.5, and with constant shaking at 280 rpm for 4 days at 26 °C. In vitro assays of biological activity indicated that rFIP-glu had significant toxicity against RAW264.7 cells, and possessed the ability to induce TNF-α mRNA expression in macrophage RAW264.7 cells. In conclusion, engineered P. pastoris showed a good fermentation property under the optimum fermentation parameters. It could be a candidate industrial strain for further study.

Ganoderma proteins and their potential applications in cosmetics.

Ganoderma have been regarded as a traditional source of natural bioactive compounds for centuries and have recently been exploited for potential components in the cosmetics industry. Besides Ganoderma polysaccharides and triterpenes, multiple proteins have been found in Ganoderma. With the in-depth study of these proteins, various pharmacological functions of Ganoderma have become important in the discovery and development of new products. In the review, we summarized and discussed the kinds and characteristics of Ganoderma proteins, especially on fungal immunomodulatory proteins (FIPs) which can be potentially developed into cosmeceuticals or nutricosmetics and are a suitable target for production using established biotechnological methods. Furthermore, we discuss their pharmacological activities of the proteins with a focus on their pharmacological functions related to cosmetics, such as antioxidant activity, inhibition of melanin, antibacterial activity, and regulation of inflammatory mediators. Numerous other questions also are addressed before the proteins can be widely accepted and used as cosmetic additives.