Enhancement of polysaccharides production using microparticle enhanced technology by Paraisaria dubia.

BACKGROUND: Polysaccharides are important active ingredients in Ophiocordyceps gracilis with many physiological functions. It can be obtained from the submerged fermentation by the anamorph (Paraisaria dubia) of Ophiocordyceps gracilis. However, it was found that the mycelial pellets of Paraisaria dubia were dense and increased in volume in the process of fermentation, and the center of the pellets was autolysis due to the lack of nutrient delivery, which extremely reduced the yield of polysaccharides. Therefore, it is necessary to excavate a fermentation strategy based on morphological regulation for Paraisaria dubia to promote polysaccharides accumulation. RESULTS: In this study, we developed a method for enhancing polysaccharides production by Paraisaria dubia using microparticle enhanced technology, talc microparticle as morphological inducer, and investigated the enhancement mechanisms by transcriptomics. The optimal size and dose of talc were found to be 2000 mesh and 15 g/L, which resulted in a high polysaccharides yield. It was found that the efficient synthesis of polysaccharides requires an appropriate mycelial morphology through morphological analysis of mycelial pellets. And, the polysaccharides synthesis was found to mainly rely on the ABC transporter-dependent pathway revealed by transcriptomics. This method was also showed excellent robustness in 5-L bioreactor, the maximum yields of intracellular polysaccharide and exopolysaccharides were 83.23 ± 1.4 and 518.50 ± 4.1 mg/L, respectively. And, the fermented polysaccharides were stable and showed excellent biological activity. CONCLUSIONS: This study provides a feasible strategy for the efficient preparation of cordyceps polysaccharides via submerged fermentation with talc microparticles, which may also be applicable to similar macrofungi.

[Study of mycelial polysaccharide from Paraisaria dubia of Ophiocordyceps gracilis asexual].

In order to provide a foundation for the development and application of Ophiocordyceps gracilis and increase the new resources of cordyceps,an asexual Paraisaria dubia was isolated from an O. gracilis fruit body. After 10 days of liquid fermentation,white globular mycelium and clear transparent fermentation were produced. The mycelium was extracted by hot water and precipitated with ethanol to obtain intracellular crude polysaccharide. The protein was deproteinized to obtain deproteinized polysaccharide. The intracellular pure polysaccharide was purified by Sepharose 4 B column chromatography and were analyzed by UV,IR,1 H-NMR,and13 CNMR data,as well as GC and HPLC. The results showed that the intracellular polysaccharide of P. dubia was composed of glucose,galactose and mannose with a molar ratio of 25. 54 ∶2 ∶1. It was a ß-configuration glycosylic bond,containing pyranoside. The initial connection of polysaccharide was ß(1→2)(1→4)(1→6) connection. This experiment provides a theoretical basis for the development and application of P. dubia.

Integration of Physiological, Transcriptomic and Metabolomic Reveals Molecular Mechanism of Paraisaria dubia Response to Zn2+ Stress.

Utilizing mycoremediation is an important direction for managing heavy metal pollution. Zn2+ pollution has gradually become apparent, but there are few reports about its pollution remediation. Here, the Zn2+ remediation potential of Paraisaria dubia, an anamorph of the entomopathogenic fungus Ophiocordyceps gracilis, was explored. There was 60% Zn2+ removed by Paraisaria dubia mycelia from a Zn2+-contaminated medium. To reveal the Zn2+ tolerance mechanism of Paraisaria dubia, transcriptomic and metabolomic were executed. Results showed that Zn2+ caused a series of stress responses, such as energy metabolism inhibition, oxidative stress, antioxidant defense system disruption, autophagy obstruction, and DNA damage. Moreover, metabolomic analyses showed that the biosynthesis of some metabolites was affected against Zn2+ stress. In order to improve the tolerance to Zn2+ stress, the metabolic mechanism of metal ion transport, extracellular polysaccharides (EPS) synthesis, and microcycle conidiation were activated in P. dubia. Remarkably, the formation of microcycle conidiation may be triggered by reactive oxygen species (ROS) and mitogen-activated protein kinase (MAPK) signaling pathways. This study supplemented the gap of the Zn2+ resistance mechanism of Paraisaria dubia and provided a reference for the application of Paraisaria dubia in the bioremediation of heavy metals pollution.

Transcriptomic Analysis of Morphology Regulatory Mechanisms of Microparticles to Paraisaria dubia in Submerged Fermentation.

Liquid submerged fermentation is an effective strategy to achieve large-scale production of active ingredients by macrofungi, and controlling mycelium morphology is a key factor restricting the development of this technology. Mining for superior morphological regulatory factors and elucidation of their regulatory mechanisms are vital for the further development of macrofungal fermentation technology. In this study, microparticles were used to control the morphology of Paraisaria dubia (P. dubia) in submerged fermentation, and the underlying regulatory mechanisms were revealed by transcriptomic. The relative frequency of S-type pellet diameter increased significantly from 7.14 to 88.31%, and biomass increased 1.54 times when 15 g/L talc was added. Transcriptome analysis showed that the morphological regulation of filamentous fungi was a complex biological process, which involved signal transduction, mycelium polar growth, cell wall synthesis and cell division, etc. It also showed a positive impact on the basic and secondary metabolism of P. dubia. We provided a theoretical basis for controlling the mycelium morphology of P. dubia in submerged fermentation, which will promote the development of macrofungal fermentation technology.

Study of mycelial polysaccharide from Paraisaria dubia of Ophiocordyceps gracilis asexual / 中国中药杂志

In order to provide a foundation for the development and application of Ophiocordyceps gracilis and increase the new resources of cordyceps,an asexual Paraisaria dubia was isolated from an O. gracilis fruit body. After 10 days of liquid fermentation,white globular mycelium and clear transparent fermentation were produced. The mycelium was extracted by hot water and precipitated with ethanol to obtain intracellular crude polysaccharide. The protein was deproteinized to obtain deproteinized polysaccharide. The intracellular pure polysaccharide was purified by Sepharose 4 B column chromatography and were analyzed by UV,IR,1 H-NMR,and13 CNMR data,as well as GC and HPLC. The results showed that the intracellular polysaccharide of P. dubia was composed of glucose,galactose and mannose with a molar ratio of 25. 54 ∶2 ∶1. It was a β-configuration glycosylic bond,containing pyranoside. The initial connection of polysaccharide was β(1→2)(1→4)(1→6) connection. This experiment provides a theoretical basis for the development and application of P. dubia.