Separation And Fermentation Cultivation Of Endophytic Fungi Producing Glycyrrhizic Acid From Ordos Plateau Licorice

Endophytic fungi is a widespread microbes special groups but has not been fully known referring to those fungi which living in healthy plant body not cause any disease symptom. They grow mainly in the plant root, stem, leaves such as plant tissues and form a relatively stable fungal colonization. The endophytic fungi of medicinal plants have rich diversity and can produce the same active ingredient with host that have important application value in medicine, agriculture, industry and other industries. It has been became a hotspot in the research of the medicine drug by using endophytic fungi fermentation to produce bioactive substances for industrial production, increasing yield and reducing the cost. In this study, 37 strains endophytic fungi were separated and purified from the root of ordos plateau licorice and four strains produced glycyrrhizic acid were identified by HPLC, LC - MS, meanwhile the fermentation culture conditions of strains B12 were optimized. Experimental results summed up as follows:(1) The surface disinfection of licorice root method that more ideal choice was tested: rinsed with 75% ethanol for 30s at first, sterile water to rinse 4 times, then rinsed with 3% H₂O₂ for 3min, sterile water rushed washing four times, each time 5min. an last rinsed with 0.1% HgCl for 1min and water to rinse 4 times and reaching both thoroughly kill the surface epiphytes, at the same time, not miscellaneous bacteria affect internal fungi separation requirements.(2) 37 strains licorice endophytic fungi were separated by preliminary identification, these strains belong to two orders, three families, six genera, they respectively belong to Fusarium, Penicillium, Aspergillus, Alternaria, trichoderma, richoderma. It can be explained that rhizosphere microorganisms may be the main source of most endophytic fungi.(3) The four endophytic fungi.produced glycyrrhizic acid were separation by HPLC screening: fungal B6, B12, B23 B17, by LC - MS further appraisal, strains of B12 can produce virtually glycyrrhizic acid, its production was 346.1μg/L, compared with other reported strains, the glycyrrhizic acid production of fungi B12 was high yield. Strains B12 was identified preliminarily the strains of mold spores for sickle. after analysis of its morphological properties.(4) With accumulated dry weight of mycelia for the index, by selecting the basic medium type, cultivate concentration of carbon source types, nitrogen source types, pH value, wave bed speed, the incubation temperature parameters, best foundation for cultivating conditions were by the production glycyrrhizic acid. The results showed that the most optimized culture conditions of endophytic fungi B12 was: potatoes sucrose medium, liquid medium carbon sources, nitrogen source for glucose for specially designed, best initial pH value for the 7.0, 4% fermentation, the optimum temperature for 28℃, rotate speed 150rpm, in these culture conditions mycelia dry weight reach a maximum for 8.7 g/L.