Screening Of Dominant Strains Of Ganoderma Lucidum For Liquid Fermentation With High Production Of Polysaccharides And Triterpenes And Exploration Of The Mechanism Of Promoting Triterpenoid Production

Ganoderma lucidum is a famous medicinal fungus in my country with a long research history.Modern research shows that Ganoderma lucidum contains a variety of active substances,among which polysaccharides and triterpenoids are the main active substances.In the 1950s,my country's Ganoderma lucidum industry developed rapidly,and then the liquid fermentation technology of Ganoderma lucidum was developed.Due to its short cycle,easy control of fermentation conditions,and stable product quality,it has gradually become the focus of research.However,my country's Ganoderma lucidum has rich germplasm resources and uneven quality,and lacks superior strains suitable for liquid fermentation.In this study,41 Ganoderma lucidum strains preserved in the laboratory were screened,and strains with high production of intracellular and extracellular polysaccharides were screened out,and then amplified and fermented in a 50 L fermenter to further explore the growth and changes of polysaccharides in liquid fermentation,which is the industrialization of polysaccharides.The application provides scientific data.The dominant strains of high yield Ganoderma lucidum triterpenes were screened out in liquid fermentation and two stage fermentation of liquid shaking and static culture,and then explore the effects of exogenous additives dexamethasone and ketoconazole on the Ganoderma lucidum triterpenes in the upper mycelium of the two stage fermentation of liquid shaking and static culture.The following are the main results of this paper:1.Screening out the dominant strains with high yield of Ganoderma lucidum mycelium polysaccharide by liquid fermentationThrough liquid fermentation,41 Ganoderma lucidum hybrid strains preserved in the laboratory were systematically analyzed in terms of dry weight of mycelium,intracellular polysaccharide content,polysaccharide yield,molecular weight distribution characteristics and cellular immune activity.The strain GZ04 with the highest dry weight of mycelium was screened out.The dry weight of mycelium was29.86 g/L,which was 95%and 63%higher than the parent G0130 and G0154respectively.The strain GZ02 with the highest intracellular polysaccharide yield was obtained.The rate was 1.34 g/L,which was 64%and 101%higher than the parental G0130 and G0154,respectively.The molecular weight distribution characteristics indicate that the intracellular polysaccharide components of the hybrid strains have no significant changes compared with the parents,but the content of different components is quite different.The aqueous extract of mycelium has a significant ability to stimulate the secretion of NO by macrophages at a concentration of 200?g/m L.A small-scale fermentation experiment on the dominant strain GZ02 with high intracellular polysaccharide production was carried out in a 50 L fermenter.After 168 hours of fermentation,the dry weight of mycelium was as high as 23.14 g/L and the intracellular polysaccharide yield was 1.43 g/L.The production of Ganoderma lucidum polysaccharides provides scientific data and provides a reference for the industrial application of Ganoderma lucidum intracellular polysaccharides by liquid fermentation,which has important economic significance.2.Screening out dominant strains of high-yield Ganoderma lucidum exopolysaccharide in liquid fermentationThrough liquid fermentation,the 41 Ganoderma lucidum hybrid strains preserved in the laboratory were systematically analyzed in terms of extracellular polysaccharide yield,polysaccharide content,molecular weight distribution characteristics and cellular immune activity.The strain GZ07 with the highest exopolysaccharide yield was screened out.The exopolysaccharide yield was 3.58 g/L,which was 72%and 87%higher than the parent G0119 and G0130,respectively.The molecular weight distribution characteristics showed that the extracellular polysaccharide component of the hybrid strain had no significant change compared with the parent,but the content of different components was quite different.Exopolysaccharides have a significant ability to stimulate macrophages to secrete NO at a concentration of 200?g/m L.A small scale fermentation experiment with a 50 L fermenter was performed on the dominant strain GZ07 with high production of extracellular polysaccharides.After 168 hours of fermentation,the yield of extracellular polysaccharides was 4.07 g/L,which provides scientific data for the production of Ganoderma lucidum polysaccharides by liquid fermentation technology.The liquid fermentation of Ganoderma lucidum exopolysaccharide provides a reference for the industrial application and has important economic significance.3.The dominant strains for high yield Ganoderma lucidum triterpenes were screened out in liquid fermentation and two stage fermentation of liquid shaking and static cultureThe 41 hybrid Ganoderma lucidum strains preserved in the laboratory were cultured through liquid fermentation and two-stage fermentation of liquid shaking and static culture respectively,and analyzed by chemical method,HPLC method and"Chinese medicine fingerprint similarity evaluation system".The content and yield of the obtained mycelial triterpenes were analyzed by liquid fermentation.The results of chemical determination showed that there were 18 strains with higher triterpene content in the hybrid offspring than the parent.The top three strains were GZ08,GZ09,and GZ36.The triterpene content was(2.66±0.04)g/100g mycelium,(2.64±0.01)g/100g mycelium,(2.58±0.02)g/100g mycelium;there are 20strains with higher triterpene polysaccharide yields than their parents.The top three strains are GZ37,GZ09,GZ01,three The terpene yields were(0.42±0.03)g/L,(0.41±0.01)g/L,and(0.41±0.01)g/L.According to the HPLC fingerprint,the peak time of different strains is basically the same,but the peak area is small and the triterpene content is low.The content and yield of triterpenoids in the upper mycelium were analyzed by two-stage fermentation of liquid shaking and static culture.The results of chemical determination showed that there were 14 strains with higher triterpene content in the hybrid offspring than the parent.The top three strains were GZ33,GZ36,and GZ32.The triterpene content was(19.56±0.12)g/100g mycelium,(18.93±0.35)g/100g mycelium,(16.92±0.16)g/100g mycelium.The similarity analysis results of fingerprints of traditional Chinese medicine showed that the similarity between different strains was above 90%,indicating that the similarity between different strains was high,and the triterpenoid species had no obvious difference.According to the HPLC fingerprint,the peak time between different hybrid strains Basically the same,but the peak area is different.There are 18 strains in the progeny of Ganoderma lucidum strains with a higher peak area than the parent.The top five hybrid strains with peak area are GZ33,GZ32,GZ22,GZ36,GZ07,with peak areas of 14.53×106 m AU·min and 14.16×10⁶ m AU·Min,13.56×10⁶m AU·min,13.38×10⁶ m AU·min,12.10×10⁶m AU·min;there are 13 strains with higher triterpene content than their parents,and the top five strains with triterpene content are GZ09,GZ36,GZ10,GZ08,GZ33,the triterpene content is 32.49×10⁶(g upper mycoderma)^-1,31.84×10⁶(g upper mycoderma)^-1,26.87×10⁶(g upper mycoderma)^-1,26.39×10⁶(g upper mycelium)^-1,26.13×10⁶(g upper mycelium)^-1.Because the peak area of GZ36 ranks first among the five strains with higher triterpene content,the plate growth rate of strain GZ36 is 5.55mm/d,and the dry weight of mycelium is 20.76 g/L,which are both at a high level.4.Exploring the influence of exogenous additives on the two stage fermentation of liquid shaking and static culture of Ganoderma lucidum triterpeneSelect GZ36 as the test bacteria beads,and explore the P450 inducer(dexamethasone)and P450 inhibitor(ketoconazole)on the 3rd day of the shaker fermentation culture,the 7th day of the shaker fermentation culture,and the 3rd day of the static culture respectively The effect of adding 50?mol/L and 250?mol/L on the triterpenoid production of Ganoderma lucidum in static culture.The results show that the addition of dexamethasone can increase the content of Ganoderma lucidum triterpenes,the total peak area in the fingerprint spectrum has a significant increase,and the effect of dexamethasone at a low concentration of 50?mol/L is better,and dexamethasone was found in this study.The effect on triterpenes is related to the concentration of addition,and the time of addition has minimal effect on the triterpenes of Ganoderma lucidum;adding ketoconazole will inhibit the production of triterpenes of Ganoderma lucidum,and the total peak area in the fingerprint is significantly reduced,and the low concentration is 50?mol/L Ketoconazole L has a good inhibitory effect.Ketoconazole at a high concentration of 250?mol/L almost completely inhibits the production of triterpenes.In this study,it was found that the concentration and time of addition of ketoconazole affect the triterpenes of Ganoderma lucidum.The effect of adding ketoconazole on the 7th day after the end of the shaking table fermentation and the 3rd day of standing is better than adding ketoconazole on the 3rd day of the shaking table fermentation.The fingerprint similarity of the samples of the two exogenous additives was evaluated,and the results showed that the similarity between different strains was at a high level,indicating that the types of triterpenes did not change.