Studies On The Nutritional Requirements And Preparation Of Liquid Inoculum For The Medicinal Fungi Including Gymnopus Sp.and Ganoderma Sp.

The development and utilization of medicinal fungi have broad prospects,but the fermentation and cultivation cycle of medicinal fungi is allow too long.As the basis and guarantee for the exploitation and utilization of medicinal fungi,the germplasm resources and the preparation of strains have many deficiencies:the germplasm resources of medicinal fungi are not clear,and the quality of liquid fungi is lack of evaluation criteria.The cultivation of medicinal fungus Ganoderma lingzhi is mainly based on solid inoculum model,and the cultivation period is up to three months,but the production of liquid inoculum model can shorten the period by about one month.The secondary metabolite of Gymnopus sp.0612-9 has strong anti-Penicillium activity and can be used as microbial preservative for Citrus preservation,however long fermentation cycle,higher energy consumption and lower efficiency are a bottleneck in production of the antifungal product.In this paper,the taxonomic status of six strains was determined by determined ITS sequence and construct phylogenetic tree method,HPLC fingerprinting and mycelium growth rate method respectively.Liquid bacteria were cultured by shaking flask culture method,single factor screening,orthogonal test to optimize the liquid culture medium and culture conditions,sampling counting method and filtration weighing method to determine the number of liquid bacteria,and iodine-amylase method to determine the activity of amylase,respectively,the number of liquid bacteria was determined by the method of sampling and counting,and the activity of amylase by iodine-starch method,and the ABTS method was used to determine the laccase activity.The following fermentation was carried out by shaking flask culture and 5L fermenter.The accumulation of active substanceⅡwas monitored by HPLC,and the activity of anti-Penicillium was evaluated by the Oxford Cup method.The yield of Ganoderma lucidum triterpenoid was determined by vanillin-perchloric acid colorimetric method.The activity of liquid bacteria was compared by mycelium growth rate.The results showed that there were significant differences in nutritional requirements among the six strains:one is Gymnopus sp.,four of them are Ganoderma lingzhi and the other is Ganoderma resinaceum（38#）.According to the characteristics of nutritional needs or the differences fingerprints of Ganoderma,5 strains of Ganoderma can be divided into four classes（one is 25#and 26#,the other three types are 38#,50#,51#by eachself）,and both results are the same.In this experiment,the feasibility of chemical classification was verified,and biological characteristics,a method of identifying varieties,was explained.The best liquid medium composition of Gymnopus sp.was:corn flour 30 g/L,maltose 10g/L,peptone 15g/L,KH₂PO₄ 2g/L,Mg SO₄·7H₂O1g/L.The best culture conditions included initial p H 5,inoculum（mycelial cake）3×Ф7mm,loaded volume 100m L/250m L,temperature 28℃,and rotation speed 160r/min.Subsequent fermentation was carried out using optimized liquid inoculum aged 9d and inoculative quantity of 7.5%.The optimized fermentation period in the fermenter was shortened from10d to 5d,and the yield increased by 8.28%over pre-optimization.The best liquid medium composition of five strains of Ganoderma were:corn powder 20g/L+soybean powder 20g/L（25#,26#）.corn flour10 g/L+soybean cake powder 30 g/L（38#,50#,51#）.The culture conditions included initial p H natural,inoculum（mycelial cake）3×Ф7mm,loaded volume100m L/250m L triangle bottle,temperature 28℃,and rotation speed 160r/min.Subsequent fermentation was carried out using optimized liquid inoculum aged4d（38#）,5d（25#,26#,50#）,6d（51#）and inoculative quantity of 10%.The optimized fermentation period in the fermenter was shortened by 1d,and the yield of 25#increased by 19.1%over pre-optimization,and the yield of 26#increased by 17.2%over pre-optimization,and the yield of 38#increased by 27.0%over pre-optimization,and the yield of 50#increased by 23.5%over pre-optimization,and the yield of 51#increased by38.2%over pre-optimization.The feeding rates of the five Ganoderma strains after optimization increased by 33.2%（25#）,29.5%（26#）,10.1%（38#）,23.5%（50#）,38.2%（51#）respectively.The feeding speed of the five Ganoderma lucidum strains was positively correlated with the number of bacteria balls,and the correlation coefficient was greater than 0.85.Therefore,the number of bacteria balls could be used as the basis for rapid evaluation of the activity of liquid strains of Ganoderma lucidum.Multivariate regression analysis was used to determine the correlation equation between the main nutrients(including crude protein,crude fiber,soluble sugar,crude fat and soluble total sugar of the medium and the polysaccharide yield of Ganoderma lucidum fruiting bodies and spores.According to the equation,the formula B of high yield of functional components of fruiting body and the formula B of high yield of functional components of spores were obtained.