Study On The Extracellular Enzyme Activity Of The Effect Of Exogenous Hormones On Liquid Culture Of Agrocybe Cylindracea

The liquid culture of Agrocybe cylindracea can get a large number of mycelia and fermentation products in a short time.At present,the submerged fermentation technology was mainly applied to two aspects.First,the production of liquid spawn.The superiorities are fast,the same age,simple inoculation,large-scale production.Second,the production of mycelia and the extraction of effective component.The liquid culture of Agrocybe cylindracea can produce biological activity matter such as polysaccharide,multi-peptides,alkaloid,sterol,enzyme,nucleic acid,aminoacid,vitamin and antibiotic.The products have the function of anti-cancer, anti-inflammation,anti-senile,anti-bacterial,anti-ulcer and so on.The nutrition ingredient of mycelium is similar with fruit body,so it can produce drink,seasoning,food additive and so on.At present,the studies on the effect of exogenous hormones on edible fungi in liquid culture mainly focus on the conventional varieties such as Pleurotus ostreatus,Lentinus edodes,Flammlina velutipes,less on the rare and precious edible fungi,such as Grifola frondosa,Pleurotus eryngi, Ganoderma Sinense and so on.And the research mainly focus on the impact of single exogenous hormone,types of hormone are fewer.From the viewpoint of application foundation,the rare and precious edible fungi,Agrocybe cylindracea,was selected as research material.For selecting the optimum cultivation conditions,this thesis studied on screening strain,carbon source,nitrogen source,carbon-nitrogen ratio,inoculum volume,pH,liquid media volume,rotation speed,cultivation time.The extracellular enzyme activity of the effect of different kinds of exogenous hormones and different concentration on liquid culture of Agrocybe cylindracea were studied emphatically.By testing mycelia biomass,the contents of protein and reducing sugar,extracellular enzyme activity related to the degradation of liquid media, the optimal exogenous hormones were selected,the effect of the optimal two kinds of exogenous hormones on liquid culture in Agrocybe cylindracea were studied.The use of exogenous hormones can improve enzyme activity and accelerate decomposition and utilization of liquid media,so which can accelerate the propagation of mycelia and cultivation cycle is short.The results laid a foundation for further research on the effect of exogenous hormones on other edible fungi.Meanwhile,it would provide an important theoretic reference for industrialized liquid culture of Agrocybe cylindracea. This thesis had done the following researches:1 By comparing mycelial growth of no.3 strain,no.5 strain,no.6 strain in the slant media and 1 level flask media,the compared results suggested that the mycelial growth rate,the number of pellets,mycelia biomass,the content of extracellular polysaccharide of no.6 strain were significantly higher than no.3 strain and no.5 strain.Therefore,no.6 strain wsa used as testing material. 2 Agrocybe cylindracea utilized carbon sources widely,monosaccharide,disaccharide, macromolecular compounds could be used.By comparing mycelia biomass and extracellular polysaccharide,glucose wsa better than other carbon sources.Peptone as nitrogen source was optimum for liquid culture of Agrocybe cylindracea.(NH₄)₂SO₄ was the worst.The results were keeping with the law of organic nitrogen better than inorganic nitrogen.Using the design of orthogonal experiment L₉(3⁴),the test of concentration optimization with the carbon source(glucose), nitrogen source(protein peptone)and inorganic salt(MgSO₄·7H₂O and KH₂PO₄)which significant influenced to liquid culture of Agrocybe cylindracea.The results of orthogonal experiment showed that,the order of each factor influence the mycelia biomass and the content of extracellular polysaccharide was:carbon source＞nitrogen source＞KH₂PO₄＞MgSO₄·7H₂O.Mycelia biomass and the content of extracellular polysaccharide were the most in the media with glucose 2%,protein peptone 0.1%,MgSO₄·7H₂O 0.2%and KH₂PO₄ 0.3%.The best C/N was 60/1.3 By Screening of the optimal culture conditions,the results showed that the inoculation was 10%, pH was natural,medium volume was 100 ml,speed was 120 r.min-1,the training time was 7 days, both mycelia biomass and metabolic products can reach a maximum.4 Among the research of effect of six kinds of exogenous hormones on Agrocybe cylindracea in liquid culture.Mycelia biomass,CMCase,Fpase,laccase,amylase activity in the media with NAA were the highest,and the optimum concentration was 2.0 mg.100ml^-1.IBA had the strongest role of promote on guaiccal activity,proteinase activity and mycelia protein content,and the optimum concentration was 1.5 mg.100ml^-1.GA had the strongest role of promote on mycelium reducing sugar content,and the optimum concentration was 1.5 mg.100ml^-1.CMC enzyme and FP enzyme were two enzymes related to degradation of cellulose and,the CMC enzyme activity wasstronger than the FP enzyme activity.The results showed that CMCase was the main extracellular cellulosease in Agrocybe cylindracea,that was similar with majority of edible fungus.Laccase activity was low,cellulase activity was high,so the capability of utilize lignin was low.Both laccase and guaiacolase were enzymes related to ligninolytic,guaiacol activity was lower than laccase activity,laccase was mainly used for the degradation of lignin of Agrocybe cylindracea.5 The mycelia biomass,CMCase,laccase,amylase,proteinase,protein content and reducing sugar content were significant different in the liquid media with NAA and IBA.NAA 2.0 mg.100ml^-1+IBA 2.0mg.100ml^-1was the best group to mycelia biomass.NAA 2.0 mg.100ml^-1+IBA 1.0 mg.100ml^-1was the best to CMCase activities.NAA 1.5 mg.100ml^-1+IBA 1.0 mg.100ml^-1can enhance laccase activities,amylase activities,proteinase activities and reducing sugar content. NAA 2.0 mg.100ml^-1+IBA 1.5 mg.100ml^-1can enhance protein content obviously.