| Tartary buckwheat is a well-known medicinal and edible coarse cereal.The tartary buckwheat sprout cultures from the germinated buckwheat seeds is a new kind of vegetable,which is rich in nutrition and has high economic value.Moreover,the tartary buckwheat sprouts is abundant in rutin,quercetin and other active flavonoids,and it has good development value and market prospect.Many studies have reported that the polysaccharides obtained from fungal endophytes could efficiently stimulate the active secondary metabolites synthesis and accumulation of their host plants.In this dissertation,the extracellular and intracellular polysaccharides of endophyte Fataf6(Bionectria piyrodes)were firstly prepared by fermentation,extraction,separation and purification procedures.And then,the effects of polysaccharides on the sprout growth and flavonoids accumulation of tartary buckwheat,as well as their stimulation mechanism were investigated further.In addition,the nutritional and functional quality of tartary buckwheat sprout cultures were also analyzed and evaluated.And the purpose of present study is to provide a helpful basis for the further development and utilization of high quality tartary buckwheat sprouts.The main research results are as follows,1.The crude polysaccharides preparation of endophyte Fataf6 and their effects on the sprout growth and flavonoids accumulation of tartary buckwheat.Three crude polysaccharides designated extracellular polysaccharides(EPS),mycelium water-extracted polysaccharides(WPS),and hyphae-extracted polysaccharides(SPS)of endophyte Fataf6 were successfully prepared by water-extraction and alcohol-precipitation method.Of them,the yield of EPS is about 0.5475 mg/mL,and that for WPS and SPS is 4.80%and 1.76%,respectively.The preliminary screening results showed that the three crude polysaccharides had certain promotion effects on the sprout growth and flavonoids accumulation of tartary buckwheat.Of them,the EPS exhibited the strongest stimulation effects,and the next one was WPS.Treatment with 200?g/mL of EPS to the sprout cultures,the main flavonoid(i.e.,orientin,isoorientin,vitexin,isovitetin,rutin,and quercetin)content of tartary buckwheat sprouts was increased to0.21 mg/g,0.27 mg/g,1.37 mg/g,1.76 mg/g,54.52 mg/g,1.36 mg/g.Compared with the control culture,it was nearly increased by 40.00%,42.11%,85.14%,37.50%,35.05%,and 115.87%,respectively.2.Isolation,purification and physicochemical characteristics analysis of the active polysaccharides from endophyte Fataf6.A total of three polysaccharides named EPS-I,WPS-I and WPS-II were successfully obtained from the crude ESP and WPS by the DEAE-52cellulose anion column chromatography method.The molecular weight distribution of three active polysaccharides was measured by the gel permeation chromatography(GPC).The results showed that the number-average molecular weight(Mn)of EPS-I was determined as 1.449×10~4,and the average molecular weight(Mw)of EPS-I was 5.152×10~4.And the Mn of WPS-I was measured to be 3.463×10~4,and the Mw of WPS-I was 6.248×10~4.Moreover,the Mn of WPS-II was 4.049×10~4,and its Mw was about 9.645×10~4.Furthermore,the high performance liquid chromatography(HPLC)and fourier transform infrared spectroscopy(FTIR)methods were used to analysis the sugar composition and structural characteristics of these polysaccharides.And the results indicated that the three active polysaccharides were mainly composed of glucose,galactose and mannose.3.The effects of active polysaccharids from endophyte Fataf6 on the sprouts growth and flavonoids accumulation of tartary buckwheat.By investigating the effects of different concentrations of EPS-I,WPS-I and WPS-II on the buckwheat sprout growth and flavonoids accumulation,it was found that the polysaccharides had positive effects on the seeds germination rate,germination potentiality,sprout length,sprout biomass and the flavonoids synthesis,and the stimulation effects were closely related with the polysaccharide species and its treatment dosage.In addition,the promotion effects of three polysaccharides were nearly consistent with that of the crude polysaccharides,which indicated the EPS-I,WPS-I,and WPS-II are the main effective components of the crude polysaccharides of endophyte Fataf6.The optimal treatment concentration of EPS-I,WPS-I,and WPS-II was determined as 150?g/mL,50?g/mL,and 100?g/mL,respectively.Of them,the EPS-I exhibited the strongest promoting effect.Applied with 150?g/mL of EPS-I to the cultures,the germination rate of tartary buckwheat seeds was increased to 95.33%,and the buckwheat sprout length was about 8.98 cm,the fresh weight of buckwheat sprouts was 11.06 g/100 sprouts,and the dry weight was as much as 0.8172 g/100 sprouts.Compared to the control cultures,that was increased by about 4.76%,25.77%,39.82%and 7.89%,respectively.For the main flavonoid(i.e.,orientin,isoorientin,vitexin,isovitetin,rutin,and quercetin)content of tartary buckwheat sprouts,it was determined to be 0.18 mg/g,0.32 mg/g,1.03 mg/g,1.51 mg/g,49.09 mg/g and 1.23 mg/g,respectively,which was increased by about 20.00%,68.42%,39.19%,17.97%,21.57%and 95.23%in comparison with the control cultures.4.The preliminary action mechanism of polysaccharides from endophyte Fataf6 in stimulating the flavonoids synthesis and accumulation of tartary buckwheat sprouts.After treatment with the active polysaccharides EPS-I(150?g/mL),WPS-I(50?g/mL),and WPS-II(100?g/mL),the defense enzyme activities such as phenylalanine ammonialyase(PAL),polyphenol oxidase(PPO)and superoxide dismutase(SOD),and the pH value,electrical conductivity,and the flavonoids accumulation of buckwheat sprout cultures were analyzed,and it could be more helpful for illustrating the potential action mechanism of endophyte polysaccharides in stimulating the flavonoids biosynthesis and accumulation of tartary buckwheat sprouts.Treatment with 150?g/mL of EPS-I,the PAL activity of buckwheat sprouts reached a peak of 81.75 U/g at 6 h,and 87.00 U/g at 36 h,which was about 1.47 and 1.56-fold in comparison with the control culture.Moreover,the PPO activity was increased to the highest peak of 23.33 U/g at 6 h,and the SOD activity was increased to 7348.45 U/g at 36 h,that was about 1.94 and 1.32-fold compared to the control.Both EPS-I and WPS-II could reduce the medium pH level of buckwheat sprout cultures.After treatment for 48 h,the medium pH value of sprout cultures reached the lowest level of 5.08 and 5.18,respectively.And that was reduced by about 11.81%and 10.07%compared to the control cultures.All the three polysaccharides could lower the conductivity of tartary buckwheat sprout cultures.After treatment with the EPS-I,WPS-I and WPS-II for 96 h,the conductivity of buckwheat sprout cultures was reduced by about 58.73%,60.75%and 81.38%,respectively in comparison with the control.After treatment with active polysaccharides,the main flavonoids accumulation trend of buckwheat sprouts was nearly the same as that of the control,but their increase amplitude were more obvious than the control cultures.Within 0-36 h of EPS-I elicitation treatment,the increase of main flavonoids of tartary buckwheat sprouts was about 30.43 mg/g,while that of the control culture was only 25.49 mg/g.All these results indicated that the polysaccharides of endophyte Fataf6 could stimulate the defense response of tartary buckwheat sprouts,and the induction signal could be gradually transmitted and amplified by the messenger molecules.Then,it led to the expression of some functional genes,and the activation of some key enzymes.And then,it could promote some flavonoids biosynthesis and accumulation of tartary buckwheat sprout cultures.5.The quality analysis of tartary buckwheat sprouts after treatment with active polysaccharides.After treatment with appropriate concentration of the three active polysaccharides,the nutritional and functional constituents such as the fat,soluble sugar,soluble protein,chlorophyll,carotenoids,amino acids,and the flavonoids of tartary buckwheat sprouts were analyzed and determined.The results showed that the polysaccharides elicitation could enhance the content of soluble sugar,soluble protein and the total flavonoids of buckwheat sprout cultures.Among these elicitation treatments,the EPS-I exhibited the most active promoting effects.With treatment of 150 mg/mL of EPS-I,the content of soluble sugar,soluble protein,and the total flavonoid of tartary buckwheat sprouts was as much as 190.29mg/g,4.11 mg/g and 58.98 mg/g,respectively,which was increased by 5.13%,11.18%and21.40%in comparison with the control cultures.The polysaccharides exhibited a slight inhibitory effect on the chlorophyll and carotenoids synthesis of tartary buckwheat sprout cultures,but had no significant effect on the content of fat.After elicitation treatments,the amino acid composition and content of tartary buckwheat sprouts were nearly the same as that of the control cultures,and they all contained seven essential amino acids such as leucine,lysine,and valine et al.These results could provide a novel strategy for the production,development and utilization of high quality tartary buckwheat sprout cultures in the future.Moreover,it will lay an important scientific basis for investigating and clarifying the action mechanism of endophytic fungi in regulating the bioactive metabolites synthesis and accumulation of their host plants.And it will be more helpful for promoting the rapid development of buckwheat industry in China. |
PDF Full Text Request