| Objective:To study the liquid fermentation conditions of Morchella esculenta rich in selenium and the corresponding changes of active substances in fermentation hyphae with different selenium concentrations,so as to provide conditions for selenium-enriched culture;The selenium collection characteristics of Morchella esculenta silk were studied to provide new ideas for the effective utilization of selenium resources.And explore the physiological and biochemical indicators of selenium-enriched hyphae and the combination of selenium and amino acid macromolecules in hyphae,so as to provide reference for understanding the physiological role and metabolic mechanism of selenium in the growth and development of Morchella esculenta,for the production of selenium-containing active substances,and for the further molecular research of Morchella esculenta in the future.The study of proteins with different solubility and saturation lays a foundation for the extraction and purification of selenium-containing proteins,and further promotes the deep processing of Morchella esculenta and the further development of medicinal and health products.At the same time,it provides a reference for further molecular research of Morchella esculenta.Methods:Taking sodium selenite as inorganic selenium source and the utilization rate of selenium source for transforming inorganic selenium into organic selenium as the investigation index,the liquid culture conditions of Morchella esculenta mycelium were optimized by orthogonal experiment.Polysaccharides in hyphae were extracted and purified by water extraction and alcohol precipitation,respectively,and the content of polysaccharides was determined by anthrone sulfate method.Soluble protein was extracted by phosphate buffer,precipitated by ammonium sulfate and purified by dialysis.The content of soluble protein was determined by Coomassie Brilliant Blue G250 method.In addition,water soluble,alcohol soluble,alkali soluble and salt soluble proteins were extracted,precipitated by ammonium sulfate and dialyzed.The selenium content of total selenium,polysaccharide and protein was determined by atomic fluorescence spectrometry.The activity of T-SOD,POD,PPO and the content of Pro were determined by kit method.HPLC and external standard method were used to determine the types and contents of hydrolyzed amino acids in hyphae of orthogonal optimal group without selenium addition SeCys and SeMet contents in the myceliumof the orthogonal optimal group without selenium addition were determined by LC-MS and external standard method.Results: 1.the best culture conditions for liquid fermentation of mycelium rich in organic selenium are as follows: selenium concentration 25μg/mL,culture time 5d,culture temperature 24℃,and liquid volume200 mL.Under these conditions,the utilization rate of selenium source for transforming sodium selenite into organic selenium from mycelium is 1.853%.2.Under the culture of selenium concentration of 10,20,30,40 and 50 μg/mL,the activity of T-SOD increased continuously,and the content of proline first increased and then decreased,which was stronger than that of the group without selenium.And PPO activity first increased and then decreased,and reached the maximum when selenium concentration was 40μg/mL.However,POD activity first decreased and then increased,and reached the lowest when selenium concentration was 30μg/mL,which was lower than that of the group without selenium.High concentration selenium would inhibit the activities of T-SOD and PPO.The activity of PPO reached the maximum when selenium concentration reached 40 μg/mL,and the activity of T-SOD reached the maximum when selenium concentration reached 50 μg/mL.Because selenium can protect phenols from oxidation to quinones to a certain extent,POD activity decreases.Then,because high concentration selenium inhibits hypha growth and hypha accelerates aging,POD activity rises.The proline content first increased and then decreased,and reached the maximum value at 30 μg/mL,indicating that the stress resistance was first strong and then weak,but both were stronger than those without selenium.After selenium enrichment,the enzyme activity and stress resistance of Morchella esculenta powder containing organic selenium were enhanced.3.Under the culture of selenium concentration of 10,20,30,40 and 50 μg/mL,the content of polysaccharide and soluble protein in hyphae increased first and then decreased.The content of polysaccharide was the highest at 20 μg/mL,and the protein content was lower than that of the group without selenium.The results indicated that selenium-enriched culture could inhibit the accumulation of soluble protein in hyphae.4.Compared with the group without selenium,the selenium content in hypha polysaccharide and soluble protein obtained by selenium-enriched culture increased in different degrees,and both increased first and then decreased,indicating that the addition of inorganic selenium was beneficial to the increase of selenium-containing polysaccharide and selenium-containing protein in hypha.5.Comparing the differentsoluble proteins in the hyphae of the orthogonal optimal group and the group without selenium,it was found that the selenium content of alkali-soluble,alcohol-soluble,salt-soluble and water-soluble proteins in the orthogonal optimal group was higher than that in the group without selenium,and the selenium content of alkali-soluble and salt-soluble proteins was relatively higher,reaching 18.15 μg/g and 9.32 μg/g respectively.The protein in mycelia of orthogonal optimal group and the group obtained by culture without selenium were precipitated by different ammonium sulfate saturation.Compared with the group without selenium,the protein selenium content in mycelia of orthogonal optimal group cultured under optimized conditions was the highest at 30% saturation.6.By measuring the hydrolyzed amino acids of protein,it was found that there were 16 kinds of amino acids in Morchella esculenta silk,among which the contents of glutamic acid,aspartic acid and serine were the highest,while the contents of histidine,methionine,isoleucine and tyrosine were low.The determination of seleno-amino acids showed that the binding form of amino acids and selenium in selenium-rich morel silk protein was mainly SeMet selenomethionine,followed by SeCys selenocysteine.Conclusion: Morchella mycelium can transform inorganic selenium into organic selenium,which is a good carrier for selenium organization,and provides a new way for the development and utilization of selenium-rich Morchella and selenium industry.Liquid fermentation hyphae of Morchella esculenta have good selenium collection characteristics,and selenium has various effects on the growth and development of Morchella esculenta silk,which indicates that liquid fermentation hyphae is an effective selenium collection method of Morchella esculenta.The addition of inorganic selenium in the culture solution promoted hyphae to produce more favorable active substances with higher selenium content in many ways,which indicated that liquid fermentation hyphae was an effective selenium collection method for Morchella esculenta,and provided a new path for the industrial development of Morchella esculenta.At the same time,the combination ways of selenium and macromolecules in Morchella silk hyphae include selenium-containing polysaccharide,selenium-containing protein,selenomethionine and selenocysteine,which provide theoretical and molecular reference for the metabolic mechanism of efficient selenium enrichment in Morchella esculenta. |
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