| In this study, anamorphic strains of Cordyceps militaris, Paecilomyces militaris were used as carrier for accumulation and transform of 3 trace elements, selenium, zinc and germanium, and enrichment and organic transform of the three trace elements, change of P. militaris mycelium composition, combination form of trace elements and P. militaris mycelium metabolites, distribution of trace elements in mycelium and bioactive and function of organic transformant were investigated for the development of healthy functional food and medical drugs of bioactivities and as an effective supplement of trace element.1. With biomass and enrichment capacity of Se, Zn and Ge as estimates,30 strains of P. militaris were screened. The result showed that different strains had different enrichment capability. The capacity was generally related with mycelium biomass and organic conversion rate of Se, Zn and Ge. Their mycelia were poorly tolerant to Se, tolerant to Zn and strongly tolerant to Ge. Se had the highest transformation rate, followed by Zn and Ge.(1) The strain PM14 showed the strongest Se-enrichment capacity. With sodium selenite concentration at 20μg/mL, the transformation rate of organic Se was up to 31.6%. At these concentration, the biomass was the highest up to 17.0 mg/mL.(2) Strains PM28 and PM14 showed strong Zn-enrichment capacity. With ZnSO4 concentration at 200μg/mL, the transformation rates of organic Se reached 6.65% and 5.78%, respectively. At these concentrations, the biomasses were the highest up to 19.5 mg/mL and 17.6 mg/mL, respectively.(3) Strains PM28 and PM14 showed the strongest Ge-enrichment capacity. With GeO2 concentration at 500μg/mL, the transformation rates of organic Ge reached 2.3% and 1.9%, respectively. At these concentrations, the biomasses were up to 15.1 mg/mL and 13.8 mg/mL, respectively.In general, because strain PM14 was chosen for further study due to its strong capacity of enrichment of Se, Zn and Ge.2. The distribution of the organic Se, Zn and Ge in the macromolecules such as protein, polysaccharide and nucleic acid was investigated. The results showed that protein, polysaccharide and nucleic acid were the main vectors and existing forms of organic Se, Zn and Ge. In addition, small portions of organic Se, Zn and Ge were combined with other substances. The content of organic Se, Zn and Ge was mainly in the protein, followed by polysaccharide and the nucleic acid. The concentration of Se, Zn and Ge had some influence on the content of the 3 elements. When the concentration was too high or too low, the content of the elements decreased.(1) As the sodium selenite concentration was at 20μg/mL in the culture medium, the organic Se content in protein, polysaccharide and nucleic acid reached the the peak,221.5μg/g,86.3μg/g and 0.6μg/g, respectively. When sodium selenite concentration was 10μg/mL, the protein-Se, polysaccharide-Se and nucleic acid-Se of the total mycelium took the highest proportion, up to 65.8%,28.1% and 0.2% of organic Se, respectively. They were 2.7,3.9 and 3.8 times of the corresponding controls. The whole organic Se content in the protein, polysaccharide and nucleic acid was 308.4μg/g when the sodium selenite concentration was 20μg/mL. As the sodium selenite concentration decreased to 10μg/g, of the total mycelium organic Se, protein Se, polysaccharide Se and nucleic acid Se showed the highest proportion, up to 94.1%.(2) Within the experiment range of Zn concentration, the organic Zn content in protein, polysaccharide and nucleic acid rose with increase of the Zn concentration in the culture medium, organic Zn proportion in protein and polysaccharide rose with increase of Zn concentration, while the proportion in nucleic acid delined, but with the change not obvious. When ZnSO4 concentration was 200μg/mL, the content of organic Zn was the highest. The distribution of the organic Zn in the mycelium was as following:the proportion of protein-Zn was the highest up to 65.6%, followed by polysaccharide Zn (7.2 %) and nucleic acid Zn (0.8%). The protein-Zn and polysaccharide-Zn were 2.2 and 1.1 times of the corresponding control, respectively, but nucleic acid Zn was decreased 33.9% compared with the control. This indicating that the Zn has promotive effect on synthesis of organic Zn in protein and polysaccharide.(3) Low concentration Ge had promotive effect on the organic Ge content in protein, polysaccharide and nucleic acid, while high concentration Ge had inhibitive effect on organic Ge formation. When GeO2 concentration was 500μg/mL, the organic Ge content in the protein, polysaccharide and nucleic acid reached the highest up to 284.0μg/g,261.5μg/g and 137.6μg/g, respectively. They were 4.4,5.4 and 8.3 folds of the control group. The proportion of the protein-Ge to total organic Ge in mycelium was similar with polysaccharide-Ge. When the GeO2 concentration was 100μg/mL, of the total organic Ge in the mycelium, protein-Ge, polysaccharide-Ge and nucleic acid-Ge showed highest proportion, which were 39.8%,36.8% and 18.2%, respectively. When GeO2 concentration was 100μg/mL, the proportion of the sum of protein-Ge, polysaccharide-Ge and nucleic acid-Ge to total organic Ge was the highest (94.8%).3. The effects of Se, Zn and Ge concentration on biomass and the main active ingredients (intracellular polysaccharide, trace elements content, cordycepin, mycelium SOD, protein content, amino acids content and so on) in mycelium were investigated. The results showed that the Se, Zn or Ge at an appropriate concentration had promotive effect on the main ingredients of mycelium. However, when the concentrations were excessive, they had inhibitive effect on the related ingredients. Different biological directors needed different Se, Zn and Ge concentrations. The most appropriate concentration of Se, Zn, and Ge were 10-20μg/mL,100μg/mL and 200μg/mL-300μg/mL, respectively.4. Antioxidation test indicated that Se, Zn or Ge-enriched polysaccharide had substantial function of scavenging superoxide radical, hydroxyl radical and DPPH. Compared with the control, the capability of scavenging radical was significantly stronger for Se-olysaccharide and Zn-polysaccharide. From the point of the radical type, the scavenging capability of DPPH was the highest, followed by those of hydroxyl radical and superoxide radical. Generally, for the capability of scavenging radical, Se-polysaccharide was the strongest, followed by Zn- and Ge-polysaccharide.5. The drosophila life-span experiment showed that the Se, Zn or Ge-polysaccharides all had significant effect on the half death time, average and maximum life span, suggesting obvious antisenescence effect. The polysaccharides dramatically elongated half death time, particularly to the males. Se-polysaccharide had no obvious effect on maximum and average life-span, but with Zn- and Ge-polysaccharide, the effect on maximum life-span was stronger than that on average life-span. In general, the effect on the males was more obvious than that on the females. The elongating effect on weak drosophila was significant with Se-polysaccharide, while that on healthy drosophila was with Zn- or Ge- polysaccharide.6. The exhaustive swimming experiments of mice and tests on contents of blood lactic acid and blood urea nitrogen, hypoxia-tolerance under normal air pressure showed that different concentrations of mycelium enriched with Se, Zn or Ge had different fatigue tolerance effect on the mice at different physiological periods. They significantly elongated exhaustive swimming time of the mice and showed the effects of reducing lactic acid production at the status of fatigue and reducing or removing blood urea nitrogen. However, the effect of hypoxia-tolerance was not obvious.7. Micronucleus test of broad bean showed that the polysaccharides enriched with Se, Zn or Ge had no mutagenic effects. Instead, they significantly inhibited the production of cell micronucleus in broad beans induced by mitomycin and ultraviolet radiation. Concentration of polysaccharide and the inhibition rate had obvious dose-effect relationship. The micronucleus inhibition rate rose with increase of the polysaccharide concentration. When concentration of the polysaccharide enriched with Se, Zn or Ge was at 100μg/mL, the inhibition rates induced by mitomycin and UV radiation were 46.5%, 37.2%,34.1% and 53.3%,48.6%,43.8%, respectively. The Se-polysaccharide had the highest inhibition, followed by Zn-polysaccharide and Ge-polysaccharide.8. In vitro antitumor experiments showed that Se-polysaccharide or Zn-polysaccharide had significant inhibition effect on lung cancer cell line, as comppared with control group. They significantly inhibited the growth of the lung cancer cell A594. When the polysaccharide concentration was 4 mg/mL, the inhibition rates of Se-polysaccharide and Zn-polysaccharide were 54.2% and 53.9%, respectively. Compared with the control of mycelium without the trace elements added, the inhibition rates increased by 38.8% and 38.0%, respectively.Se-polysaccharide, Zn-polysaccharide or Ge-polysaccharide all displayed significant inhibitionx on cell line CNE-1 of nasopharyngeal carcinoma, as compared to the control group. At concentration of 4 mg/mL, the inhibition rate of Se-polysaccharide, Zn-polysaccharide and Ge-polysaccharide were 40.2%,32.1% and 39.6%, respectively. As compared with the control of mycelium without the trace elements added, the inhibition rates increased by 128.8%,82.4% and 125.2%, respectively, suggesting strong inhibition of the growth of CNE-1.9. Acute oral toxicity test of mice showed that the mycelium of P. militaris enriched with Se, Zn and Ge belonged to nontoxic products.It is concluded that the mycelium of P. militaris produced in liquid culture enriched Se, Zn and Ge. At appropriate concentration of trace elements, the content of main active ingredients in the mycelium enriched with trace elements increased significantly and the antioxidant, antimutagenic and anti-tumor functions of the enriched mycelium rose significantly. The mycelium enriched with Se, Zn and Ge had no mutagenic effect and displayed no toxicity to mice. |
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