灰树花的深层培养工艺及其影响因素的研究

利用发酵罐进行灰树花的深层培养，以期为深层培养技术在灰树花上的应用提供理论依据，为灰树花产品的开发奠定基础。结果表明，最佳的培养条件：发酵温度25℃、起始pH值6.5、搅拌转速150 r/min、接种量10%。pH值和通气量对菌丝的生长有极显著的影响，pH值在5.50～6.50，通气量体积比(V/V)在1.00～1.40时，菌丝生物量积累最多；通过营养分析认为，可以通过深层培养进行灰树花产品的研究和开发。     

o-Orsellinaldehyde from the submerged culture of the edible mushroom Grifola frondosa exhibits selective cytotoxic effect against Hep 3B cells through apoptosis

The tumoricidal activity of a bioactive metabolite produced by submerged culture in a 2.1-L airlift fermentor of Grifola frondosa NTUS was investigated. After 14 days of cultivation, ethyl acetate extracts from the supernatant of culture broth (EES) were analyzed by cell viability assay. The IC50 of EES for cytotoxicity against human carcinoma cells (Hep 3B, Hep G2, HeLa, CL1-1) and normal human lung fibroblast MRC-5 was 78.4, 52.7, 77.6, 71.0, and 233.3 microg/mL, respectively. EES was further fractionated and a main cytotoxic compound, HE-5-5, was obtained. The IC50 of HE-5-5 based on the cell viability of Hep 3B and MRC-5 cells was 3.6 and 33.1 microg/mL, respectively. Thus, HE-5-5 showed a selective cytotoxic effect against Hep 3B cells and MRC-5. According to the UV, MS, and NMR data, HE-5-5 was identified as o-orsellinaldehyde. A DNA fragmentation assay together with the presence of a significant sub-G1 peak by flow cytometry suggested that o-orsellinaldehyde might mediate its cytotoxicity through apoptosis.     

Antioxidant properties of Antrodia camphorata in submerged culture

The biologically active compounds, antioxidant activities, and free radical scavenging effects of dry matter of cultural medium (DMCM), filtrate (DMF), and different solvent extracts of mycelia from Antrodia camphorata in submerged culture (ACSC) were investigated. DMF showed the strongest inhibition of lipid peroxidation as a function of its concentration, and was comparable to the antioxidant activity of BHA at the same concentration of 0.2 mg/mL. The hexane extract of mycelia had the weakest antioxidant ability, whereas other mycelial extracts exhibited a modest inhibition of lipid peroxidation. DMF and water extract of mycelia (WEM) showed marked activity in free radical scavenging. The antioxidant activities of filtrate and mycelial extracts were correlated with the presence of total polyphenols, the crude triterpenoids, and the protein/polysaccharide ratio of the crude polysaccharides. It was found that DMCM had lower antioxidant ability than DMF in different model systems, indicating that the major antioxidant components in DMF must be derived from the secondary metabolites of mycelia. The results presented herein indicate that DMF could possibly act as a chemopreventing agent with respect to free radical-related diseases.     

Cyclooxygenase inhibitory and antioxidant compounds from the mycelia of the edible mushroom Grifola frondosa

The bioassay-guided isolation and purification of the hexane extract of the cultured mycelia of Grifola frondosa led to the characterization of a fatty acid fraction and three compounds, ergosterol (1), ergostra-4,6,8(14),22-tetraen-3-one (2), and 1-oleoyl-2-linoleoyl-3-palmitoylglycerol (3). The composition of fatty acid fraction was confirmed as palmitic, oleic, and linoleic acids by GC-MS and by comparison with the retention values of authentic samples. The structures of compounds 1-3 were established by spectroscopic methods. The fatty acid fraction and compounds 1-3 showed cyclooxygenase (COX) enzyme inhibitory and antioxidant activities. The inhibition of COX-1 enzyme by the fatty acid fraction and compounds 1-3 at 250 microg/mL were 98, 37, 55, and 67%, respectively. Similarly, COX-2 enzyme activity was reduced by fatty acid fraction and compounds 1-3 at 250 microg/mL by 99, 37, 70, and 4%, respectively. The inhibitions of liposome peroxidation by the fatty acid fraction and compounds 1 and 2 at 100 microg/mL were 79, 48, and 42%, respectively. This is the first report of compounds 2 and 3 from the cultured mycelia of G. frondosa. The COX inhibitory activities of compounds 1-3 are reported here for the first time.     

Compositional characteristics and antioxidant properties of fresh and processed sea cucumber (Cucumaria frondosa)

The antioxidant activity of fresh and rehydrated sea cucumber (Cucumaria frondosa) samples with/without internal organs was evaluated for the first time. In addition, their proximate, amino acid, and fatty acid compositions were examined. Rehydrated sea cucumber samples in distilled water were prepared from oven-dried products. All samples contained 83-90% moisture, but showed a significant difference among groups in their protein and lipid contents. Glutamic acid was the predominant amino acid in sea cucumber, followed by glycine and aspartic acid. Essential amino acids such as leucine and lysine were also present at high levels. The trend for free amino acid was different from that of total amino acids and varied among groups. Lipids in sea cucumber were dominated by eicosapentaenoic acid (EPA, C20:5n-3), ranging from 43.2 to 56.7% of the total fatty acids. Docosahexaenoic acid (DHA, C22:6n-3) was present at a much lower concentration of 2.0-5.8%. All sea cucumber samples exhibited radical scavenging property against 2,2'-azobis(2-aminopropane) dihydrochloride (AAPH) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, with rehydrated samples, especially those with internal organs, possessing higher antioxidant activity than their fresh counterparts. No correlation existed between radical scavenging capacity and total phenolics content, suggesting that other components, in addition to phenolic compounds, contribute to the antioxidant activity of sea cucumber.     

Profiling of hepatic gene expression of mice fed with edible japanese mushrooms by DNA microarray analysis: comparison among Pleurotus ostreatus, Grifola frondosa, and Hypsizigus marmoreus

To compare and estimate the effects of dietary intake of three kinds of mushrooms (Pleurotus ostreatus, Grifola frondosa, and Hypsizigus marmoreus), mice were fed a diet containing 10-14% of each mushroom for 4 weeks. Triacylglycerol in the liver and plasma decreased and plasma cholesterol increased in the P. ostreatus-fed group compared with those in the control group. Cholesterol in the liver was lower in the G. frondosa-fed group than in the control group, but no changes were found in the H. marmoreus-fed group. DNA microarray analysis of the liver revealed differences of gene expression patterns among mushrooms. Ctp1a and Fabp families were upregulated in the P. ostreatus-fed group, which were considered to promote lipid transport and β-oxidation. In the G. frondosa-fed group, not only the gene involved in signal transduction of innate immunity via TLR3 and interferon but also virus resistance genes, such as Mx1, Rsad2, and Oas1, were upregulated.     

Protective effects of fermented filtrate from Antrodia camphorata in submerged culture against CCl4-induced hepatic toxicity in rats

The protective effects and the possible mechanisms of dry matter of fermented filtrate (DMF) from Antrodia camphorata in submerged culture (ACSC) on H(2)O(2)-induced cytotoxicity in HepG2 and carbon tetrachloride (CCl(4))-induced hepatotoxicity in Sprague-Dawley rats were investigated. The results showed that the inhibitory effect of DMF and its crude triterpenoids on lipid peroxidation occurred in a dose-response manner in an AAPH/linoleic acid system. When HepG2 cells were pretreated with DMF at the concentration of 0.10 mg/mL for 4 h and then induced by 1 h of treatment with H(2)O(2) (100 microM), lipid peroxidation was significantly (p < 0.05) decreased, as measured by the formation of malondialdehyde. The oral pretreatment with DMF [0.25 and 0.50 mg/kg of body weight (bw)] for 5 consecutive days prior to the administration of a single dose of 40% CCl(4) (0.10 mL/100 g of bw, ip) significantly prevented the increase in serum levels of hepatic enzyme markers (alanine and aspartate aminotransferase) and liver lipid peroxidation (p < 0.05). Histopathological evaluation of the rat liver revealed that DMF reduced the incidence of liver lesions, including neutrophil infiltration, hydropic swelling, and necrosis induced by CCl(4) in rats. Moreover, reduced glutathione (GSH)-dependent enzymes (glutathione peroxidase, glutathione reductase, and glutathione S-transferase) and the GSH/GSSG ratio were significantly improved in the oral pretreatment DMF of rats (p < 0.01). The results suggest that DMF may play a role in preventing oxidative damage in living systems by up-regulating hepatic GSH-dependent enzymes to preserve the normal GSH/GSSH ratio and scavenging free radicals formed during CCl(4) metabolism.     

大狼把草对猪场废水中污染物的净化效果

为了考察湿地植物大狼把草对猪场废水中污染物的净化能力,采用基质栽培法研究了大狼把草在猪场废水中的生长特性及其对氮、磷的去除率。结果表明：该植物在氨氮浓度100 mg/L左右的废水中能正常生长,单株植物的干质量达到245.8 g,其氮、磷积累量分别为5.63和0.42 g,在每株植物处理水量9 L、水力停留时间（HRT）7 d时,大狼把草在营养生长期和花果期对废水中氨氮、总氮及化学需氧量CODCr的平均去除率都在80%以上,对总磷的平均去除率在60%以上。由此可见,大狼把草对猪场废水有较强的耐污能力和较好的净化效果,可用于人工湿地处理畜禽养殖废水。     

淹水条件下有机物料对潮土外源镉形态及化学性质的影响

在室内淹水培养条件下,运用连续提取法研究稻草、紫云英和猪粪对潮土中外源镉的形态及土壤化学性质的影响。结果表明,有机物料显著增加了土壤有机碳,pH,活性氧化物的含量。有机碳的增加显著地增加紧有机结合态镉含量,而显著地降低了交换态镉的含量。并随着时间的增长,影响更显著。交换态镉的含量与Eh值呈显著的正相关,而紧有机结合态镉与Eh值呈显著的负相关。     

紫菜多糖抗氧化活性及体外免疫调节作用研究

为探讨紫菜多糖功能活性,采用超声波辅助纤维法提取分离紫菜多糖(PP),研究PP的抗氧化活性及其对小鼠体外免疫调节的影响。结果表明,PP经Cellulose DEAE-52柱层析得到7个组分PP1、PP2、PP3、PP4、PP5、PP6和PP7,得率分别为5.18%、11.21%、17.15%、19.88%、4.77%、3.02%和1.24%。选取PP和2个主要组分(PP3,PP4)进行抗氧化及小鼠体外免疫调节作用研究,发现PP、PP3和PP4均能有效清除DPPH·和·OH,抑制小鼠肝组织的脂质过氧化,对Fe~(2+)和H_2O_2诱导的肝组织过氧化物损伤具有保护作用,能减少线粒体肿胀和红细胞溶血的发生。此外,PP3和PP4不仅具有直接及协同丝裂原刀豆蛋白A(ConA)刺激淋巴细胞增殖的作用,还具有双向免疫调节的作用。本研究结果为进一步探讨紫菜多糖功能活性及其作用机制奠定了基础。     

Immunomodulatory and antitumor activities of grape seed proanthocyanidins

Proanthocyanidins are naturally occurring compounds that are widely available in many kinds of plants; particularly, the grape seeds are a rich source of proanthocyanidins. Grape seed proanthocyanidins (GSPs) have been demonstrated to possess a wide range of health beneficial properties. This study was carried out to elucidate the molecular mechanisms involved in the antitumor therapeutic and immunomodulating effects of GSPs through in vivo and in vitro models. The results showed that GSPs could significantly inhibit the growth of Sarcoma 180 tumor cells in vivo and remarkably increase thymus and spleen weight of Sarcoma 180-bearing mice and upgrade the secretion level of tumor necrosis factor-α (TNF-α) in serum. Moreover, GSPs could stimulate lymphocyte transformation, enhance lysosomal enzyme activity and phagocytic capability of peritoneal macrophages, and remarkably promote the production of TNF-α. These results suggested that GSPs could improve functional activation of the immune system, and the antitumor effects of GSPs were achieved by immunostimulating properties.     

Glass distilling collector applied for HCN recovery from submerged culture broth and fruiting body of Pleurotus eryngii for identification and quantification

Detection and surveillance of food commodities containing cyanide is a crucial issue of food safety. In this study, five strains of Pleurotus eryngii (P. eryngii) were grown in submerged culture of yeast malt broth (YMB) with the suspected production of HCN. A safety-warranted U-bent glass distilling collector with three enlarged bulbs on each arm was designed to recover the broth vapor. When AgNO(3) solution was used as an absorbent to interact with the vapor, a white precipitate was formed. The precipitate was isolated and identified as AgCN by FT-Raman spectroscopic analysis. When the absorbent was substituted by KOH, after evaporation to dryness, dissolved in D(2)O, and followed by (13)C-NMR analysis, a KCN spectrum was achieved. Formation of AgCN and KCN confirmed HCN production in the broth by P. eryngii. When a sodium picrate solution (1.4%) was used as an absorbent and various authentic KCN solutions were applied for distillation and followed by absorbance determination at 510 nm, a linear dose-dependent relationship was obtained and the procedure was applied for HCN quantification of the marketed P. eryngii mushrooms (fruiting body). As estimated, 67.3% of the products contained HCN less than 1.0 mg/kg, 17.3% between 1.0 and 2.0 mg/kg, and 15.4% higher than 2.0 mg/kg. When the mushrooms were sliced and cooked in water at 95 degrees C for 6 min, 89.1% of the original HCN was lost. When the P. eryngii strains were respectively grown by submerged cultivation in YMB or YMB supplemented with 2.5% glycine for 16 days, HCN content was slightly higher in the latter than in the former for each strain.     

Immunomodulatory Properties of the Protein Fraction from Phorphyra columbina

The phycobiliproteins from Rhodophyta , R-phycoerythrin (R-PE) and C-phycocyanin (C-PC), have been shown to exert immunomodulatory effects. This study evaluated the effects of a Phorphyra columbina protein fraction (PF) and R-PE and C-PC on rat primary splenocytes, macrophages, and T-lymphocytes in vitro. PF featured various protein species, including R-PE and C-PC. PF showed mitogenic effects on rat splenocytes and was nontoxic to cells except at 1 g L(-1) protein. IL-10 secretion was enhanced by PF in rat splenocytes, macrophages, and especially T-lymphocytes, whereas it was markedly diminished by R-PE and C-PC. The production of pro-inflammatory cytokines by macrophages was inhibited. The effect of PF on IL-10 was evoked by JNK/p38 MAPK and NF-κB-dependent pathways in macrophages and T-lymphocytes. It was concluded that PF has immunomodulatory effects on macrophages and lymphocytes that appear to be predominantly anti-inflammatory via up-regulated IL-10 production and cannot be accounted for by R-PE and C-PC.     

Formation and degradation of ethylene in submerged rice soils

Ethylene formation in submerged soils and ethylene degradation by rice roots and soils were investigated. Ethylene was formed in rice soils in amounts which may deleteriously affect the growth of rice seedlings. Ethylene formation was markedly enhanced when organic materials such as glucose or rice straw were added to the soil. The rice roots, especially those taken from lowland and rice fields, showed notable ethylene-degradation activity; the rhizosphere microorganisms of the lowland rice appeared to be responsible for the ethylene-degradation activity. Lowland soils which have been continually submerged for a long time also showed ethylene-degradation activity. Upland soil and soil recently submerged, however, showed little degradation activity.     

Nitrification and denitrification in submerged Maahas clay soil

A laboratory experiment showed that the oxidized layer of submerged soil has high nitrifying activity. Denitrification was active in soil below the soil surface. Tracer technique indicated that the nitrification and the subsequent denitrification of ammonium sulfate was most active in the layer about 2 cm below the soil surface, and that the activities extended to a depth of about 5 cm, Nitrification Inhibitors reduced the nitrogen lost through nitrification and subsequent denitrification.     

羊骨酶解物的免疫调节活性研究

以羊骨粉为底物,用木瓜蛋白酶和胰蛋白酶制备复合酶解物,用不同的剂量给小鼠灌胃(ig.),通过碳粒廓清试验、噻唑蓝法以及定量溶血法检测不同剂量酶解物对小鼠非特异性免疫、细胞免疫以及体液免疫功能的影响。结果表明:低剂量(0.5g/kg.d,ig,14d)可显著提高小鼠吞噬细胞清除碳颗粒的能力(P0.05),中剂量(1g/kg.d,ig,14d)和高剂量(3g/kg.d,ig,14d)组的吞噬能力与对照组无显著差异(P0.05)。但这2个剂量却使小鼠脾脏B淋巴细胞合成的抗体量显著高于对照组(P0.05),且中剂量组显著高于高剂量组(P0.05)。0.01mg/ml复合酶解物显著促进了T淋巴细胞对ConA的反应性(P0.05)。因此适当剂量的羊骨酶解物,能提高小鼠的非特异性免疫、体液免疫和细胞免疫能力。     

Effectiveness of tetrazolium salts in microbial ecological studies in submerged soil

Several trials to obtain information on the living microorganisms at micro-sites of the submerged soil by the novel utilization of the tetrazolium salts were described. Results are summarized as follows:

(1) The air-dried soil was incubated with TIC (triphenyl tetrazolium chloride) under submerged condition according to the modified double layer plate method. The red formazan (the reduced product of TIC) was precipitated at the biologically active sites by the dehy. drogenase activity. Using a binocular microscope, most of the biologically active sites were found to be localized on and around the organic debris.

(2) The amount of nitrogen mineralized by incubating the soil under submerged condi. tion correlated well not only with the amount of formazan formed biologically in the submerged soil but also with the amount of formazan formed non-biologically by heating the soil with TIC in Ba(OH). solution. In addition, the pattern of precipitation of the nonbiologically formed formazan was very similar to that of the biologically formed formazan. Accordingly, the substances which reduced TIC non-biologically were considered to be closely associated with the substrates for the microorganisms.

(3) The plant debris stained with the red formazan could be mounted on a slide glass and observed at high magnification for the detailed study on the process of decomposition. Addition of lactophenol cotton blue to the plant debris could visualize the microorganisms responsible for the decomposition of the plant debris.

(4) NTB (nitro tetrazolium blue) was the most suitable for staining individual microorganisms growing on cellulosic materials or plant debris. The double staining with NTB and rosebengal made the discrimination between dead or resting cells and viable ones possible when this technique was applied to the decomposed filter paper.     

水田土壤中除草剂丁草胺残留的测试方法及其应用

本研究建立了水稻田土壤中除草剂丁草胺残留的分析测试方法。研究结果表明，选用１３０℃活化４小时不加水的１ｇ氟罗里硅土净化剂，趁热装柱，同时选择含６％丙酮的石油醚为洗脱液，可提高丁草胺的氟罗里硅土柱净化效率。本方法不仅灵敏度高，重复性好，可排除有机污染物对微量丁草胺残留物测定的干扰，而且具有减少化学试剂消耗量的优点，适于土样特别含水量较高，残留量差异较大的新鲜土壤或沉积物样品中丁草胺残留的测试。     

Nitrogen transformations of ammonium sulfate and alanine in submerged Maahas clay

The fate of added nitrogen in submerged soils was studied using ¹⁵N-labelled ammonium sulfate and alanine. After 8 weeks of incubation 25 and 22%, respectively, of nitrogen from ammonium sulfate and alanine were recovered in the soil. Under the experimental conditions used nitrogen added to presubmerged soils was lost rapidly outside of the soil-water system, regardless of whether the nitrogen was organic or inorganic. Fractionation studies revealed that the amount of tagged N incorporated into exchangeable ammonium, residual fractions, volatilized as NH₃ and chemically fixed nitrogen was not enough to account for the nitrogen loss. The nitrogen loss was attributed to nitrification and subsequent denitrification during the incubation period.

The effect of N-Serve [2-chloro-6-(trichloromethyl)pyrimidine] on nitrification of ¹⁵N-labelled (NH₄)₂SO₄-in submerged soils was studied. About 15% more nitrogen was recovered from non-presubmerged soils, and less nitrate was accumulated in presubmerged soils where N-Serve coated (NH₄)₂SO₄ was applied, than from soils where (NH₄)₂SO₄ was applied without N-Serve. Presubmerged soils provided a more favorable environment for nitrification than for denitrification under the experimental conditions used.