桑黄菌原生质体的高效分离探究

以桑黄菌丝体为材料,研究不同酶解条件对桑黄菌原生质体产量的影响,以期优化出高效分离桑黄原生质体的理想条件。采用四因素三水平正交试验方法,结果表明在使用1.5%溶壁酶的酶液、菌龄为10 d的菌丝体、0.6 mol·L-1甘露醇为酶解渗透压稳定剂时,酶解时间为3 h、酶解温度为30℃、酶解转速为100 r·min-1的处理条件下,桑黄菌原生质体分离产量最高,为4625万个·m L-1。通过正交试验筛选出了分离桑黄原生质体的最佳处理方式,对于高效分离桑黄原生质体具有指导意义,为选育桑黄各种生物活性物质的高产菌株及相关遗传研究奠定基础。     

褐色双孢蘑菇原生质体制备、再生条件的研究

为了提高褐色双孢蘑菇原生质体产量和再生率,本研究以褐色双孢蘑菇棕秀一号为材料,利用Design-Expert 8.0.6软件进行二次回归分析对褐色双孢蘑菇制备原生质体的条件进行优化。结果表明,原生质体最佳制备条件为溶壁酶1.35%、蜗牛酶7.01%、酶解温度30.4℃、褐色双孢蘑菇菌丝体菌龄8.36 d、酶解时间3.00 h,在此条件下原生质体产量高达4.39×10~7个·mL^-1;当溶壁酶含量为1.32%、蜗牛酶含量为6.96%、褐色双孢蘑菇菌丝体菌龄9.00 d、酶解时间2.97 h、酶解温度31.0℃时,原生质体的再生率最高(15.1%)。利用紫外诱变和化学诱变技术对褐色双孢蘑菇原生质体进行生长特性诱变,筛选获得了18株生长速率提高的突变菌株。本研究为褐色双孢蘑菇在遗传转化、基因组重排、基因编辑技术等后续分子遗传学研究提供了理论基础。     

指状青霉(Penicillium digitatum )原生质体制备和再生条件

通过对制备材料、酶液浓度、酶解时间、酶解温度、渗透压稳定剂的种类和浓度等因素的实验研究，得到了一种制备指状青霉(Penicillium digitatum)原生质体的有效方法，并在双层培养基上初步实现了原生质体的再生，再生率可达24．9％。此外，还对原生质体的释放和再生方式进行了跟踪观察。原生质体释放有顶端、侧端和原位释放3种方式。原生质体的再生有2种方式，一种是原生质体一端突出形成酵母出芽状细胞；另一种是在原生质体一端直接长出菌丝。     

尖顶羊肚菌高生物量高氨基酸含量突变菌株选育

对尖顶羊肚菌(Morchella conicaPers.)CGAC-9506原生质体形成、再生条件进行了研究.在此基础上,诱变羊肚菌原生质体,进行高生物量高氨基酸含量突变菌株选育.原生质体形成的合适条件为①酶解系统4%蜗牛酶,5%溶壁酶,4%纤维素酶.②缓冲系统0.6mol/LKCl的磷酸缓冲液(PH6.98).③酶解时间3.5 h.④酶解温度30℃.⑤菌龄20h.原生质体产量为2.5×104个/mg.在0.3mol/LKCl+0.3mol/L环己六醇的PSA(potato sucore agar)再生培养基上,再生率为0.56%.诱变再生株M.conica CGAC-950637生物量比出发株提高7.3%,总氨基酸比出发株提高38%.     

草地早熟禾原生质体培养与融合

以草地早熟禾品种RugbyⅡ成熟种子诱导的松软胚性愈伤组织为材料,以酶解法分离出原生质体,进行了原生质体培养;利用PEG融合法对MidnightⅡ和Nuglade的原生质体进行了融合,获得了体细胞杂种愈伤组织。研究了不同酶液组成、酶解时间、愈伤组织继代时间和渗透压对草地早熟禾原生质体游离及生长的影响;预处理对亲本原生质体活力的影响和适宜的融合条件。结果表明:采用继代培养8～10d的胚性愈伤组织,在1.0%纤维素酶+1.0%离析酶+0.5%果胶酶+0.3%崩溃酶条件下,酶解14～17h可得到产量和活力较高的原生质体;原生质体在KM8P培养基中培养3d后出现第1次细胞分裂,2～3周后形成小细胞团,此时添换低渗培养基2～3次,有利于小细胞团持续分裂并形成愈伤组织;试验获得了草地早熟禾品种RugbyⅡ原生质体来源的愈伤组织;5mmol/ml碘乙酰胺处理Nuglade原生质体10min及40μg/ml罗丹明处理MidnightⅡ原生质体5min,能有效地使Nuglade和MidnightⅡ原生质体失活;两品种融合PEG(6000)适宜浓度35%,融合率为9.8%。     

黑曲霉Ni-5k原生质体的制备和再生

研究不同配比的酶解液、菌丝的菌龄、酶处理的温度和时间、培养方法及其他因素对黑曲霉(A sp erg illus n ig er)N i-5k原生质体的形成量和再生率的影响,结果表明,酶解液浓度1.0%(其中含纤维素酶0.7%,蜗牛酶0.2%,溶菌酶0.1%),菌龄18 h、酶解温度30°C、酶解时间3 h,原生质体的形成量达到106个/mL;采用双层平板法于32°C再生,再生率达39%。     

高抗重金属盐的微紫青霉菌(Penicillium janthinellum)菌株GXCR的遗传转化

我们曾分离到一株高抗多种重金属盐的微紫青霉菌(Penicillium janthinellum)菌株GXCR。本研究在该菌株中建立了基于携带苯菌灵抗性基因(Benr)的质粒pCPXBN-1、聚乙二醇(PEG)介导的原生质体转化体系。原生质体的产量和再生的最佳条件是酶解液中的酶浓度在0.6%~0.8%(纤维素酶∶蜗牛酶=1∶1)、孢子萌发后培养24 h的菌体和0.05 mol/L二硫苏半糖醇(DTT)预处理。最佳转化组合条件为3×108原生质体/mL,50μg质粒DNA组合条件下,40%PEG4000,山梨糖醇(sorbitol)-三(羟甲基)氨基甲烷盐酸盐(Tris-HCl)-氯化钙(CaCl2)缓冲液(STC)[1 mol/L sorbitol,50 mmol/L Tris-HCl(pH 7.0),50 mmol/L CaCl2],室温转化20 min。在此条件下的转化效率为0.3个转化子/μg DNA。     

海带酶解和菌解工艺优化及其降解产物对菜心抗逆性的影响

  【目的】  海藻 (海带) 降解物对作物具有抗逆促生作用,研究海带菌解和酶解的最佳工艺条件,并比较两个工艺生产的降解产物对菜心的抗逆效果。  【方法】  采用单因素和正交试验,探究不同的底物浓度、接种量、溶液pH、降解温度分别对微泡菌Microbulbifer sp. SH-1和海藻酸裂解酶AlgSH7制备海藻降解产物的影响。同时,借助光学显微镜,研究SH-1菌和AlgSH7酶降解过程中海带细胞形态变化。并在此基础上,利用盆栽试验,研究海带菌解产物、酶解产物对菜心非生物胁迫抗性的影响。  【结果】  海藻酸裂解酶AlgSH7降解海带的最佳工艺条件是底物浓度2%,酶添加量6%,降解体系初始pH 8.5,温度44℃;SH-1菌株降解海带的最佳工艺条件是底物浓度2.5%,接种量1.5%,降解体系初始pH 7.5,温度32℃。与菌解产物相比,酶解产物中海藻酸、总糖、还原糖含量分别提高141.8%、57.6%、150.5%,而褐藻多酚、甘露醇、甜菜碱含量分别下降35.3%、60.6%、62.6%,但菌解工艺的产率比酶解工艺高9.3%。细胞形态观察结果表明,海带原始细胞排列紧密,形状规则饱满,随菌解或酶解时间的延长,海带细胞可视面积变小,细胞之间的间距增大。降解24 h时,酶解海带细胞可视面积仅为菌解的77.2%。在盆栽试验中,中度干旱条件下 (土壤相对含水量50%),酶解液灌根处理的菜心生物量比菌解液处理提高13.7%;酶解液配施水溶肥 (NPK, N:P₂O₅:K₂O=110:50:60) 处理的菜心生物量比菌解液配施NPK处理明显提高10.6%。淹水条件下 (淹水层深度2 cm),酶解液处理的菜心生物量比菌解液处理降低7.1%,但均显著高于对照处理;酶解液配施NPK处理的菜心生物量比菌解液配施NPK处理提高5.6%。盐胁迫条件下 (土壤中NaCl 含量24 g/kg),海带酶解液处理的菜心生物量比菌解液处理有增加趋势,但增加不显著。  【结论】  海带酶解产物中海藻酸、总糖、还原糖含量高于菌解产物,而褐藻多酚、甘露醇、甜菜碱含量低于菌解产物。菌解海带的产率高于酶解海带的产率。逆境胁迫条件下,海带酶解液配施NPK处理的菜心生物量高于菌解液配施NPK处理,海带酶解液对农作物抗逆促生作用优于菌解产物。     

绿色木霉的原生质体制备与转化条件

对绿色木霉(Trichoderma viride)原生质体制备和再生的条件进行了较详细的分析,并在此基础上进行了木霉的转化.结果表明,木霉原生质体形成的合适条件为培养24 h的菌丝用4 mg/mL的Glucanex在磷酸盐缓冲液中(pH 6.98)、30℃、振荡(40 r/min)酶解4 h,原生质体产量达到4.7×107个/mg.原生质体在0.3 mol/L肌醇和0.3 mol/L KCl的CM培养基上再生率为14.5%.对绿色木霉原生质体的激发子基因转化,其转化率为每微克DNA得到1～2个转化子.转化子的PCR鉴定以及激发子抗体的特异酶联免疫测定(ELISA)结果,表明外源基因已转入绿色木霉并有稳定产物表达.     

蜜蜂球囊菌（Ascosphaerapis）原生质体制备及再生

为建立高效稳定的蜜蜂球囊菌似scosphaeraapis）遗传转化体系,构建具有不同表型特征的球囊菌转化子,本实验对影响蜜蜂球囊菌菌株原生质体制备及再生的因子进行了系统的研究,同时对蜜蜂球囊菌原生质体的释放及再生过程进行了显微观察。结果表明,采用液体培养基进行24h培养的蜜蜂球囊菌菌体,在28℃条件下,应用50mg／mL的崩溃酶,经4h酶解所制备的原生质体释放量最大,达到34．00×10^5／mL。在上述条件下,采用0．8mol／L柠檬酸与NaCl的混合液作为稳渗剂,原生质体的再生率也较高,达到53．06％。蜜蜂球囊菌以菌丝断裂方式释放原生质体,原生质体再生时表现为原生质体先是突出,其后延长,并最终发育成为正常菌丝。本实验首次优化了蜜蜂球囊菌原生质体制备实验流程及原生质体再生最佳条件,为深入研究蜜蜂球囊菌的致病机理及寻找蜜蜂球囊菌致病相关基因奠定基础。     

Encapsulation of essential oils in zein nanospherical particles

Three essential oils, oregano, red thyme, and cassia (100% pure oil), were encapsulated by phase separation into zein nanospheres. Topographical images indicated that the powders were made up of irregularly shaped particles ( approximately 50 mum) containing close-packed nanospheres. Approximately 31% of the oregano encapsulated particles had mean diameters greater than 100 nm compared to 19% for the zein alone particles. In vitro digestion of zein particles with pepsin at a concentration ratio of 10:1 was complete after 52 h in phosphate-citrate buffer, pH 3.5, at 37 degrees C by spectroscopic analysis. Nonenzymatic, aqueous in vitro release of essential oils from encapsulated zein particles was carried out in phosphate buffered saline at pH 7.4 and 37 degrees C. Release occurred at varying rates over 20 h probably from different locations within the closely packed nanospheres of different sizes. Gel electrophoresis SDS-PAGE of zein incubated with freeze-dried swine manure solids at 37 degrees C indicated that preformed microbial enzymes capable of digesting zein within minutes were present in the manure. Except for differences in size of nanospheres, no structural differences were resolved by several microscopic methods, suggesting that the oil and proteins phases were blended during phase separation.     

Enhanced absorption of anthocyanins after oral administration of phytic acid in rats and humans

Many studies on the bioavailability of polyphenols have been reported. However, the relative urinary excretions of AC are also low, ranging from 0.004% to 0.1%. By contrast, other polyphenols show higher urinary excretion levels. Here, we studied the enhancing effects of phytic acid (IP6) on absorption of blackcurrant anthocyanins (BCAs) in rats and humans. In rats after oral administration of BCAs (as 241 mg of AC/kg body weight) in IP6 (0%, 0.25%, 0.5%, 1%, 2.5%) solution, the ACs recovery in urine was increased dependent on IP6 dose. These results suggest that the IP6 enhances gastrointestinal absorption of ACs. At the further analysis of IP6 enhancement effect in rat, whereas BCAs were normally passed through the stomach and duodenum within 2 h, in IP6 group, after 2-6 h post-administration, stomach and jejunum content's weights were specifically heavy, and large amounts of ACs were also detected in stomach, duodenum, and jejunum. These results suggested that the mixture of BCAs and IP6 reduced the gastrointestinal motility. Prolongation of ACs residue in gastrointestinal tract then caused the enhancing effects of IP6 on absorption of AC. In the human study, each subject was orally administrated a BCA beverage containing BCA concentrate (AC 4 mg/kg body weight), 1% of IP6, and 1% of sodium citrate as a pH stabilizer. Both the plasma level and the urinary excretion of AC were increased as compared to BCA administration without IP6. AC intake with IP6 may increase the bioavailability of AC to the comparative level as other polyphenols. Yet, phytic acid, being a strong chelator of important minerals, contributes to mineral deficiencies. An interference with iron uptake has been reported. Safety tests are therefore necessary before high dose IP6 can be used in foods.     

Stabilizing behavior of soy soluble polysaccharide or high methoxyl pectin in soy protein isolate emulsions at low pH

The stability of emulsions prepared with soy protein isolates was investigated as a function of pH in the presence of two negatively charged polysaccharides: high methoxyl pectin (HMP) and soy soluble polysaccharide (SSPS). Both polysaccharides are composed of a backbone which contains galacturonic acid but, when added to soy protein isolate-stabilized emulsions, SSPS showed a different behavior than that of HMP. At neutral pH and above a critical concentration of stabilizer (0.05%), HMP caused flocculation of the emulsion droplets via a depletion mechanism. On the other hand, the emulsions containing a similar amount of SSPS did not show creaming or flocculation. At acidic pH (<4.0) the addition of pectin caused extensive droplet aggregation, while no aggregation was observed with the addition of SSPS. The differences in the stabilization behavior between the two polysaccharides can be attributed to their differences in charge, neutral sugars side chains, and molecular weight.     

纳米MnFe2O4@DE复合材料对重金属钒的钝化稳定化效果

为获得能够有效减少日益严重的土壤钒污染的稳定剂,该文合成了纳米铁锰氧化物@硅藻土复合材料(MnFe_2O_4@DE),并通过模拟试验研究该复合材料对土壤钒的稳定化效果及机理,通过植物试验探究了该复合材料对土壤钒生物有效性的影响。试验结果表明,MnFe_2O_4@DE的最佳投加量为7%,此时其对钒的稳定化率分别约为硅藻土(DE)和MnFe_2O_4的16.87及1.24倍;时间和pH值单因素试验证明MnFe_2O_4@DE对土壤钒的稳定化效果能保持较长时间,酸性及中性pH有利于复合材料对钒的稳定。表征与钒形态分析结果表明,复合材料的稳定化效果较好与其较大比表面积和孔体积、钒的残渣态含量增加以及钒价态降低有关。植物萌发试验表明,添加复合材料可使钒污染组的油菜幼苗发芽率、株高和根长较未加钒的对照组无明显差异,植物中钒含量减少约80%。因此,MnFe_2O_4@DE复合材料在重金属污染土壤修复中具有较好应用前景。     

Formation of odorants in Maillard model systems based on l-proline as affected by pH

Formation of the odorants acetic acid, 4-hydroxy-2,5-dimethyl-3-(2H)-furanone (HDMF), 6-acetyl-1,2,3,4-tetrahydropyridine (ATHP), and 2-acetyl-1-pyrroline (AP) was monitored by isotope dilution assays at pH 6, 7, and 8 in Maillard model reactions containing glucose and proline (Glc/Pro) or the corresponding Amadori compound fructosyl-proline (Fru-Pro). In general, higher yields were obtained at pH 7 and 8. Acetic acid was the major odorant with up to 40 mg/mmol precursor followed by HDMF (up to 0.25 mg/mmol), the formation of which was favored in the Fru-Pro reaction systems. On the contrary, ATHP (up to 50 microg/mmol) and AP (up to 5 microg/mmol) were more abundant in Glc/Pro. However, the sensory relevance of the two N-heterocycles was more pronounced on the basis of odor activity values, confirming their contribution to the overall roasty note of the reaction samples. It was also found that formation and decomposition of Fru-Pro were faster at pH 7 as compared to pH 6, explaining in part the preferred formation of the four odorants studied under neutral and slightly alkaline conditions. After 4 h of reaction at pH 7 in the presence of proline, about one-fourth of the glucose was consumed leading to acetic acid with a transformation yield of almost 40 mol %.     

植物吸取修复及钝化处理对后茬水稻镉吸收的影响

采集湖南湘潭县某地镉(Cd)污染酸性农田土壤及其经伴矿景天分别吸取修复两季和三季后的土壤,采用盆栽试验研究了经伴矿景天修复及钝化改良与否对土壤pH、有效态Cd、Zn以及水稻生长和稻米Cd、Zn浓度的影响。结果表明:未改良的处理,随着修复次数的增加,土壤pH显著降低,降低幅度为0.26~0.38个单位;且修复两季、三季土壤CaCl_2提取态Cd浓度较未修复土壤分别降低19.4%、24.0%;修复后土壤种植水稻品种W184,其糙米中Cd浓度显著降低,但依然超标;修复三季土壤种植低积累水稻品种IRA7190,其糙米中Cd由0.47 mg/kg降为0.03 mg/kg。施加钝化剂海泡石和石灰(10 g/kg+1 g/kg)后,修复两季、三季土壤的pH显著升高,较未施钝化剂处理土壤pH分别提高0.95、0.72;土壤CaCl_2提取态Cd浓度分别降低79.8%、79.5%;修复两季、三季土壤上水稻W184糙米的Cd浓度与未施加钝化剂相比,分别降低27.3%、44.4%,均降至国家食品安全限值0.2 mg/kg以下;无论是否添加钝化剂,伴矿景天吸取修复三季的土壤上水稻IRA7190糙米中Cd浓度均仅0.03 mg/kg。     

Osmotic Properties of Gluten

A model for dough is proposed in which the distribution of water between hydrated gluten and starch paste explains a number of practical observations such as 1) the extreme sensitivity of the consistency of dough to the amount of water in the recipe, and 2) the fact that working of the material results in an increase in consistency. The model assumes dough to be a composite material consisting of a starch paste and gluten filaments. During kneading, the starch granule paste in dough dries to become a phase with a yield stress as a result of the uptake of water by the stretching gluten filaments. This study focused on one particular aspect of this model: the osmotic properties of gluten during stretching. The results suggest that gluten can be hydrated more efficiently in the stretched state than in an unstretched conformation. Gluten hydration tends to change slowly over a period of weeks, which is accompanied by water expulsion or uptake, depending on the osmotic properties of the solvent. The rate of change does not seem to depend very much on pH and osmotic pressure for the current experimental conditions. The level of hydration of relaxed gluten depends strongly on pH, as expected. The experiments allow the construction of an osmotic pressure versus gluten concentration diagram over the range 4.6 < pH < 5.8. The level of hydration of the gluten is consistent with the proposed model for dough.