番木瓜种子原生质体制备技术的研究

以番木瓜（Carica papaya L.）成熟种子为材料,通过正交和单因素实验研究不同酶液浓度、甘露醇浓度、酶解时间、酶解pH等因素对番木瓜种子原生质体分离的影响。结果表明,高存活的番木瓜种子原生质体分离最佳条件是酶液组成为2.2％纤维素酶+0.6％离析酶、甘露醇浓度为0.7 mol/L、酶解9 h、酶解pH值为5.5。本研究奠定了原生质体遗传转化的基础,为番木瓜种子相关活性物质代谢途径的研究创造了良好的条件。     

芒果尖孢炭疽病病原菌的绿色荧光蛋白基因标记

芒果炭疽病是世界性的主要病害之一,引起芒果炭疽病的病原菌有胶孢炭疽菌和尖孢炭疽菌2种。通过溶壁酶酶解菌丝体成功制备尖孢炭疽菌原生质体,用抗潮霉素基因作为选择标记,采用PEG介导的原生质体转化法,用绿色荧光蛋白表达载体(pCT74-sGFP)转化原生质体,获得表达GFP尖孢炭疽菌的转化子菌株;转化子菌株在含潮霉素平板上经多次单孢纯化后,在无选择压下连续继代培养仍能发出稳定而强烈的绿色荧光,用GFP特异性引物PCR扩增转化子菌株基因组DNA获得预期大小片段,表明gfp基因已成功导入芒果炭疽病病原菌基因组中,且稳定遗传;GFP标记菌株生长正常,致病性和野生型菌株无明显差别,这为进一步研究该病菌在自然界的生物学及侵染方式奠定了基础。     

大豆愈伤原生质体的制备和培养方式探究

具有植物细胞全能性的原生质体是探究植物遗传转化和基因功能的理想材料,为了高效率制备大豆原生质体及其稳定培养,以交大05-133大豆未成熟子叶诱导的愈伤组织为材料,采用正交设计,对纤维素酶、果胶酶和离析酶等酶解液成分进行分析,研究原生质体高效制备方法的酶解液配比,同时探讨不同酶解时间对原生质体产量的影响,以确定最佳的原生质体酶解条件;通过对比在低熔点琼脂固体液滴培养与液体培养这两种不同培养方式下细胞的增值速率,以期建立高效的愈伤原生质体再生体系。结果显示,最佳酶解液配比为:2%纤维素酶+0. 1%果胶酶+1%离析酶,在该酶解液配比下酶解5 h,所得到原生质体产量和活力最高,达到(3. 976±0. 86)×10~6个·g~(-1)。用KP8培养基对原生质体进行培养,结果显示固体液滴的培养方式更适合原生质体的分裂,原生质体植板率高于液体培养,并且在25 d内分裂形成致密的细胞团。     

毛叶枣与冬枣原生质体分离体系的建立

研究了毛叶枣及冬枣的原生质体分离条件,为以后应用体细胞融合技术创造优异的毛叶枣体细胞杂种材料提供技术支持。结果表明:枣叶片分离出的原生质体活性显著高于悬浮培养物,但是产量低于悬浮培养物分离出的原生质体。毛叶枣酶解所需酶液最佳浓度为纤维素酶10g/L 离析酶4g/L 甘露醇0.7mol/L;冬枣酶解所需酶液最佳浓度为纤维素酶15g/L 离析酶4g/L 甘露醇0.7mol/L,酶解时间均为14￣16h。     

咖啡叶片原生质体分离条件的研究

以中粒种咖啡（Coffea. canephora）叶片组织为材料,研究酶液组合、酶解时间、酶液渗透压及预处理措施对其原生质体分离效果的影响,以期确定能够酶解出高数量且高活力的原生质体的酶解条件。结果表明：获得有活力原生质体的最佳酶液组合为2％纤维素酶+0.5％果胶酶+0.3％离析酶,同时酶解时间为20 h、酶液渗透压即甘露醇浓度为0.7 mol/L,预处理措施为黑暗24 h的咖啡叶片组织得到8.1×105个/g、活力达到73％的原生质体。     

橡胶树多主棒孢病菌原生质体转化体系的建立

由多主棒孢病菌侵染引起的棒孢霉落叶病是严重危害橡胶生产的一种世界性病害,近年来在中国也有发ⅱ生.在菌龄、细胞壁酶种类、酶作用浓度、作用时间、酶解温度、渗透压稳定剂等因素对橡胶树多主棒孢原生质体制备和再生影响分析的基础上,笔者首次建立了PEG介导的多主棒孢病菌遗传转化体系,并获得了多主棒孢病菌绿色荧光转化子.该体系中,多主棒孢病菌的原生质体再生率为19.12%,转化率为10.34个/μgDNA.     

香蕉枯萎菌原生质体形成与再生条件

通过对菌丝菌龄、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,得到了一种制备香蕉枯萎菌(Fusarium oxysporum f.sp.cubense,FOC)原生质体的有效方法,并在固体再生培养基上实现了原生质体的再生,再生率为22.5%。     

花生叶片原生质体游离和培养因子的研究

花生叶片酶解分离原生质体试验表明,植株的苗龄和叶龄对原生质体产量有很大的影响,苗龄１５～２０ｄ的顶部刚平展幼叶是获得高产原生质体的最佳材料。酶浓度和酶解时间是影响原生质体游离和培养的重要因素。通过相关和回归分析,选择较低的酶浓度,较短的酶解时间,并对原生质体产量影响不大的酶处理方法,是原生质体培养成功的关键因子之一。     

播娘蒿与甘蓝型油菜的原生质体融合与植株再生

以播娘蒿叶片和甘蓝型油菜子叶为材料提取原生质体.采用PEG-高pH、高钙附加DMSO融合方法,液体浅层静置培养,研究影响播娘蒿与甘蓝型油菜原生质体融合的因素,获得了再生植株.结果表明:4%纤维素酶 0.5%离析酶 5mmol/L MES酶解10b可获得高产率播娘蒿原生质体,1%纤维素酶 0.2%离析酶 3mmol/LMES酶解14h可获得高产率油菜原生质体;30%PEG 0.3mol/L葡萄糖 50mmoL/L CaCl2·2H2O 15%DMSO可获得10%的融合率;改良B5培养基的原生质体培养效果最好;最佳分化培养基为MS 4.0mg/L 6-BA 0.3mg/LNAA 5.0mg/L AgNO3;最佳生根培养基为MS 0.1mg/LIBA 0.1mg/L NAA.     

拮抗性大豆根瘤菌原生质体制备研究

选用具有拮抗作用的根瘤菌L396,对影响该菌原生质体制备的几个重要因素进行了研究。结果表明:制备原生质体的最佳菌龄为对数生长中期,培养液中加入0.5%青霉素可以促进原生质体的形成,在溶菌酶浓度1.5 mg.mL-1、酶解温度为35℃的情况下可以较快较好的产生原生质体,同时显微观察了原生质体的释放过程。     

Hydrolysis of glycoalkaloids fromSolanum tuberosum L. haulm by enzymes present in plant material and by enzyme preparation

Summary This paper deals with the kinetics of enzymatic hydrolysis of glycoalkaloids from potato (Solanum tuberosum L.) haulm. The hydrolysis was carried out by the action of the enzymes present in fresh haulm, juice of fresh haulm and in haulm dried at various temperatures. The highest degree of enzymatic hydrolysis of 90% was obtained during fermentation of haulm dried at 40 °C after 30 h incubation time. The enzyme preparation was obtained from the juice of fresh potato haulm by using capillary dialysator HM 16 (AQM 1681, 1.6 m² Hemofan 8 υ). The best degree of enzymatic hydrolysis by enzyme preparation, 68%, was achieved after 20 h time of incubation. The enzyme preparation from juice of fresh haulm was characterized by K_m of 0.70 mM at pH 5.5 and 35 °C.     

毛栓菌多酚氧化酶酶学性质及茶黄素的酶促合成研究

研究从毛栓菌中分离得到的多酚氧化酶(PPO)用于茶黄素的体外氧化制备。结果表明毛栓菌胞外PPO较适的反应温度范围在28~36℃之间;最适pH值为5.2;恒温反应30min内均表现出较高的活性和稳定性;50%的硫酸铵可以沉淀粗酶液中96.0%的PPO。将毛栓菌PPO加入到儿茶素反应液中进行双液相反应,可得到10.19%的茶黄素,与茶鲜叶来源的PPO相比,毛栓菌PPO制备茶黄素的总含量偏低,但其中TF-3-G的比例较高,达到总茶黄素的68.10%,占脂型茶黄素总量的92.08%。     

木薯根尖染色体制片方法的优化

以华南6号木薯根尖为材料,采用酶解去壁低渗法和压片法,制备木薯染色体标本,并对其制片过程中的预处理药品、预处理时间、酶解时间等因子进行优化。结果表明：酶解去壁低渗法能获得较好的染色体标本,α-溴代萘与对二氯苯饱和液的混合液和0．1％的秋水仙素与0.002 mol/L 8-羟基喹啉混合液的预处理效果最好,预处理时间以2 h为宜,酶解时间4 h效果最好。     

低蛋白天然胶乳的制备及其性能研究

应用2709碱性蛋白酶与稳定体系并用脱除天然胶乳（NRL）蛋白质的生物化学方法,采用多变元析因设计安排试验,研究了低蛋白天然胶乳（LPNRL）的稳定体系、酶的用量及制备条件、LPNRL的贮存及应用性能。研究结果表明:（1）2709碱性蛋白酶与稳定体系并用对鲜胶乳的脱蛋白效果较好;（2）所制备的LPNRL的氮含量为0.06％～0.07％,水溶性蛋白质（WSP）含量为1.8μg/g胶膜,达到国际上同类型产品的先进水平;（3）与正常NRL相比,LPNRL的硫化速率稍慢,但其硫化胶乳的贮存性能稳定,成膜性能良好;其干胶膜的物理性能稍低,但差别不大。      

Effect of an Enzyme Preparation Containing Pentosanases on the Bread-making Quality of Flours in Relation to Changes in Pentosan Properties

The effects of a crude pentosanase-containing enzyme preparation were studied on 12 samples of flour varying in quality for French bread making. At the optimal level of addition, the enzyme improved the dough properties, leading to a greater uniformity in quality characteristics and a higher level of quality for all samples. When an excessive amount of enzyme was added, the dough characteristics deteriorated. Changes in the properties of the flour pentosans were studied in the break-making process (kneading, shaping, end of fermentation, baking) using one of the flours, in the absence and presence of pentosanase-containing enzyme preparation at an optimal and excessive levels of addition. Part of the water-unextractable arabinoxylan became extractable during processing of the control sample, but solubilisation occurred to a greater degree with added enzyme. The specific viscosities of water extracts of doughs increased because of the arabinoxylan released from the WUP. Although more arabinoxylan was solubilised at the excessive level of addition, the apparent intrinsic viscosity of dough water-extractable arabinoxylan was greater at the optimum level of addition. These results were essentially confirmed with all the flour samples tested. The effectiveness of the enzyme preparation therefore appeared to be related to the amount and size of the extractable arabinoxylans.     

酶解菠萝蜜种子淀粉制备油脂模拟品的研究

以菠萝蜜种子淀粉为原料制备油脂模拟品,在单因素的基础上,采用响应面方法优化菠萝蜜种子淀粉油脂模拟品的制备工艺。结果表明,最佳制备工艺为：淀粉浓度为15.11％,酶用量为0.14％,酶解时间为17.10  min,酶解温度为50.10  ℃,在此工艺条件下制备的油脂模拟品的DE值为4.38;并确定了该油脂模拟品的糊化温度为80 ℃和糊化时间为10 min。     

酶解法制备大豆小肽的工艺研究

以豆粕为发酵原料,利用复合酶酶解方法制备大豆小肽,筛选碱性、中性蛋白酶和胰蛋白酶进行复配酶解豆粕。结果表明:复合酶的最佳配比为碱性蛋白酶∶中性蛋白酶∶胰蛋白酶为3∶2∶1;酶解条件:p H8.5,反应温度50℃,反应时间4.5 h,水解度为94.55%,苦味值为3;小肽显示分子量分布范围:≤1 000 Da可达74.67%以上,其中≤500 Da占55.61%以上。综上试验结果可知,对比单酶、双酶及3种酶酶解豆粕的水解度和苦味值两项指标,3种酶组合使用更适合于制备水解度高、苦味低的大豆小肽。     

酶法制备大豆多肽的研究

本文研究了以Alcalase碱性蛋白酶和风味蛋白酶水解大豆蛋白制备大豆肽的工艺。确定了酶水解的最佳温度、底物浓度、酶与底物浓度比,得到了Alcalase碱性蛋白酶和风味蛋白酶水解大豆分离蛋白的最佳水解工艺。     

Xylanase Induced Changes to Water- and Alkali-Extractable Arabinoxylans in Wheat Flour: Their Role in Lowering Batter Viscosity

The effect on the viscosity of wheat flour batter following treatments with a crude pentosanase-containing enzyme preparation and an endo-xylanase purified from the same preparation, was studied in relation to changes to the chemical features of the water- and Ba(OH)₂-extractable arabinoxylan fractions. Addition to batter of the crude enzyme or the pure xylanase caused a consistent and identical decrease in batter viscosity, demonstrating that the viscosity change was caused by the selective action of the xylanase in the crude enzyme preparation. The lowering of batter viscosity upon addition of enzyme was attributed to partial hydrolysis of the arabinoxylans in the water-extractable fraction which lead to an increase in the proportion of lower molecular weight water-extractable arabinoxylans possessing an increased xylose/arabinose ratio. There was no detectable change in the monosaccharide composition of either the water- or Ba(OH)₂-extractable arabinoxylan fraction after enzyme treatment and no indication of any increase in the amount of the water-extractable arabinoxylan fraction at the expense of the Ba(OH)₂-extractable fraction. It is concluded that the xylanase can extensively degrade arabinoxylan to small oligosaccharides in vitro. However, such degradation does not take place in situ, in the enzyme treated batters, where only a very small shift in the molecular weight profile of the water-extractable arabinoxylans was necessary to effect major changes to batter viscosity.     

Squid Pen Chitin Chitooligomers as Food Colorants Absorbers

One of the most promising applications of chitosanase is the conversion of chitinous biowaste into bioactive chitooligomers (COS). TKU033 chitosanase was induced from squid pen powder (SPP)-containing Bacillus cereus TKU033 medium and purified by ammonium sulfate precipitation and column chromatography. The enzyme was relatively more thermostable in the presence of the substrate and had an activity of 93% at 50 °C in a pH 5 buffer solution for 60 min. Furthermore, the enzyme used for the COS preparation was also studied. The enzyme products revealed various mixtures of COS that with different degrees of polymerization (DP), ranging from three to nine. In the culture medium, the fermented SPP was recovered, and it displayed a better adsorption rate (up to 96%) for the disperse dyes than the water-soluble food colorants, Allura Red AC (R40) and Tartrazne (Y4). Fourier transform-infrared spectroscopic (FT-IR) analysis proved that the adsorption of the dyes onto fermented SPP was a physical adsorption. Results also showed that fermented SPP was a favorable adsorber and could be employed as low-cost alternative for dye removal in wastewater treatment.