蘑菇中多酚氧化酶的酶学特性研究

采用二次丙酮法与75％硫酸铵沉淀法从蘑菇(Agaricus bisporus)中提取多酚氧化酶；用分光光度法对蘑菇中多酚氧化酶酶学特性进行了较为系统的研究。结果表明：二次丙酮法优于75％硫酸铵沉淀法；以邻苯二酚为底物，多酚氧化酶的最适pH为7．0，最适温度为30℃；亚硫酸钠、半胱氨酸、抗坏血酸为强抑制剂。     

Partial purification and characterization of polyphenoloxidase from peppermint (Mentha piperita)

Polyphenoloxidase (PPO) of peppermint leaves ( Mentha piperita) was isolated by (NH₄)₂SO₄ precipitation and dialysis. Its pH and temperature optima were 7.0 and 30°C, respectively. On heat-inactivation, half of the activity was lost after 6.5 and 1.5 min of treatment at 70 and 80°C, respectively. Sucrose, (NH₄)₂SO₄, NaCl and KCl appeared to be protective agents of peppermint PPO against thermal denaturation. Km of this enzyme ranged from 6.25×10⁻³ M with catechol to 9.00×10⁻³ M with L-dopa. The I₅₀ values of inhibitors studied on PPO were determined by means of activity percentage (I) diagrams. Values were 1.4×10⁻⁴ M, 1.7×10⁻⁴ M, 9.7×10⁻⁵ M, 2.45×10⁻⁴ M, 2.16×10⁻¹ M, 1.83×10⁻⁵ M, 6.5×10⁻⁵ M, 1.4×10⁻² M, 7.5×10⁻⁵ M, for potassium cyanide, glutathione, ascorbic acid, thiourea, sodium azide, sodium metabisulfite, dithioerythritol, β-mercaptoethanol and sodium diethyl dithiocarbamate respectively. Therefore, sodium metabisulfite was the most effective inhibitor.     

Effects of some thermal treatments on polyphenoloxidase and peroxidase activities of banana (Musa cavendishii,var enana)

Polyphenoloxidase (EC 1.14.18.1) and peroxidase (EC 1.11.1.7) activities were evaluated during cold storage of banana (Musa cavendishii, L var enana). The effects of some thermal treatments (blanching of peeled bananas, microwave treatment of banana slices and storage of banana slices at ?24°C) were studied. Inactivation of both enzymes by blanching was very effective (96–100 %) at all maturity levels. Microwave treatment showed differing efficiency depending on banana ripeness, and produced non-enzymic darkening due to Maillard reactions mainly in green and full-yellow bananas. Freezing of fruit slices without previous thermal treatment produced different effects in polyphenoloxidase and peroxidase activities. It was concluded that immersion in boiling water of peeled bananas with 70 % green/30% yellow peel colour maturity index is the optimal pretreatment for the preservation of frozen banana slices.     

Combined effect of high pressure,temperature and holding time on polyphenoloxidase and peroxidase activity in banana (Musa acuminata)

Polyphenoloxidase and peroxidase enzyme activities were evaluated following combined pressure, temperature and holding time treatment in banana (Musa acuminata). Using pressures of up to 110 MPa, temperatures of up to 70 °C and holding times of up to 25 min, based on a 2³ central composite design, the interactive effects were found to significantly influence the activity of both enzymes in prepared banana pulp. Temperature and pressure were found to influence the inactivation of polyphenoloxidase separately, while temperature, pressure and holding time were found to influence the loss of peroxidase in the banana, although no significant interactive effects were found. The reduction in polyphenoloxidase activity was found to be less influenced by the combined treatment than peroxidase activity, thought to be due to solubilisation of the enzyme and effects of the soluble solids content. © 2000 Society of Chemical Industry     

冬瓜多酚氧化酶的特性及抑制剂研究

以邻苯二酚为底物,采用'分光光度法在420 nm 处测定冬瓜多酚氧化酶(PPO)的活性,研究了不同温度和 pH 值对其活性的影响,并建立了酶促褐变反应动力学方程,探讨了抗坏血酸、亚硫酸氢钠、柠檬酸和 EDTA-2Na 4 种抑制剂对冬瓜酶促褐变的抑制效果.结果表明,冬瓜多酚氧化酶的最适温度为20℃,最适pH值为6.0;冬瓜多酚氧化酶的米氏方程符合单底物酶促反应动力学,相应的动力学参数为 Kin=0.0118 mol/L,Vm=0.0586 U/min;化学抑制剂抗坏血酸、亚硫酸氢钠、柠檬酸和EDTA-2Na 对多酚氧化酶都有抑制作用,其活性分别为对照的25%、32%、62%和57%.     

Studies on purification of allicin by molecular distillation

Background: With recent improvements in living standards, people have been giving more consideration to the healthcare effects of foods. In this respect, allicin, which is the most important organosulfur compound in garlic and plays a key role in physiological function, has been receiving much attention. Results: Allicin obtained from garlic by supercritical CO₂ extraction was purified by molecular distillation (MD). The effects of operating conditions such as absolute pressure (AP), distillation temperature (DT) and feed flow rate (FFR) on allicin purity and yield were studied. The optimal AP, DT and FFR levels in first‐stage MD were found to be 200 Pa, 50 °C and 15 mL min^?1 respectively. After three stages of MD the contents of allicin, diallyl disulfide (DADS) and diallyl trisulfide (DATS) were 68.04, 9.19 and 5.91% (w/w) respectively. Conclusion: This study has provided a safe and effective method for the purification of allicin. Copyright © 2011 Society of Chemical Industry     

Studies on banana fruit quality and maturity stages using hyperspectral imaging

Banana fruit quality and maturity stages were studied at three different temperatures, viz., 20, 25, and 30 °C by using hyperspectral imaging technique in the visible and near infrared (400-1000 nm) regions. The quality parameters like moisture content, firmness and total soluble solids were determined and correlated with the spectral data. The spectral data were analyzed using the partial least square analysis. The optimal wavelengths were selected using predicted residual error sum of squares. The principal component analysis was also used to test the variability of the observed data. By using multiple linear regressions (MLR), models were established based on the optimal wave lengths to predict the quality attributes. The coefficient of determination was found to be 0.85, 0.87, and 0.91 for total soluble solids, moisture and firmness of the banana fruits, respectively. The change in TSS and firmness of banana fruits stored at different temperatures, viz., 20, 25, and 30 °C during the ripening process followed the polynomial relationships and the change in moisture content followed a linear relationship at different maturity stages.     

南果梨中多酚氧化酶的动力学研究

研究了南果梨多酚氧化酶的底物专一性和不同底物的米氏常数,结果表明,最适底物为绿原酸。同时研究了不同抑制剂对南果梨多酚氧化酶的抑制效果。     

Effects of limited proteolysis on broad bean polyphenoloxidase

Partially purified broad bean polyphenoloxidase was treated with a variety of proteases. Enzyme activity increased after treatment with chymotrypsin, pepsin, subtilisin, thermolysin, trypsin, and Staphylococcus aureus V₈ protease but this activation was masked when assayed in the presence of sodium dodecylsulfate. The enzyme was resistant to proteolysis when treated with chymotrypsin, elastase, pepsin, thermolysin, and V₈ protease. Proteinase K, subtilisin and trypsin treatment resulted in partial degradation of the enzyme but only trypsin generated an active enzyme form with a slightly smaller molecular weight. Electrophoresis, followed by enzyme staining and or Western blotting, showed that trypsin treatment converted the active 45 kd enzyme into a 43 kd enzyme within 20 min. Treatment with proteinase K, subtilisin and trypsin also produced inactive higher molecular weight forms of purified polyphenoloxidase that were identified by Western blotting. The use of antibodies coupled with Western blotting also identified inactive, active, and latent polyphenoloxidase after protease treatment, and suggests that immunological methods can be used to establish the types and amounts of polyphenoloxidase present in a given tissue.     

二氧化氯对鲜切莲藕多酚氧化酶的影响研究

以莲藕为试材,研究了二氧化氯（ClO2）对鲜切莲藕在低温贮藏期间多酚氧化酶（PPO）、褐变及感官品质影响的动态变化。试验结果表明,藕的褐变与PPO有密切关系,PPO是发生酶促褐变的关键酶。50mg/L、100mg/L浓度的ClO2处理鲜切藕片5、10、15min后,在贮藏期间能抑制PPO的活性,减轻褐变,保持好的感官品质。而10mg/L浓度对PPO的活性不起抑制作用反而促进了PPO的活性。100mg/L ClO2处理鲜切藕片10min抑制PPO的活性效果最好,能使藕片在4℃下一周的时间保持最佳的感官品质和最轻的褐变度。而鲜切产品维持一周的货架寿命基本上可满足消费与生产的需求。     

大孔吸附树脂纯化栀子蓝色素的工艺研究

探讨大孔吸附树脂对栀子苷发酵液的纯化条件。以栀子蓝色素色价和含量为指标,比较了多种大孔吸附树脂对栀子蓝色素的静态吸附和洗脱效果,筛选出效果较好的D4020树脂进行动态吸附洗脱试验,对栀子蓝色素的纯化工艺进行优选。结果表明:纯化的最终条件为,上样时栀子苷发酵液A590nm为0.71,吸附流速1.5mL/min,树脂的饱和吸附量为6.5BV,用2BV的60%乙醇即可洗脱栀子蓝色素。精制的栀子蓝色素色价为130.82,得率为64.47%。     

鸭儿芹多酶氧化酶特性的研究

以鸭儿芹多酚氧化酶的基本性质为研究目标,考察了鸭儿芹多酚氧化酶的反应进程及温度、pH、底物浓度以及酶浓度对其活性的影响.结果表明:鸭儿芹多酚氧化酶在反应过程中催化速率并不随时间的增加而增大,鸭儿芹多酚氧化酶的最适活性温度为40℃,pH为6.4,酶反应底物浓度为0.1 mg·mL-1,酶浓度为0.5Iμg·mL-1.     

香蕉品种对香蕉果酱加工质量的影响

目的:探讨香蕉品种对香蕉果酱加工质量的影响,筛选适宜的加工品种;方法:以广西主栽的5个香蕉品种为原料加工成香蕉酱,对原料的理化性质、加工中多酚氧化酶的活性及产品的品质进行分析对比;结果:牛蕉含酸量和固形物含量高,褐变程度低,但没有香蕉风味;香芽蕉类香蕉具有浓郁的风味,但在加工过程中易褐变,PPO耐热性强;西贡蕉加工产品的出品率高,风味优良、色泽良好、综合评分最高;结论:牛蕉与香芽蕉类香蕉不适宜加工香蕉酱,西贡蕉是良好的香蕉酱加工品种.     

Thermal inactivation of polyphenoloxidase in pineapple puree

Prevention of browning in pineapple puree by thermal inactivation of enzyme, Polyphenoloxisase (PPO), was examined between 40 and 90 °C and in relation to exposure time. The amount of inactivation was measured as a function of time and temperature under isothermal conditions. Reaction rate constant and activation energy (E_a) as well as Decimal reduction time (D) and z-value of thermal inactivation, were determined. The rate of inactivation varied with temperatures and follows a logarithmic law. Kinetic studies showed that the thermal inactivation (40-90 °C) of the PPO followed first-order kinetics.     

藕带过氧化物酶的分离纯化及酶学性质

研究藕带中过氧化物酶(POD)的提取和纯化技术,以及温度、pH值和部分化学物质对POD酶活性的影响。结果表明:藕带中的过氧化物酶提取物经过40%饱和度的硫酸铵除杂、80%饱和度的硫酸铵沉淀、DEAE-52纤维素柱层析(100mL,0.1 mol/L NaCl的pH 7.2的磷酸缓冲溶液洗脱)、葡聚糖凝胶G-75柱层析(pH 7.2的磷酸缓冲溶液洗脱)进行分离纯化后,经SDS—PAGE鉴定为单一带,其分子量约为42kD,纯化倍数及蛋白质产率分别为32.68倍和2.11%。藕带中POD的最适温度为40℃,最适pH值为5.0。温度高于80℃基本失活,pH值小于2.0或大于8.0也基本失活。抗坏血酸对POD有明显的抑制作用;尿素、SDS、KSCN对POD具有轻微的抑制作用;草酸在低浓度时对POD具有激活作用,高于0.01 mol/L时有明显的抑制作用。     

不同对虾中多酚氧化酶的提取比较及在虾体的分布研究

用丙酮法和匀浆浸提法分别对南美白对虾和中华管鞭虾进行多酚氧化酶的提取,结果表明,在同样的提取条件下,中华管鞭虾的酶活大于南美白对虾,匀浆浸提法提取的酶活比丙酮法高1.5倍;同时对中华管鞭虾的提取条件料液比、缓冲液pH、浸提时间、水浴温度做了研究,通过正交实验得到当料液比1∶4,缓冲液pH8,浸提时间3h,水浴温度45℃条件下提取的酶具有最大的活性;进一步研究中华管鞭虾的多酚氧化酶在虾体中的分布情况,得出虾头部位多酚氧化酶含量较高,虾尾和虾身含量较低。     

绿咖啡豆中总绿原酸的纯化工艺研究

绿咖啡豆中的绿原酸共有九种同分异构体,所有同分异构体的总和称为总绿原酸,研究的主要目的是通过优化大孔吸附树脂纯化工艺将绿咖啡豆提取物中的总绿原酸含量由25%提高至70%以上,通过实验筛选合适的大孔吸附树脂,并优化树脂纯化的工艺参数如:上柱液浓度、上柱流速、解析溶剂度数、解析溶剂用量、解析流速等工艺指标,建立优化的纯化工艺路线。结果表明:上柱总绿原酸浓度为8mg/m L,上柱流速为1.0BV/h,解析用乙醇浓度为70%(v/v),乙醇用量为2BV,解析流速为1.5BV/h,经纯化后的总绿原酸经HPLC检测,总绿原酸含量可达71.23%,回收率可达87.46%。     

蜜环菌多糖分离纯化及性质的研究

进行了蜜环菌胞外多糖分离纯化及性质的研究，得到如下结果：发酵液浓缩，三们体积乙醇沉积，75％乙醇洗涤、Sevage法除蛋白、20％H2O2脱色，得到的多粗多糖上DEAE－纤维素（OH^-）柱层析，用蒸馏水、0．05－0．5mol/l的NaCl梯度洗脱，可以得到中性多糖和几种酸性多糖，中性多糖上SephadexG-150柱层析，用蒸馏水洗脱得到一种多糖A，多糖A显纯多糖，400－4000cm^-1范围内摄得多糖A的红外光谱表明其含有β－糖苷健。多糖A的完全酸水解液用硅胶GF254薄层层析，氯仿－甲醇（60：40）与丙酮－水（96：4）展层，以及纸层析，丙酮－水（96：4）展层，苯胺－二胺显色，证明其组成单糖为葡萄糖，考马斯亮兰G－250反应阴性，茚三酮反应阴性，双缩脲反应阴性，聚丙烯酰胺凝胶电泳后，考马斯亮兰R－250染色无蛋白质带，都证明其不含蛋白质。     

食品添加剂对草菇 PPO和POD活性的影响

研究了食品添加剂EDTA-2Na、Vc、酒石酸、柠檬酸、苹果酸、乳酸、醋酸、HCl、NaHSO3、NaH2PO4、Na4P2O7、CaCl2、CaSO4、CaH2PO4、FeCl3·6H2O、柠檬酸铁、乳酸亚铁对草菇多酚氧化酶(PPO)及过氧化物酶(POD)活性的影响,并对这些添加剂的作用机理进行了探讨。结果表明,10mg/L的EDTA、200mg/L的Vc、150mg/L的NaHSO3、200mg/L的Na4P2O7对PPO和POD具有十分有效的抑制作用。比较酒石酸、柠檬酸、苹果酸、乳酸、醋酸、HCl的抑制效果,以柠檬酸的抑制作用最强,HCl的抑制作用最弱。100mg/L的柠檬酸、酒石酸、苹果酸即可有效抑制PPO和POD活性。除乳酸亚铁外,CaCl2、CaSO4、Ca(H2PO4)2、FeCl3·6H2O、柠檬酸铁对草菇PPO和POD均无抑制作用,FeCl3·6H2O有较强的促进草菇PPO和POD活性的作用。     

“黑美人”马铃薯多酚氧化酶的特性研究

以邻苯二酚为底物,在413nm处测定黑美人马铃薯多酚氧化酶(PPO)的活性,研究了温度、pH、底物浓度对其活性的影响,并建立了酶促褐变反应动力学方程,探讨了L-半胱氨酸、抗坏血酸、柠檬酸、EDTA及亚硫酸氢钠五种抑制剂对酶促褐变的抑制效果。结果表明:黑美人马铃薯多酚氧化酶最适反应pH为6.3;最适反应温度为35℃;酶促褐变反应动力学符合米氏方程描述的单底物酶促反应动力学,以邻苯二酚为底物,Km=0.0011mol/L,Vmax=142.23U/min.g;动力学方程为V=142.23[S]/(0.0011+[S]);五种抑制剂均对PPO酶促褐变具有抑制作用,其强弱依次为亚硫酸氢钠>L-半胱氨酸>抗坏血酸>EDTA>柠檬酸。