Enzyme inactivation analysis for industrial blanching applications: comparison of microwave, conventional, and combination heat treatments on mushroom polyphenoloxidase activity.

Browning reactions in fruits and vegetables are a serious problem for the food industry. In mushrooms, the principal enzyme responsible for the browning reaction is polyphenoloxidase (PPO). Microwaves have recently been introduced as an alternative for the industrial blanching of mushrooms. However, the direct application of microwave energy to entire mushrooms is limited by the important temperature gradients generated within the samples during heating, which can produce internal water vaporization and associated damage to the mushrooms texture. A microwave applicator has been developed, whereby irradiation conditions can be regulated and the heating process monitored. Whole edible mushrooms (Agaricus bisporus) were blanched by conventional, microwave, and combined heating methods to optimize the rate of PPO inactivation. A combined microwave and hot-water bath treatment has achieved complete PPO inactivation in a short time. Both the loss of antioxidant content and the increase of browning were minor in the samples treated with this combined method when compared to the control. This reduction in processing time also decreased mushroom weight loss and shrinkage.     

Dose rate effect of gamma irradiation on phenolic compounds, polyphenol oxidase, and browning of mushrooms (Agaricus bisporus).

To enhance the shelf life of edible mature mushrooms, Agaricus bisporus, 2 kGy ionizing treatments were applied at two different dose rates: 4.5 kGy/h (I(-)) and 32 kGy/h (I(+)). Both I(+) and I(-) showed a 2 and 4 day shelf-life enhancement compared to the control (C). Before day 9, no significant difference (p>0.05) in L value was detected in irradiated mushrooms. However, after day 9, the highest observed L value (whiteness) was obtained for the mushrooms irradiated in I(-). Analyses of phenolic compounds revealed that mushrooms in I(-) contained more phenols than I(+) and C, the latter containing the lower level of phenols. The fluctuation of the precursors of glutaminyl-4-hydroxyaniline (GHB) was less in I(-) than in I(+). The polyphenol oxidase (PPO) activities of irradiated mushrooms, analyzed via catechol oxidase, dopa oxidase, and tyrosine hydroxylase substrates, were found to be significantly lowered (p = 0.05) compared to C, with a further decrease in I(+). Analyses of the enzymes indicated that PPO activity was lower in I(+), contrasting with its lower phenols concentration. The observation of mushrooms' cellular membranes, by electronic microscopy, revealed a better preserved integrity in I(-) than in I(+). It is thus assumed that the browning effect observed in I(+) was caused by both the decompartmentation of vacuolar phenol and the entry of molecular oxygen into the cell cytoplasm. The synergetic effect of the residual active PPO and the molecular oxygen, in contact with the phenols, allowed an increased oxidation rate and, therefore, a more pronounced browning I(+) than in I(-).     

Study and characterization of polyphenol oxidase from eggplant (Solanum melongena L.)

In this study the catecholase and cresolase activities of eggplant polyphenol oxidase (PPO) were investigated. Enzyme activity was determined by measuring the increase in absorbance using catechol as substrate and 3-methyl-2-benzothiazolinone hydrazone (MBTH) as coupled reagent. The effects of substrate specificity, heat inactivation, temperature, pH, and inhibitors were investigated to understand the enzymatic alteration of ready-to-eat preparations. Browning of vegetables was determined through a colorimeter. Decrease of lightness (L*) and increase of color difference values (ΔE*) were correlated with tissue browning. Antibrowning agents were tested on PPO under the same conditions. The enzyme activity was strongly inhibited by 0.4 M citric acid. Under natural pH conditions, the enzyme was also inhibited by tartaric acid and acetic acid. All of the results were used to understand the best conditions for food transformation (ready-to-eat and grilled eggplant slices).     

脉冲强光对多酚氧化酶活性及结构特性的影响

为探究脉冲强光(IPL)对果蔬内源酶活性的抑制效果及相关机理,采用分光光度法研究了IPL处理对多酚氧化酶(PPO)活性的影响,并通过ANS荧光探针法、内源荧光光谱法、凝胶电泳及圆二色(CD)光谱等方法探讨了不同能量IPL处理对PPO结构特性的影响。结果表明,PPO活性随着IPL单次脉冲能量、脉冲次数的增加及脉冲板间距的减小而降低。IPL处理后,PPO表面疏水性及游离巯基含量上升,色氨酸荧光强度下降,最佳发射波长红移,α-螺旋含量下降,β型结构含量上升。同时,凝胶电泳结果显示,PPO蛋白氧化降解。综上,IPL处理可改变PPO二级和三级结构,促使蛋白氧化变性,酶活性下降。本研究结果为IPL技术在抑制食品中内源酶活性及其相关机理的研究提供了一定的理论依据。     

Control of enzymatic browning in potato (Solanum tuberosum L.) by sense and antisense RNA from tomato polyphenol oxidase

Polyphenol oxidase (PPO) activity of Russet Burbank potato was inhibited by sense and antisense PPO RNAs expressed from a tomato PPO cDNA under the control of the 35S promoter from the cauliflower mosaic virus. Transgenic Russet Burbank potato plants from 37 different lines were grown in the field. PPO activity and the level of enzymatic browning were measured in the tubers harvested from the field. Of the tubers from 28 transgenic lines that were sampled, tubers from 5 lines exhibited reduced browning. The level of PPO activity correlated with the reduction in enzymatic browning in these lines. These results indicate that expression of tomato PPO RNA in sense or antisense orientation inhibits PPO activity and enzymatic browning in the major commercial potato cultivar. Expression of tomato PPO RNA in sense orientation led to the greatest decrease in PPO activity and enzymatic browning, possibly due to cosuppression. These results suggest that expression of closely related heterologous genes can be used to prevent enzymatic browning in a wide variety of food crops without the application of various food additives.     

CaCl_2和6-BA溶液处理对枣果软化褐变及相关酶活性的影响

试验探讨了鲜枣贮藏过程中果肉软化褐变与 PG活性、PPO活性的关系以及采前 Ca Cl2 和 6 - BA溶液处理对采后枣果果肉软化褐变、PG活性、PPO活性的影响。结果表明 ,枣果采后硬度逐渐下降且与贮藏时间呈极显著负相关 ,PG活性在贮藏期间呈起伏变化 ,与枣果软化无显著相关性。随贮藏时间延长果肉褐变指数逐渐增大 ,PPO活性逐渐升高 ,PPO活性与果肉软化褐变的发生相一致。采前用 1% Ca Cl2 、15 mg/kg6 - BA以及 1% Ca Cl2 +15mg/kg6 - BA对枣果进行喷雾处理 ,能降低采后枣果的 PG活性和 PPO活性 ,抑制果肉硬度的下降和果肉的褐变进程。 1% Ca Cl2 处理可使赞皇大枣贮藏末期的果肉硬度比对照增加 1.2 kg/cm2 ;1% Ca Cl2 +15 m g/kg6 - BA处理可使赞皇大枣贮藏末期的果肉硬度增加 1.5 kg/cm2 。 15 m g/kg 6 - BA处理对枣果软化褐变及 PG活性、PPO活性有一定影响 ,但效果不及 Ca Cl2 处理     

Effect of ultrahigh hydrostatic pressure on the activity and structure of mushroom (Agaricus bisporus) polyphenoloxidase

High hydrostatic pressure (HHP, treatment pressure ≤700 MPa) is approved to be the most successful commercial nonthermal processing due to its minimal modifications in nutritional and sensory quality. However, for some pressure stable enzymes such as PPO, this unique technology can hardly inactivate them at treatment pressure below of 700 MPa. This study investigated the effects of ultrahigh hydrostatic pressure (UHHP, treatment pressure >700 MPa) on the activity of Agaricus bisporus mushroom polyphenoloxidase (PPO) both in the phosphate buffer and in the mushroom puree, and on the structure of the enzyme by means of circular dichroism (CD), fluorescence emission spectra, and sulphydryl group detection. The results showed that UHHP treatment at pressure from 800 to 1600 MPa caused significant inactivation on the PPO both in the phosphate buffer and in the mushroom puree. UHHP treatment at 1400 and 1600 MPa for 1 min reduced the enzyme activity by 90.4% and 99.2% in the buffer;, however, higher enzyme activity remained in the puree after UHHP treatment at the same condition. CD and fluorescence spectra analysis showed that the secondary and tertiary structures of UHHP treated mushroom PPO were changed. The sulphydryl group (SH) detection revealed that the SH content on the surface of UHHP treated mushroom PPO was increased. It has been suggested that the inactivation of mushroom PPO by UHHP treatment at pressure higher than 1000 MPa was due to the synergistic effect of the pressure and the heat arising from pressurization, in which heat plays a major role.     

双孢菇微波冷冻干燥特性及干燥品质

为获得干燥时间短、产品质量高的蘑菇制品,采用微波冷冻干燥技术对双孢菇进行干燥处理,研究其在不同微波比功率(0.25,0.5,0.75 W/g)和系统压强(50,100,150 Pa)下的干燥曲线、有效水分扩散系数、复水比、收缩率、白度、维生素C保存率、能耗及基于模糊数学推理法下感官评定的变化规律;通过非线性拟合建立了适用于双孢菇微波冷冻干燥的数学模型;基于干燥能耗、干燥时间及部分品质指标对不同条件下双孢菇微波冷冻干燥过程进行加权综合评价。结果表明:微波比功率对干燥速率及干制品物理品质指标影响比对其他指标的影响更显著(P0.05);系统压强对干制品营养含量指标、干燥能耗以及感官评定的影响比对干燥特性的影响显著(P0.05);采用Henderson and Pabis模型能够准确(R20.9)描述干燥过程中水分变化规律;双孢菇有效水分扩散系数在10-10 m2/s数量级且受微波比功率影响更明显(P0.05);微波比功率和系统压强过高会造成双孢菇干制产品不被消费者接受;当微波比功率和系统压强分别为0.25 W/g和100 Pa时双孢菇微波冷冻干燥的综合评分值最高为0.67847,该条件较适合应用于双孢菇微波冷冻干燥中。研究探索了不同微波冷冻干燥条件下双孢菇干燥及品质特性的变化规律,为双孢菇微波冷冻干燥较优工艺参数组合的选择提供了理论依据。     

快速脱水抑制葡萄干制过程中膜脂过氧化及褐变

为探索脱水速度对无核白葡萄干制过程中膜脂过氧化作用及褐变的影响,以新疆无核白葡萄为试验材料,经过葡萄促干剂处理后,采用快速脱水和缓慢脱水2种处理,质量每减轻10%进行取样,测定脱水过程中果实干基含水率、干燥速率、褐变度、细胞膜透性、丙二醛(malonaldehyde,MDA)含量、脂氧合酶(lipoxygenase,LOX)、超氧化物歧化酶(superoxide dismutase,SOD)、过氧化氢酶(catalase,CAT)、过氧化物酶(peroxidase,POD)、多酚氧化酶(polyphenol oxidase,PPO)活性以及总酚含量的变化。结果表明:与缓慢脱水相比,快速脱水处理显著(P0.05)降低了无核白葡萄褐变度的上升,减少MDA生成量以及膜透性的增加,抑制LOX活性的升高,保持较高的活性氧清除酶SOD、CAT及POD活性及较高的总酚含量,且使PPO活性保持在一个较低的水平。因此认为,快速脱水可以有效地抑制无核白葡萄的膜脂过氧化作用对细胞膜的破坏,保持细胞的完整性,且PPO活性较低,从而减少无核白葡萄脱水褐变的发生。研究结果为快速脱水在无核白葡萄干制中的应用提供参考。     

微波预处理原料对苹果汁褐变的影响

为了防止苹果汁在贮藏过程中褐变,采用不同的微波功率和时间预处理原料苹果,研究微波预处理对苹果汁褐变的影响。结果表明,微波处理可以提高苹果汁的色值,降低多酚氧化酶的活性,引起氨基态氮含量的下降,并使苹果汁的酸度略微提高。在微波功率为720～900 W,处理时间75～125 s时,果汁色值较高。室温贮存45 d后,微波功率900 W,时间为100 s处理的果块,果汁色值为67.8,比未经微波处理的高91.5%。微波预处理是防止苹果汁褐变的一种简便安全、合理经济的加工方法。     

莲藕膨大过程中褐变酶活性的变化

本实验以3个熟性不同的莲藕品种为材料,研究了莲藕膨大过程中褐变酶活性的变化。实验表明:随着膨大节数的增加,过氧化物酶(POD)、多酚氧化酶(PPO)的活性增加;第4节藕体随着采收时间的推迟,褐变酶活性增加,且熟性不同品种之间有明显差异。并探讨了品种熟性、藕体大小和环境条件对莲藕褐变酶的影响。     

涂膜和热处理对葡萄能量和贮藏生理及品质的影响

研究不同处理对欧亚种"维多利亚"葡萄组织能量水平和生理品质的影响。采后葡萄经钙联合涂膜和热处理,即在质量分数为0.2%Ca Cl2和1%壳聚糖中浸泡20 min,45℃的0.2%Ca Cl2溶液中浸泡20 min,晾干后(4±0.5)℃贮藏,每10d测定果实的呼吸强度、褐变指数(browning index,BI)、腐烂率、硬度、丙二醛(malondialdehyde,MDA)含量、三磷酸腺苷(adenosine triphosphate,ATP)、二磷酸腺苷(adenosine diphosphate,ADP)、单磷酸腺苷(adenosine monophosphate,AMP)的含量,多酚氧化酶(polyphenol oxidase,PPO)、过氧化物酶(peroxidase,POD)、脂氧合酶(lipoxygenase,LOX)和超氧化物歧化酶(soperoxide dismutase,SOD)活性变化,以未处理为对照。结果显示对照组葡萄组织中能量物质处于亏损状态,呼吸强度减弱,细胞膜氧化增加,膜透性、BI、腐烂率增加,PPO、POD和LOX活性增加,SOD降低,果实软化。钙联合热和涂膜处理较好维持了果实的能量水平和生理品质,且涂膜优于热处理。ATP与硬度、SOD显著正相关(r=0.938,0.930,P0.01),与MDA、膜透性、LOX显著负相关(r=-0.896,-0.932,-0.940,P0.01);能荷(energy charge,EC)值与膜透性、LOX负相关(P0.05),与呼吸强度和SOD活性正相关(P0.05)。随贮藏时间的延长,葡萄组织的能量水平处于亏损状态,能量水平显著影响生理品质。经适当的采后处理可延缓能量亏损,涂膜优于热处理。     

甘薯加工过程酶促褐变及控制研究

甘薯加工过程中极易产生酶促褐变。应用分光光度法，研究了甘薯中多酚氧化酶（PPO）的特性及褐变的控制方法。结果表明，甘薯皮部的PPO活性和褐变强度是心部的2倍左右，在pH值为4.5时其活性较低。在甘薯淀粉生产过程中，结合去皮工艺，分别选择添加0.01%～0.05%柠檬酸或0.005%～0.01%抗坏血酸或0.05%～0.1%亚硫酸钠，均可有效控制PPO产生的褐变，提高甘薯淀粉的质量。     

多酚氧化酶高强度脉冲磁场灭活及动力学模型

为了找到一种有效控制果蔬中多酚氧化酶（PPO）活性的方法,该文研究了高强度脉冲磁场（PMF）对PPO活性的影响,并进行了灭酶动力学模型的研究。结果表明,当PPO于磁场强度2.5、3.5和4.5特斯拉（T）分别处理5至40个脉冲时,酶的残余活性随着磁场强度和脉冲数的增加而逐渐降低。在4.5 T处理40个脉冲时,酶的灭活率最高达到93.10%。对灭活动力学曲线分别用Bigelow模型、Weibull模型和Hülsheger模型进行拟合,发现Weibull模型对PMF下PPO的灭活的拟合度最好。可见,高强度脉冲磁场可以作为一种有效杀灭果蔬中多酚氧化酶的非热技术,且酶的灭活过程符合Weibull模型,该模型可以为实际应用提供参考。     

CaCl2和6-BA溶液处理对枣果软化褐变及相关酶活性的影响

试验探讨了鲜枣贮藏过程中果肉软化褐变与PG活性、PPO活性的关系以及采前CaCl₂和6-BA溶液处理对采后枣果果肉软化褐变、PG活性、PPO活性的影响。结果表明，枣果采后硬度逐渐下降且与贮藏时间呈极显著负相关，PG活性在贮藏期间呈起伏变化，与枣果软化无显著相关性。 随贮藏时间延长果肉褐变指数逐渐增大，PPO活性逐渐升高，PPO活性与果肉软化褐变的发生相一致。采前用1% CaCl₂、15 mg/kg 6-BA以及1% CaCl₂+15 mg/kg 6-BA对枣果进行喷雾处理，能降低采后枣果的PG活性和PPO活性，抑制果肉硬度的下降和果肉的褐变进程。1% CaCl₂处理可使赞皇大枣贮藏末期的果肉硬度比对照增加1.2 kg/cm²；1% CaCl₂+15 mg/kg 6-BA处理可使赞皇大枣贮藏末期的果肉硬度增加1.5 kg/cm²。15 mg/kg 6-BA处理对枣果软化褐变及PG活性、PPO活性有一定影响，但效果不及CaCl₂处理。     

超高压联合高密度CO2处理钝化对虾多酚氧化酶

为了弥补超高压(UHP,ultra high pressure)钝化凡纳滨对虾多酚氧化酶(PPO,polyphenol oxidase)效果差的缺点,同时利用高密度CO_2钝化凡纳滨对虾PPO的优势,初步研究UHP+CO_2处理对凡纳滨对虾PPO的钝化效果,以探讨UHP+CO_2联合处理用于开发虾类新产品的可行性。研究结果表明:UHP+CO_2联合处理比单独CO_2处理和UHP处理更能有效地钝化PPO;100 MPa UHP+CO_2联合处理30 min,PPO相对酶活降至18.92%±1.52%;200 MPa UHP+CO_2联合处理10 min,PPO相对酶活降至10.91%±1.08%;300 MPa UHP+CO_2联合处理10 min,PPO被钝化95%;400 MPa UHP+CO联合处理5 min,PPO被钝化97%;500 MPa UHP+CO联合处理10 min,PPO100%被钝化;与单独UHP处理相比,UHP+CO_2联合缩短了处理时间,提高了钝化PPO的效果;PPO经UHP+CO_2联合处理后在4℃贮藏6 d后活性未见恢复,说明PPO在处理过程中发生了不可逆的变性失活。研究结果为虾类的贮藏和加工以及开发新产品提供基础数据和技术参考。     

双孢菇高氧动态气调保鲜参数优化

为提高双孢蘑菇保鲜效果,在(2±1)℃下采用高氧动态气调方法对双孢蘑菇进行贮藏试验,选取第1阶段不同O_2/CO_2比例的气体组分、换气时间、第2阶段不同O_2/CO_2比例的气体组分作为影响因素,考察双孢蘑菇贮藏过程中总色差ΔE、硬度以及感官品质的变化,对双孢蘑菇的动态气调参数进行优化。研究结果显示,第1阶段O_2比例高于80%时,可显著抑制双孢蘑菇ΔE的上升(P0.05);第2阶段80%O_2+20%CO_2气体组分,可使双孢蘑菇的ΔE显著低于其他处理,硬度显著高于其他处理(P0.05);而换气时间则影响较小。最优的动态气调条件为:第1阶段气体组分100%O_2,第3天换气,第2阶段气体组分80%O_2+20%CO_2,双孢蘑菇的ΔE、硬度值和感官品质评分分别为:19.40、6.84×105 Pa和9.52。将此最优动态气调运用于双孢蘑菇的保鲜,研究发现,与静态气调相比,该动态气调显著抑制了双孢蘑菇的呼吸速率与膜结构的损坏,维持了较高的过氧化物酶、抗坏血酸过氧化物酶活性与较低的多酚氧化酶活性,有效抑制了贮藏过程中的酶促褐变,保持了较高的可溶性蛋白含量,并延长其保鲜期至28 d,提高了双孢蘑菇的耐贮性。     

Characterization of a tomato polyphenol oxidase and its role in browning and lycopene content

Polyphenol oxidase (PPO) was extracted from five Sicilian varieties of tomato fruit [Pizzutello, Naomi (Hazera), F1 PS212 (Peto seed), Rosa Maletto, and PO228] and assayed with a method using 3-methylbenzothyazolinone hydrazone (MBTH) as chromophore coupling agent. 3,4-Dihydroxyphenylacetic acid was chosen for tomato PPO activity determination. The tomato PPO had maximum activity at pH 4.8. The pH of juice in ripe fruits is between 4.1 and 4.4, a range in which PPO relative activity is between 74 and 87%. The optimum temperature of activity for tomato PPO was 40 degrees C; the enzyme showed a good relative activity (55% of the maximum) at cold-storage temperature (4 degrees C). PPO retained 82% relative activity at an NaCl concentration of 0.1 M; at higher concentrations the PPO became gradually inactivated. The commercial variety Naomi is more susceptible to enzymatic browning than the local varieties Pizzutello, Rosa Maletto and PO228, due to higher PPO activity levels. This result confirms the suitability of these local tomato varieties to national markets. Results from storage tests seem to relate PPO activity with color changes associated with browning and lycopene degradation, because lycopene is an antioxidant agent that reconstitutes the polyphenols oxidized by the action of PPO.     

石榴果实发育期果皮褐变及相关酶活性变化

为探明石榴果实发育期果皮褐变及其相关酶活性、总酚和抗氧化能力的变化规律及相互关系,以泰山红和泰山三白甜石榴为试材,研究各发育期果皮褐变度、多酚氧化酶(PPO)活性、过氧化物酶(POD)活性、苯丙氨酸解氨酶(PAL)活性、总酚含量和DPPH自由基清除率的变化。结果表明,2个品种果皮褐变度、PPO活性、POD活性、总酚含量和DPPH自由基清除率随着发育天数的增加逐渐降低,均在7月15日达到最高。2个品种果实发育期果皮PAL活性呈先降低后升高再降低的变化趋势,分别在7月15日和9月13日出现峰值。泰山红抗褐变能力高于泰山三白甜,二者分别在9月23日和9月13日采收能减轻褐变的发生。相关性分析表明,PAL、PPO、POD、总酚和DPPH自由基清除率均与石榴果实酶促褐变密切相关,石榴果实酚类物质含量高低与其抗氧化能力极显著正相关。本研究结果为深入揭示石榴果实褐变机理提供了理论依据。